2186 457 PB 1

Jurnal Farmasi dan Ilmu Kefarmasian Indonesia (Pharmacy and Pharmaceutical Sciences Journal)
J
Jurnal Farmasi dan urnal Farmasi dan Ilmu Kefarmasian
Indonesia (Pharmacy and Pharmaceutical
Ilmu Kefarmasian Indonesia
Sciences Journal) P-ISSN: 2406-9388; E-
Chief Editor: ISSN: 2580-8303 is an official journal
Elida Zairina, S.Si, MPH., Ph.D., Apt. published by the Faculty of Pharmacy,
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Email: jfiki@ff.unair.ac.id
i
Table of Content
No Title Page
1. Isoprinosine along with Favipiravir or Oseltamivir in Patients with Moderate 209-219

Covid-19 at RSD Dr. Soebandi Jember
Chrysnanda Maryska, Didik Hasmono, Sofiati Diah Baisuni, Ayunda Nur

Hidayatiningsih, Arina Dery Puspitasari, Retna Dwi Puspitarini, Budi Suprapti
2. The Effect of Decyl Glucoside on Stability and Irritability of Nanostructured 220-228

Lipid Carriers-Green Tea Extract as Topical Preparations
Rufaidah Azzahrah, Noorma Rosita, Djoko Agus Purwanto, Widji Soeratri
3. Effect of Alpha-Lipoic Acid on the Characteristics and Physical Stability of 229-234

NLC-Green Tea Extract
Fairuz Yaumil Afra, Widji Soeratri, Djoko Agus Purwanto
4. Optimization Method and Stability Test to Determinate Luteolin, Quercetin, 235-241

Apigenin, and Sinensetin Levels in Herbal Medicines Using TLC-Densitometry
Muhammad Hidayatullah, Mochammad Yuwono, Riesta Primaharinastiti
5. The Effect of Insulin Administration on Medication Adherence in Type 2 242-251

Diabetes Mellitus Patients with Neurological Complications
July, Rani Sauriasari, Nadia Farhanah Syafhan, Hadijah Tahir
6. The Influence of Feeling Lonely and Received Social Support on Medication 252-261
Adherence in Elderly with Hypertension
Dwi Novita Sari, Wahyu Utami, Elida Zairina
7. Sunscreen Cream Formulation of Noni Leaf Extract (Morinda citrifolia L.) with 262-271
Emulsifier Combination of Tween 80 and Lecithin
Bianca Levie Tania, Rini Dwiastuti, Agatha Budi Susiana Lestari, Dewi Setyaningsih
8. Mannitol Production from Fructose by Using Resting Cells of Methylotrophic 272-278

Yeasts
Nisa Yulianti Suprahman, Khairul Basyar, Herman Suryadi
ii
9. Jelly Candy Hydroxyapatite from Mackerel Fish Bone 279-289
Lia Anggresani, Santi Perawati, Ryan Afandi, Rahmadevi
10. Physicochemical Properties and Antioxidant Activity of Three Types of Monoflora 290-297
Honey from Indonesia
Sulistyianingsih, Achmad Toto Poernomo, Riesta Primaharinastiti
11. Assessing the Neurotoxicological Effect of the Acute Paraquat Aerosols Exposure 298-304
in Causing Parkinsonism on Mouse through Behavioral Assays
Sherlo Adha Maulana, Santi Nurul Kamilah, Choirul Muslim, Aceng Ruyani, R. R. Sri
Astuti
12. Viability and Antibacterial Activity of Bifidobacterium bifidum in Fermented 305-313

Robusta Coffee for Diarrhea Treatment
Miksusanti, Elsa Fitria Apriani, Dimas Nur Hidayat
13. Knowledge and Perception on Overclaim against the Behaviors of Implementing 314-322
the COVID-19 Prevention Protocol Communities in Indonesia
Elsa Christina, Didik Setiawan, Dina Ratna Juwita, Lianawati
iii
Jurnal Farmasi dan Ilmu Kefarmasian Indonesia Vol. 9 No. 3 December 2022, 209-219
DOI: 10.20473/jfiki.v9i32022.209-219
Available online at https://e-journal.unair.ac.id/JFIKI/
Isoprinosine along with Favipiravir or Oseltamivir in Patients with

Moderate Covid-19 at RSD Dr. Soebandi Jember
Chrysnanda Maryska1, Didik Hasmono2*, Sofiati Diah Baisuni3, Ayunda Nur Hidayatiningsih3, Arina Dery
Puspitasari2, Retna Dwi Puspitarini4, Budi Suprapti2
1
Master Program of Clinical Pharmacy, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
2
Department of Pharmacy Practice, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
3
Department of Pharmacy, RSD dr. Soebandi Teaching Hospital, Jember, Indonesia
4
Department of Pulmonology, RSD dr. Soebandi Teaching Hospital, Jember, Indonesia
*Corresponding author: didik-h@ff.unair.ac.id
Submitted: 12 February 2022

Accepted: 23 March 2022
Published: 9 December 2022
Abstract
Background: Isoprinosine is an immunomodulator that is now being used to treat Covid-19 patients. Objectives:
To evaluate Isoprinosine with Favipiravir or Oseltamivir in moderate Covid-19. Methods: In a retrospective
observational analysis, in-hospital moderate Covid-19 patients treated between June 2020 and June 2021 were
included. Results: Inclusion criteria for 364 patients were met, with 135 receiving Favipiravir-Isoprinosine
(Group 1) and 229 receiving Oseltamivir-Isoprinosine (Group 2). In group 1, the majority of patients (58.50%)
were female (35.60%), had no comorbidities (71.60%), were discharged with a positive PCR (74.80%), did not
require a breathing apparatus (99.26%), had leukocyte levels between 4,5-11,0 (82.22%), lymphocyte levels
between 25-33 (34.07%), and were discharged with no ground-glass opacity (34.07%) (54.10%), LOS was 9-13
days (50.37%), while the mortality rate was 0.70%. In group 2, the majority of patients were male (54.10%),
with the highest age range being 42-56 years (35.80%), without comorbidities (69.0%), discharged with a
positive PCR (72.50 %), and without the need for a breathing apparatus (99.13%), with leukocyte levels ranging
from 4.5 – 11.0 (81.22 %), with lymphocyte levels ranging from 25.0 – 33.0 (26.20 %), and were discharged with
no ground-glass opacity (49.34 %), LOS was 9 - 13 days (34.06 %), and the mortality rate was
0.87%.Conclusion: In this trial, it was determined that combining isoprinosine with antivirals favipiravir or
Oseltamivir could produce significant clinical improvement.
Keywords: isoprinosine, covid-19, moderate, immunomodulator
How to cite this article:

Maryska, C., Hasmono, D., Baisuni, S. D., Hidayatiningsih, A. N., Puspitasari, A. D., Puspitarini, R. D. &
Suprapti, B. (2022). Isoprinosine along with Favipiravir or Oseltamivir in Patients with Moderate Covid-19 at RSD
Dr. Soebandi Jember. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 209-219.
http://doi.org/10.20473/jfiki.v9i32022.209-219
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Jurnal Farmasi dan Ilmu Kefarmasian Indonesia Vol. 9 No. 3 December 2022 210
INTRODUCTION Thus, favipiravir can be used as an antiviral in treating

From March 2020 to the present, a virus has Covid-19.
caused a global pandemic. SARS-CoV-2 is the virus, Oseltamivir is available in oseltamivir phosphate, a
and the World Health Organization has named the prodrug metabolized by plasma and hepatic esterase to
disease coronavirus disease 2019 (Covid-19) (WHO) oseltamivir carboxylate, the active form. Oseltamivir is
(Yuki et al., 2020). The first case in Indonesia was approved for influenza A and B treatment and
discovered in March 2020, consisting of two prevention and is available in oral formulations. After
individuals, and the second case was discovered on the replication cycle, influenza viruses require the
March 6, 2020, consisting of two individuals. Thus far, enzyme neuraminidase to generate new viruses from
Covid-19 cases have continued to rise (Burhan et al., infected cells. The active metabolite of Oseltamivir,
2020). Coronavirus is a virus with a single-stranded oseltamivir carboxylate, interacts with neuraminidase,
RNA genome isolated from various animal species. altering its conformation and inhibiting its activity.
The number of cases continues to grow over time, Inhibiting neuraminidase causes viral aggregation on
confirming that this virus can be transmitted from the cell surface, reducing virus spread in the respiratory
human to human (Burhan et al., 2020). tract. Oseltamivir is in vitro activity against SARS-
Covid-19 is an infectious disease that primarily CoV-2 is unknown. In contrast to influenza,
affects respiratory and other organs. Covid-19's clinical coronavirus lacks neuraminidase. Oseltamivir was used
manifestations and severity vary considerably. as an empirical therapy during the Covid-19 pandemic
Asymptomatic, mild, moderate, severe, and critical in China before the identification of a causative agent
were used to classify the severity. As is the case with of the SARS-CoV-2 virus and because the pandemic
infectious diseases in general, the immune system occurred during the influenza season in China (Instiaty
plays a crucial role in the fight against viruses et al., 2020)
(Yazdanpanah et al., 2020). SARS-CoV-2 can suppress Immunomodulators are also used in patients with
the host immune system by inducing programmed cell Covid-19. Isoprinosine is an immunomodulator used in
death (apoptosis) (Mortaz et al., 2020). patients with Covid-19 (Dluholucky, 2020).
Numerous drugs, including lopinavir-ritonavir, Isoprinosine, also known as Inosine Pranobex or
remdesivir, hydroxychloroquine, and Azithromycin, Methisoprinol, is a synthetic purine derivative with
have been tested in clinical trials to treat Covid-19 and immunomodulatory and antiviral properties. Based on
be curative. Additional numerous medicines are in vivo studies, it is known that the use of isoprinosine
undergoing clinical trials to determine whether they results in an increase in immunity or
can be used as a definitive or adjunctive therapy (Yuki immunityIsoprinosine enhances immunity by
et al., 2020). PDPI (Perhimpunan Dokter Paru stimulating T lymphocyte differentiation into cytotoxic
Indonesia-Indonesian pulmonary doctors association) T cells and T helper cells and increasing lymphokine
has established guidelines for managing Covid-19 production. Additionally, isoprinosine enhances the
patients with varying degrees of severity in Indonesia. natural killer function of cells. Isoprinosine boosts
It has been updated several times in response to humoral immunity by stimulating B lymphocyte
scientific advances (Burhan et al., 2020). Favipiravir, differentiation into plasma cells, increasing antibody
Oseltamivir, and remdesivir are currently available and production. It increases the IgG and complement
widely used antivirals for Covid-19 patients. However, surface markers, and enhances the chemotaxis and
this study is limited to favipiravir and Oseltamivir. phagocytosis of neutrophils, monocytes, and
Favipiravir is an antiviral prodrug. Intracellularly, macrophages (Petrova et al., 2010). Numerous studies
favipiravir is ribosylated and phosphorylated to form have demonstrated that administering isoprinosine as
the active form, favipiravir ribofuranosyl-5- adjuvant therapy may benefit specific subgroups of
triphosphate (favipiravirRTP) (Instiaty et al., 2020). patients, particularly those who frequently experience
Favipiravir's mechanism of action as a selective immune dysfunction due to viral infections (Lasek et
inhibitor of RNA-dependent RNA polymerase (RdRp) al., 2015). According to a study conducted in nursing
from SARS-CoV-2, where the RdRp in SARS-CoV-2 homes in the Czech Republic, the case fatality rate was
is tenfold more active than that in other viruses significantly reduced by up to 11.9 percent, with an
(Shannon et al., 2020). FavipiravirRTP inhibits the odds ratio (OR) of 2.8 (Beran et al., 2020)
RdRp virus by binding to it and inhibiting viral genome There is a dearth of research on the antiviral
transcription and replication (Instiaty et al., 2020). and Isoprinosine effects of Covid-19. Hence, this study
P-ISSN: 2406-9388
E-ISSN: 2580-8303
evaluates Isoprinosine with Favipiravir or Oseltamivir Tools

in moderate Covid-19. The data collection process was documented in
Microsoft Excel spreadsheets. Patient identifiers have
MATERIALS AND METHODS been used in place of initials to maintain patient
Materials confidentiality. A total of 364 patients were sampled.
Study design The data analysis was then presented in a table.
This retrospective observational study used Statistical examination
medical records from patients with moderate Covid-19 In this study, both parametric and non-parametric
at the RSD dr. Soebandi Teaching Hospital in Jember. statistical tests were used.
It took place between June 2020 and June 2021.
Study population and setting RESULTS AND DISCUSSION
This study enrolled adolescent (12 years), adult, Descriptive analysis
and elderly patients with a moderate diagnosis of All data were subjected to descriptive analysis. A
Covid-19 who were hospitalized at RSD dr. Soebandi. therapy group then classified the data. Group 1
Data source and data extraction comprises patients who are receiving treatment with
This study analyzed data from 13 months of Favipiravir – Isoprinosine. While Group 2 contains
medical records (June 2020 – June 2021) with a time- patients who received Oseltamivir – Isoprinosine
limited sampling. The Faculty of Pharmacy at therapy. The analysis of the description yielded the
Universitas Airlangga has approved this study conclusions that can be seen in Table 1.
ethically. The demographic information about the Based on the data in Table 1, It is known that the
patient, the medication, the laboratory data, the clinical majority of members of group 1 are female (58.50%),
data, the PCR test, the Photo Thorax, the length of stay, the majority are between the ages of 18-60 years
and the death rate were all collected. Gender, age, (88.89%), and the majority are free of comorbidities
residence, and comorbidities are all included in (71.60%). Meanwhile, in group 2, the majority of
demographic profiles. Medication data are records of participants were male (54.10%), the median age range
medications prescribed to patients while undergoing was 18-60 years (85.59%), and the majority did not
treatment at the hospital. Laboratories data includes have any comorbidities (69.00%).
leukocyte and lymphocyte counts. Clinical data are A descriptive analysis of clinical data, laboratory
records of patients' oxygen demand levels, which are data, supporting examination data, LOS data, and
classified as mild (1 – 6 lpm), moderate (7 – 10 lpm), patient mortality rates were also carried out. Table 2
or severe (11 – 15 lpm). Patients receiving isoprinosine summarizes the analysis's findings.
therapy, the standard treatment for moderate Covid-19 As shown in Table 2, KRS patients with clinical
patients, adolescent patients, adult patients, and older conditions in groups 1 and 2 do not require oxygen.
adults, were included in this study. Prior to receiving combination therapy, most patients
in both groups had an oxygen demand of 1-6 lpm.
Table 1. The use of isoprinosine in combination with antivirals in the treatment of patients with moderate Covid-19
Favipiravir-Isoprinosine
Characteristics Oseltamivir-Isoprinosine Combination
Combination
Man 56 (41.50%) 124 (54.10%)
Gender
Woman 79 (58.50%) 105 (45.90%)
12 - 17 5 (3.70%) 4 (1.75%)
18 - 60 120 (88.89%) 196 (85.59%)
Age
> 60 10 (7.41%) 29 (12.66%)
Mean ± SD 39.6 ± 12.92 42.00 ± 14.14
No Comorbid 91 (71.60%) 147 (69.00%)
Comorbid Hypertension 25 (19.70%) 34 (16.00%)
Diabetes Mellitus 11 (8.70%) 32 (15.00%)
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Table 2. Clinical data on isoprinosine in combination with favipiravir or oseltamivir

Favipiravir-Isoprinosine Oseltamivir-Isoprinosine
Characteristics
Combination Combination
Mild 69 (51.10%) 151 (66.00%)
Oxygen Pre Moderate 49 (36.30%) 55 (24.00%)
Consumption Severe 17 (12.60%) 23 (10.00%)
Level No Requirement for
Post 134 (99.26%) 227 (99.13%)
Oxygen
Table 3. Laboratory data on isoprinosine in combination with favipiravir or oseltamivir

Characteristics
< 4.5 13 (9.63%) 18 (7.86%)
4.5 – 11.0 104 (77.04%) 177 (77.29%)
Pre
>11.0 18 (13.33%) 34 (14.58%)
mean ± SD mean 7.50 ± 3.35 mean 7.85 ± 3.69
Leukocytes
< 4.5 6 (4.45%) 4 (1.75%)
4.5 – 11.0 111 (82.22%) 186 (81.22%)
Post
>11.0 18 (13.33%) 39 (17.03%)
mean ± SD mean 8.52 ± 3.66 mean 8.90 ± 3.75
< 25.00 86 (63.70%) 141 (61.57%)
25.00 – 33.00 25 (18.52%) 50 (21.83%)
Pre
>33.00 24 (17.78%) 38 (16.60%)
mean ± SD mean 22.72 ± 11.51 mean 22.19 ± 10.76
Lymphocytes
< 25.00 52 (38.52%) 109 (47.60%)
25.00 – 33.00 46 (34.07%) 60 (26.20%)
Post
>33.00 37 (27.41%) 60 (26.20%)
mean ± SD mean 27.10 ± 9.84 mean 25.79 ± 11.34
According to Table 3, leukocyte levels in group 1 in the two combination groups. Similarly, the number
were between 4.5 -11.0 (82.22%), and lymphocyte of patients with positive PCR results decreased in both
levels were between 25.0-33.0. (34.07%). Meanwhile, combination groups.
in group 2, the leukocyte count was between 4.5 - 11.0 Parametric difference tests were performed on data
(81.22%), and the lymphocyte count was between 25.0 with interval or ratio types, such as leukocyte counts,
- 33.0 (26.2%). Normal leukocytes and lymphocytes lymphocyte counts, and patient length of stay. The
levels increased in both combinations before and after parametric difference test was also performed on
the patient received therapy. normally distributed and homogeneous data. The
According to Table 4, the LOS for group 1 was Paired t-test was used to determine the parametric
between 9 and 13 days (50.3%), and the percentage of difference in this study. The non-parametric difference
patients who died was 0.70%. While in group 2, the test can be used with data that are not normally
median LOS was 9 - 13 days (34.0%), and the distributed or homogeneous; it can also be used with
mortality rate was 0.87%. ordinal or interval/ratio scales. The Wilcoxon test is
In Table 5, it is stated that in group 1, patients used to determine non-parametric differences in this
discharged with positive PCR (74.80%) and no study. If the p-value for the paired t-test or Wilcoxon
infiltrates on chest X-rays were included (54.10%). In test is less than 0.05, the test is considered significant.
group 2, patients discharged with a positive PCR Because there is no data prior to and following the
(72.50%) and no infiltrates on chest X-rays were procedure, the LOS variable and the patient's mortality
included (49.34%). Following treatment, the number of rate cannot be tested independently. Table 6 contains
patients developing pneumonia decreased significantly the results of statistical tests conducted in this study.
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Table 4. Characteristics of LOS data and mortality rates in isoprinosine-favipiravir- or oseltamivir-combination

Characteristics
< 9 hari 3 (2.22%) 10 (4.37%)
Length 9 - 13 hari 68 (50.37%) 78 (34.06%)
of Stay > 13 hari 64 (47.41%) 141 (61.57%)
mean ± SD mean 13.0 ± 3.27 mean 17.22 ± 11.45
Death 1 (0.70%) 2 (0.87%)
Table 5. Other test data characteristics of isoprinosine in combination with favipiravir or oseltamivir
Favipiravir- Oseltamivir-
Characteristics Isoprinosine Isoprinosine
Pre Positive 135 (100%) 229 (100%)
PCR Positive 101 (74.80%) 166 (72.50%)
Post
Negative 34 (25.50%) 63 (27.50%)
Pneumonia 87 (64.40%) 159 (69.43%)
Pre
chest No Ground Glass Opacity 48 (35.60%) 70 (30.57%)
photograph Pneumonia 62 (45.90%) 116 (50.66%)
Post
No Ground Glass Opacity 73 (54.10%) 113 (49.34%)
When a patient is hospitalized for a suspected Nevertheless, only 364 medical records met the criteria
infection, isoprinosine is given. After confirming the for inclusion. The following parameters were examined
diagnosis of Covid-19 with two PCR tests, isoprinosine in this study: supporting data in the form of PCR and
was discontinued, and standard Covid-19 therapy was thorax photograph; laboratory data in the form of
initiated according to the applicable Clinical Pathway leukocyte and lymphocyte; clinical data from patients
at RSD dr. Soebandi. The standard therapy is in the form of oxygen demand level; length of stay
Azithromycin iv 1 x 500 mg, Oseltamivir po 2 x 75 mg patient mortality.
(for 7-14 days)/Lopinavir 400 mg-Ritonavir 100 mg Gender is recognized as a significant factor in the
p.o 2 x 2 tabs for 7-14 days/Favipiravir p.o loading epidemiology of various diseases. This also holds for
dose of 2 x 1600 mg (first day) and then 2 x 600 mg on Covid-19. According to studies conducted in China and
days 2 - 5, Vitamin C p.o 1 x 500 mg, and other Europe, men die at a higher rate from Covid-19
medications as indicated by the patients. After antiviral infection than women and are independently associated
therapy was completed, Isoprinosine was continued; it with poor Covid-19 disease progression. This is
was given on day six following favipiravir in group 1 because women and men have different immune
and on day eight following favipiravir in group 2. responses. Additionally, male-specific behaviours such
There was no group of patients receiving as smoking, drinking alcohol, or consuming alcoholic
Lopinavir/Ritonavir in this study. Lopinavir/Ritonavir beverages increase the risk of death from Covid-19
was used in several Covid-19 patients at dr. Soebandi, (Vahidy et al., 2021). Women are predisposed to
but the government has made the case that the antiviral autoimmune diseases, whereas men are predisposed to
should be reserved for HIV patients only. infectious diseases. Acceptable explanations include
As is the case with infectious diseases in general, the X chromosome's protection and the female
the immune system plays a critical role in the fight hormone estrogen. Estrogen is known to have the
against viruses (Yazdanpanah et al., 2020). SARS- potential to mitigate the severity of SARS-CoV-2
CoV-2, on the other hand, can impair the host immune infection. Several studies indicated that women had
system by suppressing T cell function and inducing superior anti-inflammatory, antiviral, and humoral
programmed cell death (apoptosis) (Mortaz et al., system responses during the infection process
2020). Thus, immunomodulatory agents may be compared to men. It is well-established that
considered in the treatment of Covid-19 patients. testosterone can impair a man's response to vaccines
Isoprinosine is one of the immunomodulators currently (Ciarambino et al., 2021). The majority of sexes in
being used in Covid-19 patients. group 1 were female, while most in group 2 were male.
The study included 760 medical records of
moderate Covid-19 patients as a total sample.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Table 6. Analysis of variables with dependent variables

Sig. of
Sig. of
Favipiravir
Different Test Oseltamivir and
No. Variable Significance and
Test Type Isoprinosine in
Isoprinosine in
Combination
Combination
Non-
1. PCR Wilcoxon p-value < 0.05 0.000 0.000
Parametric
Oxygen Consumption Non-
2. Wilcoxon p-value < 0.05 0.000 0.000
Level Parametric
Non-
3. Leukocytes Wilcoxon p-value < 0.05 0.010 0.000
Parametric
Lymphocytes Paired 0.000 0.000
4. Parametric p-value < 0.05
correlation coefficient t-test 0.318 0.520
Non-
5. chest photograph Wilcoxon p-value < 0.05 0.000 0.000
Parametric
6. LOS - - - -
7. Death - - - -
In contrast to numerous previous studies, the the mortality rates associated with worsening Covid-19
number of male patients infected with Covid-19 is progression (Surendra et al., 2021). This is based on
significantly greater than that of female patients. This existing research data, which indicates that group 1 has
could have occurred due to the study's focus on a mortality rate of 0.7% and group 2 has a mortality
moderate Covid-19 patients. Consequences of men's rate of 0.87%. According to a study, the mortality rate
vulnerability to disease worsening severe or critical at age 15 is 0.6%, at age 50 is 39.5%, and at all age
disease. This, however, requires additional research. groups is 6% (Beran et al., 2021).
In this study, the average age of patients with All patients in this study had been discharge in a
moderate COVID-19 infection was 42 years, with a non-oxygen-requiring state, with the highest
range of 12-81 years and a standard deviation of 14. In percentages in group 1 (96.26%) and group 2 (96.26
general, increasing age is associated with increasing percent) (99.13%). No studies demonstrate
the severity of Covid-19. Children are generally less Oseltamivir's efficacy in reducing the need for
susceptible to Covid-19 due to the still-functioning breathing apparatus in Covid-19 patients. According to
general system and lack of exposure to the surrounding one systematic review study, there is no evidence that
environment during the Pandemic (Davies et al., 2020). favipiravir can reduce patients' need for breathing
A study conducted in Italy shows that higher age is one apparatus (Özlüşen et al., 2021). However,
of the independent factors associated with the risk of Hassanipour et al. found a clinically significant
death (Aksel et al., 2021). Another study confirmed improvement in oxygen demand between the
that Covid-19 patients aged 50 years were 15.4 times favipiravir and the control groups 7 days after
more likely to die than confirmed Covid-19 patients hospitalization (RR = 1.24, 95 percent CI = 1.09-1.41;
aged 50 years (Biswas et al., 2021). P = 0.001). After 14 days of hospitalization, viral
Comorbidity is a predictor of Covid-19 patient clearance occurred in the favipiravir group but was not
progression. The percentage of samples without statistically significant (RR = 1.11, 95 percent CI =
comorbidities was highest in both groups, namely 0.98-1.25; P = 0.0094). The favipiravir group had a 7%
group 1 (71.60%) and group 2 (69.00%). In group 1, lower oxygen demand than the control group (RR =
hypertension was the most prevalent comorbidity 0.93, 95% CI = 0.67-1.28; P = 0.664). The WHO
(19.70%), while in group 2, hypertension was the least recommends oxygen therapy for patients with
prevalent (16.00%). Diabetes Mellitus is the most respiratory distress, hypoxemia, or shock with a target
common comorbid condition, with a percentage of SpO2 of greater than 94%. The favipiravir group had a
8.70% in group 1 in group 2 15.00%. Age and pre- 7% lower oxygen demand than the control group (RR =
existing comorbidities such as hypertension, diabetes, 0.93, 95% CI = 0.67-1.28; P = 0.664). Appropriate
obesity, heart disease, chronic kidney disease, and liver oxygen therapy allows for increased oxygenation of
disease worsen Covid-19 progression. According to thickened and inflamed lung tissue, effectively treating
studies conducted in China, North America, and hypoxemia (Long et al., 2021). Shortness of breath is
Europe, the patient's age and comorbidities influence caused by SARS-CoV-2-mediated mitochondrial
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damage in smooth muscle cells of the pulmonary as well as an increase in lactate dehydrogenase (LDH)
arteries, resulting in impaired pulmonary hypoxic and c-reactive protein (CRP), but normal procalcitonin
vasoconstriction (Shianata et al., 2021). Isoprinosine is (PCT). COVID-19 is classified as severe when LDH,
administered following antiviral therapy, hoping the CRP, D-dimer, and IL6 levels increase while platelets
virus-cleansing process will be accelerated by boosting and lymphocytes decrease. The percentage of
the body's immune system. Although there is no lymphocytes in the blood can be considered a reliable
literature directly related to the level of patient oxygen and accurate indicator for grading Covid-19 patients.
demand, the KRS profile of patients who no longer Low lymphocytes can express the primary SARS-CoV-
require oxygen is one of the clinical conditions that can 2 receptor, the enzyme angiotensin II (ACE2). SARS-
describe a patient's lung function improvement. CoV-2 can also enter lymphocytes via an ACE2-
The significance of groups 1 (0.010) and 2 (0.000) independent pathway. Both SARS-CoV-2 and
is well established. On this basis, it can be concluded immune-mediated immunity have mechanisms that can
that each combination results in a difference in result in lymphopenia by impairing lymphocyte
leukocyte levels prior to and following therapy, both in production, survival, and redistribution of
group 1 and group 2. The normal leukocyte count lymphocytes. In Covid-19 patients, metabolic and
ranges from 4.5 to 11.0 in RSD dr. Soebandi. biochemical changes may also affect lymphocyte
According to existing clinical reports, there are production and survival. Lymphopenia can result in
changes in the leukocyte portion of the circulation in immunosuppression and initiate a cytokine storm; these
patients with mild and severe degrees. These changes factors contribute significantly to viral persistence,
can take the form of values falling below or exceeding replication, multi-organ failure, and death (Beran et al.,
normal levels (Wang et al., 2020). Between before and 2021).
after therapy, the percentage of patients with normal There is a bias in the LOS variable due to the
leukocyte counts increases. Covid-19 management guidelines in Indonesia,
The combination of Isoprinosine Favipiravir and according to PDPI. At the pandemic's start, patients
Isoprinosine Oseltamivir is known to have a significant were permitted to KRS if their PCR results were
value of 0.000. Thus, it can be concluded that each negative for two consecutive days. Then, in PDPI ed 2,
combination results in differences in lymphocyte levels the criteria changed to allow patients to KRS if they
prior to and following treatment with Isoprinosine have been free of symptoms (requiring treatment for
Favipiravir and Isoprinosine Oseltamivir. The both Covid-19 and comorbid diseases) for three days
correlation values for each combination must be even though the PCR results remain positive (Burhan
compared to determine which combination has a et al., 2020). A different test cannot be conducted on
greater effect on the patient's lymphocyte levels. The the LOS variable because there is no prior and
correlation coefficient between isoprinosine and subsequent data.
favipiravir is 0.318, while the correlation coefficient Given that groups 1 and 2 produced statistically
between isoprinosine and Oseltamivir is 0.520. The significant results, it can be concluded that there is a
greater the correlation coefficient, the greater the difference or influence between the two combinations
impact on the patient's lymphocyte count.. on the PCR variable. The expected PCR result is
However, additional research using a control negative. The Favipiravir Isoprinosine combination had
group with and without isoprinosine is necessary to a negative PCR result rate of 25.29%, while the
determine which drug affects lymphocyte levels. Oseltamivir Isoprinosine combination had a negative
Quantitative and functional lymphocyte changes can PCR result rate of 27.50%. By examining this
impair the immune response to the virus and may percentage, it is clear that Oseltamivir Isoprinosine
develop an immunopathological response. provides superior results. Oseltamivir is no longer
Lymphopenia is a prevalent immunological disorder in recommended for Covid-19 patients who do not exhibit
severe Covid-19 patients, accounting for 96.1% of flu-like symptoms. This may have occurred because
cases. The percentage of lymphocytes in the blood is a the study's findings indicated that the data obtained
laboratory measurement significantly associated with were more favuorable to Oseltamivir Isoprinosine than
the development of Covid-19 disease (Jafarzadeh et al., to the use of Favipiravir Isoprinosine.
2021). Yusra and Natasha report that common 2020 Additionally, the criteria for patient discharge in
laboratory data abnormalities in Covid-19 include a managing Covid-19 patients who change affect. Covid-
decrease in absolute lymphocyte and albumin counts, 19 patient management PDPI ed.1, valid from April
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2020 to July 2020, states that patients may be combination of isoprinosine and favipiravir has a 54.10
discharged home if their two-time polymerase chain percent success rate in preventing infiltrates on chest x-
reaction (PCR) results are negative. As of August rays, while isoprinosine Oseltamivir has a 49.34 %
2020, it is known that patients with PDPI ed.2 Covid- success rate. In a study of the efficacy of Favipiravir
19 can be discharged with several requirements even if therapy in Covid-19 patients, it was discovered that
the PCR results remain positive. If the PCR follow-up administering Favipiravir increased the improvement in
reveals a positive result after clinical improvement and chest X-rays by 91.43 %t in patients with negative
a three-day fever-free period, a persistent positive PCR results following seven days of Favipiravir
condition caused by detecting inactivated virus administration (Özlüşen et al., 2021). This could also
fragments or particles may exist. The Cycle Threshold be related to the antibiotic Azithromycin administered
(CT) value can be used to determine whether or not a to patients with moderate-grade Covid-19 at the RSD
patient's condition is infectious, as the cut-off value dr. Soebandi Jember.Azithromycin 5 days or
varies according to the reagents and tools used. Levofloxacin seven days daily is equally effective and
According to several hypotheses, persistent positive safe for acute bacterial bronchitis exacerbations
results result from the device detecting inactivated (ABECB). Although macrolide agents continue to be
virus components. Numerous studies have discovered an effective treatment option for patients with acute
that asymptomatic patients can still have positive RT- bacterial exacerbations of ABECB and other
PCR results a few weeks after symptoms have resolved community-acquired respiratory tract infections
(Burhan et al., 2020). According to PDPI edition 3 of (Amsden et al., 2003).
2020, patients may be discharged from hospital care if Along with patients discharged from the hospital
they meet the criteria for isolation completion and with no visible infiltrate on the chest X-ray, there were
clinical criteria, namely the results of a thorough also patients discharged with pneumonia. In Covid-19
clinical study, including radiological images patients who do not experience severe respiratory
demonstrating improvement and blood tests distress during hospitalization, abnormal lung
demonstrating improvement, conducted by a physician examinations will occur ten days after the first Covid-
stating the patient is allowed to go home. Whether 19 symptoms appear. After two weeks, the lung lesions
related or unrelated, the patient requires no further are absorbed, leaving behind ground-glass opacities
action/treatment (Burhan et al., 2020). (GGO) and subpleural parenchymal bands. Clinically
Because favipiravir will not be available at RSD cured patients of Covid-19 can still be found to have
dr. Soebandi Jember until November 2020, the GGO (Pan et al., 2020). The improvement of the
magnitudes of the two percentages in the PCR results immune system can also be seen in the patient's lung
cannot be compared to determine which combination is function, and the addition of isoprinosine to the
superior. Oseltamivir lacks sufficient empirical treatment regimen is expected to aid in the immune
evidence to recommend it as a treatment for Covid-19 system's improvement so that it can fight existing
infection associated with dyspnea or hypoxia in viruses.
Wuhan. However, Oseltamivir may be used in Covid- Isoprinosine has long been recognized for its
19 patients to prevent further deterioration of the ability to treat various viral infections, including
senses of taste and smell (Chiba, 2021). Favipiravir influenza and other influenza-like illnesses.
therapy resulted in a more rapid viral clearance, as Isoprinosine contains an immunomodulatory
indicated by a more rapid negative PCR result, mechanism that promises an overall therapeutic effect
compared to the group that did not receive Favipiravir against various viral pathogenic variants. While
(Finberg et al., 2021). Isoprinosine has been shown to immunomodulators are unlikely to result in complete
enhance the immune system by increasing the number recovery, early intervention can alter the course of the
of Th1 cells and the activity of natural killer cells disease. Clinical and immunological analytical studies
(Sliva et al., 2019). With an enhanced immune system, conducted over the last six years have established that
it is hoped that the process of virus clearance in isoprinosine can treat most viral infections via natural
patients will be accelerated. killer cells and cytotoxicity; this efficacy is expected to
X-ray of the chest Each combination has a Sig be transferred to the acute respiratory infection caused
value of 0.000. Thus, isoprinosine Favipiravir or by the Covid-19 virus that is currently spreading. Early
isoprinosine Oseltamivir provides a significant isoprinosine therapy has been shown to reduce early
difference in the patient's chest X-ray results. The viral immunosuppression and lymphopenia, which are
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strongly associated with the progression of Covid-19, photos. Both combinations have a mortality rate of less
hospitalization, and death. than 1%.
However, retrospective studies and studies
conducted in Ecuador and India indicate that STUDY LIMITATION
isoprinosine may be beneficial in the treatment of This study is limited to the availability of antivirus
Covid-19 patients. Since the summer of 2020, two in RSD dr. Soebandi, causing an imbalance in the
multicenter randomized clinical trials with IP in number of participants between the two groups.
patients with moderate COVID-19 have been ongoing Additional research with a control group devoid of
in India. The first was a 60-patient open-label proof-of- isoprinosine is required.
concept study. It examined the effect of IP in COVID-
19 patients when used in conjunction with standard of ACKNOWLEDGMENT
care versus when used alone. Subgroup analysis of This study involves many authors who have an
patients in this study revealed that when inosine essential role in realizing this journal. Each author has
pranobex was added to standard of care containing expertise that is very useful in the research process
Azithromycin and Hydroxychloroquine with or without from beginning to end. All authors declare that this
Ivermectin, it resulted in a significantly higher clinical study was accomplished without any funding or
response at Day-14 (100.00 percent vs. 69.23 percent; support.
p = 0.03). At Day 7, 14, and 21, there was a trend
toward a (numerically) greater clinical response in the AUTHOR CONTRIBUTIONS
IP + standard of care group compared to the Standard Conceptualization, C. M., D. H., B. S., R. D. P.;
of care group; however, statistical significance was not Methodology, C. M., D. H., B. S., R. D. P.; Software,
reached. This could be due to the study's small sample C. M., S. D. B., A. N. H.; Validation, D. H., B. S., R.
size or to variation in the current standard of care D. P., A. D. P.; Formal Analysis, C. M., D. H., B. S.;
across sites. IP was generally well-tolerated. According Investigation, C. M., D. H., B. S., R. D. P.; Resources,
to a randomized controlled trial conducted in Ecuador C. M., D. H., B. S., R. D. P.; Data Curation, C. M., D.
from March to April 2020, 60 Covid-19 patients were H., B. S.; Writing - Original Draft, C. M., D. H., B. S.,
randomly assigned to one of two groups, with 30 R. D. P.; Writing - Review & Editing, C. M., D. H., B.
patients receiving Isopsinosine therapy and the S., R. D. P.; Visualization, C. M.; Supervision, D. H.,
remaining 30 patients receiving placebo. From the start B. S., R. D. P.; Project Administration, D. H., B. S., R.
of the study, it was known that patients in the D. P., Funding Acquisition, C. M.
Isoprinosine group improved clinically within 15 days,
as measured by swab PCR being negative faster, CONFLICT OF INTEREST
reaching the SaO2 target > 90 percent faster, and chest The authors declared no conflict of interest.
x-rays showing a greater number of no lesions in
patients in the Isoprinosine group. Additionally, REFERENCES
isoprinosine is used in the Czech Republic and possibly Aksel, G., İslam, M. M., Algın, A., Eroğlu, S. A.,
elsewhere. The analysis of healthcare reimbursement Yaşar, G. B., Ademoğlu, E. & Dölek, U. C.
data for patients treated with Isoprinosine in (2021). Early Predictors of Mortality for
combination with other standard therapies and Moderate to Severely Ill Patients with Covid-19.
mortality data reveals essential information about American Journal of Emergency Medicine; 45;
Isoprinosine's effectiveness in Covid-19 patients 290–296. doi: 10.1016/j.ajem.2020.08.076.
(Beran et al., 2021). Amsden, G. W., Baird, I. M., Simon, S. & Treadway,
G. (2003). Efficacy and Safety of Azithromycin
CONCLUSION Vs Levofloxacin in the Outpatient Treatment of
RSD dr. Soebandi Jember obtained the results Acute Bacterial Exacerbations of Chronic
from a study evaluating the combination of Bronchitis. Chest; 123; 772–777. doi:
isoprinosine and antiviral Favipiravir or Oseltamivir in 10.1378/chest.123.3.772.
treating moderate-grade Covid-19 patients with RSD. Beran, J., Špajdel, M., Katzerová, V., Holoušová, A.,
The two of varieties improved clinical conditions as Malyš, J., Rousková, J. F. & Slíva, J. (2020).
measured by PCR parameters, oxygen demand levels, Inosine Pranobex Significantly Decreased the
leukocyte counts, lymphocyte counts, and thorax Case-Fatality Rate Among PCR Positive Elderly
P-ISSN: 2406-9388
E-ISSN: 2580-8303
with SARS-CoV-2 at Three Nursing Homes in Trial of Favipiravir for the Treatment of COVID-
the Czech Republic. Pathogens; 9; 1-10. doi: 19. Open Forum Infectious Diseases; 8; 1-8. doi:
10.3390/pathogens9121055. 10.1093/ofid/ofab563.
Beran, J., Špajdel, M. & Slíva, J. (2021). Inosine Instiaty, Darmayani, I. G. A. A. P. S., Marzuki, J. E.,
Pranobex Deserves Attention as a Potential Angelia, F., William, Siane, A., Sary, L. D.,
Immunomodulator to Achieve Early Alteration of Yohanes, L., Widyastuti, R., Nova, R.,
the Covid-19 Disease Course. Viruses; 13; 1-13. Simorangkir, D. S., Lonah, Safitri, Y., Aliska, G.
doi: 10.3390/v13112246. & Gayatri, A. (2020). Antiviral Treatment of
Biswas, M., Rahaman, S., Biswas, T. K., Haque, Z. & COVID-19: a Clinical Pharmacology Narrative
Ibrahim, B. (2021). Association of Sex, Age, and Review. Medical Journal of Indonesia; 29; 332–
Comorbidities with Mortality in COVID-19 345. doi: 10.13181/mji.rev.204652.
Patients: A Systematic Review and Meta- Jafarzadeh, A., Jafarzadeh, S., Nozari, P., Mokhtari, P.
Analysis. Intervirology; 64; 36–47. doi: & Nemati, M. (2021). Lymphopenia an
10.1159/000512592. Important Immunological Abnormality in
Burhan, E., Susanto, A. D., Isbaniah, F., Nasution, S. Patients with COVID-19: Possible Mechanisms.
A., Ginanjar, E., Pitoyo, C. W., Susilo, A. et al. Scandinavian Journal of Immunology; 93; 1–16.
(2020). Pedoman Tatalaksana Covid-19 Edisi 3. doi: 10.1111/sji.12967.
In Erlina Burhan, Agus Dwi Susanto, Fathiyah Lasek, W., Janyst, M., Wolny, R., Zapała, L., Bocian,
Isbaniah, Sally Aman Nasution, E. et al. (eds). K. & Drela, N. (2015). Immunomodulatory
Jakarta: Perhimpunan Dokter Paru Indonesia Effects of Inosine Pranobex on Cytokine
(PDPI), Perhimpunan Dokter Spesialis Production by Human Lymphocytes. Acta
Kardiovaskular Indonesia (PERKI), Pharmaceutica; 65; 171–180. doi: 10.1515/acph-
Perhimpunan Dokter Spesialis Penyakit Dalam 2015-0015.
Indonesia (PAPDI), Perhimpunan Dokter Long, L., Wu, L., Chen, L., Zhou, D., Wu, H., Lu, D.,
Anestesiologi dan Terapi Intensif Indonesia Li, H., Duan, X., Han, Y., Li, X., Wang, Q. &
(PERDATIN), Ikatan Dokter Anak Indonesia Zhang, J. (2021). Effect of Early Oxygen
(IDAI). Therapy and Antiviral Treatment on Disease
Chiba, S. (2021). Effect of Early Oseltamivir on Progression in Patients with COVID-19: A
Outpatients without Hypoxia with Suspected Retrospective Study of Medical Charts in China.
COVID-19. Wiener Klinische Wochenschrift; PLoS Neglected Tropical Diseases; 15; 1–15.
133; 292–297. doi: 10.1007/s00508-020-01780- doi: 10.1371/journal.pntd.0009051.
0. Mortaz, E., Tabarsi, P., Varahram, M., Folkerts, G.,
Ciarambino, T., Para, O. & Giordano, M. (2021) Adcock, I. M. (2020). The Immune Response and
Immune System and COVID-19 by Sex Immunopathology of COVID-19. Frontiers in
Differences and Age. Women's Health; 17; 1-6. Immunology; 11; 1–9. doi:
doi: 10.1177/17455065211022262. 10.3389/fimmu.2020.02037.
Davies, N. G., Klepac, P., Liu, Y., Prem, K., Jit, M., Özlüşen, B., Kozan, S., Akcan, R. E., Kalender, M.,
CMMID COVID-19 working group & Eggo, R. Yaprak, D., Peltek, I. B., Keske, S., Gönen, M. &
M. (2020). Age-Dependent Effects in the Ergönül, O. (2021). Effectiveness of favipiravir
Transmission and Control of COVID-19 in COVID-19: A Live Systematic Review.
Epidemics. Nature Medicine; 26; 1205–1211. European Journal of Clinical Microbiology and
doi: 10.1038/s41591-020-0962-9. Infectious Diseases; 40; 2575–2583. doi:
Dluholucky, S. (2020). Inosine Pranobex (IP) - 10.1007/s10096-021-04307-1.
Possibilities of Its Use in the Treatment of Pan, F., Ye, T., Sun, P., Gui, S., Liang, B., Li, L.,
COVID19. Archives of Community Medicine and Zheng, D., Wang, Z., Hesketh, R. L., Yang, L. &
Public Health; 6; 200–201. doi: 10.17352/2455- Zheng, C. (2020). Time Course of Lung Changes
5479.000106. at Chest CT During Recovery from Coronavirus
Finberg, R. W., Ashraf, M., Julg, B., Ayoade, F., Disease 2019 (COVID-19). Radiology; 295;
Marathe, J. G., Issa, N. C., Wang, J. P., Jaijakul, 715–721. doi: 10.1148/radiol.2020200370.
S., Baden, L. R. & Epstein, C. (2021). US201 Petrova, M., Jelev, D., Ivanova, A. & Krastev, Z.
Study: A Phase 2, Randomized Proof-of-Concept (2010). Isoprinosine Affects Serum Cytokine
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Levels in Healthy Adults. Journal of Interferon Hamers, R. L. (2021). Clinical Characteristics

and Cytokine Research; 30; 223-228 doi: and Mortality Associated with COVID-19 in
10.1089/jir.2009.0057. Jakarta, Indonesia: a Hospital-Based
Shannon, A., Selisko, B., Le, N-T-T., Huchting, J., Retrospective Cohort Study. The Lancet
Touret, F., Piorkowski, G., Fattorini, V., Ferron, Regional Health - Western Pacific; 9; 1-9. doi:
F., Decroly, E., Meier, C., Coutard, B., Peersen, 10.1016/j.lanwpc.2021.100108.
O. & Canard, B. (2020). Rapid Incorporation of Vahidy, F. S., Pan, A. P., Ahnstedt, J., Munshi, Y.,
Favipiravir by the Fast and Permissive Viral Choi, H. A., Tiruneh, Y., Nasir, K., Kash, B. A.,
RNA Polymerase Complex Results in SARS- Andrieni, J. D. & McCullough, L. D. (2021). Sex
CoV-2 Lethal Mutagenesis. Nature Differences in Susceptibility, Severity, and
Communications; 11; 1–9. doi: 10.1038/s41467- Outcomes of Coronavirus Disease 2019: Cross-
020-18463-z. Sectional Analysis from a Diverse US
Shianata, C. M., Engka, J. N. A. & Pangemanan, D. H. Metropolitan Area. PLoS ONE; 16; 1–14. doi:
C. (2021). Happy Hypoxia pada Coronavirus 10.1371/journal.pone.0245556.
Disease. Jurnal Biomedik: Jbm; 13; 58–66. doi: Wang, J., Jiang, M., Chen, X. & Montaner, L. J.
10.35790/jbm.13.1.2021.31743. (2020). Cytokine Storm and Leukocyte Changes
Sliva, J., Pantzartzi, C. N. & Votava, M. (2019). in Mild Versus Severe SARS-CoV-2 Infection:
Inosine Pranobex: a Key Player in the Game Review of 3939 COVID-19 Patients in China and
Against a Wide Range of Viral Infections and Emerging Pathogenesis and Therapy Concepts.
Non-Infectious Diseases. Advances in Therapy; Journal of Leukocyte Biology; 108; 17–41. doi:
36; 1878-1905. doi: 10.1007/s12325-019-00995- 10.1002/JLB.3COVR0520-272R.
6. Yazdanpanah, F., Hamblin, M. R. & Rezaei, N. (2020).
Surendra, H. Elyazar, I. R. F., Djaafaraa, B. A., The Immune System and COVID-19: Friend or
Ekawati, L. L., Saraswati, K., Adrianf, V., Foe?. Life Sciences; 256; 1-5.
Widyastuti, Oktaviaf, D., Salamaf, N., Lina, R. Yuki, K., Fujiogi, M. & Koutsogiannaki, S. (2020).
N., Andrianto, A., Lestari, K. D., Burhang, E., COVID-19 Pathophysiology: a Review Koichi.
Shankar, A. H., Thwaites, G., Bairda, K. & Clinical Immunology Journal; 215; 1-7.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
DOI: 10.20473/jfiki.v9i32022.220-228
The Effect of Decyl Glucoside on Stability and Irritability of Nanostructured

Lipid Carriers-Green Tea Extract as Topical Preparations
Rufaidah Azzahrah1, Noorma Rosita2*, Djoko Agus Purwanto2, Widji Soeratri2
1
Master Program of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
2
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
*Corresponding author: noorma-r@ff.unair.ac.id
Submitted: 28 Februari 2022

Abstract
Background: Green Tea Extract (GTE) is a natural antioxidant compound that can protect the skin from
photocarcinogenesis (DNA damage due to ultraviolet exposure). GTE has low stability, which needs a delivery
system such as Nanostructured Lipid Carriers (NLC) with decyl glucoside (DG) as a natural surfactant that at the
right concentration can produce a significantly small particle size which can improve the stability of the NLC.
Objective: To determine the effect of DG usage on the characteristics, physical stability, and irritability of NLC-
GTE preparation. Methods: NLC-GTE preparation used the High Shear Homogenization (HSH) method with
three formulas, which contained DG 2%, 2.5%, and 3% consecutively. Afterwards, the characteristic and physical
stability tests were conducted using the thermal cycling method for three cycles with two different temperatures
(48 hours/cycle, 2 - 8°C and 40°C). The irritability test used Hen's Egg Test on the Chorioallantoic Membrane
(HET-CAM) method. Results: Characteristic test of organoleptic showed that all formulas were white, odorless,
and had a semi-solid consistency. However, the pH, particle size, and polydispersity index values from all formulas
were within the normal range of values. The physical stability test result showed that 3% DG was the most stable
formula. This formula was within the non-irritating range of values in HET-CAM. Conclusion: NLC-GTE with an
increased concentration of DG as a surfactant can improve the characteristics and physical stability of the
preparation. F3 (3% DG) is the best formula compared to other formulas and indicates non-irritating in the HET-
CAM test.
Keywords: nanostructured lipid carriers, green tea extract, decyl glucoside, thermal cycling, Hen’s Egg Test on
the chorioallantoic membrane

Azzahrah, R., Rosita, N., Purwanto, D. A. & Soeratri, W. (2022). The Effect of Decyl Glucoside on Stability and
Irritability of Nanostructured Lipid Carriers-Green Tea Extract as Topical Preparations. Jurnal Farmasi dan Ilmu
Kefarmasian Indonesia, 9(3), 220-228. http://doi.org/10.20473/jfiki.v9i32022.220-228
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION damaging the lipid membrane, which is the external

Green tea extract (GTE) is obtained from the tea protective layer on the skin, to disrupt its function
plant or Camellia sinensis (Wang et al., 2020). GTE has (Kosswig, 2012). This problem can be solved by
the highest phenolic and flavonoid content compared to selecting a surfactant that has low irritability.
other tea extracts, with an IC50 value of 0.487μg/mL Decyl glucoside (DG) is a type of natural surfactant
(Widowati et al., 2015). In addition, GTE can also be derived from plants, which is produced by the reaction
used for UVA and UVB protection (Saini et al., 2019) of glucose (from corn starch) and fatty alcohol decanol
and anti-photoaging (Siyahpoosh et al., 2022). Katiyar (from coconut). Mehling et al. (2007) conducted the
et al. (in Aaron & Robert, 2009) explained that GTE tests for DG irritability. It showed that this surfactant did
could increase penetration and absorption into the skin not show irritation in various irritability testing methods.
when used topically. This statement is supported by Dal Furthermore, based on the research of Chaiyana et al.
Belo et al. (2009) who conducted an in vitro penetration (2020), surfactant DG can produce significantly small
test on GTE cosmetic preparations, using the Franz particles in NLC preparations containing an active
diffusion cell method. The method used in this test, substance in the form of an extract, namely Ocimum
namely the Franz diffusion cell method, uses fresh sanctum Linn. In contrast, the small NLC particle size
human skin (Caucasian) as the sample application can affect various aspects, including increasing the
medium, then measured using the High-Performance stability of the preparation (Müller-Fischer et al., 2007).
Liquid Chromatography (HPLC) instrument. Based on The problem lies in the concentration of DG used. By
these tests, the results showed that this preparation has the explanation, Lasoń et al. (2018) stated in their
good skin penetration and retention capabilities. It is research article that too high a DG concentration could
characterized by detecting GTE in all skin layers, result in foam formation during the NLC preparation
especially in the dermis layer (9.2%). In addition, process using forskolin as the active substance. Based
epigallocatechin gallate (EGCG) the main phenolic on these considerations, several DG concentrations were
compound in GTE, turns out to have poor stability compared to determine their effect on the characteristics
because it is very susceptible to oxidation due to several (organoleptic, pH, particle size, polydispersity index),
factors (exposure to light, temperature, pH, and others), physical stability, and irritability of the NLC-GTE
so it must be overcome by using a suitable delivery preparation. It aims to obtain the most optimal
system (Sugihartini et al., 2016; Shi et al., 2018). preparation.
NLC or nanostructured lipid carriers is a delivery
system suitable for active antioxidant ingredients (Chen- MATERIALS AND METHODS
yu et al., 2012) and topical use (Souto et al., 2020). Materials
Manea et al. (2014) stated that green tea extract could The materials used in this research were green tea
be developed in topical preparations using an NLC extract (PT. Angler BioChemLab, Surabaya,
delivery system. In addition, Tamjidi et al. (2013) Indonesia), cetyl palmitate (BASF, Indonesia), glyceryl
explained that NLC consists of solid lipids and liquid stearate (BASF, Indonesia), grape seed oil (NHR
lipids that can form a matrix and control the entrapment Organic Oils, UK), decyl glucoside (Dow Chemical
of active substances to increase the stability of a Pacific, Singapore), synperonicF68 (PT Megasetia
preparation. In addition to the various advantages Agung Kimia, Jakarta, Indonesia), and
described above, the preparation stage of NLC has lecithin (Solae™, Amerika) were cosmetic grade.
several problems. During the crystallization process, the Aquademineralisata (Indonesia) was a technical grade
surface area of the particles will increase rapidly; thus, and fertile chicken eggs (chicken farm, West Java &
the whole system is unstable. Some literature states that East Java, Indonesia).
it can be overcome by adding surfactants (Han et al., Method
2008) which are needed to improve the quality of the Preparation of NLC-GTE
nanoparticle interface (Han et al., 2008). The quality of The method used in the preparation of NLC-GTE
the nanoparticle interface can affect the system's was a modification of the high shear homogenization
characteristics and ability to absorb the active substance. (HSH) method by Manea et al. (2014). This method was
Furthermore, the surfactant will also affect the formed carried out by preparing two different phases: the oil and
nanoparticles' physical stability (Averina et al., 2009). water phases (Table 1). The oil phase consists of cetyl
Besides that, surfactants also have disadvantages that palmitate, glyceryl stearate, and grape seed oil.
can cause irritation and various skin disorders by
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Table 1. Design formula of NLC-GTE

