Physicochemical Analyses and Antibacterial Potential of Propolis by

Stingless Bee (Homotrigona apicalis) Found in East Kalimantan,

Indonesia

Supomo

1,2 a

, Enos Tangke Arung

1,* b

, Irawan Wijaya Kusuma

and Dewi Sondari

Faculty of Forestry, Mulawarman University, Indonesia

Department of Pharmacy, Sekolah Tinggi Ilmu Kesehatan Samarinda, Indonesia

Research Center for Biomass and Bioproducts, National Research and Innovation Agency, Bogor, Indonesia

Keywords: Propionibacterium acnes, Staphylococcus epidermidis, Propolis, Homotrigona apicalis, Antibacterial

Activity.

Abstract: This study examined the antibacterial potential of Homotrigona apicalis propolis extract against

Staphylococcus epidermidis and Propionibacterium acnes, which are bacteria found on human skin that can

cause opportunistic infections. The extract was obtained through the collection, processing, extraction, and

fractionation of fresh herb samples. The antibacterial activity of the methanol extract, n-hexane fraction,

ethylacetate fraction and residual fraction, at concentrations of 2.5%, 5%, and 10% was evaluated using the

disc diffusion method, with clindamycin 0,1% and DMSO 1% as positive and negative controls respectively.

Statistical analysis was performed using ANOVA. The results revealed that both the methanol extracts and

the ethyl acetate fraction of Homotrigona apicalis propolis showed significant inhibitory effects on bacterial

growth. The greatest antibacterial activity was observed at the concentration ethyl acetate fraction of 10%,

with inhibition zones of 13.27 mm for Staphylococcus epidermidis and 12.86 mm for Propionibacterium

acnes. Importantly, Staphylococcus epidermidis exhibited higher susceptibility to the propolis extract and

fraction compared to Propionibacterium acnes. These findings suggest that Homotrigona apicalis propolis

extract has the potential to be used as an active ingredient in cosmetics targeting acne-causing bacteria,

specifically Staphylococcus epidermidis and Propionibacterium acnes. The extract demonstrated efficacy

against these pathogens suggests its potential as an alternative to conventional antibiotics.

1 INTRODUCTION

Staphylococcus epidermidis and Propionibacterium

acnes are known as commensal bacteria in human

skin which can change into opportunistic (Nakase et

al. 2014; Chessa et al. 2015). Staphylococcus

epidermidis covered various parts of the skin, while

P. acnes resides mainly at pilosebaceous skin

follicles. This microbial interplay mediated through

molecules involved in intercellular competition or

communication, may have an impact on a fine

balanced skin ecosystem. Disturbed balance or

dysbiosis significantly impacts skin health and might

initiate or contribute to events that lead to skin

https://orcid.org/0000-0002-3707-9635

https://orcid.org/0000-0002-1979-6892

https://orcid.org/0000-0002-0177-6615

https://orcid.org/0000-0002-4923-2027

disorders. One such disorder is acne vulgaris, a

multifactorial disease of pilosebaceous units of the

skin that affects adolescents (Christensen et al. 2016).

Propionibacterium acnes related significantly to

the initial stage of acne causes increasing lipogenesis

originating in sebaceous glands. It induces

inflammation and pustules on the skin (Neves et al.

2015; Blaskovich et al. 2019). Meanwhile, S.

epidermidis could act as opportunistic when it enters

the bloodstream (Nakase et al. 2014; Tabri 2019).

Skin clinic acne treatment usually uses antibiotics that

could overcome inflammation and kill such bacteria

as tetracycline, erythromycin, doxycycline, and

clindamycin (Nakatsuji et al. 2009; Doǧ an et al.

Supomo, , Arung, E. T., Kusuma, I. W. and Sondari, D.

Physicochemical Analyses and Antibacterial Potential of Propolis by Stingless Bee (Homotrigona apicalis) Found in East Kalimantan, Indonesia.

