Asia Pacific Dental Students Association Journal - APDSJV3N2

Asia Pacific Dental Students Journal | Volume 3| Number 2| June 2012
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EFFECTIVENESS OF ELLAGIC ACID THAT

CONTAINS IN STRAWBERRY FOR ACRYLIC
DISCOLORATION
D. M. Larasati1, K. N. Firsty2, M. Yogiartono3
Airlangga University, Indonesia
Asia Pacific Dental Students Journal 2012
ABSTRACT
Background: Strawberry has ellagic acid, which contains ellegitanin for teeth
whitening. While acrylic‟s properties is absorbing water slowly in a period of time, the
mechanism of absorption through the diffusion of water molecules according to the law
of diffusion. The liquid absorption in the acrylic resins is one of the factors causing
discoloration of the acrylic resin.
Objectives: The aim of this study was to determine the effectiveness of Ellagic acid
that contained in Strawberry for acrylic discoloration.
Methods: This research was an experimental in-vitro study of heat cured acrylic plate
samples of 26 mm of diameter and 0,4 mm of thickness examined under controlled post
test group design. The sample consisted of 12 acrylic plates divided equally into two
groups. The acrylic plates as sample was soaked in distilled water and strawberry juice
for 8 hours. An optical photodiode sensor was used in observing the discoloration
occured. The data were tested using one-way ANOVA which a significancy of 95% ( p
< 0,05 ).
Result: There were significant difference of acrylic plates color between 2 samples
groups ( p = 0,005 < 0,05 ).
Conclusion: These results suggest that strawberry influenced the acrylic discoloration.
Keyword: Strawberry, Ellagic acid, Acrylic, Discoloration.
INTRODUCTION
The material base of artificial tooth that is often used is polymethyl methacrylate, acrylic
resin type of heat cured. Acrylic Resin is used as a base material for artificial tooth
because it has traits do not accumulate, no irritation, no liquid is soluble in the mouth,
well, easily manipulated, easy for reparation and small dimensions change.1
Disadvantages of acrylic resin that is easily broken when falling on a hard surface or
due to fatigue ingredients because of long usage and changes color after several times
used in the mouth.2
3
Based on the results of scientific research shows that the benefits of strawberry for the
health was known. And people who consume strawberry get benefit because a lot of
nutrition by and maintain heart health. The strawberry is among others minerals, fiber,
vitamin C, potassium, folate and others. In addition there is also a red pigment
Anthocyanin compounds in strawberry can reduce blood pressure, ellagic acid a
compound phenol which can inhibit and prevent the growth of cancer and as an anti
inflammatory.3
These fruits contain elagat acids (ellagic acid) and acid malate (malic acid) that can
whiten teeth.4 Part of the strawberry plant that can be used to whiten teeth is the fruit
and leaves. The reaction occurs in the compound is ellagic acid oxidation in which
electrons that can be associated with a substance which causes change of the color on
enamel. There is a difference electronegative between O and H in the hydroxyl OH- , it
bigger than CO-and OH-COOH group causes the OH- would be easier to break and
produces H+ radical. H+ radical is formed then bound to a molecule 3 C tertiary that is
contained on the enamel of teeth that are experiencing discoloration. This bond led to
disruption of electron conjugation and the change of the absorption of energy at an
organic molecules, then organic molecules are formed, so that the enamel is the
unsaturated structure. After radical H+ is released , ellagic acid released 4 radical OH-
that can disturb structure unsaturated of the enamel into a structure saturated with
lighter color.5
From the explanation above, research is conducted to determine the discoloration
acrylic resin heat cured after soaked in strawberry juice for 8 hours. The time of soaking
is determined based on assumptions by someone who drinking strawberry juice. Once
drinks strawberry juice , assumed for 5 minutes , once in a week. The aim of this study
was to determine the effectiveness of Ellagic acid that contained in Strawberry for
acrylic discoloration.
MATERIAL AND METHODS

This research is experimental laboratory research. Measurement of discoloration is done
in the laboratory of Optical Physics Faculty of Science and Technology Airlangga
University. This research was conducted in the year 2012. This research was an
experimental in-vitro study of heat cured acrylic plate samples of 26 mm of diameter
and 0,4 mm of thickness examined under controlled post test group design.6
The manufacture of acrylic plate sample is as follows. First, prepare a cuvette that has
been covered by vaseline. Gypsum is stirred by comparison powder and water 100 gr :
30 mls (according to manufacturer's instructions) are contained within the cuvette.
Then, master model of metal brass, the form of cylinder with a diameter of 26 mm and
thick 0.4 mm3, is covered by vaseline and placed on the gypsum with the horizontal
position. After the gypsum in the lower part is hardened, cuvette surface of the gypsum
and master model is covered by vaseline. Then the cuvette opponents is fitted and
poured by gypsum dough while is placed on top of a vibrator. Then closed the cuvette
and pressed, wait until the gypsum is getting harder.
4
After gypsum hardened, opened cuvette and took out the master model. Then cleared
the Mold and covered it by a material separation (could mould seal) by using a brush
and wait until dry.
Put acrylic resin heat cured by comparison powder : a liquid = 23 mg : 10 mls
(according to manufacturer's instructions) in a porcelain pot and stirred, then closed the
porcelain pot. After reaching dough stage, filled the mold by the dough and closed with
the opponents , then pressed it with pres. Then opened the cuvette and excessed acrylic
drawn using the knife models. Next, closed the opponents and repressed cuvette with
pres, up to 22 kg/cm2 pressure Hg. with Emphasis on pres, repeated it twice until no
excess acrylic, then pressed with a clamp and pres ready for brewing. The heating
process by raising the temperature of the room temperature to 100 C for 20 minutes.
After the cuvette was getting colder then opened the cuvette and took out the acrylic
plate from cuvette , excess acrylic discarded and crushed with paper rub No. 0 under the
flow of water. Drying the acrylic plate which was smooth and no porus.
The sample consisted of 12 acrylic plates divided equally into two groups. The acrylic
plates as sample was soaked in distilled water and strawberry juice for 8 hours. The
solution of strawberry is made from 15 strawberries which is blended, then filtered the
pulps.
Discoloration is affected by the intensity of the light which is transmitted, one way to
observe the change of color is using the optical sensor photodiode, which is consist of
laser He-Ne, photodetector type OPT 101 and digital microvolt. He-Ne Laser as a light
source is directed to Photodetector type OPT 101 then the light is passed on to the
surface of the sample that is observed. Photodetector type OPT 101 is linked with the
digital microvolt. Digital microvolt reading intensity that was changed to voltage, the
more incoming light, the intensity of the received will increase and voltage on the
microvolt digital has also increased. When light enters a little, the intensity of the
received will decrease and the voltage on the digital microvolt will also decrease.
Changes the intensity of the reflected by He-Ne laser and accepted by fotodetector OPT
type 101.
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RESULTS
The Results of measurement the discoloration acrylic plate after soaking in strawberry
and distilled water for 8 hours.
Distilled Water Strawberry
6,40 mV 4,30 mV
6,50 mV 5,30 mV
6,10 mV 5,00 mV
6,80 mV 5,90 mV
6,40 mV 6,10 mV
6,00 mV 5,40 mV
First, the result is tested by Kolmogorov-Smirnov test to analysis normality of

the data. Test results can be noted that the whole group has a probability value bigger
than 0.05 (p < 0,05).,which means that the entire group of normal distribution. Then the
data were tested using one-way ANOVA which a significancy of 95% ( p < 0,05 ).
ANOVA
Millivolt
Sum of Squares df Mean Square F Sig.
Between Groups 3.203 1 3.203 12.779 .005
Within Groups 2.507 10 .251
Total 5.710 11
Based on the results of the test data analysis by using one-way ANOVA, There were
significant difference of acrylic plates color between 2 samples groups (p = 0,005 <
0,05).
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DISCUSSION
The Research has been done in acrylic plate which is soaking in strawberry juice and
distilled water for 8 hours. The research results show the discoloration that occurs
meaningful statistical, it may imply that there is a change of color on the acrylic resin
heat cured.
The principle of measurement of the experiment is using the difference in the intensity
of the light, in this case is assumed to be proportional to the value of the digital
microvolt. It is obtained from the motion of the electrons from the cathode to the anode
due to the existing differences in light intensity in photoelectric effect. The movement
of electrons can be known from the electric voltage. When the light is reflected more
than transmitted, then the values in the digital microvolt will be decreased.
Acrylic plate that is soaking in the strawberry juice obtained lower average value than
soaking acrylic plate in the distilled water. This because, the properties of the acrylic
that can absorbs colourants on strawberry fruit so that the color is darker than before.
Beside containing elagat acid (ellagic acid) and malate acid (malic acid) that can
whiten teeth, there is anthocyani which is flavonoid components, anthocyani gives red
pigment in Strawberry.3 So, the acrylic plate not only absorbs elagat acids and malate
acid that can whiten teeth , but also absorbs anthocyani that give red color in
strawberry. From the explanation above, the acrylic plate that is soaked in strawberry
juice is darker than soaked in distilled water. There is no difference color when viewed
with eye but when measured using optical sensors photodiodes, red rays from acrylic
plate that is soaked in strawberry juice less than acrylic which is soaked with distilled
water.
Acrylic plate discoloration can be caused by the absorption of liquids because of the
ability of this materials and the place around the acrylic tooth, so absorbed substances
can react with elements in acrylic resin.7 Discoloration acrylic plate not only caused by
submersion in a disinfectant solution but also because of daily food and drink that is
consumed by artificial tooth users, for example tea , coffee , drink containing colas
causing shade acrylic plate and make it dark.8
Acrylic resin materials having properties of absorbing water slowly in a period of time
with the mechanism of absorption according to the law of diffusion. 2 While the
absorption of a liquid in Acrylic resin is one of the factors causing discoloration on
acrylic resin.7
CONCLUSIONS
Based on the results of this research, can be concluded that Strawberry juice
influenced the acrylic resin heat cured discoloration. The author suggests to do more
research about the influence of strawberry for acrylic teeth.
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REFERENCES
1. Combe EC. Notes on dental material. 6th ed. Edinburg: Churchill Livingstone;
1992. p. 26–161.
2. Annusavice, K.J., 2003. Philips Science of Dental Materials, 11th ed.
W.B.Saunders Co., St Louis. Missouri.
3. Kurnia, Agus. 2005. Petunjuk praktis budidaya Stroberi. Tangerang :
Agromedia Pustaka.
4. Karina Timmel. Whiten your teeth the natural way. 2006. Available from:
http://www.healthmagazines.com. Accessed on April 28th, 2012.
5. Sarah Reksadiputro. Efek jus buah stroberi terhadap pemutihan kembali
permukaan email gigi. Skripsi. Jakarta: Universitas Indonesia; 2004.
6. McNeme SJ, Von Gonten AS, Woolsey GD. Effects of laboratory disinfecting
agents on color stability of denture acrylic resins. J Prosthet Dent 1991; 66:
132–136.
7. Crispin BJ, Caputo AA. Colour stability of temporary restorative materials. J
Prosthet Dent 1979; 42: 27–33.
8. Hanoem EK. Perubahan warna resin akrilik heat cured dan cold cured karena
minuman Coca-cola. Tesis. Surabaya: Universitas Airlangga; 2001
9. Pudjianto. Karakterisasi detektor cahaya fotosel. Surabaya: Petunjuk Praktikum
Fisika Optika, FMIPA Universitas Airlangga; 1996. h. 16-20.
10. Grieve M. Strawberry. 1995. Available from: http://www.botanical.com.
Accessed on April 28th, 2012.
9
DLINGO (Acorus calamus L) EXTRACT AGAINST

Candida albicans ISOLATES THE MAXILLARY
REMOVABLE FULL DENTURE ACRYLIC
Aisa Nirmala Setyani1, Felisha Febriane Balafif2, Revini Nuita3,
Wartadewi4
Universitas Padjadjaran, Indonesia
ABSTRACT
Objective : to obtain the value of minimum inhibitory concentration (MIC) of
ethanol extract rhizome of dlingo against Candida albicans isolates the maxillary
removable full denture acrylic.
Methods : This in vitro study method was conducted laboratory experimental with
ten samples tested by the method of serial dilution ethanol extract rhizome of dlingo
with eight different concentrations was dropped 0,1 ml Candida albicans and incubated
at 370C for 24 hours and be repeated twice. This result was grown on Sabouraud Dextro
Agar (SDA) medium that was incubated and seen its growth. The growth of Candida
albicans in every part of plate was count and then we mean it.
Results : The mean of every tube that was in treatment indicate that tube number
I with 80mg/ml concentration inhibit 99968 colonies of Candida albicans. Tube number
8 with 0,625 concentrations inhibits 26665 colonies of Candida albicans. And tube
number 6 with 2.5 mg/ml concentration inhibits 69998.5 colonies of Candida albicans.
The inhibition of tube number 6 shows that it inhibits more than fifty percent of the
inhibition colony on tube 1.
Conclusion : The usage of dlingo extract (ethanol) to Candida albicans more

effective and safe with 2.5 mg/ml concentration (MIC).
Key words: Candida albicans, rhizome of dlingo, MIC.
INTRODUCTION
Candida albicans is normal flora in mouth. The existence of Candida albicans in mouth
in patient denture used more high than others (Daniluk, 2006). Maxillary denture that
closely attached mucosal surface from saliva flow, this situation will growth up amount
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of Candida albicans and cause fungal infections that is called candidiasis. The symptom
of candidiasis is red area limited to the mucosal surface that covered by acrylic denture
(Cawson, 2002). Diffuse inflammation caused of Candida albicans in long used is
called as denture stomatitis (Usri, 2006).
Prevalance of denture stomatitis among denture used is varieties, but in population

study reported that it was approximately 50% (Greenberg and Glick, 2003). Patient with
bad denture hygiene more often to infected denture stomatitis so the patient must
instructed to keep it cleanness (Yilmaz, 2002).
The use of cleansing denture material can help to manage or remove plaque from
denture, mucosal inflammation, and denture stomatitis (Greenberg, 2003). Cleansing of
the denture with sodium hypochlorite 0,5 % concentrate as disinfectant for 10 minutes
per day (Hendrijantini, 1996), whereas 15 minutes with chlorhexidine 0,2 %
(Sukarsyah, 1999).
The submerged of denture in chemical materials have a side effect. In long submerged
in sodium hypochlorite or chlorhexidine cause the pigment of acrylic denture being fade
(David, 2005).
WHO recommends the use of traditional medicine include herbal in society health care,
prevention and medication of disease, especially chronic disease, degenerative, and
cancer. WHO also support the effort in enhancement of safety and benefit from
traditional medicine (WHO, 2003).
One of the potential plants to develop as traditional medicine is dlingo (Acorus

calamus). In countryside, this plant grows wildly in field or fishpond. Dlingo rhizome in
community is used for cough (antitusive, expectorant), antivomit, gastric and lymph
medicine (Nugrahadi, 2001).
The content of dlingo rhizome is 3 % of atsiri oil that consit of asaron, parasaron,
calamen, asarilaldehid, sescuiterpen, metileugenol, and eugenol (Nugrahadi, 2001). This
rhizome consists of β-asaron that shows the activity of antifungal more than
antibacterial (Phongpaichit, 2005). Ethyl acetate of dlingo rhizome (Acorus calamus)
has minimum inhibitory concentration about 4-5 mg/ml to Candida albicans growth
(Devi, 2009).
The specific aims of this study is to know the minimum inhibitory concentration of
ethanol extract dlingo rhizome (Acorus calamus) to Candida albicans isolate from full
maxillary acrylic denture.
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The research is experimental laboratorium and the results will be test statistically with
Kruskal-Wallis method. Dlingo rhizome as sample used is from Babakan Baru village
RT 4/RW1 Cikundul, Lembur Situ Subdistrict, Sukabumi. The ethanol extract dlingo
rhizome (Acorus calamus L) is free variable and Candida albicans is attached variable.
Minimum inhibitory concentration is the smallest concentration of antimicrobial

material that can inhibit the growth of microorganism minimally 50 %. Candida
albicans is a fungi that grows in oral cavity, characteristically colored white yellow,
smooth and slick, smelled like yeast in Sabouraud Dextro Agar. In Candida
Chromogenic Agar, the colony color of Candida albicans is green. Ethanol extract of
dlingo rhizome is dlingo rhizome which is extracted by maceration with ethanol as a
solvent and then processes it with rotary evaporator until it becomes viscous extract.
The instrument that used in this study are sterile cotton bud, object glass, spiritus lamp,
microscope, refrigerator, incubator, petridish with 10 cm of diameter and 2 cm high,
pipette, reaction tube, oese, dan sterile needle as an inoculation tool of Candida
albicans, autoclave, stopwatch, and others instruments that usually use in microbiology
laboratory.
Figure 1. The equipment
A : microscope H: absorbent pipette
B : reaction tube I : erlenmeyer 100 ml
C : tub shelf J : aquades
D : bulyon glucose K: filter paper
E : spiritus lamp L : object glass
F : petridish M: Oese
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G : sabouraud dextro agar
The proliferation medium that use in this study is Sabouraud Dextro Agar (SDA) and
the identification medium is Candida Chromogenic Agar. The experiment
microorganism is Candida albicans that isolate from maxillary removable acrylic full
denture. And the material experiment is ethanol extract of dlingo rhizome (Acorus
calamus L).
The steps that are going to do in this experiment are:
1. Taken and cultivate the examination material
It is taken by sterile cotton bud from maxillary removable acrylic full denture. And
then set it into transport media that consist of bulyon glucose in reaction tube.
Examination material cultivate in Candida Chromogenic Agar and then incubated in
37oC temperature for 24-48 hours.
2. Isolation and identification of Candida albicans
Characteristic examination of the colony in Candida Chromogenic Agar is going to

do after incubation process. Candida Chromogenic Agar is media to detect and
isolate Candida spp. The colony of Candida albicans will seen as green color,
Candida dubliniensis seen as green blue color, Candida krusei seen as purple pink
color, Candida tropicalis seen as blue color, Candida galabrata seen as light brown
color until dark brown. The colony that seen the characteristic of Candida albicans
are isolated by oese and then cultivated in Sabouraud Dextro Agar (SDA). After
that, incubate it in 37o C temperature for 18-24 hours to get pure isolate of
Candida albicans.
3. Make a suspension of Candida albicans
Suspension of Candida albicans is made equivalent with Mc Fahrland 0,5 turbidity

using bulyon glucose as a solvent. That
4. Make an extract of dlingo rhizome (Acorus calamus L)
Making extract of dlingo rhizome (Acorus calamus L) using ethanol 70 % by

maceration method for 3 x 24 hours and every 24 hours the macerate is collected
and remaceration again with new ethanol liquid 70 %. The macerate is proceeding
by rotary evaporator until it becomes viscous extract. This extract is placed into a
dish and then it steam over boiled water so it was resulted as viscous extract that
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judges with 100 % concentration. The making of extract use ethanol as a solvent
because of its ability to pull out material or non polar active component or oil class
of dlingo rhizome such as asaron, parasaron, calamen, asarilaldehid, sesquiterpen,
metileugenol dan eugenol (Mac Gaw, 2002).
5. Examination of inhibition power of dlingo extract (Acorus calamus L) according to