Formula (%)
Materials Function
F1 F2 F3
Green tea extract (GTE) Active substance 0.1 0.1 0.1
Cetyl palmitate Solid lipid 3.5 3.5 3.5
Lipid phase Glyceryl stearate Solid lipid 3.5 3.5 3.5
Grape seed oil Liquid lipid 3 3 3
Decyl glucoside Surfactant 2 2.5 3
Lecithin Cosurfactant 0.5 0.5 0.5
Water phase
Synperonic F68 Cosurfactant 0.5 0.5 0.5
Aquademineralisata Solvent Ad 100 Ad 100 Ad 100
Description:
F1 = NLC-GTE + DG 2%
F2 = NLC-GTE + DG 2.5%
F3 = NLC-GTE + DG 3%
Meanwhile, the aqueous phase consisted of were then analyzed statistically using the One Way
surfactant DG, synperonic F68, lecithin (1%, 1:1, w/w), ANOVA method.
and aquademineralisata. Furthermore, both were heated Physical stability test of NLC-GTE
with Thermo Scientific Cimarec+ at the same The stability test of NLC-GTE was performed using
temperature, namely 70°C for 30 minutes, then GTE the accelerated method, namely thermal cycling.
(which had been dissolved in 10mL aquademineralisata) According to Rohmah (2020), this method test
was added in the oil phase. Before mixing the two procedure can be conducted with a total of three cycles
phases, the aqueous phase was stirred using Ultra-Turax at two different temperatures (48 hours at a temperature
IKA®T25 Digital for 2 minutes at a speed of 15,000 of 2 - 8°C and 40°C, respectively). Moreover, the
rpm. Then the two phases were mixed for 7 minutes at a preparation characteristics such as organoleptic
speed of 15,000 rpm thus the NLC-GTE preparation was observations, pH, particle size (PS), and polydispersity
obtained. index (PI) would be monitored. The results of these tests
Characteristics test of NLC-GTE (pH, PS, and PI) were then analyzed using a statistical
Organoleptic method, namely the Paired T-test.
This organoleptic test was performed visually with Irritability test of NLC-GTE
several aspects of observation, in the form of smell, This method's test procedure began with incubating
color, and consistency of the NLC-GTE preparation and periodically rotating chicken eggs in an incubator at
(Loreta, 2015; A'yun, 2019; Indrajaya, 2021). 37°C. On the 10th day, the eggs were binoculars to
pH distinguish and select fertile eggs, marked the air cavity
The pH values of the NLC-GTE preparations were with a pencil, and then opened using sterile scissors. The
measured using a pH meter SI Analytics Lab 865 outer egg membrane was moistened with warm sterile
electrode calibrated and inserted into the preparation. 0.9% NaCl solution and incubated for 5 - 20 minutes to
The value shown on the screen was recorded as the pH facilitate the removal of the outer egg membrane. Ensure
value and replicated three times (A'yun, 2019; the chorioallantoic membrane (CAM) conditions were
Indrajaya, 2021). Furthermore, statistical analysis was still in good condition and place 0.2 mL NLC-GTE (the
carried out using the One Way ANOVA method. best formula based on the physical stability test) in the
Particle size dan polydispersity index CAM and let stand for 20 seconds (Hagino et al., 1999).
Particle size (PS) and polydispersity index (PI) As an irritant control (+C), 1% sodium lauryl sulfate
measurements were conducted by diluting the NLC- (SLS) and sterile 0.9% NaCl solution were used as
GTE preparation first and analyzing using the Beckman negative controls (-C). Then the CAM was cleaned
Coulter® DelsaTMnano C Particle Analyzer using a sterile 0.9% NaCl solution. Further observations
instrument. This measurement was replicated three began after the CAM was cleaned from the sample for
times, and the particle size and polydispersity index 300 seconds. Observed and recorded the time of
values were recorded on the monitor screen hemorrhage, lysis, and coagulation in the presence of
(Mayangsari, 2021). The data obtained from this test CAM (Yuliani, 2016).
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RESULTS AND DISCUSSION meet the NLC particle size requirements, which were in
Characteristics test of NLC-GTE the range of 10-500nm (Sharma & Baldi, 2018) and 10-
Organoleptic 1000nm (Faizatun et al., 2020). The value of each
NLC-GTE preparations with varying formula showed that F3 has the smallest size compared
concentrations of DG as a surfactant showed that all to F2 and F1. These results proved that increasing the
formulas were white, odorless, and had a semi-solid concentration of DG can reduce the PS value. This
consistency (Figure 1). statement was in line with Zirak & Pezeshki's (2015)
opinion that a high surfactant concentration can lower
surface tension. Hence, the energy obtained (stirring)
would make breaking up oil droplets (melted lipids)
easier, which can stabilize the newly formed surface to
produce a larger size. The results of statistical analysis
showed that there was a significant difference between
each formula (sig. < 0.05), so it can be said that an
Figure 1. Preparation of NLC-GTE with variations in
increase in DG concentration could affect the PS value
DG concentration (F1: 2%; F2: 2.5%; F3: 3%)
of NLC-GTE.
pH The polydispersity index (PI)
The pH value of the NLC-GTE preparation can be According to Avadi et al. (2010), this measurement
determined by measuring it using a pH meter. The aimed to determine several aspects of the colloidal
following results were obtained (Table 2). The results of solution, particle size and homogeneity distribution. The
the measurement of the pH of the NLC-GTE preparation PI value of each formula can be seen in Table 3. Table 3
described in Table 2 showed that all formulas were at a showed that all formulas have a PI value of less than 0.3
normal pH range of 4 - 7 (Souto & Műller, 2008). which meant that all formulas had a uniform and
Furthermore, statistical analysis was carried out and the homogeneous particle size distribution. This result is
results showed that the difference in DG concentration similar to Oliveira et al. (2021) in that PI value of < 0.3
did not affect the pH value of the NLC-GTE preparation a homogeneous particle size distribution. F3 produces a
(sig. > 0.05). value of 0.23 ± 0.00, the smallest PI value compared to
F1 and F2, which produced a PI value of 0.26 ± 0.01 and
Table 2. pH value of NLC-GTE preparations (n = 3)
0.25 ± 0.00, respectively. This series of values can prove
Formula Average ± SD (CV)
that variations in DG concentration can affect the PI
F1 6.19 ± 0.00 (0.02)
F2 6.20 ± 0.00 (0.10) value in NLC-GTE (Subramaniam et al., 2020).
F3 6.20 ± 0.00 (0.05) Table 3. The polydispersity index value of NLC-GTE
preparations (n=3)
Particle size (PS) Formula Average ± SD (CV)
The results of characteristic testing in the form of F1 0.26 ± 0.01 (4.19)
determining the particle size of NLC-GTE can be seen F2 0.25 ± 0.00 (3.16)
F3 0.23 ± 0.00 (1.51)
in the histogram (Figure 2). Based on the histogram in
Figure 2, it can be seen that the PS values of all formulas
600
Particle Size Value (nm)
472.30±9.10
500
385.70±13.95
400
295.96±5.41
300
200
100
0
F1 F2 F3
NLC-GTE Formula
Figure 2. Histogram of the particle size value (average ± SD) of the NLC-GTE preparation (n = 3)
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By Lullung & Suprapti's (2012) explanation, the

smaller the surfactant concentration, the greater the PI
value obtained, and vice versa. It can be caused by the
DG concentration of 3% being more optimal for
covering the lipid and water interface and reducing the
surface tension of the new particles formed during the
homogenization process so that uniform nanoparticles
can be created (Witayaudom & Klinkesorn, 2017). In
addition, an increase in surfactant concentration can
affect the energy produced in the stirring process so that
the nanoparticles formed can be distributed more
efficiently (Zirak & Pezeshki, 2015). Based on the
statistical analysis carried out, it showed that there was
no significant difference between F1 and F2 (sig. >
0.05). At the same time, for F1 and F3, there was a
significant difference between the two (sig. <0.05).
Likewise, F2 and F3 showed a significant difference
(sig. < 0.05). Based on these data, it can be said that the
PI value in F3 was significantly different when
Figure 3. Physical stability test results of NLC-GTE
compared to F1 and F2. While F1 and F2 have the same
[(a) before cycle; (b) after cycle]
PI value, there was no significant difference between the
pH
two.
The pH value was a measurement that was
Physical stability test of NLC-GTE
implemented in this physical stability test. The
Organoleptic
following was the data for each pH value before and
Physical stability testing of NLC-GTE was carried
after treatment (Table 4). The pH value of the physical
out using the thermal cycling method, and several things
stability test (thermal cycling) in all formulas was within
were observed, including organoleptic analysis, the
the range of normal skin pH values, namely 4 - 7 (Souto
results of which can be seen in Figure 3. Based on the
& Műller, 2008). The pH value data were then analyzed
thermal cycle test results of the NLC-GTE organoleptic
using statistics and showed no significant difference
preparation, which has been described in Figure 3, it can
between the pH values before and after treatment
be seen that all formulas have the same color and odor,
[sig.(2-tailed) > 0.05]. Thus, it can be said that the pH
both before and after the cycle. The difference that can
value of all NLC-GTE preparations remained stable
be seen from this observation was phase separation in
after testing the physical stability using the thermal
F1 and F2. However, there was almost no phase
cycling method.
separation at all in F3. These results are the opinion of
Zirak & Pezeshki (2015) who state that low surfactant Table 4. The pH value of NLC-GTE preparations from
concentrations can cause instability and recrystallization physical stability tests (n=3)
due to insufficient amounts in the formation of Average ± SD (CV)
Formula
nanoparticle structures. This case will trigger the Before After
formation of git, which are large particles that cannot be F1 6.19 ± 0.00 (0.02) 6.20 ± 0.00 (0.05)
F2 6.20 ± 0.00 (0.10) 6.20 ± 0.00 (0.13)
stabilized by surfactants and break into non-uniform
F3 6.20 ± 0.00 (0.05) 6.21 ± 0.00 (0.07)
particles and cause phase separation in the NLC-GTE
preparation (Lullung & Suprapti, 2012). Based on these Particle size (PS)
results, F3 (3% DG) showed better phase stability than Another measurement was applied, namely the
F1 (2% DG) and F2 (2.5% DG). Furthermore, particle size of the NLC-GTE preparation. The
measurements of PS and PI were carried out in each following are the PS values measured before and after
formula to strengthen this statement. the test cycle (Figure 4).
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600 493.00±7.92
Particle Size Value
500 401.50±3.93
400 472.30±9.10 306.46±4.50
(nm)
300 385.70±13.95
295.96±5.41 Before
200
100 After
0
F1 F2 F3
NLC-GTE Formula
Figure 4. Histogram of particle size values (average ± SD) for NLC-GTE preparations (n = 3)
from physical stability tests
PS data from each NLC-GTE formula, both before Table 5. Polydispersity index values of NLC-GTE
and after the cycle, showed that all formulas had particle preparations from physical tests (n = 3)
sizes that were still within the normal range of size Average ± SD (CV)
Formula
values, namely the range of 10-500 nm (Sharma & Before After
Baldi, 2018; Faizatun et al., 2020). Furthermore, F1 0.26 ± 0.01 (4.19) 0.32 ± 0.00 (0.94)
F2 0.25 ± 0.00 (3.16) 0.28 ± 0.00 (0.92)
statistical analysis was carried out and obtained different
F3 0.23 ± 0.00 (1.51) 0.23 ± 0.00 (3.00)
results for each formula. F1 showed a significant
difference between before and after the cycle (sig (2-
Based on the results of the physical stability tests
tailed) < 0.05). Meanwhile, F2 and F3 showed that there
(organoleptic, pH, PS, and PI) described above, it can be
was no significant difference between before and after
said that 3% DG (F3) is the most optimal concentration
the cycle (sig (2-tailed) > 0.05). Based on the results of
to obtain stable NLC-GTE. These results are by the
this analysis, it can be said that after testing the physical
explanations of Lullung & Suprapti (2012) and Han et
stability using the thermal cycling method on NLC-GTE
al. (2008), namely, the optimal amount of surfactant can
preparations, F2 and F3 had PS values that were more
cover the surface of nanoparticles formed in the
stable than F1. These results proved that increasing the
homogenization process so that it can produce and
surfactant concentration of DG can affect the physical
maintain a small particle size, prevent the formation of
stability of NLC-GTE. Following the opinion of Han et
git and can increase the stability of the nanoparticle
al. (2008) opinion, surfactants can improve the
interface in the NLC-GTE preparation. Based on these
nanoparticle interface's quality, affecting the
results, the test will be continue with an irritation test to
preparation's characteristics and physical stability
determine the safety of NLC-GTE with 3% DG as a
(Müller-Fischer et al., 2007).
topical preparation.
The polydispersity index (PI)
Irritation Test of NLC-GTE
PI values of each NLC-GTE formula obtained
The Hen's Egg Test on the Chorioallantoic
before and after the treatment cycle can be seen in Table
Membrane (HET-CAM) method tests the irritation of
5. The results indicated an increase in the PI value when
preparation, one of which was a topical preparation.
compared before and after the treatment cycle. In F1, it
According to Chaiyana et al. (2020), this method did not
showed that the PI value after the cycle was more
require ethical approval because it only used animal
significant than 0.3, so it can be ascertained that the PI
embryos for less than half the total incubation period,
value in F1 was unstable. While F2 and F3 still produce
making it quite convenient and easy. Based on this test,
PI values in the normal range even after testing, which
it can be seen that the highest irritation score was seen
was < 0.3. The data were analyzed statistically and can
in +C, which used sodium lauryl sulfate as an irritant,
be interpreted that the PI values of F1 and F2
which was 8.37±0.10. While the irritation scores -C
experienced a significant change (sig (2-tailed) < 0.05).
(sterile NaCl 0.9%) and F3 (best formula) have the same
Meanwhile, F3 did not change significantly between
value, namely 0.00. These values indicate that F3 can be
before and after treatment (sig (2-tailed) > 0.05). This
classified as non-irritant preparation (Table 6). In
analysis proved that increasing the concentration of
contrast, the results of the +C value were classified as
decyl glucoside can affect the PI value in the physical
moderate irritation (Yuliani, 2016). The results of this
stability test of NLC-GTE.
test can be seen in Figure 5, namely hemorrhage
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(bleeding) and lysis (loss of blood vessels) in the CAM ACKNOWLEDGMENT

after the application of SLS. At the same time, the -C We are grateful to PT. Megasetia Agung Kimia, for
(sterile NaCl) and F3 (best formula) groups did not show providing Synperonic F68 or Poloxamer 188. In
any difference between before and after the sample was addition, the authors would like to thank all parties who
applied. have supported this research.
Table 6. Hen's Egg Test on the Chorioallantoic
Membrane (HET-CAM) test results (n = 3) AUTHOR CONTRIBUTIONS
Conceptualization, R. A., N. R., W. S.;
Treatment Group Average ± SD (CV)
+C 8.37±0.10 (1.18) Methodology, R. A., N. R., W. S.; Validation, R. A., N.
-C 0.00±0.00 (0.00) R., W. S.; Formal Analysis, R. A.; Investigation, R. A.;
F3 0.00±0.00 (0.00) Resources, R. A., N. R., D. A. P.; Data Curation, R. A.,
N. R., W. S.; Writing - Original Draft, R. A., N. R.;
Writing - Review & Editing, R. A., N. R., W. S.;
Visualization, R. A., N. R., W. S.; Supervision, N. R.,
W. S.; Project Administration, N. R., W. S., Funding
Acquisition, R. A.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
REFERENCES
A’yun, N. Q. (2019). Pengaruh Penambahan Space
Peptide terhadap Tabilitas dan Efektivitas Krim
Freeze Dried Amniotic Membrane Stemcell
Metabolite Product (AMSC-MP). Tesis;
Universitas Airlangga, Surabaya.
Avadi, M. Z., Sadeghi, A. M. M., Mohammadpour, N.,
Figure 5. Hen's Egg Test on the Chorioallantoic Abedin, S., Atyabi, F., Dinarvand, R. & Rafiee-
Membrane (HET-CAM) test results Tehrani, M. (2010). Preparation and
Characterization of Insulin Nanoparticles Using
Based on the irritability test result used in the HET- Chitosan and Arabic Gum with Ionic Gelation
CAM method, the irritability of the NLC-GTE Method. Nanomedicine: Nanotechnology,
preparation in F3 (3% DG) can be the best formula. Biology, and Medicine; 6; 58-63. doi:
Hence, NLC-GTE, which contained 3% DG, was 10.1016/j.nano.2009.04.007.
considered safe to use and developed in various topical Averina, E. S., Seewald, G., Muller, R. H., Radnaeva, L.
cosmetic preparations. D. & Popov, D. V. (2009). Nanostructured Lipid
Carriers (NLC) on the Basis of Siberian Pine
CONCLUSION (Pinus sibirica) Seed Oil. Pharmazie; 65; 25-31.
Based on all the tests that have been carried out, it Chaiyana, W., Anuchapreeda, S., Somwongin, S.,
can be concluded that increasing the surfactant DG Marsup, P., Lee, K. H., Lin, W. C., & Lue, S. C.
concentration in the NLC-GTE preparation does not (2020). Dermal Delivery Enhancement of Natural
affect the pH of each formula. However, it can decrease Anti-Ageing Compounds from Ocimum sanctum
PS and PI and increase the NLC-GTE preparation's Extract by Nanostructured Lipid Carriers.
physical stability. Furthermore, based on the irritability Pharmaceutics; 12; 1-12. doi:
test results, F3 (3% DG) as the best formula were also 10.3390/pharmaceutics12040309.
proven safe because it did not show irritation
characteristics in the HET-CAM test.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Chen-yu, G., Chen-yu, Y., Qi-lu, L., Qi, T., Yan-wei, X., Lasoń, E., Sikora, E., Miastkowska, M., Escriano, E.,
Wei-na, L. & Guang-xi, Z. (2012). Development Garcia-Celma, M. J., Solans, C., Llinas, M. &
of a Quercetin-Loaded Nanostructured Lipid Ogonowski, J. (2018). NLCs as a Potential Carrier
Carrier Formulation for Topical Delivery. System for Transdermal Delivery Forskolin. Acta
International Journal of Pharmaceutics; 430; Biochimika Polonica; 65; 437-442. doi:
292-298. doi: 10.1016/j.ijpharm.2012.03.042. 10.18388/abp.2018_2554.
Dal Belo, S. E., Gaspar, L. R., Maia Campos, P. M. B. Loreta, L. J. (2015). Pengaruh Tween 80 sebagai
G. & Marty, J. -P. (2009). Skin Penetration of Emulsifying Agent dan Sorbitol sebagai
Epigallocatechin-3-Gallate and Quercetin from Humektan dalam Sediaan Krim Ekstrak Daun
Green Tea and Ginkgo Biloba Extract Vehiculated Jambu Biji (Psidium guajava L.) dengan Aplikasi
in Cosmetic Formulations. Skin Pharmacology Desain Faktorial. Skripsi; Universitas Sanata
and Physiology; 22; 299-304. doi: Dharma, Yogyakarta.
10.1159/000241299. Lullung, A. & Suprapti. (2012). Effect of Surfactant on
Faizatun, Gangga, E., Anindita, S., Martati, T. & Particle Diameter, Viscosity and the
Miftahurrohmah, N. (2020). Formulation of NLC Polydispersity Index of SLN Making Cocoa Fat.
Gel from Callus Mulberry Leaf Extract Induced Jurnal RisetTeknologi Industri; 6; 1-10. doi:
by NAA and BAP). Jurnal Ilmu Kefarmasian 10.26578/jrti.v6i12.1512.
Indonesia; 18; 123-129. doi: Manea, A. M., Vasile, B. S. & Meghea, A. (2014).
10.35814/jifi.v18i1.814. Antioxidant and Antimicrobial Activities of Green
Hagino, S., Kinoshita, S., Tani, N., Nakamura, T., Ono, Tea Extract Loaded Into Nanostructured Lipid
N., Konishi, K., Iimura, H., Kojima, H. & Ohno, Carriers. Comptes Rendus Chimie; 17; 331-341.
Y. (1999). Interlaboratory Validation of In Vitro doi: 10.1016/j.crci.2013.07.015.
Eye Irritation Test for Cosmetic Ingredients. (2) Mayangsari, F. D. (2021). Efek Minyak Nilam,
Chorioallantoic Membrane (CAM) Test. Pepermin dan Rosmeri pada Karakteristik Fisik,
Toxicology in Vitro; 13; 99-113. doi: Stabilitas Fisik, Iritabilitas, dan Efektivitas NLC
10.1016/s0887-2333(98)00065-4. Coenzyme G10 Sleeping Mask sebagai Kosmetik
Han, F., Li, S., Yin, R., Liu, H., & Xu, L. (2008). Effect Antiaging. Tesis; Universitas Airlangga,
of Surfactant on the Formation and Surabaya.
Characterization of a New Type of Colloidal Drug Mehling, A., Kleber, M. & Hensen, H. (2007).
Delivery System: Nanostructured Lipid Carriers. Comparative Studies on the Ocular and Dermal
Colloids and Surfaces A: Physicochem. Eng. Irritation Potential of Surfactants. Food and
Aspect; 315; 210-216. doi: Chemical Toxicology; 45; 747-758. doi:
10.1016/j.colsurfa.2007.08.005. 10.1016/j.fct.2006.10.024.
Indrajaya, S. (2021). Pengaruh Carbopol940 dan CMC- Müller-Fischer, N., Suppiger, S. & Windhab, E. J.
Na terhadap Karakteristik, Stabilitas Fisik, (2007). Impact of Static Pressure and Volumetric
Efektivitas dan Iritabilitas Ubiquinone – Energy Input on the Microstructure of Food Foam
Nanostructured Lipid Carriers (NLC) dalam Whipped in a Rotor-Stator Device. Journal of
Hydrogel Sleeping Mask sebagai Kosmetik Food Engineering; 80; 306-316. doi:
Antiaging. Tesis; Universitas Airlangga, 10.1016/j.jfoodeng.2006.05.026.
Surabaya. Oliveira, R. N. d., Campos, P. M., Pinto, R. M. C.,
Katiyar, S., Elmest, C. A. & Katiyar, S. K. (2009). Mioduski, J., Santos, R. D., Justus, B., Paula, J. d.
Green Tea and Skin Cancer: Immunological F. P. d., Klein, T., Boscardin, P. M. D., Corrêa, S.
Modulation and DNA Repair. In: Aaron Tabor & d. A. P., Allegretti, S. M. & Ferrari, P. C. (2021)
Robert Blair (ed.) Nutritional Cosmetics Beauty The Promising Antischistosomal Activity of Oleic
from Within; 472-491. Oxford, USA: William Acid-Loaded Polymeric Nanocapsules for Oral
Andrew. Administration. Journal of Drug Delivery Science
Kosswig, K. (2012). Surfactants. In Ullmanns (ed.) and Technology; 63; 1-9. doi:
Encyclopedia of Industrial Chemistry; 35; 431- 10.1016/j.jddst.2021.102429.
505. Rohmah, N. N. (2020). Pengaruh Peppermint Essential
Oil terhadap Karakteristik dan Stabilitas Fisik
P-ISSN: 2406-9388
E-ISSN: 2580-8303
(Thermal Cycle) Sistem Nanostructured Lipid Galat dalam Krim The Hijau dengan Variasi
Carriers Coenzyme Q10. Skripsi; Universitas konsentrasi Antioksidan Vitamin C 1% dan
Airlangga, Surabaya. Vitamin E 1%. Jurnal Farmasi Sains dan
Saini, S., Gupta, A., Singh, N. & Sheikh, J. (2019). Komunitas; 13; 52-56. doi:
Functionalization of Linen Fabric Using Layer by 10.24071/jpsc.132186.
Layer Treatment with Chitosan and Green Tea Tamjidi, F., Shahedi, M., Varshosaz, J. & Nasirpour, A.
Extract. Journal of Industrial and Engineering (2013). Nanostructured Lipid Carriers (NLC): A
Chemistry; 4811; 1-6. doi: Potential Delivery System for Bioactive Food
10.1016/j.jiec.2019.10.005. Molecules. Innovative Food Science and
Sharma, A. & Baldi, A. (2018). Nanostructured Lipid Emerging Technology; 19; 29-43. doi:
Carriers: A Review. Journal of Developing Drugs; 10.1016/j.ifset.2013.03.002.
7; 1-12. doi: 10.4172/2329-6631.1000191. Wang, T., Zhang, F., Zhao, R., Wang, C., Hu, K., Sun,
Shi, M., Shi, Y. L., Li, X. M., Yang, R., Cai, Z. Y., Li, Y., Politis, C., Shavandi, A. & Nie, L. (2020).
Q. S., Ma, S. C., Ye, J. H., Lu, J. L., Liang, Y. R. Polyvinyl Alcohol/Sodium Alginate Hydrogels
& Zheng, X. Q. (2018). Food-Grade Incorporated with Silver Nanoclusters via Green
Encapsulation Systems for (-)-Epigallocatechin Tea Extract for Antibacterial Applications.
Gallate. Molecules; 23; 1-17. doi: Designed Monomers and Polymers; 23(1); 188-
10.3390/molecules23020445. 133. doi: 10.1080/15685551.2020.1804183.
Siyahpoosh, A., Sheikhi, A. R., Jafarzadeh, E. & Widowati, W., Herlina, T., Ratnawati, H., Constantia,
Pipelzadeh, M. (2022). Effectiveness of Green G., Deva, I. D. G. S. & Maesaroh, M. (2015).
Tea Cream in Comparison with Silver Antioxidant Potential of Black, Green and Oolong
Sulfadiazine Cream in Treatment of Second Tea Methanol Extracts. Biology, Medicine, &
Degree Burn in Human Subject. Journal of Herbal Natural Product Chemistry; 4; 35-39. doi:
Medicine; 32; 1-8. doi: 10.14421/biomedich.2015.42.35-39.
10.1016/j.hermed.2022.100533. Witayaudom, P. & Klinkesorn, U. (2017). Effect of
Souto, E. B. & Muller, R. H. (2008). Cosmetic Features Surfactant Concentration and Solidification
and Application of Lipid Nanoparticles (SLN, Temperature on the Characteristics and Stability
NLC). International Journal of Cosmetic Science; of Nanostructured Lipid Carrier (NLC) Prepared
30; 157-165. doi: 10.1111/j.1468- from Rambutan (Nephelium lappaceum L.)
2494.2008.00433.x. Kernel Fat. Journal of Colloid and Interface
Souto, E. B., Baldim, L., Oliveira, W. P., Rao, R., Science; 505; 1082-1092. doi:
Yadav, N., Francisco M. G. & Sheefali, M. 10.1016/j.jcis.2017.07.008.
(2020). SLN and NLC for Topical, Dermal, and Yuliani, S. H., Rahmadani, Y. & Istyastono, E. P.
Transdermal Drug Delivery. Expert Opinion on (2016). Irritation Test of Wound Healing Gel of
Drug Delivery; 1-12. doi: Ethanolic Extract of Binahong Leaf Using Slug
10.1080/17425247.2020.1727883. Irritation Test. Jurnal Ilmu Kefarmasian
Subramaniam, B., Siddik, Z. H. & Nagoor, N. H. (2020). Indonesia; 14; 135-140.
Optimization of Nanostructured Lipid Carriers: Zirak, M. B. & Pezeshki, A. (2015). Effect of Surfactant
Understanding the Type, Design and Parameters Concentration on the Particle Size, Stability and
in the Process of Formulation. Journal of Potential Zeta of Beta Carotene Nano Lipid
Nanoparticle Research; 22(141); 1-29. doi: Carrier. International Journal of Current
10.1007/s11051-020-04848-0. Microbiology and Applied Sciences; 4(9); 924-
Sugihartini, N., Susanti, H., Zaenab, Hanifah, H. & 932.
Marlina, S. A. (2016). Stabilitas Epigalokatekin
P-ISSN: 2406-9388
E-ISSN: 2580-8303
DOI: 10.20473/jfiki.v9i32022.229-234
Effect of Alpha-Lipoic Acid on the Characteristics and Physical Stability of

NLC-Green Tea Extract
Fairuz Yaumil Afra1, Widji Soeratri2*, Djoko Agus Purwanto3
1
2
*Corresponding author: widjisoeratri@yahoo.com
Submitted: 28 February 2022

Abstract
Background: The addition of alpha-lipoic acid in Nanostructured Lipid Carrier-Green Tea Extract (NLC-GTE)
has potential to increase effectiveness of anti-aging preparations. It happened because alpha-lipoic acid can
increase stability and antioxidant activity. Objective: Comparing the physical characteristics and stability of NLC-
GTE with or without alpha-lipoic acid. Methods: NLC-GTE manufactured using the High Shear Homogenization
method. NLC-GTE was divided into two formulas, without the addition of alpha-lipoic acid for F1 and with the
addition of alpha-lipoic acid for F2. The characteristics and physical stability were tested, including organoleptic,
pH, particle size, and polydispersity index. Stability test was held using the thermal cycling method. Results: Based
on characteristic test, it was found that F2 had larger particle size value than F1. The average particle size value
of F1 is 313.9 ± 0.76 nm and 423.4 ± 0.75 nm for F2. The F1 and F2 preparations had a polydispersity index value
below 0.3, so they were homogeneous. The average pH value of F1 is 5.998 ± 0.01, and F2 is 4.798 ±
0.004. The physical stability test showed a difference before and after the sixth day in particle size and pH, but it
was still in the range, so it was safe. However, there was a separation in F1 after day 6. Conclusion: Based on the
characteristics and physical stability tests, F1 (without alpha-lipoic acid) and F2 (with alpha-lipoic acid) had
differences in particle size and pH. From the physical stability test, it can be concluded that F2 is more stable than
F1.
Keywords: nanostructured lipid carrier (NLC), green tea extract, alpha-lipoic acid

Afra, F. Y., Soeratri, W. & Purwanto, D. A. (2022). Effect of Alpha-Lipoic Acid on the Characteristics and Physical
Stability of NLC-Green Tea Extract. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 229-234.
http://doi.org/10.20473/jfiki.v9i32022.229-234
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION of being quite complex in preparation and low instability

At this time, the development of the world's (Manea et al., 2014). Meanwhile, SLN and NLC showed
population has entered a period called the aging high drug loading capacity (Frias et al., 2016). SLN only
population, an increase in life expectancy followed by consists of solid lipids that form perfect crystallinity.
an increase in the number of older people in all countries Thus, the entrapment power (EE) is lower. Meanwhile,
in the world. Indonesia experienced an increase in the NLC is a modification of SLN, which forms lower
number of older people from 18 million people (7.56%) crystallinity so that EE is higher and has better stability
in 2010 to 25.9 million people (9.7%) in 2019, and it is than SLN (Frias et al., 2016). It is because NLC contains
expected to increase in 2035 to 48.2 million people liquid lipids, which can reduce crystallinity to
(15.77%) (Kemenkes, 2020). It will be the reason for accommodate more active ingredients (Mayangsari et
health problems, including aging (Ahmad & Damayanti, al., 2021).
2018). Green tea extract also has a disadvantage, known as
Aging is a decrease in the size and number of skin photodegradation which causes it is easy to lose active
cells and changes in skin function resulting from a compounds, reducing stability. In the research of Chen
decline in the standard structure and function of normal et al. (2017), EGCG has a deficiency, which is proven
skin. Physiological changes in elderly skin are shown in with the presence of reactivity that causes oxidation,
impaired barrier function, slowed epidermal cell hydrolysis, epimerization, and polymerization reactions.
turnover, decreased blood vessel network around the To overcome this, needed the addition of co-
base of the hair and glands, decreased cell turnover antioxidants (Scalia et al., 2013). One of the co-
function, and decreased sweat production antioxidants that can be used is alpha-lipoic acid, where
(Anggowarsito, 2014). Cosmeceuticals are cosmetic alpha-lipoic acid has antioxidant activity that effectively
products with active ingredients that aim to have removes free radicals by reducing alpha-lipoic acid to
medical or medicinal benefits and protect against dihydrolipoic acid. Alpha-lipoic acid is soluble in fat
deteriorating skin conditions. Cosmeceuticals claim to and water to reach all parts of the cell (Perricone, 2000).
improve skin tone, function, and texture by promoting Alpha-lipoic acid is a weak acid with a pKa of 4.7,
collagen growth and reducing wrinkles (Vaishali et al., therefore it will affect the characteristics of the
2013). preparation (Cichewicz et al., 2013). In addition, alpha-
Camellia sinensis is a plant that produces various lipoic acid is an antioxidant that can keep the preparation
types of tea, including green tea. Green tea extract has stable. Alpha-lipoic acid has the ability neutralize free
pharmacological effects, including antioxidants and radicals in watery and fatty areas of cells. Alpha-lipoic
photoaging, that have potential in developing acid can neutralize various free radicals, such as singlet
cosmeceutical products (Ganesan & Dong, 2016). Green oxygen, superoxide, peroxyl and hydroxyl radicals. In
tea extract has a high solubility in water but very low the study of Hu et al. (2015), alpha-lipoic acid can
solubility in lipids, making it difficult to penetrate the reduce lipid oxidation in order to protecting EGCG from
stratum corneum. Green tea extract contains the main degradation during carrier process. EGCG can be
catechins such as epicatechin gallate (ECG), epicatechin degraded by pH so acidic state is needed to be stable.
(EC), epigallocatechin (EGC), and epigallocatechin Alpha-lipoic acid is a weak acid with a pKa of 4.7, it will
gallate (EGCG). The most active and abundant catechin lower the pH and increase the stability of EGCG.
in green tea is epigallocatechin gallate (EGCG) (Namita In a study by Bianchi et al. (2013), alpha-lipoic acid
& Kumar, 2012). as a photo stabilizer for EGCG was much more efficient
EGCG is highly soluble in water (40 g/l at 20 0C) and effective at lower concentrations than vitamins E, C,
and has good stability at pH 3.7 (250C) and pH 3.9 and BHT (Butylated hydroxytoluene). Alpha-lipoic acid
(400C). EGCG has low lipophilicity, with a log P value inhibits EGCG photodegradation and maintains
of 1.1 at pH 4.0, while the optimal log P value for functional activity under solar radiation. The addition of
optimal penetration ranges from 2 - 3 (Rosita et al., alpha-lipoic acid in NLC green tea extract can increase
2019). A nanocarrier system can be used to overcome the effectiveness of anti-aging preparations. It happened
this problem. Nanocarrier consists of Nanoparticle (NP), because alpha-lipoic acid can increase stability and
Solid Lipid Nanoparticle (SLN), and Nanoparticle Lipid increase antioxidant activity. For the introduction in this
Carrier (NLC). When compared in cost, SLN and NLC preparation, the characteristics and physical stability
are more affordable than Liposomes. In addition to tests were carried out on NLC-Green Tea Extract and
being more expensive, liposomes have the disadvantage NLC-Green Tea Extract with 0.5% alpha-lipoic acid.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
The preparation of this research using high shear put into a beaker and heated at 700C for 22 minutes.
homogenizer (HSH) method, in order to avoid the use of After that, the aqueous phase was homogenized using
organic solvents that needed in the other technique. Ultra Turrax IKA®T25 Digital High Shear
Homogenizer at 15000 rpm for 2 minutes. After finished
MATERIALS AND METHODS with homogenization, the water phase was added to the
Materials oil phase and stirred using Ultra Turrax IKA® T25
The ingredients used in this study were Meditea Digital High Shear Homogenizer at 16000 rpm for 7
Green Tea Extract (PT. Angler BioChemab, Indonesia), minutes. It was continued with stirring using a hotplate
cetyl palmitate (BASF, Indonesia), glyceryl stearate stirrer at 300 rpm for 12 minutes or until reaching room
(BASF, Indonesia), poloxamer 188 (PT. Megasetia temperature. In F2 preparations, alpha-lipoic acid is
Agung Kimia, Indonesia), lecithin (Solae, England), added to the oil phase gradually while stirring using a
grape seed oil (Brighton, UK), tween 20 (Zhang Yan, hotplate stirrer. The formula can be seen in Table 1.
Singapore), alpha-lipoic acid (Tokyo Chemical
Industry, Tokyo, Japan), NaH2PO4 (SAP, Indonesia), Physical characteristic testing
and Na2HPO4 (SAP, Indonesia). Organoleptic observations
Instrumentation Organoleptic observations were carried out by
This research used Ultra Turrax IKA®T25 Digital conducting color, odor, consistency, and visual phase
High Shear Homogenizer, OHAUS analytical balance, separation tests.
Beckman Coulter DelsaTM Nano C Particle Analyzer, Particle size and polydispersity index (PI)
Thermo Scientific Hotplate, SI Analytics Lab 865 pH Particle size and polydispersity index (PI) were
meter, and other supporting tools. measured using the DelsaTM Nano C Particle Size
Methods Analyzer. The F1 and F2 preparations were diluted first
NLC-GTE manufacture by weighing 50 mg of the preparation plus 50 mL of
The manufacture of NLC-GTE used the HSH (High distilled water and then stirred with a 500 rpm hotplate
Shear Homogenizer) method consist of an oil phase and stirrer for 10 minutes. After that, the preparation was
a water phase mixed. The oil phase consisted of solid diluted again by taking 2 mL of the preparation and
lipids (cetyl palmitate and glyceryl stearate) and liquid adding 8 mL of distilled water. The solution was stirred
lipids (grape seed oil), melted at 700C for 30 minutes. In again at 100 rpm for 10 minutes. Lastly, the solution was
the 20th minute, the green tea extract, which was put into a cuvette and measured using a DelsaTM Nano C
previously dissolved in a pH 5.0 buffer phosphate, was Particle Size Analyzer.
heated at the same temperature. After 30 minutes, the pH value measurement
green tea extract liquid was added gradually into the oil pH testing was performed by measuring the
phase and then stirred using a hotplate stirrer at 300 rpm preparation using a pH meter which was previously
for 10 minutes. The aqueous phase consisted of calibrated with a standard solution of pH 4. The name of
surfactants (tween 20), co-surfactants (Lecithin and the tool used is SI Analytics pH Meter Lab 865.
Poloxamer 188), and buffer pH 5.0. All ingredients were
Tabel 1. F1 and F2 test formula

Material Amount used (%)
Composition
Function F1 F2
Green tea extract Active Ingridients 0.1 0.1
Cetyl Palmitate Solid Lipid
Glyceryl Stearate 1.16 : 1.16 : 1 (10%)
Grape Seed Oil Liquid Lipid
Tween 20 Surfactant 2 2
Lecithin Co-surfactant 1:1
Poloxamer 188 (1%)
Alpha-lipoic acid Co- antioxidant - 0.5
Water Solvent Ad 100 Ad 100
*F1 = NLC-GTE without Alpha-lipoic acid
F2 = NLC-GTE with Alpha-lipoic acid
P-ISSN: 2406-9388
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Physical stability test 0.272 ± 0.033 for F2. With the addition of alpha-lipoic
Physical stability testing was carried out using the acid, the result showed that the greater the
thermal cycling method. The preparation was stored in concentration, the greater the value of polydispersity
1,5 cycles, two days in the oven at 400C , in the index, but it is still below the range therefore the formula
refrigerator at 2 - 80C, and in the oven for six days. is still homogeneous. The literature of Lason et al.
Organoleptic, pH, particle size, polydispersity index, (2017) stated that a polydispersity index value below 0.3
and the presence or absence of separation testing was means it was homogeneous. For statistical test using
carried out, before and after the physical stability Paired-Sample T-Test with α = 0.05, the value of Sig. (2
testing. tailed) > 0.05, it can be concluded that there was no
significant difference between F1 and F2.
RESULTS AND DISCUSSION The average pH value of F1 was 5.998 ± 0.01, and
Physical characteristic test F2 was 4.798 ± 0.004. It was in line with the literature,
Organoleptic observation i.e. 4.5 - 6.8 (Mayangsari et al., 2021). The NLC-GTE
In the manufacturing process of NLC-GTE preparation with the addition of alpha-lipoic acid has
preparations with two formulas, NLC-GTE without the more acidic pH. After statistical testing with the Paired-
addition of alpha-lipoic acid for F1 and with the addition Sample T-Test with α = 0.05, information was obtained
of alpha-lipoic acid for F2. The results of the that the value of Sig. (2 tailed) < 0.05, it can be
characteristic test showed that the NLC-GTE concluded that there was a significant difference.
preparations (F1 and F2) had white color for F1 and Differences in the concentration of alpha-lipoic acid
broken white for F2, odorless, liquid consistency, and between formulas affect the pH value, which make the
soft texture. Visually, the homogeneous NLC-GTE preparation relatively stable under acidic conditions.
preparations can be seen in Table 2. This can protect the preparation from being degraded by
Particle size and polydispersity index were pH, resulted in the preparation remains stable. The
important factors in the parameter characteristics of addition of alpha-lipoic acid which is a weak acid with
nanoparticle preparations. The average particle size of a pKa value of 4.7 resulted in a conclusion that the
NLC GTE was 313.9 ± 0.76 nm for F1 and 423.4 ± 0.75 greater the concentration, the lower the pH value
nm for F2. A statistical test was performed using the (Cichewicz et al., 2013). However, all formulas still
Paired-Sample T-Test with α = 0.05, and the result was meet the normal pH range of the skin (4.5 – 6.8) so it
the Sig value. (2 tailed) < 0.05, it can be concluded that does not cause any side effect on the skin.
there was a significant difference. The particle size of F2 Physical stability testing
was larger than F1 due to the addition of alpha-lipoic Physical stability testing used the thermal cycling
acid. It was supported by Lason et al. (2017) research method by storing the preparations in 1.5 cycles, two
that the particle size of the NLC base without alpha- days in the oven, in the refrigerator, and in the oven for
lipoic acid was smaller than the NLC preparation six days. Organoleptic, pH, particle size, polydispersity
containing the active ingredient alpha-lipoic acid. index, and the presence or absence of separation testing
However, the particle size of F2 was still within the was carried out, before and after the physical stability
range for NLC preparations, below 1000 (Mayangsari et testing.
al., 2021). For the results of the polydispersity index, the
results were below 0.3, i.e., 0.263 ± 0.007 for F1 and
Tabel 2. Characteristic observation results of F1 and F2

Observation F1 F2
Color White White
Consistency Liquid Liquid
Organoleptic
Texture Soft Soft
Odor Odorless Odorless
Particle Size 313.9 ± 0.76 423.4 ± 0.75
Polydispersity index 0.263 ± 0.007 0.272 ± 0.033
pH 5.998 ± 0.01 4.798 ± 0.004
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Table 3. Results of observation of particle size, polydispersity index, pH, and the presence or absence of separation in
the physical stability test
T=0 T = 6 days
Formula Particle Particle
Separation Size (nm) PDI ± SD pH ± SD Separation Size (nm) PDI ± SD pH ± SD
± SD ± SD
F1 313.9 ± 0.263 ± 5.998 ± 385.26 ± 0.265 ± 5.730 ±
(Without - 0.76 0.007 0.01 + 2.49 0.012 0.007
Lipoic)
F2 (With 423.4 ± 0.272 ± 4.798 ± 435.16 ± 0.260 ± 4.847 ±
Lipoic) - 0.75 0.033 0.004 - 3.65 0.024 0.012
Information:
- = absence of separation
+ = presence of separation
After six days, there were no significant changes in (Mayangsari et al., 2021). F1 and F2 were statistically
color and odor based on organoleptic testing. However, tested with the Paired-Sample T-Test, and the results
in F1, there was separation after six days, while in F2, obtained were Sig values (2 tailed) < 0.05, so there was
there was no separation, so it remained homogeneous. It a significant difference in pH F1 and F2. The results of
meant that F1 was unstable at extreme temperatures observations for particle size, polydispersity index, pH,
while F2 remained stable. In the physical stability test, and the presence or absence of separation were in Table
F2 contains alpha-lipoic acid and resulted with no phase 3. However, all formulas were still in the skin range of
separation occurs. This is supported by the research of 4.5 - 6.8 so it still safe to be used. ALA is a weak acid
Lason et al. (2017), that NLC containing the active with a pKa of 4.7 therefore it can lower the pH of the
ingredient alpha-lipoic acid when observed with an formulation but increase the stability of EGCG.
optical microscope showed that the preparation was
homogeneous and no agglomerates or crystals were CONCLUSION
found. Based on the physical characteristics test, it was
Observations of particle size values in F1 and F2 concluded that F1 (without alpha-lipoic acid) and F2
were conducted on days 0 and 6, resulted that there was (with alpha-lipoic acid) had differences in particle size
a change in particle size but still according to the and pH. From the physical stability test, it can be
literature, which has a value below 1000 nm concluded that F2 is more stable than F1.
(Mayangsari et al., 2021). In F1 and F2, statistical tests
were carried out using the Paired-Sample T-Test with α AUTHOR CONTRIBUTIONS
= 0.05. The results obtained were the Sig values (2 Conceptualization, F. Y. A., W. S., D. A. P.;
tailed) < 0.05, it can be concluded that there was a Methodology, F. Y. A., W. S., D. A. P.; Validation, F.
significant difference before and after 1.5 cycles of Y. A., W. S., D. A. P.; Formal Analysis, F. Y. A.;
thermal cycling. This difference is indicated by an Investigation, F. Y. A.; Resources, F. Y. A., W. S., D.
increase in particle size before and after storage. A. P.; Data Curation, F. Y. A., W. S.; Writing - Original
The results of the observation from the F1 and F2 Draft, F. Y. A.; Writing - Review & Editing, F. Y. A.,
polydispersity indices after the sixth day were below W. S., D. A. P.; Visualization, F. Y. A.; Supervision, F.
0.3, and this was in accordance with the literature (Lason Y. A., W. S., D. A. P.; Project Administration, F. Y. A.,
et al., 2017), if the value was below 0.3, the preparation W. S., D. A. P.; Funding Acquisition, F. Y. A.
was homogeneous. The polydispersity index before and
after 1.5 cycles of thermal cycling in F1 and F2 was CONFLICT OF INTEREST
statistically tested using the Paired-Sample T-Test, and The authors declared no conflict of interest.
the results obtained were Sig values. (2 tailed) > 0.05, so
there was no difference and the preparation remained REFERENCES
stable. Ahmad, Z & Damayanti. (2018). Penuaan Kulit
Related to pH observation on days 0 and 6, the Patofisiologi dan Manifestasi Klinis. Berkala Ilmu
results were relatively stable for F1 and F2. The pH Kesehatan Kulit dan Kelamin-Periodical of
value obtained is still in the skin pH range of 4.5 - 6.8
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Dermatology and Venereology; 30; 208-215. doi: 004/indonesia-masuki-periode-aging-

10.20473/bikk.V30.3.2018.208-215. population.html. Accessed : 11 March 2020.
Anggowarsito, J. L. (2014). Aspek Fisiologi Penuaan Lason, E., Elzbieta, S., Malgorzata, M., Paulina, S. &
Kulit. Jurnal Widya Medika; 2; 56-61. doi: Jan, O. (2017). NLC Delivery Systems for Alpha
10.33508/jwm.v2i1.1652. Lipoic Acid: Physicochemical characteristics and
Bianchi, A, Valentina, T. & Santo, S. (2013). Release Study. Colloids and Surfaces A:
Photostability of Epigallocatechin-3- Gallate in Physicochemical and Engineering Aspects; 6; 1-6.
Topical Formulation. Monographio Supplement doi: 10.1016/j.colsurfa.2017.06.083.
Series: Sun Care. 8; 18-22. Manea, A. M, Bogdan, S. V. & Aurelia, M. (2014).
Chen, J., Ning, W., Maria, L. G., Dianna, A., Jason, L., Antioxidant and Antimicrobial Activities of
Colin, A. & Teresa, P. (2017). Development and Green Tea Extract Loaded Into Nanostructured
Evaluation of Resveratrol, Vitamin E, and Lipid Carriers. Comptes Rendus Chimie; 17; 331-
Epigallocatechin Gallate Loaded Lipid 341. doi: 10.1016/j.crci.2013.07.015.
Nanoparticles for Skin Care Applications. Mayangsari, F. D, Tristiana, E, Widji, S. & Noorma, R.
European Journal of Pharmaceutics and (2021). Karakteristik dan Stabilitas Fisik NLC-
Biopharmacetics; 117; 286-291. doi: Koenzim Q10 dalam Sleeping Mask dengan
10.1016/j.ejpb.2017.04.008. Minyak Nilam. Jurnal Farmasi dan Ilmu
Cichewicz, A., Chelsea, P., Ashley, C., Martha A. H. & Kefarmasian Indonesia; 8; 178-186. doi:
Luciana, B. L. (2013). Cutaneous Delivery of α- 10.20473/jfiki.v8i22021.178-186.
Tocopherol and Lipoic Acid Using Namita, P. R. M & Kumar, V. (2012). Camellia sinensis
Microemulsions: Influence of Composition and (Green Tea): a Review. Global Journal of
Charge. Journal of Pharmacy dan Pharmacology; Pharmacology; 6; 52-59.
65; 817-826. doi: 10.1111/jphp.12045. Perricone, N. V. (2000). Topical 5% Alpha Lipoic Acid
Frias, I., Ana, R. N., Marina, P. & Salette, R. (2016). Cream in the Treatment of Cutaneous Rhytids.
Design, Development, and Characterization of Aesthetic Surgery Journal; 20; 208-222. doi:
Lipid Nanocarriers-Based Epigallocatechin 10.1016/S1090-820X(00)70020-3.
Gallate Delivery System For Preventive and Rosita, N., Vinta, A. M., Mirna, C. R., Dewi, M. H. &
Therapeutic Supplementation. Dovepress; 10; Andang, M. (2019). Enhancing Skin Penetration
3519-3528. doi: 10.2147/DDDT.S109589. of Epigallocatechin Gallate by Modifying
Ganesan, P. & Dong, K. C. (2016). Current Application Partition Coefficient using Reverse Micelle
of Phytocoumpound-Based Nanocosmeceuticals Method. Therapeutic Delivery; 15; 1-9. doi:
for Beauty and Skin Therapy. International 10.4155/tde-2019-0015.
Journal of Nanomedicine; 11; 1987-2007. doi: Scalia, S., Nicola, M. & Anna, B. (2013). Comparative
10.2147/IJN.S104701. Evaluation of Different Co- Antioxidants on the
Hu, C., Guodong, Z., Qiang, X. & Ria, S. (2015). Photochemical and Functional-Stability of
Development and Characteristic of a Self-Double- Epigallocatechin-3-gallate in Topical Creams
Emulsifying Drug Delivery System Containing Exposed to Simulated Sunlight. Molecules; 18;
Both Epigallocatechin-3-Gallate and α-Lipoic 574-587. doi: 10.3390/molecules18010574.
Acid. Journal of Materials Science; 50; 6567- Vaishali, K., Chintale, A. G., Deshmukh, K. P. &
6577. doi: 10.1007/s10853-015-9194-7. Nalwad, D. N. (2013). Cosmeceuticals an
Kementerian Kesehatan (Kemenkes). (2020). Indonesia Emerging Concept: A Comprehensive Review.
Masuki Periode Aging Population. International Journal of Research in Pharmacy
https://www.kemkes.go.id/article/view/19070500 and Chemistry; 3; 308-316.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
DOI: 10.20473/jfiki.v9i32022.235-241
Optimization Method and Stability Test to Determinate Luteolin,

Quercetin, Apigenin, and Sinensetin Levels in Herbal Medicines Using TLC-
Densitometry
Muhammad Hidayatullah1, Mochammad Yuwono2*, Riesta Primaharinastiti2
1
2
*Corresponding author: yuwono05@yahoo.com
Submitted: 20 April 2022

Accepted: 8 September 2022
Abstract
Background: Nephrolithiasis is a condition in which there are one or more kidney stones in the pelvis or calyces.
Luteolin, quercetin, apigenin, and sinensetin are marker compounds in the extracts of Plantago major, Sonchus
arvensis, Strobilanthes crispus and Orthosiphon stamineus which have nephrolithiasis activity. To control the
quality of herbal medicines, a TLC-Densitometry method was developed in this study using luteolin, quercetin,
apigenin, and sinensetin as phytochemical markers. Objective: The present work aimed to develop optimal
conditions for analyzing luteolin, quercetin, apigenin, and sinensetin. Methods: Determination of optimal
conditions for analysis is carried out by determining the composition of the mobile phase, chamber saturation
time, and analysis wavelength. Silica gel 60 F254 was used as the stationary phase. Stability tests were carried out
by analyzing standards and samples at 0, 4, 8, and 24 hours. Results: The best separation that produces
symmetrical peaks of herbal medicine was achieved under isocratic conditions using the composition of the mobile
phase chloroform : acetone: dichloromethane : acetonitrile : formic acid (6 : 2: 2 : 0,05 : 0.05 v/v/v/v/ v) with a
wavelength of 335 nm with a saturation time of 30 minutes. Conclusion: In this study, the optimal conditions for
the analysis of luteolin, quercetin, apigenin, and sinensetin. Luteolin, quercetin, apigenin, and sinensetin are
unstable during 8 hours of storage. Therefore, standard solutions and samples must be made fresh to maintain
stability.
Keywords: TLC-densitometry, luteolin, quercetin, apigenin, sinensetin

Hidayatullah, M., Yuwono, M. & Primaharinastiti, R. (2022). Optimization Method and Stability Test for
Determination of Luteolin, Quercetin, Apigenin, Sinensetin Levels in Herbal Medicines Using TLC-Densitometry.
Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 235-241. http://doi.org/10.20473/jfiki.v9i32022.235-241
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION medicines. The analytical method commonly used to

