Pharmacogn J.

2020; 12(4): 777-783

A Multifaceted Journal in the field of Natural Products and Pharmacognosy
Original Article
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Standardization Study of Simplicia and Extract of Calamondin

(Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and
Antibacterial Assay
Elidahanum Husni*, Friardi Ismed, Dony Afriyandi

ABSTRACT
Introduction: Calamondin (Citrus microcarpa Bunge) is a commodity which is widely grown in Indonesia,
including in western Sumatera. Aim: This study was conducted to Standardization Study of Simplicia and
Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay.
Material and Methods: The semi-solid extract of Calamondin peel was made by the maceration method
Elidahanum Husni*, Friardi using 70% ethanol solvent. Then standardization study chamomile extract (Organoleptic examination
Ismed, Dony Afriyandi of extracts, Chromatographic analysis, Total Ash, Acid-insoluble ash, Water content), quantification
of hesperidin by TLC-densitometry method and antibacterial activity assay for diffusion method. The
Faculty of Pharmacy, University Andalas, antibacterial activity of extracts against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis,
INDONESIA. and Pseudomonas aeruginous. Results: The organoleptic properties of the calamondin peel showed that
the outer surface was brown, and the inside was yellow, slightly smelly, and sour taste. Microscopic
Correspondence
characterizations obtained identifiers of calcium oxalate crystal fragments, fibers, parenchyma with oil
Elidahanum Husni cells, ladder-shaped transport tissue. Water and alcohol-soluble extractive are not less than 19.73% ±
Faculty of Pharmacy, University Andalas, 0.97% and 10.26% ± 0.25%, loss on drying is not more than 10.78% ± 0.05%, and the total and acid-
INDONESIA. insoluble ash is not more than 4.33% ± 0.03% and 1.01% ± 0.07%. The calamondin peel extract is
E-mail: elidahanumhusni@phar.unand.ac.id described in the form of thick extract, a specific smell, a black color, bitter taste, and yield not less than
History 25.33% ± 1.3%. Quantification of hesperidin obtained not less than 4.78% ± 0.09%, a water content of
• Submission Date: 04-04-2020; no more than 17.47% ± 0.82%, and the total ash content and acid insoluble ash are not more than 4.65%
• Review completed: 23-04-2020; ± 0.06% and 0.13% ± 0.04%. Antibacterial activity of extracts against Staphylococcus aureus, Escherichia
• Accepted Date: 06-05-2020;
coli, Enterococcus faecalis, and Pseudomonas aeruginous at 15% concentration with inhibitory diameter
range of 7.65 mm ± 0.36 mm to 9.96 mm ± 0.52 mm and at a concentration of 20% with inhibitory
DOI : 10.5530/pj.2020.12.111 diameter ranges of 9.26 mm ± 0.72 mm to 13.39 mm ± 0.28 mm. Conclusion: Calamondin (Citrus
Article Available online microcarpa Bunge) peel have antioxidant and antibacterial activity.
Key words: Antibacterial, Antioxidants, Citrus microcarpa Bunge, Hesperidin, Standardization.
http://www.phcogj.com/v12/i4