Kirby Bauer diffuse method
The determining of antifungal activity by diffuse method is a method to see the

inhibition power that occurs around the test liquid to Candida albicans colony. First,
draw up the SDA plate, and then the suspension of Candida albicans that equivalent
with Mc.Fahrland 0.5 turbidity is taken about 0.1 ml is cultivates in SDA plate by
sterile cotton bud. Heat up the punching tools that use for perforator in SDA plate
about five holes. The first hole is dropped 0.1 ml extract of dlingo rhizome 10 %
concentrate, second hole is 8 %, third hole is 6 %, fourth hole is 4 %, and the fifth
hole is 0.1 ml of aquades for controlling or comparison. And the SDA plate
incubates in 37oC temperature for 24 hours according to anaerobe facultative and
control the inhibition area around the hole. The inhibition area is measured by
counting the diameter of inhibition area minus the hole diameter, and the result is
divided into two (Cappuccino, 2001).
6. Diluting test of testing substance and the determination of minimum inhibitory

concentration (MIC)
Preparation of standard solution by diluting the extract of the rhizome dlingo in

dissolving1 gramof rhizome extract dlingo into 12.5 ml ofglucose broth to obtain co
ncentrations of 80 mg/ml.
Determination of minimum inhibitory concentration dlingo rhizoma extract toward

the growth of Candida albicans is done by serial dilutions of eight different
concentrations as well as positive controls and negative controls. MIC of testing
substance is determined base on a twice dilution series method in 10 tubes. A total
of 2 ml of broth is filled into tubes 2 through 8 and 10. The first tube is filled with 4
ml of standard solution, and then the total of 2 ml of pipette is filled into the tube 2
and shaken until homogeneous.
Table 1. Preparation of the rhizome extract dlingo
Tube Bulyon The suspension of Concentration Suspensition

Glucose Dlingo Rhizome extract of Candida
albicans
1 - 4 ml from standard solution 80 mg/ml 0,1 ml
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2 2 ml 2 ml from tube 1 40 mg/ml 0,1 ml

6 2 ml 2 ml from tube 5 2,5 mg/ml 0,1 ml
9 - 2 ml from tube 8 0,625 mg/ml -
10 2 ml - - 0,1 ml
2 ml solution is taken from tube 2 then inserted into tube 3, and shaken until
homogeneous, and so on until the tube 9. Candida albicans suspension in
a broth made with turbidity equivalent 0,5 Mc. Fahrland is pipette in each tubes 0.1
ml to tubes 1 to 8 and 10. Tube 9 is containing only broth and rhizome extract
solution dlingo constitute a negative control and tube 10 which containing a solution
of broth and Candida albicans is positive control. Entire tubes are incubated
at 370C for 18-24 hours.
Turbidity of each tube is recorded and taken an oese from each

tube for cultures grown on SDA and incubated in the same temperature
and time. Culture sector is observed to look how many a growing number of
colonies. MIC is shown in the sector which showing the least amount of
colony growth.
7. Statistical Analysis
The results obtained are tested statistically using the Kruskal-Wallis by the formula:
12 𝑘
𝑁 𝑁+1 𝑗 =1 𝑛𝑗 𝑅𝑗2 - 3(N+1)
KW =
𝑞
𝑡 𝑖3 −𝑡 𝑖
1− 𝑖=1
𝑁 3 −𝑁
Keys: N= Number of all data
nj = Number of j treatment of data
𝑅𝑗 = average ranking of the j treatment
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T i= Number of points equal to-i
K = Number of treatments
If the two treatments revealed different (significantly), then further tested by

using the formula:
𝛼 𝑁(𝑁+1) 1 1
𝑅𝑢 − 𝑅𝑣 ≥z 𝑘(𝑘−1) 12 𝑛𝑢
+
𝑛𝑣
𝑅𝑢 − 𝑅𝑣 = Difference in average ranking of the two treatment
𝛼
z 𝑘(𝑘−1)
= Value of Z table
RESULTS
The result of sector cultivated in SDA plate seen that sector 1 until 4 there are a few
growth of Candida albicans colony. The growth of colony more happening from sector
5 until 8 (Figure 4.6).
Figure 2. The determining of minimum inhibitory concentration of Candida albicans

growth in Sabouraud Dextro Agar
The test of minimum inhibitory concentration ethanol extract odf dlingo rhizome
(Acorus calamus L) to Candida albicans give a result that seen in table 2.
Table 2. The result of Candida albicans proliferation by sector method in SDA plate in
many varieties concentration of ethanol extract of dlingo rhizome.
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The total colony of Candida albicans in every tube

Funga Repe K K
l Test titio 1 2 3 4 5 6 7 8
n (-) (+)
1 A 0 5 5 1 0 1 3 5 0 105
B 0 10 6 2 6 2 4 11 0 105
2 A 0 0 7 21 16 1 1 1 0 105
B 0 0 11 37 21 2 2 21 0 105
3 A 1 0 8 5 7 9 52 6,7.1 0 105
04
B 0 1 6 16 0 16 44 6,7.1 0 105
04
4 A 2 1 15 18 3,3.1 105 105 6,7.1 0 105
04 04
B 6 0 13 25 3,3.1 3,3.1 6,7.1 6,7.1 0 105
04 04 04 04
5 A 0 6 9 14 3,3.1 3,3.1 6,7.1 105 0 105
04 04 04
B 0 3 20 36 3,3.1 3,3.1 6,7.1 105 0 105
04 04 04
6 A 14 204 3,3.1 3,3.1 3,3.1 3,3.1 6,7.1 105 0 105
0 04 04 04 04 04
B 14 232 3,3.1 3,3.1 3,3.1 3,3.1 6,7.1 105 0 105
8 04 04 04 04 04
7 A 0 203 3,3.1 3,3.1 3,3.1 3,3.1 105 105 0 105
04 04 04 04
B 0 240 3,3.1 3,3.1 3,3.1 3,3.1 105 105 0 105
04 04 04 04
8 A 68 148 150 3,3.1 3,3.1 6,7.1 105 105 0 105
04 04 04
B 60 120 132 3,3.1 3,3.1 6,7.1 105 105 0 105
04 04 04
9 A 76 85 90 3,3.1 3,3.1 3,3.1 105 105 0 105
17
04 04 04
B 13 129 90 3,3.1 3,3.1 3,3.1 105 105 0 105
1 04 04 04
10 A 0 1 1 82 3,3.1 3,3.1 6,7.1 105 0 105
04 04 04
B 0 1 1 82 3,3.1 3,3.1 6,7.1 105 0 105
04 04 04
The concentration of ethanol extract of dlingo rhizome in tube :
1 : 80 mg/ml 4 : 10 mg/ml 7 : 1,25 mg/ml
2 : 40 mg/ml 5 : 5 mg/ml 8 : 0,625 mg/ml
3 : 20 mg/ml 6 : 2,5 mg/ml K (-) : negative control
K (+): positive control
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Table 3. The mean of Candida albicans proliferation by sector method in SDA plate in
many varieties concentration of ethanol extract of dlingo rhizome
The total colony of Candida albicans in every tube

Fungal
K K
Test 1 2 3 4 5 6 7 8
(-) (+)
1 0 7,5 5,5 1,5 3 1,5 3,5 8 0 105

2 0 0 9 29 21 1,5 1,5 11 0 105
3 0,5 0,5 7 10,5 3,5 12,5 48 6,7.104 0 105
4 4 0,5 14 21,5 3,3.104 6,7.104 8,3.104 6,7.104 0 105
5 0 4,5 14,5 25 3,3.104 3,3.104 6,7.104 105 0 105
6 144 218 3,3.104 3,3.104 3,3.104 3,3.104 6,7.104 105 0 105
18
7 0 221,5 3,3.104 3,3.104 3,3.104 3,3.104 105 105 0 105

8 64 134 141 3,3.104 3,3.104 6,7.104 105 105 0 105
9 103,5 107 90 3,3.104 3,3.104 3,3.104 105 105 0 105
10 0 1 1 82 3,3.104 3,3.104 6,7.104 105 0 105
The concentration of ethanol extract of dlingo rhizome in tube :
1 : 80 mg/ml 4 : 10 mg/ml 7 : 1,25 mg/ml
2 : 40 mg/ml 5 : 5 mg/ml 8 : 0,625 mg/ml
3 : 20 mg/ml 6 : 2,5 mg/ml K (-) : negative control
K (+): positive control
In Kruskal Wallis statistic analysis with error level (α) about 0.05, the hypothesis is
given :
H0 : There is no difference among the growth of Candida albicans from the ten tubes.
H1 : There is a difference among the growth of Candida albicans from the ten tubes.
Table 4. The result of Kruskal Wallis test
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The results from Kruskal Wallis test table indicate a significant result. It because of P-
value (0.00) smaller than, α (0,05), so the decision is refuse H0 hypothesis and accept
H1 hypothesis. It means there is difference among the growth of Candida albicans from
the ten tubes. The difference from every treatment is shown the recapitulation of
analysis for every set of treatment.
Table 5. The mean of total colony in every treatment
Treatment Group n The mean

Tube Concentration of colony
9 K(-) 10 0
1 80 mg/ml 10 31,60
2 40 mg/ml 10 69,45
3 20 mg/ml 10 6694,80
4 10 mg/ml 10 13350,15
5 5 mg/ml 10 23335,85
6 2,5 mg/ml 10 30001,5
7 1,25 mg/mg 10 58338,7
8 0,625 mg/ml 10 73335,30
10 K(+) 10 100000
The table above shown that tube 1 with 80 mg/ml has the highest inhibition power, that
is 99,968 colony. Tube 8 with 0.625 concentration has the smallest inhibition power,
that is 26,665 colony. Tube 6 with 2.5 mg/ml concentration has inhibition power that
caninhibit more than 50 % of the initial colony, that is 69998.5 colony. Tube 1 until 6
have inhibition power more than 50 % of initial colony.
DISCUSSION
The material examination that used is five maxillary removable acrylic full denture that
get from five respondent with more than 50 aged. And the denture minimally was used
20
over 5 year. Denture that has been used for many years possibly a plaque deposition in
denture surface, so we can isolate the microorganism easily.
The presences of Candida albicans infection or colonization on denture can possibly a

way in for inflammation extension. Candida spp. Isolation on denture is connected with
unclean condition of denture, the long use, the use of denture in night hour, and
modification of hard tissue support (Daniluk, 2006).
Candida Chromogenic Agar is selective medium to identificate and isolate Candida. The
colony color of Candida that was formed cause of degradation of chromogenic substrate
by specific enzyme from the fungi. X-NAG substrate (5-bromo-4-chloro-3-indolyl N
acetil ß-D-glucosaminide) degradation by hexosaminidase enzyme and BCIP (5-bromo-
6-chloro-3-phospat indolyl p-toluidine salt) by phosphate alkali. Colony that show
charactheristic of Candida albicans in Candida Chromogenic Agar seen as light green
color (Ferreira,2008).
The test result of minimum inhibitory concentration of ethanol extract of dlingo rhizome
to Candida albicans can observe visually or sector. Visually in tube 1 until 5 the color
of suspension are brown until yellow, so the viscouscity level of suspension is difficult
to detect. Tube 6 until tube 8 the color of suspension is transparent, but from tube 6
until 8 they show the viscousity level is more higher, it means from tube 1 until 8 there
is a Candida albicans growth.The visual observation must be reinforced with sector
observation in table 4.1 from statistically test and quality test.
Fifth sector seen a inhibition growth of Candida albicans colony more than half of
initial colony in among ten fungal test, but in sector 6 there is a inhibition more than
half of initial colony that is not far enough from sector 5, but in smaller concentration.
The sensitivity of fungal test to ethanol extract of dlingo rhizome is caused of the
difference of morphologic or the structure of fungal wall cell (Devi, 2009).
The Kruskal Wallis test is 0.000 < 0.05 so it can be concluded that there is a
significance difference in all tubes. It means that all of the eight concentration have an
effect significantly to the growth of Candida albicans. The growth of Candida albicans
will more higher if the concentration of extract is smaller, and vice versa. The
concentration of rthanol extract of dlingo rhizome is inversely with the amount of
Candida albicans growth.
The medicine that is tested to microorganism such as bacteria or fungal, one of the
response that suitable to observe is letal condition. All of the medicine can cause a death
and its show on the last certain point that can be identified rapidly and definitely. Letal
dose (or LD50) is the total of antimicrobial material or a medicine that will kill more
than 50 % of microorganism in colony (Yagiela, 2004). The study of dlingo rhizome
21
extract is expected capable to inhibit the growth of total Candida albicans, minimally
50 %.
The result of sector cultivated show that the amount of Candida albicans colony
have a difference in every concentration. The concentration that can inhibit the growth
of Candida albicans more than 50 % of initial colony is on tub 6, 5,4,3 ,2, an 1. The
minimum concentration from the sixth tube is tube 6, so the minimm inhibitory
concentration of ethanol extract of dlingo rhizome to Candida albicans is 2,5 mg/ml.
This result is different with the study about it in Thailand, whereas the MIC is 4-
5 mg/ml. This can cause by the difference of the solvent that use to extract the dlingo
rhizome. In this study we use ethanol as a solvent, not ethyl acetate.
The research by Phongpaichit (2005) found that the fraction of β-asaron has
a minimum inhibitory concentration value of 0.1 to 1 mg / ml. A
research in India by Devi (2009) found that the ethyl acetate extract of
dlingo rhizomes (Acorus Calamus) has an inhibitory concentrations
of antifungal through the test of minimum inhibitory concentration about 4-5 mg / ml to
the growth of Candida albicans. The results of this study is different from the results of
the research that is conducted by Devi and Phongpaichit. This can be caused
by differences in the location of growing plant, climate, and geographical
differences can lead to differences in levels of the active substance such as α- and β-
asaron in the dlingo rhizome (Devi, 2009).
The difference use of solvents in this study with previous studies that use methanol and
ethyl acetate as a solvent causes the differencity of the amount of active material that
are extracted from dlingo rhizome. According to Phongpaichit (2005) found that the
minimum inhibitory concentration of the methanol extract of dlingo 0.1-1 mg/ml with
methanol solvent has a minimum inhibitory concentration greater than this study. So the
use of methanol as a solvent is better to attract the active ingredient than ethanol. The
research in India by Devi (2009) found that the ethyl acetate extract of dlingo rhizome
(Acorus calamus) have a minimum inhibitory concentration of 4-5 mg/ml with ethyl
acetate as a solvent. The MIC value is smaller that compared with this study so the use
of ethanol as a solvent to extract the active component is more suited than the ethyl
acetate.
The antifungal characteristic of ethanol extract of dlingo rhizome is got from α-asaron
and β-asaron fraction that have a pretty good antifungal to Candida albicans. β-asaron
has an ntifungal activity greater than α-asaron. Ethanol extract of dlingo rhizome with
smaller concentration contain a small content of α-asaron and β-asaron which is an
antifungal fraction of Candida albicans. The influence of this fraction against fungal
hyphae and conidia showed a drastic change of morphologic, that get into shrink and
22
disintegrate. This can be caused by a leak in the cell wall or the change of permeability
membrane (Pongpaichit, 2005).
This disorder occurs because of the attack of cell membrane by α-asaron dan β-asaron
with non polar charactheristically that capable to induces the change of membrane
permeability via the interaction between the active side of the cell membrane, especially
the ergosterol of Candida albicans. This interaction results in a change of kinetic energy
which results in changes in membrane permeability and form a pore, so through this
pore the essential component of fungal cell such as K ion, inorganic phosphate,
carboxylic acids, amino acids, and esther phosphate that leak out and cause the death of
fungal cells (Banfi et al, 2006).
CONCLUSIONS
From the results of this study it can be concluded minimum inhibitory concentration of ethanol
extract dlingo rhizome against to Candida albicans isolate from maxillary removable acrylic
full denture is 2,5 mg/ml.
23
REFERENCES
Banfi, et all. 2006. Antifungal and antimycobacterial activity of new imidazole and
triazole derivatives A combined experimental and computational approach.
J.Antimicrobial Chemotheraphy 58 (1): 76-84.
Cappuccino.J.G and N.Sherman. 2001. Microbiology :for the Health Sciences 5thed.
New York.Lippincott. 211-225 pp.
Cawson, R.A, and E.W.Odell. 2002. Oral Pathology and Oral Medicine. 7th. London.
Churchill Livingstone. 178-179 pp.
Daniluk T, et al. 2006. Occurrence rate of oral Candida albicans in denture wearer
patients. Advances in Medical Sciences. Vol 51: 77-80.
Devi, A and Deepak. 2009. Antimicrobial activity of Acorus calamus (L.) rhizome and
leaf extract. Acta Biologica Szegediensis.Vol. 53(1) :45-49.
Greenberg, M. S. and M. Glick. 2003. Burket’s Oral Medicine Diagnosis and Treatment
10thed. Ontario.BC Decker Inc. 85-101 pp.
Hendrijantini, N. 1996. Pengaruh konsentrasi larutan sodium hypochloride sebagai
desinfektan gigi tiruan resin akrilik terhadap Candida albicans. Jurnal
Kedokteran Gigi. Vol 30: 73–7.
McGaw I.J, Jager AK, Van Staden J. 2002. Isolation of β-asarone, an antibacterial and
anthelmintic compound, from Acorus calamus in South Afrika. South Afrika J
Bot 68:31-35.
Nugrahadi, Trias, dkk. 2001. Tanaman Obat di Propinsi Jawa Barat Karakteristik dan
Khasiatnya. Bandung. Unpad Press. hlm 48-49.
Phongpaichit, S et al. 2005. Antimicrobial activities of the crude methanol extract of
Acorus calamus Linn. Songklanakarin J. Sci. Technol., 27(Suppl. 2) : 517-523.
Putra, T. 2001. Isolasi Candida albicans dan uji kerentanan obat anti jamur. Dental
Journal, Vol. 34, No. 3a, hlm 174-176.
Silverman, S., L. R. Eversole, E. L. Truelove. 2002. Essentials of Oral Medicine.
London. BC Decker Inc.170-173 pp.
Sukarsyah, P.M. 1999. Pengenceran bahan desinfektan untuk sanitasi gigi tiruan secara
optimal. Majalah Ilmiah Kedokteran Gigi FKG Usakti. hlm 16-21.
Usri, K dkk. 2006. Diagnosis dan Terapi Penyakit Gigi dan Mulut. Bandung. Lembaga
Studi Kesehatan Indonesia. hlm 61-62.
24
WHO. 2003. Traditional medicine, http://www.who. int/mediacentre/