Over the decades, herbal plants and their active identify marker compounds is TLC-Densitometry
constituents have been developed and used to treat (Kartini & Azminah, 2012; Kim et al., 2010; Bertrams
various ailments. The increasing development and use et al., 2013; Attarde et al., 2017). Identification and
of herbal medicines are based on several reasons, quantitation of extracts in polypharmacy herbal
namely: not all modern medicines can efficiently medicines is a challenge. Therefore, developing and
overcome/cure all human pathologies, there is validating the TLC-Densitometry method are essential
increasing interest and attention to the guarantee and to obtain a simple and fast procedure applied to a quality
safety of synthetic medicines, and many natural control laboratory. The research that has been carried
products are proven to have better quality than synthetic out is limited to determining marker compounds in
medicines. Plantago major, Sonchus arvensis, single herbal plants and has not been applied to the
Strobilanthes crispus, and Orthosiphon stamineus, formulation of herbal medicinal preparations using the
which are empirically used to treat or prevent TLC-Densitometry method (Abdullah et al., 2012;
nephrolithiasis have been developed in many countries. Hossain & Ismail, 2016; Kuppusamy et al., 2017).
Extracts of leaves, stems, and roots have long been used Several analytical methods for determining luteolin,
as a treatment for kidney stones, antifungal, bladder, quercetin, apigenin, and sinensetin have been developed
antioxidant, gastrointestinal infections, diabetes, and using the HPLC method (Kuppusamy et al., 2017; Grek
anticancer (Kartini & Azminah, 2012; Hossain & Ismail, et al., 2019; Ghiasian et al., 2021). however, this
2016). Several studies have proven that Plantago major, instrument is expensive and require expertise
Sonchus arvensis, Strobilanthes crispus, and specialized in instrument operation.
Orthosiphon stamineus have nephrolithiasis effects This study aimed to obtain an alternative method of
(Singh et al., 2009; Alkreathy et al., 2014; Chun et al., TLC-densitometry which is cheaper and more
2020; Ghiasian et al., 2021). Jimoh et al. (2021) proved straightforward for the simultaneous analysis of
that luteolin, quercetin, apigenin, and sinensetin luteolin, quercetin, apigenin, and sinensetin in herbal
compounds play a role in controlling the crystallization medicines containing extracts of Plantago major,
process of kidney stones (jimoh et al., 2021). Hossain & Sonchus arvensis, Strobilanthes crispus, and
Ismail, (2012) also proved that luteolin, quercetin, Orthosiphon stamineus.
apigenin, and sinensetin compounds could inhibit
electrolyte reabsorption in the loop of Henle so that it MATERIALS AND METHODS
has a diuretic effect (Hossain & Ismail, 2016). Diuretics Materials
effectively reduce urinary calcium excretion and kidney Luteolin standard (Sigma Aldrich), quercetin
stone recurrence in nephrolithiasis patients by standard (Sigma Aldrich), apigenin standard (Sigma
controlling urine supersaturation (Ahmed et al., 2018). Aldrich), sinensetin standard (Sigma Aldrich), and
The structure of luteolin, quercetin, apigenin, and Herbal medicinal products obtained from the
sinensetin can be seen in Figure 1. pharmaceutical industry in Indonesia (PT. Interbat,
Sidoarjo), ethanol (Merck, US/Canada), formic acid
(Merck, US/Canada), chloroform (Merck, US/Canada),
dichloromethane (Merck, US/Canada), acetone (Merck,
US/Canada), acetonitrile (Merck, US/Canada).
Tools
Silica gel TLC plate 60 F254 aluminum sheet 20 x
20 cm (E, Merck, Darmstadt, Germany), Camag vessel
10 x 10 cm (Camag), TLC Scanner 3 with UV detector
(Camag), winCATS software version 1.4.8.2012
Figure 1. Structure of luteolin (a), quercetin (b), (Camag).
apigenin (c) dan sinensetin (d) Method
Preparation of standard solution
In developing polypharmacy herbal medicines, the
Standards (1.0 mg) were each dissolved in 10 mL
selection of marker compounds and methods of
of ethanol in a measuring flask. The stock standar
identification/quantification of marker compounds play
solutions were then diluted to working solutions of 20 -
an important role in ensuring quality control of herbal
100 ppm.
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Table 1. Variation of mobile phase composition tested to obtain optimal conditions

No. Mobile phase composition Ref.
1. Chloroform - dichloromethane - ethyl acetate (7:4:1, v/v/v) (Kartini et al., 2020)
2. Isopropyl alcohol - n-butanol (5:5, v/v) (Choudhary et al., 2020)
3. Toluene - ethyl acetate - formic acid (6:4:0,15, v/v/v) (Attarde et al., 2017)
Chloroform - acetone - dichloromethane - acetonitrile - formic acid
4. (Singh et al., 2009)
(6:2:2:0,05:0,05, v/v/v/v/v)
Table 2. Variations in the composition of the mobile phase composition of a mixture of luteolin, quercetin, apigenin,
and sinensetin
Mobile phase Compound Retardation factor (Rf) Rs Peak
1 Luteolin 0.19 0.94 Fronting
Apigenin 3.34 -
Sinensetin 0.82 2.4
2 Luteolin 0.34 - Fronting
Quercetin 0.42 1.2
Apigenin 0.51 1.4
Sinensetin 0.61 2.1
3 Luteolin 0.18 - Fronting
Quercetin 0.21 -
4 Luteolin 0.23 2.3 Symmetric
Quercetin 0.24 2.2
Apigenin 0.34 1.8
Sinensetin 0.60 3.7
Sample preparation RESULTS AND DISCUSSION

Herbal medicine was weighed 1 g and dissolved 20 The data in Table 2 are based on experiments
ethanol in a measuring flask. The solution was sonicated conducted on variations in the composition of the
for 30 minutes filtered through a 0.45 µm membrane mobile phase.
filter and spotted on a TLC plate. The composition of the mobile phase 1 shows a
Preparation of mobile phase resolution < 1.5 so that the peaks of luteolin, quercetin,
For optimization of the mobile phase, the mobile apigenin, and sinensetin are not entirely separated from
phase is made in several compositions as shown in Table the peaks of impurities. In addition, the peak is also
1. fronting so mobile phase 1 is not selected. The
Optimization of the analytical conditions composition of phase 2 also showed poor resolution
Optimization of analytical conditions is done by between luteolin, quercetin, apigenin, and sinensetin,
changing the composition of the mobile phase, the namely < 1.5. This could affect the analysis results, so
saturation time of the vessel, and determining the mobile phase 2 was not selected. The composition of
maximum wavelength. The development method uses mobile phase 3 was unable to completely separate
various mobile phase processes. The mobile phase was luteolin, quercetin, apigenin, and sinensetin, which
sonicated for 15 minutes before use. Parameters showed a resolution < 1.5 and had a fronting peak, so
observed in selecting optimal conditions are the the composition of mobile phase 3 was not chosen. The
retardation factor (Rf), and the best resolution (Rs). composition of the mobile phase 4 shows the most
Stability test of the test solution optimal resolution of > 1.5 and an asymmetrical peak,
The stability test was carried out by dividing the so this mobile phase was selected and used for further
standard solution and the sample into four different analysis. The optimal conditions obtained in this study
tubes. Each tube is labeled 0 hours, 4 hours, 8 hours, and can be used to test the stability of the pre-validation
24 hours. The test solution was analyzed according to stage method. The composition of the mobile phase 4
the time specified on the label. All tubes were stored at shows the most optimal resolution of > 1.5 and an
4oC. asymmetrical peak, so this mobile phase was selected
and used for further analysis. The optimal conditions
obtained in this study can be used to test the stability of
the pre-validation stage method.
P-ISSN: 2406-9388
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Figure 2. The Spectrum of apigenin compounds and its maximum wavelength using solvents chloroform - acetone -
dichloromethane - acetonitrile - formic acid (6:2:2:0.05:0.05, v/v/v/v/v)
Table 3. Sample load optimization results
Concentration (ng/ µl) Sample load (µl) Compound Area Peak
12,18 4 Luteolin 6686.1 Symmetrical
Quercetin 20802.7
Apigenin 12071.5
Sinensetin 9858.0
12,18 8 Luteolin 7563.3 Symmetrical
Quercetin 2187.7
Apigenin 1398.3
Sinensetin 9987.0
12,18 14 Luteolin 10987.3 Fronting
Quercetin 26234.6
Apigenin 16345.3
Sinensetin 13345.6
The maximum wavelength of the selected analysis The saturation time of the vessel has been optimized
was observed from the peak, which gave the largest area in this study. The optimization results are listed in Table
value in the spectra of luteolin, quercetin, apigenin, and 4. From the optimization results, it is found that the
sinensetin. Besides, it did not cause interference peaks saturation time of the vessel can affect Rf. The optimal
that could affect the analysis results of luteolin, vessel saturation time in this study is at least 30 minutes
quercetin, apigenin, and sinensetin compounds. At 335 because the area and Rf are stable after 30 minutes.
nm, the maximum wavelength of apigenin compounds
Table 4. Optimization of apigenin saturation time
produced by high peaks of luteolin, quercetin, apigenin,
Saturation time (minutes) Area Rf
and sinensetin and the resulting interference peak is very 15 956,6 0,19
small, so the selected wavelength used is 335 nm (Figure 30 1098,8 0,23
2). 60 1095,0 0,23
Complete saturation of the vessel is required so that
the elution and separation process can run smoothly Stability of the test solution
good. The results of the sample load optimization are A stability test on standard solutions and samples is
presented in Table 3. Based on the results obtained, the one of the tests carried out at the pre-validation stage.
sample loads that give a symmetrical peak shape are 4 This stability test is carried out to evaluate the stability
µl and 8 µl. At a sample load of 14 µl, the peak of the test solution at a certain storage time. Parameters
experienced fronting. This could be because the observed in the selection of optimal conditions are the
injection volume is too large. The optimal separation in retardation factor (Rf), and the best resolution (Rs); a
thin-layer chromatography will be obtained if the good Rf value indicates a good separation is in the range
sample is spotted with the smallest and narrowest of 0.2-0.8, and a good separation if the Rs value meets
possible spot size. As in other chromatographic the requirements, namely > 1.5 (AOAC, 2015). The
procedures, the resolution will decrease if too many results of the stability test of the test solution at several
samples are used (Chun et al., 2020). times of measurements can be seen in Table 5 and Table
6.
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Table 5. The results of stability tests of standard solutions in the range 0 - 24 hours
Observation Time (hours) Standard Average Area Area Difference (%) Rf Difference Rf (%)
Luteolin 76861 - 0.23 -
Quercetin 218027 - 0.24 -
0
Apigenin 13071.5 - 0.34 -
Sinensetin 9958 - 0.6 -
Luteolin 7543.2 1.86 0.23 -
Quercetin 21513.5 1.33 0.24 -
4
Apigenin 13042.8 0.22 0.34 -
Sinensetin 9843.4 1.15 0.61 1.64
Luteolin 7435.9 3.26 0.23 -
Quercetin 20978.1 2.49 0.25 4.00
8
Apigenin 12878.2 3.78 0.35 2.86
Sinensetin 9634.7 1.48 0.62 3.23
Luteolin 6928.9 9.85 0.22 4.55
Quercetin 19913.3 8.67 0.24 -
24
Apigenin 11976.3 8.38 0.34 -
Sinensetin 8967.9 9.94 0.61 1.64
Table 6. The results of stability tests of sample solutions in the range 0 - 24 hours
Observation Time (hours) Sample Average Area Area Difference (%) Rf Difference Rf (%)
Luteolin 8542.2 - 0.23 -
Quercetin 7128.3 - 0.24 -
0
Apigenin 7893.4 - 0.34 -
Sinensetin 19077 - 0.6 -
Luteolin 8512.1 0.35 0.23 -
Quercetin 7098.3 0.42 0.24 -
4
Apigenin 7856.9 0.46 0.34 -
Sinensetin 18893.5 0.97 0.6 -
Luteolin 8467.9 0.88 0.23 -
Quercetin 6983.2 2.08 0.25 4.00
8
Apigenin 7398.4 6.69 0.35 2.86
Sinensetin 18349.1 3.97 0.61 1.64
Luteolin 8119.3 5.21 0.22 4.55
Quercetin 6723.3 6.02 0.24 -
24
Apigenin 7239.9 9.03 0.34 -
Sinensetin 18122.1 5.27 0.62 3.23
Figure 3. Chromatogram of standard (1) luteolin (2) quercetin (3) apigenin and (4) sinensetin (A) and herbal drug
sample (B) with mobile phase composition of chloroform : acetone : dichloromethane : acetonitrile : formic acid (6 : 2 :
2 : 0.05 : 0.05 v/v/v/v/v)
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Based on the data obtained, the test solution is of Eisai Kucing (Orthosiphon stamineus) Leaves.
known to be unstable after 8 hours of storage. Then an Apcbee Procedia; 2; 178-182. doi:
analysis was carried out using SPSS to determine 10.1016/j.apcbee.2012.06.032.
whether there was a significant difference between the Ahmed, S., Hasan, M. M., Khan, H., Mahmood, Z. A. &
area and factor retardation at each observation time. The Patel, S. (2018). The Mechanistic Insight of
data obtained showed a normal and homogeneous Polyphenols in Calcium Oxalate Urolithiasis
distribution so two-way ANOVA tested it. The results Mitigation. Biomedicine & Pharmacotherapy;
of the analysis show that there are significant differences 106; 1292-1299. doi:
in the area mean and Rf at each time of observation. This 10.1016/j.biopha.2018.07.080.
indicates that the longer the standard solution is stored, Alkreathy, H. M., Khan, R. A., Khan, M. R. & Sahreen,
the lower the concentration will be (Figure 3). S. (2014). CCl4 Induced Genotoxicity and DNA
Oxidative Damages in Rats: Hepatoprotective
CONCLUSION Effect of Sonchus arvensis. BMC complementary
The optimal conditions obtained in this study for and Alternative Medicine; 14; 1-7. doi:
philanthropic analysis were the composition of the 10.1186/1472-6882-14-452.
mobile phase chloroform: acetone: dichloromethane: Attarde, D. L., Pal, S. C., & Bhambar, R. S. (2017).
acetonitrile: formic acid (6 : 2 : 2 : 0.05 : 0.05 v/v/v/v/v), Validation and Development of HPTLC Method
with saturation time 30 minutes, wavelength analysis for Simultaneous Estimation of Apigenin and
335 nm. The stability of the solution was tested in this Luteolin in Selected Marketed Ayurvedic
study as a pre-validation stage. The stability of the test Formulations of ‘Dashmula’ and in Ethyl Acetate
solution decreased with storage for a certain time, Extract of Premna integrifolia L. Journal of
indicated by a decrease in area and a retardation shift in Analytical & Bioanalytical Techniques; 8; 1-9.
the chromatogram. It is recommended that the test doi: 10.4172/2155-9872.1000343.
solution be made fresh when the analysis is carried out. AOAC. (2015). Guidelines for Dietary Supplements and
The optimal conditions obtained can be continued with Botanicals. Rockville: AOAC International.
method validation for further research. Bertrams, J., Kunz, N., Müller, M., Kammerer, D. &
Stintzing, F. C. (2013). Phenolic Compounds as
ACKNOWLEDGMENT Marker Compounds for Botanical Origin
Thanks to ULP Universitas Airlangga who has Determination of German Propolis Samples
provided the means for this research to run, and thanks Based on TLC and TLC-MS. Journal of Applied
to PT. Interbat Sidoarjo provides research-related Botany and Food Quality; 86; 143 - 153. doi:
samples. 10.5073/JABFQ.2013.086.020.
Chun, S. C., Gopal, J., Iyyakannu, S., & Muthu, M.
AUTHOR CONTRIBUTIONS (2020). An Analytical Retrospection of Mass
Conceptualization, M. H., M. Y., R. P.; Spectrometric Tools Established for Plant Tissue
Methodology, M. H., M. Y.; Software, M. H.; Culture: Current Endeavours and Future
Validation, M. H., M. Y., R. P.; Formal Analysis, M. H., Perspectives. TrAC Trends in Analytical
M. Y., R. P.; Investigation, M. H., M. Y., R. P.; Chemistry; 126; 1-16. doi:
Resources, M. H., M. Y., R. P.; Data Curation, M. H.; 10.1016/j.trac.2020.115843.
Writing - Original Draft, M. H.; Writing - Review & Choudhary, N. K., Gupta, K., Jain, A. K. & Pal, A.
Editing, M. H., M. Y., R. P.; Visualization, M. H., M. (2020). Quantitative Estimation of Marker
Y., R. P.; Supervision, M. H., M. Y., R. P.; Project Compound (Lupeol and Diosgenin) in Polyherbal
Administration, M. H., M. Y., R. P.; Funding Formulation by HPTLC Method. Plant Archives;
Acquisition, M. H., M. Y., R. P. 20; 4337-4342.
Ghiasian, M., Niroomandi, Z., Dastan, D., Poorolajal, J.,
CONFLICT OF INTEREST Zare, F. & Ataei, S. (2021). Clinical and
The authors declared no conflict of interest. Phytochemical Studies of Plantago major in
Pressure Ulcer Treatment: a Randomized
REFERENCES Controlled Trial. Complementary Therapies in
Abdullah, S., Shaari, A. R. & Azimi, A. (2012). Effect Clinical Practice; 43; 1-6. doi:
of Drying Methods on Metabolites Composition 10.1016/j.ctcp.2021.101325.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Grek, O., Chubenko, L., Kumar, A., Khareba, V. Kartini, K., Jayani, N. I. E., Hadiyat, M. A. & Avanti, C.
Tymchuk, A., & Onopriichuk, O. (2019). (2020). Thin Layer Chromatography
Polyphenolic Compounds Transition into Protein- Fingerprinting and Clustering of Orthosiphon
Plant Concentrates During the Deposition of Milk stamineus Benth from Different Origins.
Proteins by Plantago major L. Ukrainian Food Pharmacognosy Journal; 12; 1683-1691. doi:
Journal; 8; 745-754. 10.5530/pj.2020.12.257.
Hossain, M. A. & Ismail, Z. (2016). Quantification and Kim, H. J., Jee, E. H., Ahn, K. S., Choi, H. S. & Jang,
Enrichment of Sinensetin in the Leaves of Y. P. (2010). Identification of Marker Compounds
Orthosiphon stamineus. Arabian Journal of in Herbal Drugs on TLC with DART-MS.
Chemistry; 9; S1338-S1341. doi: Archives of Pharmacal Research; 33; 1355-1359.
10.1016/j.arabjc.2012.02.016. Kuppusamy, P., Lee, K. D., Song, C. E., Ilavenil, S.,
Jimoh, S. O., Ha, M., Arowolo, L. A., Badmos-Oladapo, Srigopalram, S., Arasu, M. V. & Choi, K. C.
R. B. & Akinlade, R. Y. (2021). Invitro Activity (2018). Quantification of Major Phenolic and
of Phyllanthus amarus Extract on Nephrolithiasis Flavonoid Markers in Forage Crop Lolium
and Urea-Splitting Bacteria. Fountain Journal of multiflorum using HPLC-DAD. Revista
Natural and Applied Sciences; 10; 25-32. Brasileira de Farmacognosia; 28; 282-288. doi:
Kartini & Azminah. (2012). Chromatographic 10.1016/j.bjp.2018.03.006.
Fingerprinting and Clustering of Plantago major Singh, B., Mungara, P., Nivsarkar, M. & Anandjiwala,
L from Different Areas In Indonesia. Asian S. (2009). HPTLC Densitometric Quantification
Journal of Pharmaceutical and Clinical of Glycyrrhizin, Glycyrrhetinic Acid, Apigenin,
Research; 4; 191-195. Kaempferol and Quercetin from Glycyrrhiza
glabra. Chromatographia; 70; 1665-1672.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
DOI: 10.20473/jfiki.v9i32022.242-251
The Effect of Insulin Administration on Medication Adherence in Type 2

Diabetes Mellitus Patients with Neurological Complications
July1, Rani Sauriasari2*, Nadia Farhanah Syafhan2, Hadijah Tahir1
1
National Brain Center Hospital, Jakarta, Indonesia
2
Department of Pharmacy, Faculty of Pharmacy, University Indonesia, Jakarta, Indonesia
*Corresponding author: rani@farmasi.ui.ac.id
Submitted: 1 June 2022

Accepted: 17 October 2022
Abstract
Background: Medication adherence is essential to achieving controlled blood sugar in diabetic patients. Insulin
generally provides better glycemic control but is considered painful and requires special techniques. Insulin
administration in patients with neurological complications requires particular consideration because these
complications can cause physical and cognitive barriers. Objective: This study analyses the effect of insulin
administration on medication adherence in diabetic patients with neurological complications and the influence of
various confounding variables (baseline characteristics, medical and medication history). Methods: This
observational study was conducted with a cross-sectional design at a government hospital in East Jakarta from
September 2021 to January 2022. The sample was type 2 diabetes mellitus patients with neurological
complications who received antidiabetics for at least six months. The neurological complications include central
nervous disorders (such as stroke) and peripheral nervous disorders (such as neuropathy). The independent
variable was insulin administration, while the dependent variable was adherence, measured using subjective
methods [Adherence to Refills and Medications Scale (ARMS)] and objective methods (Medication Refill
Adherence (MRA) and HbA1c]. Results: Of 175 respondents, based on the three methods (MRA, ARMS, HbA1c),
13 respondents (7.4%) were adherent, namely one respondent (1.8%) in the insulin group and 12 respondents
(10.1%) in the non-insulin group. Insulin administration affects adherence to antidiabetics by 0.123 times (95%
CI: 0.015 - 1.024), or patients who use insulin have 87.7% lower adherence controlled by antidiabetic changes
and the total number of medicines used. Conclusion: Insulin administration significantly affects medication
adherence in diabetes mellitus patients with neurological complications.
Keywords: diabetes mellitus, insulin, adherence, neurological complications

July, Sauriasari, R., Syafhan, N. F. & Tahir, H. (2022). The Effect of Insulin Administration on Medication
Adherence in Type 2 Diabetes Mellitus Patients with Neurological Complications. Jurnal Farmasi dan Ilmu
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION neurological complications by using subjective methods

Diabetes mellitus is among the ten leading causes [Adherence to Refills and Medications Scale (ARMS)]
of death globally, with a 70% increase since 2000. and objective methods [Medication Refill Adherence
Around 2.3 million women and 1.9 million men aged 20 (MRA) and HbA1c measurement], as well as the effect
– 79 are estimated to die from diabetes and its of a various confounding variable on medication
complications in 2019 (WHO, 2020). Indonesia had the adherence.
seventh-highest number of people with diabetes in the
world in 2019; the number of people with diabetes is MATERIALS AND METHODS
around 10.7 million (International Diabetes Federation, This observational study used a cross-sectional
2019). design and was conducted at a government hospital in
Pharmacological therapy in patients with type 2 East Jakarta from September 2021 to January 2022. The
diabetes mellitus consists of oral antidiabetic and sampling technique used was the consecutive sampling
injection antidiabetic (insulin) or a combination. Insulin method. The inclusion criteria were typed 2 diabetes
is given if the target blood glucose level has not been mellitus patients with neurological complications who
achieved with two types of oral antidiabetics (Perkeni, had received at least six months of antidiabetes with a
2021). payment system by Badan Penyelenggara Jaminan
Medication adherence is essential in the effective Sosial (BPJS)/Indonesian Universal Covered Health
management of diabetes mellitus. Adherence to Insurance and willingness to be respondents in this
antidiabetic use is associated with improved control of study.
blood sugar levels (Doggrell & Warot, 2014). Neurological complications are divided into central
Uncontrolled blood sugar levels increase the risk of and peripheral nerve disorders. Central nervous
recurrent stroke by 1.45 times compared to patients with disorders were divided into patients with and without
controlled sugar levels (Shou et al., 2015). Insulin stroke, while peripheral nervous disorders were divided
generally provides better glycemic control, improving into patients with and without. Both of these conditions
quality of life and reducing diabetic complications. On are determined based on the doctor's diagnosis in the
the other hand, it causes discomfort, pain, and medical records.
aggravation and limits the patient's daily activities, Patients who redeemed antidiabetics outside the
affecting adherence and ultimately the success of hospital where the study was conducted, whose medical
therapy. In particular, the administration of drugs with record data were incomplete, and who did not fully
special techniques, such as insulin, requires special answer the ARMS questionnaire were excluded from
consideration in patients with neurological this study.
complications because these complications can cause Using the Lemeshow formula (Lachenbruch et al.,
the patient to have physical and cognitive barriers and 1991) and previous research data (Osborn & Gonzalez,
limitations due to disorders of the central and/or 2016), 95% confidence interval, and 70% test power, the
peripheral nerves. This may be will affect medication minimum number of samples required is 53 samples for
adherence. Complications are secondary diseases or each group.
conditions that develop during the primary disease or Data were collected in two ways, interviews with
condition (Complication Definition & Meaning - outpatients who had received medication for at least six
Merriam-Webster, 2022). In patients with diabetes months based on data obtained from the patient's
mellitus, neurological complications can include central medical records. Secondary data were medical history,
nervous disorders (such as stroke) and peripheral nerve medicine, and HbA1c from medical records.
disorders (such as neuropathy) (Ireland et al., 2010). Measurement of adherence with the subjective
Various studies in Indonesia have previously been method was carried out using the Indonesian version of
undertaken to examine medication adherence with oral the Adherence to Refills and Medications Scale
antidiabetics without considering the patient's (ARMS) questionnaire, in which validity and reliability
comorbidities and generally use one or two measuring were determined. This questionnaire contains twelve
instruments (Salistyaningsih et al., 2011; Alfian, 2015; questions: eight items from the drug use subscale to
Adikusuma & Qiyaam, 2017; Nanda et al., 2018; Bulu assess the patient's adherence to using prescribed drugs
et al., 2019). The purpose of this study was to analyse as directed, and four items from the prescription refill
the effect of insulin administration on medication subscale to assess the patient's adherence to refilling his
adherence in diabetes mellitus patients with prescription. The ARMS questionnaire has been
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translated and validated in several countries, such as medication history (duration of diabetes diagnosed,
Iran, Korea, China, Spain, and Poland (Jin et al., 2016; antidiabetic changes, number of antidiabetic drugs,
Kim et al., 2016; González-Bueno et al., 2017; Barati et number of comorbidities, diagnosis of central and
al., 2018; Lomper et al., 2018; Park et al., 2018 Chen et peripheral nervous disorders, total number of
al., 2020; Kripalani et al., 2009; Zairina et al., 2022). medications, herbal consumption, allergies, prescription
Measurement of adherence with the objective iterations, and family/caregiver assistance). Diagnosis
method was carried out based on changes in HbA1c of central and peripheral nervous disorders data was
values in the last two HbA1c examinations with a obtained from the patient's medical record. Central
minimum distance of three months and using the nervous disorders were categorised into respondents
percentage of Medication Refill Adherence (MRA) with with a history of ischemic or hemorrhagic stroke (stroke
the formula: category) and did not have a history of stroke (non-
stroke category). Non-stroke respondents include
Number of prescribed treatment days for each refills patients diagnosed with Parkinson's, dementia,
MRA = × 100
Number of days between the refills
depression, and others. We divided it into stroke and
(1)
non-stroke because more than 80% of respondents had a
Respondents are considered to be adherent if they
stroke history. Peripheral nerve disorders were divided
met the criteria for adherence in all tests (overall),
into patients with peripheral nerve disorders and those
namely having an ARMS score of 12 (Kripalani et al.,
without. Diagnosis of peripheral nerve disorders
2009), an MRA percentage of 80 - 120% (Kindmalm et
includes neuropathy and peripheral pain.
al., 2007), and a decrease in the HbA1c value in the last
two measurements 0.2% (Sherwani et al., 2016). Data
RESULTS AND DISCUSSION
were analysed using SPSS 26, namely descriptive
Characteristics of respondents
analysis, different proportion analysis, and logistic
Table 1 shows the 175 respondent characteristics,
regression analysis using the backward method.
consisting of 56 respondents who used insulin (insulin
This study also analysed the effect of various
group) and 119 respondents who did not use insulin
confounding variables derived from the patient's
(non-insulin group). Patients taking insulin can take
characteristics (age, gender, education, occupation, and
insulin alone or in combination with oral antidiabetics.
body mass index) as well as the patient's medical and
Table 1. Characteristics of respondents
Characteristics Insulin n (%)a Non-Insulin n (%)a Total b p-value (inter-group)
Age group 1 (35 - 44 years) 3 (5.4) 5 (4.2) 8 (4.6) 0.951 c
2 (45 - 54 years) 10 (17.9) 23 (19.3) 33 (18.9)
3 (55 - 64 years) 25 (44.6) 47 (39.5) 72 (41.1)
4 (65 - 74 years) 14 (25.0) 33 (27.7) 47 (26.9)
5 (75+) 4 (7.1) 11 (9.2) 15 (8.6)
Gender Man 30 (53.6) 74 (62.2) 104 (59.4) 0.179 c
Woman 26 (46.4) 45 (37.8) 71 (40.6)
Education Did not finish 1 (1.8) 2 (1.7) 3 (1.7) 0.478 c
elementary school/did
not go to school
Elementary 4 (7.1) 9 (7.6) 13 (7.4)
Junior High School 4 (7.1) 8 (6.7) 12 (6.9)
Senior High School 14 (25.0) 46 (38.7) 60 (34.3)
College 33 (58.9) 54 (45.4) 87 (49.7)
Work Retired/not working 22 (39.3) 52 (43.7) 74 (42.3) 0.801 c
PNS/TNI/POLRI 7 (12.5) 8 (6.7) 15 (8.6)
Self-employed/ 2 (3.6) 7 (5.9) 9 (5.1)
trader
Private employees 5 (8.9) 9 (7.6) 14 (8.0)
Housewife 18 (32.1) 37 (31.1) 55 (31.4)
Other 2 (3.6) 6 (5.0) 8 (4.6)
Body mass index (Kg/m2) 25.77 ± 4.49 25.57 ± 4.15 25.64 ± 4.25 0.775 d
a b
Information: Value is expressed in n (%), the percentage in one category; Values are expressed in n (%), percentage of
all respondents; c Chi-squared test, d unpaired T-test because body mass index data is normal.
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Table 1 shows that the respondents in this study addition of antidiabetics. Sixty-five respondents
consisted of respondents aged 55 - 64 years (41.1%), (37.1%) got three antidiabetics, 59 respondents (33.7%)
followed by 65 - 74 years (26.9%), 45 - 54 years got two antidiabetics, and the rest received a single
(18.9%), over 75 years (8.6%), and 35 - 44 years old antidiabetic or a combination of four antidiabetics. The
(4.6%). Male respondents were 59.4%, while female antidiabetic can be insulin and/or oral antidiabetic.
respondents were 40.6%. Most respondents were Respondents who got four antidiabetics commonly
undergraduate (49.7%) and high school (34.3%). As received insulin; only one received four oral
many as 42.3% of respondents are not working or antidiabetics, which consisted of metformin,
retired, and 31.4% are homemakers. Only eight vildagliptin, gliclazide, and pioglitazone. Most
respondents (4.6%) showed drug allergy but were not respondents had stroke prevously (86.95%, n = 152).
antidiabetic. Most respondents were overweight, whit a This rate is in line with the meta-analysis of 102
body mass index of 25.64 ± 4.25 Kg/m2. prospective studies involving 698782 people showed
Respondent's medical and medication history that diabetes increased the risk of ischemic stroke by
The respondent's medical and medication history is 2.27 times and hemorrhagic stroke by 1.56 times.
summarised in Table 2. Of the 175 respondents, 71 (Sarwar et al., 2010; Bloomgarden & Chilton, 2021). A
respondents (40.6%) had received antidiabetes for 6 - 12 total of 56 respondents (32.0%) were diagnosed with
months, 55 respondents (31.4%) for 12-24 months, and peripheral nerve disorders. A small proportion of
49 respondents (28%) for more than 24 months. A small respondents (20.0%, n = 35) received more than ten
proportion of respondents (37.1%, n = 65) experienced medicines, categorised as major polypharmacy (Kim et
changes in antidiabetics, which is the replacement or al., 2014).
Table 2. Medical and medication history of respondents
Insulin n Non-Insulin n
Characteristics Totalb p-value (inter-group)c
(%)a (%)a
Duration of < 12 months 27 (48.2) 44 (37.0) 71 (40.6) 0.315
diagnosed diabetes 12 - 24 months 14 (25.0) 41 (34.5) 55 (31.4)
> 24 months 15 (26.8) 34 (28.6) 49 (28.0)
Antidiabetic changes Yes 30 (53.6) 35 (29.4) 65 (37.1) 0.002
in the last six months Not 26 (46.4) 84 (70.6) 110 (62.9)
Number of Single drug 0 (0.0) 33 (27.7) 33 (18.9) < 0.001
antidiabetics Combination of 9 (16.1) 50 (42.0) 59 (33.7)
(insulin and/or 2 drugs
antidiabetic oral) Combination of 30 (53.6) 35 (29.4) 65 (37.1)
3 drugs
Combination of 17 (30.4) 1 (0.8) 18 (10.3)
4 drugs
Number of ≤3 25 (44.6) 52 (43.7) 77 (44.0) 0.517
comorbidities >3 31 (55.4) 67 (56.3) 98 (56.0)
Central nervous Stroke 53 (94.6) 99 (83.2) 152 (86.9) 0.027
system disorders Non-stroke 3 (5.4) 20 (16.8) 23 (13.1)
Peripheral nerve Yes 20 (35.7) 36 (30.3) 56 (32.0) 0.290
disorders Not 36 (64.3) 83 (69.7) 119 (68.0)
Total amount of ≤10 37 (66.1) 103 (86.6) 140 (80.0) 0.002
medicine >10 19 (33.9) 169 (13.4) 35(20.0)
Consumption of Yes 17 (30.4) 29 (24.4) 46 (26.3) 0.254
herbs in the last six Not 39 (69.6) 90 (75.6) 129 (73.7)
months
Allergy Yes 3 (5.4) 5 (4.2) 8 (4.6) 0.500
Not 53 (94.6) 114 (95.8) 167 (95.4)
Recipe iteration Yes 34 (60.7) 95 (79.8) 129 (73.7) 0.007
Not 22 (39.3) 24 (20.2) 46 (26.3)
Family/ Yes 41 (73.2) 69 (58.0) 110 (62.9) 0.037
Caregiver support Not 15 (26.8) 50 (42.0) 65 (37.1)
Description: a Value is expressed in n(%), percentage in one category; b Values are expressed in n(%), percentage of all
respondents; c Chi-square test.
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Medication adherence examinations, taken from medical record data.

Adherence to Refills and Medications Scale (ARMS) Respondents were declared adherent if they showed a
The Indonesian version of the ARMS questionnaire 0.2% decrease in HbA1c value because it reduced
in this study has obtained permission from the owner mortality by 10% (Sherwani et al., 2016). Based on the
(Kripalani et al., 2009). Validity and reliability tests reduction in HbA1c, there were 63 respondents (36%)
were carried out on the first 30 respondents and obtained who were adherent, namely 26 respondents (46.4%) in
valid results; each question on the questionnaire showed the insulin group and 37 respondents (31.1%) in the non-
r results (correlated item-total correlation) greater than insulin group.
the r table (α = 0.05, df 28 (n-2)), and reliable, means The medication adherence results using each of
having Cronbach's Alpha above 0.6, which is 0.829. The these measuring instruments and the combination of the
reliability test result of the ARMS questionnaire is close three measuring instruments are summarised in Table 3.
to Cronbach's value in the reliability test of the The effect of insulin administration and
questionnaire translation conducted by previous confounding variables was analysed using the backward
researchers, which is 0.865 (Zairina et al., 2018). The method in multivariate logistic regression. Before
ARMS score of the insulin group respondents was 12 - multivariate analysis, bivariate analysis was performed
24, while the respondents in the non-insulin group were for each confounding variable (Table 4).
12 - 30. Based on the ARMS score, respondents were Confounding variables that had a p-value less than
declared adherent if they had a score of 12 (Kripalani et 0.25 were included in the multivariate logistic
al., 2009); as many as 43 respondents (24.6%) were regression analysis, which included body mass index,
adherent, consisting of 10 respondents (17.9%) in the changes in antidiabetic in the last six months, number of
insulin group and 33 respondents (27.7%) in the non- comorbidities, diagnosis of peripheral nerve disorders,
insulin group. the total number of medicines consumed by
Medication Refill Adherence (MRA) respondents, and consumption of herbs. In addition, age
The percentage of MRA is 20.73-114.86%. and gender were also included in the multivariate
Respondents are declared adherent if the MRA is 80- analysis. The effect of insulin administration and the
120% (Kindmalm et al., 2007). Based on the MRA confounding variables were analysed by logistic
percentage, there were 87 respondents (49.7%) who regression analysis using the backward method. The
were adherent, namely 25 respondents (44.6%) in the logistic regression analysis showed that the variables
insulin group and 62 respondents (52.1%) in the non- that needed to be controlled in determining the effect of
insulin group. insulin administration on medication adherence were
Glycosylated hemoglobin (HbA1c) antidiabetic changes in the last six months and the total
Adherence based on HbA1c was determined based number of medications used by patients (Table 5).
on changes in HbA1c values in the last two
Table 3. Medication adherence

Characteristics Insulin n (%)a Non-Insulin n(%)a Total n (%)b p-value (inter-group)c
ARMS Adherent 10 (17.9) 33 (27.7) 43 (24.6) 0.109
Non-adherent 46 (82.1) 86 (72.3) 132 (75.4)
MRA Adherent 25 (44.6) 62 (52.1) 87 (49.7) 0.224
Non-adherent 31 (55.4) 57 (47.9) 88 (50.3)
HbA1c Adherent 26 (46.4) 37 (31.1) 63 (36.0) 0.036
Non-adherent 30 (53.6) 82 (68.9) 112 (64.0)
Overall Adherent 1 (1.8) 12 (10.1) 13 (7.4) 0.042
Non-adherent 55 (98.2) 107 (89.9) 162 (92.6)
Information: a Value is expressed in n(%), a percentage in one category; b Values are expressed in n(%), percentage of all
respondents; c Chi-square test; Overall, adherence is measured using the three measurement methods, the patient is
declared adherent if the results of the three methods show adherence.
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Table 4. Differences in respondent adherence based on characteristics, medical history, and medication history
Adherent Non-adherent p-value
Confounding Variables Total n (%)b
n (%)a n (%)a (inter-group)c
Age 35 - 44 years old 0 (0.0) 8 (100.0) 8 (4.6) 0.419
45 - 54 years old 2 (6.1) 31 (93.9) 33 (18.9)
55 - 64 years old 5 (6.9) 67 (93.1) 72 (41.1)
65 - 74 years old 6 (12.8) 41 (87.2) 47 (26.9)
75+ 0 (0.0) 15 (100.0) 15 (8.6)
Gender Man 8 (7.7) 96 (92.3) 104 (59.4) 0.559
Woman 5 (7.0) 66 (93.0) 71 (40.6)
Education Did not finish 1 (33.3) 2 (66.7) 3 (1.7) 0.406
elementary
school/did not go to
school
Elementary 1 (7.7) 12 (92.3) 13 (7.4)
Junior High School 0 (0.0) 12 (100.0) 12 (6.9)
Senior High School 5 (8.3) 55 (91.7) 60 (34.3)
College 6 (6.9) 81 (93.1) 87 (49.7)
Work Retired/ 6 (8.1) 68 (91.9) 74 (42.3) 0.817
Doesn't work
PNS/TNI/ 1 (6.7) 14 (93.3) 15 (8.6)
POLRI
Self-employed/ 1 (11.1) 8 (88.9) 9 (5.1)
trader
Private employees 0 (0.0) 14 (100.0) 14 (8.0)
Housewives 5 (9.1) 50 (90.9) 55 (31.4)
Other 0 (0.0) 8 (100.0) 8 (4.6)
Body mass index Normal 5 (14.7) 29 (85.3) 34 (19.4) 0.081 *
Abnormal 8 (5.7) 133 (94.3) 141 (80.6)
Duration of < 12 months 7 (9.9) 64 (90.1) 71 (40.6) 0.594
diagnosed 12-24 months 3 (5.5) 52 (94.5) 55 (31.4)
diabetes > 24 months 3 (6.1) 46 (93.9) 49 (28.0)
Antidiabetic Yes 8 (12.3) 57 (87.7) 65 (37.1) 0.058*
changes in the last
six months Not 5 (4.5) 105 (95.5) 110 (62.9)
Number of Single drug 4 (12.1) 29 (87.9) 33 (18.9) 0.415
antidiabetics Combination of 2
2 (3.4) 57 (96.6) 59 (33.7)
drugs
Combination of 3
6 (9.2) 59 (90.8) 65 (37.1)
drugs
Combination of 4
1 (5.6) 17 (94.4) 18 (10.3)
drugs
Number of ≤3 4 (5.2) 73 (94.8) 77 (44.0) 0.242 *
comorbidities >3 9 (9.2) 89 (90.8) 98 (56.0)
Central nervous stroke 11 (7.2) 141 (92.8) 152 (86.9) 0.532
system disorders Non-stroke 2 (8.7) 21 (91.3) 23 (13.1)
Peripheral nerve Yes 6 (10.7) 50 (89.3) 56 (32.0) 0.201 *
disorders Not 7 (5.9) 112 (94.1) 119 (68.0)
Total amount of ≤10 12 (8.6) 128 (91.4) 140 (80.0) 0.223*
medicine >10 1 (2.9) 34 (97.1) 35 (20.0)
Recipe iteration Yes 10 (7.8) 119 (92.2) 129 (73.7) 0.540
Not 3 (6.5) 43 (93.5) 46 (26.3)
Consumption of Yes 5 (10.9) 41 (89.1) 46 (26.3) 0.233 *
herbs Not 8 (6.2) 121 (93.8) 129 (73.7)
Family/ Yes 8 (7.3) 102 (92.7) 110 (62.9) 0.569
caregiver support Not 5 (7.7) 60 (92.3) 65 (37.1)
Information: a Value is expressed in n(%), percentage in one category; b Values are expressed in n(%), percentage of all
respondents. * p-value < 0.25, the variable was included in the multivariate logistic regression analysis.
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Table 5. Effect of insulin administration and confounding variables on medication adherence

95% Confidence Interval
Model Confounding Variables Category p-value OR
(Min-Max)
Crude Antidiabetic Insulin 0.084 0.162 0.021 - 1.279
Non-Insulin
Multivariate Antidiabetic Insulin 0.054 0.113 0.012 - 1.041
Non-Insulin
Age 35 - 44 years old 0.628
45 - 54 years old 0.999 0.000 0.000
55 - 64 years old 0.999 0.000 0.000
65 - 74 years old 0.999 0.000 0.000
75+ 1.000 1.573 0.000
Gender Man 0.783 0.821 0.202 - 3.335
Woman
Body mass index Normal 0.045 4.345 1.031 - 18.311
Abnormal
Antidiabetic changes Yes 0.020 5.431 1.305 - 22.595
Not
Number of comorbidities ≤ 3 0.201 0.374 0.083 - 1.690
>3
Peripheral nerve Yes 0.305 2.104 0.508 - 8.719
disorders Not
Total amount of ≤ 10 0.208 5.008 0.408 - 61.445
medicine > 10
Consumption of herbs Yes 0.075 3.739 0.877 - 15.942
Not
Adjusted Antidiabetic Insulin 0.053 0.123 0.015 - 1.024
Non-Insulin
Antidiabetic changes Yes 0.020 4.171 1.254 - 13.878
Not
Total amount of ≤ 10 0.487 2.131 0.253 - 17.960
medicine > 10
This study shows that respondents' characteristics neuropathy was the most patient-reported complication
(age, gender, education, occupation, and body mass affecting adherence (Zhang et al., 2021). Patients with
index) did not impact medication adherence statistically more than three comorbidities showed a higher
significantly. The results shown on the variables of age proportion of adherence. This result contradicts the
and sex are following a study conducted by Sham et al. research conducted by Saadat et al., which states that the
in Pakistan which showed that age and gender were not more comorbidities, the lower the patient's adherence
significantly associated with patient adherence (Shams (Saadat et al., 2015). This is due to increased patient
et al., 2016). Regarding education, medication comorbidities followed by increased visits to different
adherence appears to improve as the respondent's specialists, so patients' adherence is better (Capoccia et
education level increases. This is in line with the review al., 2016). However, the effect of these three variables
of articles and meta-analyses by Al Shaikh et al. (2016) on medication adherence was not statistically
that education improves patient adherence. significant. This is in line with research on the impact of
Bivariate analysis showed that respondents with a comorbidities on adherence to antihypertensive use
stroke history showed lower adherence than patients (Saadat et al., 2015).
without a stroke history. The proportion of stroke Respondents who experienced antidiabetic changes
respondents adherent was 7.2%, while the non-stroke showed higher adherence than those who did not
respondents were 8.7%. This is in accordance with a experience antidiabetic changes (12.3% vs 4.5%). The
study conducted by Bauler et al. (2014) which stated antidiabetic change in the adherent respondents was the
that adherence to medication after a stroke was addition of antidiabetics, which improved the patient's
influenced by various barriers and facilitators. On the HbA1c.
other hand, respondents with peripheral nerve disorders Based on the results of measuring adherence using
show a higher proportion of adherence. This contrasts MRA, ARMS, and HbA1c values changes, there were
with previous studies showing that peripheral 13 respondents (7.4%) who were adherent, namely one
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respondent (1.8%) in the insulin group and 12 Visualization, J., R. S., N. F. S., H. T.; Supervision, J.,
respondents (10.1%) in the non-insulin group. Based on R. S., N. F. S., H. T.; Project Administration, J., R. S.,
the results of multivariate analysis, insulin N. F. S., H. T.
administration affected patient adherence in using the
antidiabetics by 0.123 times (95% CI: 0.015 - 1.024) or CONFLICT OF INTEREST
patients who received insulin had 87.7% lower The authors declared no conflict of interest.
adherence than patients who did not receive insulin after
being controlled by antidiabetic changes and amount of REFERENCES
the total drug used by the patient at the time of data Adikusuma, W. & Qiyaam, N. (2017). Hubungan
collection from the medical records. Tingkat Kepatuhan Minum Obat Antidiabetik
Based on the three measuring instruments, only 13 Oral Terhadap Kadar Hemoglobin Terglikasi
out of 175 respondents were adherent. Therefore, further (HbA1c) pada Pasien Diabetes Melitus Tipe 2.
research is needed to analyses variables other than Jurnal Ilmiah Ibnu Sina; 2; 279–286.
insulin administration that cause low medication Al AlShaikh, S., Quinn, T., Dunn, W., Walters, M. &
adherence in diabetes mellitus patients with Dawson, J. (2016). Predictive Factors of Non-
neurological complications. Adherence to Secondary Preventative Medication
The limitations of this study include the sample of After Stroke or Transient Ischaemic Attack: a
only 175 respondents with patients using insulin only 56 Systematic Review and Meta-Analyses. European
respondents, the sampling location was only one Stroke Journal; 1; 65–75. doi:
hospital, and using the indirect adherence measurement 10.1177/2396987316647187.
method. In the MRA method, the patient is assumed to Alfian, R. (2015). Korelasi Antara Kepatuhan Minum
use the drug every day since the antidiabetic prescription Obat dengan Kadar Gula Darah pada Pasien
was received until the next visit. Another limitation is Diabetes Melitus Rawat Jalan dI RSUD DR. H.
that this study has not analysed other variables that may Moch. Ansari Saleh Banjarmasin. Jurnal
affect the HbA1c value. Pharmascience; 2; 15–23.
Barati, M., Taheri-Kharameh, Z., Bandehelahi, K., Yeh,
CONCLUSION V. M. & Kripalani, S. (2018). Validation of the
The results showed that the adherence of diabetes Short form of the Adherence to Refills and
mellitus patients was low, especially in patients who Medications Scale in Iranian Elders with Chronic
used insulin. The administration of insulin significantly Disease. Journal of Clinical and Diagnostic
affects patient adherence in diabetes mellitus patients Research; 12; 10–13. doi:
with neurological complications, which is influenced by 10.7860/JCDR/2018/37584.12305.
the confounding variable of antidiabetic changes and the Bauler, S., Jacquin-Courtois, S., Haesebaert, J., Luaute,
total number of medicines used by the patients. The J., Coudeyre, E., Feutrier, C., Allenet, B.,
results of this study are expected to help hospital Decullier, E., Rode, G. & Janoly-Dumenil, A.
decision-makers and health care providers when (2014). Barriers and Facilitators for Medication
initiating insulin administration and improve medication Adherence in Stroke Patients: A Qualitative Study
adherence in patients using insulin. Conducted in french Neurological Rehabilitation
Units. European Neurology; 72; 262–270. doi:
ACKNOWLEDGMENT 10.1159/000362718.
The authors would like to thank the nurses and Bloomgarden, Z. & Chilton, R. (2021). Diabetes and
pharmacists at the national brain center hospital who Stroke: an Important Complication. Journal of
helped carry out this research. Diabetes; 13; 184–190. doi: 10.1111/1753-
0407.13142.
AUTHOR CONTRIBUTIONS Bulu, A., Wahyuni, T. D. & Sutriningsih, A. (2019).
Conceptualization, J., R. S., N. F. S.; Methodology, Hubungan antara Tingkat Kepatuhan Minum Obat
J., R. S., N. F. S.; Software, J.; Validation, J., R. S., N. dengan Kadar Gula Darah pada Pasien Diabetes
F. S., H. T.; Formal Analysis, J., R. S., N. F. S.; Melitus Tipe II. Nursing News; 4; 181–189.
Investigation, J.; Resources, J., H. T.; Data Curation, J., Capoccia, K., Odegard, P. S. & Letassy, N. (2016).
R. S., N. F. S., H. T.; Writing - Original Draft, J.; Medication Adherence with Diabetes Medication:
Writing - Review & Editing, J., R. S., N. F. S., H. T.; a Systematic Review of the Literature. The
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Diabetes Educator; 42; 34–71. doi: Kindmalm, L., Melander, A. & Nilsson, L. (2007).
10.1177/0145721715619038. Refill Adherence of Antihyperglycaemic Drugs
Chen, Y. J., Chang, J. & Yang, S. Y. (2020). Related to Glucose Control (HbA 1c) in Patients
Psychometric Evaluation of Chinese Version of with Type 2 Diabetes. Acta Diabetologica; 44;
Adherence to Refills and Medications Scale 209–213. doi: 10.1007/s00592-007-0006-7.
(ARMS) and Blood-Pressure Control Among Kripalani, S., Risser, J., Gatti, M. E. & Jacobson, T. A.
Elderly with Hypertension. Patient Preference (2009). Development and Evaluation of the
and Adherence; 14; 213–220. doi: Adherence to Refills and Medications Scale
10.2147/PPA.S236268. (ARMS) Among Low-Literacy Patients with
Complication Definition & Meaning. (2022). Merriam- Chronic Disease. Value in Health; 12; 118–123.
Webster. (n.d.). https://www.merriam- doi: 10.1111/j.1524-4733.2008.00400.x.
webster.com/dictionary/complication#learn- Lachenbruch, P. A., Lwanga, S. K. & Lemeshow, S.
more. Accessed: 15 July 2022. (1991). Sample Size Determination in Health
Doggrell, S. A. & Warot, S. (2014). The Association Studies: A Practical Manual. Journal of the
Between the Measurement of Adherence to American Statistical Association; 86; 1149. doi:
Antidiabetes Medicine and the HbA1c. 10.2307/2290547.
International Journal of Clinical Pharmacy; 36; Lomper, K., Chabowski, M., Chudiak, A.,
488–497. doi: 10.1007/s11096-014-9929-6. Białoszewski, A., Dudek, K. & Jankowska-
González-Bueno, J., Calvo-Cidoncha, E., Sevilla- Polańska, B. (2018). Psychometric Evaluation of
Sánchez, D., Espaulella-Panicot, J., Codina-Jané, the Polish Version of the Adherence to Refills and
C. & Santos-Ramos, B. (2017). Spanish Medications Scale (ARMS) in Adults with
Translation and Cross-Cultural Adaptation of the Hypertension. Patient Preference and Adherence;
ARMS-Scale for Measuring Medication 12; 2661–2670. doi: 10.2147/PPA.S185305.
Adherence in Polypathological Patients. Atencion Nanda, O. D., Wiryanto, B. & Triyono, E. A. (2018).
Primaria; 49; 459–464. doi: Hubungan Kepatuhan Minum Obat Antidiabetik
10.1016/j.aprim.2016.11.008. dengan Regulasi Kadar Gula Darah pada Pasien
International Diabetes Federation. (2019). IDF Diabetes Perempuan Diabetes Mellitus. Amerta Nutrition;
Atlas (S. Karuranga, B. Malanda, P. Saeedi, & P. 2; 340-348. doi: 10.20473/amnt.v2i4.2018.340-
Salpea (Eds.); 9th ed.). Brussels: International 348.
Diabetes Federation. Osborn, C. Y. & Gonzalez, J. S. (2016). Measuring
Ireland, S., Endacott, R., Cameron, P., Fitzgerald, M. & Insulin Adherence Among Adults with Type 2
Paul, E. (2010). Association of Glycaemia with Diabetes. Journal of Behavioral Medicine; 39;
Macrovascular and Microvascular Complications 633–641. doi: 10.1007/s10865-016-9741-y.
of Type 2 Diabetes: Prospective Observational Park, H. Y., Seo, S. A., Yoo, H. & Lee, K. (2018).
Study. Resuscitation; 35; 18–23. doi: Medication Adherence and Beliefs About
10.1016/j.resuscitation.2010.10.016. Medication in Elderly Patients Living Alone with
Jin, H. K., Kim, Y. H. & Rhie, S. J. (2016). Factors Chronic Diseases. Patient Preference and
Affecting Medication Adherence in Elderly Adherence; 12; 175–181. doi:
People. Patient Preference and Adherence; 10; 10.2147/PPA.S151263.
2117–2125. Perkeni. (2021). Pedoman Pengelolaan dan Pencegahan
https://doi.org/10.2147/PPA.S118121 Diabetes Melitus Tipe 2 di Indonesia. Jakarta:
Kim, C.-J., Park, E., Schlenk, E. A., Kim, M. & Kim, D. Perkeni.
J. (2016). Psychometric Evaluation of a Korean Saadat, Z., Nikdoust, F., Aerab-Sheibani, H.,
Version of the Adherence to Refills and Bahremand, M., Shobeiri, E., Saadat, H.,
Medications Scale (ARMS) in Adults With Type Moharramzad, Y. & Morisky, D. E. (2015).
2 Diabetes. Diabetes Educator; 42; 188–198. doi: Adherence to Antihypertensives in Patients with
10.1177/0145721716632062. Comorbid Condition. Nephro-Urology Monthly;
Kim, H. -A., Shin, J. -Y., Kim, M. -H. & Park, B. -J. 7; 1-6. doi: 10.5812/numonthly.29863.
(2014). Prevalence and Predictors of Salistyaningsih, W., Puspitawati, T. & Nugroho, D.
Polypharmacy among Korean elderly. PLoS ONE; (2011). Hubungan Tingkat Kepatuhan Minum
9; 1-7. doi: 10.1371/journal.pone.0098043. Obat Hipoglikemik Oral dengan Kadar Glukosa
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Darah Pada Pasien DM Tipe 2. Universitas Recurrence in Stroke Patients: A Meta-analysis.

