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Characterization and antibacterial activity of cocos Nucifera L. Meat extract

and powder as a drug and cosmetic agent

Article in International Journal of Research in Pharmaceutical Sciences · January 2020

DOI: 10.26452/ijrps.v11i1.1864

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 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

ORIGINAL ARTICLE

INTERNATIONAL JOURNAL OF RESEARCH IN

PHARMACEUTICAL SCIENCES
Published by JK Welfare & Pharmascope Foundation Journal Home Page: www.pharmascope.org/ijrps

Characterization and antibacterial activity of cocos Nucifera L. Meat

extract and powder as a drug and cosmetic agent
Dewi Melani Hariyadi*1 , Sisunandar2 , Suciati3 , Isnaeni4 , Noorma Rosita1
1
Pharmaceutics Department, Faculty of Pharmacy, Airlangga University, Jl. Mulyorejo, Surabaya
60286, Indonesia
2
Biology Department, Universitas Muhammadiyah Purwokerto, Indonesia
3
Pharmacognosy and Phytochemistry Department, Faculty of Pharmacy, Airlangga University, Jl.
Mulyorejo, Surabaya 60286, Indonesia
4
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Airlangga University, Jl. Mulyorejo,
Surabaya 60286, Indonesia

Article History: ABSTRACT

Received on: 20.09.2019 Cocos nucifera L. or Kopyor coconut is the natural material plant that has
Revised on: 12.12.2019 nutrient content, including carbohydrates, proteins, fats and fatty acids. The
Accepted on: 18.12.2019 potential of Kopyor coconut is mainly produced from water and soft lesh or
meat. Some bene its of the meat have not been widely studied as an active drug
Keywords:
agent. This study was aimed to identify and characterize kopyor meat extract
and to test activity as an antibacterial agent. The maceration method was used
Characterization, to extract kopyor meat. The optimized extraction resulted in a yield of 23%
Antibacterial activity, ef iciency. Kopyor meat extract was identi ied in terms of loss on drying, total
Kopyor meat, ash, acid-insoluble ash, extract content and saponi ication value. Evaluation
Extract of the antibacterial activity of both dried meat kopyor and extract were con-
ducted. The standardized extract had a loss on drying of 35%, total ash of
8.95%, acid-insoluble ash of 31%, and extract content soluble in water and
ethanol of 56.9% and 0.6% respectively. The saponi ication value showed a
value of 56. It was shown that both powder and Cocos nucifera extract had
as same high activity as an antibacterial agent against Staphylococcus aureus,
Pseudomonas aeruginosa and Staphylococcus epidermidis therefore this rec-
ommended for further formulation and evaluation as drug and cosmetic.

*
Corresponding Author humans. Therefore, it is considered a versatile
Name: Dewi Melani Hariyadi plant, especially for coastal communities and is a
Phone: type of plant from the Arecaceae. The most widely
Email: dewi-m-h@ff.unair.ac.id used main component of coconut kopyor is young
coconut water and soft lesh. Some of the bene its
ISSN: 0975-7538 are kopyor coconut water for antipyretic and anti-
in lammatory activities (Mantena et al., 2003; Xiao
DOI: https://doi.org/10.26452/ijrps.v11i1.1864 et al., 2017). While the bene its of the fruit lesh
Production and Hosted by themselves have not been widely studied as medic-
inal or cosmetic ingredients. The content of the
Pharmascope.org main bigger components in the fruit lesh is hypoth-
© 2020 | All rights reserved.
esized to produce pharmacological activity as a
medicinal and cosmetic ingredient anti-oxidants,
INTRODUCTION anti-osteoporosis, antidiabetic, antineoplastic, bac-
tericidal, antihelminthic, antimalarial, leishmanici-
Cocos nucifera L. or coconut kopyor is a plant that dal, antifungal, and antiviral activities (Ajeet et al.,
almost all parts of the plant have been used by