factsheets/fs134/en/(diakses Agustus 2010).
Yagiella, Dowd, and Neidle. 2004. Pharmacology and Therapeutics for Dentistry. 5th
Ed. St.Louis. Mosby. 62-64 pp.
Yilmaz, H and Ulkem. 2002. Is denture stomatitis related with denture hygiene.
Gulhane Tip Dergisi 44(4) : 412-414.
25
EFFECTS OF Curcuma xanthorrhiza Roxb.

IN WOUND AND BONE HEALING IN RATS
Camelia Dewi H*, Citra Kartika Y*, Denanda Adyandara S*, Destiawaty
Nathania A*
*Faculty of Dentistry, Prof. Dr. Moestopo University, Jakarta, Indonesia
ABSTRACT
Introduction: The main substances of Curcuma xanthorrhiza Roxb. are starch, fiber,
volatile oil and also curcuminoid. Curcuma xanthorrhiza Roxb. is known as Javanese
turmeric, also has been traditionally used in Indonesia for food and medicinal purposes.
However, there was no studies have been carried out so far to observe the influence of
Curcuma xanthorrhiza Roxb. in wound and bone healing. Thus, the aim of this study
was to indicate potencial therapeutic benefits of Curcuma xanthorrhiza Roxb. by oral
on impaired wound and bone healing in rats. The results of this study may provide
useful information for further hard and soft tissues repair consuming the herbal plants
which can be reached by each person in traditional Indonesian medicine.
Methods: 80 male Spraque-Dawley rats (150-250 g) were used and a bone drill defect
was created in the mandibular of each animals. Rats divided into 3 groups : the first
group was a control group, the second group was given Clindamycin (150 mg/kg BW
per day; oral route; two times daily), the third groups were administrated by Curcuma
xanthorriza Roxb. (250 mg/kg BW, 500 mg/kg BW and 1000 mg/kg BW; oral route;
once daily). Each animals from each group were sacrificed on days 5, 9, 15, 18 and 22.
By histological examination, the bone healing was evaluated from the regeneration of
osteoblasts, osteoclasts and osteocytes. On the wound healing, the tissues repair was
observed by the regeneration of fibroblast and collagen.
Result: Wounds of rats treated with Curcuma xanthorrhiza Roxb. showed significant
(p< 0.05) increases in bone turnover and activity (reflected by osteoblasts, osteoclasts
and osteocytes) and also in wound healing (performed by fibroblasts and collagen)
compare the others groups. Curcuma xanthorrhiza Roxb. can prevent further
deterioration of the bone structure and produce beneficial changes in bone repair.
Conclusion: This result showed that Curcuma xanthorrhiza Roxb. had enhanced wound
and bone repair, and could be developed as a pharmacological agent in such clinical
setting.
Keywords : Curcuma xanthorrhiza Roxb., wound healing, bone healing
26
INTRODUCTION
Curcuma xanthorrhiza Roxb. (Zingiberaceae family, commonly known as temu lawak
or Javanese turmeric in Indonesia), which is found both wild and cultivated in
Indonesia, has been traditionally used for medicinal purposes.1 Either the fresh rhizomes
or a decoction of dried sliced rhizomes have been used to treat various stomach diseases
and liver disorders such as jaundice and gall stones, and promote the flow of bile.2 C.
xanthorrhiza Roxb. is also used as a tonic in Indonesia.3
This plant has effect in anti analgesic,4 anti bacteria,5 anti diabetic,6 anti inflammation,7
anti oxidant8 and anti tumor.9 Through the activity of anti inflammatory, Curcuma
xanthorrhiza Roxb. is effective for treating arthritis, rheumatism and inhibiting growth
of bacteria as well as staphylococcus, streptococcus and salmonella.10-12 The rhizomes
of Curcuma xanthorrhiza Roxb. contain volatile oil, saponin, flavonoid and tannin.13-15
Volatile oil such as phelandren, camphor, tumeron, sineol, borneol, and xanthorrhizol
(1.48-1.63%) and also curcuminoid like, curcumin and desmetoxicurcumine (1.6-
2.2%).13-14 Xanthorrhizol isolation from C. xanthorrhiza Roxb ethanol extract could
inhibit the growth of bacteria S.mutans.16,17 Through many studies, another benefit of C.
xanthorrhiza Roxb as anti-microba has also been found.13-16,18,19
Infections and diseases in the oral cavity such as cysts or neoplasms, can cause local
tissue damage of the hard (alveolar bone) and soft tissues, as well as on several
measures in dentistry such as tooth extraction, odontectomy, implants and orthodontics
appliances. Destruction of bone and soft tissues normally takes time for healing which
may inhibit following treatments. Therefore, the faster healing process will extremely
support treatments, thereby providing satisfactory to both the patient and dentist
because patients are eager to reach optimum outcome with less pain.
Therefore, we wanted to examine the effects of Curcuma xanthorrhiza Roxb. in bone

and wound healing process.

Experimental Design
Male Sprague-Dawley rats weighing between 150-250 g (2-3 months old) were used as
experimental animals. A bone drill (d= 2 mm) defect was created in the mandibular of
eighty male Spraque-Dawley rats (150-250 g). Rats were divided into 3 groups. The
first group was a control group, the second group was given Clindamycin (150 mg/kg
BW per day; oral route; two times daily for 5 days), the third groups were administrated
by Curcuma xanthorriza Roxb. (250 mg/kg BW, 500 mg/kg BW and 1000 mg/kg BW;
oral route; once daily). Each animals from each group were sacrificed on day 5, 9, 15,
18 and 22.
Preparation of extract Curcuma xanthorrhiza Roxb

The coarsely powdered material (800g) of Curcuma xanthorrhiza Roxb. was macerated
with ethanol 70% (8L) for 72 h with occasional shaking. The maceration was repeated
27
thrice. The extract was filtered and concentrated at reduced pressure on rotary
evaporator resulting in brown colored semisolid mass (yield 5.2%).
Histological Observation
Immediately after rats sacrificed, the mandibulars were removed and fixed in formalin
within 3 days. The tissues were embedded in Formic Acid 90% 100 cc, aquadest 1000
cc and HCl 80 cc for 5 days. Afterward, the tissues were sectioned by microtome,
immersed in distilled water for 3 hours and the sections were stained with hematoxylin
and eosin (H and E). The bone healing was evaluated from the regeneration of
osteoblasts, osteoclasts and osteocytes. On the wound healing, the tissues repair was
observed by the regeneration of fibroblast and collagen.
Statistically Analysis
Data were presented as mean and the result were analyzed statistically using Kruskal-
Wallis test. The differences was considered significant when p < 0.05.
RESULTS
In the Table 1, until day 5, there was no statistically significant difference of
osteoblasts, osteocytes, osteoclasts, fibroblasts and collagen, neither on day 9 between
Curcuma xanthorrhiza Roxb. group, Clindamycin as a control positive group and
control negative group. Followed on day 15 and 18, the numbers of osteoblasts,
osteocytes, osteoclasts, fibroblasts and also collagens in the Curcuma xanthorrhiza
Roxb group was significantly higher compare other groups during bone formation phase
as callus starting formatted. There was also a statistically significant difference on day
22 of the number of osteoblasts, osteocytes, osteoclast, fibroblasts and collagens in
between groups, which is the Curcuma xanthorrhiza Roxb group showed higher the
number of osteoblasts, osteocytes, osteoclasts, fibroblasts and collagens compare the
other groups.
28
Table 1. The effect extract of Curcuma xanthorrhiza Roxb. to Bone and Wound
Healing Against The Control Group
DAY
5 9 15 18 22
CELLS
OSTEOBLAST 0.136 0.066 0.032* 0.013* 0.014*
OSTEOCYTE 0.097 0.186 0.027* 0.010** 0.090**
OSTEOCLAST 0.071 0.945 0.023* 0.023* 0.018*
FIBROBLAST 0.067 0.174 0.023* 0.029* 0.027*
COLLAGEN 0.096 0.294 0.033* 0.015* 0.015*

*p<0.05, **p<0.01, evaluated by Kruskal-Wallis Test
29
30
A B
C D
Fig 6. Histological view of bone healing from

day 22, control negative group (A),
Clindamycin group (B), treated with
C.xanthorrhiza Roxb 250mg/kgBW (C),
treated with C.xanthorrhiza Roxb
500mg/kgBW (D), and treated with
C.xanthorrhiza Roxb 1000mg/kgBW (E).
31
A B
C D
Fig7. Histological view of wound healing

from day 22, control negative group (A),
Clindamycin group (B), treated with
C.xanthorrhiza Roxb 250mg/kgBW (C),
treated with C.xanthorrhiza Roxb
E 500mg/kgBW (D), and treated with
C.xanthorrhiza Roxb 1000mg/kgBW (E).
32
DISCUSSSION
Overall according to our study, it showed Curcuma xanthorrhiza Roxb. increased

wound and bone healing in rats. Many studies of bone healing used rats as a specimen
and the rat is the most frequently used animal for studies of fracture healing,20,21
therefore we used rats as a specimen in this study.
For positive control group, we used Clindamycin as a drug to repair bone and wound
healing. Because Clindamycin is a drug of choice for the treatment for joint and bone
infections.22 Extraction with ethanol 70% was conduced since with ethanol 70% is a
versatile solvent for preliminary extraction and it has universal characteristic so it can
attract polar compounds in the rhizome especially alkaloid curcuminoid and terpenoid
and also non polar compounds. Another component as a result of the extraction using
23
ethanol is phenol compound.
Large defects caused by trauma, cysts, neoplasms, infections or congenital

malformations may not regenerate spontaneously and the use of surgical or
pharmacological measures is required for complete regeneration. Bone deficiencies are
of major concern and affect therapies in all dental and medical fields. 24
Alveolar bone healing has been extensively studied in several animal models and its
sequence is relatively well understood. 25,26 The healing process in rats can be divided
into 3 phases: early phase/inflammatory phase (1 to 5 days), bone formation phase (5 to
20 days) and remodelling phase (20 to 60 days). 25,27
While in wound healing, in general, there are three major stages of wound healing:
inflammation, proliferation and remodeling.28 In the inflammatory stage, hematoma
develops within the fracture site during the first few hours and days. Inflammatory cells
(macrophages, monocytes, lymphocytes, and polymorphonuclear cells) and fibroblasts
infiltrate the bone under prostaglandin mediation. This results in the formation of
granulation tissue, ingrowth of vascular tissue, and migration of mesenchymal cells.29
Notably, the early stage of inflammation is regarded as a critical period of the wound
healing process, essential for clearing the contaminating bacteria and creating an
environment conducive to succeeding events involved in tissue repair and
regeneration.30-32 Inflammation at the wound site is marked initially by the infiltration of
neutrophils. These are the predominant inflammatory cells during the early
inflammatory stage that serve to prevent infection through phagocytic processes and
propagate the inflammatory response by releasing cytokines and chemokines.33 Clinical
signs: Rubor (redness), Calor (heat), Tumor (swelling or edema), Dolor (pain), Functio
Laesa (loss of function).34,35 Therefore in our study on day 5 until 9, we didn‟t find the
significant differences between curcuma and other groups. Because this period was
inflammatory stage, there were not increasing production of bone cells in the first week.
During the repair stage in bone healing process, fibroblast begin to lay down a stroma
that helps support vascular ingrowth. As vascular ingrowth progresses, a collagen
33
matrix is laid down while osteoid is secreted and subsequently mineralized, which leads
to the formation of a soft callus around the repair site.29 The second stage of wound
healing is proliferation. In this stage, these following happened: 1. Ephitelialization,
2.Wound contraction, and 3.Collagen deposition. Epithelialization is a requirement for
orderly progression into the proliferative phase. It starts in the inflammatory phase, it
requires de-differentiation, mitosis, migration and then re-differentiation by basal cells
of epidermis. While in wound contraction wounds heal from side to side but contract
from end to end% and its mediated by myofibroblast which is can produce collagen but
also contains smooth muscle filaments. In collagen deposition, collagen is deposited.
After that, collagen deposition is in balance with collagen resorption and no further net
collagen deposition occurs.36 According to that phase in this research we found that on
day 15 and 18, the numbers of osteoblasts, osteocytes, osteoclasts, fibroblasts and also
collagen in curcuma group significantly higher compare another groups during this
phase as callus starting formatted. It showed that in repair stage of bone healing and
proliferation stage of wound healing in Curcuma xanthorhiza Roxb. group was better
than the control groups.
In remodeling phase on day 22 the result of this research showed significant difference
between osteoblasts, osteocytes, osteoclasts, fibroblasts, also collagen in Curcuma
xanthorhiza Roxb. group compare another groups. Fracture healing is completed during
the remodeling stage in which the healing bone is restored to its original shape,
structure, and mechanical strength. In the remodeling phase, of wound healing
represents time during which type III collagen is replaced by type I collagen, and is re-
oriented across lines of tension with the creation of more stable bonds between fibers -
net results decreases the amount of collagen required to maintain wound integrity.36
Triterpenoids in rhizome of C. xanthorrhiza Roxb promote the wound healing process
mainly due to their astringent and antimicrobial property, which seems to be responsible
for wound contraction and increased rate of reepithelialization.37
Bone tissue has two remarkable properties that distinguish it from other structural
material: it can alter its mechanical characteristics in response to changes in functional
demand, and it also has the capacity to heal itself through a healing process resulting not
in a scar, but in an actual reconstitution of the injured tissue. This healing process is
affected by many variables, including the extent of damage to soft and hard tissue and
the vascular supply caused by both the fracture and the fracture treatment. 38
In our research that doses of Curcuma xanthorrhiza Roxb. improve bone formation and
wound repair. With the fact that the number of active osteoblasts, osteocytes and active
osteoclasts increased, meanwhile both fibroblasts increased and collagen fibers became
more tightly since day 15, 18 and 22. In figure 6 and 7 the bone formatted incomplete
yet but both bone cells and fibroblasts signicantly increased and the collagen fibers
became tightly which was caused by the administration of Curcuma xanthorrhiza
Roxb.1000 mg/BW compare the other groups.
CONCLUSIONS
In conclusion, rats treated with Curcuma xanthorrhiza Roxb. showed significant
(p<0.05) increases in bone turnover and activity and also in wound healing started day
34
15. Curcuma xanthorrhiza Roxb. had enhanced wound and bone repair, also could be
developed as a pharmacological agent in such clinical setting.
ACKNOWLEDGEMENTS
The authors are thankful to Dr. Evie Lamtiur Pakpahan as the head of research center in
faculty of dentistisy, Prof. Dr. Moestopo University for supporting this research. Also to
Dr. Ahmad Aulia, Ph.D, the staff of histology department in medical faculty of
Indonesia University for providing the necessary facilities. No financial support was
received.
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Continuous Clindamycin Infusion, an Innovative Approach to Treating Bone and
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23. Anbinder AL, Junquiera JC, Mancini Maria NG, Balducci I, Fernandes da Rocha R,
amd Carvalho YR. Influence of simvastatin on bone regeneration of tibial defects
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25. Brandao AC, Brentegani LG, Novaes AB Jr., et al. Histomorphometric analysis of
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26. Smith, N. A comparative histological and radiographic study of extraction socket
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27. Wemer S, Grose R. Regulation of wound healing by growth factors and cytokines.
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28. Kalfas, IH. Principles of Bone Healing. Neurosurg Focus 2001; 10(4):1.
29. Abbot RE, Corral CJ, Maclvor DM, Lin X, Ley TJ, Mustoe TA. Augmented
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91-7.
37
PTHrP INDUCES STROMAL FIBROBLASTS INTO