DOI: 10.5220/0013667100003873

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Medical Science and Health (ICOMESH 2023), pages 143-150

ISBN: 978-989-758-740-5

143

2017). However, these drugs' side effects are

identified as irritation and allergy and long-term

consumption of antibiotics causes resistance, organ

damage, and immune hypersensitivity (Adawiyah et

al. 2010; Tan et al. 2018; Dikicier 2019). The entire

side effect leads researchers to discover and develop

new sources for antimicrobial agents of natural

products, e.g. medicinal plants (Abdallah 2011).

Sadeek & Abdallah (2019), some phytochemical

compounds extracted from medicinal plants showed

effective antibacterial potential against multi-drug-

resistant pathogens and these compounds could be

exploited as antibacterial drugs.

Antibiotic resistance is the changing sensitivity of

microorganisms due to antibiotics, therefore, higher

concentrations are needed to inhibit the growth of

resistant bacteria compared to susceptible strains

(Galderm, 2014). A significant factor that contributes

to increasing resistance is the irrational consumption

of antibiotics and antibiotics (Walshet al, 2016).

Related to this fact other alternatives are sought to

treat infections using natural ingredients (Utamiet al,

2021). The natural ingredient mostly found as an

antibacterial is Propolis from the stingless bee

Homotrigona apicalis.

The bee Homotrigona apicalis is a species of

stingless honey-producing bee part of the

Meliponidae family and it has no sting and is small in

size (Francoy et al., 2019). These insects make nests

in tree holes, wall cracks, and bamboo cavities, a

source of food for bees in the form of pollen, nectar,

and resin, naturally. Bees are herbivorous animals

(Achyani and Wicandra, 2019) and Bee hive

homotrigone useful benefit for the health of the

human body and can produce honey, royal jelly, bee

pollen, and propolis (Syafrizalet al, 2016). This

natural bee product is known to have antibacterial,

antifungal, anticancer, anti-inflammatory, and anti-

asthmatic benefits (Campos et al., 2015; Lopez et al.,

2019; Farias et al., 2014).

It is described that propolis is one of the

substances produced by bees and consists of a

mixture of bee saliva and plant exudates that they

collect (Mardiah, 2017). Propolis is trusted as a

natural ingredient, empirically and relatively safe

completed plenty of benefits (Lutpiatina, 2015). The

common benefits of propolis are as a medicine or

supplement, mouthwash, anti-inflammatory, disease

therapy, and accelerating wound healing. Based on

Rosyidi et al (2018), it has plenty of chemical

compounds and varies depending on the environment

surrounding the bee farm, therefore, plenty of

differences in compounds in propolis in Indonesia are

found. Propolis consists of amino acids, terpenoids,

and polyphenols (phenolic acids, esters, and

flavonoids), in general (Pujirahayu et al, 2014) and

Flavonoids are one of the significantly important

ingredients in propolis which have antioxidant,

anticancer, anti-inflammatory, anti-allergic, antiviral

and antibacterial effects (Rismawati et al, 2017;

Alexandra, 2018; Hermalinda, 2019).

Lutpiatina (2015) found that propolis has

antibacterial properties, the inhibitory zone of the

ethanol extract of kelutut bee propolis (Trigona sp)

originating from the South Kalimantan area at

concentrations of 20%, 40%, 60%, 80% and 100%

against Staphylococcus aureus is 6.4 mm; 10mm;

12.6 mm; 14.4mm; 16.4mm, meanwhile,

Gusmawarni et al, (2021) concluded that the ethanol

extract of bee propolis (Trigona sp) originating from

Pekanbaru area has inhibitory zone activity against

bacterial growth Streptococcus mutans at

concentration of 40%, 60% and 80% respectively 8

mm, 9.3 mm and 11 mm. Methanol extract of propolis

shows the highest inhibitory rate at concentrations of

750 μg/mL (6 mm) and 1000 μg/mL (10 mm) against

E. Coli and Staphylococcus aureus were compared by

hexane and ethyl acetate extracts (Yusop et al, 2018).

Empirical research by Mayangsari (2013) found that

Lawang propolis extract against Fusobacterium

nucleatum obtained MIC (Minimum Inhibitory

Concentration) of 1.48% and MBC (Minimum

Bactericidal Concentration) of 1.54%.