© International Journal of Research in Pharmaceutical Sciences 611

 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

2017; Lima et al., 2015; Alleyne et al., 2005). To method. A sample of 3 ml was placed in a test tube
be able to provide great bene its in developing its and then 5 ml of methanol was added, followed by
potential as a medicinal and cosmetic ingredient, the addition of 3 ml of ammonia to a pH value of 8-9.
Kopyor coconut extract needs to be extracted with The methanol extract was iltered and 2 ml of sul-
the proper method, which needs to be identi ied furic acid was added and was shaken to get 2 lay-
and characterized by all the main components con- ers. The top layer (sulfate) was taken 5 drops, then 1
tained. The utilization was mostly still in water or drop of Dragendorf reagent was added and the for-
oil. Extracts of Kopyor coconut were hypothesized mation of orange deposits showed the presence of
and indicated for anti-itching, anti-bacterial, antiox- alkaloids (Rao and Mohd, 2016).
idant, anti-viral, analgesic and anti-in lammatory Saponin Test
drugs (Lima et al., 2015). While the bene its in cos-
metics for skin and hair care can also reduce psori- The Saponin test was carried out using the Forth
asis, eczema, soften the skin, reduce skin dryness, method by inserting 2 ml of the sample into a test
prevent wrinkles and black spots as well as anti tube and then 10 ml of distilled water was added.
UV radiation (Esquenazi et al., 2002). The extrac- The mixture was heated for 2-3 minutes and was
tion process is the irst process before kopyor being cooled down. After being shaken for 30 seconds,
tested for pre-formulation, formulation and further changes occurred. If the permanent solid foam was
activity testing. Extraction is a process of extract- formed (permanently for 30 seconds), this indicated
ing an active compound from material or simplicia the presence of saponins (Rao and Mohd, 2016).
using a suitable solvent. Extraction can be done Flavonoid Test
by various methods, according to nature and pur-
The lavonoid test was carried out using the Shinoda
pose. This research will study the identi ication,
method. A total of 0.5 ml of the sample was dropped
characterization and antibacterial activity of kopyor
on a glass preparation. Next, 3 drops of methanol
coconut extract (Cocos nucifera L.).
were added and were stirred until homogeneous.
Following that, a small piece of Mg tape was added,
MATERIALS AND METHODS
then 3 drops of concentrated HCl were added. The
formation of yellow, orange, red, or blue indicated
Cocos nucifera L. extract and dried kopyor, aquadest,
the presence of lavonoid compounds.
methanol (p.a), hexane (p.a), HCl (p.a), NaOH (p.a),
FeCl3 (p.a), ilter paper (Whatman No. 1). Phenolic Test
Sample Preparation A total of 0.50 ml of sample was dropped on a glass
preparation, then 3 drops of methanol were added
Samples of kopyor powder (Cocos nucifera Linn)
and were stirred until homogeneous, followed by
were obtained from the Coconut Research Center in
the addition of 3 drops of 5% FeCl3 . The formation
Purwokerto. Samples of this research were divided
of green, red, purple, or blue indicated the presence
into kopyor powder and extract.
of phenolic compounds (Rao and Mohd, 2016).
Extraction of Cocos nucifera L. by maceration
Flavonoid Test using Thin Layer Chromatography
method using 70% ethanol
(TLC)
Kopyor powder samples (Cocos nucifera L) were
The iltrate on phytochemical screening was plated
weighed as much as 300 grams. Samples were put
on silica gel 60 F254 plates, then it was rubbed with
in a maceration container and 70% ethanol contain-
a mixture of butanol: acetic acid: water at ratio 3:
ing 1% HCl with a ratio of 1: 4 (w/v) was added until
1: 1, then it was dried and was observed using 254
all submerged and tightly closed, followed by keep-
nm and 366 nm UV light. Furthermore, the plates
ing for 24 hours and stirring several times during
were sprayed with ammonia, were dried and were
maceration. Samples were iltered using ilter paper
re-observed with 254 nm and 366 nm UV light (Rao
Whatman No. 1 and were separated. The iltrate was
and Mohd, 2016).
then macerated again with a new solution of 70%
ethanol containing 1% HCl. This process was done Anthocyanine Content Test
three times with each time of 24 hours. The extract This method was used to test the existence of antho-
obtained was evaporated at 50◦ C and a speed of 80 cyanin. The irst method was by heating with 2M
rpm until a thick extract was obtained. HCl for 2 minutes at 100◦ C, then the sample color
Phytochemical Screening Analysis was observed. If the red color in the sample did
not change (steady), this showed the presence of
Alkaloid Test anthocyanin. The second way was by adding sam-
The Alkaloid Test was carried out by the Dragendorff ples with drops of 2M NaOH. If the red color turns