Respati Yogyakarta; 27; 215–221. Journal of Stroke and Cerebrovascular Diseases;
Sarwar, N., Gao, P., Kondapally Seshasai, S. R., Gobin, 24; 1961–1968. doi:
R., Kaptoge, S., Di Angelantonio, E., Ingelsson, 10.1016/j.jstrokecerebrovasdis.2015.04.004.
E., Lawlor, D. A., Selvin, E., Stampfer, M., WHO. (2020). The Top 10 Causes of Death. Geneva:
Stehouwer, C. D. A., Lewington, S., Pennells, L., WHO.
Thompson, A., Sattar, N., White, I. R., Ray, K. K., Zairina, E., Khotib, J., Ardianto, C. & Sulaiman, S. A.
Danesh, J., Tipping, R. W. & Wormser, D. (2010). (2018). Unity in Diversity and the Standardisation
Diabetes Mellitus, Fasting Blood Glucose of Clinical Pharmacy Services. Florida: CRC
Concentration, and Risk of Vascular Disease: A Press.
Collaborative Meta-Analysis of 102 Prospective Zairina, E., Nugraheni, G., Sulistyarini, A., Mufarrihah,
Studies. The Lancet; 375; 2215–2222. doi: Setiawan, C. D., Kripalani, S. & Lestari, S. I.
10.1016/S0140-6736(10)60484-9. (2022). Factors Related to Barriers and
Shams, N., Amjad, S., Kumar, N., Ahmed, W. & Medication Adherence in Patients with Type 2
Saleem, F. (2016). Drug Non-Adherence In Type Diabetes Mellitus: A Cross-Sectional Study.
2 Diabetes Mellitus; Predictors and Associations. Journal of Diabetes and Metabolic Disorders; 21;
Journal of Ayub Medical College, Abbottabad; 28; 219–228. doi: 10.1007/s40200-021-00961-6.
302–307. Zhang, Z. P., Premikha, M., Luo, M. & Venkataraman,
Sherwani, S. I., Khan, H. A., Ekhzaimy, A., Masood, A. K. (2021). Diabetes Distress and Peripheral
& Sakharkar, M. K. (2016). Significance of Neuropathy are Associated with Medication Non-
HbA1c Test in Diagnosis and Prognosis of Adherence in Individuals with Type 2 Diabetes in
Diabetic Patients. Biomarker Insights; 11; 95– Primary Care. Acta Diabetologica; 58; 309–317.
104. doi: 10.4137/Bmi.s38440. doi: 10.1007/s00592-020-01609-2.
Shou, J., Zhou, L., Zhu, S. & Zhang, X. (2015). Diabetes
is an Independent Risk Factor for Stroke
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DOI: 10.20473/jfiki.v9i32022.252-261
The Influence of Feeling Lonely and Received Social Support on Medication

Adherence in Elderly with Hypertension
Dwi Novita Sari1, Wahyu Utami2, Elida Zairina2*
1
2
Department of Pharmacy Practice, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
*Corresponding author: elida-z@ff.unair.ac.id
Submitted: 11 July 2022

Accepted: 28 October 2022
Abstract
Background: Hypertension is currently a non-contagious disease that primarily affects the elderly population in
Indonesia. Medication adherence is critical in managing hypertension and reducing the risk of morbidity and
mortality. Previous research has found that loneliness and support received by older adults with hypertension
influence medication adherence. Objective: This study aimed to examine the impact of feeling lonely and receiving
social support on medication adherence in the elderly with hypertension at the Community Health Center in
Surabaya. Methods: The study design of this research was a descriptive cross-sectional study from December
2021 to March 2022. A total of 235 eligible subjects fulfilled the inclusion criteria. The instruments used in the
data collection were the patient's information form, UCLA-Loneliness Scale, MOS-Social Support Survey, and the
ARMS (Adherence to Refill and Medication Scale). Results: The results revealed that the correlation between
loneliness and social support was significantly associated with medication adherence in the elderly with
hypertension (p < 0.05). In addition, other factors, such as occupation status, living status, comorbidity, the
number of drugs taken, and antihypertensive drug therapy, showed a significant correlation with medication
adherence (p < 0,05). The most influential factor on medication adherence was loneliness (35.5%), followed by
social support (24.4%), the number of drugs taken (7.1%), antihypertensive drug therapy (monotherapy or
combination therapy (2.5%), occupation status (2.4%), comorbidity (1.6%), and living status (0.2%). Conclusion:
This study confirms that feeling lonely and receiving social support affect medication adherence in the elderly with
hypertension at the Community Health Center in Surabaya.
Keywords: adherence, elderly, hypertension, loneliness, social support

Sari, D. N., Utami, W. & Zairina, E. (2022). The Influence of Feeling Lonely and Received Social Support on
Medication Adherence in Elderly with Hypertension. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 252-
261. http://doi.org/10.20473/jfiki.v9i32022.252-261
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INTRODUCTION loneliness and social support on medication adherence

The ageing process induces various kinds of decline in the elderly with hypertension is still limited. This
in body functions, such as physical, psychological, and study aims to see if feeling lonely and receiving social
social conditions (Suadirman, 2011). The decreased support affects medication adherence.
physical condition due to the degenerative process of
ageing causes non-contagious diseases like MATERIALS AND METHODS
hypertension, heart disease, Diabetes Mellitus, dental Materials
problem, etc. (Kementerian Kesehatan RI, 2019). In This study was acquitted on the elderly with
Indonesia, the most common degenerative disease hypertension at the Community Health Centers in
suffered by the elderly population is hypertension Surabaya. The Community Health Centers are
(Kementerian Kesehatan RI, 2019). Puskesmas Gading, Puskesmas Rangkah, and
Long-term drug therapy is needed to treat chronic Puskesmas Mojo. The criteria for inclusion must be met:
diseases such as hypertension, even for the rest of life elderly aged 60 – 79 years diagnosed with hypertension
(Dzau & Balatbat, 2019). Hypertension can be and have started antihypertensive medication therapy
prevented and treated with treatment adherence, whether for at least three months or more, can communicate well,
by taking medication therapy, following a diet, or can read and complete self-administered questionnaires.
having a good lifestyle (World Health Organization, The exclusion criteria were: elderly hypertensive
2003). Adherence to antihypertensive medication patients who also had dementia, schizophrenia,
therapy should be done regularly and continuously with tuberculosis, tested positive for covid-19, or had
the result that the 'patient's blood pressure can be well physical disorders such as eye, hand movement, and
controlled (World Health Organization, 2019). The hearing loss.
therapeutic goal of medication adherence is to prevent Tools
and reduce the likelihood of morbidity and mortality in Patient information form, University of California
elderly patients (Corrao et al., 2017). Los Angeles (UCLA)-Loneliness Scale, and Medical
Several factors influence adherence to taking Outcome Study-Social Support Survey (MOS-SSS)
medication in the elderly with hypertension, one of were used to collect data. Patient information forms
which is demographic factors such as age, gender, level include demographic and clinical data. The UCLA-
of education, economic status, marital status, living Loneliness Scale (Version 3) was used to measure 'one's
status, and occupation status (Gast & Mathes, 2019; subjective feelings of loneliness (Russell, 1996). The
Hazwan & Pinatih, 2017; Liberty et al., 2018; Sinuraya Medical Outcome Study-Social Support Survey (MOS-
et al., 2018; Uchmanowicz et al., 2018). Psychosocial SSS) determined the availability of support which was
factors influencing medication adherence in the elderly created to assess perceived social support for patients
include loneliness and social support (Hacihasanoglu et with chronic conditions (Sherbourne & Stewart, 1991).
al., 2020; Lu et al., 2020). A study conducted on 1,233 The ARMS (Adherence to Refill and Medication Scale)
elderly in Indonesia aged ≥ 60 years showed that one in measured a patient's adherence to antihypertensive
five elderly lives alone, and half experience loneliness medication therapy (Kripalani et al., 2009). The ARMS
(Widhowati et al., 2020). was translated and validated into Indonesian by a
Loneliness can be caused by the lack of previous study (Mubasir et al., 2017). The authors have
interpersonal relationships due to disconnection or obtained permission from the creators of the instruments
social contact with family, friends, or partners. Those in this study. In this study, the original English version
can lead a person to be unable to feel social support of the UCLA-Loneliness Scale and MOS-Social
(Donovan & Blazer, 2020). Sufficient support has a Support Survey was translated and adapted into
crucial role in the treatment of hypertension, especially Indonesian by native Indonesian speakers who were
support from family, healthcare providers, friends, and fluent in English, using forward and backward
others such as peer groups (Shahin et al., 2021). Elderly translations according to Basic Guidelines for
hypertensive patients who live with their families have Translating Survey Research and Development
better adherence than those who e live alone or in a (RAND) (RAND, 2021). The procedure is as follows:
nursing home (Uchmanowicz et al., 2018). (1) forward translation, the English version of the
Previous research has helped us understand how UCLA-Loneliness Scale and MOS-Social Support
loneliness and social support affect medication Survey were translated into Indonesian by a professional
adherence. However, research on the impact of translator from the Pusat Bahasa (Language Center) of
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Universitas Airlangga, (2) review, identifying the Sample size

differences in items, terms, and concepts and the The Lemeshow formula was used to calculate
completion by the expert panel, (3) back-translation, the sample size at a confidence level of 95%, an error
reviewed Indonesian version of the UCLA-Loneliness tolerance of 5%, and the estimated population
Scale and MOS-Social Support Survey were translated proportion as the most significant proportion of data
back into original English by another professional (50%). Based on the formula, it was obtained that the
translator from the Pusat Bahasa (Language Center) of sample size was 235.
Universitas Airlangga, (4) accomplish discrepancies or The ARMS (Adherence to Refills and Medication
problems in the translation by a committee that includes Scale)
the forward translator, backward translator, and The ARMS is a valid and reliable medication
reviewer. The Indonesian version of the UCLA- adherence scale when used in 'patients with chronic
Loneliness Scale and MOS-Social Support Survey were disease, with good performance characteristics, and
pre-tested on 70 elderly with hypertension with the same even in patients with low literacy skills (Kripalani et al.,
inclusion criteria in this study. Data were analyzed, and 2009). This questionnaire consists of 12 question items,
the results showed that the Corrected Item-Total 8 are sub-scales for medication adherence and 4 are sub-
Correlation for all items were greater than the r value (r scales for drug refill adherence. Each item was rated
UCLA-Loneliness Scale = 0.2992; r MOS-Social using a Likert scale, ranging from 1 (none of the time)
Support Survey = 0.3007) and the ' 'Cronbach's alpha to 4 (all of the time). Especially for the last question, the
was above 0.7 (UCLA-Loneliness Scale = 0.882; MOS- scale is reversed from 1 (for all of the time) to 4 (none
Social Support Survey = 0.937), which indicates high of the time) (Kripalani et al., 2009). The overall
internal consistency. The Indonesian version of the adherence score may range from 12 to 48, with lower
UCLA-Loneliness Scale and MOS-Social Support scores indicating better adherence (Kripalani et al.,
Survey is valid and reliable for measuring loneliness and 2009).
support in elderly patients with hypertension. The UCLA-loneliness scale (version 3)
Method The UCLA-Loneliness Scale (Version 3) is the
Study design latest version of the UCLA-Loneliness Scale developed
This study used a descriptive cross-sectional study by Russell (1996). This is a 20-item scale that has
from December 2021 and March 2022. The research attempted to simplify the response format and the
began with the recruitment of respondents. First, the wording of the item. The scale has been used in studies
researcher explained the objectives and benefits of the of various populations, including the elderly (Russell,
study to each respondent; then, the respondent was 1996). Each item has been rated using the Likert scale
asked to sign an informed consent if they were willing from 1 (never) to 4 (always), 11 negative words (lonely)
to participate in the study. Second, the respondent filled encoded directly and nine positively (non-
out the self-administered questionnaire. lonely)inverted. Higher scores indicate a higher degree
Researchers met 368 elderly patients with of loneliness (Russell, 1996).
hypertension during the research. A total of 133 patients The MOS-social support survey
were excluded because of not willing to participate (n = The MOS-Social Support Survey is a 19-item
21), being illiterate (n = 21), did not bring glasses, so multidimensional instrument for patients with chronic
they did not read (n = 19), having physical impairment conditions in the Medical Outcomes Study developed by
(n = 44), resigned at the time of filling out the self- Sherbourne & Stewart (1991). This questionnaire
administered questionnaire (n = 3), and tested for measures the availability of support in various
positive Covid-19 (n = 9). About 235 elderly with dimensions of social support, not only the sources of
hypertension fulfilled the inclusion criteria and social support but also overall perceived support,
completed the self-administered questionnaire. The including emotional/informational support, tangible
respondents were recruited from 3 Community Health support, affectionate support, and positive social
Centers in Surabaya (Puskesmas Gading, Puskesmas interaction (Sherbourne & Stewart, 1991). Each item
Rangkah, and Puskesmas Mojo). Community health was rated using a Likert scale from 1 (none of the time)
centers were chosen because they are included in the to 5 (all of the time). A higher score indicates more
five largest health centres with the highest estimated support than perceived by ' 'participants (Sherbourne &
number of hypertension patients in Surabaya (Dinas Stewart, 1991).
Kesehatan Kota Surabaya, 2019).
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Statistical analysis with medication adherence (Agung et al., 2021; Gast &
The data were presented in the Statistical Package Mathes, 2019; Liberty et al., 2018; Sinuraya et al., 2018;
for the Social Sciences (SPSS) version 24.0. Statistical Wan et al., 2022), there are other having similar findings
analyses used descriptive analysis to have the to our study. The studies in Thailand, China and Korea
distribution of frequency of demographic and clinical exhibit that age, gender, marital status, education level,
characteristics. The normality of the distribution of occupation status, the number of drugs taken, and the
loneliness, social support, and adherence variables was duration of hypertension were not statistically
examined using the Kolmogorov-Smirnov test. The test significant to medication adherence in the elderly with
results showed that the data were not normally hypertension (Cho et al., 2018; Wan et al., 2022;
distributed, so the differences in the proportion of Woodham et al., 2018). A meta-analysis study in the
adherence between two groups based on demographic Asian region revealed that gender and education level
and clinical factors examine by using the Mann-Whitney were not associated with medication adherence in
test and between three groups or more using Kruskal- patients with hypertension (Akbar et al., 2021).
Wallis. The 'Spearman's rank correlation coefficient was In this study, the first factor related to medication
used to acquire the correlation between loneliness, adherence in elderly hypertensive patients was
social support, and adherence. Linear regression was occupation and living status. Previous research
used as a multivariate analysis to determine how much conducted in China and Indonesia (Bandung, Magelang,
influence each variable has on adherence. The results are and Semarang) showed that occupation status had a
statistically significant with P-value (< 0,05). statistically significant effect on medication adherence
Ethics consideration in adult to elderly hypertensive patients (Agung et al.,
The Human Research Ethics Committee Faculty of 2021; Nurhanani et al., 2020; Pan et al., 2019; Sinuraya
Nursing Universitas Airlangga has approved this study et al., 2018). Patients who are not working or retired
(No.: 2392-KEPK). tend to adhere less to the treatment of hypertension (Pan
et al., 2021; Woodham et al., 2018). Living status in this
RESULTS AND DISCUSSION study included living alone, with a spouse, or with
The comparison between demographic and clinical family. Another study in China showed that elderly
characteristics to adherence scores hypertensive patients with hypertension who lived with
The detailed demographic and clinical spouses and offspring had a much higher level of
characteristics of the patients are shown in Table 1. In medication adherence than those who lived alone (Wan
this study, most participants were aged 60 - 79 years et al., 2022). Elderly hypertensive patients who live with
(74%) with the mean (SD) was 65.8 ± 8.3, female (61%), others and have social interactions are more motivated
married (64%), living with family (81%), primary to adhere to antihypertensive medication therapy (Lu et
school graduate (36%), not working (77%), the income al., 2020).
per month less than Rp 1,500,000.00 (88%), the duration Several studies revealed a significant association
of hypertension 1 to 5 years (51%) with a mean (SD) between the number of drugs taken and medication
was (59.3 ± 67.3) (months), the number of drugs taken adherence in elderly hypertensive patients (Shareinia et
is one pill a day (38%), not have comorbidity (39%), and al., 2020), and patients with more complicated
having monotherapy antihypertensive drug (84%). The prescriptions had better medication adherence (Thuy et
results in Table 2 showed that age, gender, marital al., 2020). In this study, clinical factors related to
status, level of education, and the duration of medication adherence were comorbidities,
hypertension showed no significant difference in antihypertensive medication therapy, and the total
adherence to antihypertensive (p > 0.05). Table 2 number of daily drugs taken. Similar results were
presents the mean rank of adherence level for each obtained from the studies in Romania and Korea on
group. The group with a lower mean rank score indicates elderly hypertensive patients. Comorbidities such as
better adherence. However, occupation status, living diabetes mellitus, heart disease, kidney disease,
status, comorbidity, antihypertensive medication, and dyslipidemia, cancer, and stroke had high medication
the number of drugs taken significantly differed with adherence (Cho et al., 2018; Tilea et al., 2018). A study
adherence to hypertension medication (p < 0.05). in Wuhu, China, conducted antihypertensive medication
Although there are still differences between therapy (single or combination), and the total number of
previous studies, which indicated that demographic and drugs taken did not affect antihypertensive medication
duration of hypertension are significantly associated adherence (Wan et al., 2022). In this study, however, the
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opposite results were obtained; elderly hypertensive antihypertensive drugs taken was related to medication
patients who received antihypertensive monotherapy adherence; when the number of medicines taken
demonstrated better adherence (Uchmanowicz et al., increased, a person had better medication adherence
2018). Another study found that the number of (Pan et al., 2021).
Table 1. Demographic and clinical characteristics (N=235)
Variable Category N (%)
Age (years), 60 - 69 173 (74%)
mean ± SD (65.8 ± 8.3) 70 - 79 62 (26%)
Gender Female 144 (61%)
Male 91 (39%)
Marital status Not married 2 (1%)
Married 150 (64%)
Divorced and not remarried 8 (3%)
Widowed and not remarried 75 (32%)
Living status Living alone 13 (5%)
With spouse 32 (14%)
With family 190 (81%)
Education None 54 (23%)
Primary school 84 (36%)
Secondary school 40 (17%)
High school 47 (20%)
Vocational 2 (1%)
Bachelor 8 (3%)
Occupation status Working 54 (23%)
Not working 181 (77%)
Income per month < Rp 1,500,000.00 207 (88%)
Rp 1,500,000,00 – Rp 2,500,000.00 16 (7%)
Rp 2,500,000,00 - Rp 5,000,000.00 11 (5%)
> Rp 5,000,000.00 1 (0%)
Duration of hypertension (in years), <1 61 (26%)
mean ± SD (59.3 ± 67.3) (in months) 1–5 120 (51%)
6 – 10 31 (13%)
11 – 15 6 (3%)
16 – 20 13 (6%)
> 20 4 (2%)
The number of drugs taken 1 89 (38%)
2 40 (17%)
3 68 (29%)
4 28 (12%)
5 8 (3%)
>5 2 (1%)
Comorbidities None 92 (39%)
Diabetes Mellitus 84 (36%)
Heart disease 10 (4%)
Hypercholesterolemia 6 (3%)
Gastritis 4 (2%)
Vertigo 4 (2%)
Others 35 (15%)
Antihypertensive drug therapy Monotherapy 197 (84%)
Combination of 2 drugs 37 (16%)
Combination of 3 drugs 1 (0%)
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Table 2. Medication adherence based on demographic and clinical characteristics

Medication Adherence
Variable Category
Mean rank P-value
Age (years)a 60 - 69 119.34 0.610
70 - 79 114.27
Female 116.39 0.643
Gendera
Male 120.55
Not married 127.00 0.523
Marital status Married 113.31
Divorced and not remarried 116.69
Widowed and not remarried 127.46
Living alone 164.19 0.007*
Living status With spouse 94.66
With family 118.77
None 121.07 0.461
Primary school 109.58
Secondary school 111.56
Level of Education
High school 132.97
Vocational 151.75
Bachelor 120.06
Working 137.67 0.014*
Occupation status
Not working 112.13
Income per month < Rp 1,500,000.00 114.20 0.087
Rp 1,500,000,00 – Rp 2,500,000.00 136.97
Rp 2,500,000,00 – Rp 5,000,000.00 161.55
> Rp5,000,000.00 122.50
<1 128.76 0.739
1–5 112.67
Duration of 6 – 10 121.58
hypertension (years)b 11 – 15 115.83
16 – 20 115.62
> 20 99.38
1 132.28 0.001*
2 136.38
The number of drugs 3 106.16
taken 4 96.32
5 65.81
>5 30.00
None 132.87 0.000*
Diabetes Mellitus 113.36
Heart disease 57.18
Comorbidities Hypercholesterolemia 128.20
Gastritis 186.25
Vertigo 148.30
Others 99.03
Monotherapy 123.44 0.015*
Antihypertensive drug
Combination of 2 drugs 93.04
therapy
Combination of 3 drugs 30.00
*statistically significant (P < 0.05)
a
using the Mann-Whitney test
b
using the Kruskal-Wallis test
c
the group with a lower mean rank score indicates better adherence
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The relationship between loneliness and social social support was significantly associated with
support with adherence medication adherence in hypertensive patients (Shahin
The total score of the UCLA-Loneliness Scale et al., 2021). The existence of adequate social support
questionnaire indicates that the lower the score obtained, from the social environment around the patient can
the lower the perceived loneliness, and the lower improve medication adherence (Shahin et al., 2021).
ARMS-adherence score indicates that the adherence is Low social support can lead to suboptimal medication
lower, so the two scores from the two questionnaires are adherence in elderly hypertensive patients with or
in line (positive correlation coefficient). There was a without comorbidities (Lu et al., 2020). Another study
positive correlation coefficient value (0.618) in Table 3, on adult and elderly hypertensive patients showed that
which means that the two variables have a positive the more significant support received, the better
direction. There was a significant association between adherence to medication (Gast & Mathes, 2019; Pan et
loneliness and medication adherence in this study. In our al., 2021; Turan et al., 2019). Practical support such as
study, medication adherence is improved when medical assistance (reminders to take medication, direct
loneliness is less of a concern. In addition, people who instructions for drug use, and prescription) and financial
experience loneliness, especially the elderly, tend to be support lead to better medication adherence (Adisa et
more at risk of hypertension, cardiovascular disease, and al., 2017; Ashoorkhani et al., 2018; Jung & Lee, 2017;
respiratory disease (Golaszewski et al., 2022). Yazdanpanah et al., 2019). A study that provides social
support through SMS intervention (medication
Table 3. The connection between loneliness and social
support with medication adherence reminders, diet, and control schedules) can improve
medication adherence and control blood pressure in
Medication Adherence
Variable patients with hypertension (Nursalam et al., 2020).
Correlation Coefficient Sig.
Loneliness 0.618** 0.000 The impact of demographic, clinical, loneliness, and
Social support -0.558** 0.000 social support on medication adherence
**statistically significant (< 0,01) This study evaluated the factors influencing
The higher score of the MOS-Social Support medication adherence in the elderly with hypertension.
Survey indicates the higher support received, while the The results of the linear regression test revealed the R
lower ARMS-adherence score indicates that the square value on medication adherence in Table 4. The
adherence is getting better, so the two scores are most influential factor on medication adherence based
opposite or inverted (negative correlation coefficient). on R square values is loneliness (35.5%), followed by
Social support has a negative correlation coefficient social support (24.4%), the number of drugs taken
value (-0.558) in Table 3., it means that the more (7.1%), antihypertensive drug therapy (2.5%),
support the elderly felt, the better medication adherence. occupation status (2.4%), comorbidity (1.6%), and
This study's findings are similar to the current study that living status (0.2%).
investigated the association between loneliness and This study is inseparable from limitations; there are
medication adherence; loneliness is statistically three limitations. First, this study only reached patients
significantly related to medication adherence; the lower who could communicate well and read, so it did not
perceived loneliness, the better medication adherence reach patients who could not speak well and were
(Hacihasanoglu et al., 2020; Jankowska-Polańska et al., illiterate. Second, this study excluded patients who
2020; Lu et al., 2020). could read but had limited physical impairment such as
Social support can be defined as support from hearing loss, body movement disorders (history of
anyone, including family, friends, neighbours, and stroke in hand; unable to hold a pen), and vision
health care providers, containing emotional or impairment (not carrying glasses, low vision, blurred
informational support, tangible support, affectionate eyes, cataract, glaucoma, etc.) so that patient cannot read
support, and positive social interaction (Sherbourne & the letters. Third, this research was conducted in the
Stewart, 1991). The results of this study revealed that urban area; it did not reach rural or remote areas. In the
social support is significantly related to medication future, further research is needed to find a design that
adherence. A systematic review study explained that can be applied to all conditions of elderly hypertensive
patients.
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Table 4. The connection between Loneliness and Social Support with medication adherence
Medication adherence
Variable
R R square P value
Loneliness 0.596 0355 0.000*
Social support 0.494 0.244 0.000*
Living status 0.040 0.002 0.545*
Occupation status 0.155 0.024 0.018*
Comorbidity 0.126 0.016 0.053*
Antihypertensive drug therapy 0.157 0.025 0.000*
The number of drugs taken 0.266 0.071 0.000*
*statistically significant (< 0.05)
CONCLUSION Agung, W. T., Nugraheni, A., Wahyudi, F. & Ardhianto,

Adherence to medication therapy may significantly P. (2021). Factors Associated with Medication
impact treatment goals in the elderly with hypertension. Adherence in Hypertension in The Ngluwar
Feelings of loneliness and lack of received social Public Health Center in Magelang Regency.
support may decrease medication adherence in the Diponegoro Medical Journal; 10; 426-432.
elderly with hypertension. In the future, strategies such Akbar, I., Isa, M., Arifin, S., Barlian & Husaini. (2021).
as appropriate treatment or intervention are needed to Meta-Analysis: Relationship of Age, Gender, and
overcome loneliness and increase social support to Education Level with Medication Adherence of
increase medication adherence among elderly patients. Hypertension Patients. The International Journal
of Travel Medicine and Global Health; 7; 20–24.
ACKNOWLEDGMENT doi: 10.20469/ijhms.7.3004.
The authors would like to thank Prof. Daniel W. Ashoorkhani, M., Majdzadeh, R., Gholami, J., Eftekhar,
Russel, Dr. Cathy D. Sherbourne, Dr. Anita L. Stewart, H. & Bozorgi, A. (2018). Understanding Non-
who have permitted them to use the UCLA-Loneliness Adherence to Treatment in Hypertension: A
Scale dan MOS-Social Support Survey. In addition, the Qualitative Study. International Journal of
authors would like to express gratitude to the Community Based Nursing and Midwifery; 6;
Community Health Centers staff who helped during the 314–323.
research process. Cho, M. H., Shin, D. W., Chang, S. -A., Lee, J. E.,
Jeong, S. -M., Kim, S. H., Yun, J. M. & Son, K.
AUTHOR CONTRIBUTIONS (2018). Association between Cognitive
Conceptualization, D. N. S., W. U., E. Z.; Impairment and Poor Antihypertensive
Methodology, D. N. S., W. U., E. Z.; Software, D. N. S.; Medication Adherence in Elderly Hypertensive
Validation, W. U., E. Z.; Formal Analysis, D. N. S., E. Patients Without Dementia. Scientific Reports; 8;
Z.; Investigation, D. N. S.; Resources, D. N. S., E. Z.; 1-7. doi: 10.1038/s41598-018-29974-7.
Data Curation, D. N. S., E. Z.; Writing - Original Draft, Corrao, G., Rea, F., Monzio Compagnoni, M., Merlino,
D. N. S., W. U., E. Z.; Writing - Review & Editing, D. L. & Mancia, G. (2017). Protective Effects of
N. S., W. U., E. Z.; Visualization, D. N. S., E. Z.; Antihypertensive Treatment in Patients Aged 85
Supervision, E. Z.; Project Administration, D. N. S., W. Years or Older. Journal of Hypertension; 35;
U., E. Z.; Funding acquisition, E. Z. 1432-1441. doi:
10.1097/HJH.0000000000001323.
CONFLICT OF INTEREST Dinas Kesehatan Kota Surabaya. (2019). Profil
The authors declared no conflict of interest. Kesehatan Kota Surabaya Tahun 2019. Surabaya:
Dinas Kesehatan Kota Surabaya.
REFERENCES Donovan, N. J. & Blazer, D. (2020). Social Isolation and
Adisa, R., Olamide, O. & Fakeye, T. (2017). Social Loneliness in Older Adults: Review and
Support, Treatment Adherence and Outcome Commentary of a National Academies Report.
among Hypertensive and Type 2 Diabetes Patients The American Journal of Geriatric Psychiatry;
in Ambulatory Care Settings in Southwestern 28; 1233–1244. doi: 10.1016/j.jagp.2020.08.005.
Nigeria. Ghana Medical Journal; 51; 64–77. Dzau, V. J. & Balatbat, C. A. (2019). Future of
Hypertension. Hypertension (Dallas, Tex. : 1979);
P-ISSN: 2406-9388
E-ISSN: 2580-8303
74; 450–457. doi: Liberty, I. A., Pariyana, P., Roflin, E., & Waris, L.
10.1161/HYPERTENSIONAHA.119.13437. (2018). Determinan Kepatuhan Berobat Pasien
Gast, A., & Mathes, T. (2019). Medication Adherence Hipertensi pada Fasilitas Kesehatan Tingkat I.
Influencing Factors—an (updated) Overview of Jurnal Penelitian Dan Pengembangan Pelayanan
Systematic Reviews. Systematic Reviews; 8; 1-17. Kesehatan; 1; 58–65. doi:
doi: 10.1186/s13643-019-1014-8. 10.22435/jpppk.v1i1.428.
Golaszewski, N. M., LaCroix, A. Z., Godino, J. G., Lu, J., Zhang, N., Mao, D., Wang, Y. & Wang, X.
Allison, M. A., Manson, J. E., King, J. J., (2020). How Social Isolation and Loneliness
Weitlauf, J. C., Bea, J. W., Garcia, L., Kroenke, C. Effect Medication Adherence among Elderly with
H., Saquib, N., Cannell, B., Nguyen, S. & Chronic Diseases: An Integrated Theory and
Bellettiere, J. (2022). Evaluation of Social Validated Cross-Sectional Study. Archives of
Isolation, Loneliness, and Cardiovascular Disease Gerontology and Geriatrics; 90; 1-7. doi:
Among Older Women in the US. JAMA Network 10.1016/j.archger.2020.104154.
Open; 5; e2146461–e2146461. doi: Mubasir, M., Setiawan, C. D., Wathaniah, N.,
10.1001/jamanetworkopen.2021.46461. Rahmaniah, A., Maharani, P., Mahmudah, A. &
Hacihasanoglu, A. R., Yildirim, A., Saglam, R., Achmad, G. N. V. (2017). The Influence of
Demirturk Selcuk, E., Erduran, Y. & Sarihan, O. Medication 'Days' Supply on Adherence and Cost
(2020). The Effect of Loneliness and Perceived of Hypertensive Patients at Primary Health Care
Social Support on Medication Adherence Self- Centre. Indonesian Journal of Pharmacy; 28; 213
Efficacy in Hypertensive Patients: An Example of – 220. doi:
Turkey. Journal of Vascular Nursing; 38; 183- 10.14499/indonesianjpharm28iss4pp213.
190. https://doi.org/10.1016/j.jvn.2020.07.003. Nurhanani, R., Susanto, H. S. & Udiyono, A. (2020).
Hazwan, A. & Pinatih, G. N. I. (2017). Gambaran Hubungan Faktor Pengetahuan dengan Tingkat
Karakteristik Penderita Hipertensi dan Tingkat Kepatuhan Minum Obat Antihipertensi (Studi
Kepatuhan Minum Obat di Wilayah Kerja Pada Pasien Hipertensi Essential di Wilayah Kerja
Puskesmas Kintamani I. Intisari Sains Medis; 8; Puskesmas Bandarharjo Kota Semarang). Jurnal
130–134. doi: 10.1556/ism.v8i2.127. Kesehatan Masyarakat; 8; 114–121.
Jankowska-Polańska, B., Świątoniowska-Lonc, N., Nursalam, N., Dewi, L. C., & Widhiastuti, E. (2020).
Sławuta, A., Krówczyńska, D., Dudek, K. & The Effect of Social Support Based on Short
Mazur, G. (2020). Patient-Reported Compliance Message Service (Sms) on Compliance in
in Older Age Patients with Chronic Heart Failure. Treatment and Reduction of Blood Pressure in
PLOS ONE; 15; 1-16. doi: Hypertension Patients in Betun Puskesmas and
10.1371/journal.pone.0231076. Besikama Puskesmas. Critical Medical and
Jung, H. & Lee, J. -E. (2017). The Impact of Surgical Nursing Journal (CMSNJ); 9; 10–16.
Community-Based eHealth Self-Management doi: 10.20473/cmsnj.v9i1.17802.
Intervention among Elderly Living Alone with Pan, J., Hu, B., Wu, L. & Li, Y. (2021). The Effect of
Hypertension. Journal of Telemedicine and Social Support on Treatment Adherence in
Telecare; 23; 167–173. doi: Hypertension in China. Patient Preference and
10.1177/1357633X15621467. Adherence; 15; 1953–1961. doi:
Kementerian Kesehatan RI. (2019). Laporan Nasional 10.2147/PPA.S325793.
RISKESDAS 2018. Jakarta: Kementerian Pan, J., Wu, L., Wang, H., Lei, T., Hu, B., Xue, X. & Li,
Kesehatan RI. Q. (2019). Determinants of Hypertension
Kripalani, S., Risser, J., Gatti, M. E. & Jacobson, T. A. Treatment Adherence Among a Chinese
(2009). Development and Evaluation of the Population using the Therapeutic Adherence
Adherence to Refills and Medications Scale Scale for Hypertensive Patients. Medicine; 98; 1-
(ARMS) Among Low-Literacy Patients with 7. doi: 10.1097/MD.0000000000016116.
Chronic Disease. Value in Health: The Journal of RAND. (2021). Basic Guidelines for Translating
the International Society for Pharmacoeconomics Surveys. California: RAND.
and Outcomes Research; 12; 118–123. doi: Russell, D. W. (1996). UCLA Loneliness Scale
10.1111/j.1524-4733.2008.00400.x. (Version 3): Reliability, Validity, and Factor
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Structure. Journal of Personality Assessment; 66; Wan, J., Wu, Y., Ma, Y., Tao, X. & Wang, A. (2022).
20–40. doi: 10.1207/s15327752jpa6601_2. Predictors of Poor Medication Adherence of Older
Shahin, W., Kennedy, G. A. & Stupans, I. (2021). The People with Hypertension. Nursing Open; 9;
Association Between Social Support and 1370-1378. doi: 10.1002/nop2.1183.
Medication Adherence in Patients Widhowati, S. S., Chen, C. -M., Chang, L. -H., Lee, C.
with Hypertension: A Systematic Review. -K. & Fetzer, S. (2020). Living Alone, Loneliness,
Pharmacy Practice; 19; 1-8. doi: and Depressive Symptoms among Indonesian
10.18549/PharmPract.2021.2.2300. Older Women. Health Care for Women
Sherbourne, C. D. & Stewart, A. L. (1991). The MOS International; 41; 984-996. doi:
Social Support Survey. Social Science & 10.1080/07399332.2020.1797039.
Medicine; 32; 705–714. doi: 10.1016/0277- Woodham, N., Taneepanichskul, S., Somrongthong, R.,
9536(91)90150-b. & Auamkul, N. (2018). Medication Adherence
Sinuraya, R., Destiani, D., Puspitasari, I. M. & Diantini, and Associated Factors among Elderly
A. (2018). Medication Adherence among Hypertension Patients with Uncontrolled Blood
Hypertensive Patients in Primary Healthcare in Pressure in Rural Area, Northeast Thailand.
Bandung City. Indonesian Journal of Clinical Journal of Health Research; 32; 449–458. doi:
Pharmacy; 7; 124–133. doi: 10.1108/JHR-11-2018-085.
10.15416/ijcp.2018.7.2.124. World Health Organization. (2003). Adherence to Long-
Suadirman, S. P. (2011). Psikologi Usia Lanjut. Term Therapies: Evidence for Action. Geneva:
Yogyakarta: Gadjah Mada University Press. World Health Organization.
Turan, G. B., Aksoy, M. & Çiftçi, B. (2019). Effect of World Health Organization. (2019). Hypertension by
Social Support on the Treatment Adherence of WHO. https://www.who.int/news-room/fact-
Hypertension Patients. Journal of Vascular sheets/detail/hypertension. Accessed: 13
Nursing; 37; 46–51. doi: September 201.
10.1016/j.jvn.2018.10.005 Yazdanpanah, Y., SalehMoghaddam, A., Mazloum, S.,
Uchmanowicz, B., Chudiak, A., Uchmanowicz, I., Hajialibeigloo, R. & Mohajer, S. (2019). Effect of
Rosińczuk, J. & Froelicher, E. S. (2018). Factors an Educational Program based on Health Belief
Influencing Adherence to Treatment in Older Model on Medication Adherence in Elderly
Adults with Hypertension. Clinical Interventions Patients with Hypertension. Evidence Based Care
in Aging; 13; 2425–2441. doi: Journal; 9; 52-62. doi:
10.2147/CIA.S182881. 10.22038/EBCJ.2019.35215.1895.
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DOI: 10.20473/jfiki.v9i32022.262-271
Sunscreen Cream Formulation of Noni Leaf Extract (Morinda citrifolia L.)

with Emulsifier Combination of Tween 80 and Lecithin
Bianca Levie Tania, Rini Dwiastuti, Agatha Budi Susiana Lestari, Dewi Setyaningsih*
Departement of Pharmaceutical Sciences and Pharmaceutical Technology, Faculty of Pharmacy, Sanata Dharma
University, Yogyakarta, Indonesia
*Corresponding author: dewi@usd.ac.id

Accepted: 22 November 2022
Abstract
Background: Noni leaf (Morinda citrifolia L.) extract is a natural product that can be used as a sunscreen. The
extract contains flavonoids which function act as an antioxidant. In this work, sunscreen cream formulated with
noni leaf extract was prepared using a combination of tween 80 and lecithin. Objective: The purpose of this study
is to evaluate how the combination of tween 80 and lecithin affects the physical qualities of the cream, such as
organoleptic, homogeneity, emulsion type, spreadability, adhesion, pH, and stability over 28 days of storage at
room temperature, and to find the best formula. Methods: This study used 10% of noni leaf extract in the cream
formulation. The Simplex Lattice Design (SLD) method was used to determine the effect of different concentrations
of the two emulsifiers on the cream's spreadability, adhesion, and pH. Furthermore, the SLD was used to find the
best formula. Results: The results showed that different concentrations of the emulsifier, which are the tween 80
and lecithin combination, affected the physical properties and storage stability of cream preparations. The
interaction of tween 80 and lecithin is having a significant impact on the cream's adhesion and spreadability;
however, the effect of the interaction on the pH value was not significant. Conclusion: The formula containing 2.5
% tween 80 and 2.5 percent lecithin was found to be the most effective in fulfilling the cream physical properties
while remaining stable during storage.
Keywords: noni leaf extract (Morinda citrifolia L.), sunscreen, cream, tween 80, lecithin

Tania, B. L., Dwiastuti, R., Lestari, A. B. S. & Setyaningsih, D. (2022). Sunscreen Cream Formulation of Noni Leaf
Extract (Morinda citrifolia L.) with Emulsifier Combination of Tween 80 and Lecithin. Jurnal Farmasi dan Ilmu
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INTRODUCTION 80 molecules can be desorbed from the oil droplet to the

Ultraviolet (UV) rays are one type of radiation aqueous phase, leaving an uncovered region of the oil
emitted by sunlight. UV rays are classified as UV-A droplet at the interface and can cause flocculation or
(320 - 400 nm), UV-B (290-320 nm), and UV-C (200- coalescence (Athas et al., 2014).
290 nm) (Morita, 2018). UV-A and UV-B radiation are The use of a combination of emulsifiers, one more
important factors in skin ageing (Park et al., 2017). hydrophobic and one more hydrophilic, can improve
Long-term sun exposure can have negative effects on emulsion stability (Yamashita et al., 2017). Tween 80, a
the skin, such as causing degenerative changes in skin hydrophilic emulsifier, can be combined with a more
cells that lead to premature ageing, sunburn, and skin hydrophobic emulsifier, such as lecithin, to improve
cancer (Rohmah et al., 2020). Sweating, melanin emulsion stability. With an HLB value of 4, lecithin is
formation, and other natural mechanisms protect the less toxic and more biocompatible than polymer
skin from the harmful effects of sunlight (Putri et al., surfactants (Chuacharoen et al., 2019; Estiasih et al.,
2019). However, skin tissue can be damaged if the 2015). Lecithin is an emulsifier with two hydrophobic
body's defense system is insufficient to protect it from tails, one of which has unsaturated cis bonds, while
such radiation. tween 80 (HLB 15) has 1 oleyl tail with one unsaturated
Sunscreen can protect the skin against the harmful cis bond. The presence of unsaturated cis bonds in the
effect of UV radiation from the sun. The sunscreen's tail of the emulsifier causes the tail to remain flexible
component(s) can absorb, reflect or scatter the sunlight and liquid at room temperature. By combining the
(Goswami et al., 2013). The effectiveness of sunscreen unsaturated with the saturated bond in lecithin and
protection against UV rays can be determined by the Sun Tween 80; the tails of the saturated bond tend to stiffen
Protection Factor (SPF) of the range 0 - 100, at which a at room temperature; this combination renders
value of more than 15 is categorized as adequate flexibility in the molecular organization in the cream
protection (Mulyani et al., 2015; Suhaenah et al., 2019). formulation. The flexible lecithin and tween 80 tails will
The molecular structure of flavonoids contains help pack the molecules close together. The interactions
more than one phenol group (-OH and aromatic groups) between the hydrocarbon tails of tween 80 and lecithin
and conjugated double bonds to protect against free include van der Waals forces, whose interactions
radicals (Kamilatussaniah et al., 2015). The leaf of the become more vital when the molecules are close
noni tree (Morinda citrifolia L.) contains more together (Athas et al., 2014). The aim of this study was
flavonoids than the fruit. According to Rubens et al., to understand the effect of the combination of emulsifier
(2018), the noni leaf has 89.1063 mg/L of rutin tween 80 and lecithin on the cream physical properties
flavonoid. Besides flavonoids, other phytochemical and to obtain the optimum formulation of the cream
compounds are present in the noni leaf extract. containing noni leaf extract.
Phytochemical screening of noni leaf extract by Ly et al.
(2020) resulted from maceration on 70% ethanol MATERIALS AND METHODS
demonstrates the existence of alkaloids, tannins, Materials
triterpenoids, saponins, coumarins, anthraquinones, A dry powder of the noni leaf ethanol extract was
carotenoids, organic acids, and reducing agents. obtained from PT. Bina Agro Mandiri, Tirtonirmolo,
In this study, the oil-in-water (O/W) cream was Kasihan, Bantul, Yogyakarta. Ethanol pro analysis were
chosen as a sunscreen dosage form because it has obtained from Merck (Darmstadt, Germany). Liquid
advantages such as being comfortable to use, easy to paraffin, distilled water, and glycerin were purchased
apply, non-sticky, and easy to wash with water from CV. Sentra Teknosains Indonesia. Stearic acid,
compared to ointment or paste preparations (Sharon et cetyl alcohol, methylparaben, and tween 80 were
al., 2013). In cream preparations, an emulsifier or obtained from PT. Brataco, Indonesia. Lecithin was
surfactant is required to stabilize the emulsion. purchased from Tokyo Chemical Industry (TCI)
Surfactant is also chemical enhancer to accelerate the (Japan).
absorption of active substances (Ramadon et al., 2021). Method
Tween 80 is one of the most commonly used Total flavonoid content
emulsifiers. Tween 80 is used because it is relatively The total flavonoid content was determined using
stable to electrolytes, does not irritate the skin, is non- rutin as the standard compound. The measurement was
toxic, and can produce stable emulsions (Syamsuddin et carried out at Gadjah Mada University's Integrated
al., 2016). However, due to its hydrophilic nature, tween
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Research and Testing Institute (LPPT) using a UV-Vis The EE X I value in calculation of SPF value can be
spectrophotometer instrument. seen in Table 1.
Determination of noni leaf extract SPF value Sunscreen cream formulation
The noni leaf extract samples were prepared in The formula refers to Mugitasari & Rahmawati
ethanol at 5%, 10%, and 20% concentrations. The UV (2020) research on the formulation of noni leaf extract
absorption of the samples was measured using a UV-Vis sunscreen cream using various extract concentrations
spectrophotometer at the wavelength of 290 - 320 nm (Table 2).
with 5 nm intervals (Mugitasari & Rahmawati, 2020; The ingredients that will be used in making the
Mulyani et al., 2015). The Mansur formula in equation cream are weighed first. The oil phase in the (stearic
(1) (Mansur et al., 1986), which was updated by a later acid, cetyl alcohol, liquid paraffin, and lecithin) was
researcher (Yulianti et al., 2015) was used to determine melted in a water bath at a temperature of 70 - 80ºC. The
the Sun Protection Factor (SPF) value. The SPF formula aqueous phase (glycerin, propylene glycol, and tween
involves the value of the constant of Effectiveness of 80) was dissolved in distilled water heated at a
Erythema due to light (EE x I). The value of EE x I were temperature of 70 - 80ºC in a beaker glass. The oil phase
according to Table 1. is added to the water phase gradually in the stainless
Calculation of SPF value bowl and stirred using a mixer at 5800 rpm until a cream
The SPF value of noni leaf extract was is formed. Noni leaf extract powder that has been
calculated using the Mansur formula: weighed is put into a mortar and ground until smooth.
SPF = 𝐶𝐹 × ∑320 290 𝐸𝐸 (𝜆) × 𝐼 (𝜆) × 𝐴𝑏𝑠(𝜆) Half of the distilled water used in the formula was added
(1) gradually into the mortar containing the extract and
Information: ground until the extract was dissolved. Noni leaf extract,
EE : The effectiveness of erythema due to UV light which had been dissolved, was added to the base at
at a wavelength of nm 45ºC. The cream is stirred again and put into a closed
I : Intensity of UV light at wavelength nm container (Mugitasari & Rahmawati, 2020; Safitri et al.,
Abs : Extract absorbance 2014).
CF : Correction factor (10) (Yulianti et al., 2015). Physical properties test
Emulsion type. The dilution method conducted the
Table 1. EE X I value in calculation of SPF value test by dissolving the oil-in-water (O/W) cream in water.
No Wavelength (λ nm) EE x I If the cream is soluble in water, then the cream is O/W
1 290 0.015 type (Elcistia & Zulkarnain, 2018). Spreadability test. A
2 295 0.0817 total of 0.5 grams of cream was weighed, placed in a
3 300 0.2874
round glass, then covered with another glass, and left for
4 305 0.3278
5 310 0.1864 1 minute. The diameter of the spread cream was
6 315 0.0839 measured by taking the average diameter from several
7 320 0.018 sides. Then the load is added every 1 minute, 50 grams
Total 1 to 250 grams, and the diameter of the spread is measured
(Elcistia & Zulkarnain, 2018).
Table 2. Noni leaf extract cream formula modified from Mugitasari & Rahmawati, (2020)
Concentration (%w/v)
Ingredients
F1 F2 F3 F4 F5
Noni leaf extract 10 10 10 10 10
Stearic acid 4 4 4 4 4
Liquid paraffin 10 10 10 10 10
Cetyl alcohol 3 3 3 3 3
Glycerine 10 10 10 10 10
Methyl paraben 0.1 0.1 0.1 0.1 0.1
Tween 80 5 3.75 2.5 1.25 0
Lecithin 0 1.25 2.5 3.75 5
Distilled Water ad 100 mL ad 100 mL ad 100 mL ad 100 mL ad 100 mL
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The spreadability test requirements for topical results of the tests, a 70% ethanol extract of noni leaves
preparations are 5 - 7 cm (Mugitasari & Rahmawati, had a total flavonoid content of 4768.18 µg/g,
2020). Adhesion test. A total of 0.1 grams of cream was standardized as rutin.
weighed and placed on two glass objects whose area had Determination of extract concentration
been determined. Then press with a load of 1 Kg for 5 The concentration of noni leaf extract is being
minutes. The glass object is mounted on the test determined in order to determine the concentration of
equipment, and a 20-gram load is dropped. The time was the extract to be used in the preparation of sunscreen
recorded until the two glass objects were released cream. According to the results, extracts with
(Elcistia & Zulkarnain, 2018). The adhesion test concentrations of 5%, 10%, and 20% (equal to 0.024,
requirement for topical preparations is 4 seconds 0.048, 0.095% flavonoid) in ethanol solvent had SPF
(Mugitasari & Rahmawati, 2020). pH measurement. A values of 39.46, 39.59, and 39.68, respectively. All
total of 1 gram of cream was weighed and then diluted extract concentrations are ultra-protective, with SPF
with 10 mL of distilled water. The pH of the preparation values greater than 15 (Damogalad et al., 2013).
was measured using a pH meter. The pH requirement for When large quantities of plant-based derivatives are
good topical preparation is 4.5 - 6.5 (Lumentut et al., employed to increase the product's effectiveness, the
2020). research on the interactions between the active
Storage stability test ingredients and the vehicle components should always
The stability test of the cream was carried out by be considered. Finding the ideal balance between
storing the cream in a tightly-closed container for 28 rheological characteristics, such as the usability and
days at 75% Relative Humidity and 30°C temperature stability of the emulsion, and the efficacy of the product,
(Phetmung & Sawatdee, 2019). Changes in the cream such as the concentration of its active ingredients, is the
physical properties such as organoleptic (color, shape, most difficult task for cosmetic formulators Numerous
odor), spreadability, adhesion, and pH were observed. If literature reports discuss how plant extracts' activity
there is no significant change during the storage period, decreases when they are combined with topical bases
its characterization remains within acceptable limits, and when stored, which emphasizes the significance of
and there is no phase separation, the cream preparation thorough formulation development and optimization
is stable (Dewi et al., 2014; Mailana et al., 2016). process (Almeida et al., 2014). In this study, we
Data analysis examined the strength of the tween 80 emulsifier and
The Design-Expert programme version 13.0 lecithin in sustaining and supporting the stability of the
Trial was used to analyse quantitative data on the cream product, so we chose 10% extract rather than 5%
physical properties of sunscreen cream, such as extract for our formulation in the tween 80-lecithin
spreadability, adhesion, and pH. The stability test results emulsion system. Therefore, through this study, we
of sunscreen cream, including spreadability, adhesion, convey that the carrying capacity of the tween 80-
and pH after 28 days of storage, were analysed with a lecithin system can still produce stable emulsions at
95% confidence level using paired samples T-Test on 10% extract, so we predict that this system can be used
IBM SPSS Statistic version 25 Trial programme. The at lower concentrations.
data was analysed on the first and 28th days. There is no Initially, the cream was made with extract
significant difference during storage if the p-value is concentrations of 10% and 20% (Table 3). The
greater than 0.05. sunscreen cream with 10% extract had a smooth texture
Using Design-Expert version 13.0 Trial, the with no coarse grains. Meanwhile, grains of noni leaf
optimum emulsifier composition was determined by extract powder feel rough on the skin when sunscreen
inserting the criteria for spreadability, adhesion, and pH cream with 20% extract is applied. The presence of
that met the requirements for a good cream preparation, powder granules in sunscreen cream containing 20%
and then selecting the formula that approached the extract is due to the extract powders not being
highest desirability. completely dispersed in the emulsion with the emulsifier
used. Based on the preliminary results, a concentration
RESULTS AND DISCUSSION of 10% extract was used in subsequent experiments,
Noni leaf extract total flavonoid content test resulting in an SPF value of 39.57.
This test aimed to determine the total flavonoid
content of noni leaf extract powder. According to the
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Table 3. Sunscreen cream formula using noni leaf extract 10% and 20%
Concentration (%w/v)
Ingredients
10% Extract Cream 20% Extract Cream
Noni leaf extract 2 4
Stearic acid 0.8 0.8
Liquid paraffin 2 2
Cetyl alcohol 0.6 0.6
Glycerin 2 2
Methyl paraben 0.02 0.02
Tween 80 0.5 0.5
Lecithin 0.5 0.5
Distilled Water ad 20 mL ad 20 mL
Table 4. Organoleptic test results of F1, F2, F3, F4, F5
Organoleptic F1 F2 F3 F4 F5
Shape Slightly liquid Semisolid Semisolid Semisolid Semisolid
Yellowish Yellowish Yellowish
Colour Light green Light green
green green green
Typical noni Typical noni Typical noni Typical noni Typical noni
Odour
leaves leaves leaves leaves leaves
Texture Smooth Smooth Smooth Smooth Smooth
Table 5. Spreadability test results of F1, F2, F3, F4, F5
Spreadability F1 (cm) F2 (cm) F3 (cm) F4 (cm) F5 (cm)
𝑥̅ ± SD 7.45 ± 0.05 6.66 ± 0.15 6.3 ± 0.2 5.61 ± 0.12 4.47 ± 0.13
Organoleptic test Physical appearance

