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PUBLICATION TEXT

THE EFFECT OF BILIMBI FRUIT (Averrhoa bilimbi) ETHANOLIC EXTRACT


CONCENTRATION VARIATION AS A NYLON THERMOPLASTIC DENTURE
CLEANSER ON THE GROWTH OF Candida albicans

THESIS

By :
MUHAMMAD HIRZI NUGRAHA
11/311664/KG/08807

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION


GADJAH MADA UNIVERSITY
FACULTY OF DENTISTRY
YOGYAKARTA
2015

THE EFFECT OF BILIMBI FRUIT (Averrhoa bilimbi) ETHANOLIC EXTRACT


CONCENTRATION VARIATION AS A NYLON THERMOPLASTIC DENTURE
CLEANSER ON THE GROWTH OF Candida albicans
Muhammad Hirzi Nugraha*, Harsini**, Purwanto Agustiono**
*Program S-1 Fakultas Kedokteran Gigi, Universitas Gadjah Mada
**Bagian Ilmu Biomaterial Kedokteran Gigi, Fakultas Kedokteran Gigi, Universitas Gadjah
Mada
Abstract: Thermoplastic nylon denture are always in contact with oral cavity
promoting the growth of Candida albicans. Bilimbi fruit could be used as a denture cleanser
for its antifungal compound. The purpose of this study is to identify the effect of bilimbi fruit
ethanolic extract concentration variation as thermoplastic nylon denture cleanser against
Candida albicans growth.
Bilimbi fruit was extracted using maceration method with ethanol 70%. Sixteen (16)
disc-shaped thermoplastic nylon of 10X2 mm were made. The whole samples were soaked in
saliva for one hour and then put into a suspension of Candida albicans 108 CFU/mL for as
long as 24 hours with the temperature set at 37o C. These samples were then divided into 4
groups. Each group would then be soaked for 5 minutes in an ethalonic extract of bilimbi
with different concentrations of 0.00%, 0.05%, 0.10%, and 0.15% under a room temperature.
After being soaked, these samples were rinsed using aquades, moved into a conical tube, and
then shaken using a vortex. The cultivation of Candida albicans was carried out by taking 10
L of it and incubated as long as 48 hours under a room temperature. After that, fungi
calculation was done. The data were then analyzed using one way ANOVA and LSD0.05.
The result show that the average numbers of fungi of groups 0.00%, 0.05%, 0.10%,
and 0.15% respectively were 564.7585.496; 350.5047.592; 245.7522.036; and
141.7511.236. The One way ANOVA has showed that the effect of ethanolic extract
concentration variation as thermoplastic nylon denture cleanser against the growth of
Candida albicans (p<0.05). The test of LSD0.05 resulted a significant difference among all
groups of treatment (p<0.05). The conclusion of this study was the billimbi fruit ethanolic
extract with concentration 0,05%, 0,1% and 0,15% as thermoplastic nylon denture cleanser
have significant differences against Candida albicans growth.
Introduction
Dental caries and periodontal disease are causative factors of tooth loss 1. Tooth loss
could alter anatomical changes of oral cavity, aesthetic loss, and reduction of vertical
dimension. One of the way to restore the function of teeth are by using prosthetic using
removable partial denture2.
Thermoplastic nylon is a removable denture base generated from condensation
reaction between diamine and dibasic acid 3. This material have advantages that are higher
flexibility, better aesthethic (non-metal clasp) and could be used as an alternative for patient
that have an allergic reaction to polymethilmetacrylate 4.
Thermoplastic nylon have its drawback on poor color stability, easily discolored and
increasing roughness after weeks of use5. Denture base roughness is a factor that affect
adhesion and microorganism growth like Candida albicans (C. albicans)6.
One way to control C. albicans growth is by immersing it in a denture cleanser.
Synthetic denture cleanser chemically have disadvantages on thermoplastic nylon physical
properties as hardness change, increase of roughness and reducing color stability8. The use of
herbal material is starting to developed for economical reason and relatively low side effect.

Squeezed bilimbi fruit (Averrhoa bilimbi) is known widely as a nutritious herbal.