612 © International Journal of Research in Pharmaceutical Sciences

 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

blue-green and fades slowly, it was indicated antho- was calculated in %w/w.
cyanin (Anggriani et al., 2017). Determination of loss on drying
Characterization of Kopyor Extract 1 gram of extract was weighed in a weighing bot-
Moisture Content tle that has been anchored and dried at 105◦ C. The
smooth extract was lattened by tapping the weigh-
The moisture content of the extract was measured
ing bottle onto the loor. The weighing bottle was
using the Moisture Content Analyzer after the drying
inserted into the oven at 105 ◦ C. Weight up to con-
process with Freeze Dryer.
stant weights and was expressed in % w/w
Extract standardization and characterization
Saponi ication value
Determination of Ash Levels 2.5 grams of sample in Erlenmeyer 250 mL was
2 grams of the re ined test material was weighed, weighed. 25.0 mL of KOH - ethanol 0.5 NLV was
and then it was inserted into the silicate crucible, added. The mixture was continued to re lux for 30
which has been glowed and anchored. Spread it minutes. Then 1 mL of the PP indicator was added.
slowly until the charcoal runs out in the furnish tem- The excess of KOH was titrated using 0.5 N HCl. The
perature of 800◦ C. The crucible was then cooled and saponi ication value was calculated.
weighed. If the charcoal cannot be removed, enough Characterization of fatty acid content
hot water was added. Hot water was stirred and
then iltered using ash-free ilter paper. The iltrate The identi ication of fatty acid content in kopyor
was re ined along with ilter paper in the same sil- extract by the GC-MS method was conducted. Methyl
icate crucible in the 800◦ C and it was furnished to esters from fatty acids were made by dissolving sam-
a ixed weight. The total ash content was measured ples in HCl (1.5 M, 15 mL) in methanol. The sam-
and was expressed in % w/w. ple solution was then re luxed at 60ºC for 2 hours
using a water bath. Then toluene (1 mL) was added,
Determination of Non-Soluble Acid Levels the solvent was then evaporated in the rotary evap-
The ash obtained in the determination of total ash orator. The FAME reaction was puri ied with SiO2
content was boiled with 25.0 mL dilute HCl for 5 eluted with hexane / EtOAc (1/1) to obtain FAME
minutes. Insoluble parts were gathered in acid using derivatives, which were then analyzed by GC-MS.
ash-free ilter paper. The ilter paper was rinsed The GC-MS analysis was performed on GC-FID with
with enough hot water and spread the ash-free ilter Agilent Technologies 6890N and GC-MSD with the
paper into the crucible in the furnish temperature of Agilent 6973 series equipped with the Willey 7n.1
800◦ C to a ixed weight. The insoluble ash content database in the HP-5 column (30 mx 0.250 mm
in the acid was calculated against the weight of the x 0.25 µm). The temperature for GC-MS starts
tested samples and was expressed in % w/w. at 100ºC, then increased to 250 º at a rate of
Determination of Water-Soluble Extract Content 16ºC/minute, and was held for 20 minutes.
5 grams of dried powder was weighed and inserted The total plate number of dried kopyor and
into Erlenmeyer. 100.0 mL of chloroform saturated kopyor extract
water (water: chloroform = 1: 1) was added and A total of 25 grams of dried coconut was inserted
was shaken many times for 6 hours. This mixture into 225 mL of sterile saline solution, and it was
was then left for 18 hours and was iltered. The 20.0 shaken overnight on a shaking incubator on 30 ◦ C
mL of iltrate was iltered in a dish that has been milk with a speed of 150 rpm. 1 mL of suspension
anchored at 105◦ C. Heating the iltrate into the oven was pipetted, 9 mL of saline solution was added and
at 105◦ C until the weight remains and the level of vortexed, then it was made diluted to 10−4 . From 1
the water-soluble extract was calculated in %w/w. mL was pipetted and was put into sterile petridisk,
Determination of Ethanol Soluble Extract Con- 10 mL nutrient o
media was added until the tempera-
tent ture was 45 C, then was incubated at 37o C for 24-48
hours. The number of colonies was then calculated.
5 grams of dried powder was weighed and inserted
into Erlenmeyer. 100.0 mL of concentrated ethanol Antibacterial activity test
pro analysis was added and was shaken many times For microbial test preparation, a representation of
for 6 hours. This mixture was then left for 18 hours gram-negative and positive bacteria was used. Pure
and was iltered. The 20.0 mL of iltrate was iltered culture of test microbes was rejuvenated in nutri-
in a dish that has been anchored at 105◦ C. Heating ent agar sloping media. Setelang was incubated 24
the iltrate into the oven at 105◦ C until the weight hours at 37◦ C plus 10 mL of saline solution, shaken
remains and the level of the ethanol-soluble extract with a vortex to release the culture from agar.