Copyright
© 2020 Phcogj.Com. This is an open- INTRODUCTION activity8. One of the potentials of hesperidin can be
access article distributed under the terms developed as a chemotherapy drug because it has
of the Creative Commons Attribution 4.0 Calamondin (Citrus microcarpa Bunge) is a been known to have a cytotoxic effect. The research
International license. commodity that is widely grown in Indonesia, of Adam et al., showed that hesperidin can increase
even promoted by the One Village One Product the cytotoxic effect of doxorubicin9. The combination
(OVOP) of calamondin in Bengkulu1. However, of hesperidin and vitamin C can overcome blood
wastewater from calamondins such as peel, flow disorders and accelerate the healing of bruises10.
pulp, seeds, and extracted water has not been Hesperidin has anti-hyperglycemic potential and
processed into products which has a high sale improves heart function in mice with type two
value2. Calamondin contains vitamin C 7.3 gr, diabetes mellitus11, an antibacterial activity, such as
vitamin A 57.4 mg IU, calcium 8.4 mg, water 15.5 against Helicobacter pylori12.
gr, potassium 37 mg, and fiber 1.2 gr. Calamondin
peel contains essential oils, 0.15% ascorbic acid, The community widely likes this plant as a flavoring
and 5.5% citric acid3. Calamondin peels and leaves and scented food and drink in Indonesia13. Almost
contain about 1.2% and 0.93% volatile oil4. The all parts of the calamondin has the potential as a
medicine. The methanol extract of the leaves has
study results of Cheong et al., dichloromethane and
efficacy as an anti-inflammatory and inhibits the
hexane extracts from calamondin, identified 79
growth of E.coli, Citrobacter freundii, Aeromonas
compounds representing 98% of compounds that
hydrophila, P.aeruginosa, S.agalatiae, and Yersinia
evaporate with N-methylantranylate were the most
enterocolitica14, an antioxidant activity and has
detected compounds. Methanol extract contains a
anti-inflammatory activity15. Calamondin juice can
lot of phenolic acids, especially p-Coumaric and
stimulate appetite, improve digestive disorders,
ferulic acid5. The study results of Bhat et al., stated
reduce blood pressure and cholesterol16,17.
that calamondin contains flavonoids around 1.41
mg / 100 mL with antioxidant capacity around 777 Monographs on calamondin peels are still minimal.
mg / 100 mL and ascorbic acid around 40.2 mg / Therefore, it is necessary to standardize specific
100 mL6. The main flavonoid found in calamondin and non-specific parameters and determine levels
is hesperidin7. Hesperidin has been shown to have of hesperidin which is the identity compound of
antioxidant, anti-cancer, and anti-inflammatory calamondin. Specific parameters are aspects of

Cite this article: Husni E, Ismed F, Afriyandi D. Standardization Study of Simplicia and Extract

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of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial
Assay. Pharmacogn J. 2020;12(4):777-83.

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 Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

qualitative chemical content and quantitative aspects of the levels 5. Acid-insoluble ash: Ash obtained from determination of total ash
of chemical compounds that are directly responsible for certain content was boiled with 25 mL of 2N HCl for 5 minutes. Collect
pharmacological activities, while non-specific parameters are useful insoluble acid parts, filter using ash-free filter paper, wash with hot
for maintaining compound content, safety, and stability of extracts water, incandesce ash until the weight remains. Acid insoluble ash
to have a safety and efficacy consistency in consumers18. Because content is expressed in% w/w.
hesperidin has been shown to have antibacterial activity, so researchers
6. Alcohol-soluble extractive: Simplicia powder of about 5 grams that
are interested in testing the antibacterial activity of the calamondin peel
have been dried in the air. Put in the clogged pumpkin, add 100 mL
extract against the bacteria S.aureus, E.coli, E.faecalis, and P.auruginosa.
of ethanol-water, shake it repeatedly for the first 6 hours and leave it
P. auruginosa and E. coli represent Gram-negative bacteria whereas E.
for 18 hours. Then strain and evaporate 20 mL of the filtrate to dry
faecalis and S. aureus represent Gram-positive bacteria
in a vaporizer cup that has been incubated at 105° C until the weight
MATERIAL AND METHOD remains.
7. Water-soluble extractive: Simplicia powder of about 5 grams that
Place and time have been dried in the air. Put in the clogged pumpkin, add 100 mL
The research conducted in two months in April-September 2019. The of saturated chloroform water, repeatedly shake for the first 6 hours
preparation and extraction of calamondin peel to determinating the and leave it for 18 hours. Then strain and evaporate 20 mL of the
standardization from calamondin peel’s extract were conducted in filtrate to dry in a vaporizer cup that has been incubated at 105 ° C
three weeks at Sumatra Biota Laboratory Andalas University. until the weight remains.