Based on the significant benefits of propolis and

easy bee farm Homotrigona apicalis, this study

implementing bee propolis Homotrigona apicalis by

breeders at Palaran District, City of Samarinda, East

Kalimantan to identify antibacterial characteristics of

propolis methanol extract through bacteria

Propionibacterium acnes and Staphylococcus

epidermidis.

2 METHOD

2.1 Instruments and Materials

This study implemented glassware (pyrex), blender,

vortex, hotplate (Ceran®), petri dishes, spirit lamps,

autoclaves, incubators, magnetic ironer,

micropipette, analytical balance, caliper, water bath,

laminator airflow cabinet (LAF), maserator, rotary

evaporator, spectrophotometer, knife, cutting board.

Meanwhile, materials contributes for this research are

methanol, n-hexane, ethyl-acetate, bee propolis

Homotrigona apicalis, filter paper, sterile cotton,

distilled water, Muller Hinton Agar (MHA) media,

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

144

Nutrient Agar media, clindamycin 0.1%, DMSO,

NaCl 0.9%.

2.2 Procedure

2.2.1 Simplicia Setup

Propolis is taken from Tani Harapan Loa Janan, Kutai

Kartanegara, East Kalimantan, which is then wet

sorted and targeted to separate propolis from the nest

and impurities.

2.2.2 Proceed Extracts

The simplicia obtained was macerated for three days

using methanol. Remaceration is carried out on the

simplicial dregs and it is filtered to obtain macerate,

then evaporated using a rotary evaporator and

evaporated over a water bath until a thick extract is

obtained (14).

2.2.3 Fractionation

Weighed 5 grams of extract and partitioned using

distilled water and n-hexane with a ratio of 1:1 v/v.

The sample was shaken repeatedly in a separating

funnel until homogeneous and left until a layer of

water and a layer of n-hexane were formed. The n-

hexane layer was collected in a different container.

The n-hexane layer was then heated over a water bath

until thick and the n-hexane fraction was obtained.

The water layer was partitioned over using an

ethylacetate completed ratio of 1:1 v/v. the following

phase is shaken in a separating funnel until

homogeneous, left to stand until two layers are

formed, namely the water layer and the ethyl acetate

layer. Each layer is collected into a different

container, heated until thick and the ethyl acetate

fraction and residual fraction are obtained

(Mujipradana, 2018)

2.2.4 Phytochemical Screening

Alkaloid

0.5 g sample was added to 1 mL of 2 N hydrochloric

acid and 9 mL of distilled water heated over a water

bath for two minutes and finally cooled and filtered.

The filtrate obtained was used for alkaloid testing.

Take 3 test tubes, add 0.5 mL of filtrate to each tube.

Add 2 drops of reagent of each test tubemayer,

bouchardat and dragendorf. Alkaloid is positive if

precipitate occurs and at least 2 of the 3 reagents

above are positive then sample is defined to contain

alkaloids, namely the formation of white precipitate

at reagent mayer, brown at reagent Bouchardat and

brick red deposits on the fixer dragendorf

(Handayaniet al, 2019; Nafisah et al, 2014).

Saponin

0.5 g sample is put into a test tube then 10 mL of hot

water is added, cooled briefly after cooling, and

shaken vigorously for 15 minutes, if a stable foam

form for 10 minutes and the foam is 1-10 cm high and

when dripped 1 drop 2 N hydrochloric acid foam is

still present, then the sample contains saponin

compounds (Supomo, et al, 2019).

Flavonoid

1 gram of sample is added to 10 ml of hot water then

boiled for 5 minutes, filtered while it is still hot. 5 mL

of the filtrate obtained was taken then 0.1 g of

magnesium powder, 1 mL of HCl, and 2 mL of amyl

alcohol were added, then shaken and allowed to

separate. Samples contain flavonoids if there is a red

color change at the amyl alcohol layer (Supomo et al,

2019).

Tannin

1 mL sample solution is reacted with 10% iron (III)

chloride solution, if dark blue, blackish blue or

greenish black color occurs, it indicates the presence

of tannins (Supomo et al, 2019).