CANCER-ASSOCIATED FIBROBLASTS.
Rikuka Shimizu, Keishi Nagao, Yae Ohata, Aya Matsuda, Tetsuya

Kitamura, Fumihiro Higashino, Masanobu Shindoh
Hokkaido University Graduate School of Dental Medicine
ABSTRACT
Objective: The microenvironment surrounding tumor cells attracts attention. We have
reported that tumor endothelial cells have abnormality and fibroblasts in cancer stromal tissue
were also shown to have different phenotype and termed as “Cancer-Associated Fibroblast
(CAF)”. CAF was shown to concern with epithelial-mesenchymal transition (EMT) that
correlates with malignant phenotype of cancer cells. Parathyroid hormone-related protein
(PTHrP) is known to induce bone resorption by activating RANKL as well as PTH, and
PTHrP expression has been reported to be closely associated with bone metastasis of breast
carcinoma. We supposed that PTHrP might affect stromal fibroblasts to change into CAF.
Methods: We investigated PTHrP expression by Western blotting in oral squamous cell
carcinoma cell lines. siRNA for PTHrP was used for downregulating PTHrP.
Immunohistochemical detection of PTHrP was performed in mucoepidermoid carcinoma, and
surveyed the significance in clinico-pathological features. The effects of PTHrP on fibroblast
cell lines were investigated culturing with supernatant of carcinoma cells.
Results: PTHrP was identified in all squamous cell carcinoma cell lines examined. PTHrP
was highly detectable in intermediate cells and epidermoid cells of mucoepiedrmoid
carcinoma, and cancer with rich PTHrP expressed intermediate cells showed significant
relation of cancer malignancy. αSMA-positive CAF was seen in stromal tissue around PTHrP
expressing cancer cells. We next examined the effects of PTHrP on fibroblast cell lines. Rapid
proliferation of fibroblasts was observed when fibroblasts were treated with supernatant of
PTHrP highly producing oral carcinoma cells, and reduced proliferative activity was seen
when fibroblasts were incubated with supernatant of siRNA of PTHrP incorporated cancer
cells. The number of αSMA-positive fibroblasts was also increased when fibroblasts were
cultured with PTHrP highly producing cancer cell supernatant.
Conclusions: Our results indicate that PTHrP producing carcinoma induce CAF in stroma
that may feed back to malignant phenotype of cancer cells.
Keywords: PTHrP, CAF, mucoepidermoid carcinoma, malignant phenotype
38
INTRODUCTION
Parathyroid hormone-related protein (PTHrP) was originally identified as a major factor
responsible for humoral hypercalcemia in malignant tumors such as lung and breast
carcinomas1,2). PTHrP binds to the common PTH/PTHrP receptor in osteoblasts and
induce bone resorption and hypercalcemia3,4). PTHrP is produced by some malignant
tumors, and involved in malignant conversion of breast, colon, and prostate cancer by
increasing cell proliferation, survival, adhesion, migration, and invasion 5,6,7). We have
reported that PTHrP was highly expressed in oral carcinoma cell lines and PTHrP
promoted malignancies of oral cancers 8).
Mucoepidermoid carcinoma is the most common malignant salivary gland tumor 9,10),
and Its clinical behavior is highly variable and ranges from slow growing and indolent
to locally aggressive and highly metastatic 11,12). Histologically, mucoepidermoid
carcinoma is comprised of 3 different cell types: mucinous cells, intermediate cells, and
epidermoid cells. Growth patterns range from cystic to solid to infiltrative. These
parameters have been incorporated into several different grading systems that have been
correlated with prognosis and therefore play an important role in treatment decisions9).
However, there are some mucoepidermoid carcinoma cases of poor prognoses that were
estimated as low-grade malignancy in histological examination.
The microenvironment surrounding tumor cells attracts attention. We have reported that
tumor endothelial cells have abnormality and fibroblasts in cancer stromal tissue were
also shown to have different phenotype and termed as “Cancer-Associated Fibroblast
(CAF)”. CAF was shown to concern with epithelial-mesenchymal transition (EMT) that
correlates with malignant phenotype of cancer cells.
We examined the PTHrP expression in mucoepidermoid carcinoma and its concern on
alteration of stromal fibroblasts into CAF.
Materials and methods

Patients and tissue samples
Twenty-one patients who consulted the Department of Oral Surgery, Hokkaido

University Hospital, and diagnosed as mucoepidermoid carcinoma were examined.
Informed consent was obtained from the patients prior to the samples being used. The
experiment was conducted under the ethics rules of Hokkaido University Hospital.
TNM classification was done according to the UICC criteria and tumors were graded
according to the World Health Organization guidelines 2005.
Western blotting
Human oral squamous cell carcinoma cell lines HSC2, HSC3, HSC4 (JCRB, Osaka,
Japan) and PC-3, a prostate carcinoma cell line were used for PTHrP expression study.
Fibroblast cell lines, MRC5, NIH3T3, Cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells
39
were lysed in lysis buffer, and protease inhibitor cocktail for 20 min on ice and clarified
by microcentrifugation. The supernatant was subjected to SDS-PAGE, and transferred
to polyvinylidene difluoride membranes. A PTHrP rabbit polyclonal antibody was used
for detection of PTHrP in cultured oral squamous cell carcinoma cell lines, and αSMA
was detected by using monoclonal antibody.
Immunohistochemical analysis
Immunohistochemical detection of PTHrP, αSMA and CD34 was conducted using

paraffin embedded tissue sections. Four-μm sections were deparaffinized and
rehydrated. They were immersed in 3% hydrogen peroxide in distilled water to block
endogenous peroxidase activity. Then they were exposed to the primary rabbit
polyclonal antibody for PTHrP, αSMA and CD34 for 1 hour at room temperature. They
were visualized by peroxidase reaction method, and counterstained with haematoxylin.
PTHrP expression ratio of mucoepidermoid carcinoma was calculated by counting the

number of positive tumor cell and areas involved in tumor tissue.
Statistical analysis
Data concerning the PTHrP expression ratio of mucoepidermoid carcinoma were e

analyzed and compared with the two-sample t-test for differences in means. The
criterion for statistical significance was P<0.05.
RESULTS
Clinical features of cases examined.
Clinical features of the cases are shown in Table 1. There were 12 male and 9 female
patients. The mean age of the patients was 56 years. Primary sites of tumors are 4 in
parotid gland, 1 in submandibular gland, 1 in sublingual gland, and other 15 cases were
occurred in minor salivary glands. TNM classification was performed according to the
guidelines of the International Union Against Cancer TNM classification system.
Patients were followed up in 5 years concerning the prognosis including lymph node
metastasis and/or tumor recurrence. TNM classification of tumor size was T1 in 5
patients (24%), T2 in 10(47%), T3 in 4 (19%), and T4 in 2 (10%). Nodal status was N0
in 17 patients, N1 in 1 and N2 in 3 patients. 5 patients had subsequent metastases
including regional lymph nodes or distant organ, and tumor recurrence in the 5-year
follow up period.
40
PTHrP protein is expressed in oral squamous cell carcinoma cell lines.
To address the role of PTHrP in oral epithelial tumors, we first examined whether it was
expressed in oral carcinoma cell lines HSC2, HSC3, HSC4, which were analyzed using
Western blotting. All cell lines expressed PTHrP protein, which were almost equal or
higher than the level of PC-3, a positive control of prostate carcinoma cell line.
PTHrP expression in mucoepidermoid carcinomas is related to cancer metastasis
and recurrence.
Immunohistochemical detection of PTHrP was performed and cytoplasmic PTHrP-

positive signals were observed in 17 of the 21 cases. PTHrP positive signals were seen
predominantly in intermediate cells and epidermoid cells. There was no significance in
PTHrP expression ratio in epidermoid cells and tumor malignancy; however, significant
correlation was observed between PTHrP expression ratio in intermediate cells and 7
cases of primary and subsequent tumor metastasis and/or recurrence.
Cancer associated fibroblast (CAF) induction was estimated byαSMA expression in

cancer stromal tissue. In normal mucosa, aSMA expression was almost equal to the
distribution of CD34-positive vascular tissue, and no obvious induction of CAF was
observed. In contrast, αSMA-positive CAF was induced in stromal tissue of
mucoepidermoid carcinoma. CAF was more abundant in stromal tissue around
epidermoid cancer cells and intense aSMA-positive CAF was widely observed in
stromal tissue around intermediate cancer cells.
DISCUSSION
Mucoepidermoid carcinoma is formerly classified as benign tumor termed as
mucoepidermoid tumor. Most patients have a favorable outcome, but some patients died
of disease and mucoepidermoid carcinoma replaced into malignant tumor in 1992 WHO
classification because of the capacity of metastasis regardless of the macroscopic and
histologic appearance13). Mucoepidermoid carcinoma was categorized into low- and
high-grade of malignancy with respect to local recurrence and metastatic ability13).
These criteria continued to 2005 classification 9), and the grading system with low- to
high-grade malignancy using five histopathologic features are now utilized. It could be
reproducible in defining prognosis of mucoepidermoid carcinoma patients; however,
there are some exceptions concerning tumor grading and prognoses, and it is needed to
establish new methods that may estimate exact tumor malignancies.
41
PTHrP was purified from a human lung cancer cell line, and was shown to have
biological activities similar to parathyroid hormone (PTH) that correlate with humoral
hypercalcemia of malignancy2). Clinical evidence supports another important role for
PTHrP in malignancy as a mediator of the bone destruction associated with osteolytic
metastasis 7,14). PTHrP expression by breast carcinoma cells may provide a selective
growth advantage in bone due to its ability to stimulate osteoclastic bone resorption 1).
Furthermore, growth factors such as transforming growth factor-beta (TGF-beta), which
are abundant in bone matrix, are released and activated by osteoclastic bone resorption
and may enhance PTHrP expression and tumor cell growth 14,15). Moreover, PTHrP
overexpression increases mitogenesis and decreases apoptosis in the human breast
cancer cell line. Clones of MCF-7 cells that overexpress wild-type PTHrP showed
significantly higher laminin adhesion and migration. It indicate that PTHrP may play a
role in breast cancer metastasis by upregulating proinvasive integrin expression, and
controlling PTHrP production in breast cancer may provide therapeutic benefit 7).
In this study, PTHrP was detected in oral squamous cell carcinoma cell lines, and it
raised the possibility that PTHrP might be involved in oral cancer malignancies.
Mucoepidermoid carcinoma is composed of mucous producing, epidermoid (squamoid)
and cells of intermediate type. We supposed that epidermoid (squamoid) cells might
show higher level of PTHrP expression, and PTHrP expression was predominantly
observed both in epidermoid cells and intermediate cells. Few signals were observed in
mucous producing cells in mucoepidermoid carcinoma. PTHrP expressing cell volumes
were measured by morphometry, and cancer with rich PTHrP expressed intermediate
cells showed significant relation of cancer malignancy including lymph node metastasis
and/or tumor recurrence in 5-year-follow up period. These results indicate that PTHrP is
actually expressed in mucoepidermoid carcinoma, and PTHrP expression in
intermediate cells would be closely related to the cancer malignancy.
Recently, the microenvironment surrounding tumor cells attracts attention. Stromal cells
have been thought to be composed of normal cells; however, Hida et al have reported
that tumor endothelial cells have abnormality and have different phenotype compared to
normal endothelial cells 16,17,18). Fibroblasts in cancer stromal tissue were also shown to
have different phenotype and termed as “Cancer-Associated Fibroblast (CAF)” 19,20).
CAF was shown to produce TGF-βthat induces epithelial-mesenchymal transition
(EMT)21). We supposed that PTHrP might affect extracellular matrix, and play a role in
changing extracellular matrix to CAF. CAF was induced in stromal tissue in
mucoepidermoid carcinoma, and CAF was most often seen around intermediate cells of
mucoepidermoid carcinoma. The precise mechanism of mucoepidermoid carcinoma
development was still obscure. However, intermediate cells are thought to be less
differentiated than these other cell types, and afterwards the source of the other cell
types in mucoepidermoid carcinoma22). Our results indicate that PTHrP producing
mucoepidermoid carcinoma induce CAF in stromal tissue, especially around in
42
intermediate cancer cell. It may account for the malignant potential of intermediate cell,
and would suggest that PTHrP expression could be a prognostic factor in
mucoepidermoid carcinoma.
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GA, Katsilieris I, Patsouris E: Mucoepidermoid carcinoma of the salivary glands,
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44
ALLERGIC ASTHMA PREVENTION WITH ASSISTED

DRAINAGE THERAPY, MINOCYCLINE, AND
TOOTHPICK TOOTHBRUSHING METHOD
Kimberly Clarissa Oetomo1, Antonius Lucky Arnando2, Ernie
Maduratna Setiawati3, Daniel Haryono Utomo4
ABSTRACT
Background : Based on classic literatures, allergic asthma is an inherited disease.
Nevertheless, new paradigm which is considered as “out of the box” revealed that
allergic asthma could be transmitted. This phenomenon is in accordance with the
transmission of periodontopathic bacteria concept. Bacterial toxin might stimulate
immunocompetent cells, thus aggravating allergic asthma; thus, local immune response
should be increased to prevent infection. Since oral and nasal cavity have the same
innervation, according to “one airway one disease” concept, this toxin might induced
allergic asthma because of immunogenic and neurogenic inflammation interaction. The
immune response system in children under 10 are still not well-developed, especially
Immunoglobulin A, which is the first barrier to fight infection. Therefore, children were
more susceptible to periodontal infection such as gingivitis. Based on current research,
chronic gingivitis could trigger allergic asthma symptoms and the Assisted Drainage
Therapy (ADT) was able to increase respiratory quality in minutes. ADT as well as
tooth pick technique could reduce the inflammation. In addition, minocycline, aside
from being an antibacterial substance also possesses cytoprotective effect. Last but not
least, toothpick toothbrushing method may increase epithelial keratinization. Purpose :
To investigate and provide solutions to prevent allergic asthma transmission through
periodontopathic bacteria with ADT, toothpick toothbrushing method and 0.2 %
minocycline mouthrinse. Methods: Children with mild allergic asthma were conducted
ADT as well as tooth pick technique, several rinse with Minocycline. Statistic analysis
uses paired-t test. Result: Tooth pick technique increased respiratory quality after ADT
even more in 2 hours despite insignificant (p=0.054). Nevertheless, after 2 weeks,
toothpick combined with minocycline increased significantly (p= 0.018). Conclusion:
ADT, toothpick technique and rinsing with 0.2% minocycline were able to reduced
asthmatic symptoms effectively if worked in concert, because gingiva become less
susceptible to infection which leads to the decrease of allergic asthma symptoms.
Keyword : children, allergic asthma, assisted drainage therapy, minocycline
mouthrinse, toothpick toothbrushing method
45
INTRODUCTION
Asthma is a chronic respiratory disease characterized by inflammation of the
airways and lungs. Asthma prevalence of all ages throughout the world is increasing,
including in Indonesia. Epidemiological studies during 1991-2003 in Indonesian
children (6-15 years) revealed variable results, from 2.6 % (in Bandung, 1997) up to
17.4 % (in Jakarta, 1996).1 Approximately two-thirds of asthma is allergic and >50% of
patients with severe asthma have allergic asthma. Allergic asthma or immunoglobulin E
(IgE) mediated asthma is characterized by the presence of IgE antibodies against one or
more common environmental allergens. Asthma attacks can cause a variable symptoms
ranging in severity from mild to life-threatening. These symptoms are wheezing,
breathlessness, chest tightness, and coughing. Asthma can be controlled by taking
bronchodilators or corticosteroids; mast cell stabilizers, immunotherapy etc.
Nevertheless, there is still no exact cure for asthma; even after treated with recent
established asthma management protocol there were still 5-10% patients who
unsuccessfully controlled.2 In addition, some etiologies of asthma were still unknown;3
therefore, other possibility such as oral focal infection as a trigger of asthma
comorbidities (i.e. rhinosinusitis)4,5 should be considered.
Although asthma management had been updated every year, until the beginning
of 2009 control of oral infection was not included in asthma management protocol.6,7
Probably, it could be related to an established concept by David Strachan with a catchy
name the “hygiene hypothesis”. It proposed that early infections may enhance the T-
helper 1 (Th1) immune response development which is allergy-resistant.8 It is
interesting that this concept also agreed by Arbes et al.9 and Friedrich et al.10 who were
dental researchers. Moreover, according to Steinbacher and Glick,11 dental treatment
increases anxiety which trigger asthma exacerbation. Therefore, the concept of reducing
asthmatic symptoms through elimination of oral infection is not easily accepted either
by medical or dental professionals.
Several case reports revealed that asthmatic symptoms could be diminished by
elimination of oral infection12 and dental plaque control therapy.13 In addition, rapid
resolution of rhinosinusitis, one of asthma comorbidities, had been reported resulted
after intra-oral approach that proposed as the “assisted drainage” therapy (ADT) .14
Therefore, to verify the concept, a collaborated research was done by dental practitioner
and pediatric allergy consultant in Department of Child Health RSUD Dr. Soetomo,
Surabaya (Utomo dan Harsono). Evaluation of respiratory quality for forced expiratory
volume in one second (FEV1) was done using a computerized spirometer (Vitalograph
Spirotrac IV).15 It was interesting that the assisted drainage therapy was able to increase
FEV1 within minutes; this quick response actually resulted from β-adrenergic inhaler.
However, it could be elucidated with several theories: the neurogenic switching
mechanism16 and the axon reflex (i.e. nasobronchial reflex).17 Nevertheless, ADT could
only conducted by dental practitioners, daily maintenance of oral hygiene to avoid
asthma exacerbation was not yet evaluated.
46
It was interesting that according to Yoo et al,18 recent asthma researches