Bilimbi fruit could produce antibacterial power that is higher compared to bilimbi leaf 10.
Bilimbi fruit ethanolic extract contains phenolic compound like flavonoid and tannin, that
have antimicrobial potential11. Flavonoid on bilimbi fruit have the potentiality to condensate
microbial protein cell wall12. Tannin could inhibit the generation of microbes cell wall
generation13. Concentration is a factor that determined antimicrobial effectivity.
Materials and Methods
Bilimbi fruit extraction were done in Laboration of Pharmacy Biology GadjahMada
University. Small cuts of 2 kilograms of bilimbi fruit dried using oven on 24C for 24 hours.
Dried bilimbi fruit then grinded into simplisia powder, after that immersed in 70% ethanol.
Liquid extract concentrated by using water bath so that viscous extract is acquired. The
bilimbi fruit ethanolic extract were made by adding 100 mL of aquades into 0,05 grams of
bilimbi fruit ethanolic extract. The same applies for 0,1% and 0,5% concentration with the
weight of 0,1 grams and 0,15 grams respectively.
Sixteen (16) disc-shaped thermoplastic nylon of 10X2 mm were made. Wax model
implantation were done with sprue in a cuvet then injected with thermoplastic nylon, then
trimmed with cross-cut carbide bur with no polished done.
This research were done by using diluting method. Sixteen thermoplastic nylon
samples immersed in saliva for 1 hour and then moved into C. albicans suspension for 24
hours on 37oC. The whole samples were divided into four groups, immersing it into bilimbi
fruit ethanolic extract with 0% concentration on the first group, 0,05% on the second group,
0,1% on the third group, and 0,15% on the fourth group. Immersion were done for 5 minutes
at room temperature. All samples then rinsed by using aquades and moved into 16 conical
tube each contains 10 mL of aquades then vibrated using vortex for 1 minute and diluted until
10-3 then dripped onto saboraud dextrose agar inside petri dish then incubated for 48 hours on
37oC. Fungal count were done on each bilimbi fruit ethanolic extract and control.
Result
Mean and standard deviation of C. albicans fungal count after immersion in billimbi
fruit ethanolic extract on 0%, 0,05%, 0,1% and 0,15% concentrationis presented in table 1.
Table 1. Fungal Count Mean and Standard Deviation after immersion in different
concentration of billimbi fruit ethanolic extract.
Concentration
0%

N
4

Mean
564,75

Standard Deviation
85,496

0,05%
0,1%
0,15%

4
4
4

350,50
245,75
141,75

47,592
22,036
11,236

Table 1 showed the highest mean of fungal count is 564,7585,496 in 0%


concentration and the lowest mean of fungal count is 141,7511,236 in 0.15% concentration.
One way ANAVA showed p<0,05 which means there was a significant effect of
billimbi fruit ethanolic extract in different concentration level as thermoplastic nylon denture
cleanser on the growth of C. albicans. LSD0,05 test was conducted to determine the mean
difference of fungal count between treatment groups.

Table 2. The result of LSD0,05 mean difference of fungal count numbers in thermoplastic
nylon between treatment groups.
Group
BW0,05
BW0,1
BW0,15

BW0,05

BW0,1

BW0,15

BW0

104,750*

208,750*

214,250*

104,000*

319,000*
423,000*

BW0
* significant difference

Results of LSD0,05 test (Table 2) indicate that there were significant differences of
fungal count mean in thermoplastic nylon between all treatment groups with variation of
billimbi fruit ethanolic extract concentration.
Discussion
The result showed decreasing fungal count tendency after exposure of bilimbi fruit
ethanolic extract. One way ANOVA test showed the effect of bilimbi fruit ethanolic extract
concentration variation as a denture cleaner on C. albicans fungal count.
The reduction of fungal count after exposured with bilimbi fruit ethanolic extract for 5
minutes showed that bilimbi fruit ethanolic extract has an antifungal property. This result
were not different with the result of Karon et al15 stated that bilimbi fruit has an antifungal
property. Bilimbi fruit has secondary metabolite compound that is phenolic (antifungal)
consist of flavonoid and tannin11.
The mechanism of flavonoid as an antifungal is by denaturating microbes cell wall
12
protein . The outermost layer of C. albicans cell wall contains negative charged phosphate
cluster. This enable C. albicans to have the capability for binding positive charged ion16.
Flavonoid is fenol compound derivate that have OH- cluster and could donor H+ ion when
reacting water, making phenoxide and oxonium ions17. C. albicans cell wall could be
denaturated because of phenolic compound that has acidic nature bind with the outermost
layer of C. albicans cell wall. H+ ion diffused into cytoplasm because denaturated cell wall
decrease cytoplasmic pH. The reduction of cytoplasmic pH could harms microorganisms in
ways of inhibiting enzyme activities and membrane transport proteins 18.
Tannin could disturb cell wall integrity from microbe in ways of inhibiting chitin
synthesis13. Chitin synthesis need the activity of carbohydrate enzyme as
glucosyltransferase19. Chitin work to keep C. albicans cell wall integrity20.
Glucosyltransferase will be activated on meeting the substrate and cofactor 21. Cofactor from
glucosyltransferase is a divalent metal like Mn2+ 22. Tannin could bind metal ion so that the
activation of glucosyltransferase is inhibited23. The broken cell wall compromise the adhesion
of C. albicans24.
The result of LSD0,05 test (Table 2) showed that there are significant differences of
fungal count among all treatment groups (p<0,05). It happened because of the difference of
phenolic substance inside various bilimbi fruit ethanolic extract concentration 25. The increase
of antifungal concentration (phenolic) inside bilimbi fruit will give more significant effect
against fungal26. According to Pelzar and Chan14, the higher antifungal consentration will
affect the number of killed fungal.

Conclusion
According to the result of this study, it could be concluded that: bilimbi fruit ethanolic
extract with 0,05%, 0,1%, and 0,15% as nylon thermoplastic denture cleanser have
significant effect against the growth of Candida albicans.
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