Phenol

Several dissolved samples were extracted by 20 mL

of 70% ethanol. 1 mL of the resulting solution was

taken and then 2 drops of 5% FeCl

were added. A

positive reaction is indicated by the presence of a

green or blue-green color (Anisa et al, 2022).

Quinones

5 mL sample of the experimental solution obtained

from the identification of flavonoids was put into a

test tube, and a few drops of 1 N NaOH solution were

added. The formation of a red color indicates the

presence of quinine group compounds (Lestari and

Andriani, 2021).

2.2.5 Antibacterial Testing

Making positive and negative controls

This study used Clindamycin as a positive control of

0.1% and DMSO as a negative control of 1%.

Preparation of extract and fraction concentration

series

This study used a series concentration of methanol

extract, n-hexane fraction, ethyl acetate fraction and

residual fraction of propolis, namely 2.5%, 5%, and

10% completed calculations (w/v), using 1% DMSO

as a solvent.

Physicochemical Analyses and Antibacterial Potential of Propolis by Stingless Bee (Homotrigona apicalis) Found in East Kalimantan,

Indonesia

145

Sterilization of tools

The first phase is using sterilized glassware for

antimicrobial activity research at an autoclave at 121

ºC for 15 minutes, the tweezers were burned by

burning over a direct flame (Mujipradhana et al,

2018).

Slanted agar media preparation

Weighed 5 g of Nutrient Agar (NA), dissolved in 250

mL of distilled water (20 g/1,000 mL). The

homogenized media was then sterilized in an

autoclave at 121˚C for 15 minutes. Pour 5 ml of NA

media into a test tube, let it sit Nutrient Agar at room

temperature till preparation solidifies at a 45-degree

tilt position for 10 minutes.

Bacterial rejuvenation

Bacteria Propionibacterium acnes and

Staphylococcus epidermidis swabbed into slanted

agar media, and incubated in an incubator at 37ºC for

2x24 hours.

Preparation of Mac. Farland solution

Solution H

1% as much as 0.25 ml is mixed

completed using BaCl

solution

1% as much as 0.1

gram in an Erlenmeyer. Followed by shaking until a

cloudy solution is formed, then the turbidity standard

is measured with a spectrophotometer.

Preparation of bacterial suspensions

The test bacterial were taken ± 1 cycle then suspended

in a tube containing 10 ml of 0.9% NaCl solution. The

turbidity of the solution was then measured using a

spectrophotometer uv-vis with absorbance that has

been determined with standard solutions Mac.

Farland at a wavelength of 600 nm

(Mujipradana,2018).

Antibacterial Activity Test of methanol extract, n-

hexane fraction, ethyl acetate fraction and residual

fraction of propolis

The paper discs were soaked in methanol extract, n-

hexane fraction, ethyl acetate fraction and residual

fraction of propolis with concentrations 2,5%, 5%,

and 10%, clindamycin 0.1% as positive control, and

DMSO 1% as negative control for 3 minutes. Prepare

72 petri dishes, then pour in 20 mL of MHA media,

then let it solidify. Dip a sterile cotton swab into each

Propionibacterium acnes and Staphylococcus

epidermidis suspension, while it is absorbed, lift the

stick and squeeze it by pressing against the wall of the

tube. One hundred microliter bacterial suspension

was taken using a micropipette and placed in a petri

dish containing MHA media, then smeared using a

sterile cotton swab until the entire surface of the agar

media was tightly covered and left for 5-15 minutes,

therefore, bacterial suspension seeped into the agar

medium. The soaked paper disc is placed on the

surface of the MHA media which has been inoculated

with bacteria. The petri dishes were left at room

temperature for an hour before being incubated at

37°C for 24 hours and this test was repeated three

times (Pratiwi, et al., 2016). Antibacterial activity

showed by Inhibition Zone Diameter (mm).

MIC (Minimum Inhibitory Concentration) and

MBC (Minimum Bactericidal Concentration)

While observing the disc paper test, we could see an

inhibition zone that forms around the disc paper and

after obtaining the inhibition zone, the concentration

range of the inhibition zone is used to determine the

MIC and MBC using the solid dilution method.