revealed that it could be transmitted, and coincidentally, Lee et al.19 reported the
possibility of periodontopathic bacteria tranmission from caregivers to children.
Therefore, oral hygiene improvement of children, thus periodontal health, especially in
asthmatic children and their caregivers should be maintained to reduce asthma
symptoms.
Poor gingival resistance to infection which may lead to gingivitis and asthma
susceptibility could be improved by toothpick toothbrushing method that had been
proven to increase gingival resistance to infection by increase keratinization and
immunoglobulin A. Additionally, .2 % Minocycline mouthrinse had antibacterial
activity as well as cytoprotective effect to gingival epithelial cells.20
The objective of this research is to evaluate the effectiveness of the of tooth pick
toothbrushing method and minocycline mouthrinse to maintain the result of the assisted
drainage therapy in improving respiratory quality in allergic asthma. Hopefully, this
study encourage other medical and dental practitioners to conduct researches, which
then could lead this dental approach of asthma therapy in children‟s asthma
management protocol.
The Assisted Drainage Therapy (ADT)
Assisted drainage therapy is a term for the process of subgingival plaque
cleaning accompanied by a massaging movement of gingiva sulcus for three minutes
using a sickle shaped scaler which is a modification of scaling-root planning (SRP)
procedure (Fig. 1). The scaler will be conducted as SRP procedure on the buccal,
interdental, and palatinal of posterior maxillary teeth with added massaging strokes on
gingiva sulcus using the blunt side (red arrow) so there will be blood seeping out. The
pressure must not cause any pain.
Figure 1. The Assisted Drainage Therapy movement, scaling-root planing (SRP)

with gingival sulcus massage (adapted from Newman et al., 2006).21
On chronic gingivits, light pressure on gingiva sulcus may cause mild bleeding.
For prevention of unstoppable bleeding complication, a blood clotting test must be
done. While for the infection caused by the action, application of hexetidine 0,1% must
be done in a minute before the process. If presumed that there is congenital heart
disease and acute infection, the action should be cancelled and referred to a heart
specialist. The assisted drainage therapy also succeeded in relieving children
47
rhinosinusitis cases rapidly and by keeping the oral hygiene the symptoms will not
appear anymore.22
Utomo and Harsono23 reported that ADT significantly increase respiratory
quality based on FEV1 in minutes, it also reduced histamine level in blood serum.
Respiratory Function test in this research in done by computerized spyrometer Spirotrac
IV®.
A simple indicator for direct evaluation of the successfulness of the ADT is the
“paper blowing” test. It is done after about 3 minutes after the ADT by blowing a piece
of paper with nose in the treated side, concomitantly with closing the mouth and the
other nostril with the operator‟s finger. If the patient is able to blow the paper without
effort, the procedure is successful. Subsequently, it was followed by dental health
education (DHE) for conducting toothpick toothbrushing method and also told to rinse
with 0.2 % minocycline mouthrinse twice daily.
Toothpick Toothbrushing Method
Toothpick toothbrushing method is a relatively new method, with an advantage
of plaque cleansing on interproximal more than other methods, where the part is the first
area for periodontal disease route anda it can stimulate IgA that functions as mucosal
defense. IgA secretory in oral cavity functions as an inhibitor of bacterial or virus on
epithelial surface and aglutinate antigen.24
Toothpick toothbrushing method is done by putting the tip of the brush on the
edge of gingiva facing the crown on 30-45 degrees according to the length of teeth axis.
The brush will be pushed into the interdental gap and pulled as the way a toothpick
would on the buccal and lingual side. This back and forth movement is repeated eight
until nine times in one region. The application of this technique allows interproximal
plaque cleansing without the help of dental floss or interproximal toothbrush. A small
amount of brush will enter the narrow interdental gap, while one or two bunch will enter
the wide interdental gap.25.
The toothbrushing mechanic stimulation affects on the activity of basal
junctional epithelium cell proliferation, synthesis of collagen and gingiva cells,
increasing gingiva oxygen saturation and stimulates the secretion of IgA on mucous
membrane as a humoral defense towards antigen. The toothbrushing stimulation effect
varies towards periodontal tissue, one of them is stimulating the keratinization of
epithelial oral, stimulating the circulation of gingiva capiler, fibroblast proliferation, and
reducing the amount of inflamed cell.24
Minocycline Mouth Rinse
Minocycline, a wide spectrum antibiotics derived from the second generation of
Tetracycline, has been improved for periodontal therapy. Minocycline 0. 2 % was
considered had anti bacterial activity as well as antiinflammatory. Moreover, it had
cytoprotective effect, thus it was able to prevent gingivitis.20
48

Children 5 – 12 years old who suffered from mild persistent asthma were
selected by pulmonogist.The study was clinical experimental, pre-post control design. It
had been done in Periodontology Clinic Airlangga University, Surabaya; Pulmonology
Clinic Mardi Rahayu Hospital, Kudus and Pediatric Clinic, Kartini Hospital Mojosari.
According to sample formula it was found that minimal samples were 6. However, In
this study, 12 children were included. The parents were signed informed consent to be
included in this study. Ethical Clearance was approved by local ethics committee at
each study center.
Sample were boys and children aged 5-12 who suffered from mild
persistent asthma, lung function reversibility based on FEV1 > 12%, pre and
post bronchodilator. Exclusion criteria were: (a) suffer from acute pulpitis; (b)
CLP; (c) using prosthesis or orthodontic appliance; (d) suffer from systemic
disorder; (e) consuming antibiotics during research; (f) consuming allergic
medicines; (g) Patient is not an active or passive smoker. Samples were
selected by Simple Random Sampling.
Variables investigated were: toothpick tooth brushing technique and
minocycline mouth rinse (independent variable); and respiratory quality lung
function test, FEV1 (dependent variable).
Procedures: Samples who are asthmatic patients were conducted the assisted drainage
therapy (ADT) first, then 2 hours later were taught the tooth pick toothbrushing
technique. They were evaluated before and after treatment (Table 1). The result was
very good, the differential significancy using paired-t test was p: 0.001, and the Number
Needed to Treat (NNT) was 1.09; it was meant that every one patient treated, the
successful result was nearly 100% (100 : 109).
Table 1. Mean and SD

Group Mean SD
of FEV1 (%)
Prior to treatment (control) 67.25 7.63
Treatment 1 (SRP+ADT) 81.58 12.52
Treatment 2 (Toothpick Technique) 83.75 9.72

2 weeks post Toothpick Technique 81.16 10.57
Notes: Cut of point of controlled asthma (asymptomatic asthma) is 80%.
49
Tabel 2. Paired t-test pre-ADT (control) with treatment group 1 SRP+ADT,

treatment group 2 toothpick technique, and 2 week post toothpick.
Treatment Group P Significancy

Pre ADT (control) and Treatment 1 (SRP+ADT) 0.001 Significant
Pre- ADT(control) and Treatment 2 toothpick 0.001 Significant
Pre-ADT(control) and 2 week post toothpick 0.001 Significant
Tabel 3. Paired t-test Tretmen Group 1 (SRP+ADT) with Treatment Group 2

toothpick and 2 week post toothpick, and Treatment Group2 2 toothpick and 2
weekpost toothpick
Group P Significancy
Treatment 1 (SRP+ADT) and Treatment 2 toothpick 0.054 insignificant
Treatment 1(SRP+ADT) and 2 weeks post toothpick 0.586 insignificant
Treatment 2 toothpick and 2 weeks post toothpick 0.018 significant
DISCUSSION
For better understanding of this discussion, a glance of basic medical sciences
such as anatomy, physiology; immune and neural system should be reviewed.
Additionally, according to literatures, there was an interaction between the
immunogenic and neurogenic inflammation so called the “neurogenic switching
mechanism”16,26that should be considered in the asthma etiopathogenesis. Therefore, in
our concept, an ideal asthma management protocol should include an integrated
management of both inflammations.
The assisted drainage therapy has the propensity for reducing both the trigger of
the immunogenic and neurogenic inflammation (by removing subgingival plaque and
toxins), and their products (i.e. pro-inflammatory mediators and neuropeptides).12
These mediators were capable to stimulate the maxillary nerve innervating the oral
cavity and propagate antidromically (in reverse direction to the regular neural
impulse)27 via the Sphenopalatine ganglion (SPG) (Fig.2) which then spread the
inflammation to systemic or distant organs.26,28
50
Sphenppalatine
Ganglion
Sphenopalatina
Figure 2. The Connection of Maxillary Nerve (CN V2) with sphenopalatine ganglion
According to Chih Feng and Baraniuk,29 the neural system has an important role
in the sensitivity of respiratory organs, hence the nose. In addition, the SPG, a
parasympathetic ganglion has an important role as a relay center between the afferent
nerves, the autonomic (the parasympathetic) and sensory nerve (maxillary nerve) which
related to multiple chemical sensitivity (MCS) upon stimulation of allergens, cold,
smokes etc. Moreover, further analysis of the interrelationship of the maxillary nerve
and asthma was the existence of receptors in the nose and pharynx and, presumably, in
the paranasal sinuses that had afferent fibers which became part of maxillary nerve.
They passed to the brain stem and connected with the reticular formation of the dorsal
vagal nucleus. From the vagal nucleus, parasympathetic efferent fibers travel in the
vagus nerve to the bronchi.28 The interneural relationship between upper and lower
respiratory tract was able to explain the “one airway-one disease” concept.30 Therefore,
down-regulation of SPG sensitivity via ADT which led to rapid improvement of
respiratory quality (FEV1) is logical.
Rapid improvement of FEV1 after ADT that was showed in Table 1. could be
the effect of rapid reduced expression of pro-inflammatory cytokines (i.e. tumour
necrosis factor-α), neuropeptides which involved in the neurogenic switching
mechanism (substance P, SP and calcitonin gene-related peptide, CGRP)16,26 that were
“drained out” within the oozed blood caused by ADT. Substance P half life was <6 min
and CGRP was 6-10 min after degraded by neuropeptidase.16 This interesting
phenomenon had been verified by Utomo31 in an animal study, which revealed a sudden
fall in SP and CGRP expression 20 min after ADT, with significant difference
(p=0.001; CI 95%) compared to control.
Mast cells and basophils are the “traditional” major sources of asthma mediators,
Bacterial dental plaque and their toxins, the proteoglycans, PGN (Gram-positive) and
lipopolysaccharides, LPS (Gram-negative), are able to stimulate these
immunocompetent cells to produce asthma mediators such as histamine and
leukotrienes.20 However, this stimulation is not limited via the cross-linking mechanism
51
that is only for mast cells and basophils; nevertheless, most immunocompetent cells
could be stimulated via the complement receptors (i.e. C3aR for bacteria), and toll-like
receptors (TLRs).32
Our study was contradictory to Arbes et al.9 who showed that lower allergy
prevalence existed in people with higher serum IgG to Porphyromonas gingivalis, and
Friedrich et al.10 who revealed that periodontitis patients were allergy-resistant; these
studies were in accordance with the hygiene hypothesis. However, this contradiction
could be explained by understanding our concept. Following ADT and oral hygiene
maintenance, oral pathogenic bacteria, toxins and pro-inflammatory mediators
diminished which led to decrease the neurogenic switching mechanism and stimulation
of immunocompetent cells which are responsible in allergic asthma reaction.
In day one, 2 hours after ADT, tooth pick technique was able to increase FEV1
even more, even rather insignificant (p=0.054; p<0.05) (Table 2), thus had an
advantageous effect to improve respiratory quality. This phenomenon was confirmed
since after 2 weeks samples doing toothbrushing with toothpick technique the difference
was significant (p=0.018) (Table 3). The use of minocycline mouthrinse may also
support the successful therapy, it was used for its antibacterial as well as cytoprotective
effect to gingival epithelial cells. Nevertheless, future researches for evaluating the oral
flora environment after a long period of mouthrinse use should also be done.
The effect of duration and strength of mechanical stimulation on brushing teeth
affect the proliferation activity of basal cell in junctional epithelium, synthesis of
collagen and gingival cells, increases the oxygen saturation of gingiva as well as
stimulating the secretion of IgA in mucous membrane as humoral defense against
antigens. Immunoglobulin A is one class of immunoglobulin secreted by gingival
crevicular fluid which acts as a defense of the mouth mucous membranes.24.
Secretory IgA at the mouth cavity inhibits the attachment of bacteria or viruses
on the s epithelial surface and agglutinates antigen. It also triggers epithelial
keratinization.24 Moreover, The “first barrier” of defense that were gingival epitehelial
cells were strengthened by the cytoprotective effect of 0.2 % minocycline rinse.
This research supports the concept of respiration quality improvement with ADT
which is pioneered by Utomo and Harsono23 as therapy to mild allergic asthma
persistent with Number Needed to Treat value (NNT)= 1,09 (95%; CI 0,9-1,3;), it
means to get relief on 1 patient required 1 subject. Utomo 31 in an animal study
reported the value NNT = 1,04± 0,4 (CI 95%).
This research also support the fulfillment of four stages of clinical application
research in humans so that the results of this research can be applied widely in asthma
sufferers to reduce corticosteroid drug dependence.
52
CONCLUSION AND SUGGESTION
For the concluding remarks, toothpick tooth brushing method and minocycline
0.2% mouthrinse had the ability to support the assisted drainage therapy in maintaining
the increase of FEV1 in asthmatic children 5-12 years old. Therefore, this home
maintenance could be suggested as an adjuvant in asthma management protocol. In
order to achieve long lasting result, ongoing collaborated evaluation with a pediatric
allergy expert or pulmonologist; concomitant with parents‟ cooperation for supervising
children‟s oral hygiene maintenance are mandatory.
Furthermore, for wide acceptance of this concept, which was considered
contradictory with the established hygiene hypothesis, multi-centered collaborative
study should be conducted. Additionally, widespread of this invention through journals
and media in Indonesia and abroad is compulsory to be included in the global strategy
for asthma management and prevention: Global Initiative for Asthma (GINA).
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7. Gupta RS, Weiss KB. The 2007 National Asthma Education and Prevention Program
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10. Friedrich N, Volzke H, Schwahn C, Kramer A, Junger M, Schafer T et al. Inverse
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11. Steinbacher DM, Glick M. The dental patient with asthma: An update and oral
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behavioral intervention and children‟s immunity. J Indonesian Dent Assoc.
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Surabaya, August 2007. p. 5-10.
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16. Lundy W, Linden R. Neuropeptides and neurogenic mechanism in oral and
periodontal inflammation. Crit Rev Oral Biol 2004; 15(2): 82-98.
17. Yaprak M. The axon reflex. Neuroanatomy 2008; 7: 17-9
18. Yoo J1, Tcheurekdjian H, Lynch SV2, Cabana M3, Boushey HA. Microbial
Manipulation of Immune Function for Asthma Prevention Inferences from
Clinical Trials. Proc Am Thorac Soc 2007; 4: 277–82,
19. Lee Y, Straffon LH, Welch KB, and W.J. Loesche WJ. The Transmission of
Anaerobic Periodontopathic Organisms. J Dent Res 2006; 85(2):182-6
20,Agustina EF, The comparison of minocycline oral rinse and gel to reduce pocket
depth, Dent J 2010; 43(1): 21-5
21. Newman MG, Takei H, Klokkevold PR and Carranza FA, 2006. Carranza‟s
Clinical Periodontology. 10th ed. Elsevier-Saunders. p.146
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approach in children’s rhinosinusitis management. Indonesian J of Dentistry
2006; 13:133-6.
23.Utomo H, Harsono A. Rapid improvement of respiratory qualityin asthmatic children
after assisted drainage therapy. Paediatr Indo 2010; 50: 199-206
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of toothbrushing frequency on proliferation of gingival cells and collagen
synthesis, J Clin Period 2004; 31(1): 40-4
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Accessed April 20, 2012.
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29. Chih-Feng T, Baraniuk JN. Upper airway neurogenic mechanisms. Cur Al Clin
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Bronconeomol 2005; 41: 569-78
31. Utomo H. The immunoneuromodulatory effect of the assisted drainage therapy
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Airlangga University. Unpublished data. 2009: 90
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Philadelphia. Saunders-Elsevier 2007: 24-5, 343
55
ANTIBACTERIAL EFFECT OF MORINDA

