EFFECTIVENESS OF EXTRACT PROPOLIS KELULUT BEE

(Trigonaspp) ON GROWTH OF Candida

Albicans
PutuYettyNugraha
Departement Pedodontia,Faculty of Dentistry, Mahasaraswati-Denpasar University
Email : putuyetty@gmail.com

ABSTRACT
Oral thrush is a childhood disease caused by poor oral hygiene in the child's
oral cavity so that the normal flora in the oral cavity develops into a pathogen. The main
microorganisms that cause oral thrush are Candida Albicans. Bee propolis kelulut is a
material that has anticandida active content that is, flavonoid, saponin, tannin, phenol and
alkanoid. This content can damage Candida Albicans cell membrane. The purpose of this
study was to determine the effectiveness of propolis bee extract of kelulut in inhibiting
Candida Albicans growth. This research is an experimental research with Posttest only -
control design. This test uses 24 samples with 8 treatments and three repetitions. The
treatments were extract of bee propolis larvae (Trigona Spp) with concentration of 10%,
20%, 40%, 50%, 80% and 100%, ketoconazole as positive control, and aquadest as
negative control. Inoculation of Candida Albicans on SDA medium (Sabouraud Dextrosa
Agar) concentration of 10% was 6 mm, concentration of 20% was 6 mm, concentration of
40% was 6,7 mm, concentration 50% equal to 6,7 mm, concentration 80% equal to 8, 7
mm and 100% concentration of 8 mm. Concentrations of 40%, 50% and 80% can inhibit
Candida Albicans, the conclusion of the extract of bee propolis (Trigona spp) that is most
effective in inhibiting Candida Albicans is 80% concentration.

Keywords: Extract of bee propolis kelulut (Trigona spp), Candida Albicans, anticandida

Introduction

Oral Thrush is an infected mucous membrane of the child's mouth by

Candida Albicans characterized by the appearance of whitish spots on the

entire surface of the tongue and plaques in the mouth. On Oral Thrush the

surface of the tongue is covered by white pseudomembrane which can be

lifted and leave the red area below the surface and bleed easily (Yusnita and

Julyarni, 2017). Oral Thrush is not a deadly disease for children, but if not

treated will cause discomfort and disrupt the growth and development of

children. This disease causes the child losing appetite because of the pain

experienced, the child refused to eat and drink milk so fluids and caloric

intake decreased and children will lose weight, development, and growth. This
acidic white sediment is evenly on the surface of the tongue and lips, thus

reducing the appetite of drinking in children. Oral Thrush must be treated so

as not to interfere with drinking activity in children (breast milk or bottle

milk), this infection will causes the child refuse to eat and drink milk so that

make the general condition of children’s health decreased (Aldhie, 2012).

Oral Thrush Treatment can be either topical or systemic form derived from

synthetic (chemical) drugs. Chemical drugs used, such as nystatin,

amphotericin B and clotrimazole. The use of chemical drugs can have quite

serious side effects, such as nausea, vomiting, diarrhea, seizures,

gastrointestinal infections, itching, headache, poor penetration of certain

tissues and the emergence of resistant Candida. Based on the side effects that

exist in antifungal drugs, encourage the use of alternative materials as a

substitute for chemical drugs (Hakim and Ramadhian, 2015).

Indonesia has a lot of biodiversity that has potential as a medicine. Ancient

society empirically using herbal ingredients as a cure of disease and even the

last decade the use of herbal ingredients as a drug is in great demand by the

public. Traditional medicine is used as an alternative treatment because it has

relatively small side effects, low toxicity level, easy to obtain and relatively

cheaper price when compared with chemical drugs (synthesis). Therefore it is

necessary to do a scientific research of herbal medicine related to its function

as a medicine (Wahyudi, 2010).

Medicinal herbs are medicines derived from plants that are processed in

such a way become powders, pills, or liquids which in the process there is no
addition of chemicals in it (Andareto, 2015). World Health Organization

(WHO) in 2003 suggested the use of herbal is to maintain public health,

especially in the prevention and treatment of diseases. Herbs believed to be

empirically have many efficacy and are relatively safe are propolis from bees

(Margaretta et al, 2011), propolis produced by bees is to defend themselves,

but many people use propolis as an herbal medicine that can treated various

diseases (Lutpiatina, 2015 ). Propolis that often used in the last year is

propolis of Apis spp bee, but not many people know that the propolis from bee

Trigona spp has a better content (Margaretta, 2012).