Variations in solid dilution concentration were made

based on the smallest concentration which still

provided an inhibition zone for the antibacterial

potential test. Test bacterial suspensions and extracts

that have been dissolved according to varying

concentrations are inoculated for flat in MHA media

completed ratio of bacterial suspension, such extract

(1:1) incubated at 37°C for 24 hours. Meanwhile, the

clearest media is test media completed by the smallest

concentration accompanied by no bacterial growth

designated as MIC. The results determined as KHM

are confirmed by doing the results streak on MHA

media and incubated at 37°C for 24 hours. On the

results streak observed based on the turbidity of

bacterial growth at media and when the media still

looks clear then the results are designated as MBC

(Murtiwi, 2014).

3 RESULT AND DISCUSSION

The nest criteria of propolis use at this study which

having brittle texture and dark color, taken from Desa

Tani Harapan Loa Janan, Kutai Kartanegara, East

Kalimantan. Propolis is extracted using methanol.

The secondary metabolite groups testing were aimed

to determine the presence of secondary metabolites in

natural material samples. The results of

phytochemical screening tests on total methanol

extract of propolis can be seen in Table 1.

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

146

Table 1: Phytochemical Screening Test Result.

Com

ounds Test Result

Alkaloid

(

)

Bouchardat

(

)

Dragendorf (-)

Saponins HCl 2N (+)

Flavonoid

HCl + Mg + Amyl

Alcohol

(+)

Tannin FeCl

(+)

Fenol FeCl

5% (+)

Quinone NaOH 1 N (+)

Notes:

(-) = Negative result

(+) = Positive result

Groups of compounds which are suspected of

being potential antioxidants in the ethanol extract of

propolis are including flavonoids. Alkaloid, saponin.

Tannin, fenol and quinones. Flavonoid compounds in

their structure contain hydroxyl groups that can

donate hydrogen atoms to free radicals, so flavonoid

compounds have the potential as antioxidants.

Flavonoids are reducing compounds that can inhibit

many oxidation reactions. Moreover, flavonoids have

the ability as antioxidants since they can transfer an

electron to free radical compounds as well as

quinones (Ridho, 2013).

The method is based on disc diffusion for

antibacterial activity. Paper discs containing the

antimicrobial agent methanol extract of propolis were

placed on MHA media which had previously been

planted by bacteria Propionibacterium acnes and

Staphylococcus epidermidis, paper discs that have

been soaked in antimicrobial agents from methanol

extract of propolis will diffuse on MHA agar media.

The antibacterial test results on Propionibacterium

acnes and Staphylococcus epidermidis completed

various concentrations, the inhibition zone is obtained

shown at table 2.

Identified at antibacterial test, the enterely

variations in concentration showed that the propolis

extract and fraction had a very active inhibitory

response to bacterial growth Propionibacterium

acnes and Staphylococcus epidermidis. Based on

measurements of the result bacterial inhibition zone,

it shows that the bacteria Staphylococcus epidermidis

which is a type of gram-positive bacteria has a larger

inhibition zone than bacteria Propionibacterium

acnes.

Based on Table 2, seems that the concentration of

propolis methanol extract has the largest inhibitory

zone diameter against the bacteria Staphylococcus

epidermidis namely a concentration of 10% which

has an average inhibitory diameter of 0 mm and the

smallest concentration at 2.5% is 9.63 mm.

Table 2: Antibacterial Activity Test Results Average Zone

of Inhibition (mm)

Bacteria Treatment

Average Inhibition

Zone Diameter (mm)

2,5% 5% 10%

P. acnes

Extract 9,34 9,67 10,17

n-Hexane

Fraction

11,83 12 12,67

Ethylacetate

Fraction

11,87 12,26 12,86

Residual

Fraction

7,03 8 9,16

Clindamycin

0.1%

22,27

DMS0 1% 0

epidermidis

Extract 9,63 9,86 10

n-Hexane

Fraction

11 11,67 13,17

Ethylacetate

Fraction

11 11,85 13,27

Residual

Fraction

8,35 9,21 9,78

Clindamycin

0.1%

22,27

DMSO 1% 0

Table 2 shows the inhibitory power of propolis

extracts and fractions on bacterial growth of

Propionibacterium acnes and Staphylococcus

epidermidis has a different level of sensitivity.