CITRIFOLIA.L ETHANOL EXTRACT TOWARDS THE
GROWTH OF PORPHYROMONAS GINGIVALIS AND
AGGREGATIBACTER ACTINOMYCETEMCOMITANS
(IN VITRO)
Anindyajati Nuralifiana S, Riztika A Devi, Robbykha Rosalien, Sanny
Tulim
Faculty of Dentistry Universitas Indonesia
ABSTRACT
Objective: To reveal antibacterial active compounds in Morinda citrifolia.L fruit and
leaf ethanol extract and to analyze the antibacterial effect of Morinda citrifolia.L fruit
and leaf towards the growth of two dominant bacteria in periodontal diseases:
Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.
Method: Maceration method was used to extract M. citrifolia fruit and leaf using
ethanol 96% as the solvent. Phytochemical test was performed to reveal the antibacterial
active compounds contained in both extracts. Different concentrations of extracts were
tested towards the growth of P.gingivalis and A.actinomycetemcomitans with dilution
method to determine MIC and MBC and diffusion method to determine zone of
inhibition.
Result: 11 antibacterial active compounds were found in M. citrifolia fruit and leaf
ethanol extracts: anthraquinone, terpenes, alkaloids, flavonoids, steroid, glycoside,
saponins, essential oil, phenols, tannins, terpenoids. MIC and MBC of M. citrifolia leaf
ethanol extracts are at 5% and 25% towards the growth of P.gingivalis and
A.actinomycetemcomitans. While the MIC and MBC of M. citrifolia fruit ethanol
extracts are at 10% and 20% towards the growth of P.gingivalis and
A.actinomycetemcomitans. The widest inhibitory zone is 3.65mm at 100%
concentration of M. citrifolia fruit ethanol extracts towards the growth of P.gingivalis.
While the narrowest inhibition zone is 2.0 mm at 45% concentration of M. citrifolia leaf
ethanol extracts towards the growth of P.gingivalis.
Conclusion: Ethanol extracts of both M. citrifolia leaf and fruit have antibacterial effect
towards the growth of P.gingivalis and A.actinomycetemcomitans due to the presence of
antibacterial active compounds in it. Therefore, M. citrifolia is a potential candidate for
treatment of periodontal diseases.
56
Keywords : Extract ethanol of Morinda citrifolia.L, Porphyromonas gingivalis,

Aggregatibacter actinomycetemcomitans, antibacterial effect, phytochemical test.
INTRODUCTION
Traditional medicine has taken a major role as primary health care needs of more than
80% of the world‟s population according to World Health Organization (WHO).
Substances found in plants especially the secondary metabolite products have taken a
lot of attention of scientists in order to get a wider variety of drugs as the antibiotic-
resistant bacteria is raising. The chemical substances in the medicinal plants produce a
definite physiological action on human body that over the years have been used to treat
various ailments in traditional medicine.1,2
M. citrifolia has been classified as a medicinal plant due to its therapeutic properties.
These plants grow from India through Southeast Asia and Australia to Eastern
Polynesia and Hawaii. Previous researches have revealed that almost all parts of the
plant such as roots, stems, leaves and fruit have many therapeutic properties, including
anti-inflammatory, antibacterial, anti-oxidant, anti-fungal, etc.3,4 In the present study,
M. citrifolia antibacterial property has been screened and the results showed effect on
the growth of most of the tested bacteria. It also indicate that M. citrifolia can be used in
the treatment of infectious disease. The active compounds in M. citrifolia works alone
or in a combination towards the growth of bacteria.5
Pseudomonas aeruginosa, Staphyloccocus aureus, Bacillus subtilis, Escherichia coli,
Salmonella, Shigella, M. pyrogenes, Enterobacter aerogenes ATCC 13048, Proteus
vulgaris and Proteus morgaii were some of bacteria had been tested that can be
inhibited or killed by antibacterial active compounds in M. citrifolia plants.4,5
To the best of our knowledge, there hasn‟t been any research about antibacterial effect
of M. citrifolia plants against the growth of dominant bacteria in periodontal diseases
which affect 70-80% of the population worldwide. Porphyromonas gingivalis and
Aggregatibacter actinomycetemcomitans are most dominant bacteria found in chronic
and aggressive periodontitis that according to the recent study showed some resistance
to metronidazole, amoxicillin and clyndamicin.6,7
Thus, the objectives of this research were to reveal the antibacterial active compounds
in both M. citrifolia leaves and fruits and also to investigate the antibacterial effect of
M. citrifolia plant against the growth of P. gingivalis and A. actinomycetemcomitans.
57

Extraction of Plant Materials :
Fresh yellowish green fruits and dark green leaves were collected from a M. citrifolia
plantation of PT Trias Sukses DInamika in Bogor West Java Indonesia. The plants were
identified and confirmed as Morinda citrifolia. L by Research Center for Biology of
Indonesian Institute of Science.
A total of 5150 grams of fresh yellowish green M. citrifolia fruit and 2085 grams of
fresh dark green M. citrifolia leaf were washed and chopped into thin small pieces then
air dried in shade for 24hrs, moved into cabinet dryer for another 24hrs, dried in hot
oven at 50oC 30mnts.
563 grams of dry M. citrifolia fruit and 465 grams of dry M. citrifolia leaf were grinded
into fine powder using waring blender, macerated with 96% ethanol solution as solvent
in 1:4 ratio of powder:solvent. The powder and solvent were mixed together for 4 hr
and stored for 20hr.
Filtrate was separated from the residue and then using rotary evaporator in 40-50oC
175atm 70rpm, the solvent was evaporated from the filtrate leaving 165 grams and 95
grams of paste of M. citrifolia fruit and leaf extract as final products.
Identification of Qualitative Antibacterial Compounds in extracts:

Tannin (Extracts were dissolved in ethanol 96%. Reaction with 5ml of gelatin 10% or
5ml of 10% NaCl ad 1% gelatin will results in the formation of sediment which shows
the presence of tannin). Alkaloids (Extracts were mixed with 1ml HCL and 9ml
aquadest, steamed for 2 minutes. Mixing three drops of mixture with 2 drops of
Bouchardat LP will result in the formation of brown to black sediment if alkaloid is
present in the extract). Saponins (Mixing and shaking extracts with 10ml of hot water
will results in the formation of persistence frothing which shows the presence of
saponins). Terpenoid (few drops of eter filtrate from extract mixed with 2 drops of
acetic anhydride and 1 drops of H2SO4. A change of color into red, orange and yellow
shows the presence of terpenoid, while a change of color into green shows the presence
of steroid). Flavonoid (reaction with Mg-HCl:red velvet color, reaction with NaOH
10%:yellow color, reaction with H2SO4 :orange color, all indicate the presence of
flavonoid). Phenol (extract+aquadest+ FeCl3: bluish green color indicates the presence
of phenol). Anthraquinone (reaction with H2SO4 heated and then cooled, shake and
filtered, yellow residue on paper indicates the presence of anthraquinone). Essential Oil
(heat the mixture of extract and ethanol. Fragrance from the heated extract indicates
presence of essential oil). Terpenn (extract mix with hexane dropped to thin layer
chromatograph plate. Plate soaks into H2SO4 then dried and heated. Bluish purple color
appear on the plate indicates the presence of terpenn).
58
Bacterial Strains
In vitro antibacterial activity was examined for ethanol extracts from the fruits and
leaves of M. citrifolia. Bacteria Aggregatibacter actynomicetemcomitans ATCC 43718
and Porphyromonas gingivalis ATCC 33277 were obtained from Oral Biology
Laboratory of Faculty of Dentistry Universitas Indonesia. All cultures were tested for
purity.
Media Preparation and Antibacterial Activity

Dilution method:
Two ml of ethanol extracts of different concentrations were put into test tubes and 0.2
ml of microorganism suspension (106 CFU/ml of A.actinomycetemcomitans and 108
CFU/ml of P.gingivalis) poured into the test tube. The tubes were incubated
anaerobically at 37oC. After the period of incubation, the tube containing the least
concentration of extract showing no visible sign of growth was considered as the MIC.
To determine the MBC, after 3x24hrs, one drop of every tube was scratched on a Brain
Heart Infusion (BHI) agar media and incubated again for another 3x24hrs. The least
concentration scratched that showed no bacterial growth was considered as the MBC
Diffusion method:
0,2ml of bacteria suspension (106 CFU/ml of A.actinomycetemcomitans and 108 CFU/ml
of P.gingivalis) poured on BHI agar media enriched with vitamin K. Ten 6mm-
diameter-blank disc inoculated with 20μl of different concentration of extracts were put
on the agar and incubated for 2x24hrs 37oC. Antibacterial activity was determined by
measuring the inhibition zone.
Results
Phytochemical test revealed the presence of antibacterial active compounds in the fruits
and leaves ethanol extracts shown in table 1.
59
Table. 1 Antibacterial active compounds in leaves and fruits extract

Antibacterial Leaves Fruits
compounds Extract extract
Terpen + +
Tannin + +
Phenol + +
Essential oil + +
Alkaloids + -
Saponins + +
Flavonoids + +
Anthraquinone + +
Glycoside + +
Steroid + -
Terpenoids - +
Antibacterial activity test

Lack of growth of bacteria on BHI agar media at concentration 25% of leaves extract
showed that MBC of M. citrifolia leaf ethanol extracts are at 5% towards the growth of
P.gingivalis and A.actinomycetemcomitans (Table 1). The MIC of leaf extracts was at
5% concentration towards the growth of P.gingivalis and A.actinomycetemcomitans, it
could be seen from the presence of inhibition zone at 5% concentration in the diffusion
method.
The MBC of M. citrifolia fruit ethanol extracts are at 20% concentration towards the
growth of P.gingivalis could be seen from lack of growth of bacteria, while the MIC
was at 10% concentration towards the growth of A.actinomycetemcomitans. it could be
seen from the re-growth of bacteria on BHI agar media at 10% concentration that did
not turn cloudy on dilution method showing no bacterial growth. (Table 2).
The widest inhibitory zone is 3.65mm at 100% concentration of M. citrifolia fruit
ethanol extracts towards the growth of P.gingivalis. While the narrowest inhibition zone
is 2.0 mm at 45% concentration of M. citrifolia leaf ethanol extracts towards the growth
of P.gingivalis. The inhibition zone was greater as the concentration of extracts were
also higher due to the increasing quantity of antibacterial active compounds consist in
the extracts (Graphic 1 and 2).
60
Table 2. MIC and MBC of leaves extract towards the growth of P.gingivalis and
A.actinomycetemcomitans
Uji Konsentrasi (%) K K

Dilusi
5 10 15 20 25 30 35 40 45 (+) (-)
I + + + + - - - - - + -
II + + + + - - - - - + -
Table 3. MIC and MBC of fruits extract towards the growth of P.gingivalis and
A.actinomycetemcomitans
Uji Konsentrasi (%) K K

Dilusi 10 20 30 40 50 60 70 80 90 100 (+) (-)
I + - - - - - - - - - + -
II + - - - - - - - - - + -
Graphic 1. Inhibition zone of leaves extract

towards the growth of P.gingivalis and A.actinomycetemcomitans in mm
2.5
1.5 Pg
Aa
1
0.5
0
5% 10% 15% 20% 25% 30% 35% 40% 45% 61
Graphic 2. Inhibition zone of fruits extract

towards the growth of P.gingivalis and A.actinomycetemcomitans in mm
4
3.5
3
2.5
2 Pg
1.5 Aa
1
0.5
0
10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
DISCUSSION
The increased of resistant bacteria towards many commonly used antibiotic leads
scientists toward medicinal plants for a new, cheap, effective and lower risk drugs to
treat infectious diseases. Plant herbal mixture as therapeutic treatment have made large
contribution to human health and well being.5,8
Technique of extraction and the choice of solvent are two of the most important
methods to consider because the quantity and quality of antibacterial active compounds
extracted from the plant relies on it.9 Maceration technique using ethanol 96% as a
solvent was chosen considering the low temperature of heat during maceration and the
ability of ethanol 96% in extracting both polar and non-polar compounds.
Preliminary phytochemical test revealed the presence of 11 antimicrobial active
compounds in fruits and leaves extracts of M. citrifolia plants. This presents are the
reasons behind the ability of fruits and leaves extract to kill and inhibit the growth of
P.gingivalis and A.actinomycetemcomitans. The decision to test those 11 antibacterial
active compounds was taken due to previous literature research by Wang, et al and
Jayarman, et al confirming the existence of phenol, tannin, alkaloid, anthraquinone,
saponins, glycoside, flavonoid, terpenoid, essential oil, steroid and terpenn in M.
citrifolia.3,4
62
It is not surprising that fruits extract showed a higher antibacterial activity in the result
shown above due to the higher concentration of extracts tested in the diffusion method,
which was from 10%-100% concentration, compared to leaves extract, which was only
5%-45% concentration. Since phytochemical test was not able to reveal the quantity of
each antibacterial active compound in the extract, higher quantity of compounds in the
fruits extract was considered as the reason behind its higher antibacterial activity
compared to leaves extract.
The antibacterial active compounds in leaves and fruits extracts of M. citrifolia plant
work in many ways. Winjsma and Verpoorte (1986) reviewed various anthraquinone
present in Morinda spp. About 90% of these compounds occur as derivatives of 9, 10-
anthracenedione with several hydroxy and other functional groups such as methyl,
hydroxy methyl or carboxyl side chain. Hydroxy anthraquinones are the active
principles of many phyto-therapeutic drugs.10 The presence of this compound in M.
citrifolia might be the reason behind its medicinal values.
Besides anthraquinone, other antibacterial active compounds also have their own roles
as antibacterial agents. Alkaloids, steroids, essential oil and flavonoids inhibit the
formation of cell wall. Phenols, terpen and saponins disturb the function of cell
membranes, while tannins inhibit the production of protein and nucleic acid and also
inhibit metabolism of the bacteria.11,12
Those antibacterial activities varied related to the test organisms and acted differently
against A.actinomycetemcomitans and P.gingivalis because the structure of cell wall
envelope and cytoplasm of both bacteria are also different.2,13
Further investigation is needed to determine the quantity of each antibacterial active
compound in the extracts, to reveal which antibacterial active compound works most
effectively against bacteria, and to analyze the possibility of using M. citrifolia fruits
and leaves as a candidate of treatment for periodontal disease in human being.
CONCLUSION
Ethanol extracts of both Morinda citrifolia.L leaf and fruit have antibacterial effect
towards the growth of P.gingivalis and A.actinomycetemcomitans due to the presence of
antibacterial active compounds in it. The overall result of this research indicate
promising baseline of information for the potential use of M.citrifolia in the formulation
for treatment of periodontal diseases. However, it is necessary to determine the toxicity
of the M. citrifolia extract, their side effects and pharmaco-kinetic properties.
63
REFERENCES
1. Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity of some
ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC
Complementary and Alternative Medicine 2006;6:35
2. Pirbalouti et al. Ethnobotany and antimicrobial activity of medicinal plants of Bakhtiari
Zagross mountains, Iran. Journal of Medicinal Plants Research. 2012; 6:5. p 675-679
3. Peter PI. Review on The Current Scenario of M. citrifolia Research: Taxonomy,
Distribution, Chemistry, Medicinal and Therapeutic Values of Morinda citrifolia.
International Journal of M. citrifolia research. 2005. 1(1): p. 1-12.
4. Wang MY, et al. Morinda citrifolia (M. citrifolia): A literature Review and Recent
Advances in M. citrifolia Research. Acta Pharmacol Sin. 2002;23:12 p.1127-1141.
5. Natheer et al. Evaluation of antibacterial activity of Morinda citrifolia, Vitex trifolia and
Chromolaena odorata. African Journal of Pharmacy and Pharmacology. 2012; 6:11, pp.
783-788.
6. Samaraya LP. Microbiology of Periodontal Disease. Essential microbiology for
dentistry. 2ed. Edinburg: Churchil Livingstone 2002: 229-30.
7. Ardila CM, López MA, Guzmán IC. High resistance against clindamycin,
metronidazole and amoxicillin in Porphyromonas gingivalis and Aggregatibacter
actinomycetemcomitans isolates of periodontal disease. Med Oral Patol Oral Cir Bucal.
2010;1:15 (6):e947-51..
8. Sharma A, Verma R, Ramteke P. Antibacterial Activity of Some Medicinal Plants Used
by Tribals Against Uti Causing Pathogens. World Applied Sciences Journal. 2009; 7:3
pp 332-339.
9. Praveen K et al. Antioxidant activity, total phenolic and flavonoid content of morinda
citrifolia fruit extracts from various extraction processes. Journal of Engineering
Science and Technology. 2007;2:1 pp 70 - 80
10. S. Sreeranjini, E.A. Siril. Evaluation of anti-genotoxicity of the leaf extracts of Morinda
citrifolia Linn. Plant Soil Environ. 2011;57:5 p. 222–227
11. Sarida M, Tarsim, and Faizal I. Pengaruh Ekstrak Buah Mengkudu (Morinda citrifolia
L.) dalam Menghambat Pertumbuhan Bakteri Vibrio harveyi secara In Vitro. Jurnal
Penelitian Sains. 2010; 13:2. p. 59-63.
12. Zuhud EAM, et al. Aktivitas Antimikroba Ekstrak Kedawung (Parkia roxburghii
G.Don) terhadap Bakteri Patogen. Jurnal Teknologi dan Industri Pangan. 2001; 12:1. p.
6-12.
13. P. Barber et al. Identification of Porphyromonas gingivalis and Actinobacillus
actinomycetemcomitans in Apical Border Plaque. Microbial Ecology in Health and
Disease. 1991;4:3. p 159-167
64
Carica papaya LATEX EXTRACT AS