Tools and materials 8. Water content: Weigh the extract which is estimated to contain 1-4
mL of water put in a dry flask. For substances that can cause sudden
The tools used in the research were Rotary evaporator (Buchi®), fluctuation when boiling add a boiling stone. Add about 200 mL of
sonicator (Elma®), analytical scales (Kern®), curs, vaporizer cup, water-saturated toluene to the flask, heat the flask carefully for 15
desiccator, chromatographic vessel, aluminium foil, microscope minutes. After the toluene boils, set the distillation to about two
(Bel Engineering®), cover and slide glass, filter paper (Whatman ™ ), drops per second until most of the water is distilled and increase the
measuring flask (Pyrex®), measuring cup (Pyrex®), UV lamps 254 and distillation speed to 4 drops per second. After all the water has been
366 nm, UV-Vis spectrophotometer (Shimadzu®), capillary tube, TLC distilled, the inside of the condenser is washed with water-saturated
Scanner (Camag®), grinder, oven (Memmert®), furnaces (1500 furnaces toluene while cleaning with a tube brush connected to a copper
Barnstead thermolyne®), micropipettes (Biokit proline®), tweezers, wire and soaked with water-saturated toluene. Continue distillation
Laminar Air Flow (LAF) (Biobase®), paper discs, autoclaves (All for 5 minutes. Cool the receiver tube to room temperature. If there
American®), hot plates, magnetic stirrers, ose needles, long bars, Petri are drops of water attached, rub the coolant tube and receiver tube
dishes, corn yarn, spatel, cotton, gauze, vortex (Etech®), plastic wrap, with rubber that is attached to a copper wire and moistened with
spiritus lamps, standard glassware. water-saturated toluene until the water drops drop. Read the volume
The materials used in the research were calamondin peel, Ethanol of water after the water and toluene separate. Water content is
(Merck®), aqua dest, hesperidin, hydrochloric acid (Merck®), chloral calculated in% v / w.
hydrate, chloroform (Merck®), toluene (Merck®), ethyl acetate (Merck®), 9. Chromatographic analysis: The material used in this work is the 60
formic acid (Merck®) ), citroborate, Silica gel 60 F254 plate (Merck®), F254 silica gel plate as the stationary phase and ethyl acetate: formic
Nutrient Agar (NA) (Merck®), physiological NaCl, Mc Farland Solution, acid: water (100: 15: 17) as the mobile phase. The test solution is an
dimethyl sulfoxide (DMSO), Test microbes: Staphylococcus aureus extract with a concentration of 5% in 2 ml of aqua dest and sufficient
ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC methanol up to 10 ml. The comparison used was hesperidin with
29212, and Pseudomonas auruginosa ATCC 27853 a concentration of 1000 µg / ml in a suitable solvent. Then, 5 µl of
the test solution and 5 µl of the comparison were made spots on the
Extracting TLC plate using capillary tubes. The TLC plate is placed on a buffer
Calamondin peel is sorted wet, then dried at 45 °C. Simplicia is made rack until the making spots station is located below and put into the
into powder using a grinder. Simplicia powder macerated using 70% vessel until it reaches the bottom absorbent layer where the spots is
ethanol for a day. Then filtered using filter paper and the filtrate was not submerged. The vessel is closed and left until the mobile phase
concentrated using a rotary evaporator, to obtain a thick extract. The propagates to the limit at the creepage distance. Then, the TLC plate
rendemen obtained is the weight percentage (w/w) between the extract is removed from the vessel and dried. Blemish detection under UV
obtained and the weight of the simplicia used. light with a wavelength of 365 nm.