Research finding by Saputera, et al (2016), the higher

the concentration of the extract used, the larger the

inhibition zone produced and it related significantly

through research results of each concentration of

2.5% < 5% < 10% completed diameter response of

the inhibition zone being successively larger.

That empirical fact shows that the methanol

extract and fractions of propolis have antibacterial

activity against acne-causing bacteria. It related

significantly to previous research by Abdullah et al,

that propolis from bees (Heterotrigona itama) from

Brunei Darussalam has antibacterial activity from 20

gram/L propolis extract against bacteria

Pseudomonas aeruginosa dan Staphylococcus

aureus. The antibacterial activity is thought to be due

to the presence of chemical compounds at the

methanol extract of propolis, according to Hotnida et

al (2011), the chemical content contained in propolis

could differ between regions, places where propolis

is honeycombed and its biological activity. It

described the flora ecosystem surrounding which

influences the chemical content of propolis.

Based on the results of phytochemical screening,

the methanol extract of propolis contains alkaloids,

saponins, tannins, flavonoids, phenols, and quinones.

Physicochemical Analyses and Antibacterial Potential of Propolis by Stingless Bee (Homotrigona apicalis) Found in East Kalimantan,

Indonesia

147

Alkaloids have an antibacterial mechanism by

inhibiting the peptidoglycan components in cells so

that the cell walls are not intact and cause cell death

(Riyanto et al, 2019). The mechanism of flavonoids

as antibacterials is by forming complex compounds

with extracellular and dissolved proteins, causing

phospholipids to be unable to maintain the shape of

the bacterial cell membrane, as a result, the bacterial

cell membrane will leak and the growth of the

bacteria will be hampered until death (Malanggi et al,

2012). Based on find research of Hotnida et al,

(2011), types of flavonoids found in propolis are

pinocembrin, respectable, quercetin, pinostrobin,

kaempferol, pinobaxin.

The mechanism of tannin as an antibacterial is to

disrupt peptidoglycan could be proved that cell wall

formation becomes imperfect and causes bacterial

cells to lyse, while the mechanism of action of

saponin is to reduce the surface tension of the

bacterial cell wall, resulting in

increased permeability or cell leakage causing

intracellular compounds to come out (Malanggi et al,

2012). The mechanism of action of high

concentrations of phenol as an antibacterial is by

penetrating and disrupting bacterial cell walls and

precipitating proteins in bacterial cells. Phenol in

lower concentrations inactivates important enzyme

systems in bacterial cells (Purwatiningsih et al 2014).

The mechanism of action of quinones as

antibacterials is by forming complex compounds that

have properties irreversible completed by

nucleophilic amino acid residues on plasma

transmembrane proteins, cell wall polypeptides, and

enzymes found on the surface of cell membranes,

thereby disrupting the life of bacterial cells (Sapara et

al, 2016).

The SPSS statistical tests show the data tested

implemented test Kolmogorov Smirnov normally

distributed at normality test, namely completed

significance value (p>0.05). meanwhile, the

normality test on the data Pseudomonas aeruginosa

shows a significance value of 0.810 while in

Staphylococcus aureus The significance value is

0.895. The normality test with normally distributed

data is a requirement for carrying out further tests on

One Way ANOVA.

Meanwhile, homogeneity of variance test shows

the data is homogeneously related to each

significance value of Pseudomonas aeruginosa data

(p>0.005) p = 0.054, and the significance value of

Staphylococcus aureus data shows p= 0.081.

Homogeneity testing completed homogeneous data is

requirements for carrying out the One Way Anova test

and while One Way Anova at test results shows

Pseudomonas aeruginosa and Staphylococcus aerus

of each research data indicated each significance

value (p<0.05), namely p= 0.000 and it concluded

there are no similarities in each treatment group or

each concentration. This finding related significantly

to the hypothesis which described that there are

differences in the effectiveness of the antibacterial

power of the methanol extract of propolis of each

concentration on bacterial growth Pseudomonas

aeruginosa and Staphylococcus aureus.