ACCELERATING AGENT FOR ORAL MUCOSA
LESION HEALING
Priska Angelia, Yovita Kusnadi, Nathania

Trisakti university, Indonesia
ABSTRACT
Background : Papaya is one of Indonesian famous fruits. However,Currently, people
only consume the fruit without using its latex. Whereas, papaya latex contains a lot of
beneficial nutrients such as saponin, flavonoid, and several kinds of vitamin that could
accelerate tissue healing process. oral mucosa lesions which is not treated immediately
often causes inflammation, clinically characterized by swelling and discoloration of the
mucosa and serologically increase the levels of IL-1 β.
Objective : The aim of this study is to determine the effect of carica papaya latex in
accelerating the healing process of oral mucosa lesion, by observing the clinical
symptoms and IL-1β.
Material and methods : The method which is used is experimental laboratory. Papaya
latex was made into powder by using centrifuge tube and freeze dry machine. Twelve
Spraque dawleys had their labial mucosa rubbed by the hydrogen peroxide (H2O2) 3%
and divided equally into two groups, treatment and negative control group. The papaya
latex extract powder was applied twice a day on the treatment group. Each subject from
each group were executed on the second, fifth, and seventh day to make specimen for
healing process evaluation. Followed score the colour of mucosa : 1. Dark red 2. Red 3.
Light red 4. Normal gingiva
Results : On the second day, subjects of both group‟s oral lesion scored 1, while on the
fifth day, treatment group scored 3, negative control group scored 2. On the seventh
day, both groups scored 4. The interleukin (IL) measurement showed that after one day,
the level of IL-1β in blood of treatment group and negative control group are 3.12 pg/
mL and 1.19 pg/mL respectively, and on the seventh day are 9.07 pg/mL and 14.11
pg/mL respectively.
Conclusion : This study concludes that Carica papaya latex extract may accelerate the
healing of oral mucosa lesion.
Keywords : healing of oral mucosa lesion, carica papaya extract, IL-1β

65
INTRODUCTION
Papaya is one of Indonesian most favourite fruits. Papaya (Carica papaya) is a
fruit that originated from southern Mexico and northern parts of South America1,
Currently, papaya is well widespread and widely grown in tropical. Commonly, people
only consume the meat of the fruit. 2
In tropical country such as Indonesia, this fruit can be easily grown either in
lowland and highland with a simple treatment and low cost maintanance. Papaya has
many benefits for the human body, whereas papaya latex contains a lot of beneficial
nutrients such as alkaloid, saponin, flavonoid, and several kinds of vitamin.
Papaya also has an antibiotics function which can be used for some
treatments without any side effects.3 It helps digestion process, prevention of
ulcer and short-sighted. The seeds are used as an helmintic 4 Since ancient times, the
root of papaya gives a good effect in treatment of kidney, bladder and
soreness. The leaves have the benefit of treating malaria, stomach cramps 4 Stems,
leaves and fruit of papaya contain white latex. This latex contains protein-breaking
enzyme or proteolytic enzyme called papain (Moehd,1999) . 6
Component Amount
Calories 46 cal
Protein 0.5 gr
Fat 0 gr
Carbohydrate 12.2 gr
Calsium 23 mg
Phosphor 12 mg
Iron / Fe 17 mg
Vitamin A 365 SI
Vitamin C 78 mg
Table 1. Content of Carica papaya (100g) 5
Papaya (Carica papaya) are classified as:
Kingdom : Plantae
Divisio : Spermatophyta
Subdivisio : Angiospermae
66
Class : Dicotyledonae
Ordo : Caricales
Familia : Caricaceae
Genus : Carica
Species : Carica papaya L.
Papain in papaya latex contains a proteolytic enzyme. Commonly it is used for

meat tenderizer, silk cloth washing material, beer purifying beverages, and milk
coagulant
Papain is well stabilized in pH 5.00 solution. Papain has a synthetic activity and
the characteristic of heat resistant is higher than any other enzymes. Besides to break the
activity of proteins, papain has also an ability to form new protein called plastein or
hydrolized protein. 8 Papain is a protease enzyme that can destroy protein primary
structure 9 and catalyze the reaction of peptide hydrolisis.10
The latex also contains alkaloids, saponins, flavonoids and ascorbic acid. Papaya
latex has a benefit effect to tissues especially the soft tissue that found in the human oral
cavity.
Saponins are found in various animals and plants. Saponins‟ character is similar
to soap because of its surfactant characteristic . Saponins‟ biological activities that can
accelerate wound healing process are hemolytic activity, anti – inflammation,
and immune system stimulation . Moreover, it showed anti-microbial character, not
only against fungi but also bacteria and protozoa. The chemical structure of saponin is
a complex compound consist of saccharide, attached to a steroid or triterpene. 11.
Flavonoids (or bioflavonoids), also known as Vitamin P and Citrin, is a class of

secondary metabolites plant 12. Flavonoid, a type of phenolic compounds, is a subtitude
benzene with-OH groups. Flavonoids mostly derived from plants, its colours
are usually red, purple, blue, and yellow. Flavonoids basic structure consist of two
benzene rings which are bonded to three carbon atoms (propane). Based on its basic
structure, flavonoids can be divided into three type,
namely flavonoids, isoflavonoids, and neoflavonoids.
Flavonoids are phenolic compounds derivied from various types of vascular

plants, with more than 8,000 individual compounds known. In plant, flavonoids act as
antioxidants, antimicrobials, photoreceptors, visual attractor,repellants
feeder, and for light filter. Many studies have shown that flavonoids has
biological activity, including anti-allergenic, antiviral, anti-
inflammatory, and vasodilator. 13
Vitamin C or ascorbic acid is a vitamin with molecular weight 178

and molecular formula C6O8H8 14. Vitamin C is more stable at low pH compared
to high pH. It oxidized easily , especially if it catalyzed by Fe, Cu, ascorbate
67
oxidase enzyme, light, high temperatures. Vitamin C aqueous solutions at pH <7.5 is

still stable when there is no catalysts. Oksidated Vitamin C will
form dehydroascorbic acid 15. Vitamin C deficiency can cause gum bleeding, canker
sores, muscle pain or neurological disorders. Further deficiencies may cause
anemia, frequent infections and rough skin. While the excess vitamin C can cause
diarrhea. Excess vitamin C due to the use of supplements in a long time can cause
kidney stones, whereas the excess vitamin C derived from fruits generally do not cause
side effects 16. The following table shows content of vitamin C in fruits every
100 grams 17.
Fruits Vitamin C content

of fruit (mg/100 g)
Guava 183
Kiwi 100
Longan 84
Papaya 62
Mandarins 31
orange
Melon 42
Mango 28
Pineapple 15
Banana 9
Avocado 8
Breadfruit 29
Wine 32
Tables 2. Content of vitamin C in fruits
One of the most important function of Vitamin C is associated with collagen

synthesis. Collagent is the main protein of connective tissue, bone, cartilago, dentin, and
the layer of vascular endothelium. Vitamin C defieciency intake causing the scurvy.
Spontan bleeding is the manifestation of scurvy as the result of gingivitis and systemic
disease such as anemia, which may be related to the specific functions of ascorbic acid
in the synthesis of hemoglobin. Scurvy is associated with imperfect synthesis of
collagen which manifested by poor healing wounds imperfect the formation of teeth,
capillaries rupture 18
68
Based on the theory, we conduct a research that papaya latex can heal the damaged
tissue in the oral mucosa. The aim of this study is to determine the effect of carica
papaya latex in accelerating the healing process of oral mucosa lesion, by observing the
clinical symptoms and IL-1β.

Sample Selection
The study was approved by the ethical committee of the Animal Hospital of
Bogor Agricultural University. Twelve rats (Spraque dawleys) divided equally into two
groups, treatment group and negative control group. All rats were 2 months old, with
average weight 200 gr
Study Protocol
The way to collect the carica papaya latex
The equipments which is used are
Knife
is used to strike the papaya to get it’s latex. It should be sharp, and stainless
Bowl
is used to collect all the latex from the papaya. The bowl must be made from
stainless steel, light, and unbreakable. The diameter is 6-7 cm, heigh 4-5cm
Holder
is used to place the bowl when the latex taking process.
After all the equipments are well prepaired, the taking process can be started. the
most suitable time to collect the latex is 5.00am before the sun rises. The skin of the
papaya is stroken, the depth is about 0,5cm from the top to the bottom. Each papaya get
4 times stroken. While doing this process, avoid latex get contact to the skin, it can
make the skin irittated. Papayin which is contained in the latex can get the skin itchy
and irritated. Gloves is needed during the process.
From the striking process, the drop of the latex is put in the bowl. The taking
process is done in 2 or 3 days. The important things must be remembered is new stroke
69
process must be spaced 2cm from the previous stroke. Usually the latex drop will be
stopped in 1 hour.
The Way to Purify The Carica Papaya Latex Extract
The latex which is already collected is mixed with 27mL natrium carbonate
devided into six centrifuge tube 1,5mL (volume ratio of the natrium carbonate and
papaya latex is 3:1)
Place the centrifuge tube in a laboratory refrigerated centrifuge with 6000rpm

(the cooling type is used to avoid the stuctural damage which is causing by the
centrifuge process)
The latex will be separated, the dense part will be at the bottom and the liquid
will be at the top. Throw away the liquid by using the pipet to reduce the water. The
dense part is dried by the freeze dry in -40C in about 24 hours until it perfectly dried.
Six rats (Spraque dawleys) had their labial mucosa rubbed by the hidrogen
peroxide (H2O2) 3% and divided equally into two groups, treatment group and negative
control group. The papaya latex extract powder was applied twice a day on the
treatment group. One sprague dawley from each group were executed on the second,
third, fifth, and seventh day to make specimen for healing process evaluation
Evaluation of serology
The steps of "indirect" ELISA follows the mechanism below:
 A buffered solution of the antigen to be tested for is added to each well of

a microtiter plate, where it is given time to adhere to the plastic through charge
interactions.
 A solution of non-reacting protein, such as bovine serum albumin or casein, is
added to block any plastic surface in the well that remains uncoated by the antigen.
 Next the primary antibody is added, which binds specifically to the test antigen that
is coating the well. This primary antibody could also be in the serum of a donor to
be tested for reactivity towards the antigen.
 Afterwards, a secondary antibody is added, which will bind the primary antibody.
This secondary antibody often has an enzyme attached to it, which has a negligible
effect on the binding properties of the antibody.
 A substrate for this enzyme is then added. Often, this substrate changes color upon
reaction with the enzyme. The color change shows that secondary antibody has
bound to primary antibody, which strongly implies that the donor has had an
70
immune reaction to the test antigen. This can be helpful in a clinical setting, and in
R&D.
 The higher the concentration of the primary antibody that was present in the serum,
the stronger the color change. Often a spectrometer is used to give quantitative
values for color strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies
remain bound, the enzyme molecules will produce many signal molecules. Within
common-sense limitations, the enzyme can go on producing color indefinitely, but the
more primary antibody is present in the donor serum the more secondary antibody +
enzyme will bind, and the faster color will develop. A major disadvantage of the
indirect ELISA is that the method of antigen immobilization is non-specific; when
serum is used as the source of test antigen, all proteins in the sample may stick to the
microtiter plate well, so small concentrations of analyte in serum must compete with
other serum proteins when binding to the well surface. The sandwich or direct ELISA
provides a solution to this problem, by using a "capture" antibody specific for the test
antigen to pull it out of the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results
provide a simple positive or negative result (yes or no) for a sample. The cutoff between
positive and negative is determined by the analyst and may be statistical. Two or three
times the standard deviation (error inherent in a test) is often used to distinguish positive
from negative samples. In quantitative ELISA, the optical density (OD) of the sample is
compared to a standard curve, which is typically a serial dilution of a known-
concentration solution of the target molecule. For example, if a test sample returns an
OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same
analyte concentration as your sample.
Phytochemical test
Flavonoids test
A little amount of water added into 4 grams of papaya latex extract powder, boiled for 5
minutes. The filtrate mixed together with a little Mg powder and 1cc concentrated
HCL. The positive result shown by the formation of red, yellow, or orange colour.
Saponin Test
4 grams of papaya latex extract added with hot water and concentrated HCl was boiled
for 5 minutes. 10 mL of the Fitrate was taken and then being shake for 10 seconds. The
positive results shown by formation of foam
71
RESULTS
Clinical
Negative
Days Treatment Group Score control Score
2 P1 1 N1 1
P2 1 N2 1
5 P3 3 N3 1
P4 3 N4 2
7 P5 4 N5 4
P6 3 N6 3
Day 2
1.2
1
0.8
0.6
p1
0.4 p2
0.2 n1
n2
0
Day 5 day 2
72
Day 3
3.5
3
2.5
2
p3
1.5
p4
1
0.5 n3
Day 7
0 n4
day 5
Day 5
3
p5
2 p6
n5
1
n6
0
day 7
The clinical symptom was evaluated by observing the colour of mucosa with score as
followed: 1. Dark red ; 2. Red ; 3. Light red ; 4. Normal gingiva
73
On the second day, subjects of both group‟s oral lesion had score 1, while on the fifth
day, treatment group had score 3, negative control group had score 2. On the seventh
day, both groups had score 4. On the second day, subjects of both group‟s oral lesion
scored 1, while on the fifth day, treatment group scored 3, negative control group scored
2. On the seventh day, both groups scored 4.
Interleukin -1β
Negative Controp
Days Treatment Group Group
1 3,12 pg/mL 1,19 pg/mL
7 9,07 pg/mL 14,11 pg/mL
15
10
day 1
5 day 7
0
treatment control
The interleukin (IL) measurement showed that after one day, the level of IL-1β in blood
of treatment group and negative control group are 3.12 pg/ mL and 1.19 pg/mL
respectively, and on the seventh day are 9.07 pg/mL and 14.11 pg/mL respectively.
74
Fitochemical examination results
Alkaloid +++
Saponin +
Tanin -
Fenolik +
Flavonoid ++
Triterpenoid -
Steroid -
Glikosida +
- : negative
+ : positive
++ : strong positive
+++ : very strong positive
DISCUSSION
Although based on the statistic data there isn‟t any significant differences
between the treatment group and control group, based on the scoring , the conclusion
and discussion should be:
Clinical observation = on the fifth day,the healing process of the treatment group
is more significant than the control group (score 3 for the treatment group and score 1,2
for the control group)
The observation of the inflammatory cells with the interleukin 1 beta showed on
the first day, the treatment group showed more inflammatory response compared to
control group (3,12 pg/mL compared to 1,19pg/ mL). It means the healing process is
significantly more active on the treatment group. It can be seen from the release of the
interleukin 1 beta
75
In contrary, on the 7 days, the IL-1 beta score on the control group is higher than
on the treatment group (14.11 pg / mL compared to 9:07 pg / mL). This means the
healing process is faster on the treatment group compared to the control group so that
the amount of chemical inflammatory mediator released by the control group was
less than the treatment group‟s
CONCLUSION
This study concludes that Carica papaya latex extract may accelerate the healing
of oral mucosa lesion which is observed by clinical and serology
REFERENCES
1. http://www.kesehatan123.com/2111/manfaat-pepaya/
2. Kalie, Moehd Baga, Bertanam Pepaya (Revisi) .2008. Jakarta : Penebar
Swadaya
3. Suprapti, M. Lies. 2005. Teknologi Pengolahan Pangan ANEKA OLAHAN

PEPAYA MENTAH. Kanisius : Yogyakarta
4. Suprapti, M. Lies. 2005. Teknologi Pengolahan Pangan ANEKA OLAHAN

5. Direktorat Gizi Departemen kesehatan RI. 1996 .Daftar Analisis Bahan

Makanan. Jakarta
6. Kalie, Moehd Baga, Bertanam Pepaya (Revisi) .2008. Jakarta : Penebar Swadaya
7 . Suprapti, M. Lies. 2005. Teknologi Pengolahan Pangan ANEKA OLAHAN

8. Begot, Santoso. 2007. Pelajaran Biologi untuk SMA , Jakarta : Interplus
9. Hasbullah, 2004. Teknologi Tepat Guna Industri Kecil Sumatera Barat.