Simplicia and extract standardization19,20 Quantification of hesperidin by TLC-densitometry

1. Organoleptic properties: Identifying simplicia include color, method 21
appearance, size, surface properties, texture, and shape of the cross- 1. Determination of the maximum wavelength of absorption of
section. hesperidin: 10 mg/ml hesperidin solution was determined for its
2. Microscopic characterizations: Identifying the simplicia powder maximum absorption wavelength using a UV-Vis spectrophotometer.
identification fragment using aqua dest and chloral hydrate reagents. 2. Preparation of calibration curve: Perform TLC on the comparison
3. Loss on drying: Simplicia powder of 1-2 grams is put into a weighing made in concentrations of 50, 100, 150, 200, 250, 500, and 1000 (µg /
bottle that has been measured. Smooth over the simplicia powder in mL) make spots with a volume of 5 μl each. Then, it is scanned using
the weighing bottle. Then dry it at 105 ° C in an open container using a densitometer with a maximum absorption wavelength. The curve
an oven until the weight remains. between concentration and AUC that is formed must be linear (R2>
0.99) and determine the regression equation.
4. Total Ash: The simplicia powder or extract of 2-3 grams is inserted
into the curs that has been measured, then incandesce sample until 3. Determination of Hesperidin Levels in Extracts: Scan the TLC plate
the charcoal is used up. Ash content is expressed in % w / w. that has eluted the sample with a concentration of 5% and comparison

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 Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

using a densitometer at its maximum absorption wavelength. Then The missing compound is a volatile compound such as water, essential
obtained histogram area data from hesperidin compounds in the oils, and others. It is intended that the simplicia is not easy to moldy
test solution (y). The histogram area data of the test compound is so it does not affect the chemical content of the simplicia. In addition,
entered into the regression equation y = a + bx. Thus the level of loss on drying parameters can be used to ensure the drying process of
compound (x) can be determined. This test is carried out using a plants has been carried out perfectly. Loss on drying value of simplicia
triple system. calamondin peel obtained no more than 10.76%. The next parameter
is the determination of water-soluble and ethanol extracts content.
Antibacterial activity assay for diffusion method Determination of the extract content in certain solvents provides an
1. Preparation of Nutrient agar (NA) media: Nutrient agar powder initial picture of the amount of compounds present in the sample. The
as much as 20 grams dissolved with 1 L aqua dest in Erlenmeyer, water used is chloroform saturated water to avoid microbial growth in
then heated on a hot plate using a magnetic stirrer to form a clear the water. Ethanol used is 96% ethanol. The results of the determination
solution. Next, it was sterilized in an autoclave at 121 ° C with a of water-soluble extract content and ethanol-soluble extract content of
pressure of 15 lbs for 15 minutes. calamondin peel obtained no less than 19.73% ± 0.97% and 10.26% ±
0.25%. From the results of the percentage of water-soluble and ethanol
2. Preparation of microbial suspensions: As much as one microbial
extract content, it can be concluded that secondary metabolites are
test loop was taken from pure culture and suspended into sterile
physiological NaCl, then homogeneous using a vortex. The turbidity
of the suspension is compared using the standard 0.5% Mc-Farland
on a black background and bright light. A 0.5% Mc-Farland strength
standard was created by adding 99.5% mL of 1% H2SO4 to 0.5
mL of 1.175% BaCl2 solution. The test extract was dissolved with
DMSO solution with concentrations of 15 and 20%. A total of 100
µL of the microbial test suspension was inserted into the petri dish
and then added with enough NA media to cover the lower surface
of the petri dish and then homogenized. After the solid media is
placed, a sterile blank disc that has been dropped 10 µL of the test
solution. For positive control, chloramphenicol is used. While the
negative control was used a sterile blank disc that dropped by 10 µL
DMSO. The Petri dishes were incubated at 35-37°C for 18-24 hours.
Antibacterial activity is positive if there is a barrier zone in the form
of a clear zone around the blank disc. Barriers are measured using
callipers. The smallest concentration that still shows the clear zone is
used to determine the Minimum Inhibitory Concentration (MIC).
All tests were done in triplicate.