Propolis from Trigona spp bee has efficacy as antifungal, anticancer,

antiviral and antibiotic (Haryanto et al., 2012). Propolis Trigona spp has

phenol content (including Caffeid Acid and Ferulic Acid), flavonoids (which

include Pinocembrin and Galangin), propylene glycolphenolic ester (Caffeic

Acid Phenetyl Ester or CAPE), and esters. It shows that Trigona spp propolis

has the potential to act as an antifungal to suppress the growth of various types

of fungi, especially Candida Albicans and can be used as a natural antifungal

(Maharani et al., 2011).

Based on this background, the author is encouraged to conduct research on

"The Effectiveness of Propolis Bee Extract (Trigona spp) on the growth of

Candida Albicans". Therefore this research is important to make people more

easily reach and use propolis to reduce the frequency of Oral Thrush

especially in children.
 MATERIALS AND METHODS

The research method that used in this research is laboratory experimental.

The research design used is Posttest Only-control Design (Sugiyono, 2013)

The sample subjects used in this study were extracts of bee propolis

kelulut (Trigona spp) and Candida Albicans ATCC (American Type Culture

Collection) 10231.

Large Sample used in this study based on the formula of Federer (1997),

i.e.:

(n-1)(t-1)≥15

Description:

n = number of samples per treatment

t = many treatment groups

In this experiment the concentration of the ingredients is divided into

8 groups :

1. Group I: 10%

2. Group II: extract of bee propolis 20%

3. Group III: extract of 40% bee propolis

4. Group IV: honey bee extract propolis 50%

 5. Group V: extract of bee propolis 80%

6. Group VI: extract of 100% smooth bee propolis

7. Group VII: positive control (ketoconazole 0.1%)

8. Group VIII: negative control (Suspension Candida Albicans)

So the treatment (t) is 8

(n-1)(8-1)≥15

7(n-1)≥15

7n-7≥15

7n≥22

n≥22/7

n≥3

Based on the calculation above, it takes as many as 3 samples in each

treatment, so the total number of samples required is 24 samples.

1. Procedure of Making Extract Propolis Kelulut Bees

Extraction method used is maseration technique. Maseration is a simple

technique. The extraction stage are:

a) Propolis that previously refrigerated in refrigerator, put into oven

for three days with a temperature of 40 ° C.

b) Added 70% ethanol liquid as much as 2 Liters into 1000 grams of

propolis that have been put into the oven

 c) To speed up dissolution, propolis is destroyed by stirrer.

d) Let the propolis in ethanol solution for 48 hours. During that, stir

the solution every day.

e) The dried propolis is then filtered by filtration and the resultant

filter is allowed for a certain amount of time to precipitate

unnecessary substances but not dissolved in ethanol.

f) Then the rest of the filtration mixed back into a 70% ethanol

solution, and then repeat stages d and e as much as 3-4 repetitions.

2. Making Sabouraud Dextrose agar media (SDA)

Sabouraud Dextrose Agar 19.5 grams dissolved in 300 ml of aquades

using erlenmeyer. After that, homogenized with a strawer over a water

heater until it boils. The medium was sterilized in the autoclave at 121 atm

for 15 minutes. Then put in waterbath at 50ºC for ± 30 minutes. Next, pour

into a sterile petri dish volume of 20-25 ml and left at room temperature

until solidified. Incubation of total amount of medium (5%) at 37ºC for 18-

24 hours. After it is sterile, the medium is made to ± 6 mm in diameter and

the media can be directly used to test the propolis of bee moist extracts on

the growth of Candida Albicans ATCC 10231.

3. Preparation of Material Test Concentration

The extract that has been made is the extract of bee kelulut with 100%

concentration. Making this concentration using liquid dilution method by

preparing 8 bottles first. Bottle 1 contains 2 ml of propolis extract with

 100% concentration. Bottle 2 contains 0.5 ml of aquades plus 2 ml of bee

extract extract of 100% moisturizing concentration, then homogenized by

rotating to a concentration of 80%. Bottle 3 contains 2 ml of aquades plus

2 ml of bee propolis moist with 100% concentration, then homogenized by

rotating to 50% concentration. Bottle 4 contains 3 ml of aquades plus 2 ml

of bee propolis with 100% concentration, then homogenized by rotating to

a concentration of 40%. Bottle 5 contains 8 ml of aquades plus 2 ml of bee

moist propolis with 100% concentration, then homogenized by rotating to

a concentration of 20%. Bottle 6 contains 12 ml of aquadas plus 2 ml of

bee moist propolis with 100% concentration, then homogenized by

rotating to a concentration of 10%. Solution on bottle 2, bottle 3, bottle 4,

bottle 5 and bottle 6 altogether smooth to contain liquid as much as 2 ml.