The organoleptic test aims to determine the Physical appearance was used to monitor whether
characteristics of the cream preparations that have been the cream product was smooth, and free of coarse
made. The organoleptic test was performed visually by particles or lumps. According to the observations, all
observing the cream preparation's shape, color, smell, formulas were free of coarse particles and lumps.
and texture (Table 4). Emulsion type test
According to observations, all sunscreen creams The dilution test of water determines the type of
have a typical noni leaf aroma, a green colour, and a emulsion used in the sunscreen cream. Based on the
smooth texture. F1 has a slightly liquid consistency. observations, it was determined that all formulas were
Meanwhile, F2, F3, F4, and F5 have semisolid forms, soluble in water, implying that all formulas were of the
and the consistency becomes denser as the concentration O/W type.
of lecithin increases. This is due to a lecithin emulsifier Spreadability test
in F2, F3, F4, and F5, but not in F1. The presence of The spreadability test determines how easily the
lecithin in the oil phase reduces the size of the oil cream can be used or applied (Lumentut et al., 2020).
droplets and narrows the particle size distribution, The spreadability test for topical preparations is 5-7 cm
allowing for increased viscosity. Some lecithin (Mugitasari & Rahmawati, 2020). Table 5 shows the
molecules can migrate to the aqueous phase and form results of the spreadability test.
vesicles, increasing the volume fraction of the According to the spreadability test results, F2, F3,
dispersion phase in the system and the emulsion's and F4 have diameters in the range of 5 - 7 cm and thus
viscosity (Luo et al., 2017). meet the requirements for good cream spreadability
There is a slight colour difference between the (Mugitasari & Rahmawati, 2020). Meanwhile, F1 does
formulas due to differences in the composition of the not meet the requirements for good cream spreadability
lecithin concentration. Because it lacks lecithin, F1 because it has an average of more than 7 cm whereas F5
cream is typically light green. While F5 has the highest has an average spread of less than 5 cm. The analysis
concentration of lecithin, the cream is yellowish-green. results using the Simplex Lattice Design method are
Lecithin is a brownish-yellow coloured emulsifier (Agu given in equation (2).
et al., 2021). The higher the concentration of lecithin
used, the more yellow the cream.
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Table 6. Adhesion test results of F1, F2, F3, F4, F5

F1 F2 F3 F4 F5
Adhesion
(seconds) (seconds) (seconds) (seconds) (seconds)
𝑥̅ ± SD 2.00 ± 0.00 5.00 ± 0.00 5.67 ± 0.58 6.67 ± 0.58 8.33 ± 0.58
Y = 7.46A + 4.49B + 1.07AB (2) As shown in Table 6, F2, F3, F4, and F5 have met
Information: the requirements for good cream adhesion, which are
Y : spreadability (cm) greater than 4 seconds. Meanwhile, F1 does not meet the
A : concentration tween 80 adhesive power requirements because the stickiness is
B : lecithin concentration less than 4 seconds. This has to do with the spreadability
AB : interaction between tween 80 and lecithin of each cream formulation formula. The adhesion value
is inversely proportional to the spreadability value—the
In equation (2), it is discovered that tween 80 has a greater the spreadability, the lower the adhesion value
higher coefficient than lecithin. As a result, tween 80 has (Lumentut et al., 2020). The analysis results using the
a greater effect than lecithin in increasing spreadability. Simplex Lattice Design method are given in equation
(3).
Y = 2.03A + 8.37B + 2.67AB (3)
The tween 80 coefficient and lecithin have a
positive value according to equation (3). As a result, the
two emulsifiers affect the adhesion of sunscreen cream
preparations. Because lecithin has a higher coefficient
than tween 80, it can be said that lecithin has a more
significant effect on cream adhesion than tween 80.
Figure 1. Spreadability test results contour plot graph

of F1, F2, F3, F4, F5
According to the contour plot graph in Figure 1, the

higher the spreadability value, the higher the
concentration of tween 80 and the lower the
concentration of lecithin. The spreadability value is
inversely proportional to the viscosity; the greater the
viscosity, the lower the spreadability value (Yusuf et al., Figure 2. Adhesion test results contour plot graph of
2017). Lecithin can increase cream viscosity due to the F1, F2, F3, F4, F5
formation of phospholipid vesicles in the aqueous phase
(Arancibia et al., 2017). As a result, the higher the The contour plot of the adhesion test results in
concentration of lecithin, the higher the viscosity and the Figure 2 shows that the higher the concentration of
lower the spreadability value. tween 80 and the lower the concentration of lecithin, the
Adhesion test lower the adhesion value. The adhesion contour plot has
The adhesion test aims to determine the cream's an inversely proportional to the spreadability contour
ability to adhere to the skin (Mailana et al., 2016). The plot. This follows the statement that the greater the
adhesion test time for good topical preparations must be spreadability, the lower the adhesive power (Lumentut
at least 4 seconds (Mugitasari & Rahmawati, 2020). The et al., 2020).
results of the adhesion test can be seen in Table 6.
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Table 7. pH value of F1, F2, F3, F4, F5

pH F1 F2 F3 F4 F5
𝑥̅ ± SD 5.07 ± 0.06 5.03 ± 0.06 5.03 ± 0.06 5.1 ± 0.00 5.13 ± 0.06
pH measurement indicates this is greater than 0.05. The adhesion test

The pH measurement aims to determine the results of each formula still met the requirements for
preparation's pH value in order to meet the pH good cream adhesion on the 28th day because it can stick
requirements of topical preparations. A good topical for more than 4 seconds.
preparation must have a pH between 4.5 and 6.5 There was no significant change in each formula
(Lumentut et al., 2020). A very low pH or acidic based on the analysis of the pH test data from the first to
preparation can irritate the skin, whereas a high pH or the 28th day. The pH of all formulas is within the
alkaline preparation can make the skin scaly (Iskandar required range of 4.5 - 6.5. As a result, the pH of each
et al., 2021). According to Table 7, all formulas met the formula remained stable after 28 days of storage.
requirements for the pH of the cream preparation, which Interaction between lecithin and tween 80
ranged from 4.5 to 6.5, and there was no statistically Tween 80 and lecithin may bind strongly at the O/W
significant difference between each formula. cream preparation's interface and stabilize the cream.
Storage stability test The interaction between the tail and head of each
The appearance of F4 and F5 changes after 28 days emulsifier results in this tight packaging. Both
of storage. On day 14, both formulas began to release oil emulsifiers have hydrocarbon tails; tween 80 has one
on the surface of the cream preparation. This indicates oleyl tail, and lecithin has two C16 - C18 tails. There is
that the resulting cream is unstable. Because F4 contains a van der Waals or dispersion force interaction between
more lecithin than tween 80 and F5 only contains the hydrocarbon tails, which becomes much more potent
lecithin as an emulsifier, oil may be released onto the when the tails are close. Because of the presence of
cream's surface. Excess lecithin concentration cannot be unsaturated cis bonds in the emulsifier's tail, it remains
adsorbed at the oil droplet interface, resulting in a less flexible and liquid at room temperature. On the other
stable emulsion. As a result, when the lecithin hand, Saturated tails tend to stiffen or "freeze" at room
concentration is too high, the oil droplets are completely temperature. The flexible lecithin and tween 80 tails will
covered, and excess lecithin can cause droplet aid in packing the molecules (Figure 3a) (Athas et al.,
interactions or coalescence, resulting in the formation of 2014).
an oil layer on the cream preparation's surface (Dammak Because of its low HLB, lecithin is hydrophobic and
& Sobral, 2017). During storage, F1, F2, and F3 have oil-soluble. As a result, when lecithin reaches the O/W
consistent physical appearances. The odour and colour cream interface, it has a low tendency to desorb into the
of each formula did not change significantly during aqueous phase and remains on the oil droplets (Figure
storage. 3b). The lecithin will cover the oil droplets, preventing
There were significant changes in F1 and F4 the cream preparation from coalescing. Tween 80, on
marked with a significance value of 0.05 based on the the other hand, provides steric repulsion on oil droplets
analysis of the spreadability test data on the first and (Figure 3c). This is due to tween 80's highly water
28th day. Spreadability in F1 is decreasing while soluble oxyethylene side chain, which prefers to be
spreadability in F4 is increasing. F2, F3, and F5 have surrounded by solvent rather than interpenetrated when
significance values greater than 0.05, indicating that droplets collide (Athas et al., 2014).
there is no significant change in the spreadability of the If the O/W cream only contains lecithin, there will
cream after 28 days of storage. F1 to F4 still had a be only a weak barrier to prevent oil droplet
spreadability value within the required limits of 5 - 7 cm coalescence. If only tween 80 is present, a steric barrier
on the 28th day, indicating that they were stable during will be present initially, but desorption of tween 80 into
storage, whereas F5 had a spreadability value of less the aqueous phase will leave the droplet uncovered, and
than 5 cm, indicating that they did not meet the coalescence may occur (Athas et al., 2014). This helps
requirements for the cream spreadability value. to explain why an emulsifier combination of lecithin and
There was no significant difference of each formula tween 80 is required in the emulsion to ensure the
based on the analysis of the adhesion test data on the first stability of the O/W cream.
and 28th days. The significance value of each formula
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E-ISSN: 2580-8303
Figure 3. (a) The unsaturated cis tail of tween 80 and lecithin provide a tight packing in the oil droplet. (b) Lecithin
does not desorb to the water phase because its low HLB. (c) Tween 80 prevent the droplets from coalescence by steric
repulsion (Athas et al., 2014)
Determination of optimum composition stability of the noni leaf extract sunscreen cream,
The highest desirability value was discovered to be including spreadability and adhesion, but does not
between F3 and F4 based on data analysis using the affect pH value. F3 with 2.5 % tween 80 and 2.5 %
Design Expert version 13.0 Trial. F3 and F4 are the best lecithin is a sunscreen cream with an optimal formula
formulas based on the desirability value (Figure 4). that has met the requirements for good cream
However, according to the stability test, the oil began to preparation and good physical stability of the cream
leak from the F4's surface on the 14th day. This indicates based on data analysis and storage stability for 28 days.
that the cream preparation is unstable due to the
presence of coalescence, which occurs in the O/W cream ACKNOWLEDGMENT
preparation, and thus F4 was not chosen as the optimum Sanata Dharma University funded the research
formula. F3 was selected as the best formula because it under project number 017/Penel./LPPM-USD/IV/2022.
has a desirability value close to one and meets the
criteria for organoleptic tests, homogeneity, emulsion AUTHOR CONTRIBUTIONS
type, and storage stability. Conceptualization, D. S., B. L.T.; Methodology, D.
S.; Software, B. L. T.; Validation, D. S., A. B. S. L.;
Formal Analysis, D. S., B. L. T., A. B. S. L.;
Investigation, B. L. T.; Resources, D. S., R. D.; Data
Curation, B. L. T.; Writing - Original Draft, D. S., B. L.
T., A. B. S. L., R. D.; Writing - Review & Editing, D.
S.; Visualization, B. L. T.; Supervision, D. S.; Project
Administration, D. S.; Funding acquisition, D. S., B.
L.T.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
REFERENCES
Agu, H. O., Ejike, R. C., Obiegbuna, J. E. & Ezegbe, C.
C. (2021). Production and Physicochemical
Figure 4. Desirability value of F1, F2, F3, F4, F5
Properties of Cake with Different Ratios of
Production and Physicochemical Properties of
CONCLUSION
Cake with Different Ratios of Soy Lecithin. Asian
Based on the findings, it can be concluded that 10%
Food Science Journal; 20; 1–9. doi:
noni leaf extract is the best extract concentration, with
10.9734/afsj/2021/v20i930337.
an SPF value of 39.59. The tween 80/lecithin emulsifier
combination affects the physical properties and physical
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Almeida, I. F., Costa, P. C. & Bahia, M. F. (2014). Goswami, P. K., Samant, M., Srivastava, R., Kantivan,
Optimization of A Surfactant-free Antioxidant P. & Email, G. (2013). Natural Sunscreen Agents:
Formulation Using Response Surface A Review. Scholars Academic Journal of
Methodology. Drug Development and Industrial Pharmacy (SAJP); 2; 458–463.
Pharmacy 40; 120–125. doi: Iskandar, B., Sidabutar, S. E. B. & Leny. (2021).
10.3109/03639045.2012.750666. Formulasi dan Evaluasi Lotion Ekstrak Alpukat
Arancibia, C., Riquelme, N., Zúñiga, R. & Matiacevich, (Persea Americana) sebagai Pelembab Kulit.
S. (2017). Comparing the Effectiveness of Natural Journal of Islamic Pharmacy; 6; 14–21. doi:
and Synthetic Emulsifiers on Oxidative and 10.18860/jip.v6i1.11822.
Physical Stability of Avocado Oil-based Kamilatussaniah, Yuniastuti, A. & Iswari, R. . (2015).
Nanoemulsions Comparing the Effectiveness of Pengaruh Suplementasi Madu Kelengkeng
Natural and Synthetic Emulsifiers on Oxidative terhadap Kadar TSA dan MDA Tikus Putih yang
and Physical Stability of Avocado Oil. Innovative Diinduksi Timbal (Pb). Jurnal MIPA; 38; 108–
Food Science and Emerging Technologies; 44; 114.
159-166. doi: 10.1016/j.ifset.2017.06.009. Lumentut, N., Jaya, H. & Melindah, E. (2020).
Athas, J. C., Jun, K., McCafferty, C., Owoseni, O., John, Formulasi dan Uji Stabilitas Fisik Sediaan Krim
V. T. & Raghavan, S. R. (2014). An Effective Ekstrak Etanol Kulit Buah Pisang Goroho (Musa
Dispersant for Oil Spills Based on Food-Grade acuminafe L.) Konsentrasi 12.5% sebagai Tabir
Amphiphiles. Langmuir; 30; 9285–9294. doi: Surya. Jurnal MIPA; 9; 42–46. doi:
10.1021/la502312n. 10.35799/jmuo.9.2.2020.28248.
Chuacharoen, T., Prasongsuk, S. & Sabliov, C. M. Luo, X., Zhou, Y., Bai, L., Liu, F., Zhang, R., Zhang, Z.,
(2019). Effect of Surfactant Concentrations on Zheng, B., Deng, Y. & McClements, D. J. (2017).
Physicochemical Properties and Functionality of Production of Highly Concentrated Oil-In-Water
Curcumin Nanoemulsions under Conditions Emulsions Using Dual-Channel
Relevant to Commercial Utilization. Molecules; Microfluidization: Use of Individual and Mixed
24; 1–12. doi: 10.3390/molecules24152744. Natural Emulsifiers (Saponin and Lecithin). Food
Dammak, I. & Sobral, P. J. do A. (2017). Formulation Research International; 96; 103–112. doi:
and Stability Characterization of Rutin-Loaded 10.1016/j.foodres.2017.03.013.
Oil-in-Water Emulsions. Food and Bioprocess Ly, H. T., Pham Nguyen, M. T., Nguyen, T. K. O., Bui,
Technology; 10; 926–939. doi: 10.1007/s11947- T. P. Q., Ke, X., & Le, V. M. (2020).
017-1876-5. Phytochemical Analysis and Wound-Healing
Damogalad, V., Jaya Edy, H. & Sri Supriati, H. (2013). Activity of Noni (Morinda citrifolia) Leaf Extract.
Formulasi Krim Tabir Surya Ekstrak Kulit Nanas Journal of Herbs, Spices and Medicinal Plants;
(Ananas comosus L Merr) dan Uji In Vitro Nilai 26; 379–393. doi:
Sun Protecting Factor (SPF). Pharmacon Jurnal 10.1080/10496475.2020.1748159.
Ilmiah Farmasi; 2; 39–44. Mailana, D., Nuryanti & Harwoko. (2016). Antioxidant
Dewi, R., Anwar, E. & Yunita, K. S. (2014). Uji Cream Formulation of Ethanolic Extract from
Stabilitas Fisik Formula Krim yang Mengandung Avocado Leaves (Persea americana Mill.). Acta
Ekstrak Kacang Kedelai (Glycine max). Pharmaciae Indonesia; 4; 7–15.
Pharmaceutical Sciences and Research; 1; 194– Mansur, J. de S., Breder, M. N. R., Mansur, M. C.
208. d’Ascençäo, & Azulay, R. D. (1986).
Elcistia, R., & Zulkarnain, A. K. (2018). Optimasi Determinaçäo do Fator de Proteçäo Solar Por
Formula Sediaan Krim O/W Kombinasi Espectrofotometria. Anais Brasileiros de
Oksibenzon dan Titanium Dioksida serta Uji Dermatologia; 61; 121–124.
Aktivitas Tabir Suryanya secara In Vivo. Majalah Morita, A. (2018). Current Developments in
Farmaseutik; 14; 63–78. Phototherapy for Psoriasis. Journal of
Estiasih, T., Ahmadi, K. & Alifatur Rizqiyah, L. (2015). Dermatology; 45; 287–292. doi: 10.1111/1346-
Mikroemulsifikasi Fraksi Tidak Tersabunkan 8138.14213.
Distilat Asam Lemak Minyak Sawit. Jurnal Mugitasari, D. M. & Rahmawati, B. (2020). Formulasi
Teknologi dan Industri Pangan; 26; 189–200. doi: Krim Ekstrak Daun Mengkudu (Morinda
10.6066/jtip.2015.26.2.189. citrifolia L.) sebagai Sediaan Pelindung Sinar
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Ultraviolet. Jurnal Keperawatan dan Kesehatan Safitri, N. A., Puspita, O. E. & Yurina, V. (2014).
Masyarakat; 9; 109–119. Optimasi Formula Sediaan Krim Ekstrak Stroberi
Mulyani, Syamsidi, A. & Putri, P. (2015). Penentuan (Fragaria x ananassa) sebagai Krim Anti Penuaan.
Nilai SPF (Sun Protecting Factor) Ekstrak N- Majalah Kesehatan FKUB; 1; 235–246. doi:
Heksan Etanol dari Rice Bran (Oryza Sativa) 10.22146/jps.v1i1.23429.
secara In Vitro Dengan Metode Spektrofotometri Semenzato, A., Costantini, A., Meloni, M., Maramaldi,
UV-VIS. Online Jurnal of Natural Science; 4; 89– G., Meneghin, M. & Baratto, G. (2018).
95. Formulating O/W Emulsions with Plant-Based
Park, J. H., Lee, J. E., Choi, S. S., & Park, T. H. (2017). Actives : A Stability Challenge for an Effective
Protective Effects of Silkworm Hemolymph Product. Cosmetics; 5; 1–11. doi:
Extract and Its Fractions on UV-induced 10.3390/cosmetics5040059
Photoaging. Biotechnology and Bioprocess Sharon, N., Anam, S. & Yuliet. (2013). Formulasi Krim
Engineering; 22; 37–44. doi: 10.1007/s12257- Antioksidan Ekstrak Etanol Bawang Hutan
016-0588-4. (Eleutherine palmifolia L. Merr). Online Jurnal of
Phetmung, H. & Sawatdee, S. (2019). Development of Natural Science; 2; 111–122.
Cosmetic Cream Containing a Pharmaceutical Suhaenah, A., Tahir, M. & Nasra, N. (2019). Penentuan
Cocrystal of Salicylic and Phenylalanine. Nilai SPF (Sun Protecting Factor) Ekstrak Etanol
International Journal of Applied Pharmaceutics; Jamur Kancing (Agaricus bisporus) secara In
11; 1–11. doi: 10.22159/ijap.2019v11i5.33869. Vitro dengan Metode Spektrofotometri UV-VIS.
Putri, Y. D., Kartamihardja, H., & Lisna, I. (2019). Jurnal Ilmiah As-Syifaa; 11; 82–87. doi:
Formulasi dan Evaluasi Losion Tabir Surya 10.33096/jifa.v11i1.523.
Ekstrak Daun Stevia (Stevia rebaudiana Bertoni Syamsuddin, A., Zulkarnain, I. & Hasyim, N. (2016).
M). Jurnal Sains Farmasi & Klinis; 6; 32-36. doi: Formulasi Losio Emolien dari Ekstrak Etanol
10.25077/jsfk.6.1.32-36.2019. Sarang Semut (Myrmecodia pendens) dengan
Ramadon, D., McCrudden, M. T. C., Courtenay, A. J., Menggunakan Emulgator Anionik dan Nonionik.
& Donnelly, R. F. (2021). Enhancement Strategies Journal Og Pharmaceutical and Medicinal
for Transdermal Drug Delivery Systems: Current Sciences; 1; 79–82.
Trends and Applications. Drug Delivery and Yamashita, Y., Miyahara, R. & Sakamoto, K. (2017).
Translational Research; 12; 758-791. doi: Emulsion and Emulsification Technology. In
10.1007/s13346-021-00909-6. Cosmetic Science and Technology: Theoretical
Rohmah, J., Wulandari, F. E., & Rini, C. S. (2020). In Principles and Applications; 2017; 489-506. doi:
Vitro: Sunscreen Activity of Red Lettuce Extract 10.1016/B978-0-12-802005-0.00028-8
(Lactuca sativa var. crispa L.). IOP Conference Yulianti, E., Adelsa, A. & Putri, A. (2015). Penentuan
Series: Earth and Environmental Science; 519; 1- Nilai SPF (Sun Protection Factor) Ekstrak Etanol
7. doi: 10.1088/1755-1315/519/1/012009. 70 % Temu Mangga (Curcuma mangga) dan Krim
Rubens, P. de B., Ligia, S. R., Joao, G. da C., Amanda, Ekstrak Etanol 70 % Temu Mangga (Curcuma
C. L., Isabelle, C. S. M., Claudio, G. da S., mangga) secara In Vitro Menggunakan Metode
Aldenir, F. dos S., Jhonatan, D. S. das N., Adriana, Spektrofotometri. Majalah Kesehatan FKUB; 2;
G. D., Gabriela, S. V. de M., Johnnatan, D. de F., 41–50.
Jonas, dos S. S. & Simone, P. B. F. (2018). Yusuf, A. L., Nurawaliah, E. & Harun, N. (2017). Uji
Bioactivity and Phenolic Composition of Extracts Efektivitas Gel Ekstrak Etanol Daun Kelor
of Noni (Morinda citrifolia L., Rubiaceae) in (Moringa oleifera L.) sebagai Antijamur
Tomato Moth (Tuta absoluta Meyrick, 1917) Malassezia furfur. Kartika: Jurnal Ilmiah
(Lepidoptera: Gelechiidae). African Journal of Farmasi; 5; 62-67. doi: 10.26874/kjif.v5i2.119.
Agricultural Research; 13; 2063–2069. doi:
10.5897/ajar2018.13447.
P-ISSN: 2406-9388
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DOI: 10.20473/jfiki.v9i32022.272-278
Mannitol Production from Fructose by Using Resting Cells of

Methylotrophic Yeasts
Nisa Yulianti Suprahman1*, Khairul Basyar2, Herman Suryadi2
1
Department of Pharmacy, Faculty of Science, Institut Teknologi Sumatera, Lampung, Indonesia
2
Department of Pharmacy, Faculty of Pharmacy, Universitas Indonesia, Jakarta, Indonesia
*Corresponding author: nisa.suprahman@fa.itera.ac.id

Abstract
Background: Mannitol is a polyol sugar widely used in pharmaceutical and food industries which can be produced
by bioconversion. Using of resting cells and methanol as a carbon source are strategies to increase the efficiency
of mannitol production by increasing NAD(P)H needed in the reduction process. Objectives: This research aimed
to optimize bioconversion condition by using resting cells of methylotrophic yeasts with methanol and fructose as
carbon source and substrate, respectively. Methods: Several isolates were used including Candida sp,
Debaryomyces nepalensis and Debaryomyces hansenii and three species suspected to be yeast isolated from a
local paddy field. The methylotrophic characteristic of the yeasts was screened by turbidometry. The optimization
of fermentation condition was conducted by varying cultivation time (24-96 hours), resting cell concentration (30-
140 mg/mL), fructose concentration (7.5-15%), ammonium sulphate concentration (0.25-0.75%) and aeration
condition (50-80%). Quantitative analysis of the mannitol was conducted by HPLC with NH 2 column and
Refractive Index Detector. Results: D. hansenii showed the highest yield value in mannitol production (23.17%),
followed by D. nepalensis, Isolate A and Candida sp. (6.52%, 6.50% and 4.38%, respectively). Variation of
bioconversion condition using D. hansenii showed that the highest resting cell concentration (140 mg/mL)
incubated for 72 hours, moderate fructose concentration (10%), the highest ammonium sulphate concentration
(0.75%) and moderate aeration condition (70%) would result in the highest yield value of mannitol (60%).
Conclusion: This finding showed the potency of D. hansenii in mannitol production and gave preliminary
information of its optimum fermentation condition.
Keywords: resting cell, methylotrophic yeast, mannitol, fructose, methanol

Tania, B. L., Dwiastuti, R., Lestari, A. B. S. & Setyaningsih, D. (2022). Sunscreen Cream Formulation of Noni Leaf
Extract (Morinda citrifolia L.) with Emulsifier Combination of Tween 80 and Lecithin. Jurnal Farmasi dan Ilmu
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION production could be increased, especially on an

Mannitol is a polyol sugar with the lowest glycemic industrial scale.
index (Msomi et al., 2021). Mannitol is used not only in
the food industry but also in the pharmaceutical MATERIALS AND METHODS
industry, as an active ingredient and excipient (Shi et al., Materials
2020; Yang et al., 2021), particularly as tablet diluent. Three strain of yeasts were used in this study,
The advantages of mannitol as a diluent are its low including Candida sp. UICC Y216, Debaryomyces
hygroscopicity and pleasant taste (Kosugi et al., 2020; hansenii UICC Y276 and Debaryomyces nepalensis
Martau et al., 2021). Currently, industrial production of UICC Y328 (University of Indonesia Culture
mannitol is done with catalytic hydrogenation. Collection). In addition, species expected to be yeast,
However, this method provides low efficiency and isolated from local paddy field (Jl. Sindang Barang,
comprises complex purification steps (Mérillon & Dramaga, Bogor Barat, Bogor, Indonesia), was also
Ramawat, 2018). used.
Bioconversion technology for producing mannitol Four types of medium were used in this study,
using lactic acid bacteria, yeast and fungi has been including medium for isolation, cultivation, preculture
studied to increase its efficiency on industrial scale. In and fermentation. An isolation medium was used for
microorganism cells, mannitol is produced with a yeast isolation from paddy soil. It was a selective media,
reduction process from fructose, catalyzed by mannitol composed of 0.5 g of ammonium sulphate, 0.05 g of
dehydrogenase (MDH) enzyme (Gonçalves et al., 2019; magnesium sulphate, 0.35 g of disodium hydrogen
Lu et al., 2019). phosphate, 0.3 g of kalium dihydrogen phosphate, 0.05
Several research have been conducted to optimize g of chloramphenicol and 0.01 g of yeast extract
the efficiency of the bioconversion by using (Asthana et al., 1971). Chloramphenicol was added to
methylotrophic yeasts, which are able to use methanol the isolation media. The composition of each 100 mL of
as carbon source (Yurimoto et al., 2011). Compared to cultivation medium was 2 mL of glucose, 2mL of
sugars, methanol has higher reduction degree by which peptone, 1 g of yeast extract and 1.5 g of agar (Lai et al.,
the yield value will increase. In the bioconversion 2019).
process, Nicotinamide Adenine Dinucleotide According to Suryadi et al. (2000), the
(Phosphate) Hydrogen (NAD(P)H) is required to reduce fermentation was done in two steps with some
fructose into mannitol. Using methanol, NAD(P)H is modifications. The media used in the first steps was
produced from oxidation of methanol into formaldehyde preculture media composed of 1 mL of methanol, 1 mL
catalyzed by methanol dehydrogenase (Wang et al., of fructose, 0.5 g of ammonium sulphate, 0,05 g
2019). magnesium sulphate, 0.35 g disodium hydrogen
Another strategy to increase the efficiency of phosphate, 0.3 g potassium dihydrogen phosphate and
mannitol production is resting cells. Resting cells are 0.01 g yeast extract in 100 mL of distilled water. At the
cells that are not active in growing, but cell metabolisms second step, the media used was fermentation media
are still going on. This characteristic allows minimum composed of 1% methanol and 10% fructose.
carbon and energy use for biomass production. Instead, Chemicals used in this study were methanol (JT
those are used for oxidation-reduction (Jackson et al., Baker), acetonitrile (JT Baker), fructose (Merck),
2019; Ng, 2020). Mannitol production by resting cell of glucose (Merck), mannitol (Merck), ammonium
Escherichia coli BL21 and Candida magnoliae has been sulphate (Merck), magnesium sulphate (Merck),
studied previously and showed 82% and 45% yield dinatrium hydrogen phosphate (Merck), kalium
value, respectively (Khan et al., 2009; Koko et al., dihydrogen phosphate (Merck), chloramphenicol, yeast
2021). However, studies on utilizing the resting cell of extract (Bacto) and 1.5 g of agar (Wako), glucose
methylotrophic yeast for mannitol production have not (Merck), peptone (Liofilchem), yeast extract (Bacto)
been published. Therefore, this research was performed and 1.5 g of agar (Wako).
to identify the ability of resting cells from several Instruments
methylotrophic yeasts to make mannitol, including Shaking bath incubator (Labline), pH meter
optimising the fermentation condition. The change in (Eutech), microscope (Euromex), centrifuge (Kubota
yield value with varying fermentation conditions 6800), HPLC (Shimadzu LC-20AD), refractive index
showed the significance of each variable in mannitol detector (Shimadzu RID-10A), degasser (Shimadzu
production, by which efficiency of the mannitol DGU-20A5), HPLC oven column (Shimadzu CTU-
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6AS), Carbohydrate Analysis NH2 column (3,9 mm x 96 hours); resting cell concentration (300, 500, 100 and
300 mm, 10 µm) (Waters) and Spectrophotometry UV- 1400 mg), fructose concentration (7,5; 10; 12.5 and
Vis (Shimadzu 1601). 15%), ammonium sulphate concentration (0.25; 0.50
Methods and 0.75%.) and aeration condition (varying the volume
Isolation of yeast from paddy soil of fermentation media into 10, 15, 20 and 25 mL in 50
The soil was taken from 2-10 cm depth of the paddy mL Erlenmeyer.).
field. Paddy soil was suspended with serial Quantitative analysis of substrate and product
concentrations (10-1, 10-2, 10-3, 10-4 g/mL). The Fructose and mannitol calibration curves had
suspension (0.1 mL) was then incubated in an isolation previously been prepared. The substrate (fructose) and
medium for 2-3 days. Then, the cells grown were product (mannitol) were then quantified from 1 mL of
recultivated in new isolation medium with the addition fermentation culture. The culture was centrifuged at
of 1% methanol, for 2-3 days in at room temperature. 6000 rpm for 10 minutes, and the supernatant was
The isolation medium was adapted from Asthana et al., filtered through a 0.22m filter. HPLC was then used to
(1971). analyze the supernatant. (Shimadzu model LC-20AD),
Screening of paddy soil isolates refractive index detector (Shimadzu RID-10A),
Isolates grown from paddy soil were screened for degasser (Shimadzu DGU-20A5), oven column
the methylotrophic ability as well as the ability to (Shimadzu CTU-6AS), Waters Carbohydrate Analysis
produce mannitol. Screening for methylotrophic ability NH2 column (3.9 mm x 300 mm, 10 µm), with mobile
was conducted by cultivation of the isolates in the phase acetonitrile:distilled water (97:3). The column
preculture media while screening for mannitol was run at a temperature of 298 K, the flow rate of 1.0
production was done in the preculture media containing mL/min and and injection volume of 20 𝜇L.
5% of fructose.
Determination of cell concentration RESULTS AND DISCUSSION
Cell suspension in the preculture media was Macroscopic and microscopic visualization of
centrifuged and the supernatant was filtered through isolates were depicted in Figure 1. All isolates have
0.22 µm filter. The biomass was determined glossy surfaces and circular edges. The color of isolate
turbidometrically at 600 mm with Spectrophotometry A, B and C colonies were yellow, bright yellow and
UV-Vis (Shimadzu UV 1601) (Suryadi et al., 2000). white, respectively. Microscopically, all isolates were
Dry cell weight was determined by centrifugation and circular. Similar media was used by other research
wash of cell suspension. The supernatant was then dried previously and isolated 2 yeast species and 6
at 110°C for 7 hours and then triplicate weighing was Actinomycetes bacteria (Asthana et al., 1971).
performed. From this procedure, one OD unit was
shown corresponded to 2.26 mg/mL dry cells.
Fermentation
Three loopful of cells were cultivated in 100 mL of
preculture media for 2 days with 175 rpm shaking in
room temperature (Kasbawati et al., 2022). Resting cells
were then made from preculture cell suspension by
centrifugation at 8000 rpm, 5°C for 10 minutes. The
cells were resuspended in sterile NaCl 0.9% solution and
then centrifuged at 8000 rpm, 5°C for 10 minutes. This
procedure was done with two replicates to get clean
resting cells (Khan et al., 2009). The resting cells were
then cultivated in the fermentation media, incubated at
175 rpm with shaking incubator in room temperature.
Optimization of fermentation condition was conducted Figure 1. Yeast colonies (A) and microscopic (100 x
by the following factors: cultivation time (24, 48, 72 and magnification) examination of isolates (B, C, D)
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Table 1. Yield value of mannitol from fermentation with different yeasts

Yeast Yield Value
Debaryomyces hansenii 23.17%
Debaryomyces nepalensis 6.52%
Isolate A 6.50%
Candida sp. 4.38%
Isolate B Undetected
Isolate C Undetected
The cell's ability to use methanol as a carbon source

60000
was screened based on the change in Optical Density
50000
(OD) after incubation in preculture media for three days
Concentration (mg/l)
40000
at room temperature. It was found that all isolates were
30000
methylotrophs. However, the ability to produce
20000
mannitol was only shown by UICC strains and isolate
10000
A. The highest mannitol yield was achieved by D.
hansenii (23.17%), followed by D. nepalensis, isolate A 0
0 24 48 72 96 120
Incubation Time (hours)
and Candida sp (Table 1). The D. hansenii has long been
Mannitol Produced
known for its potency in polyol production, including
Fru ctose Detected
arabitol and xylitol (López-Linares et al., 2018;
Mardawati et al., 2019). The yeast was reported as a
Figure 2. Mannitol and fructose concentration detected
higher xylitol producer than Candida sp (Breuer &
in fermentation culture of Debaryomyces hansenii with
Harms, 2006; Loman et al., 2018; Rice et al., 2020).
resting cell concentration 140 mg/mL
As D. hansenii showed the highest yield value of
mannitol among the 5 other tested yeasts, variation of This is probably due to the negative feedback
fermentation condition was then only conducted with mechanism, in which the mannitol inhibits mannitol
this microorganism. The result of bioconversion to dehydrogenase, as reported by research (Lee et al.,
produce mannitol with each fermentation condition 2003). This was supported by the different resultss
variation using D. hansenii was presented in Table 2. obtained when less mannitol was produced in the third
Addition of the resting cell at the optimum concentration day of fermentation using less resting cell concentration.
(140 mg/mL) leads to an increase in the mannitol yield The addition of 30, 50 and 100 mg/mL resting cells
from the first to the third day. However, the yield value caused the mannitol production on the third day to be
of mannitol was decreased on the fourth day. On the smaller than the mannitol produced by 140 mg/mL
contrary, the amount of fructose decreased from the first resting cells. In this scenario, the mannitol yield value
to the third day but increased on the fourth day (Figure increased from the first to the fourth day, indicating that
2), indicating that mannitol had been re-converted into product inhibition was not shown with less mannitol
fructose on day 4. (Figure 3).
Table 2. Yield value of mannitol from fermentation with varying resting cell concentration, fructose and ammonium
concentration and aeration condition
Ammonium Sulphate
Resting Cells Variation Fructose Variation Aeration Variation
Variation
Yield Yield Ammonium Yield Yield
Resting Cell Fructose Aeration
Value Value Sulphate Value Value
Concentration Concentration Concentration
(%) (%) Concentration (%) (%)
300 mg 5.24 7.50 15.65 0.25 3.47 80.00 10.67
500 mg 14.18 10.00 16.33 0.50 9.57 70.00 61.15
0.75 40.99
1000 mg 18.44 12.50 11.0 60.00 31.85
1400 mg 20.49 15.00 9.35 - - 50.00 6.14
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30000
Mannitol Concentration (mg/l)
25000
Resting cell 30 mg/ml
20000
15000
10000
5000
0
48 72 96 120
Incubation Time (hours)
Figure 3. Mannitol concentration detected in fermentation culture of Debaryomyces hansenii with different resting cell
concentration
The effect of biomasses variation on mannitol yield (Figure 4). Previous research studied galacticol
valuhas been studied previously in mannitol production by yeast Rhodosproridium toruloides
dehydrogenase-transfected Escherichia coli BL21. In IFO0880 with variations in galactose concentration (20,
this study, increasing biomass from OD (600 nm) 20 to 40 and 60 g/L). This research reported that increasing
60% decreased mannitol production, which may have galactose concentration up to 60 g/L increased biomass
been caused by lower substrate availability with higher production. However, the yield value of galactitol was
cell amount (Koko et al., 2021). This study did not increased only by increasing galactose concentration up
examine fermentation with resting cell concentrations to 40 g/L but decreased by higher galactose. This
greater than 140 mg/mL.. However, given the behavior occurrence indicated that increasing biomass production
of decreased mannitol yield value on the fourth day by increasing galactose concentration did not correlate
compared to the third day, increasing resting cell with galactitol yield value (Jagtap et al., 2019).
concentration to more than 140 mg/mL without
increasing substrate concentration may not result in 25000
Concentration (mg/l)
increasing product yield value. 20000

The decrease in mannitol yield value was also 15000
obtained by using fructose concentration higher than 10000
10%. This was probably because of the fructose osmotic 5000
effect on biomass production. Osmotic pressure 0
equivalent to NaCl 3-5% w/v has been reported as the 6 8 10 12 14 16
ideal osmotic condition for D.hansenii growth as Fructose Concentration Added (%)
moderate halophilic yeast (Breuer & Harms, 2006;
Navarrete et al., 2022). The fructose is an osmotic agent. Mannitol Detected Fructose Detected
Optimal growth will shift the use of substrate to fulfil
Figure 4. Mannitol and fructose concentration detected
the biomass production, therefore, the mannitol yield
in fermentation culture of Debaryomyces hansenii with
value was decreased. This was supported by less amount
different resting cell concentration
of fructose detected on the fourth day of fermentation
using fructose 12.5%, compared to 7.5 and 10% Optimization of aeration level was conducted by
fructose. Moreover, fructose detected on the fourth day varying the volume of culture media (10, 15, 20 dan 25
of fermentation was higher than the added amount in mL) with the use of the same amount of resting cell (500
fermentation using 15% fructose, indicating the high use mg), the same concentration of fructose (10%) and
of fructose caused mannitol to be converted into fructose methanol (1%) and the same Erlenmeyer volume (50
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mL). The variation in culture media volume will allow Project Administration, N. Y. S., K. B., H. S.; Funding
different aeration level (80%, 70%, 60% and 50% for acquisition, N. Y. S., K. B., H. S.
media culture volume of 10, 15, 20 and 25 mL,
subsequently). Based on this optimization, the highest CONFLICT OF INTEREST
mannitol yield value was achieved by the moderate The authors declared no conflict of interest.
aeration (70%, resulted from the use of 15 mL media in
50 mL Erlenmeyer flask). REFERENCES
This founding was in concordance to research Asthana, H., Humphery, A. E. & Moritz, V. (1971).
conducted previously on bioconversion of xylose to Growth of Yeast on Methanol as The Sole Carbon
xylitol by D. hansenii (Breuer & Harms, 2006). In the Substrate. Biotechnology and Bioengineering; 13;
highly aerobic condition, the yield value of xylitol was 923–929. doi: 10.1002/bit.260130615.
decreased because the reoxidation of NADH into NAD + Breuer, U., & Harms, H. (2006). Debaryomyces
needed for biomass production was increased with the hansenii - an Extremophilic Yeast with
high availability of oxygen in the respiratory chain. In Biotechnological Potential. Yeast; 23; 415–437.
contrast, with moderate aeration, the available oxygen doi: 10.1002/yea.1374.
was only used for the regeneration of NADH into NAD + Gonçalves, C., Ferreira, C., Gonçalves, L. G., Turner, D.
required for xylitol production. In minimal oxygen, L., Leandro, M. J., Salema-Oom, M. &
xylitol yield value is low, owing to low NADH- Gonçalves, P. (2019). A New Pathway for
dependent Xylitol Dehydrogenase activity (Breuer & Mannitol Metabolism in Yeasts Suggests a Link to
Harms, 2006). the Evolution of Alcoholic Fermentation.
Frontiers in Microbiology; 10; 1–15. doi:
CONCLUSION 10.3389/fmicb.2019.02510.
Debaryomyces hansenii is a promising Jackson, E., Ripoll, M. & Betancor, L. (2019). Efficient
microorganism for mannitol production. Variations in Glycerol Transformation by Resting
fermentation conditions will highly determine the yield Gluconobacter Cells. Microbiology Open; 8; 1–
value of mannitol production. The balance between cell 10. doi: 10.1002/mbo3.926.
amount and substrate will provide an optimum number Jagtap, S. S., Bedekar, A. A., Liu, J. J., Jin, Y. S. & Rao,
of cell production and substrate availability for mannitol C. V. (2019). Production of Galactitol from
production. Limiting biomass production will direct the Galactose by the Oleaginous Yeast
use of substrate for metabolism to produce mannitol Rhodosporidium toruloides IFO0880.
rather than cell growth. Therefore, the use of resting cell Biotechnology for Biofuels; 2019; 1–13. doi:
and applying osmotic and aeration condition that is less 10.1186/s13068-019-1586-5.
in favor of biomass production will increase mannitol Kasbawati, K., Mardawati, E., Samsir, R. & Suhartini,
production efficiency. S. (2022). An Appropriate Unstructured Kinetic
Model Describing the Batch Fermentation Growth
ACKNOWLEDGMENT of Debaryomyces hansenii for Xylitol Production
The authors would like to acknowledge University Using Hydrolysis of Oil Palm Empty Fruit Bunch.
of Indonesia for providing yeasts culture for this Biotechnology & Biotechnological Equipment,
research and for Prof. Dr. Harmita, Apt for his expertise 36; 462–471. doi:
and assistance in quantitative analysis. 10.1080/13102818.2022.2098816.
Khan, A., Bhide, A. & Gadre, R. (2009). Mannitol
AUTHOR CONTRIBUTIONS Production from Glycerol by Resting Cells of
Conceptualization, N. Y. S., K. B., H. S.; Candida magnoliae. Bioresource Technology;
Methodology, N. Y. S., K. B., H. S.; Software, N. Y. S., 100; 4911–4913. doi:
K. B., H. S.; Validation, N. Y. S., K. B., H. S.; Formal 10.1016/j.biortech.2009.04.048.
Analysis, N. Y. S., K. B., H. S.; Investigation, N. Y. S., Koko, M. Y. F., Sami, R., Muhoza, B., Khojah, E. &
K. B., H. S.; Resources, N. Y. S., K. B., H. S.; Data Mansour, A. M. A. (2021). Promising Pathway of
Curation, N. Y. S., K. B., H. S.; Writing - Original Draft, Thermostable Mannitol Dehydrogenase (MtDH)
N. Y. S.; Writing - Review & Editing, H. S.; from Caldicellulosiruptor Hydrothermalis 108 for
Visualization, N. Y. S., K. B., H. S.; Supervision, H. S.; D-mannitol Synthesis. Separations; 8; 1-11. doi:
10.3390/separations8060076
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Kosugi, A., Leong, K. H., Urata, E., Hayashi, Y., Reviews in Biotechnology; 40; 608–622. doi:
Kumada, S., Okada, K. & Onuki, Y. (2020). Effect 10.1080/07388551.2020.
of Different Direct Compaction Grades of Mérillon, J. -M. & Ramawat, K. G. (2018). Sweeteners.
Mannitol on the Storage Stability of Tablet Pharmacology, Biotechnology and Applications.
Properties Investigated Using a Kohonen Self- Berlin: Springer.
Organizing Map and Elastic Net Regression Msomi, N. Z., Erukainure, O. L. & Islam, M. S. (2021).
Model. Pharmaceutics; 12; 1–14. doi: Suitability of Sugar Alcohols as Antidiabetic
10.3390/pharmaceutics12090886. Supplements: A Review. Journal of Food and
Lai, Y. T., Cheng, K. C., Lai, C. N., & Lai, Y. J. (2019). Drug Analysis; 29; 1–14. doi: 10.38212/2224-
Isolation and Identification of Aroma Producing 6614.3107.
Strain with Esterification Capacity from Yellow Navarrete, C., Sánchez, B. J., Savickas, S. & Martínez,
Water. PLoS ONE; 14; 1–16. doi: J. L. (2022). DebaryOmics: an Integrative –Omics
10.1371/journal.pone.0211356. Study to Understand the Halophilic Behaviour of
Lee, J. K., Koo, B. S., Kim, S. Y. & Hyun, H. H. (2003). Debaryomyces hansenii. Microbial
Purification and Characterization of a Novel Biotechnology; 15; 1133–1151. doi:
Mannitol Dehydrogenase from a Newly Isolated 10.1111/1751-7915.13954
Strain of Candida magnoliae. Applied and Ng, W. (2020). Metabolism of Non-Growing Cells and
Environmental Microbiology; 69; 4438–4447. Their Application in Biotransformation. Preprints;
doi: 10.1128/AEM.69.8.4438-4447.2003. 2020; 1-6. doi:
Loman, A. A., Islam, S. M. ., & Ju, L. -K. (2018). 10.20944/preprints202007.0402.v1.
Production of Arabitol from Enzymatic Rice, T., Zannini, E., K. Arendt, E. & Coffey, A. (2020).
Hydrolysate of Soybean Flour by Debaryomyces A Review of Polyols–Biotechnological
hansenii Fermentation. Biotechnological Production, Food Applications, Regulation,
Products and Process Engineering; 102; 641– Labeling and Health Effects. Critical Reviews in
653. doi: 10.1007/s00253-017-8626-5. Food Science and Nutrition; 60; 2034–2051. doi:
López-Linares, J. C., Romero, I., Cara, C., Castro, E. & 10.1080/10408398.2019.1625859.
Mussatto, S. I. (2018). Xylitol Production by Shi, J., Tan, L., Ye, J. & Hu, L. (2020). Hypertonic
Debaryomyces hansenii and Candida Saline and Mannitol in Patients with Traumatic
guilliermondii from rapeseed Straw Brain Injury: A Systematic and Meta-Analysis.
Hemicellulosic Hydrolysate. Bioresource Medicine; 99; 1-7. doi:
Technology; 247; 736–743. doi: 10.1097/MD.0000000000021655.
10.1016/j.biortech.2017.09.139. Suryadi, H., Katsuragi, T., Yoshida, N., Suzuki, S., &
Lu, F., Xu, W., Zhang, W., Guang, C. & Mu, W. (2019). Tani, Y. (2000). Polyol Production by Culture of
Polyol Dehydrogenases: Intermediate Role in the Methanol-Utilizing Yeast. Journal of Bioscience
Bioconversion of Rare Sugars and Alcohols. and Bioengineering; 89; 236–240. doi:
Applied Microbiology and Biotechnology; 103; 10.1016/S1389-1723(00)88825-8.
6473–6481. doi: 10.1007/s00253-019-09980-z. Wang, X., Wang, X., Lu, X., Ma, C., Chen, K. &
Mardawati, E., Andoyo, R., Muhaemin, M., Nurjanah, Ouyang, P. (2019). Methanol Fermentation
S., Natawigena, D., Herwanto, T. & Increases the Production of NAD(P)H-Dependent
Kramadibrata, A. M. (2019). Fermentation Chemicals in Synthetic Methylotrophic
Process of Glycerol to Arabitol from Byproducts Escherichia coli. Biotechnology for Biofuels; 12;
of Reutalis trisperma Biodiesel Using Yeast of 1–11. doi: 10.1186/s13068-019-1356-4.
Debaryomyces hansenii. IOP Conference Series: Yang, H., Fu, Y., Zhang, Y., Zhou, J., Wang, D., Gao,
Earth and Environmental Science; 347; 1-8. doi: Z. & Wang, X. (2021). Evaluation of Mannitol
10.1088/1755-1315/347/1/012132 and Xylitol on the Quality of Wheat Flour and
Martau, G. A., Coman, V. & Vodnar, D. C. (2020). Extruded Flour Products. Internation Journal of
Recent Advances in the Biotechnological Food Science and Technology; 56; 4119–4128.
Production of Erythritol and Mannitol. Critical doi: 10.1111/ijfs.15040.
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DOI: 10.20473/jfiki.v9i32022.279-289
Jelly Candy Hydroxyapatite from Mackerel Fish Bone

Lia Anggresani1,2, Santi Perawati3*, Ryan Afandi3, Rahmadevi4
1
Department of Midwifery, STIKes Syedza Saintika Padang, Padang, Indonesia
2
Graduate School of Natural Science and Technology, Gifu University, Japan
3
Department of Pharmacy, STIKes Harapan Ibu Jambi, Jambi, Indonesia
4
Department of Pharmacy, Adiwangsa University, Jambi, Indonesia
*Corresponding author: santiperawati@gmail.com
Submitted: 7 May 2022

Abstract
Background: Calcium is a mineral that is needed by bones and teeth. Calcium needs have not been achieved
evenly among children, about 1000mg/day. Hydroxyapatite contains Ca and apatite which is good for the
maintenance of bones and teeth. Hydroxyapatite is made from the bones of mackerel, where the bones of mackerel
have a high source of calcium. For easy being consumed for children, the hydroxyapatite made in the form of jelly
candy. Objective: The aim of this research was Hydroxyapatite Mackerel Fish Bone can be made as Jelly Candy.
Methods: Hydroxyapatite from mackerel bones is made by the precipitation method. Then, hydroxyapatite was
formulated into jelly candy with concentrations of hydroxyapatite was 18%, 19% and 20% respectively for
Formula I, II and III. Jelly candy evaluation includes organoleptic test, gel strength, khamir and ALT ochre, weight
uniformity, pH, hedonics, homogeneity and storage. Results: The evaluation showed that all jelly candy formulas
are safe for consumption according to SNI 3547.2-2008. Conclusion: In conclusion, hydroxyapatite mackerel fish
bone can be made as Jelly Candy with the best concentration in Formula 1.
Keywords: calcium, hydroxyapatite, jelly candy, mackerel fish bone, precipitation

Anggresani, L., Perawati, S. Afandi, R. & Rahmadevi. (2022). Jelly Candy Hydroxyapatite from Mackerel Fish
Bone. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 279-289. http://doi.org/10.20473/jfiki.v9i32022.279-
289
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E-ISSN: 2580-8303
INTRODUCTION market. However, because hydroxyapatite has not been

Calcium is an important mineral in the human body, made in candy preparations, there needs to be efforts to
which can affect the development and growth of bones develop hydroxyapatite preparations in a form that can
and teeth in humans, where 99% of calcium in the be consumed more easily by the community, especially
human body is found in bones (Shita & Sulistiyani, children. Children become unacceptable when directly
2010). Age 9 to 18 years is the age that most need contact with the tongue taste receptor, which has
calcium with an average amount of calcium of 1000 - partially dissolved in it. Jelly candy is a kind of oral drug
1200 mg/day compared to other ages (Jauhari et al., that many pharmaceuticals have been used nowadays for
2019). Unfortunately, many children in Indonesia do not patients who lost their primary teeth (such as children),
have enough calcium intake every day. Calcium sources and patients who have Parkinson’s, stroke, nausea, and
are divided between animals and vegetables (Shita & thyroid disorder disease (Sunil et al., 2020). Jellies are
Sulistiyani, 2010). translucent and transparent and can be used for internal
Hydroxyapatite (HAp) with molecular formula and external application. Jellies offers efficacy, safety,
Ca5(PO4)3(OH) or better known as Formula low cost of treatment. In general, jelly candy reveals
Ca10(PO4)6(OH)2 is bio ceramic calcium apatite that can pleasant taste, magnificent appearance and convenient
be found in human teeth and bones. Hydroxyapatite is to handle.
the most similar to the mineral part of the bone, since the Based on the description, researchers were
salt calcium phosphate (CaP) is the main mineral that interested in making hydroxyapatite jelly candy from
composes bones and teeth (Mozartha, 2000). It is widely mackerel fish bone due to the easy to be consumed. Jelly
used as an adsorbent for removing heavy metals due to candy is made from water or juice of plants and gel-
high-efficiency ion exchange between ion Ca and metal, forming materials (Alridho et al., 2017).
purification of wastewater treatment and biomedical
applications with excellent physical and mechanical MATERIALS AND METHODS
properties. Many studies investigated the preparation Materials
and characterization of Hydroxyapatite from natural Reagents and chemicals used in this research were
sources, such as cockle shells, cattle bones, and fish mackerel bone waste (Scomberomorus guttatus) were
bones with various methods (Stötzel et al., 2009). collected from Traditional Market in Jambi City,
One source of hydroxyapatite is mackerel bones. distilled water (Brataco® Indonesia), phosphoric acid
Fish bones have the most calcium content among other (Merck® Germany), 85% 0.1 M, Ethanol (Merck®
parts of the fish body because the main elements of fish Germany) 50%, hydrochloric acid (Merck® Germany)
bones are calcium, phosphorus and carbonate (Chadijah 37% 1 M, diammonium hydrogen phosphate (Merck®
et al., 2018). Mackerel bones (Scomberomorus guttatus) Germany) 10 M, sodium hydroxide 0.1 %, and acetone
can be processed into products that have a high (Merck® Germany), coco pandan syrup, gelatin, water,
economic value in the form of hydroxyapatite citric acid, benzoic acid, sodium benzoate, menthol and
(Ca5(PO4)3(OH)) (Anggresani et al., 2018). Aside from HAp.
mackerel bones, hydroxyapatite can be found in egg Equipment
shells (Noviyanti et al., 2017), blood shells (Ahmad, The equipment were used in this study were furnace
2017), and tuna fish bones (Mutmainnah et al., 2017). (Sh scientiac® Canada), oven, balance analytics
The development of science and technology in the (Shimadzu® Japan), stirrer hot plate (Ika c-mag hs7®
field of pharmacy encourages pharmacists to make the Germany), soaking container, sift mesh 80, porcelain
right formulations to process hydroxyapatite into a form cup, beaker glass, spatula, measuring pipette, burette,
of preparation that is easily accepted by the community measuring glass, X-ray Analysis Fluorescence
(Andasari, Zukhri and Nurjanah, no date). Spectrometer (XRF) UK, United Kingdom, X-Ray
Pharmaceutical preparations made from hydroxyapatite Diffraction (XRD) (Xpert pro analytical ® United
active that has been made into a pharmaceutical product Kingdom, United Kingdom), Scanning Electron
are the form of toothpaste (Anggresani et al., 2021; Microscopy (SEM) (Tabletop Microscope tm3000
Hernawan et al., 2021; Wadu et al., 2015), biomaterials United States).
(Hanura et al., 2017), and injectable bone substitutes Method
(Budiatin et al., 2016). There are also jelly preparations The powder formation of mackerel fish bone waste
made from active calcium (Lesmana et al., 2008) and Mackerel fish bones (Scomberomorus guttatus)
there are preparations of calcium jelly candy in the 1 Kg was cleaned and boiled. Then washed it, immersed
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in 10 L of 0.1% NaOH solution for 7 hours, drained then Weight uniformity test. Thirty pieces of jellies, each
soaked in a container containing 50% acetone for 8 of them was weight and the average weight from each
hours. Then, the bones are drained and dried for 7 days. formula was calculated. Organoleptic test. Organoleptic
Then crush it into powder. The powder was calcinated tests included the observed of the color, smell, texture,
for 3 hours at 800°C. Then the powder is crushed then and taste. pH test. This test used pH meter. Jelly candy
sieved with 80 mesh. Powder of mackerel fish bone hydroxyapatite mackerel fish bone was diluted with hot
(CaO) was formed and then analyzed using XRF distilled water 10 mL. Then measure the pH using pH
(Anggresani et al., 2021). meter. Hedonic test. This test included color, taste and
The synthesis of hydroxyapatite smell of the jelly candy hydroxyapatite mackerel fish
CaO powder 180 g was diluted with 1 L distilled bone made by the panelists. Kapang khamir test. This
water and then stirrer at speed 300 rpm for 30 minutes test was carried out to determine the shelf life of the
until the suspension Ca(OH)2 was formed. Add product. Kapang khamir test was carried out by counting
diammonium hydrogen phosphate (NH4)2HPO4 to the the colonies of kapang growth on jelly candy. According
suspension Ca(OH)2 using the variation of mol Ca/P was to the National Standardization Agency, the limit of
1,67 and for 1 hour heated at 90oC. Adjust pH using kapang maximum was 1 x 102 colony/gram. Total plate
NaOH 1 M until it reaches 12. Aging the solution for 24 number test. This test refers to National Standardization
hours at room temperature. Then the formed deposition Agency (SNI) 3557.2-2008. Gel strength. Gel strength
was filtered and dried using an oven at 120°C for 5 was measured using a texture analyzer with a load
hours. The dry precipitated was calcinated at 900°C for strength of 4500 grams and a cylindrical TA5 probe with
5 hours. Then analyzed with XRD and SEM a length of 40.25 mm. Gel strength was measured as the
(Anggresani et al., 2021). force required by the probe to compress a thick gel 5 mm
The formulation of jelly candy hydroxyapatite from at a speed 2.5 mm/second. Homogeneity test. This test
mackerel fish bone waste was observed using a piece of glass, and then jelly candy
Hydroxyapatite from Mackarel Fish Bone Waste. was smeared in it. Storage test. The test was carried out
The formulation of jelly candy can be seen in Table 1. by storing jelly candy at room temperature for 21 days.
The evaluation of jelly candy hydroxyapatite from Then, jelly candy was observed on the 7th day, 14th day
mackerel fish bone waste and 21st day.
The evaluation of raw material
The raw material evaluation includes organoleptic RESULTS AND DISCUSSION
and the solubility of hydroxyapatite, sodium benzoate, Calcium oxide (CaO) formation from mackerel fish
citric acid, gelatin, syrup, and menthol. All materials bone
used in this formulation conform to the standard. Mackerel fish bones was boiled for 45 min to
The evaluation of jelly candy cleanse the bones from the meat. After that, the bones
The evaluation of jelly candy includes a weight were soaked with NaOH 0.1% and acetone 50% to
uniformity test, organoleptic test, pH test, and hedonic remove the fat compound. Then crusher the bones and
test with ethical clearance No. LB.02.06/2/007/2021, calcinated using a furnace at 800oC for 3 hours to get
kapang khamir test and total plate number test, gel CaO powder. The function of calcination is to remove
strength, homogeneity test and storage test (Badan calcium carbonate (CaCO3) and becomes calcium oxide
Standarisasi Nasional, 2008) (from SNI Standard No. (CaO) (Mutmainnah et al., 2017).
3547-2-2008).
Table 1. The formulation of jelly candy
Concentration (%)
Formula
Formula 0 Formula 1 Formula 2 Formula 3
Hydroxyapatite - 18 19 20
Sodium Benzoate 0.15 0.15 0.15 0.15
Benzoic Acid 0.1 0.1 0.1 0.1
Citrate Acid 0.8 0.8 0.8 0.8
Gelatin 19 19 19 19
Syrup 35 35 35 35
Menthol 1 1 1 1
Distilled water Ad 100 Ad 100 Ad 100 Ad 100
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Figure 2. SEM analysis of hydroxyapatite with magnification (a) 500x, (b) 1000x, (c) 1500x
CaCO3  CaO + CO2