RESULTS AND DISCUSSION

In this study, samples were used in the form of calamondin peel from
three different regions based on administrative differences (at least
districts) in West Sumatera randomly selected, namely Bukittinggi
and Padang, and Pesisir Selatan District. The sample is dried in an
Figure 1: TLC calamondin peel extract with
oven at 45 ° C for several days until the sample is dry. The use of an Eluent: Ethyl acetate, formic acid, aquadest
oven with this temperature to keep the simplicia from impurity and (100: 15: 17). Description: a: Sample
guarantee the quality22. Dry samples are then mashed using a grinder Bukittinggi; b: sample Padang; c: sample
so that the simplicia powder is obtained. Furthermore, standardization Pesisir Selatan; d: Hesperidin (comparison).
is performed on simplicia (Figure 1).
The first parameter determined is macroscopic simplicia. This
examination is carried out by directly observing the physical form of the
simplicia and extract of the calamondin peel, aiming to determine the
characteristics of the calamondin peel. The next parameter that is carried
out is microscopic observation, which is an examination to identify the
identification fragment of the simplicia powder using a microscope to
prevent the falsification of the simplicia19. The observation is carried out
using a microscope at 400 times magnification with a chloral hydrate
reagent. The addition of chloral hydrate aims to eliminate the contents
of cells such as starch and protein so that other cells can be seen clearly
under a microscope. The simplicia calamondin peel had brownish outer
surface, and the inside is yellow, slightly smelly and sour taste (Figure 2).
Microscopic examination obtained identifiers of calcium oxalate crystal
fragments, fibers, parenchyma with oil cells, ladder-shaped transport
tissue. Microscopic examination can be seen in Figure 3.
The next step is to determine the simplicia standardization parameters,
namely loss on drying. The purpose of the loss on drying parameter is to Figure 2: Macroscopic simplicia of calamondin peel.
give a range of magnitude of the lost compound to the drying process23.

779 Pharmacognosy Journal, Vol 12, Issue 4, July-Aug, 2020

 Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

found more in water solvents than ethanol solvents. So calamondin addition, the use of toluene is intended so that fats do not evaporate
simplicia contains a higher polar compound than semipolar-nonpolar when distilled because non-polar fats will dissolve in toluene which is
compounds. The result of all parameter simplicia can be seen in Table 1. also a non-polar solvent27. Water content obtained from the extract of
the calamondin peel is not more than 17.47% ± 0.82%. The result of all
The extract is made by maceration of simplicia powder by using ethanol
parameter extract can be seen in Table 1.
solvent because it is able to dissolve almost all substances, both polar,
semi-polar and non-polar in nature and can precipitate proteins and The next parameter in extract standardization is TLC. The TLC
inhibit enzymes work so that they can avoid the process of hydrolysis profile is a qualitative analysis to show the presence of certain marker
and oxidation24. Ethanol used is ethanol 70% which is commonly compounds present in the sample and to describe the composition of
used for the extraction of dry samples. Maserat is evaporated under the chemical compounds contained in the sample. The TLC profile
vacuum so that air pressure on the surface is reduced so that the boiling of the ethanol extract of calamondin peel used the comparative
point drops. This condition can reduce the possibility of decomposing compound hesperidin. It is because hesperidin is a compound that
the compounds contained in the sample at high temperatures. After is mostly contained in oranges28. In detecting patches of hesperidin,
evaporating, the extract was obtained not less than 25.33% ± 1.3%. citroborate stain reagents are used, assisted with heating and detected
The results of giving calamondin peel extract in the form of thick by UV with wavelength 365 nm. Citroborate is used as a stain-viewer
extract, special odor, black color and bitter taste. The standardization to identify flavonoid compounds29. The results of TLC calamondin
of extracts, the water content was determined. The water content in the peel extract was Rf1: 0.34; Rf2:0.43; Rf3: 0.58; Rf: 0.68 and Rf5: 0.78
extract also determines the stability of the extract, because high water while hesperidin (comparison) Rf was 0.68. From this pattern, it can
content results in the ease of bacteria, mold, and yeast to multiply so be concluded that hesperidin is found in the calamondin peel extract
that there will be changes to the extract25,26. Determination of extract (Figure 3).
water content is done by distillation. One of the chemicals that can be
used in determining the extract water content is toluene. The use of Other parameters in the standardization of simplicia and extracts are
toluene as a distillation-carrying chemical liquid because toluene has a a determination of total ash content and acid insoluble ash content.
boiling point higher than water that is 110 ° C, and toluene cannot mix Determination of total ash content aims to provide an overview of the
with water because toluene is non-polar while water is more polar and internal and external mineral content and organic matter originating
toluene has a lower specific gravity than water that is 0.867 gr / mL. In from the initial process until the extract is formed. Determination