© International Journal of Research in Pharmaceutical Sciences 613

 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

The suspension or inoculum was measured by a Characterization of kopyor coconut extract

spectrophotometer and was diluted to 25% trans- Organoleptic characterization
mittance. A total of 5 µL of suspension was inocu-
lated into 8 mL nutrient seed media at 45◦ C. It was Kopyor coconut extract after maceration and dry-
shaken and was poured over the surface of the nutri- ing with freeze dryer, the results were obtained and
ent base media, which had been compacted in ster- were then organoleptically characterized, as shown
ile petridisk. The hole was made to use a perforator in Table 3.
with a diameter of 0.8 cm and a height of 0.5 cm. Test
Table 3: Characteristics of the kopyor coconut
media was ready for use. extract
In the testing hole, 50 µL of the test solution both Parameter Observation
from the powder and kopyor extract was loaded and
Form Hard sticky
was dissolved in DMSO with a concentration of 50
mg/10 mL. For comparison, a 200 ppm standard Color Brownish
solution was used. Incubation was carried out for Smell Typical coconut
◦
24 hours at 37 C. The observed inhibition and zone
diameter were measured (mm).
Moisture Content
RESULTS AND DISCUSSION Results of the moisture content of freeze-dried
kopyor meat were 6.42%. The extract did not extract
Characterization of kopyor Cocos nucifera L. because it was a thick extract and contained high
The physical performance of the organoleptic char- amounts of oil.
acterization of fresh kopyor and dried kopyor Standardization of extracts and characterization
coconut meat, as seen in Table 1 and Table 2. Results of standardization of extracts referred to
the Indonesian Pharmacopoeia 5th edition. Stan-
Table 1: Characteristics of Fresh Kopyor
dardization was included a loss on drying, total ash
Parameter Observation content, acid insoluble ash content, water-soluble
Form Soft extract content and ethanol-soluble extract content
Color White obtained was shown in Table 4.
Smell Typical coconut
Table 4: Standardization of cocos Nucifera meat
Taste Sweet extract
Parameter of Standard- Measurement results
ization (% w/w)
Table 2: Characteristics of dried kopyor
Average ± SD
resulting from a frozen, dried process
Loss on drying 34.96 ± 0.30
Parameter Observation
Total ash content 8.95 ± 0.10
Form Hard Levels of acid-insoluble 30.94 ± 2.67
Color White yellowish ash
Smell Typical coconut Water-soluble extract 56.85 ± 0.97
Taste Sweet content
Level of a soluble extract 0.60 ± 0.20
of ethanol
Extraction
Simplicia of dried kopyor meat was extracted by the
maceration method. A sample of 272 grams of dried Saponi ication Value Test
kopyor was added by one Litre of 70% ethanol and The results of the saponi ication value are as follows
was soaked for 24 hours. The iltrate and the residue in Table 5 and Table 6. The saponi ication value was
were separated using a Buchner funnel. The residue 56.00.
was then macerated again twice. All iltrates of
extract were collected, the solvent was evaporated Characterization of fatty acid content
using a rotary evaporator until the remaining water Characterization of the kopyor extract has been car-
phase and then was dried with freeze-drying. The ried out and showed that in the derivatization pro-
extract was obtained was 62.35 grams and this was cess, the extract was not stable, produced a blackish-
equal to extract yield of 22.9 %. brown liquid. Therefore the process of identi ication

614 © International Journal of Research in Pharmaceutical Sciences

 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

Table 5: Calculation of standard solution for Table 8: MIC of standard against Pseudomonas
saponi ication aeruginosa
m KHP NaOH NaOH HCl N HCl Concentration Inhibition Zone Diameter
(99.5%); vol- vol- volume (ppm) (mm)
Mr = 204 ume ume Average ± SD
(Vt1 ) (Vt2 ) (VHCl ) (mol / L) 5.00 15.70 ± 0.96
(gram) (mL) (mL) (mL) 2.75 9.23 ± 0.95
1.0165 10.30 10.45 10.0 0.5025 2.50 8.10 ± 0.22
1.0170 10.30 10.30 10.0 0.4955 2.25 -
Average N HCl 0.4990 2.00 -