Then the powder analyzed with XRF (X-Ray
Diffraction) to determined calcium oxide content. XRF
analysis obtained CaO content of 49.846%. In Tuna
Bones (Mutmainnah et al., 2017), the CaO content as
much as 62.31%, cow bones the CaO content of 31.48%
(Yuliana et al., 2017), and mackerel fish bones
(Anggresani et al., 2018) as 50.814%. In this study has
smaller CaO content from the previous studies. It is
because when calcination occurs there is an organic
material degradation process (Mutmainnah et al., 2017).
The synthesis of hydroxyapatite
Figure 1. XRD analysis of hydroxyapatite
Hydroxyapatite synthesis was made by calcium
precursor and phosphate precursor in precipitation XRD analysis was conducted to see the
methods. In this study, the calcium precursor is CaO composition of atoms in a crystalline material so that it
from mackerel fish bones and the phosphate precursor is can be known the structure, orientation and size of
(NH4)2HPO4. Precipitation methods is an alkaline acid crystals (Munasir et al., 2012). The crystal size of the
reaction that produces crystalline solids as well as water. XRD analysis can be known using the Scherrer method,
This process is simpler, using cheap raw materials and the crystal size in this synthesis was 77.47 nm. Products
homogeneity (Haris et al., 2016). that have a low FWHM will result in a larger crystal size
In synthesis, CaO from mackerel fish bones was (Warna et al., 2015).
dissolved in distilled water to get Ca(OH)2 calcium SEM analysis was conducted to look at the
hydroxide. Reactions that occur as follows: morphology of particle surfaces of hydroxyapatite
CaO + H2O  Ca(OH)2 compounds. SEM analysis was done at magnification
500x, 1000x and 1500x (Figure 2).
Further, to synthesize hydroxyapatite, needed
This study with a mole ratio of Ca/P 1.67 particles
phosphate as a source of phosphate. In this study, we
formed granular like a sphere with an even particle
used diammonium hydrogen phosphate (NH4)2HPO4
distribution and the particle size is in the range of 0.1 to
with a mole ratio of Ca/P 1.67. Hydroxyapatite with this
0.3 μm. Particles shaped like spheres with a size of 20 –
mole ratio has the same crystalline arrangement as
30 μm will form at pH 10, while most hydroxyapatite
animal/human bones (Rana et al., 2017). The reaction
synthesized at pH 8 are needle-like with a length of 0.25
that occurs in the process of hydroxyapatite synthesis is:
μm (Wang et al., 2010), but if there is the interaction
10Ca(OH)2 + 6(NH4)2HPO4  Ca10(PO4)6(OH)2 + between substances, it can affect the crystal morphology
6H2O + 12 NH4OH of a substance (Fadhila et al., 2020).
The hydroxyapatite was analyzed using X-Ray Using calcination temperatures of 800°C and
Diffraction (XRD). High intensity was found in 2Ꝋ: 1,000°C for 5 hours impacts the morphological changes
32.58; 33.71; 33.14; 23.13; and 31.82 where the peak of of hydroxyapatite particles that bond between particle
this 2Ꝋ was in accordance with ICSD standard of granules and form irregular rods (Setiawan & Basit,
hydroxyapatite No. 96-900-3549 (Figure 1). 2012). The particle size obtained is 0.19433 μm, which
is in the range of 0.1 to 0.3 μm. This particle size is
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almost the same as the previous research conducted by Jelly candy without hydroxyapatite has a very
researchers who obtained particle sizes of 0.127 - 0.538 striking red color, for jelly candy that uses
μm with an average of 0.396 μm (Siregar & hydroxyapatite has a pink color, this is due to the
Sulistyawati, 2019). addition of hydroxyapatite substances affect the color
Jelly candy evaluation quality of jelly candy (Figure 3). The addition of
a) Raw Material Inspection Results calcium carbonate to milk jelly candy affects the
Examination of raw materials carried out Brightness of milk jelly candy (Lesmana et al., 2008)
organoleptic and solubility in hydroxyapatite as an which means the addition of calcium carbonate affects
active substance with white powder and practically the Brightness of milk jelly candy which is getting
insoluble in aquadest and ethanol 95%. Sodium murky due to insoluble calcium. The observation result
benzoate as a preservative, white in color, odorless and of organoleptic test present in Table 3.
soluble in aquadest. Benzoic acid as a preservative,
Table 3. Organoleptic test
hablur powder, colorless, and odorless and soluble in
The Observation Result
aquadest. Citric acid as a taster of acid, white powder, Formulation
Smell Color Taste Texture
odorless and very easily soluble in aquadest. Gelatin as Sweet Chewy
F0 Mint Red
a gel shaper and candy base, pale yellowish in color and Mint
soluble in hot water. Cocopandan syrup as a sweetener Pink Sweet Chewy
F1 Mint
Mint
and dye, red liquid, and menthol as a flavor giver and
Pink Sweet Chewy
mint aroma. F2 Mint
Mint
b) Jelly Candy Evaluation Sweet
F3 Mint Pink Chewy
Evaluation on Jelly Candy preparations was Mint
conducted to see which formula was best and meets the
requirements of Jelly Candy in the National Agency
Standard (SNI) No. 3547.2-2008 including weight
uniformity test, organoleptic test, pH test, hedonic test,
kapang khamir test, total plate number test, gel strength,
homogeneity test and stability test.
A weight uniformity test was conducted to see the
uniformity of the size of gummy candy preparations
made, all formulas of hydroxyapatite jelly candy
preparations 6 candies deviate from the condition of
weight uniformity that should not be more than 5%.
Factors that affect the uniformity of weights are shape,
print and temperature (Firdaus et al., 2014). In addition,
during the heating process causes the amount of water
that evaporates in each formula is not the same and at
the time of pouring printed or printing techniques. If the
diameter and thickness are not uniform it will affect the
number of doses of active substances (Syukri et al.,
2018). Weight uniformity test in all formulation
summarizes in Table 2.
Figure 3. Jelly candy hydroxyapatite fish bone
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Table 2. Weight uniformity test in all formulation

No F0 %D F1 %D F2 %D F3 %D
1 1.6021 5.5309%* 1.9212 4.5745% 1.7285 4.7133% 1.9934 8.6084%*
2 1.6594 2.1522% 1.9242 4.4255% 1.7685 2.5082% 1.9647 7.0447%*
3 1.6675 1.6746% 1.9133 4.9669% 1.7866 1.5104% 1.8205 0.8118%
4 1.6369 3.4789% 1.9937 0.9735% 1.7743 2.1888% 1.8421 0.3650%
5 1.6325 3.7384% 1.9258 4.3460% 1.8216 0.4189% 1.8737 2.0867%
6 1.6028 5.4897%* 1.9649 2.4040% 1.8525 2.1223% 1.8174 0.9807%
7 1.6203 4.4878% 2.0653 2.5828% 1.7039 6.0694%* 1.8252 0.5557%
8 1.6228 4.3103% 2.0227 0.4668% 1.8645 2.7839% 1.8058 1.6127%
9 1.7554 3.5084% 2.0562 2.1308% 1.8223 0.4575% 1.8347 0.0381%
10 1.6664 1.7394% 2.0445 1.5496% 1.7851 1.5931% 1.8093 1.4220%
11 1.7098 0.8196% 2.0164 0.1539% 1.7627 2.8280% 1.8694 1.8524%
12 1.6817 0.8373% 2.0586 2.2500% 1.8951 4.4707% 1.8139 1.1714%
13 1.7474 3.0367% 2.0518 1.9122% 1.7317 4.5369% 1.8240 0.6211%
14 1.7140 1.0672% 2.0403 1.3410% 1.8611 2.5964% 1.8764 2.2338%
15 1.6892 0.3950% 2.0972 4.1672% 1.8436 1.6328% 1.8043 1.6944%
16 1.7081 0.7193% 2.0458 1.6142% 1.7583 3.0705% 1.8341 0.0708%
17 1.7414 2.6829% 2.0813 3.3775% 1.8087 0.2921% 1.8217 0.7464%
18 1.6485 2.7949% 2.0099 0.1688% 1.8503 2.0011% 1.8065 1.5745%
19 1.7941 5.7904%* 1.9156 4.8527% 1.8360 1.2127% 1.8173 0.9861%
20 1.7546 3.4612% 1.9824 1.5347% 1.8171 0.1708% 1.8273 0.4413%
21 1.6654 1.7984% 2.0436 1.5049% 1.8437 1.6372% 1.8176 0.9698%
22 1.7446 2.8716% 2.0538 2.0116% 1.8382 1.3340% 1.8007 1.8905%
23 1.7655 4.1040% 2.0111 0.1092% 1.8071 0.3803% 1.8325 0.1580%
24 1.7196 1.3974% 2.0788 3.2533% 1.8749 3.3572% 1.8089 1.4438%
25 1.7344 2.2701% 2.0470 1.6738% 1.8468 1.8081% 1.8263 0.4958%
26 1.7673 4.2101% 2.0081 0.2582% 1.8215 0.4134% 1.8223 0.7137%
27 1.7438 2.8244% 2.0354 1.0977% 1.8067 0.4024% 1.8150 1.1114%
28 1.7408 2.6475% 1.9817 1.5695% 1.8156 0.0882% 1.8139 1.1714%
29 1.6993 0.2004% 2.0933 3.9735% 1.8285 0.7993% 1.8173 0.9861%
30 1.6427 3.1369% 1.9138 4.9421% 1.8642 2.7673% 1.8260 0.5121%
Avera 1.6959 ± 2.0133 ± 1.8140 ± 1.8354 ±
ge 0.0544 0.0574 0.0462 0.0435
Note : F0 : Formulation jelly candy without hydroxyapatite
F1 : Formulation jelly candy with hydroxyapatite 18%
%D : % deviation
*
: Deviation > 5%
The test results of each hydroxyapatite jelly candy 8

formula have a pH value range between 4.8 - 6.7 (Figure 6
4). The pH test is conducted to determine the pH value
pH
4
of the preparations that have been made. pH testing is
2
important to know the acidity level of a product because
0
acidity levels can affect consumer preference (Susanti et F0 F1 F2 F3
al., 2019). Formulation
pH testing in F1 obtained a more acidic pH
compared to formulas that use hydroxyapatite, this is Figure 4. pH test of jelly candy hydroxyapatite
thought to be influenced by hydroxyapatite, because mackerel fish bone
hydroxyapatite produced is made in a state of pH 12
The hedonic test due to observe the respondent’s
which means more alkaline and can increase the pH
preference in jelly candy hydroxyapatite mackerel fish
value in hydroxyapatite jelly candy. The pH value of
bone. The hedonic test in smell indicated that F2 has the
jelly candy is pH 4.3 to pH 6 (Lees, 1973), which means
highest percentage in like jelly candy around 40%. On
that hydroxyapatite jelly candy preparations enter the
the other hand, F1 has the most significant percentage in
criteria.
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dislike jelly candy hydroxyapatite, about 30% (Figure limit of the time according to SNI 3547.2-2008 (Badan
5). Standarisasi Nasional, 2008).
Figure 7. The result of hedonic test in taste

Figure 5. The result of the hedonic test in smell
Based on the result of hedonic test in color, the
really like respondents were in F0 (formulation without
hydroxyapatite) and the middle like respondents of the
jelly candy were in F2 and F3 about 40%. The more
hydroxyapatite added in the formulation, the jelly candy
become soft pink and not eye-catching anymore like F0.
However, the percentage of respondents dislike jelly
candy hydroxyapatite were in F1 (Formulation 1)
around 20% (Figure 6). Figure 8. The result of hedonic test in texture
Regarding the results of the test of khamir time, it
turns out that the sample produced the number of
colonies that met the limit of khamir cup contamination
on jelly candy preparations. Based on SNI, a jelly candy
was safe if the total contamination of khamir time is not
more than 1 x 102 colonies/g and the maximum ALT is
5 x 104 colonies/g, while in the test results of weak
contamination and ALT is in the range of 1 x 101 to 2 x
101 and for ALT ranges from 2x101 to 8.5 x 102 then
Figure 6. The result of hedonic test in color from the test results obtained shows that the preparation
Regarding this result of hedonic test in taste (Figure of hydroxyapatite jelly candy was not dangerous to be
7), the respondents like the taste of formulation 3 (F3) consumed because it does not exceed the standard limit
around 40%. However, the taste of jelly candy that the of SNI 3547.2-2008 (Badan Standarisasi Nasional,
respondents disliked was in F1 and F2, about 40%. 2008). Based on gel strength test results, the more
Meanwhile, the really like respondents in the texture of hydroxyapatite added the higher the strength of the gel
jelly candy (Figure 8) were in F0 by 40% and the dislike (Table 5).
respondents was in F2 and F3 around 30%. Based on the Table 5. Gel strength test result
result of hedonic test, F0 was the best formulation in
No Formula Gel Strength (N)
color, taste, and texture. 1. F0 0.2173 N
The test of the kapang khamir test and total plate 2. F1 1.8689 N
figures (Table 4) were obtained that all formulas of 3. F2 5.0647 N
hydroxyapatite jelly candy are still below the standard 4. F3 6.3446 N
Table 4. Kapang Khamir Test and Total Plate Number Test Result
Kapang Khamir Result Total Plate Number Test
Formula
Test Result (koloni/g) Standard (koloni/g) Test Result (koloni/g) Standard (koloni/g)
F0 0 1 x 102 2 x 101 5 x 104
F1 1 x 101 1 x 102 10 x 101 5 x 104
2 1
F2 0 1 x 10 3 x 10 5 x 104
1 2 1
F3 2 x 10 1 x 10 85 x 10 5 x 104
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The addition of different calcium concentrations mentions the addition of calcium has an effect on the
may increase the strength value of the gel (Dewi et al., hardness of jelly candy (Lesmana et al., 2008).
2020). Increased moisture content can decrease The addition of calcium concentration impacts the
hardness, where water will diffuse into the gel gel's strength (Dewi et al., 2020), and the moisture
(Muhandri & Subarna, 2009). So that the gel formed content increases the gel's hardness (Muhandri &
becomes softer and causes the hardness to decrease. Subarna, 2009). In this study, formulas 2 and 3 have less
A homogeneity test (in Figure 9) is done to find out moisture content than the formula 0 and 1, causing the
whether the spread evenly or not hydroxyapatite in jelly jelly candy to harden on the 7th and 21st days. For the
candy. The test results were obtained that taste of hydroxyapatite jelly candy obtained changes on
hydroxyapatite is distributed evenly in jelly candy even the 14th and 21st days in formulas 2 and 3; the change is
though jelly candy. the decrease in the taste of mint in preparations that
cause the taste of sweet candy only; this is thought to be
due to the addition of highly relevant hydroxyapatite
resulting in the fading of mint on preparations on days 2
- 14 and 21. On the pH examination of hydroxyapatite,
jelly candy obtained a stable pH value at pH 4 to 6.
The storage test of jelly candy hydroxyapatite was
observed in the stability of the formulation, which stored
at room temperature for 3 weeks and tested every week;
where the observations consisted of several parameters
are, including organoleptic test color, smell, texture and
taste) and pH test (Figure 10). This is examined to
guarantee that the jelly candy before and after the
preparation was still in good parameters during the
storage time. Based on Table 6, the changes occurred
from the smell, texture and taste after the 7th day until
the 21st day. However, there were no differences in the
Figure 9. Homogeneity test color from the 0 day until the 21st day. Formulations 2
Jelly candy hydroxyapatite conducted observations and 3 were altered on the 7th day and 21st day,
stored at room temperature for three weeks, the testing respectively, due to the more hydroxyapatite quantity in
of which included organoleptic examination of the taste, this formula. This research is in accordance with
color, aroma, texture, and pH of jelly candies. Lesmana et al. (2008), who stated that the addition of
Hydroxyapatite jelly candy changes every week. calcium affects the hardness of jelly candy.
Storage of jelly candy changes on the 7th to 2nd day,
where the change occurs in aroma, shape and taste while
for the color in the jelly candy hydroxyapatite does not
change from the 0th day to the 21st day. On the 14th day
the aroma of mint begins to fade because the mint
derived from menthol evaporates with increasing days.
Formula 2 and formula 3 on the 7th and 21st days undergo
changes in texture, this is because formulas 2 and 3 have
more hydroxyapatite additions compared to formula 1.
This is in line with the research that has been done that
Figure 10. Storage test in pH
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Table 6. Storage test in organoleptic

Day
Temperature Observation Formulation
0 7 14 21
Color F0 Red Red Red Red
F1 Pink Pink Pink Pink
Smell F0 Mint Mint Mint Syrup
Room Temperature
F1 Mint Mint Mint Syrup

F2 Mint Mint Syrup Syrup
F3 Mint Mint Syrup Syrup
Texture F0 Chewy Chewy Chewy Chewy
F1 Chewy Chewy Chewy Chewy
Taste F0 Sweet Mint Sweet Mint Sweet Mint Sweet Mint
F1 Sweet Mint Sweet Mint Sweet Mint Sweet Mint
F2 Sweet Mint Sweet Mint Sweet Sweet
F3 Sweet Mint Sweet Mint Sweet Sweet
CONCLUSION Karagenan. Cerata Jurnal Ilmu Farmasi; 9; 26–

Based on the results of this study, we can conclude 38.
that Hydroxyapatite Mackerel Fish Bone can be made as Anggresani, L., Perawati, S. & Rahayu, I. J. (2018).
Jelly Candy, with the best concentration of Limbah Tulang Ikan Tenggiri (Scomberomorus
Hydroxyapatite was 18%. guttatus) sebagai Sumber Kalsium pada
Pembuatan Hidroksiapatit. Jurnal Katalisator; 4;
AUTHOR CONTRIBUTIONS 153–161.
Conceptualization, L. A., S. P., R. A., R.; Anggresani, L., Sari, Y. N. & Rahmadevi, R. (2021).
Methodology, L. A.; Validation, L. A., S. P., R.; Formal Hydroxyapatite (HAp) from Tenggiri Fish Bones
Analysis, L. A., S. P., R. A., R.; Investigation, L. A., S. as Abrasive Material In Toothpaste Formula.
P., R. A., R.; Resources, L. A., S. P., R. A., R.; Data Jurnal Kimia Valensi; 7; 1–9. doi:
Curation, L. A., S. P., R. A., R.; Writing - Original Draft, 10.15408/jkv.v7i1.19165.
L. A., S. P., R. A., R.; Writing - Review & Editing, L. Noviyanti, A. R., Haryono & Rinal, P. D. R. E. (2017).
A., S. P., R. A., R.; Visualization, R.A.; Supervision, L. Cangkang Telur Ayam sebagai Sumber Kalsium
A., S. P.; Project Administration, L. A., S. P., R. A., R.; dalam Pembuatan Hidroksiapatit untuk Aplikasi
Funding acquisition, L. A., S. P., R. A., R. Graft Tulang. Chimica et Natura Acta; 5; 107–
111.
CONFLICT OF INTEREST Badan Standarisasi Nasional. (2008). Kembang gula –
The authors declared no conflict of interest. Bagian 2: Lunak. Jakarta: Badan Standarisasi
Nasional.
REFERENCES Budiatin, A. S., Khotib, J., Hasmono, D. & Samirah.
Ahmad, I. (2017). Pemanfaatan Limbah Cangkang (2016). Injektabel Komposit Hydroksiapatit-
Kerang Darah (Anadara granosa) Sebagai Bahan Gelatin sebagai Sistem Penghantaran Alendronat.
Abrasif Dalam Pasta Gigi. Jurnal Galung Jurnal Farmasi dan Ilmu Kefarmasian Indonesia;
Tropika; 6; 49–59. doi: 10.31850/JGT.V6I1.210. 3; 1-6.
Alridho B, Ali, A. & Rossi, E.. (2017). Pembuatan Chadijah, S., Hardiyanti & Sappewali. (2018). Sintesis
Permen Jelly Ektrak Jahe Merah dengan dan Karakterisasi Hidroksiapatit dari Tulang Ikan
Penambahan Karagenan. Jom Faperta Ur; 4; 1– Tuna (Thunnus albacores) dengan XRF, FTIR,
13. dan XRD. Al-Kimia; 6; 178-184.
Andasari, S. D., Zukhri, S. & Nurjanah, P. (2018). Dewi, I. K., Wijayanti, I. & Kurniasih, R. A. (2020).
Formulasi Permen Jelly Bunga Turi (Sesbania Pengaruh Nanokalsium terhadap Kekuatan Gel
grandiflora L.) dengan Variasi Kadar Gelatin dan Kamaboko Ikan Mujair (Oreochromis
mossambicus). AgriTech; 40; 91–101.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Fadhila, M., Umar, S. & Zaini, E. (2020). Pembentukan Aplikasinya (JPFA); 2; 20-29. doi:
Kokristal Asam Usnat – N-Methyl-DGlucamine 10.26740/jpfa.v2n1.p20-29.
dengan Metode Penguapan Pelarut dan Mutmainnah, M., Chadijah, S. & Rustiah, W. O. (2017)
Pengaruhnya terhadap Penurunan Interleukin-8 Hidroksiapatit dari Tulang Ikan Tuna Sirip
pada Tikus Inflamasi. Jurnal Sains Farmasi & Kuning (Tunnus albacores) dengan Metode
Klinis; 7; 23-30. doi: 10.25077/jsfk.7.1.23- Presipitasi. Al-Kimia; 5; 119–126. doi:
30.2020. 10.24252/al-kimia.v5i2.3422.
Firdaus, F., Islamaya, W. & Fajriyanto, F. (2014) Rana, M., Akhtar, N., Rahman, S., Jamil, H. M. &
Formulasi Nutraseutikal Sediaan Gummy Candies Asaduzzaman, S. M. (2017). Extraction of
Sari Buah Belimbing Manis (Averrhoa carambola Hydroxyapatite from Bovine and Human Cortical
L) dengan Variasi Kadar Manitol dan Corn Syrup Bone by Thermal Decomposition and Effect of
sebagai Basis. Teknoin; 20; 1–11. doi: Gamma Radiation: A Comparative Study.
10.20885/teknoin.vol20.iss1.art5. International Journal of Complementary &
Hanura, A. B., Trilaksani, W. and Suptijah, P. (2017) Alternative Medicine; 8; 1-10. doi:
Karakterisasi Nanohidroksiapatit Tulang Tuna 10.15406/ijcam.2017.08.00263.
Thunnus sp Sebagai Sediaan Biomaterial. Jurnal Sunil, S., Sharma, U. K. & Arathy, S. A. (2020).
Ilmu dan Teknologi Kelautan Tropis; 53; 619– Pharmaceutical Jellies: a Novel Way of Drug
629. doi: 10.1017/CBO9781107415324.004. Delivery. Journal of Pharmaceutical Sciences and
Haris, A., Fadli, A. and Yenti, S. R. (2016). Sintesis Research; 12; 904–909.
Hidroksiapatit dari Limbah Tulang Sapi Setiawan, D. & Basit F, M. (2012). Sintesis dan
menggunakan Metode Presipitasi dengan Variasi Karakterisasi Hidroksiapatit Untuk Aplikasi
Rasio Ca/P dan Konsentrasi H3PO4. Jom Fteknik; Sinovektomi Radiasi. Jurnal Forum Nuklir; 6;
3; 1–11. 120-126. doi: 10.17146/jfn.2012.6.2.3338.
Hernawan, A. D., Anggresani, L. & Meirista, I. (2021). Shita, A. D. P. & Sulistiyani. (2010). Pengaruh Kalsium
Formulasi Pasta Gigi Hidroksiapatit dari Limbah terhadap Tumbuh Kembang Gigi Geligi Anak.
Tulang Ikan Tenggiri (Scomberomorus guttatus). Stomatognatic (J. K. G Unej); 7; 40–44.
Chempublish Journal; 6; 34–45. Siregar, R. F. and Sulistyawati, E. (2019). Karakteristik
Jauhari, M., Thonthowi & Santoso, S. A. (2019). Hidroksiapatit Porous dari Prekursor Cangkang
Asupan Protein dan Kalsium Serta Aktivitas Fisik Keong Sawah dan Bahan Porogen Pati Sukun.
pada Anak Usia Sekolah Dasar. Ilmu Gizi Eksergi; 16; 59-63. doi: 10.31315/e.v16i2.3082.
Indonesia; 2; 79–88. Stötzel, C., Müller, F. A., Reinert, F., Niederdraenk, F.,
Lees, R. (1973) Sugar Confectionary and Chocolate Barralet, J. E. & Gbureck, U. (2009). Ion
Manufacture. New York: Springer. Adsorption Behaviour of Hydroxyapatite with
Lesmana, S. N., Putut, T. I. & Kusumawati, N. (2008) Different Crystallinities. Colloids and Surfaces B:
Pengaruh Penambahan Kalsium Karbonat sebagai Biointerfaces; 74; 91–95. doi:
Fortifikan Kalsium terhadap Sifat Fisikokimia dan 10.1016/j.colsurfb.2009.06.031.
Organoleptik Permen Jeli Susu. Jurnal Teknologi Susanti, K. I. A., Tamrin & Asyik, N. (2019). Pengaruh
Pangan dan Gizi; 7; 28–39. doi: Penambahan Sari Jahe Gajah (Zingiber officinale)
10.33508/JTPG.V7I1.148. terhadap Organoleptik, Sifat Fisik dan Kimia
Mozartha, M. (2000). Hidroksiapatit dan Aplikasinya di dalam Pembuatan Permen Jelly Daun Katuk
Bidang Kedokteran Gigi. Journal of Visual (Sauropus androgynus). Jurnal Sains dan
Languages & Computing; 11; 287–301. Teknologi Pangan; 4; 2073–2085.
Muhandri, T. & Subarna. (2009). Pengaruh Kadar Air Syukri, Y., Wibowo, J. T. & Herlin, A. (2018).
NaCl dan Jumlah Passing terhadap Karakteristik Pemilihan Bahan Pengisi untuk Formulasi Tablet
Reologi Mi Jagung. Jurnal Teknologi dan Industri Ekstrak Buah Mahkota Dewa (Phaleria
Pangan; 20; 71-77. macrocarpa Boerl). JSFK (Jurnal Sains Farmasi
Munasir, Triwikantoro, T., Zainuri & Darminto. (2012). & Klinis); 5; 66–71. doi: 10.25077/JSFK.5.1.66-
Uji XRD dan XRF pada Bahan Meneral (Batuan 71.2018.
dan Pasir) sebagai Sumber Material Cerdas Wadu, I. et al. (2015). Pasta Gigi Pencegah Gigi
(CaCO3 dan SiO2). Jurnal Penelitian Fisika dan Berlubang Berbahan Aktif Mikro Hidroksiapatit
(Hap) dari Limbah Kerabang Telur Pasar Raya
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Kota Salatiga. Prosiding Seminar Nasional; 2018; Warna, N. S., Fadli, A. & Irdoni (2015). Sintesis
116–124. Hidroksiapatit dari Cangkang Telur dengan
Wang, P., Li, C., Gong, H., Jiang, X., Wang, H. & L, K. Metode Presipitasi. Jurnal Online Mahasiswa
(2010). Effects of Synthesis Conditions on the Fakulas Teknik; 2; 1–11.
Morphology of Hydroxyapatite Nanoparticles Yuliana, R., Rahim, E. A. & Hardi, J. (2017). Sintesis
Produced by Wet Chemical Process. Powder Hidroksiapatit dari Tulang Sapi dengan Metode
Technology; 203; 315–321. doi: Basah pada Berbagai Waktu Pengadukan dan
10.1016/j.powtec.2010.05.023. Suhu Sintering. KOVALEN: Jurnal Riset Kimia;
3; 201–210.
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DOI: 10.20473/jfiki.v9i32022.290-297
Physicochemical Properties and Antioxidant Activity of Three Types of

Monofloral Honey from Indonesia
Sulistyianingsih1, Achmad Toto Poernomo2, Riesta Primaharinastiti2*
1
2
*Corresponding author: r.nastiti@gmail.com

Abstract
Background: In addition to minerals, honey contains carbohydrates (glucose and fructose), protein, amino acids,
water, enzymes, ash, vitamins, and other substances. Compounds of honey can affect the chemical properties of
honey. Knowing the physicochemical properties of honey is very important because physicochemical properties
affect the quality of honey. One of the biological activities of honey is an antioxidant. Antioxidants can interfere
with oxidative processes, prevent disease, and play an important role in the body's defence system. Objective: to
determine and compare physicochemical properties (color, viscosity, ash content, water content, reducing sugar
(glucose), total phenolic compound, HMF) and antioxidant activities of monofloral honey samples from Indonesia.
Methods: The color of honey are categorized using the Pfund scale. Viscosity measurement is carried out using a
Brookfield viscometer. The water content is carried out using a refractometer. Phenolic content and antioxidant
activities analysis were carried out by UV-VIS spectrophotometer. Results: The results show that rambutan honey
from Malang has the highest physicochemical properties and antioxidant activity, which had an amber color,
water content of 21.7% b/b, acidity 20.7 mL NaOH/Kg, viscosity of 33.08 poise, ash content of 0.17% b/b, reducing
sugar 69.38%b/b, total phenolics content 533.7 mg GAE/Kg sample and IC50 0.111 µg/mL. Conclusion: The
quality of honey varies from region to region. The best honey (according to SNI) is rambutan honey from Malang.
Keywords: honey, physicochemical properties, antioxidant activity, total phenolics

Sulistyianingsih, Poernomo, A. T. & Primaharinastiti, R. (2022). Physicochemical Properties and Antioxidant
Activity of Three Types of Monoflora Honey from Indonesia. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia,
9(3), 290-297. http://doi.org/10.20473/jfiki.v9i32022.290-297
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION for cosmetic treatments (Suranto, 2004). Vallianoul et

Honey is a natural liquid, that is generally sweet al., (2014), stated that antioxidants in manuka honey
tasting and produced by bees (Apis sp.) from play a role in cleaning the skin, eliminating skin
floranectars or other parts of the plant (SNI, 2013). discoloration, and increasing skin elasticity. Research
Honey is widely circulated and produced in Indonesia, (Chayati & Isnatin, 2015) shows differences in the
and can be divided into monofloral and multifloral. antioxidant activity of monofloral honey originating
Monofloral honey is obtained from Apis cerana or Apis from Central Java, which is rambutan, kelengkeng,
mellifera bees with feed derived from one type of nectar coffee, randu, and calliandra honey, respectively, 11.9;
source. This honey is commonly named based on the 8.73; 5.56; 13.1 and 48.0%.
source of nectar, such as kelengkeng honey, rambutan Given the many benefits of honey to human health,
honey, randu honey, and other. Monofloral honey has a the purpose of this study is to identify and compare
specific aroma, flavor and color based on the source of physicochemical parameters (viscosity, color, ash
honey (Suranto, 2007). Results from several studies content, water content, reducing sugar (glucose),
showed that monofloral honey, which is randu honey, acidity, total phenolic content, Hydroxymethylfurfural
kelengkeng honey, and rambutan honey had different (HMF), and antioxidant activities of monofloral honey
physicochemical properties (Chayati, 2008). from Indonesia.
Honey contains not only minerals but also
carbohydrates, protein, water, ash, small of vitamins, MATERIALS AND METHODS
enzymes, amino acids, and other substances (Buba et al., Materials
2013). The mineral contents in honey are Cr, Na, Al, Ni, Randu honey from Kediri, kelengkeng honey from
Ca, K, Mg, Zn, Co, Cu, and Fe (Conti et al., 2014. The the cultivation of National Beekeeping Center
compounds in honey will affect physicochemical (Pusbahnas) Bogor, and rambutan honey from Malang,
properties of honey. Knowing the physicochemical DPPH (2,2-difenil-1-pikrilhidrazil) p.a (Sigma-
properties of honey is very important because Aldrich.), aquadest, gallic acid p.a (Sigma. Co.), Folin-
physicochemical properties affect the quality of honey. Ciocalteau (E. Merck), ethanol p.a (E. Merck),
Several areas in Indonesia are known to produce hydrochloric acid 37% (E. Merck), dan AlCl3 10% (E.
monofloral honey are Kediri, Malang and Bogor. Ceiba Merck).
pentandra (randu) honey, Dimocarpus longan Instruments
(kelengkeng) honey, and Nephelium lappaceum Spektrofotometer UV-Vis Shimadzu UV 260 and
(rambutan) honey are types of honey that are produced Brookfield DV3TLV digital viscometer.
continuously in Indonesia. Method
Empirically, honey has long been used as a Color analysis
component in traditional medicine in various countries. One hundred milliliter of honey was placed in a
One of the biological activities of honey is an clear glass jar with a bright enough light and compared
antioxidant. Natural antioxidants protect the body from with the standard. The colors of honey are categorized
the attack of free radicals, and can delay the onset of with the Pfund scale.The color scale are divided into
chronic diseases (Wahdaningsih et al., 2011). Based on seven levels, which are dark amber, amber, light amber,
RISKESDAS (2007), the prevalence of degenerative extra light amber, white, extra white, and water white
diseases such as heart disease, hypertension, stroke, (White, 1984).
tumors, and diabetes were 16.1; 53.7; 20.2; 8.8; and Viscosity
3.7%. Several studies have been done to determine the Viscosity measurements were carried out using the
antioxidant activity of honey against chronic diseases. Brookfield DV3TLV Digital Viscometer. The honey
Research by Saputra & Wulan (2015) proves that honey sample was put into a special container on the
with antioxidant activity can reduce the risk of chronic viscometer. The rotor is dipped in the honey sample.
obstructive pulmonary disease and lung cancer. The Ash content
antioxidant potential of manuka honey, a type of honey The cup that has been heated in the oven at 105°C
that has been registered as a wound care product, contain for 24 hours is cooled in a desiccator and weighed. Two
methyl syringate (a type of phenolic compound), which grams of honey were weighed and placed into the cup
is considered to be quite potential to interfere with the and weighed, then burned on a hot plate at 400°C until
process of amplification of inflammation by ROS smokeless and turn to ashes. Then put into the furnace
(Molan, 2011). Manuka honey is also commonly used at a temperature of 550°C for 6 hours, then removed and
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cooled in a desiccator. The cup was weighed, and the replicated twice (SNI, 2018). The results were expressed
weight was recorded. Ash content analysis was in mg HMF / 100 g honey using the following formula:
replicated twice (Sudarmadji, 2007). 𝑚𝑔 𝐴284 −𝐴336 𝑥 14,97 𝑥 5
HMF = ( 𝑔 𝑚𝑎𝑑𝑢) =
Water content 100 𝑏𝑜𝑏𝑜𝑡 𝑠𝑎𝑚𝑝𝑒𝑙 (𝑔)
The water content measurement was carried out by Acidity

a refractometer to read the index of refraction of a honey Acidity analysis was carried out by weighing 10
sample at a temperature of 20°C. The water content grams of honey, then dissolving with 75 mL of CO2-
analysis was replicated twice. The water content of free water in a 250 mL beaker, stirring with a stirring
honey is determined by comparing the refractive index rod and inserting a pH meter to record the pH, titrated
values of honey and water (SNI, 2018). with 0.05 M NaOH at a rate of 5.0 mL/min. The titration
Reducing sugar (glucose) was stopped when the pH reached 8.50. 0.05 M NaOH
Anhydrous Na2CO3 was weighed and dissolved in ± was taken with a 10 mL pipette and immediately titrated
300 mL of distilled water to make Luff's solution. Fifty with 0.05 M HCl to pH 8.30. In the blank test, 75 mL of
grams of citric acid was added in 50 mL of distilled CO2-free water was titrated with NaOH to pH 8.5.
water while stirring. Twenty-five grams of CuSO4.5H2O Acidity analysis was replicated twice (SNI, 2018).
was added and dissolved in 100 mL of distilled water. Total phenolics content
The solution was transferred to a 1000 mL volumetric Procedure for preparing a calibration curve: A
flask, and squeezed to the mark with distilled water, standard solution of gallic acid (H2O solvent) was
stored for 24 hours. Reducing sugar is determined by prepared with a concentration of 5 to 25 ppm. One mL
weighing 1.5 grams of honey and then putting it into a of each concentration was pipetted into a test tube. 0.5
500 mL erlenmeyer. 100 mL of 3% HCl was added and mL of Folin-Ciocalteu was added and left for 5 minutes,
heated for 3 hours in an upright cooler. Then cooled and and then 2 mL of 10% sodium carbonate solution was
neutralized using 30% NaOH solution and a little 3% added.
CH₃COOH. The solution was transferred to 500 mL Sample preparation: 0.5 g honey was weighed into
volumetric flask to volume and filtered. 10 mL of the a 25 mL volumetric flask and added water to the
filter results in a 500 mL Erlenmeyer, then added 25 mL measuring line. 1.0 mL aliquot was pipetted into the vial
of luff solution and 15 mL of distilled water. Heated for and 0.5 mL Folin-Ciocalteu was added. The mixture was
3 minutes, then cooled. 15 mL of 20% KI solution and incubated for 5 minutes followed by addition of 2 mL
25 mL of 25% H2SO4 were added. Titrated with 0.1 N 10% sodium carbonate. The mixture was further
sodium thiosulfate solution and added a small amount of incubated for 10 minutes followed by absorbance
0.5% starch solution. The same treatment was carried measurement of 770 nm. The total content of phenolic
out for the blanks. Glucose level analysis was replicated compounds was expressed as gallic acid equivalents (mg
twice. (SNI, 1992). GAE/Kg honey) using the standard curve for gallic acid.
HMF Content Total phenolics content analysis was replicated twice
The honey sample was weighed as much as 5 g (Alfian & Susanti, 2012).
and put into a 50 mL volumetric flask, then dissolved Antioxidant activity
with distilled water until the volume of the solution was One milliliter of the aqueous honey solution was
50 mL. The Carrez I solution and the Carrez II solution mixed with 1 mL DPPH ethanol. The mixture was left
were added as much as 0.50 mL, shaken and diluted with in the dark for 30 minutes, and the absorbance was
aquadest to the line mark. Added a drop of alcohol to spectrophotometrically read at 519 nm. Ascorbic acid
remove the foam on the solution’s surface. The solution was used for calibration, and the results were expressed
was filtered and the first 10 mL of the filter was as mmol of ascorbic acid per Kg of honey. Antioxidant
discarded. The filter results were pipetted as much as activity analysis was replicated twice. The antioxidant
5 mL and put into 18 mL x 150 mL test tube. 5 mL of activity of honey can be read from the IC 50 value. The
aquadest was pipetted and put into a tube for the sample IC50 value is the sample concentration value to measure
solution and as a comparison, solution 5 mL of 0.20% the ability of the sample to consider its antioxidant
sodium bisulfate was added, then homogenized until activity to be 50% free radical. Antioxidant activity
completely mixed, and the absorbance of the sample analysis was replicated twice (Chayati & Isnatin, 2015).
against the comparison was determined at a wavelength
of 284 nm and 336 nm. HMF level analysis was
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RESULTS AND DISCUSSION viscosity) can also cause a faster fermentation process
Honey color which can change the taste of honey to become sour
Using the Pfund scale, honey can be categorized (Apriani, 2013).
into dark amber, amber, light amber, extra light amber, Ash Content
white, extra white, and water white (Pontis et al., 2014). Ash content is a mixture of inorganic or mineral
Analysis of the test results for honey color parameters in components in a food ingredient. If the ash content is
Table 1 and Figure 1 shows that monofloral honey high, the mineral content is also high. Testing the ash
(randu, kelengkeng and rambutan) has a color from content in honey needs to be done to determine the total
amber to extra light amber. The color of honey consists mineral content of honey because each honey has a
of water-soluble and fat-soluble fractions. Brightly different mineral content. It depends on the source of
colored honey has less water soluble than fat-soluble soil and nectar around the bees (Sihombing, 2005). The
honey. The existence of an oxidation process causes a honey quality requirement for ash content based on SNI
color change in honey (Adriani, 2011). The dye's 8664:2018 is a maximum 0.5% w/w. The higher the ash
persistence is caused by a mixture of several amino acids content of the sample, the higher the mineral content of
with iron from packaging or processing equipment the honey (Qadar et al., 2015).
(Sihombing, 2005). In this research, rambutan honey As a result of ash analysis, Randu honey from
was darker than randu and kelengkeng honey. Indonesia Kediri, kelengkeng honey from the cultivation of the
has a very diverse vegetation that blooms regularly. This National Beekeeping Center (Pusbahnas) Bogor, and
enables beekeepers to gather various single or multi- rambutan honey from Malang, have the appropriate ash
flowered honey in different colors. Previous studies content with the standard set by SNI 8664:2018 which
have shown that transition metals react with organic are 0.17; 0.10; 0.17 %w/w. This matter indicates that the
compounds in honey to form colorful complexes. The mineral content in those areas is still quite good because
darker honey color indicates higher total phenolic still in accordance with the standards that have been set.
content and antioxidant activity (Harris, 2014). Water content
The results of the water content analysis show that
each type of honey from different regions has different
water content. The results show that kelengkeng honey
from Bogor has moisture of 19% w/w. The reason is that
the environmental temperature in the Bogor area is
higher by 31.9°C (Badan Pusat Statistik, 2021), so
Figure 1. Color of (A) randu; (B) kelengkeng and (C) honey in that area has low hygroscopic properties.
rambutan honey Honey in this area is following with the water content
Viscosity standard in SNI 8664:2018. Therefore, honey in the
The viscosity requirement based on SNI Bogor area has good quality. Good quality honey is
(Indonesian National Standard) is minimum 10 poise. honey that contains water, about 17 - 21% (Sihombing,
The value of test results for viscosity parameter (Table 2005).
1) randu honey, kelengkeng and rambutan was 4.85, 75, The water content of randu honey from Kediri is
and 3.31 poise. The viscosity of rambutan and 27.30%. These results indicate that the water content in
kelengkeng honey the label has a viscosity value this area is slightly higher than the honey quality
according to SNI standards. The viscosity of honey can requirements in SNI 8664:2018. This can be influenced
be affected by temperature and water content. The by the environmental temperature in Kediri which is
higher the water content, the higher the liquid content of around 22.5°C (Badan Pusat Statistik, 2020). The water
honey, and the lower the water content, the higher the content of rambutan honey from Malang has a water
density of honey. The viscosity of randu honey from content of 21.7%. The results of the water content in this
Kediri is lower than the SNI standard. . This is because area are in accordance with the water content standard
the water content in randu honey from Kediri is higher in SNI 8664:2018. This is because Malang has an
than kelengkeng and rambutan honey. Honey with high altitude of 498.48 above sea level and an environmental
water content is very susceptible to fermentation temperature of around 22.38°C (Badan Pusat Statistik,
because water can stimulate the growth and 2021). Low temperatures cause honey to absorb more
development of yeast cells. The condition of honey that water (Evahelda et al., 2017). Therefore, the lower the
has a higher water content or is more dilute (lower
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ambient temperature, the higher the moisture content in of reducing sugar (glucose) (Prica & Balos, 2014).
honey. Storage at high humidity also affects the acidity of
In addition, the level of maturity of honey that has honey (Savitri, et al., 2017). In addition, high acidity in
not been perfect also affects the water content (Savitri, honey can also be caused by unhygienic post-harvest
et al., 2017). This is in accordance with the taking of processing, which can lead to honey being easily
honey in Kediri, which is not in accordance with the contaminated. It is very important to pay attention to the
harvest time so that the maturity is not perfect. acidity level of honey to keep honey hygienic and safe
Generally, the honey harvest time that has been for consumption (Karnia et al., 2019).
determined is at 11-12 days marked by a nest covered HMF Content
with beeswax (Fatma, et al., 2017). HMF content analysis has been performed as a
Reducing sugar (glucose) measure of honey quality and has served as a reference
Reducing sugar is an important parameter to for some studies to determine the authenticity of honey.
determine the quality of honey. Honey has two The HMF value is an indicator for measuring the
important components, namely sugar and water. freshness of honey, the heating process, and the shelf
However, two types of sugar are more dominant, life. If the honey is stored for too long, HMF levels will
glucose and fructose as much as 70 - 80% and water 10 increase (Suranto, 2004). This is because the C atoms of
- 20% (Evahelda et al., 2017). The results for the glucose, fructose, and monosaccharides decompose
analyzed level of reducing sugar (glucose) honey can be when heated and undergo levulinic oxidation to formic
seen in Table 1. Requirements for reducing sugar acid (Anjana, 2014). The permissible HMF level for
content based on SNI are at least 65% w/w. The honey is 40 mg/Kg according to Indonesian National
reducing sugar content produced in this study from Standard (SNI 01-8664-2018). Based on the presented
randu honey, kelengkeng, and rambutan were 63.74; results Table 1 HMF content of kelengkeng honey
72.36 and 69.53%b/w. These results stated that (27.86 mg/Kg) and rambutan honey (36.59 mg/Kg) were
kelengkeng and rambutan honey complied with the in accordance with the mentioned standards. Higher
requirements for glucose-reducing sugar levels. Randu levels of HMF (76.75 mg/Kg) have been found in randu
honey with a value of 63.74% w/w does not meet the honey, and this significant increase can be attributed to
requirements for reducing sugar content because it is inferior products due to overheating, improper storage,
less than 65%/w. Alcohols reacting with oxygen can or the addition of inverted sugar syrup.
form reactions with acetic acid. The formation of acetic Excessive heating can cause HMF levels to increase
acid can cause an increase in acidity in honey (Kuntadi, (Minarti et al., 2016). High levels of HMF in honey will
2013). The different types of plants that are a food reduce honey quality because the HMF content is related
source for bees to produce honey will affect the to several other chemical characteristics of honey, such
characteristics of honey, such as taste, aroma, color, as water content, pH, free acid content, reducing sugar
quality, and sugar content in honey (Mulu et al., 2004). content, and enzymatic activity in honey (Kowalski et
Acidity al., 2013). HMF levels increased during the heating
Based on SNI 8664:2018 the maximum acidity process, with a decrease in water content, reducing
value is 50 mL NaOH/Kg. The results of the acidity test sugar, diastase enzyme activity, and increased free acid
on some honey samples Table 1 showed that kelengkeng levels.
and rambutan honey have acidity levels of 11.63 and Total phenolics content
20.67; mL NaOH/Kg that had the acidity value Total phenol content was determined using the
according to SNI standards. Kelengkeng and rambutan Folin-Ciocalteu method and standard gallic acid by UV-
honey have good quality because it indicates that Vis spectrophotometry. Gallic acid plays an important
microbes will not grow in the honey. This can be seen in role, so we use it as a comparative substance because it
the slightly thick honey texture (Savitri, et al., 2017). has a heteropolymer with three hydroxy phenol groups.
Meanwhile, randu honey, has high acidity values, 70.0 Phenolic hydroxy groups will be oxidized by the Folin-
mL NaOH/Kg. These results are not following SNI Ciocalteu reagent under alkaline conditions. Folin-
8664:2018. High acidity values can be affected by the Ciocalteu reagent will oxidize gallic acid in its phenolic
water content of honey. Honey is acidic and has a high hydroxy group to form a molybdenum-tungsten
water content which will increase fermentation. complex with a blue color (Alfian & Susanti, 2012).
Increasing the fermentation process can produce an
increasingly sour taste of honey and decrease the value
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Table 1. Analysis results of physicochemical properties and antioxidant activity

Antioxidant
Physicochemical properties
Honey Activity
Samples Ash Water HMF Phenolic
Color Viscosity Glucose Acidity
content content
Randu Light 4.85 0.17 ± 27.3 ± 63.74 ± 70.0 ± 76.75 ± 465.9 ± 0.096 ±
amber 5.6 2.1 4.5 4.8 1.6 7.3 2.1
Kelengkeng Light 72.2 0.10 ± 19.0 ± 72.35 ± 11.6 ± 27.86 ± 272.4 ± 0.251 ±
amber 0.14 2.1 3.7 0.2 3.8 45.7 8.8
Rambutan Amber 33.08 0.17 ± 21.7 ± 69.38 ± 20.7 ± 36.59 ± 533.7 ± 0.111 ±
1.0 0.9 1.2 5.4 0.2 14.0 2.7
Note: The data represent the mean ± RPD (Relative Percent Different) data of the two replications
Total phenolic content is influenced by greater the antioxidant activity (Shahwar et al., 2010).
environmental factors such as light, rainfall, soil Capacity of phenolic antioxidants is affected by several
nutrients, altitude, and humidity. Besides that, total factors, one of which is the functional group associated
phenolic content is also affected by the cultivation with the main structure. The study by Mohsen & Ammar
process, such as fertilization, irrigation, and post-harvest (2009), showed that the radical scavenging activity
treatment (Malinikova et al., 2013). The results in this tested on Phenolics is related to the number and position
study showed that total phenolic compounds in of hydroxyl group bonds (OH) in the molecule. The
rambutan honey (533.7 mg GAE/Kg honey) were higher more hydroxyl groups are substituted in the molecule,
than in randu honey and kelengkeng honey were 465.9 the stronger the antioxidant capacity becomes because
and 272.4 mg GAE/Kg honey (Table 1). According to more hydrogen atoms can be generated (Yu Lin et al.,
Ferreira et al. (2009), dark honey was richer in phenolic 2009).
compounds, and this was also confirmed in our study.
Phenolic compounds are associated with antioxidant CONCLUSION
activity. Plants that have high phenolic compounds also The quality of honey varies from region to region.
have high antioxidant activity. Phenolic compounds The best honey (according to SNI) is rambutan honey
protect antioxidants because phenolic compounds can from Malang has the highest physicochemical properties
scavenge the action of free radicals and react with and antioxidant activity and has an amber color, water
reactive oxygen species (ROS) so that they no longer content of 21.7% b/b, acidity 20.7 mL NaOH/Kg,
damage cells in the human body. viscosity 33.08 poise, ash content 0.17 %b/b, reducing
Antioxidant Activity sugar 69.38 % b/b, total phenolics content 533.7 (mg/Kg
Antioxidant activity was determined by the DPPH GAE) and IC50 0.111 ppm.
method with a UV-Vis spectrophotometer. DPPH is a
molecule containing unstable nitrogenous radicals that ACKNOWLEDGMENT
can bind with hydrogen ions, so it was used to test The authors would like to deliver to gratitude to
antioxidant activity. The presence of antioxidant Indonesia Endowment Found for Education (LPDP)
compounds in the sample caused a color change of the Scholarship from the Ministry of Finance of the
methanol DPPH solution, which was initially dark Republic of Indonesia. for the financial support.
purple to pale yellow. This color change occurs because
DPPH is reduced, leading to electrons becoming paired AUTHOR CONTRIBUTIONS
(Zuraida et al., 2010). Conceptualization, R. P.; Methodology, S., R. P., A.
In testing the antioxidant activity Table 1, IC50 T. P.; Sotfware, A. T. P.; Validation, S.; Formal
values of randu, kelengkeng and rambutan were 0.095; Analysis, S.; Investigation, S.; Resources, S.; Data
0.240 and 0.109 ppm. Kelengkeng honey from Bogor Curation, S., R. P., A. T. P.; Writing - Original Draft, S.;
showed the highest DPPH radical scavenger activity Writing - Review & Editing, R. P., A. T. P.;
compared with randu honey from Kediri and rambutan Visualization, R. P.; Supervision, R. P.; Project
honey from Malang. Pointis et al, (2014) demonstrated Administration, R. P.; Funding acquisition, R. P.
a positive relationship between phenol concentration,
antioxidant capacity and color of monoflora honey. The CONFLICT OF INTEREST
higher the concentration of phenolic compounds, the The authors declared no conflict of interest.
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REFERENCES Honey for Identification of The Botanical Origin.