(1) (2) (3)

(4) (5)

Figure 3: Microscopic simplicia of calamondin (1) calcium oxalate crystal (2) fibers (3) parenchyma with cells oil (4) ladder-
shaped transport tissue (5) crystalline parenchyma calcium oxalate.

Table 1: Simplicia standardization data and calamondin peel extract.

Results (%)
Sample No Standardization parameter
Pesisir Selatan Padang Bukittinggi
Specific Parameters
1 Water Soluble extractive 23.06 ± 0.44 19.73 ± 0.97 21.89 ± 0.77
2 Alcohol Soluble extractive 14.49 ± 0.38 11.08 ± 0.9 10.26 ± 0.25
Simplicia Non Specific Parameters
3 Loss on Drying 5.15± 0.06 4.83 ± 0.28 10.78 ± 0.05
4 Total Ash Content 4.33 ± 0.03 4.31 ± 0.05 4.06 ± 0.03
5 Acid insoluble ash content 0.94 ± 0.1 1.01 ± 0.07 0.95 ± 0.15
Specific Parameters
1 Hesperidin levels 4.78 ± 0.09 5.23 ± 0.23 5.58 ± 0.24
Non Specific Parameters
Extract
2 Rendemen 25.33 27.93 26.82
3 Total Ash Content 2.61 ± 0.06 4.65 ± 0.06 3.78 ± 0.43
4 Acid insoluble ash content 0.1 ± 0.02 0.12 ± 0.02 0.13 ± 0.04

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 Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

of total ash content is done by heating at high temperatures where The agar Nutrient Media is chosen because it has complete nutritional
the organic compounds and their derivatives are decomposed and content for the growth of bacteria such as peptone, NaCl, yeast extract,
evaporated at a temperature of 700 0C until only mineral and inorganic and beef extract31. The antibacterial activity test of calamondin peel
elements remain23. Total ash content obtained from simplicia and extract extract was carried out at a concentration of 20% and 15% in DMSO.
of calamondin peel is not more than 4.33% ± 0.03% and 4.65% ± 0.06%. DMSO is used as a test extract solvent because it can dissolve polar and
Ash obtained from the determination of total ash content is used to nonpolar compounds32. The negative control used is DMSO aimed to
determine acid insoluble ash content. Determination of acid-insoluble prove that DMSO which is to dissolve the test extract does not have
ash content aims to evaluate the material against contamination of antibacterial activity. The positive control used in antibacterial testing
ingredients which contain silica like soil and sand. Acid insoluble ash is chloramphenicol at 0.3%. Chloramphenicol is used as a positive
content obtained from simplicia and extracts calamondin peel no more control because it belongs to a broad spectrum of antibiotics that can
1.01% ± 0.07% and 0.13% ± 0.04%. The result of all parameter of extract inhibit the growth of Gram-positive and Gram-negative bacteria33.
can be seen in Table 2. The test is carried out three times repeated for the accuracy of the
The next parameters was determination of chemical content test and minimize errors. The bacterial suspension was made in 0.9%
(hesperidin) in the extract. It used TLC-densitometry method with NaCl solution. NaCl as a source of bacterial minerals because one of
a maximum absorption wavelength of hesperidin was 284nm. The the factors that influence bacterial growth. NaCl 0.9% is used to keep
linear regression of the hesperidin comparison calibration curve bacterial cells in an isotonic state. 0.9% NaCl used must be sterile
for the calculation of hesperidin levels was y = 15.914x - 49.778 to avoid contamination of other microbes34. Bacterial suspension
R² = 0.996 and the heperidin level of calamondin peel extract was not compared to turbidity using the Mc Farland 0.5% standard. Cell density
less 4.78%. The spectrum λ max of hesperidin can be seen in Figure 4. of Mc Farland 0.5% was 1.5 x 108 CFU / mL. This means that in the
bacterial suspension made, it is estimated that there are a number of
Furthermore, the antibacterial activity of the calamondin peel extract bacterial cells of 1.5 x108 cells in each mL of solution35.
was tested using the agar diffusion method. The principle of the agar
diffusion method is that the test substance is dripped on a blank disc, The results of the antibacterial activity test of calamondin peel
then placed on the agar media which has been inoculated by bacteria. extract from three regions namely Bukittinggi, Padang and Pesisir
The test substance will diffuse into the agar medium, if a clear area Selatan against S. aureus, E. coli, E. faecalis, and P. auruginosa at 15%
is formed around the disc on the media indicating the presence of concentration with inhibitory diameter range of 7.65 mm ± 0.36 mm
bactericidal activity by antibacterial agents on the surface of the agar to 9.96 mm ± 0.52 mm and at a concentration of 20% with a diameter
media30. range of 9.26 mm ± 0.72 mm to 13.39 mm ± 0.28 mm.