Table 6: Calculation of saponi ication value

Table 9: The diameter of the inhibition zone of
m Titration Sample Saponi ication extract sand powders against Staphylococcus
Sam- Blank titration Number aureus and Pseudomonas aeruginosa
ple
Samples Inhibition Zone Diameter (mm)
(gram) (mL) (mL)
Staphylococcus Pseudomonas
1.5205 19.80 16.75 56.15 aureus aeruginosa
1.5519 19.80 16.70 55.92
Cocos nucifera powder
Average 56.00
Positive 12.68 10.65
x 0.23 Control
RPD 0.40 Powder 14.22 12.25
sample 1
Powder 13.37 11.32
with MS GC was not carried out. sample 2
Minimum Inhibitory Concentration (MIC) test Powder 14.17 12.28
sample 3
MIC test on Staphylococcus aureus
Average 13.92 11.95
MIC test of standard against Staphylococcus aureus,
as shown in Table 7. Cocos nucifera extract
Table 7: The MIC of standard against Positive 12.14 10.88
Staphylococcus aureus Control
Concentration Inhibition Zone Diameter Extract sam- 14.25 11.45
(ppm) (mm) ple 1
Average ± SD Extract sam- 12.38 12.21
ple 2
5.00 19.03 ± 1.89
Extract sam- 13.30 13.27
2.75 11.40 ± 0.54 ple 3
2.50 10.73 ± 0.71 Average 13.31 12.31
2.25 9.42 ± 1.51
2.00 8.65 ± 2.19

The zone inhibition diameter of extracts and pow-

MIC test on Pseudomonas aeruginosa der samples against the Staphylococcus aureus and
MIC test of standard against Pseudomonas aerugi- Pseudomonas aeruginosa both showed equal activ-
nosa, as shown in Table 8. ity and signi icantly showed higher activity com-
pared to the standard solution. This showed the ini-
Antibacterial activity test on Staphylococcus tial information that the powder and extract both
aureus,Pseudomonas aeruginosa and Staphylo- have potential as antimicrobials. Antimicrobial
coccus epidermidis activity can be applied to the skin or other route
The activity results against Staphylococcus aureus administration. To further strengthen the results for
and Pseudomonas aeruginosa were presented in skin diseases, the activity of extracts and powder to
Table 9. the skin or topical disease was then conducted using

© International Journal of Research in Pharmaceutical Sciences 615

 Dewi Melani Hariyadi et al., Int. J. Res. Pharm. Sci., 2020, 11(1), 611-616

skin bacteria such as Staphylococcus epidermidis. REFERENCES

Antibacterial activity against Staphylococcus Ajeet, A., Aggarwal, B., Lamba, H., Sharma, P.
epidermidis 2017. Various Pharmacological Aspects of Cocos
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The antibacterial activity test against Staphylococ- cological Sciences, 5(2):25–30.
cus epidermidis bacteria was shown in Table 10.
Alleyne, T., Roache, S., Thomas, C., Shirley, A. 2005.
The control of hypertension by the use of coconut
Table 10: Antibacterial activity against water and mauby: two tropical food drinks. West
Staphylococcus epidermidis Indian Medical Journal, 54(1):3–8.
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3 9.80 Alviano, C. S. 2002. Antimicrobial and antivi-
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The kopyor Cocos Nucifera L. extract and powder ing, Total Flavonoid and Phenolic Content Assays
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Cocos nucifera meat extract has been character-
ized compared to dried powder Cocos nucifera.
The antibacterial activity results showed the poten-
tial of cocos Nucifera extract against Staphylococcus
aureus, Pseudomonas aeruginosa and Staphylococ-
cus epidermidis bacteria. This potential active agent
can be suggested for further evaluation for topical
disease application or other route administration.

ACKNOWLEDGEMENT

The authors are very thankful toUniversitas Air-

langgafor providing the research grant and also
thank the Faculty of Pharmacy Airlangga University
(UNAIR) for supportingresearch facilities.

616 © International Journal of Research in Pharmaceutical Sciences

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