Alfian, R. & Susanti, H. (2012). Penetapan Kadar Acta Scientiarum Polonorum Technologia
Fenolik Total Ekstrak Metanol Kelopak Bunga Alimentaria; 12; 51-59.
Rosella Merah (Hibiscus sabdariffa Linn) dengan Kuntadi. (2013). Pengaruh Umur Larva terhadap
Variasi Tempat Tumbuh Secara Spektrofotometri. Potensi Kualitas Ratu yang Dihasilkan pada
Jurnal Ilmiah Kefarmasian; 2; 73-80. Penangkaran Lebah Ratu Apis cerana L.
Apriani. (2013). Studi Tentang Nilai Viskositas Madu Hymenoptera: Apidae dengan Teknik Grafting.
Hutan dari Beberapa Daerah di Sumatera Barat Jurnal Entomologi Indonesia; 10; 1-6.
untuk Mengetahui Kualitas Madu. Pillar Of Malinikova, E., Kukla, J., Kuklová, M. & Balazova, M.
Physics Journal; 2; 91-98. (2013). Altitudinal Variation of Plant Traits:
Badan Standardisasi Nasional (SNI). (2013). SNI Morphological Characteristics in Rosaceae
3545:2013. Madu. Jakarta: Badan Standardisasi (Fragaria vesca L.). Annals of Forest Research;
Nasional. 56; 79–89. doi: 10.1108/09533239510093215.
Badan Standardisasi Nasional (SNI). (2018). SNI Minarti, S. (2010). Ketersediaan Tepungsari dalam
8664:2018. Madu. Jakarta: Badan Standardisasi Menopang Perkembangan Anakan Lebah Madu
Nasional. Apis mellifera di Areal Randu (Ceiba pentandra)
Buba F, Gidado A & Shugaba A. (2013). Analysis of dan Karet (Hevea brasilliensis). Jurnal Ternak
Biochemical Composition of Honey Samples Tropika; 11; 54-60.
from North-East Nigeria. Biochemistry and Mohsen, S. M. & Ammar, A. S. M. (2009). Total
Analytical Biochemistry; 2; 1-7. doi: Phenolic Contents and Antioxidant Activity of
10.4172/2161-1009.1000139. Corntassel extracts. Journal of Food Chemistry;
Chayati, I. (2008). Sifat Fisikokimia Madu Monoflora 112; 595–598.
dari Daerah Istimewa Yogyakarta dan Jawa Molan, P. C. (2011). The Evidence and the Rationale for
Tengah. Agritech; 28; 9-14. the Use of Honey as Wound Dressing. Wound
Chayati, I. & Isnatin, M. (2015). Kajian Kadar Practice and Research; 19; 204-220.
Flavonoid, Aktivitas Antioksidan, dan Kapasitas Mulu, A., Tessema, B. & Derbie, F. (2004). In Vitro
Antioksidan Madu Monoflora. Mgmi; 6; 11-24 Assesment of the Antimicrobial Potential of
Conti, M. E., Maria, G. F., Luc, F., Giustino, M., Honey on Common Human Pathogens. The
Francesco, B. & Ivo, L. (2014). Characterization Ethiopian Journal of Health Development; 18;
of Argentine Honeys on The Basis of Their 107-112.
Mineral Content and Some Typical Quality Pontis, J.A., Costa, L.A.M.A.D., Silva, S.J.R.D. &
Parameters. Chemistry Central Journal; 8; 1-10. Flach, A. (2014). Color Phenolic and Flavour
Evahelda, E., Filli, P., Nura, M. & Budi, S. (2017). Sifat Content and Antioxidant Activity of Honey from
Fisik dan Kimia Madu dari Nektar Pohon Karet di Roraima Brazil. Journal of Food Science and
Kabupaten Bangka Tengah, Indonesia. Agritech; Technology; 34; 69-73.
37; 363-368. doi: 10.22146/agritech.16424. Prica, N. & Balos, M. Z. (2014). Moisture ad Acidity as
Fatma, S. Haryanti & Agung, S. W. (2017) Uji Kualitas Indicator’s of The Quality of Honey Originating
Madu pada Beberapa Wilayah Budidaya Lebah from Vojvodina Region. Arhiv Veterinarske
Madu di Kabupaten Pati. Jurnal Biologi; 6; 58-65. Medicine; 7; 99-109.
Ferreira, I. C. F. R., Aires, E., Barreira, J. C. M. & Qadar, S., Noora, A. & Maminga. (2015). Karakteristik
Estevinho, L. M. (2009). Antioxidant Activity Of Fisika Kimia madu Hutan Desa Terasa. Jurnal
Portuguese Honey Samples: Different Techno; 4; 37-41.
Contributions of The Entire Honey and Phenolic RISKESDAS. (2007). Laporan Hasil Riset Kesehatan
Extract. Food Chemistry; 114; 1438-1443. doi: Dasar 2007. Jakarta: Badan Penelitian dan
10.1016/j.foodchem.2008.11.028. Pengembangan Kesehatan, Riset Kesehatan
Karnia, I., Hamidah, S. & Thamrin, G. A. R. (2019). Dasar, Departemen Kesehatan Republik
Pengaruh Masa Simpan Madu Kelulut (Trigona Indonesia.
sp) terhadap Kadar Gula Pereduksi dan Saputra, T. & Wulan, A. (2015). Madu ssebagai
Keasaman. Jurnal Sylva Scienteae; 2; 1094-1099. Pencegah Penyakit Paru Obstruksi Kronik.
Kowalski, S., Łukasiewicz, M. & Berski W. (2013). Majority; 5; 37-42.
Applicability of Physicochemical Parameters of
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Savitri, N. P. T., Hastuti, E. D. & Suedy, S. W. A. AntiInflammatory, Antibacterial and Antioxidant

(2017). Kualitas Madu Lokal dari Beberapa Properties. General Medicine; 2; 1 -5.
Wilayah di Kabupaten Temanggung. Buletin Wahdaningsih, S., Setyowati, E. P. & Wahyuono, S.
Anatomi dan Fisiologi; 2; 58-66. (2011). Aktivitas Penangkap Radikal Bebas dari
Shahwar, D., Ahmad, N., Ullah, S. & Raza, M. A. Batang Pakis (Alsophila glauca J . Sm). Majalah
(2010). Antioxidant Activities of the Selected Obat Tradisional; 16; 156–160.
Plants from the Family Euphorbiaceae, Lauraceae, White, J. W. (1984). Instrumental Color Classification
Malvaceae and Balsaminaceae. Journal of of Honey: Collaborative study. Journal of the
Biotechnology; 9; 1086–1096. doi: Association of Official Analytical Chemists; 67;
10.5897/AJB09.1622. 1129–1131.
Sihombing, D. T. H. (2005). Ilmu Ternak Lebah Madu. Yu, L. H., Kuo, Y. H., Lin., Y. L. & Chiang, W. (2009).
Yogyakarta: Gajah Mada University Press. Antioxidative Effect and Active Components
Sudarmadji, S. (2007). Analisa Bahan Makanan dan from Leaves of Lotus (Nelumbo nucifera).
Pertanian. Yogyakarta: Liberty. Journal of Agricultural and Food Chemistry; 57;
Suranto, A. (2004). Khasiat dan Manfaat Madu Herbal. 6623–6629. doi: 10.1021/jf900950z.
Jakarta: Agromedia Pustaka. Zuraida., S., Sajuthi, D. & Irma H. S. (2017). Fenol,
Suranto, Adji. (2007). Terapi Madu. Jakarta: Penebar Flavonoid, dan Aktivitas Antioksidan pada
Swadaya. Ekstrak Kulit Batang Pulai (Alstonia scholaris R.
Vallianoul, N. G., Gounaril, P., Skourtis, A., Penagos, J. Br). Penelitian Hasil Hutan; 35; 211-219.
& Kazazis, C. (2014). Honey and Its
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DOI: 10.20473/jfiki.v9i32022.298-304
Assessing the Neurotoxicological Effect of the Acute Paraquat Aerosols

Exposure in Causing Parkinsonism on Mouse through Behavioral Assays
Sherlo Adha Maulana1*, Santi Nurul Kamilah1, Choirul Muslim1, Aceng Ruyani2, R. R. Sri Astuti1
1
Department of Biology, Faculty of Mathematics and Natural Sciences, University of Bengkulu, Bengkulu, Indonesia
2
Graduate School of Science Education, Faculty of Teacher Training and Education, University of Bengkulu, Bengkulu,
Indonesia
*Corresponding author: sherlo.a.maulana@gmail.com

Abstract
Background: In the scientific community, there is no consensus that paraquat, a widely used herbicide, has a
strong relationship with the occurrence of Parkinson’s disease. A reliable epidemiological explanation of how
paraquat can induce parkinsonism is urgently needed because it relates to the agriculture community's potential
public health problem. Objective: In this study, mice exposed to aerosols of paraquat solution were assessed by
behavioral assays designed to observe whether mice exposed to paraquat aerosols develop cardinal symptoms of
Parkinson’s disease, such as tremor-at-rest, bradykinesia, rigidity, and postural instability. Methods: To obtain
the intended information, we carried out the observation on distal extremities, catalepsy test, wire suspension test,
and swimming test consisting of the head position sub-test, the involvement of limbs sub-test, and the swimming
direction test, respectively, to both the group of mice exposed to paraquat aerosols and the one which is not.
Results: According to the result of the independent-samples t-test calculation on the data obtained from behavioral
assays, a significant difference is shown only by the wire suspension test used to assess the development of forelimb
rigidity and not the others. Conclusion: Therefore, this study showed that daily exposure for a week to paraquat
aerosols insignificantly causes tremor-at-rest, bradykinesia, and postural instability in studied mice but
dramatically affects their forelimb performance in the form of rigidity.
Keywords: paraquat, Parkinson’s disease, parkinsonism, aerosol, behavioral assay

Maulana, S. A., Kamilah, S. N., Muslim, C., Ruyani, A. & Astuti, A. R. R. (2022). Assessing the Neurotoxicological
Effect of the Acute Paraquat Aerosols Exposure in Causing Parkinsonism on Mouse through Behavioral Assays.
Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 9(3), 298-304. http://doi.org/10.20473/jfiki.v9i32022.298-304
P-ISSN: 2406-9388
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INTRODUCTION MATERIALS AND METHODS

To eradicate undesired weeds and grasses from the Animals
plantation field during the planting period, farmers This study is based only on BALB/c male mice 8-
usually apply herbicides by spraying them in the field 12 weeks in age bred at Andalas University. Mice were
for a period (Konthonbut et al., 2020). However, in most caged individually and provided standard rodent chow
developing countries where deficient working and water ad libitum. After being acclimatized (12 hours
conditions, such as the type of equipment being used are day and 12 hours night) for a week at Animal Anatomy
poor, improper maintenance of equipment by farmers, and Physiology Laboratory, University of Bengkulu,
climatic conditions in which farmers usually stand mice were randomly divided into two groups containing
upwind while spraying, and illiteracy make controlled eight mice each. The first group was the control group
and safe use of herbicide is complex (Watts, 2011). (P0), which was the group that is only being maintained
Among the widely used herbicides in agriculture is in their cage during the study, whereas the other group
paraquat (1,1-dimethyl-4,4-bipyridinium dichloride). was the mice exposed to aerosols of paraquat solution
As one of the highly toxic nonselective contact (P1). All mice used in this study were treated ethically
ammonium herbicides, paraquat is associated with the with procedures approved by the Committee of Ethics at
occurrence of Parkinson’s disease (Tanner et al., 2011: the Faculty of Medicine and Health Sciences, University
Gao et al., 2020), a neurological movement disorder of Bengkulu.
characterized by the loss of the nigrostriatal Paraquat exposure
dopaminergic pathway (Tieu, 2011). The source of paraquat used in this study is
Even though there are several pathways that Gramoxone containing 276 g/L paraquat dichloride
paraquat can exist in the body and give rise to health (equal to 200 g/L paraquat ions) produced by Syngenta
problems, those are ingestion of the residue in water or Indonesia. Before being used in the study, 5 mL of
food, dermal penetration, and inhalation of the aerosols, Gramoxone was mixed with aqua dest until the volume
a recent finding by Anderson et al. (2021) showed that of the solution reached 500 mL. The paraquat solution
paraquat could be found and translocated to various was then poured into a spray bottle.
brain regions of mice exposed to paraquat aerosols. It is Paraquat exposure was conducted by spraying the
known that paraquat can trigger oxidative stress that aerosols of paraquat solution onto the face of each P1
leads to neuronal damage (Guo et al., 2018). However, mouse three times (±2.62 mL of paraquat solution
it is still unclear how paraquat can specifically attack received by each mouse) in the isolation cage. After 10
dopaminergic neurons in substantia nigra pars compacta minutes of isolation, each mouse will be placed back
(Tieu, 2011) to induce parkinsonism. into its maintenance cage. This procedure was done
In our laboratory, we exposed aerosols of paraquat daily for a week for P1 mice.
solution onto the face of mice and isolated them for 10 Behavioral assays
minutes in an isolation cage. After daily paraquat Behavioral assays were done in a quiet, dimly lit
exposure for a week, we carried out behavioral assays room in the evening time three days after the last
on studied mice to know whether acute paraquat exposure of paraquat aerosols applied to P1 mice.
aerosols exposure can induce the manifestation of Behavioral assays that were carried out in this study
movement disorders associated with Parkinson’s consist of the catalepsy test, inclined plane test, wire
disease (parkinsonism), such as tremor-at-rest, suspension test, and swimming test, which comprises a
bradykinesia (slowness of a performed movement), head position sub-test, limb movement sub-test, and
rigidity, and postural instability (inability to lance due to swim direction sub-test. The detail of how each
loss of postural reflexes) (Berardelli et al., 2001; behavioral assay was done is as follows:
Jankovic, 2008). In contrast to many studies on the The catalepsy test was carried out based on Grabow
effect of paraquat on the occurrence of Parkinson's & Dougherty (2001), with the scoring method used in
disease, which were conducted via intraperitoneal this study as follows: 0 if the mouse went ahead toward
injection, we attempted to demonstrate whether the the gap when it was placed at the edge of the table; 1 if
result would be consistent if one of the actual conditions the mouse kept staying in its position at the edge of the
in which most farmers are exposed to paraquat in their table more than 10 seconds; and 2. if the mouse turned
plantation field, namely exposure to paraquat aerosols backward in less than 10 seconds to avoid the gap.
inhaled when applying that herbicide by spraying, was The wire suspension test was performed by
mimicked. allowing the mouse grasps a horizontal wire and then
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evaluating its ability to pull its hanged body. It got 0 if et al., 1970). The loss of spontaneous movement
it could not hold the wire provided to it and pull its indicates that the mice have difficulty initiating a
hanged body up by flexing its forelimbs, and one if vas movement and/or slowly performing it (Jankovic,
versa. 2008). Therefore, this test could show whether mice
The inclined plane test was conducted based on exposed to paraquat aerosols in this research developed
Grabow & Dougherty (2001), with the scoring method bradykinesia.
as follows: 0 if the mouse slipped down from the As illustrated by Figure 1a, paraquat aerosol
inclined plane; 1 if the mouse could keep its body in the exposure could slightly decrease the score for the
inclined plane; and 2, if the mouse was not only able to catalepsy test of the mice exposed to paraquat compared
keep its body in the inclined plane but also performed to the control group. However, the result of the
negative geotaxis movement. independent-sample t-test (p = 0.149) shows that there
The swimming test was conducted by placing the is no significant difference between the mean score of
mouse in a water-filled aquarium and evaluating the paraquat-exposed mice and the control group (p > 0.05).
response shown by the mouse while in the water. This A significant decrease in the mean score of the wire
test observed three aspects: head position, swimming suspension test for paraquat-exposed mice
direction, and whether the mouse uses its limbs to swim. Rigidity in Parkinson’s disease manifests in flexor
For evaluating the head position, the mouse would get 0 muscle inhibition and extensor muscle facilitation
as its score if the entire head of the mouse sank in the (Andrews et al., 1972). In this study, the wire suspension
water; 1 if the nose of the mouse was above the surface test score represents the ability of a mouse to flex its
of the water; 2 if the nose and upper head of the mouse forelimbs when it pulls up its body which hung with its
were above the surface of the water; 3 if nose and eyes, forelimbs grasp a string of wire. Figure 1b shows that
as well as ¾ of mouse’s ears, were above the surface of the mean score of the wire suspension test in paraquat-
the water; and 4, if nose, eyes and entire ears of the exposed mice highly decreases compared to the control
mouse were above the surface of the water. For group's mean score. Further independent-sample t-test
evaluating the swimming direction, the mouse would get on this data (p = 0.004) confirmed that the difference
0 as its score when the mouse sank, 1 when the mouse between the mean score of both the control and
floated in the water, 2 when the mouse swam circularly, paraquat-exposed group is significant (p < 0.05).
and 3 when the mouse straightly swam toward its A statistically insignificant decline was observed in
desired direction. For evaluating the involvement of the mean score of the inclined plane test for mice
limbs, the mouse would get 0 if the limbs were not exposed to paraquat
involved in swimming; 1 if only hindlimbs were To maintain the balance of the body in an inclined
involved; and 2 if both fore- and hindlimbs were plane, mice need to have good postural reflexes, and to
involved. assess whether paraquat-exposed mice lose their
Statistical analyses postural reflexes, this research observed the response of
Statistical analyses were performed using IBM both the group of mice exposed to paraquat aerosols and
SPSS Statistics version 28.0.1.1 (IBM SPSS, Inc,) for the control group when they were placed in an inclined
Windows. Statistically significant differences between plane. Figure 1c presents the bar graph of the mean score
two normally distributed groups were analyzed by of both groups, showing that paraquat exposure could
performing the Independent-Samples T-Test, in which decline the score of the inclined plane test result in
statistical significance was set at p < 0.05. All data were several mice exposed to paraquat aerosols, even though
presented as mean + standard deviation. if it was compared to the control group, the difference
between both groups is statistically insignificant (p =
RESULTS 0.060) based on the independent-samples t test
A slight, insignificant decline in the mean score of the calculation (p > 0.05).
catalepsy test for the group of mice exposed to
paraquat
The catalepsy test is a behavioral assay designed to
assess whether mice lose spontaneous mobility (Simon
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Figure 1. The result of behavioral assays of mice exposed to paraquat aerosols (P1, pink) and the control group (P0,
red). Each bar represents the mean score of the assessed behavioral assays (see Materials and Methods). Behavioral
assays tested for both groups comprises the catalepsy test (a), wire suspension test (b), inclined plane test (c), and
swimming test. The swimming test assessed for both groups consists of the head position sub-test (d), swimming
direction sub-test (e), and limbs-using sub-test (f). Error bars on the graphs show the standard deviation. Asterix sign
means the test result shows a significant difference statistically
An insignificant difference in the result of the when it swims will inform that its flexor and extensor
swimming test between the group of mice exposed to limb muscles can function properly, indicating that there
paraquat aerosols and the control group is no rigidity.
Swimming is a complex movement that includes As depicted by Figure 1d, the mean score for the
the ability to coordinate each limb to keep the body's observation of mouse head position is slightly decreased
balance and move the body to the desired direction while in mice exposed to paraquat aerosols compared to the
in water. In this study, the ability to maintain body control group, which was confirmed insignificant (p >
stability in water was assessed by scoring the ability of 0.05) by the independent-samples t-test calculation (p =
a mouse to keep its head out of the water, whereas the 0,334). In the ability to swim properly in any direction,
ability to perform a movement at a required speed to both the control group and mice exposed to paraquat
avoid drowning was assessed by scoring the ability to show the exact same mean score (Figure 1e). For the
swim toward a certain direction. Furthermore, to check result of the use of limbs sub-test (Figure 1f), the
whether the mouse can easily use its limbs for statistical calculation (p = 0.120) proved that the
swimming properly, we observe the movement of limbs difference between the control group and the group of
during swimming activity. mice exposed to paraquat aerosols is insignificant (p >
Each behavioral assay score represents the mouse's 0.05).
ability to conduct normal movements required to swim There is no observed tremor-at-rest in the group of
when the mouse does not develop parkinsonism. When mice exposed to paraquat aerosols
a mouse swims and can maintain its head out of the Tremor-at-rest, or rest tremor, is the most common
water, it is more likely to have no problem balancing its and easily recognized symptom of Parkinson’s disease
posture in the water and vice versa. Moreover, if the and almost always be prominent in the distal part of
mouse has no problem swimming properly toward any extremities. Observation on the distal part of mice's
direction it wants, it means the mouse does not develop extremities was done during this research, and there is
bradykinesia because if it does, the mouse will be no observed tremor-at-rest in both mice exposed to
drowning. The ability of the mouse to move its limbs paraquat aerosols or the control group.
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DISCUSSION damage because it can cause rigidity to emerge in the

Many studies showed that cardinal symptoms of forelimb of paraquat aerosols exposed mice. One of the
Parkinson’s disease only emerge when the dysfunction possibilities underlying rigidity in Parkinson’s disease is
of the basal ganglia circuit involved in motor control, brainstem degeneration (Bologna & Paparella, 2020).
including locomotor movement and posture, ensued A study by Esposito et al. (2014) found that the
from the degeneration of 50-80% of substantia nigra brainstem nucleus medullary reticular formation ventral
dopaminergic neurons, which are part of the input level part, which its high synaptic density was found contacts
of the circuit (Grillner et al., 2013; DeMaagd & Philip, to the biceps, extensor carpi radialis, and extensor digiti
2015; Yttri and Dudman, 2018). Tremor-at-rest, for quarti motor neurons, is a key brainstem area
example, is a condition caused by the failure of the specifically connecting to a subset of forelimb-
circuit's direct pathway output level (which includes the innervating spinal motor neuron. Degeneration of those
GABAergic substantia nigra pars reticulata and the brainstem nuclei might affect forelimb motor
globus pallidus internal) as a result of the dysfunctional performance, such as causing rigidity. Therefore, the
input level, with the effect on the indirect pathway result of our study in which forelimb rigidity is observed
output level (which includes the subthalamic nucleus) also supports the current view that was established by
causing bradykinesia (Wichmann & DeLong, 1996; Tai the study by Braak et al. (2003), that the early stage of
et al., 2012; Grillner et al., 2013; Yttri & Dudman, Parkinson’s disease is started in the brainstem and will
2018). Moreover, because of the reciprocal connection only show its full clinical manifestation when
between the basal ganglia and the mesencephalic neurodegeneration has reached substantia nigra.
locomotor region cholinergic neurons involved in the However, as far as our knowledge, there is no
control of locomotion, posture, and balance, the research focused on the effect of paraquat on brainstem
dysfunction of the basal ganglia circuit also gives rise to degeneration either anatomically or
postural instability in Parkinson’s disease patients immunohistochemically, so we suggest that future
(Grabli et al., 2012; Pahapill & Lozano, 2000; Caggiano research may focus on this area to fully understand how
at al., 2018). paraquat can gradually induce neuronal damage in the
In the previous study by Fahim et al. (2013), rats brain leading to Parkinson’s disease. Moreover, since
which were administered by intraperitoneal injection of farmers who worked in plantation fields are regularly
paraquat for three weeks in a row showed a significant exposed to herbicide throughout their lifetime, to fully
reduction in motor activity and difficulty in movement understand the effect of paraquat aerosols in inducing
following the degeneration of dopaminergic neurons in the occurrence of Parkinson’s disease, unlike this study
substantia nigra pars compacta. However, our study on which focused only on the impact of acute exposure, we
mice exposed to paraquat aerosols for a week showed a need to know whether the chronic exposure of paraquat
different result. As shown in Figure 1 (except Figure aerosols may, too, lead to parkinsonism.
1b), the mean score for behavioral assays designed to
assess mouse motor performance in this study showed a CONCLUSION
statistically insignificant reduction or none in the group In summary, our study shows that daily exposure
of mice acutely exposed to paraquat aerosols compared for a week to paraquat aerosols insignificantly causes
to the control group. This result may imply that the tremor-at-rest, bradykinesia, and postural instability in
typical degeneration of dopaminergic neurons in studied mice but dramatically affects their forelimb
substantia nigra that led to a significant reduction in performance in the form of rigidity.
motor activity and difficulty in movement is absent in
mice exposed to paraquat aerosols in our study. ACKNOWLEDGMENT
Furthermore, it also suggests that the degree of paraquat The authors acknowledge Deni Parlindungan for his
neurotoxicity may depend on the route paraquat comes assistance in purchasing the mice used in this research.
into the body, how a body of different species deals with
it, and the duration of exposure, since those three aspects AUTHOR CONTRIBUTIONS
are what differs our study from Fahim et al. (2013). Conceptualization, S. A. M.; Methodology, C. M.;
However, the result of the wire suspension test for Validation, S. N. K., R. R. S. A.; Formal Analysis, S. A.
both the control group and the group of mice acutely M.; Investigation, S. A. M.; Data Curation, S. A. M.;
exposed to paraquat aerosols in this study (Figure 1b) is Writing - Original Draft, S. A. M.; Writing - Review &
prominent evidence that paraquat can induce neuronal
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Editing, S. N. K.; Visualization, S. A. M.; Supervision, Gao, L., Yuan, H. & Xu, E. (2020). Toxicology of
C. M., A. R.; Project Administration, C. M., S. N. K. Paraquat and Pharmacology of the Protective
Effect of 5-Hydroxy-1-Methylhydantoin on Lung
CONFLICT OF INTEREST Injury Caused by Paraquat Based on
The authors declared no conflict of interest. Metabolomics. Scientific Reports; 10; 1-16.
Grabli, D., Karachi, C., Welter, M. L., Lau, B., Hirsch,
REFERENCES E. C., Vidailhet, M. & François, C. (2012).
Anderson, T., Merrill, A. K., Eckard, M. L., Marvin, E., Normal and Pathological Gait: What We Learn
Conrad, K., Welle, K., Oberdörster, G., from Parkinson's Disease. Journal of Neurology,
Sobolewski, M., Cory-Slechta, D. A. (2021). Neurosurgery, and Psychiatry; 83; 979-985. doi:
Paraquat Inhalation, a Translationally Relevant 10.1136/jnnp-2012-302263.
Route of Exposure: Disposition to the Brain and Grabow, T. S. & Dougherty, P. M. (2001).
Male-Specific Olfactory Impairment in Mice. Cervicomedullary Intrathecal Injection of
Toxicological Sciences; 180; 175-185. doi: Morphine Produces Antinociception in the
10.1093/toxsci/kfaa183. Orofacial Formalin Test in the Rat.
Andrews, C. J., Burke, D. & Lance, J. W. (1972). The Anesthesiology; 95; 1427-1434. doi:
Response to Muscle Stretch and Shortening in 10.1097/00000542-200112000-00023.
Parkinsonian Rigidity. Brain; 95; 795-812. doi: Grillner, S., Robertson, B. & Stephenson-Jones, M.
10.1093/brain/95.4.795. (2013). The Evolutionary Origin of the Vertebrate
Berardelli, A., Rothwell, J. C., Thompson, P. D. & Basal Ganglia and Its Role in Action Selection.
Hallett, M. (2001). Pathophysiology of The Journal of Physiology; 591; 5425-5431. doi:
Bradykinesia in Parkinson's Disease. Brain; 124; 10.1113/jphysiol.2012.246660.
2131-2146. doi: 10.1093/brain/124.11.2131. Guo, J. D., Zhao, X., Li, Y., Li, G. R. & Liu, X. L.
Bologna, M. & Paparella, G. (2020). Pathophysiology (2018). Damage to Dopaminergic Neurons By
of rigidity in Parkinson's disease: Another Step Oxidative Stress in Parkinson's Disease (Review).
Forward. Clinical Neurophysiology; 131; 1971- International Journal of Molecular Medicine; 41;
1972. doi: 10.1016/j.clinph.2020.05.013. 1817-1825. doi: 10.3892/ijmm.2018.3406.
Braak, H., Del Tredici, K., Rüb, U., de Vos, R. A., Jankovic, J. (2008). Parkinson's Disease: Clinical
Jansen, S. E. N. & Braak, E. (2003). Staging of Features and Diagnosis. Journal of Neurology,
Brain Pathology Related to Sporadic Parkinson's Neurosurgery, and Psychiatry; 79; 368-376. doi:
Disease. Neurobiol Aging; 24; 197-211. doi: 10.1136/jnnp.2007.131045.
10.1016/s0197-4580(02)00065-9. Konthonbut, P., Kongtip, P., Nankongnab, N.,
Caggiano, V., Leiras, R., Goñi-Erro, H., Masini, D., Tipayamongkholgul, M., Yoosook, W., &
Bellardita, C., Bouvier, J., Caldeira, V., Fisone, G. Woskie, S. (2020). Paraquat Exposure of
& Kiehn, O. (2018). Midbrain Circuits that set Backpack Sprayers in Agricultural Area in
Locomotor Speed and Gait Selection. Nature; Thailand. Human and Ecological Risk
553; 455-460. doi: 10.1038/nature25448. Assessment: an International Journal; 26; 2798-
DeMaagd, G. & Philip, A. (2015). Parkinson's Disease, 2811. doi: 10.1080/10807039.2019.1684187.
and Its Management (Part 1): Disease Entity, Risk Pahapill, P. A. & Lozano, A. M. (2000). The
Factors, Pathophysiology. Clinical Presentation, Pedunculopontine Nucleus and Parkinson's
and Diagnosis; 40; 504-532. Disease. Brain; 123; 1767-1783. doi:
Esposito, M. S., Capelli, P. & Arber, S. (2014). 10.1093/brain/123.9.1767.
Brainstem Nucleus MdV Mediates Skilled Simon, P., Malatray, J. & Boissier, J. R. (1970).
Forelimb Motor Tasks. Nature; 508; 351-356. doi: Antagonism by Amantadine of
10.1038/nature13023. Prochlorpemazine-Induced Catalepsy. Journal of
Fahim, M. A., Shehab, S., Nemmar, A., Adem, A., Pharmacy and Pharmacology; 22; 546-547. doi:
Dhanasekaran, S. & Hasan, M. Y. (2013). Daily 10.1111/j.2042-7158.1970.tb10567.x.
Subacute Paraquat Exposure Decreases Muscle Tai, C. H., Pan, M. K., Lin, J. J., Huang, C. S., Yang, Y.
Function and Substantia Nigra Dopamine Level. C. & Kuo, C. C. (2012). Subthalamic Discharges
Physiological Research; 62; 313-321. doi: as a Causal Determinant of Parkinsonian Motor
10.33549/physiolres.932386.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Formulation
Really like Like Middle like Dislike Really dislike
Deficits. Annals of Neurology; 72; 464-476. doi: Perspectives in Medicine; 1; 1-20. doi:
10.1002/ana.23618. 10.1101/cshperspect.a009316.
Tanner, C. M., Kamel, F., Ross, G. W., Hoppin, J. A., Watts, M. (2011). Pesticide action network Asia and the
Goldman, S. M., Korell, M., Marras, C., Pacific by Watts.
Bhudhikanok, G. S., Kasten, M., Chade, A. R., http://wssroc.agron.ntu.edu.tw/note/Paraquat.pdf.
Comyns, K., Richards, M. B., Meng, C., Priestley, Accessed: 7 January 2022.
B., Fernandez, H. H., Cambi, F., Umbach, D. M., Wichmann, T. & DeLong, M. R. (1996). Functional and
Blair, A., Sandler, D. P. & Langston, J. W. (2011). Pathophysiological Models of the Basal Ganglia.
Rotenone, Paraquat, and Parkinson's Disease. Current Opinion in Neurobiology; 6; 751-758.
Environmental Health Perspectives; 119; 866- doi: 10.1016/s0959-4388(96)80024-9.
872. doi: 10.1289/ehp.1002839. Yttri, E. A. & Dudman, J. T. (2018). A Proposed Circuit
Tieu, K. (2011). A Guide to Neurotoxic Animal Models Computation in Basal Ganglia: History-
of Parkinson's Disease. Cold Spring Harbor Dependent Gain. Movement Disorders Journal;
33; 704-716. doi: 10.1002/mds.27321.
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DOI: 10.20473/jfiki.v9i32022.305-313
Viability and Antibacterial Activity of Bifidobacterium bifidum in

Fermented Robusta Coffee for Diarrhea Treatment
Miksusanti1, Elsa Fitria Apriani2*, Dimas Nur Hidayat2
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Padang, Indonesia
2
Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Padang, Indonesia
*Corresponding author: elsafitria@mipa.unsri.ac.id

Abstract
Background: Diarrhea can be treated with probiotic bacteria such as Bifidobacterium bifidum, which decreases
the intestinal environment's pH to become acidic so that pathogenic bacteria cannot thrive. Objective: To make
fermented coffee that can increase the number of probiotic bacteria Bifidobacterium bifidum and has antidiarrheal
activity against pathogenic bacteria Escherichia coli. Methods: Robusta coffee (20.25% and 19.75%) was
fermented using Saccharomyces cerevisiae, and then the probiotic bacteria Bifidobacterium bifidum was added.
Unfermented coffee was compared with the same concentration (20.25% and 19.75%) to obtain four formulas.
Organoleptic panelists tested all formulas to determine the best formula for fermented and non-fermented coffee.
The number of Bifidobacterium bifidum and antibacterial activity was calculated on the optimum formulation
using the Total Plate Count and Disc Diffusion Method. Result: The optimum formula obtained at fermented and
unfermented coffee concentration was 20,25%. The number of probiotic bacteria Bifidobacterium bifidum growing
in fermented and non-fermented coffee was 7.3 x 108 ± 32.4 and 3.1 x 108 ± 30.7 (p < 0.05). The diameter of the
inhibition zone of the best fermented and non-fermented coffee was 11.5 ± 0.5 mm and 8.5 ± 0.5 mm, respectively
(p < 0.05). Conclusion: Fermented coffee can increase the growth of the probiotic bacteria Bifidobacterium
bifidum and has strong antibacterial activity against Escherichia coli bacteria.
Keywords: Bifidobacterium bifidum, disc diffusion, Escherichia coli, fermented coffee, total plate count

Miksusanti, Apriani, E. F. & Hidayat, D. N. (2022). Viability and Antibacterial Activity of Bifidobacterium bifidum
in Fermented Robusta Coffee for Diarrhea Treatment. Jurnal Farmasi Dan Ilmu Kefarmasian Indonesia, 9(3), 305-
313. http://doi.org/10.20473/jfiki.v9i32022.305-313
P-ISSN: 2406-9388
E-ISSN: 2580-8303
INTRODUCTION 2019), Salmonella enterica (Symonds et al., 2012), and

Diarrhea is a disease associated with environmental E. coli (Abdelhamid et al., 2018).
hygiene. Diarrhea is mainly caused by food Based on the description above, researchers are
contamination of pathogenic bacteria such interested in making a fermented robusta coffee as a
as Escherichia coli, Salmonella, and Shigella (Wang et symbiotic product containing the probiotic bacteria B.
al., 2015). According to research by Zhou et al. bifidum and testing the growth of the probiotic bacteria
(2018), E. coli is the most common bacteria that causes using the total plate count method. Then, the
diarrhea. Diarrheal disease management can be treated antibacterial activity was tested against pathogenic
with antibiotics such as metronidazole, vancomycin, bacteria E. coli using the Disc Diffusion Method. Non-
ciprofloxacin, tetracycline, and doxycycline (Giannelli, fermented robusta coffee was used as a comparison.
2017). In addition, there are other antidiarrheal agents,
such as probiotics (Guarino et al., 2015). MATERIALS AND METHODS
Probiotics are live microorganisms that provide the Materials
host health benefits when administered appropriately. The materials used consisted of robusta coffee
Microorganisms that can act as probiotics are non- (South Sumatra, Indonesia), S. cerevisiae (buy at
pathogenic microorganisms tolerant of acids and bile, Sriwijaya University, Indonesia), distilled water
modulate the immune system, and can produce lactic (Bratachem, Indonesia), Man Rogosa and Sharpe media
acid (Riddle et al., 2016). One of the most widely used (Merck, Indonesia), B. bifidum BRL-130 (buy at Gadjah
microorganisms as probiotics is Bifidobacterium Mada University, Indonesia), E. coli ATCC-25922 (buy
bifidum (B. bifidum) (Pandey et al., 2015). Probiotics at Sriwijaya University, Indonesia), ciprofloxacin,
can prevent the attachment of pathogenic bacteria to the sodium alginate (Merck, United States), barium chloride
intestinal mucosa, increase the immune response, and (Sigma-Aldrich, United States), sodium chloride
increase the microflora in the intestine (Plaza-Diaz et (Sigma-Aldrich, United States), hydrochloric acid
al., 2019). The growth of probiotic bacteria can be (Bratachem, Indonesia), and concentrated sulfuric acid
assisted with a prebiotic agent. (Bratachem, Indonesia).
Prebiotics are a nutrient source rich in simple Equipment
carbohydrates (Arslanoglu et al., 2008). The The equipment used consisted of Freeze Dryer
fermentation process can obtain simple carbohydrates (Nuaire® NU9483GC), glassware (Pyrex® and
(Pokuesava et al., 2011). In this study, robusta coffee Iwaki®), magnetic stirrer (IKA® C-MAG HS 4), micro
was used as a source of carbohydrates. Based on the pipette (DragonLab®), analytical balance (Ohaus®),
research of Mindarti et al. (2020), Robusta coffee autoclave (Lequitron®), incubator (Biosan®), Oven
contains carbohydrates up to 62.78% w/w. The main (Memmert®), Furnace (Thermolyne®), butyrometer.
carbohydrate in robusta coffee is sucrose (Wulandari et Methods
al., 2021). Complex carbohydrates in robusta coffee will Production of Bifidobacterium bifidum probiotic
be fermented using Saccharomyces cerevisiae, which powder
can break down complex carbohydrates into simpler The suspension of the probiotic bacteria B.
components to be used as a bifidum in de Man, Rogosa & Sharpe (MRS) broth
source of nutrition for B. bifidum (Rizal et al., 2020). S. media was dried using the freeze-drying method. The
cerevisiae can break down sucrose into glucose and probiotic powder was made by
fructose (Marques et al., 2016). mixing Bifidobacterium suspension into 10% skim milk
The fermented coffee will be added with the solution and 4% sodium alginate solution, then
probiotic bacteria B. bifidum to become a symbiotic incubated at 37oC for 8 hours. The mixed suspension
product. In fermented products, the probiotic bacteria B. was then dried at -23oC for 24 hours (Holkem et al.,
bifidum will degrade carbohydrates into organic acids 2016).
such as lactic acid, succinic acid, and acetic acid Production of fermented robusta coffee
(Chichlowski et al., 2011; Stiverson et al., 2014; Wang Robusta coffee was fermented using 3% S.
et al., 2021). These organic acids have a strong cerevisiae yeast for 5 hours. After that, it is washed
antimicrobial effect against bacterial pathogens (Makras thoroughly and dried in the sun. After drying, the coffee
et al., 2006). Bifidobacterium can kill pathogenic beans are cleaned and then roasted at a temperature of
bacteria such as Clostridioides difficile (Yang & Yang, 120oC. Then the coffee beans are ground to obtain
fermented robusta coffee powder (Pereira et al., 2014).
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Table 1. The formula of probiotic coffee

Formula Concentration (%)
Samples
F1 F2 F3 F4
Fermented coffee 20.25 - 19.75 -
Non-fermented coffee - 20.25 - 19.75
Probiotic powder 1.00 1.00 1.00 1.00
Glucose 3.75 3.75 3.75 3.75
Production of robusta coffee symbiotic temperature of 65 - 70oC for 5 minutes and then mixed.
Robusta coffee symbiotic products are made from Put the mixture upside down and reheat for 2 - 3
fermented and non-fermented coffee with added minutes. Calculate the percent fat on the butyrometer
probiotic powder containing the B. bifidum. Fermented line.
coffee powder or non-fermented coffee is added with Carbohydrate content test
probiotic powder. Then add glucose and mix until The carbohydrate content of the sample was
smooth. The formula for probiotic coffee can be seen in calculated by subtracting 100% of the nutritional content
Table 1. of the sample from the moisture content, total ash
Organoleptic test content, protein content, and fat content.
Organoleptic tests were conducted to determine the Probiotic bacteria growth test
best formula with assessments including color, smell, The growth test of the probiotic bacteria B.
taste, and texture from samples shown by 30 untrained bifidum was carried out using the Total Plate Count
panelists with a target age of 15 to 50 years. Samples are Method. In this test, a suspension of B. bifidum was used
placed in containers and coded according to the formula. as a positive control, and distilled water as a negative
Panelists were asked to rate each sample on the control. Selected probiotic coffee formula from
questionnaire sheet. The scale used in this study fermented and non-fermented coffee was brewed with
consisted of five numerical scales, namely strongly warm water. Then the dilution was performed from 10-1
dislike (1), dislike (2), neutral (3), like (4), and like very to 10-8 using 0.9% NaCl solution. Then the solution was
much (5). poured into MRS Agar media and incubated at 37oC for
Proximate test of robusta coffee beans and fermented 48 hours (Rosburg et al., 2010). The amount of growth
coffee of probiotic bacteria was calculated using equation 1.
The proximate test refers to the rules of SNI 01-
2891-1992, including water, ash, protein, fat, and ∑𝐶
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑜𝑙𝑜𝑛𝑦 (𝑁) =
carbohydrate content. (1𝑥𝑛1)+(0.1𝑥𝑛2)+(0.01𝑥𝑛3)𝑥𝑑𝑓
Water content test [1]

Moisture content was measured using the Information:
Thermogravimetric Method. The best probiotic coffee N = Number of product colonies (cfu/mL or cfu/g)
preparations were dried in an oven at 105oC for 5 hours ƩC = Number of colonies in all counted plates
and then weighed until a constant weight was obtained. n1 = Number of cups in the first dilution is calculated
Total ash content test n2 = Number of cups in the second dilution is calculated
Total ash content was measured by calculating the n3 = Number of cups in the third dilution is calculated
constant weight of the sample, which had been heated at Fp = First dilution calculated
600oC for 2 hours using a furnace. Antibacterial activity test against E. coli
Protein content test The antibacterial activity test was carried out using
A number of samples were mixed with 1.9 g K2SO4, the Disc Diffusion Method (Mohammed et al., 2020).
40 mg HgO, and 2 mL concentrated H2SO4, then boiled The antibiotic ciprofloxacin was used as a positive
until the solution became clear. The solution was then control, and distilled water was used as a negative
distilled with 10 mL of 60% NaOH, 5 mL of boric acid, control. Filter paper with a diameter of 6 mm was dipped
and 5 mL of MB:MM indicator for 15 minutes. The in each sample (positive control, negative control, and
solution was then titrated with 0.02 N HCl. coffee samples) for 5 minutes and then planted on MRS
Fat content test Agar media which already contained the pathogenic
A total of 10 mL of H2SO4 and a number of samples bacterium E. coli. The Petri dish was then incubated at
were put into the butyrometer. Add amyl alcohol and stir 37oC for 24 hours. Antibacterial activity was measured
until homogeneous. The mixture was heated at a from the diameter of the resulting inhibition zone.
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Data analysis bacteria B. bifidum are simple carbohydrates such as

Data analysis was carried out statistically using inulin, fructooligosaccharides (FOS),
SPSS version 23. The normality test was carried out galactooligosaccharides (GOS), and lactulose
using the Shapiro-Wilk method. If the data were (Gibson et al., 2010). In this study, robusta coffee will
normally distributed (p > 0.05), then it was continued be fermented using the yeast Saccharomyces
with the one-way ANOVA test to see the differences cerevisiae to help the degradation process of complex
between the test groups. carbohydrates into prebiotic compounds. Fermentation
is a chemical process that can convert complex
RESULTS AND DISCUSSION compounds into simpler compounds. S. cerevisiae is
Probiotic powder containing B. bifidum bacteria known to have the enzyme Fructosyl-transferase
has a smooth and dry texture. Drying was carried out (FTase), which is an enzyme capable of converting
using the freeze-drying method with the help of coating fructose into inulin and fructooligosaccharides (FOS)
materials such as sodium alginate and skim milk. (Louis et al., 2016; Mohkam et al., 2016). S.
According to research by Chandramouli et al. (2004), cerevisiae can also hydrolyze sucrose into glucose and
sodium alginate coating has several advantages, such as fructose (Marques et al., 2016).
being non-toxic, easy to form a gel matrix around Organoleptic is an essential parameter in functional
bacterial cells, and easy release of active substances food products to increase consumer attractiveness.
when contact with the intestine fluid. In addition, skim Fermented and non-fermented robusta coffee was
milk, which contains high protein, can also prevent carried out by organoleptic tests using 30 panelists to
damage to bacterial cell membranes (Amine et al., determine the best formula. Based on this test,
2014). fermented coffee formula one and non-fermented
The growth and metabolic activity of the probiotic formula two were chosen as the best formula according
bacteria B. bifidum can be selectively increased by the to Figure 1. The results of statistical analysis showed
presence of carbohydrates in the environment. Robusta that there were no significant differences in taste and
coffee is used as a carbohydrate source for the growth of odor parameters (p > 0.05) while color and texture had
the probiotic bacteria B. bifidum. Robusta coffee is significant differences (p < 0.05). Formula 1 generally
known to contain mucilage consisting of pectin and has the best results from all parameters. It indicates that
carbohydrates (Haile & Kang, 2019). Based on the the fermentation process in coffee can improve the
research of Mindarti et al. (2020), Robusta coffee quality of the coffee produced. Robusta coffee
contains carbohydrates up to 62.78% w/w. In this study, fermented with S. cerevisiae has been proven to make
the robusta coffee beans used had a water content of 6.63 coffee that has good taste qualities such as the presence
± 0.14%, ash content of 3.68 ± 0.18%, protein content of a caramel aroma in the coffee, a sweet taste at the
of 7.84 ± 0.11%, fat content of 0.86 ± 0.03%, and beginning and bitter at the end, and a fresh aroma
carbohydrate content of 75.98 ± 0.07% (Table 2). In (Bressani et al., 2020; Evangelista et al., 2014; Silva et
addition, S. cerevisiae also contains β-Galactosidases al., 2013). This change is because the coffee
which can convert lactose into galactooligosaccharides fermentation process will produce new byproducts such
(GOS) (Macfarlane et al., 2008; Osman et al., 2012). as acetic acid, citric acid, malic acid, lactic acid, and
Carbohydrates that can be used as nutrients for probiotic succinic acid (Da Mota et al., 2020).
Figure 1. Organoleptic results of symbiotic coffee (n = 30, assessed using questionnaire)

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Table 2. Test results of coffee beans and fermented coffee the best formula (n = 5)
Parameter (%) Robusta coffee beans Fermented coffee SNI 6685, 2009
Water content 6.63 ± 0.14 8.76 ± 0.13 Max 7
Ash content 3.68 ± 0.18 4.48 ± 0.21 Max 1
Protein content 7.84 ± 0.11 38.11 ± 0.49 Min 1
Fat content 0.86 ± 0.03 1.77 ± 0.02 Min 0.6
Carbohydrate content 75.98 ± 0.07 46.87 ± 0.61 -
Table 4. Inhibition zone diameter in samples (n = 5)

Treatment Group Inhibition zone diameter Category
(mm)
Positive control 26.5 ± 0.5 Very strong
Negative control 0.0 ± 0.0 None
F1- Fermented coffee 11.5 ± 0.5 Strong
F2- Non-fermented coffee 8.5 ± 0.5 Moderate
The proximate results of the best-fermented robusta Statistical analysis showed significant differences
coffee have met the standards of SNI 6685, 2009, between groups (p < 0.05). It indicates that fermented
namely water content of 8.76 ± 0.13%, ash content of coffee and non-fermented coffee produce different
4.48 ± 0.21%, the protein content of 38.11 ± 0.49%, fat antibacterial activities. Fermented coffee makes a larger
content was 1.77 ± 0.02%, and carbohydrate content was diameter of inhibition zone than non-fermented coffee
46.87 ± 0.61 (Table 2). and is included in the category of antibacterial solid
Formula 1 and 2 were continued by testing the compounds, namely 11.5 ± 0.5 mm. It proves that the
viability of probiotic bacteria. The results of the viability fermentation process can positively affect antibacterial
test of the probiotic bacteria B. bifidum can be seen in activity. As described above, the robusta coffee
Table 3. fermentation process will produce prebiotic compounds
such as FOS and GOS. FOS and GOS will then be
Table 3. Viability of the probiotic bacteria B. bifidum
in samples (n = 5) further fermented by the bacterium B. bifidum. FOS will
be fermented into organic acids such as lactic acid and
Treatment Number of colonies
Group (cfu/mL) succinic acid, while GOS will be fermented into acetic
Positive control 7.4 x 108 acid, which can inhibit the growth of E. coli bacteria
Negative control 1 x 108 (Bondue & Delcenserie, 2015; Stiveson et al., 2014).
F1-Fermented coffee 7.3 x 108 Organic acids will make the environment acidic so E.
F2-Non-fermented coffee 3.1 x 108
coli bacteria cannot grow. In addition, the undissociated
form of organic acids will enter the E. coli bacterial cell
Based on the results of statistical analysis for the
and dissociate in the cytoplasm, which causes the
viability of probiotic bacteria, there was no significant
bacterial cell to lysis (Bermudez-Brito et al., 2012). B.
difference between the positive control and the
bifidum probiotics can also produce bacteriocins in the
fermented coffee formula 1 (p > 0.05). In contrast, the
form of Bifidocin A. Bifidocin A enters the cells of E.
non-fermented coffee formula 2 differed significantly
coli bacteria to form pores that cause leakage of
from the positive control and formula 1 (p < 0.05). It
intracellular compounds such as proteins, nucleic acids,
proves that the fermentation process increases the
and ions (Liu et al., 2015). B. bifidum can stimulate
growth of the probiotic bacteria B. bifidum. According
mucus secretion, strengthening intestinal epithelial
to the description above, S. cerevisiae can convert
defenses (Denkova et al., 2017). Pathogenic bacteria
complex carbohydrates such as sucrose and lactose into
attach to the intestinal epithelium due to the interaction
FOS and GOS. FOS and GOS have been shown to
between Microbe-Associated Molecular Pattern
increase the growth of the bacterium B.
(MAMP) and Pattern Recognition Receptor (PRR)
bifidum (Saulnier et al., 2008).
(Madsen, 2012). B. bifidum can recognize these
Furthermore, Formula 1 and 2 were continued with
receptors to replace pathogens attached to the intestinal
antibacterial activity tests against E. coli pathogenic
epithelium (Sarkar & Mandal, 2016).
bacteria. The results of the antibacterial activity test can
In addition to the probiotic effect, the antibacterial
be seen in Table 4.
activity of fermented and non-fermented robusta coffee
can also be caused by the content of robusta coffee.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Robusta coffee contains flavonoid compounds, Pharmaceutical Journal; 26; 603–607. doi:
alkaloids, tannins, and terpenoids (Less & Kamengon, 10.1016/j.jsps.2018.03.004.
2021). Flavonoids can act as antibacterial by inhibiting Amine, K. M., Champagne, C. P., Salmieri, S., Britten,
nucleic acid synthesis, inhibiting porin formation, M., St-Gelais, D., Fustier, P. & Lacroix, M.
disrupting membrane structure, and changing membrane (2014). Effect of Palmitoylated Alginate
permeability (Xie et al., 2015). Alkaloids can act Microencapsulation on Viability of
antibacterial by inhibiting ATP-dependent transport of Bifidobacterium longum during Freeze-drying.
compounds across the cell membrane (Mabhiza et al., Food Science and Technology; 56; 111-117. doi:
2016). Tannins work as antibacterial by interfering with 10.1016/j.lwt.2013.11.003.
the metabolism of bacterial cells (Kaczmarek, 2020). Arslanoglu, S., Moro, G. E., Schmitt, J., Tandoi, L.,
Meanwhile, terpenoids act as antibacterial by disrupting Rizzardi, S. & Boehm, G. (2008). Early Dietary
the integrity of cell membranes (Guimarães et al., 2019). Intervention with a Mixture of Prebiotic
However, the antibacterial activity of fermented and Oligosaccharides Reduces the Incidence of
non-fermented coffee was not as strong as the antibiotic Allergic Manifestations and Infections during the
ciprofloxacin. But for fermented coffee, the antibacterial First Two Years of Life. Journal of Nutrition; 138;
activity is in a strong category. 1091–1095. doi: 10.1093/jn/138.6.1091.
Bermudez-Brito, M., Plaza-Diaz, J., Muñoz-Quezada,
CONCLUSION S., Gómez-Llorente, C. & Gil, A. (2012).
The fermented robusta coffee produced in this study Probiotic Mechanisms of Action. Annals of
meet with SNI requirements in characteristics parameter Nutrition and Metabolism; 61; 160–174.
namely water content, ash content, the protein content, Bondue, P. & Delcenserie, V. (2015). Genome of
the fat content, and carbohydrate content. Fermented Bifidobacteria and Carbohydrate Metabolism.
robusta coffee also has a strong antibacterial activity Korean Journal for Food Sciences of Animal
against E. coli while the non-fermented coffee has Resources; 35; 1-9. doi:
moderate antibacterial activity. Fermented robusta 10.5851/kosfa.2015.35.1.1.
coffee can also increase the growth of B. Bressani, A. P. P., Martinez, S. J., Sarmento, A. B. I.,
bifidum bacteria due to the presence of prebiotic Borém, F. M. & Schwan, R. F. (2020). Organic
compounds such as FOS and GOS. So, fermented Acids Produced during Fermentation and Sensory
robusta coffee can be used as an alternative symbiotic Perception in Specialty Coffee using Yeast Starter
product for diarrhea management. Culture. Food Research International; 128;
108773. doi: 10.1016/j.foodres.2019.108773.
AUTHOR CONTRIBUTIONS Chandramouli, Y., Gandhimati, R., Yasmeen, B. R.,
Conceptualization, M., E. F. A.; Methodology, M., Vikram, A., Mahitha, B. & Imroz, S.M. (2004).
E. F. A.; Sotfware, M., E. F. A.; Validation, M., E. F. Review On Cocrystal as An Approach with Newer
A., D. N. A.; Formal Analysis, M., E. F. A., D. N. A.; Implication in Pharmaceutical Field. International
Resources, M., E. F. A., D. N. A.; Investigation, M., E. Journal of Medicinal Chemistry and Analysis; 2;
F. A., D. N. A.; Data Curation, M., E. F. A., D. N. A.; 91-100.
Writing - Original Draft, M., E. F. A.; Writing - Review Chichlowski, M., German, J. B., Lebrilla, C. B. & Mills,
& Editing, M., E. F. A.; Visualization, M., E. F. A; D. A. (2011). The Influence of Milk
Supervision, M., E. F. A.; Project Administration, M., Oligosaccharides on Microbiota of Infants:
E. F. A. Opportunities for Formulas. Annual Review of
Food Science and Technology; 2; 331-351. doi:
CONFLICT OF INTEREST 10.1146/annurev-food-022510-133743.
The authors declared no conflict of interest. Da Mota, M. C. B., Batista, N. N., Rabelo, M. H. S.,
Ribeiro, D. E., Borém, F. M. & Schwan, R. F.
REFERENCES (2020). Influence of Fermentation Conditions on
Abdelhamid, A. G., Esaam, A. & Hazaa, M. M. (2018). the Sensorial Quality of Coffee Inoculated with
Cell Free Preparations of Probiotics Exerted Yeast. Food Research International; 136; 109482.
Antibacterial and antibiofilm Activities Against doi: 10.1016/j.foodres.2020.109482.
Multidrug Resistant E. coli. Saudi Denkova, R., Goranov, B., Teneva, D., Denkova, Z. &
Kostov, G. (2017). Antimicrobial Activity of
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Probiotic Microorganisms: Mechanisms of Control; 62; 309-316. doi:

Interaction and Methods of Examination. 10.1016/j.foodcont.2015.10.033.
Antimicrobial Research: Novel Bioknowledge and Louis, P., Flint, H. J. & Michel, C. (2016). How to
Educational Programs; 34; 201-212. Manipulate the Microbiota: Prebiotics. Advances
Evangelista, S. R., Miguel, M. G., de Souza Cordeiro, in Experimental Medicine and Biology; 902; 119-
C., Silva, C. F., Pinheiro, A. C. & Schwan, R. F. 142. doi: 10.1007/978-3-319-31248-4_9.
(2014). Inoculation of Starter Cultures in a Semi- Macfarlane, G., Steed, H. & Macfarlane, S. (2008).
Dry Coffee (Coffea arabica) Fermentation Bacterial Metabolism and Health-Related Effects
Process. Food Microbiology; 44; 87–95. of Galacto-Oligosaccharides and Other Prebiotics.
Giannelli, F. R. (2017). PA-C Antibiotic-associated Journal of Applied Microbiology; 104; 305–344.
Diarrhea. Journal of the American Academy of Mabhiza, D., Chitemerere, T. & Mukanganyama, S.
Physician Assistants; 30; 46-47. doi: (2016). Antibacterial Properties of Alkaloid
10.1097/01.JAA.0000524721.01579.c9. Extracts from Callistemon citrinus and Vernonia
Gibson, G. R., Scott, K. P., Rastall, R. A., Tuohy, K. M., adoensis against Staphylococcus aureus and
Hotchkiss, A., Dubert-Ferrandon, A., Gareau, M., Pseudomonas aeruginosa. International Journal
Murphy, E. F., Saulnier, D. & Loh, G. (2010). of Medicinal Chemistry; 2016; 1-7. doi:
Dietary Prebiotics: Current Status and New 10.1155/2016/6304163.
Definition. Food Science & Technology Bulletin Madsen, K. L. (2012). Enhancement of Epithelial
Functional Foods; 7; 1–19. Barrier Function by Probiotics. Journal of
Guarino, A., Guandalini, S. & Vecchio, A. (2015). Epithelial Biology & Pharmacology; 5; 55–59.
Probiotics for Prevention and Treatment of Makras, L., Triantafyllou, V., Fayol-Messaoudi, D.,
Diarrhea. Journal of Clinical Gastroenterology; Adriany, T., Zoumpopoulou, G., Tsakalidou, E.,
49; 37-45. doi: Servin, A. & DeVuyst, L. (2006). Kinetic
10.1097/MCG.0000000000000349. Analysis of the Antibacterial Activity of Probiotic
Guimarães, A. C., Meireles, L. M., Lemos, M. F., Lactobacilli Towards Salmonella enterica Serovar
Guimarães, M., Endringer, D. C., Fronza, M. & typhimurium Reveals a Role for Lactic Acid and
Scherer, R. (2019). Antibacterial Activity of Other Inhibitory Compounds. Research in
Terpenes and Terpenoids Present in Essential Microbiology; 157; 241–247. doi:
Oils. Molecules; 24; 2471. doi: 10.1016/j.resmic.2005.09.002.
10.3390/molecules24132471. Marques, W., Raghavendran, V., Stambuk, B. U. &
Haile, M. & Kang, W. H. (2019). The Role of Microbes Gombert, A. K. (2016). Sucrose and
in Coffee Fermentation and Their Impact on Saccharomyces cerevisiae: A Relationship Most
Coffee Quality. Journal of Food Quality; 2019; 1- Sweet. Federation of European Microbiological
16. doi: 10.1155/2019/4836709. Societies Yeast Research; 16; 1-16. doi:
Holkem, A., Raddatz, G., Nunes, G., Cichoski, A., 10.1093/femsyr/fov107.
Jacob-Lopes, E., Grosso, C. & Menezes, C. Mindarti, S., Zalizar, L., Damat, Saati, E. A. & Fajriani,
(2016). Development and Characterization of S. (2020). Characterization of Fiber Fraction,
Alginate Microcapsules Containing Physical and Chemical Properties of Coffee Flour
Bifidobacterium BB-12 Produced by (Coffea sp.) as Functional Foodstuff for Diabetes
Emulsification/Internal Gelation Followed by Mellitus Patient. IOP Conferences Series: Earth
Freeze Drying. Food Science and Technology; 71; and Environmental Science; 462; 1-10.
302-308. doi: 10.1016/j.lwt.2016.04.012. Mohammed, M., Raman, N., Alhoot, M. & Alwan, R.
Kaczmarek, B. (2020). Tannic Acid with Antiviral and (2020). Antibacterial Activities of Allium sativum
Antibacterial Activity as A Promising Component (Garlic) Extracts Against Staphylococcus aureus
of Biomaterials-A Minireview. Materials; 13; and Escherichia coli. European Journal of
3224. Molecular & Clinical Medicine; 7; 526-534.
Liu, G., Song, Z., Yang, X., Gao, Y., Wang, C. & Sun, Mohkam, M., Nezafat, N., Berenjian, A.,
B. (2015). Antibacterial Mechanism of Bifidocin Negahdaripour, M., Behfar, A. & Ghasemi, Y.
A, a Novel Broad-Spectrum Bacteriocin Produced (2016). Role of Bacillus genus
by Bifidobacterium animalis BB04. Food in the Production of Value-Added Compounds.
Basel: Springer.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Osman, A., Tzortzis, G., Rastall, R. A. & Silva, C. F., Vilela, D. M., de SouzaCordeiro, C.,
Charalampopoulos, D. (2012). BbgIV is an Duarte, W. F., Dias, D. R. & Schwan, R. F. (2013).
Important Bifidobacterium β-galactosidase for the Evaluation of a Potential Starter Culture for
Synthesis of Prebiotic Galactooligosaccharides at Enhances Quality of Coffee Fermentation. World
High Temperatures. Journal of Agriculture and Journal of Microbiology & Biotechnology; 29;
Food Chemistry; 60; 740–748. 235–247. doi: 10.1007/s11274-012-1175-2.
Pandey, K. R., Naik, S. R. & Vakil, B. V. (2015). Stiverson, J., Williams, T., Chen, J., Adams, S.,
Probiotics, Prebiotics and Symbiotics - a Hustead, D., Price, P., Guerrieri, J., Deacon, J. &
Review. Journal of Food Science and Yu Z. (2014). A Comparative Evaluation of
Technology; 52; 7577–7587. Prebiotic Oligosaccharides using In Vitro
Pereira, G., Thomaz-Soccol, V., Pandey, A., Madeiros, Cultures of Infant Fecal Microbiome. Applied and
A., Lara, J., Gollo, A. & Soccol, C. (2014). Environmental Microbiology; 80; 7388-7397. doi:
Isolation, Selection and Evaluation of Yeasts for 10.1128/AEM.02200-14.
Use in Fermentation of Coffee Beans by the Wet Symonds, E. L., O'Mahony, C., Lapthorne, S.,
Process. International Journal of Food O'Mahony, D., Sharry, J. M., O'Mahony, L. &
Microbiology; 188; 60-66. doi: Shanahan, F. (2012). Bifidobacterium infantis
10.1016/j.ijfoodmicro.2014.07.008. 35624 Protects Against Salmonella-Induced
Plaza-Diaz, J., Ruiz-Ojeda, F. J., Gil-Campos, M. & Gil, Reductions in Digestive Enzyme Activity in Mice
A. (2019). Mechanisms of Action of by Attenuation of the Host Inflammatory
Probiotics. Advances in nutrition; 10; 49–66. doi: Response. Clinical and Translational
10.1093/advances/nmy063. Gastroenterology; 3; 15.
Pokusaeva, K., Fitzgerald, G. F. & van Sinderen, D. Wang, X., Wang, J., Sun, H., Xia, S., Duan, R. & Liang,
(2011). Carbohydrate metabolism in J. (2015). Etiology of Childhood Infectious
Bifidobacteria. Genes & Nutrition; 6; 285–306. Diarrhea in a Developed Region of China:
doi: 10.1007/s12263-010-0206-6. Compared to Childhood Diarrhea in a Developing
Riddle, M. S., DuPont, H. L. & Connor, B. A. (2016). Region and Adult Diarrhea in a Developed
ACG Clinical Guideline: Diagnosis, Treatment, Region. PLoS ONE; 10; 1-14. doi:
and Prevention of Acute Diarrheal Infections in 10.1371/journal.pone.0142136.
Adults. American Journal of Gastroenterology; Wang, Y., Wu, J., Lv, M., Shao, Z., Hungwe, M., Wang,
111; 602-622. doi: 10.1038/ajg.2016.126. J., Bai, X., Xie, J., Wang, Y. & Geng, W. (2021).
Rizal, S., Murhadi, Kustyawati, M. A. & Hasanudin, U. Metabolism Characteristics of Lactic Acid
(2020). Growth Optimization of Saccharomyces Bacteria and the Expanding Applications in Food
cerevisiae and Rhizopus oligosporus during Industry. Frontiers in Bioengineering and
Fermentation to Produce Tempeh with High β- Biotechnology; 9; 1-19. doi:
glucan Content. Biodiversitas; 21; 2667-2673. 10.3389/fbioe.2021.612285.
doi: 10.13057/biodiv/d210639. Wulandari, S., Ainuri, M. & Sukartiko, A. C. (2021).
Rosburg, V., Boylston, T. & White, P. (2010). Viability Biochemical Content of Robusta Coffees Under
of Bifidobacteria Strains in Yogurt with Added Fully-Wash, Honey, and Natural Processing
Oat Beta-Glucan and Corn Starch During Cold Methods. IOP Conferences Series: Earth and
Storage. Journal of Food Science; 75; 439–444. Environmental Science; 819; 1-9.
doi: 10.1111/j.1750-3841.2010.01620.x. Xie, Y., Yang, W., Tang, F., Chen, X. & Ren, L. (2015).
Sarkar, A. & Mandal, S. (2016). Bifidobacteria - Insight Antibacterial Activities of Flavonoids: Structure-
into Clinical Outcomes and Mechanisms of Its Activity Relationship and Mechanism. Current
Probiotic Action. Microbiological Research; 192; Medicinal Chemistry; 22; 132–149. doi:
159-171. doi: 10.1016/j.micres.2016.07.001. 10.2174/0929867321666140916113443.
Saulnier, D. M., Gibson, G. R. & Kolida, S. (2008). In Yang, J. & Yang, H. (2019). Antibacterial Activity of
Vitro Effects of Selected Symbiotics on the Bifidobacterium breve Against Clostridioides
Human Faecal Microbiota Composition. difficile. Frontiers in Cellular and Infection
Federation of European Microbiological Microbiology; 9; 1-12. doi:
Societies Microbiology & Ecology; 66; 516–527. 10.3389/fcimb.2019.00288.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Zhou, Y., Zhu, X., Hou, H. et al. (2018). Characteristics Diarrhea: A Hospital Based Study. BMC
of Diarrheagenic Escherichia coli among Infectious Disease; 18; 1-10. doi:
Children Under 5 Years of Age with Acute 10.1186/s12879-017-2936-1.
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DOI: 10.20473/jfiki.v9i32022.314-322
Knowledge and Perception on Overclaim against the Behaviors of

Implementing the COVID-19 Prevention Protocol Communities in
Indonesia
Elsa Christina1, Didik Setiawan1,2*, Dina Ratna Juwita1, Lianawati2
1
Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Purwokerto, Indonesia
2
Center for Health Economic Studies, Universitas Muhammadiyah Purwokerto, Purwokerto, Indonesia
*Corresponding author: d.didiksetiawan@gmail.com
Submitted: 31 May 2022

Abstract
Background: COVID-19 occurs in various countries and has been declared a pandemic by WHO. Multiple efforts
have been made to reduce the number of cases of COVID-19. However, the incidence of COVID–19 continues to
increase, along with control efforts carried out by various parties, causing overclaims for the prevention or
treatment of COVID–19. Objective This study aimed to determine the relationship between knowledge and public
perception of the behavior of implementing the COVID-19 prevention protocol in Central Java Province. Methods:
This cross-sectional study used primary data collected online via WhatsApp, Telegram, Instagram, and Facebook
in December 2020. A total of 1,098 of 1,115 respondents passed the inclusion and exclusion criteria. Backward
Elimination is used to determine factors related to behavior in the multivariable model stage using multiple logistic
regression. Results: The knowledge, perception, and behaviors prevalence of implementing COVID-19 prevention
protocols were good & enough 79.1% (95% CI 76.63 – 81.45), 96.6% (95% CI 95.38 – 97.55), and 92.3% (95%
CI 90.62 – 93.78) respectively. The result revealed that Knowledge (adjOR = 2.034, 95% CI 1.253 - 3.302, P =
0.004) and Perception (adjOR = 4.064, 95% CI 1.859 - 8.882, P = < 0.001) were possibly associated with
behaviors of implementing COVID-19 prevention protocols among communities in Central Java Province.
Conclusion This study found a slight prevalence of good & enough knowledge, perception, and behaviors of
implementing COVID-19 prevention protocols in a representative sample among Communities in Central Java
Province. Knowledge and perceptions were statistically significant with behaviors.
Keywords: knowledge, perception, behaviors, COVID–19

Christina, E., Setiawan, D., Juwita, D. R. & Lianawati. (2022). Knowledge and Perception on Overclaim against the
Behaviors of Implementing the COVID-19 Prevention Protocol Communities in Indonesia. Jurnal Farmasi dan Ilmu
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INTRODUCTION be effective as a COVID-19 drug but has not shown

COVID-19 (Corona Virus Disease 2019) is a significant resultsUniversitas Airlangga’s COVID-19
deadly disease, and the number of patients continues to combination drug, which was declared capable of curing
increase rapidly throughout the world (Manchia et al., COVID-19 patients, has not yet gone through the
2022; WHO, 2020). It was originally discovered in clinical trial stage (Mawalia, 2020; Wibowo, 2020). The
Hubei Province, China, and quickly spread to 215 other news about "The COVID-19 vaccine test in Indonesia
countries around the world. Furthermore, on March 20, was successful" shows that the vaccine test has not been
2020, WHO declared COVID-19 a pandemic. The completed (Li, 2021; PT Bio Farma, 2020).
number of cases of COVID-19 continues to increase. As To put it another way, claiming to be able to prevent
of 4 October 2020, there were 34 million cases and more and cure COVID-19 can lead to various interpretations
than 1 million deaths worldwide. In Indonesia, on in society. These exaggerated claims can potentially
November 2022, there were 6,627,538 confirmed influence risk perception patterns and public behavior.
cases of COVID-19 with 159,524 deaths, reported to Studies on the relationship between risk perception
WHO. In Central Java Province, 23,521 confirmed patterns and responsive behaviors that emerged during
cases and 2,018 confirmed deaths. The positive number the pandemic yield inconsistent and insignificant
for COVID-19 in Indonesia is 17.51%, this figure is the results, which influence people's behavior in response to
highest in Southeast Asia (Johns Hopkins University COVID-19 (Xu & Peng, 2015). Previous research
and Medicine, 2022; WHO, 2022). claimed that inaccurate information could mislead the
Control of COVID-19 in Indonesia has been public. Besides, people can lose their sense of crisis, so
pursued by issuing the 5th revision of the Corona Virus they do not want to prevent, treat, or participate in
Disease (COVID-19) Prevention and Control vaccinations, and even lose trust in health workers and
Guidelines by the Ministry of Health of the Republic of the government (Cha et al., 2021; Fanelli 2009). This
Indonesia. One of the keys to breaking the chain of could make the COVID-19 pandemic worse and more
transmission of COVID-19 is not to create a source of difficult to control (Islam et al., 2020;
transmission by implementing new habits or by Tangcharoensathien et al., 2020). Since it was declared
implementing health protocols in every activity (MoH a pandemic, COVID-19 has become the biggest threat
Indonesia, 2020). However, data showed that the to society and has been exacerbated by the overclaims
incidence and mortality due to COVID-19 continue to made by various parties. This can make the community
increase. The implementation of protocols and feel safe and neglect the COVID-19 prevention protocol
screening programs has not provided optimal results. so that the Overclaim can affect people's perception and
This is demonstrated by the number of COVID-19 cases behaviors in implementing the COVID-19 transmission
that have occurred since September 8, 2020, which is prevention protocol.
consistently greater than 3,000 cases per day (Islam et
al., 2020; Susanna, 2020; WHO, 2020). MATERIALS AND METHODS
Ineffective COVID-19 control has led to many Tools
control measures that have resulted in overclaims that This study will determine the relationship between
can mislead and harm the public (Cheng et al., 2021; knowledge and public perception of the behavior of
Stewart et al., 2022; Vijaykumar et al. 2021). This is implementing the COVID-19 prevention protocol in
evidenced by claims of prevention and treatment that do Central Java Province. After reviewing the literature, the
not yet have a solid scientific basis, including; The researcher designed this research questionnaire.
Corona Herbavid-19, a herbal medicine by the DPR-RI Communities in other provinces outside Central Java
COVID-19 Resistance Task Force claimed to have province carried out validation. Evaluation of the
succeeded in curing positive COVID-19 patients reliability of the questionnaire was carried out using a
(Garnesia, 2020). Various Indonesian spices such as pilot study on 40 respondents (Riwidikdo & Setiawan
ginger and turmeric are believed to increase the body's 2006). Test the validity and internal consistency of the
immunity to ward off the coronavirus transmission. The questionnaire using Cronbach Alpha and set the
Ministry of Agriculture claims that eucalyptus necklaces minimum acceptable validity value α = 0.32 (Budiman
or eucalyptus plants can ward off the coronavirus & Riyanto 2013). The validity test results on the
(Wibowo, 2020). Eucalyptus is still in the research- knowledge, perception and behavior questionnaire were
based stage of computational analysis (in silico). 0.539 - 0.773; 0.525 - 0.644; 0.592 - 0.808. The results
Hydroxychloroquine (an antimalaria drug) is claimed to
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of the reliability test on the knowledge, perception and smartphone users, can read and fill out questionnaires,
behavior questionnaire were 0.912, 0.786, and 0.919. and have a telephone number that can be contacted. The
The final questionnaire consisted of four sections. exclusion criteria were health workers, or the
The first part of the questionnaire contains demographic respondent's occupation is related to health sciences.
information of the respondents, including gender, age, Overclaimed is that various forms of
education level, and profession. The second part misinformation during a pandemic, as well as science
contains 15 statements to evaluate respondents' communication strategies that confuse the public, fake
knowledge about the overclaiming of COVID-19 news and misinformation or exaggerated information
prevention and treatment. The third part consists of 6 about the outbreak, can thrive on social media with
statements assessing respondents' perceptions of potentially dangerous consequences.
overclaiming COVID-19 prevention and treatment. The All questionnaires were labelled, including the
last section contains ten statements about the respondent's date, time, and location. Informed consent
respondent's behavior regarding the behavior of was taken before distributing the questionnaire to each
implementation COVID-19 health prevention. All respondent, and the confidentiality of the respondent's
questions were closed except for the demographic information was maintained. No incentives are given to
questionnaire. any respondent. Informed consent was done through
Respondents were asked to choose the option online media before filling in the data. Respondents
“True” or “False” for the statements of knowledge and were explained and filled out a consent form before
perception. The correct answer (yes) is given a score of being able to access the questionnaire.
one (1), while the wrong answer (no) is given a score of Statistical analysis
zero. A three-point Likert scale was used for behavioral This study used an observational analytic study
statements (constantly = 3, sometimes = 2, never = 1). with a cross-sectional design. The descriptive stage was
Therefore, the minimum and maximum scores for used to determine the characteristics of this study, and
knowledge, perception, and behavior are 0 to 15, 0 to 6, comparative statistics were used to define whether
and 10 to 30, respectively. The questionnaire was differences between both (good and poor behavior)
distributed online via WhatsApp, Telegram, Instagram groups existed. Furthermore, the inferential stage is used
and Facebook in December 2020. The behavior, to determine the relationship between knowledge and
knowledge, and perception were calculated from the public perception of the behavior of implementing the
total score as a continuous data and then transformed by COVID-19 prevention protocol in Central Java
x-tile (3q) into a categorical data (ordinal) good, enough, Province. In the bivariate stage, we used chi-square
and poor. The variable category was regenerated to good analysis to evaluate the association of knowledge and
& enough and poor (dichotomous). perception on behavior. The backward elimination
Method method was used to determine factors related to
Study design and sampling behavior in the multivariable model stage using multiple
A cross-sectional study design was applied in this logistic regression using the SPSS version 23. The
study. Data collection in this study was conducted online results are presented as an adjusted odds ratio (adjOR)
from communities in Central Java Province in and 95% confidence interval (CI).
December 2020. About 1,098 of 1,115 respondents
passed the inclusion and exclusion criteria selection. RESULTS AND DISCUSSION
The sampling technique used is non-probability Demographic characteristic
sampling with an accidental sampling method. The Among a total of 1,098 respondents who passed our
sample size was calculated using the Rao software inclusion and exclusion criteria, most of them were
online sample size calculator with a 95% CI and 5% female (65.3%), aged 18 - 29 years old (92.5%),
margin of error. A total of 1,098 respondents finishing their secondary school (53.5%), and their
participated in the survey. Analysis was performed on current status as a student (73.4%) (Table 1). Generally,
385 usable forms. there are no statistical differences in characteristics
Inclusion criteria in this study were people who live between people with excellent and poor behavior (p-
in the Central Java Province are over the age of 18 years, value of > 0.05).
are willing to be a respondent, and have read or heard
the news about the prevention or treatment of COVID-
19, which is claimed to be excessive (Overclaim),
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Table 1. The Respondent characteristics between good and poor behaviors groups implementing COVID-19 Prevention
protocols in Central Java
Behaviors
Total (%)
Variable Good (%) Poor (%) P-value
(n = 1134)
(n = 1,014) (n = 84)
Gender
Male 348 34.32 33 39.29 381 34.70 0.358
Female 666 65.68 51 60.71 717 65.30
Age (years)
18 – 29 938 92.50 78 92.86 1016 92.53 0.956
30 – 59 75 7.40 6 7.14 81 7.38
≥ 60 1 0.10 0 - 1 0.09
Education level
Primary education 10 0.99 2 2.38 12 1.09 0.377
Secondary education 541 53.35 46 54.76 587 53.46
University education 76 7.50 3 3.57 79 7.19
Post graduation 387 38.17 33 39.29 420 38.25
Occupation
Student 750 73.96 56 66.67 806 73.41 0.671
Govt. Employee 50 4.93 5 5.95 55 5.01
Private employee 181 17.85 20 23.81 201 18.31
Military 1 0.10 0 - 1 0.09
Housewife 32 3.16 3 3.57 35 3.19
Table 2. Factor associated with behaviors of implementing COVID-19 prevention protocols in Central Java Province
Behaviors (%)
Variable OR P-value
Good (n = 1,014) Poor (n = 84)
Knowledge
Good & enough 815 80.37 54 64.29 2.275 < 0.001
Poor 199 19.63 30 35.71
Perception
Good & enough 987 97.34 74 88.10 4.940 < 0.001
Poor 27 2.66 10 11.90
Table 3. Factor associated with behaviors of implementing COVID-19 prevention protocols among communities in
Central Java Province by using multiple logistic
Model
Variable 1 2
Pseudo R2 = 0.0392 Pseudo R2 = 0.0353
Perception 4.30 (1.95 – 9.50) *** 4.06 (1.86 – 8.88) ***
Knowledge 2.05 (1.26 – 3.33) ** 2.03 (1.25 – 3.30) **
Gender 0.78 (0.49 – 1.24)
Age 0.83 (0.34 – 2.03)
Education level 0.62 (0.23 – 1.65)
Occupation 1.58 (0.60 – 4.18)
Factor associated with behaviors Multiple logistic regression was used for
Our study showed that although more people have multivariable analysis to define the relationship between
better knowledge (80.37% vs. 64.29%) and perception respondent characteristics, knowledge, perception, and
(97.34% vs. 88.10%) among both good and poor behavior in implementing the COVID-19 prevention
behavior groups, those minor differences in knowledge protocol. The initial result showed that respondent
(OR of 2.275, p-value 0.000) and perception (OR of characteristics do not significantly influence their
4.940, p-value 0.000) resulting significant increase on behavior (Table3). Furthermore, the final result revealed
how much people will have good behavior on that Knowledge (adjOR = 2.034, 95% CI 1.253 - 3.302,
implementing COVID-19 prevention protocols on their P = 0.004) and Perception (adjOR = 4.064, 95% CI
daily life (Table 2). 1.859 - 8.882, P = < 0.001) were possibly associated
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with behaviors of implementing COVID-19 prevention This present study revealed that Knowledge (adjOR
protocols among communities in Central Java Province. = 2.034, 95%CI 1.253 - 3.302, P = 0.004) and Perception
(adjOR = 4.064, 95%CI 1.859 - 8.882, P = <0 .001) were
Discussion possibly associated with behaviors of implementing
The knowledge, perception, and behaviors COVID-19 prevention protocols among communities in
prevalence of implementing COVID-19 prevention Central Java Province. This study is similar to previous
protocols was Good & Enough 79.1% (95% CI 76.63 – study, which stated that most students had inadequate
81.45), 96.6% (95% CI 95.38 – 97.55), and 92.3% (95% knowledge about COVID-19 (good knowledge about
CI 90.62 – 93.78) respectively. It was different from the COVID-19 = 23.5%, 95% CI 19.5% to 28.1%) and were
previous study in Cameron that showed the result of the less involved in COVID-19 prevention behavior
prevalence of high overall knowledge score, perception, (Handebo et al., 2021). Risk perception and knowledge
and behaviors/practice towards COVID-19 were related to COVID-19 can influence protective behavior
84.19%, 69%, and 60.8%, respectively (Ngwewondo et (Rattay et al., 2021). Perception of cognitive and
al., 2020). A study in Ethiopia found that 62.3% of particularly effective risk is a further significant
respondents had good knowledge, 56.6% had positive predictor of behavior in the face of COVID-19 (Betsch
attitudes/perceptions of COVID-19, and 47.5% had et al., 2021). Previous studies in China highlighted the
good behavior/practices towards COVID-19 (Adhena & usefulness of cognitive assessment (i.e., perceived
Hidru 2020). Cross-Sectional Study from Nigeria found severity, perceived controllability, and knowledge of
that 88.59% of respondents had good knowledge about COVID-19), as a core process in dealing with stress, in
COVID-19, and most of the health workers had bad explaining public emotions and behaviors in the face of
attitudes (n = 101, 25.06%) or were indifferent toward public health issues (Li et al., 2020). A qualitative study
work (n = 233, 57.82). %) in the COVID-19 era, and in Ghana found that health knowledge has increased due
81.39% have a high level of practice to prevent COVID- to COVID-19 regarding access to health information
19 infection. (Pauline et al., 2020) Previous research and increased understanding of health issues. Increased
from Uganda showed that 91% of research respondents knowledge and access to information reduce the risk of
had good knowledge, 74% had positive being misinformed or claimed to be redundant from
attitudes/perceptions towards COVID-19, and 57% had pandemic protocols (Saah et al., 2021).
good practices/behaviors towards COVID-19 (Olum et Sufficient knowledge provides an understanding of
al., 2020) It may be different from this research because certain situations, including dealing with the COVID-19
the research from Cameron, Ethiopia, Nigeria, and pandemic. Understanding the situation provides self-
Uganda has a different region and culture from confidence so that it is not easily provoked or
Indonesia. Indonesia is an archipelagic country with manipulated by invalid or overclaimed information
hundreds of cultures in it. Information is often biased (Mao et al., 2021) Behavioral changes require
due to the acceptance of understanding between each information about potential threats to the health of
tribe. The prevalence of Knowledge, Perception, and oneself or others. Valid information and increased
Behavior has comparable numbers compared to knowledge affect handling the COVID-19 pandemic
previous studies. This is important to consider for (Azlan et al., 2020; Chesser et al., 2020; Šuriņa et al.,
handling pandemic in Indonesia. 2021).
The importance of knowledge impacting attitudes This knowledge investigation of a new infectious
and behavior in the application of health protocols disease (COVID-19) is needed to identify knowledge
during the COVID-19 pandemic (Zegarra-Valdivia et gaps and sources of misinformation that can assist
al., 2020). Previous studies have shown evidence of a public health efforts in designing and implementing
high prevalence of COVID-19-related knowledge more focused intervention measures (Sallam et al.,
among all participants included in this study. Similar to 2020). Sufficient knowledge, and accurate
this study, there are still significant gaps in perception understanding, can minimize the occurrence of
and behavior towards COVID-19 (Nwagbara et al., misinformation and overclaims and speed up the
2021). Research from North Sulawesi Indonesia completion of the pandemic (Guan et al., 2020; Rothan
revealed that most respondents had good knowledge, & Byrareddy, 2020; Sallam et al., 2020).
positive attitudes, and good practices toward COVID-19 A previous study among university students in the
prevention. However, knowledge of specific topics is UK found that perception (β = 0.13, p = 0.016) was
still insufficient (Simanjorang et al., 2021). statistically significant for the unique variance in hand
P-ISSN: 2406-9388
E-ISSN: 2580-8303
hygiene behavior during the pandemic (Barrett & Editing, D. S., D. R. J., L.; Visualization, D. S., D. R. J.,
Cheung 2021). Previous study with 633 participants L.; Supervision, D. S., D. R. J.; Project Administration,
revealed that better understanding shapes perceptions D. S., L.; Funding Acquisition, D. S.
that affect behavior (Lim et al., 2021). Another previous
study revealed that the perception of COVID-19 can CONFLICT OF INTEREST
positively predict behavior indirectly (Mahmoud et al., The authors declared no conflict of interest.
2021).
Public perception of COVID-19 affects health REFERENCES
behavior. Previous qualitative studies conducted among Adhena, G., & Hidru, H. D. (2020). Knowledge,
communities in Kathmandu, Kanchanpur, Bajura and Attitude, and Practice of High-Risk Age Groups
Jhapa districts in Nepal revealed a very good general to Coronavirus Disease-19 Prevention and
understanding among respondents about COVID-19, Control in Korem District, Tigray, Ethiopia:
personal precautions and population level strategies. Cross-Sectional Study. Infection and Drug
Respondents recognized the important role of the media Resistance 13; 3801–3809. doi:
in increasing awareness, knowledge, and straightening 10.2147/IDR.S275168.
perceptions. Participants also expressed concern over Albaqawi, Mohammad, H., Alquwez, N., Balay-odao,
the misleading (overclaimed) news that was spread by E., Bajet, J. B., Alabdulaziz, H., Alsolami, F.,
several media (Bhatt et al., 2020). Tumala, R. B., Alsharari, A. F., Hanan, M. M. T.,
Planning an effective educational intervention for Felemban, E. M. & Cruz, J. P. (2020). Nursing
handling the COVID-19 pandemic requires knowledge, Students’ Perceptions, Knowledge, and
perception, and preventive behavior as awareness of the Preventive Behaviors Toward COVID-19: A
health risks posed by this disease (Albaqawi et al., Multi-University Study. Frontiers in Public
2020). This is important to increase the general public’s Health; 8; 1–9. doi: 10.3389/fpubh.2020.573390.
knowledge in preventing the spread of COVID-19. Azlan, A. A., Hamzah, M. R., Sern, T. J., Ayub, S. H. &
Knowledge can make people aware of the seriousness of Mohamad, E. (2020). Public Knowledge,
this pandemic situation (Gohel et al., 2021). Human Attitudes and Practices towards COVID-19: A
behavior plays an important role as the world grapples Cross-Sectional Study in Malaysia. PLoS ONE;
with public health threats such as the COVID-19 15; 1–15. doi: 10.1371/journal.pone.0233668.
pandemic. Appropriate behavior can be an important Barrett, C. & Cheung, K. L. (2021). Knowledge, Socio-
factor in reducing cases of the COVID-19 pandemic Cognitive Perceptions and the Practice of Hand
(Jalloh et al., 2021). Hygiene and Social Distancing during the
COVID-19 Pandemic: A Cross-Sectional Study of
CONCLUSION UK University Students. BMC Public Health; 21;
This study found a slight prevalence of good & 1–18. doi: 10.1186/s12889-021-10461-0.
enough knowledge, perception, and behaviors of Betsch, C., Korn, L., Burgard, T., Gaissmaier, W.,
implementing COVID-19 prevention protocols in a Felgendreff, L., Eitze, S., Sprengholz, P., Böhm,
representative sample among Communities in Central R., Stollorz, V., Ramharter, M., Promies, N., De
Java Province. Knowledge and perceptions were Bock, F., Schmid, P., Renner, B., Wieler, L. H. &
statistically significant with behaviors. Bosnjak, M. (2021). The Four Weeks before
Lockdown during the COVID-19 Pandemic in
ACKNOWLEDGMENT Germany: A Weekly Serial Cross-Sectional
The authors would like to acknowledge all the Survey on Risk Perceptions, Knowledge, Public
respondents from Central Java province willing to Trust and Behaviour, 3 to 25 March 2020. Euro
respond to this research. Surveillance: Bulletin Europeen Sur Les Maladies
Transmissibles = European Communicable
AUTHOR CONTRIBUTIONS Disease Bulletin; 26; 1–13. doi: 10.2807/1560-
Conceptualization, E. C., D. S.; Methodology, E. 7917.ES.2021.26.42.2001900.
C., D. S.; Sotfware, E. C., D. S.; Validation, D. S., D. R. Bhatt, N., Bhatt, B., Gurung, S., Dahal, S., Jaishi, A. R.,
J.; Formal Analysis, E. C., D. R. J.; Investigation, D. S., Neupane, B. & Budhathoki, S. S. (2020).
D. R. J.; Resources, E. C., D. R. J., L.; Data Curation, E. Perceptions and Experiences of the Public
C..; Writing - Original Draft, E. C.; Writing - Review & Regarding the COVID-19 Pandemic in Nepal: A
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Qualitative Study Using Phenomenological Study. BMJ Open; 11; 1–10. doi:
Analysis. BMJ Open; 10; 1–11. doi: 10.1136/bmjopen-2021-050189.
10.1136/bmjopen-2020-043312. Islam, M. S., Sarkar, T., Khan, S. H., Kamal, A. H. M.,
Budiman & Agus Riyanto. (2013). Kapita Selekta Hasan, S. M. M., Kabir, A., Yeasmin, D., Islam,
Kuesioner Pengetahuan dan Sikap Dalam M. A., Chowdhury, K. I. A., Anwar, K. S.,
Penelitian Kesehatan. Jakarta: Salemba Medika. Chughtai, A. A. & Seale, H. (2020). COVID-19-
Cha, M., Cha, C., Singh, K., Lima, G., Ahn, Y. Y., Related Infodemic and Its Impact on Public
Kulshrestha, J. & Varol, O. (2021). Prevalence of Health: a Global Social Media Analysis. American
Misinformation and Factchecks on the COVID-19 Journal of Tropical Medicine and Hygiene; 103;
Pandemic in 35 Countries: Observational 1621–1629. doi: 10.4269/ajtmh.20-0812.
Infodemiology Study. JMIR Human Factors; 8; Jalloh, M. F., Nur, A. A., Nur, S. A., Winters, M.,
1–6. doi: 10.2196/23279. Bedson, J., Pedi, D., Prybylski, D., Namageyo-
Cheng, M., Yin, C., Nazarian, S. & Bogdan, P. (2021). Funa, A., Hageman, K. M., Baker, B. J., Jalloh, M.
Deciphering the Laws of Social Network- B., Eng, E., Nordenstedt, H. & Hakim, A. J.
Transcendent COVID-19 Misinformation (2021). Behaviour Adoption Approaches during
Dynamics and Implications for Combating Public Health Emergencies: Implications for the
Misinformation Phenomena. Scientific Reports; COVID-19 Pandemic and Beyond. BMJ Global
11; 1–14. doi: 10.1038/s41598-021-89202-7. Health; 6; 1–9. doi: 10.1136/bmjgh-2020-004450.
Chesser, A., Ham, A. D. & Woods, N. K. (2020). Johns Hopkins University and Medicine. (2022). Johns
Assessment of COVID-19 Knowledge Among Hopkins Coronavirus Resource Center.
University Students: Implications for Future Risk Baltimore: The Johns Hopkins University School
Communication Strategies. Health Education and of Medicine.
Behavior; 47; 540–43. doi: Li, J. B., Yang, A., Dou, K., Wang, L. X., Zhang, M. C.
10.1177/1090198120931420. & Lin, X. Q. (2020). Chinese Public’s
Fanelli, D. (2009). How Many Scientists Fabricate and Knowledge, Perceived Severity, and Perceived
Falsify Research? A Systematic Review and Controllability of COVID-19 and Their
Meta-Analysis of Survey Data. PLOS ONE; 4; 1- Associations with Emotional and Behavioural
11. doi: 10.1371/journal.pone.0005738. Reactions, Social Participation, and Precautionary
Garnesia, I. (2020). Menguji Klaim Herbavid-19 Behaviour: A National Survey. BMC Public
Sebagai Obat COVID-19. Jakarta: Tirto Id. Health; 20; 1–14. doi: 10.1186/s12889-020-
Gohel, K. H., Patel, P. B., Shah, P. M., Patel, J. R., 09695-1.
Pandit, N. & Raut, A. (2021). Knowledge and Li, Q. (2021). The Efficacy, Safety and Immunogenicity
Perceptions about COVID-19 among the Medical Study of Inactivated SARS-CoV-2 Vaccine for
and Allied Health Science Students in India: An Preventing Against COVID-19.
Online Cross-Sectional Survey. Clinical https://clinicaltrials.gov/ct2/show/NCT04659239.
Epidemiology and Global Health; 9; 104–109. Accessed: 19 March 2021.
doi: 10.1016/j.cegh.2020.07.008. Lim, V. W., Lim, R. L., Tan, Y. R., Soh, A. S. E., Tan,
Guan, W., Ni, Z., Hu, Y., Liang, W., Ou, C., He, J., Liu, M. X., Othman, N. B., Dickens, S. B., Thein, T.
L., Shan, H., Lei, H., Hui, D. S. C., Du, B., Li, L., L., Lwin, M. O., Ong, R. T. H., Leo, Y. S., Lee, V.
Zeng, G., Yuen, K., Chen, R., Tang, C., Wang, T., J. & Chen, M. I. C. (2021). Government Trust,
Chen, P., Xiang, J., Li, S., Wang, J., Liang, Z., Perceptions of Covid-19 and Behaviour Change:
Peng, Y., Wei, L., Liu, Y., Hu, Y., Peng, P., Wang, Cohort Surveys, Singapore. Bulletin of the World
J., Liu, J., Chen, Z., Li, G., Zheng, G., Qiu, S., Health Organization; 99; 92–101. doi:
Luo, J., Ye, C., Zhu, S. & Zhong, N. (2020). 10.2471/BLT.20.269142.
Clinical Characteristics of Coronavirus Disease Mahmoud, A. B., Polay, D. H., Reisel, W. D., Fuxman,
2019 in China. New England Journal of Medicine; L., Grigoriou, N., Mohr, I. & Aizouk, R. (2021).
382; 1708–1720. doi: 10.1056/nejmoa2002032. Who’s More Vulnerable? a Generational
Handebo, S., Adugna, A., Kassie, A. & Shitu, K. (2021). Investigation of COVID-19 Perceptions’ Effect
Determinants of COVID-19-Related Knowledge on Organisational Citizenship Behaviours in the
and Preventive Behaviours among Students in MENA Region: Job Insecurity, Burnout and Job
Reopened Secondary Schools: Cross-Sectional
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Satisfaction as Mediators. BMC Public Health; Pauline, M., Ngozichukwu, C., Azubuike, C. M. C.,
21; 1–17. doi: 10.1186/s12889-021-11976-2. Mbachu, I. I., Ndukwu, C. I., Ezeuko, A. Y. A.,
Manchia, M., Gathier, A. W., Yapici-Eser, H., Schmidt, Udigwe, I. B., Nnamani, C. P., Umeh, U. M.,
M. V., de Quervain, D., Amelsvoort, T. V., Ezeagwuna, D. A., Onah, S. K., Eze, H. O.,
Jonathan, B., Cryan, J. F., Howes, O. D., Pinto, L., Okereke, U. C. & Orji-Ifeanyi, E. N. (2020).
van der Wee, N. J., Domschke, K., Branchi, I. & COVID-19 Infection: Knowledge, Attitude,
Vinkers, C. H. (2022). The Impact of the Practices, and Impact among Healthcare Workers
Prolonged COVID-19 Pandemic on Stress in a South-Eastern Nigerian State. Journal of
Resilience and Mental Health: A Critical Review Infection in Developing Countries; 14; 943–52.
across Waves. Annals of Oncology; 55; 22–83. doi: 10.3855/JIDC.13248.
Mao, Y., Chen, H., Wang, Y., Chen, S., Gao, J., Dai, J., PT Bio Farma. (2020). Efficacy, Safety and
Jia, Y., Xiao, Q., Zheng, P. & Fu, H. (2021). How Immunogenicity Study of SARS-CoV-2
Can the Uptake of Preventive Behaviour during Inactivated Vaccine (COVID-19). Rockville: U.S.
the COVID-19 Outbreak Be Improved? An National Library of Medicine.
Online Survey of 4827 Chinese Residents. BMJ Rattay, P., Michalski, N., Domanska, O. M., Kaltwasser,
Open; 11; 1–11. doi: 10.1136/bmjopen-2020- A., de Bock, F., Wieler, L. H. & Jordan, S. (2021).
042954. Differences in Risk Perception, Knowledge and
Mawalia, K. A. (2020). Peneliti UNAIR Temukan Lima Protective Behaviour Regarding COVID-19 by
Kombinasi Obat Efektif Lawan Corona. Education Level among Women and Men in
https://news.unair.ac.id/2020/06/12/peneliti- Germany. Results from the COVID-19 Snapshot
unair-temukan-lima-kombinasi-obat-yang- Monitoring (COSMO) Study. PLoS ONE; 16; 1–
efektif-lawan-corona-2/?lang=id. Accessed: 10 26. doi: 10.1371/journal.pone.0251694.
March 2021. Riwidikdo, H. & Setiawan, A. (2006). Statistik
MoH Indonesia. (2020). Pedoman Kesiapsiagaan Kesehatan: Belajar Mudah Teknik Analisis Data
Menghadapi Coronavirus Disease (Covid-19). Dalam Penelitian Kesehatan (plus Aplikasi
Jakarta: Kementerian Kesehatan Republik SPSS). Yogyakarta: Mitra Cendekia Press.
Indonesia. Rothan, H. A. & Byrareddy, S. N. (2020). The
Ngwewondo, A., Nkengazong, L., Ambe, L. A., Ebogo, Epidemiology and Pathogenesis of Coronavirus
J. T., Mba, F. M., Goni, H. O., Nyunaï, N., Disease (COVID-19) Outbreak. Journal of
Ngonde, M. C. & Oyono, J. L. E. (2020). Autoimmunity Journal; 109; 1-4.
Knowledge, Attitudes, Practices of/towards Saah, F. I., Amu, H., Seidu, A. A. & Bain, L. E. (2021).
COVID 19 Preventive Measures and Symptoms: Health Knowledge and Care Seeking Behaviour in
A Cross-Sectional Study during the Exponential Resource-Limited Settings amidst the COVID-19
Rise of the Outbreak in Cameroon. PLoS Pandemic: A Qualitative Study in Ghana. PLoS
Neglected Tropical Diseases; 14; 1–15. doi: ONE; 16; 1–15. doi:
10.1371/journal.pntd.0008700. 10.1371/journal.pone.0250940.
Nwagbara, U. I., Osual, E. C., Chireshe, R., Bolarinwa, Sallam, M., Dababseh, D., Yaseen, A., Al-Haidar, A.,
O. A., Saeed, B. Q., Khuzwayo, N. & Hlongwana, Taim, D., Eid, H., Ababneh, N. A., Bakri, F. G. &
K. W. (2021). Knowledge, Attitude, Perception, Mahafzah, A. (2020). COVID-19
and Preventative Practices towards COVID-19 in Misinformation: Mere Harmless Delusions or
Sub-Saharan Africa: A Scoping Review. PLoS Much More? A Knowledge and Attitude Cross-
ONE; 16; 1–20. doi: Sectional Study among the General Public
10.1371/journal.pone.0249853. Residing in Jordan. PLoS ONE; 15; 1–18. doi:
Olum, R., Kajjimu, J., Kanyike, A. M., Chekwech, G., 10.1371/journal.pone.0243264.
Wekha, G., Nassozi, D. R., Kemigisa, J., Simanjorang, C., Tooy, G., Wuaten, G. &
Mulyamboga, P., Muhoozi, O. K., Nsenga, L., Pangandaheng, N. (2021). Knowledge, Attitudes,
Lyavala, M., Asiimwe, A. & Bongomin, F. and Practices Towards COVID-19 among North
(2020). Perspective of Medical Students on the Sulawesi Indonesia Residents. Journal of Health
COVID-19 Pandemic: Survey of Nine Medical Education; 6; 57–64. doi:
Schools in Uganda. JMIR Public Health and 10.1080/10556699.1994.10603001.
Surveillance; 6; 1–12. doi: 10.2196/19847.
P-ISSN: 2406-9388
E-ISSN: 2580-8303
Stewart, R., Madonsela, A., Tshabalala, N., Etale, L. & Peers Sharing COVID-19 Misinformation on
Theunissen, N. (2022). The Importance of Social WhatsApp in Brazil. Journal of the American
Media Users’ Responses in Tackling Digital Medical Informatics Association: JAMIA; 29; 33–
COVID-19 Misinformation in Africa. Digital 42. doi: 10.1093/jamia/ocab219.
Health; 8; 1-15. doi: WHO. (2020). WHO Director-General’s Opening
10.1177/20552076221085070. Remarks at the Media Briefing on COVID-19:
Šuriņa, S., Martinsone, K., Perepjolkina, V., WHO Director General’s Speeches.
Kolesnikova, J., Vainik, U., Ruža, A., Vrublevska, https://www.who.int/director-
J., Smirnova, D., Fountoulakis, K. N. & Rancans, general/speeches/detail/who-director-general-s-
E. 2021. “Factors Related to COVID-19 opening-remarks-at-the-media-briefing-on-covid-
Preventive Behaviors: A Structural Equation 19---11-march-2020. Accessed: 11 March 2021.
Model. Frontiers in Psychology; 12; 1–15. doi: WHO. (2022). COVID-19 Weekly Epidemiological
10.3389/fpsyg.2021.676521. Update. World Health Organization; 58; 1–23.
Susanna, D. (2020). When Will the COVID-19 Wibowo, Y. C. (2020). 4 Alasan Kita Perlu Tolak Klaim
Pandemic in Indonesia End?. Kesmas; 15; 160– Keampuhan Obat Herbal Di Tengah Pandemi
162. doi: 10.21109/KESMAS.V15I4.4361. COVID-19: The Conversation.
Tangcharoensathien, V., Calleja, N., Nguyen, T., https://theconversation.com/4-alasan-kita-perlu-
Purnat, T., D’Agostino, M., Garcia-Saiso, S., tolak-klaim-keampuhan-obat-herbal-di-tengah-
Landry, M., Rashidian, A., Hamilton, C., pandemi-covid-19-140642. Accessed: 11 March
AbdAllah, A., Ghiga, I., Hill, A., Hougendobler, 2021.
D., van Andel, J., Nunn, M., Brooks, I., Sacco, P. Xu, J. & Peng, Z. (2015). People at Risk of Influenza
L., de Domenico, M., Mai, P., Gruzd, A., Pandemics: The Evolution of Perception and
Alaphilippe, A. & Briand, S. (2020). Framework Behavior. PLoS ONE; 10; 1–14. doi:
for Managing the COVID-19 Infodemic: Methods 10.1371/journal.pone.0144868.
and Results of an Online, Crowdsourced Who Zegarra-Valdivia, Vilca, C. & Guerrero, A. (2020).
Technical Consultation. Journal of Medical Knowledge, Perception and Attitudes in Regard to
Internet Research; 22; 1–8. doi: 10.2196/19659. COVID-19 Pandemic in Peruvian Population.
Vijaykumar, S., Rogerson, D. T., Jin, Y. & Costa, M. S. Pediatria Integral; 24; 69–70.
O. (2021). Dynamics of Social Corrections to
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Jurnal Farmasi dan Ilmu Kefarmasian Indonesia (Pharmacy and Pharmaceutical Sciences Journal)

1. Isoprinosine along with Favipiravir or Oseltamivir in Patients with Moderate 209-219

Chrysnanda Maryska, Didik Hasmono, Sofiati Diah Baisuni, Ayunda Nur

2. The Effect of Decyl Glucoside on Stability and Irritability of Nanostructured 220-228

Rufaidah Azzahrah, Noorma Rosita, Djoko Agus Purwanto, Widji Soeratri

3. Effect of Alpha-Lipoic Acid on the Characteristics and Physical Stability of 229-234

Fairuz Yaumil Afra, Widji Soeratri, Djoko Agus Purwanto

4. Optimization Method and Stability Test to Determinate Luteolin, Quercetin, 235-241

Muhammad Hidayatullah, Mochammad Yuwono, Riesta Primaharinastiti

5. The Effect of Insulin Administration on Medication Adherence in Type 2 242-251

July, Rani Sauriasari, Nadia Farhanah Syafhan, Hadijah Tahir

Dwi Novita Sari, Wahyu Utami, Elida Zairina

8. Mannitol Production from Fructose by Using Resting Cells of Methylotrophic 272-278

Nisa Yulianti Suprahman, Khairul Basyar, Herman Suryadi

9. Jelly Candy Hydroxyapatite from Mackerel Fish Bone 279-289

Lia Anggresani, Santi Perawati, Ryan Afandi, Rahmadevi

Sulistyianingsih, Achmad Toto Poernomo, Riesta Primaharinastiti

12. Viability and Antibacterial Activity of Bifidobacterium bifidum in Fermented 305-313

Miksusanti, Elsa Fitria Apriani, Dimas Nur Hidayat

Elsa Christina, Didik Setiawan, Dina Ratna Juwita, Lianawati

Isoprinosine along with Favipiravir or Oseltamivir in Patients with

*Corresponding author: didik-h@ff.unair.ac.id

Submitted: 12 February 2022

Keywords: isoprinosine, covid-19, moderate, immunomodulator

How to cite this article:

INTRODUCTION Thus, favipiravir can be used as an antiviral in treating

evaluates Isoprinosine with Favipiravir or Oseltamivir Tools

Table 2. Clinical data on isoprinosine in combination with favipiravir or oseltamivir

Table 3. Laboratory data on isoprinosine in combination with favipiravir or oseltamivir

Table 4. Characteristics of LOS data and mortality rates in isoprinosine-favipiravir- or oseltamivir-combination

Table 6. Analysis of variables with dependent variables

Levels in Healthy Adults. Journal of Interferon Hamers, R. L. (2021). Clinical Characteristics

The Effect of Decyl Glucoside on Stability and Irritability of Nanostructured

*Corresponding author: noorma-r@ff.unair.ac.id

Submitted: 28 Februari 2022

How to cite this article:

INTRODUCTION damaging the lipid membrane, which is the external

Table 1. Design formula of NLC-GTE

By Lullung & Suprapti's (2012) explanation, the

(bleeding) and lysis (loss of blood vessels) in the CAM ACKNOWLEDGMENT

Effect of Alpha-Lipoic Acid on the Characteristics and Physical Stability of

*Corresponding author: widjisoeratri@yahoo.com

Submitted: 28 February 2022

How to cite this article:

INTRODUCTION of being quite complex in preparation and low instability

Tabel 1. F1 and F2 test formula

Tabel 2. Characteristic observation results of F1 and F2

Dermatology and Venereology; 30; 208-215. doi: 004/indonesia-masuki-periode-aging-

Optimization Method and Stability Test to Determinate Luteolin,

*Corresponding author: yuwono05@yahoo.com

Submitted: 20 April 2022

Keywords: TLC-densitometry, luteolin, quercetin, apigenin, sinensetin

How to cite this article:

INTRODUCTION medicines. The analytical method commonly used to

Table 1. Variation of mobile phase composition tested to obtain optimal conditions

Sample preparation RESULTS AND DISCUSSION

The Effect of Insulin Administration on Medication Adherence in Type 2

*Corresponding author: rani@farmasi.ui.ac.id

Submitted: 1 June 2022