Table 2: Test data for antibacterial activity of calamondin peel extract.

Inhibition zone
Sample Bacteria
20% 15%
Pseudomonas aeruginosa 9.70±0.69 8.58±0.38
Escheria Coli 11.41±1.22 9.53±0.94
Bukittinggi
Staphylococcus auerus 10.74±0.43 8.44±0.57
Enterococcus Faecialis 10.47±0.71 8.03±1.02
Pseudomonas aeruginosa 10.46±1.43 7.65±0.36
Escheria coli 10.03±0.82 8.08±0.62
Padang
Staphylococcus auerus 10.78±0.93 7.82±0.81
Enterococcus faecialis 9.79±0.68 8.37±0.66
Pseudomonas aeruginosa 13.39±0.44 9.96±0.52
Escheria coli 9.26±0.72 7.96±0.65
Pesisir Selatan
Staphylococcus auerus 11.18±0.44 9.39±1.12
Enterococcus faecialis 10.84±0.93 8.20±1.08

Figure 4: The spectrum λ max of hesperidin.

781 Pharmacognosy Journal, Vol 12, Issue 4, July-Aug, 2020

 Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

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Husni, et al.: Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay

GRAPHICAL ABSTRACT

Calamondin
(Citrus microcarpa Bunge)

Dried

SIMPLICIA
Macerated

EXTRACTS

Profile TLC
a: Bukittinggi
b: Padang
c: Pesisir Selatan
p: Comparison (Hesperidin)

ABOUT AUTHORS
• Dr. Elidahanum Husni, M.Si, Apt.: Currently, as a lecturer at the Faculty of Pharmacy, University Andalas. Graduated from
Faculty of Pharmacy Universitas Andalas in 1986, then Master Program in 1995 at School of Pharmacy Institut Teknologi
Bandung (ITB Bandung) and Doctoral Program in Department Biomedical, Faculty of Medicine, University Andalas in 2015.
The research and expertise are Pharmacognosy.
• Dr. Friardi Ismed, Apt.: Graduated Faculty of Pharmacy Universitas Andalas, Indonesia (B.Sc. Hons.; 2004); Universite de
Rennes 1, France (Doktoral, 2012). Currently, as a lecturer at the Faculty of Pharmacy, since 2015. Research interests are
natural product chemistry and medicinal chemistry, including Sumatran medicinal plants.
• Dony Afriyandi, S.Farm: Graduate student in Faculty of Pharmacy Universitas Andalas who involved in assisting the
research and collecting data.

Cite this article: Husni E, Ismed F, Afriyandi D. Standardization Study of Simplicia and Extract of Calamondin (Citrus microcarpa
Bunge) Peel, Quantification of Hesperidin and Antibacterial Assay. Pharmacogn J. 2020;12(4):777-83.

783 Pharmacognosy Journal, Vol 12, Issue 4, July-Aug, 2020