Preparation of positive control solution that is by using ketoconazole 0,1%

and negative control of Candida Albicans by using aquadest.

4. Preparation of Candida Albicans suspension

Candida Albicans ATCC 10231 was taken using a heated ose and

suspended into a tube containing 0.9% physiological NaCL solution. This

suspension is compared to Mc Farland's 0.5% standard turbidity by

holding both jars at each other and turbidity compared. If the turbidity of

Candida Albicans suspension has equaled 0.5% of Mc Farland's opacity

then Candida Albicans suspension is ready for research.

5. The cultivation Candida Albicans In Saboraud Dextrose agar Media

(SDA)
The cultivation of Candida albicans uses a 10μl ratio of Candida albicans

suspension with 100 ml of bee extract moisture propolis. Enter 10 μl of

Candida albicans suspension on 7 new bottles which have been provided

then added 100 μl of bee extract of bees kelulut with concentration of 10%

into bottle 1 and homogenized, added 100 μl of bee extract of kelulut bee

with concentration 20% into bottle 2 and homogenized, added 100μl

extract of propolis bee kelulut with concentration 40% into bottle 3 and

homogenized, added 100μl extract of bee moist propolis with

concentration 50% into bottle 4 and homogenized, added 100 ml extract of

bee moist with moisture concentration 80% into bottle 5 and

homogenized, added 100μl extract of bee propolis kelulut with

concentration 100 % into bottle 6 and homogenized, added 0.1%

ketoconazole control of 100 μl into bottle 7 and homogenized.

Extracts of bee propolis kelulut with concentration of 10%, 20%, 40%,

50%, 80%, 100%, ketoconazole 0.1% control and homogenized Candida

Albicans suspension, taken as 10μl and planted into SDA media (Saboraud

Dextrose So that). Cultivation using ose that has been heated until the

color become red. Ose is applied 4 times from the tight quadrant I, then to

the second quadrant by turning again the SDA media (Saboraud Dextrose

Agar) as much as 90 º and drag the line from quadrant I then apply it with

spacing. The third quadrant is apply by turning the SDA media (Saboraud

Dextrose Agar) as much as 90 º and take the line from quadrant ll and

giving the wider distance from the second quadrant during the smearing.

The fourth quadrant that is by turning again the SDA media (Saboraud
 Dextrose Agar) as much as 90 ° and apply it by giving distance without

concerning quadrant I, II and III. After all is cultivated into SDA media

(Saboraud Dextrose Agar), then proceed by inserting SDA media

(Saboraud Dextrose Agar) into the incubator and incubated for 48 hours.

6. Conduct a measurement of Candida Albicans inhibit zone

After incubation for 48 hours, measurements were made

The production of Sabouraud Dextrose

Agar

Propolis extract from kelulut bees

Production of extract with various consentration 10%, 20%, 40%, 50%, 80%, 100%

Production of Candida Albicans 0,5% Mc Farland suspension

Liquid diffusion method

Propolis Propolis Propolis Propolis Propolis Propolis Ketokonazol Aquadest

extract of extract of extract of extract of extract of extract of 0,1% (positive (negative
kelulut bees kelulut bees kelulut bees kelulut bees kelulut bees kelulut bees control) control)
consentration consentration consentration consentration consentratio consentration
10% 20% 40% 50% n 80% 100%

Candida Albicans inhibit zone measured manually with calipers

Tabulation

Data analysis
RESULT

A. Laboratory Results

The result of Candida Albicans inoculation with SDA media (Saboraud

Dextrosa Agar) on positive control showed no growth of Candida Albicans

while in negative control showed the growth of Candida Albicans.

10%
20%
Control (-)
40%
Control (+)

50% 100%

80%

Figure 4.1. Inoculation of Candida Albicans concentrations of 10%, 20%, 40%,

50%, 80% and 100% in the first repetition

Control (-)
100%

80%
10%
Control (+)

20% 50%

40%

Figure 4.2. Inoculation of Candida Albicans concentrations of 10%, 20%, 40%,

50%, 80% and 100% in the second repetition.