The following test carried out after this LSD

which shows the antibacterial activity of propolis

against bacteria Pseudomonas aeruginosa, shows

significant differences at the entire concentrations

and there was a significant difference between the

antibacterial activity of propolis extract and the

positive control clindamycin. Meanwhile, bacteria

Staphylococcus aureus there was no significant

difference in the inhibition zone between

concentrations of 2.5%, 5%, and 10%. The zone of

inhibition of all propolis extract concentrations was

significantly different from the positive control

clindamycin.

MIC (Minimum Inhibitory Concentration) and

MBC (Minimum Bactericidal Concentration)

Determination of MIC (Minimum Inhibitory

Concentration) and MBC (Minimum Bactericidal

Concentration) used solid dillution method. This

main advantage method is more practical because one

concentration of the test antimicrobial agent can be

used to test more than one test microbe (Supomo et

al, 2021). The MIC and MBC testing targeted to

determine the number of doses of propolis methanol

extract that could inhibit and kill bacteria that cause

infection.

Based on finding research Nadhilla (2014),

antibacterial substances are divided into two, namely

bacteriostatic and bactericidal. Antibacterials that

have bacteriostatic activity are substances that could

inhibit the growth of bacteria, while bactericides are

substances that can kill bacteria. Variations in the test

concentrations of propolis extract and methanol

fraction were 2.5%, 5%, and 10%.

Table 3: Bacterial Growth After 1x24 hours.

Concentration

(%)

Bacteria

P. acnes S. epidermidis

2,5 --

5 - -

10 - -

Note : (-)=No bacterial growth

(+)=There is bacterial growth

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148

Results of the first incubation show that there was

no bacterial growth at all concentrations and indicated

that MIC is the smallest concentration that could

inhibit bacterial growth, thus the smallest

concentration is 2.5% without bacterial growth

Propionibacterium acnes and Staphylococcus

epidermidis. The empirical finding shows the MIC of

methanol extract of propolis for bacteria

Propionibacterium acnes and Staphylococcus

epidermidis (w/v) is 25 mg/mL. Next, a confirmation

test was carried out by streaking again on the media

with the bacterial suspension Propionibacterium

acnes and Staphylococcus epidermidis then incubated

for 1 x 24 hours at 37°C in a row.

Table 4: Bacterial growth after re-streaking.

Concentration

(%)

Bacteria

P. acnes S. epidermidis

2,5 - -

5 - -

10 - -

Note: (-) = No bacterial growth

(+) = There is bacterial growth

Table 4 shows that after re-streaking the media

remains clear or there is no bacterial growth at all

variations in concentration, then MBC is the

minimum concentration that could kill bacteria at a

concentration of 2.5%.

4 CONCLUSIONS

Research indicated that bee propolis extracts and

fractions of Homotrigona apicalis can inhibit

bacterial growth. Methanol extract of propolis has

inhibitory power against bacteria Propionibacterium

acnes and Staphylococcus epidermidis at

concentrations of 2.5%, 5%, and 10%. MIC and MBC

of propolis methanol extract on bacteria

Propionibacterium acnes and Staphylococcus

epidermidis of 25 mg/mL. Propolis extract has the

potential to be an active ingredient in cosmetics as an

antibacterial Propionibacterium acnes and

Staphylococcus epidermidis cause of acne.

ACKNOWLEDGEMENT

Special thanks to Mr. Prof. Enos Tangke Arung, MP,

Ph.D., as the Promoter who has provided a lot of

direction, guidance, motivational contribution, and

very valuable suggestions. Mr. Prof. Irawan Wijaya

Kesuma, MP, Ph.D. and Mrs. Dr. Dewi Sondari, M.Si

as the promoter who guided and directed the author.

Lecturers and staff of the Forestry Science Doctoral

Study Program, Faculty of Forestry, Mulawarman

University.

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