Dewan Ilmu Pengetahuan, Teknologi dan Industri.
10. Muchtadi, 1992. Enzim dalam Industri Pangan. Departemen Pendidikan

dan Kebudayaan Direktorat Jenderal Pendidikan Tinggi, Pusat
Antar Universitas Pangan dan Gizi Institut Pertanian Bogor.
11. Academic Press.”Toxic constituents of plant foodstuffs”. 1969, New York
12. http://dictionary.reference.com/browse/vitamin+p)
13 (Pietta PG, 2000) http://www.ncbi.nlm.nih.gov/pubmed/10924197
76
14. deMann, John M. 1989. Principles of Food Chemistry. Wadsworth,Inc : Canada
15. Sudarmadji, S, dkk. 2003. Analisa Bahan Makanan dan Pertanian. Liberty:
Yogyakarta.
16. http://www.anneahira.com/vitamin-c.htm
17. http://herball.net/kandungan-vitamin-c-pada-buah/
18. Ltd Michael J. Gibney, dkk. 2005, Public health nutrition, Blackwell publishing
Ltd : Oxford
77
CORRELATION BETWEEN ORAL CONTRACEPTION

CONSUMPTION AND PERIODONTAL HEALTH: A
LITERATURE STUDY
William Adi Santoso1, Madjidah2
ABSTRACT
Background: Homeostasis of the periodontium includes many factors and the
endocrine system has an important role in homeostasis. Hormone is a specific molecule,
which regulates the reproductive, growth and differentiation functions in our body.
Estrogen and progesterone are essential sex hormones for female. They work
biologically and affecting other organic systems incorporating the oral cavity. The
estrogen and progesterone receptors are found on the gingival tissue, thus becoming the
target organ for those two hormones. The hormonal contraception is a way to inhibit
ovulation, which mainly contains of estrogen and progestin, and the most common way
is ingesting per oral. Conclusion: The usage of oral contraception on fertile women can
cause disruption of the periodontium. In contrary, this kind of contraception is found to
be beneficial for maintaining bone density of menopausal women.
Keywords: oral contraception, periodontal health, estrogen, progesterone
INTRODUCTION
Periodontitis can be defined as inflammation of the tooth supporting tissues
followed by progressive destructions of the periodontal ligament and alveolar bone.
These destructions will disrupt the stability of the tooth resulting in premature loss of
the tooth, thus making periodontitis as a major cause of tooth loss and edentulous in
adult patients.1
Gingivitis and periodontitis are common periodontal diseases that are related to
the dental plaque accumulation. Metabolic systemic diseases, namely diabetes mellitus
and HIV, can disturb the somatic immune system while environmental factors such as
smoking habit and stress can interrupt the body response adjacent to dental plaque
accumulation.2
78
Some of inflammation response can cause gingival swelling and furthermore

encourage the infiltration of bacteria and their metabolites, i.e. lipopolysaccharides,
peptidoglycans and hydrolytic enzymes, into the blood circulation. The bodily response
towards the periodontal infection is by releasing cytokine and its mediators (PGE2 and
IL), which produce antibody serum.3
Hormones are chemical substances carrying messages, which are produced by

the body to command somatic cells when to work, to differentiate, to replicate and to
die. Hormones regulate multiple functions, including growth, sexual libido, hunger,
thirst, body metabolism, lipid catabolism and anabolism, blood glucose rate, blood
cholesterol rate and reproduction.4
Estrogen hormone is one of the reproductive steroid hormones based on its

chemical structure with steroid as the nucleus of the hormone, which physiologically
produced by the female reproductive endocrine system. The specific article for this
hormone is that its secretion from the ovary is tied to the menstrual periods and it has a
tremendous importance in preparing the gestation. Natural essential estrogens are estron
(E1), estradiol (E2) and estriol (E3).5
The exogenous estrogen has been used by more than 100 million women in the
world either as an oral contraception (OC) or as a hormonal therapy for postmenopausal
women. General practitioners are quite often prescribing oral contraception regarding its
safety, efficacy and high tolerance dose.6
Bacteria have been established as the main cause of periodontal diseases. The
physical conditions of the patients must be considered to determine prognoses and the
severity of periodontal diseases. One of these physical conditions is the usage of oral
contraception, which suspected to be a risk factor in periodontal diseases.
LITERATURE REVIEWS
Homeostasis of the periodontium consists of many factors and endocrinal
system has an important role in that homeostasis. Hormones can be classified into four
groups based upon their chemical structure including steroids, glycoproteins,
polypeptides and amines. As well as being the regulators of reproductive functions, sex
steroid hormones have potent effects on the nervous and cardiovascular system and on
major determinants of the development and integrity of the skeleton and oral cavity
including periodontal tissues.7
Estradiol is a main premenopausal estrogen produced by the ovary. This
estrogen has a significant effect on secondary sexual marks, uterine growth, luteinizing
hormone (LH) release and skeletal growth peripherally and axially. Another important
hormones for female body are progesterone produced by luteal body, placenta and
adrenal cortex. Progesterone affects osseous metabolism and has a significant role in
bone absorption and reformation directly using the osteoblastic receptor.8
79
Estrogen and progesterone act biologically throughout the body, including the
oral cavity. Estrogen and progesterone receptors are found on gingiva. Estrogen
receptors in human gingiva provide the first direct evidence that human gingiva may
function as a target organ for estrogens. Estrogen receptors can be identified in
periosteal fibroblast, fibroblast in lamina propria, periodontal ligament and osteoblast.7
The effects of progesterone and estrogen on periodontium are showed in Figure
1 and 2.
Figure 1. Effects of Progesterone on Periodontium8
 Enhances vascular dilatation and permeability.

 Enhances prostaglandin production.
 Enhances PMN leukocytes (PMNL) and PGE2 in gingival crevicular fluid (GCF).
 Diminishes the anti-inflammatory effect of glucocorticoid.
 Inhibits the proliferation of gingival fibroblast.
Figure 2. Effects of Estrogen on Periodontium8
 Diminishes keratinization but increasing the epithelial glycogen, thus decreasing

the efficacy of barrier epithelium.
 Enhances proliferation of blood vessel.
 Stimulates phagocytosis of PMNL.
 Inhibits chemotactic of PMNL.
 Diminishes spinal production of leukocytes.
 Inhibits pro-inflammatory cytokine release.
 Diminishes T-cell inflammatory mediator.
 Stimulates proliferation of gingival fibroblast.
 Stimulates synthesis and maturation of gingival connective tissues.
 Enhances gingival inflammation.
A. Oral Contraception
Oral contraception (OC) is a way to stop ovulation. It contains a low-dose

estrogen (0.05 mg/day) and progestin (1.5 mg/day). Progestin prevents the leap of
luteinizing hormone, which is required in releasing ovum; thickening cervical mucus
and creating an environment inside the womb, making it harder for the sperm to
80
penetrate; thinning the endometrium, thus allowing no implantation for the ovulated
ovum.9
The component of estrogen inside the OC increases the potency in controlling

the menstrual cycle by inhibiting the release of follicle stimulating hormone (FSH) from
the pituitary gland, prohibiting the development of dominant follicles and in
cooperation with progestin inhibiting LH. Estrogen enhances the menstrual cycle
control by stabilizing endometrium and minimalizing bleeding occurrence.10
Figure 3. Influence of OC Usage Clinically and Microbiologically on

Periodontium8
 Inflammation, starts from mild edema and erythema up to severe inflammation
with hemorrhage.
 An increase of gingival tissue volume up to 50%.
 An increase of bacterial count from Bacteroides sp.
Hormones can give physiological or pathological effects onto many tissues in

the body. The target for particular hormones, such as estrogen, progesterone and
androgen, is also toward the periodontal tissue. Therefore, an imbalance of endocrinal
system can impact significantly the pathogenesis of periodontitis.
A woman who takes OC can give a similar response to a pregnant woman.

Estrogen and progesterone levels during gestation affect the microcirculation system
and cause some alterations, i.e. swelling of the venous endothelium and periocytes;
adhesion of granulocytes and platelets on endothelium, formation of microthrombi;
disruption on perivascular mast cells; increasing permeability and proliferation of the
blood capillary. These alterations cause gingival swelling and increase of GCF. A 50
per cent increase in gingival fluid volume in women using oral contraceptives for a
period of 12 months compared to those who were not on birth control pills, the response
might be due to alterations of microvasculature, increased gingival permeability and the
increasing synthesis of prostaglandins.11
In a clinical study by Lloyd et al (2000), the usage of OC during the adolescents

(20 years old) does not have a negative effect on bone mass.12 Polaneczky (2002) has
cited on their research paper that the peak of low bone mass and osteoporosis of the
young ages may occur in adolescents who are hypoestrogenic due to athletics, eating
disorder or hypothalamus amenorrhea.13 Another study by Geetha (2010) has shown a
worse periodontal health of OC consumer compared with non-OC consumer. The
change of the periodontium can be assessed from OC consumer after 1.5 years.14
Mullaly et al (2007) has reported worse periodontal health indicated by
hemorrhage during probing, increasing depth of periodontal pocket and severe loss of
81
gingival attachment.15 Another research conducted by Brusca et al (2010) has resulted

on an increase of periodontitis prevalence in the OC consumer group. They had deeper
pockets and had higher GI and CAL scores compared with non-OC consumer.16
B. Oral Contraception and Menopause
Menopause can cause alterations in connective tissue, as a result of decreasing

steroid hormones. Bone loss on women is accelerated by the menopausal condition
when the normal rate of estrogen is diminished. Estrogen and progesterone hormonal
therapies are used for restoring the hormones in order to maintain bone density and
decreasing fracture risks.17
A research conducted by Marcos et al (2009) has shown the result from post-
menopausal women, that hormonal therapy can decrease periodontal disruption on
menopausal women.18 Another research by Kuohung et al (2000) on menopausal
women shows a significant correlation between the usage of OC and increasing bone
mass. The benefit of OC is parallel to the duration of usage. Estrogen protects the bone
mass, thus making it beneficial for women with estrogen deficiency.19 Gambacciani et
al (2004) found the 20 mg EE OC preparations can prevent the decrease in bone density
observed in premenopausal women.20
CONCLUSION
The usage of oral contraception on fertile women can cause alterations in
periodontium. On the contrary, oral contraception has a beneficial effect on maintaining
bone mass of the menopausal women.
REFERENCES
1. Pejčić A, Kojović D, Grigorov I, Stamenković B. Periodontitis And Osteoporosis.
Facta Universitatis - Series: Medicine and Biology. 2005;12(2):100-103.
2. Taggart EJ, Perry DA in Perry DA, Beemsterboer PL. Periodontology for the
dental hygienist. 3rd Ed. Missouri: Elsevier Publ. 2007:124-53.
3. Krejci CB, Bissada NF. Women‟s health issues and their relationship to
periodontitis. J Am Dent Assoc 2002;133:323-9.
4. http://transhealth.vch.ca/resources/library/tcpdocs/consumer/hormones-MTF.pdf
(accessed December 15, 2011).
5. Koreeda N, Iwano Y, Kishida M, Otsuka A, Kawamoto A, Sugano N, Ito K.
Periodic exacerbation of gingival inflammation during the menstrual cycle. J Oral
Sci 2005;47(3):149-54.
82
6. Rosendaal FR, Helmerhorst FM, Vandenbroucke JP. Female hormones and

thrombosis. Arterioscler Thromb Vasc Biol 2002;22:201-10.
7. Najwa AN, Wafa AA, Waheed AS. The effects of the oral contraceptive pill
lofemenal on the gingival and periodontal health. J Royal Med Serv 2010;17(1):7-
9.
8. Guncu GN, Tozum TF, Ca‟glayan F. Effects of endogenous sex hormones on the
periodontium –Review of literature. Aust Dent J 2005;50:3.
9. Speroff L, Darney P. A clinical guide for contraception. 4th Ed. Philadelphia PA:
Lippincott Williams & Wilkins. 2005:21–138.
10. Hatcher RA, Trussell J, Stewart FH. Contraceptive technology. 18th Ed. New York,
NY: Ardent Media. 2004:391–460.
11. Newman MG, Takei HH, Klokkevold PR, Carranza FA. Carranza's Clinical
Periodontology. 11th Ed. Missouri: Elsevier Inc. 2012.
12. Lloyd T, Taylor DS, Lin HM, Matthews AE, Eggli DF, Legro RS. Oral
contraceptive use by teenage women does not affect peak bone mass: a
longitudinal study. Fertil Steril 2000;74:734-8.
13. Polaneczky M. Oral Contraceptives: Part 1. Am Acad Pediatrics 2002;14(3):1-9.
14. Vijay G. Relationship of duration of oral contraceptive therapy on human
periodontium - A clinical, radiological and biochemical study. Indian J Dent Adv
2010;2(2):168-173.
15. Mullally BH, Coulter WA, Hutchinson JD, Clarke HA. Current oral contraceptives
status and periodontitis in young adults. J Perio 2007;78(6):1031-6.
16. Brusca MI, Rosa A, Albaina O, Moragues MD, Verdugo F, Ponton J. The impact
of oral contraceptives on women‟s periodontal health and the subgingival
occurrence of aggressive periodontopathogens and candida species. J Perio
2010;81(7):1010-8.
17. Jeffcoat MK, Lewis CE, Reddy MS, Wang CY, Redford M. Post-menopausal bone
loss and its relationship to oral bone loss. Perio 2000 2000:23:94–102.
18. Marcos JFL, Valle SG, Iglesias AAG. Periodontal aspects in menopausal women
undergoing hormone replacement therapy. Med Oral Patol Oral Cir Bucal
2009;10:132-41.
19. Kuohung W, Borgatta L, Stubblefield P. Low-Dose Oral Contraceptives And Bone
Mineral Density: An Evidence-Based Analysis. Contraception 2000;61:77-82.
20. Gambacciani M, Monteleone P, Ciaponi M, Sacco A, Genazzani AR. Effects of
oral contraceptives on bone mineral density. Treat Endocrinol 2004;3:191-6.
83
Description of Patient Loyalty Level in Dental

Polyclinic at Cibabat General Hospital, Town of
Cimahi in Year 2010
Priscilla Daniego Pahlawan; Randita Diany Yordian

Faculty of Dentistry Padjadjaran University
ABSTRACT
Objective: The purpose for this research was to determine the level of loyalty among
the patients from Dental Polyclinic at Cibabat General Hospital, Town of Cimahi in
year 2010.
Results: The result of this research showed that 198 patients (79,84%) had a strong
preferences level, 188 patients (75,81%) high differentiation level,and 196 patients
(79,03%) high repetition level. In addition, 188 patients (75,81%) had a high attachment
level. Mean time, 159 patients (64,11%) had a high loyality level.
Conclusion: The study concluded that the majority of patients who visited Dental
Polyclinic Cibabat General Hospital Town of Cimahi had a Premium Loyalty level
(high customer loyalty level).
INTRODUCTION
A health care services requires good management which is reflected in the
planning. Good planning is determined by means of good planning and knowledge
management. Good planning have to be implement appropriately by the human
resources who have the competence to control the systematic procedure. Thus, hospitals
are expected to be able to provide excellent service and quality. This condition will help
the hospital achieve customer satisfaction. Customer satisfaction will create loyal
customers of using the services of certain hospital care when needed at other times
(Budiman, 2003). Cibabat General Hospital Town of Cimahi is the only a government
hospital available in town, moreover, the three other hospital were belong to private.
That means health care competition in Town of Cimahi is very tight. From the
inverviewed, The author found out that Cibabat General Hospital as well as dental
policlinic have never measured the level of loyalty of patients who came to the hospital.
Patients who were loyal would recommend the place of health care to someone else
who can be an excellent promotional tool that can increase profit to the hospital that
may affect the future development of the hospital (Griffin, 2005). Patients who are loyal
84
will always be expected by a health service, in order for a health service may still exist,
loyal customers will tend to be bound and will continue to use health services at the
hospital, although many other alternatives (Tjiptono, 2007). Patient loyalty that the
author had measured was a commitment to patient who visited the Dental Polyclinic
Cibabat General Hospital, Town of Cimahi between April 2010 to June 2010. Study
always shows that loyalty was created because of the Attachment (Preferences and
differentiation) to a product or service, and Repetition was a repeat purchase by the
patient (Griffin, 2005). Based on the description above, it is very importance loyalty of
the patient have to be measured. The authors were interested in knowing the level of
loyalty of patient in Dental Polyclinic Cibabat General Hospital, Town of Cimahi.

Tools and materials used in this study are as follows:
1. Questionnaire form
2. Notes and stationery
The type of this research was descriptive. Meantime, by using sampling techniques through
purposive sampling method the number of samples required in this study were 248 participates.
Purposive sampling was sampling based on the criteria / considerations or
considerations individual researchers (Sudjana, 2005).
Criteria of the population are as follows:
1. Man or woman
2. Not illiterate
3. Patient visited
4. Informed consent
Number of samples was determined by calculating the average patient visit in a
month. The calculation formula (Notoatmodjo,2005) :
n= N
1 + N (d )²
Explanation:
n = the samples
N = the number of population = 650 (25 person = 26 days)
d = (0,05)
n = 650
1 +650 (0,05) ²
= 248 participates
85
A. DATA COLLECTION TECHNIQUE

The data used in this study were the data obtained from the first source which the
respondents who have committed at least two visits to the Dental Polyclinic Cibabat
General Hospital, Town of Cimahi. In this study, the authors use data collection
techniques in the form of a questionnaire which was defined as a list of questions,
good order in which the respondent in this case the patient not only can give an
answer, but also can give some comments (Notoatmodjo, 2005).
B. RESEARCH PROCEDURE : The procedures of this research were :
1. Writer observed by interviews with the nurses at Dental Polyclinic Cibabat
General Hospital, Town of Cimahi to obtain the initial data
2. Authors compiled a questionnaire which contains questions to the respondent.
Before filling out the questionnaire carried out, then do a trial questionnaire in
advance to find out if there were terms that do not understand or mean sentence
was interpreted differently by the author
3. Filling in the questionnaire conducted during research period
4. The data obtained were processed and presented as a result of the research
C. PROCESSING, ANALYSIS, AND PRESENTATION OF DATA

Type of data used in this questionnaire was ordinal by using Lickert scale that will
be accumulated and systematically arranged. Lickert scale to measure attitudes,
opinions, and perceptions of a person / group of people on social phenomena. This
scale were a bipolar scale method of measuring both positive and negative responses
to a question (Sugiyono, 2007).
Measuring the results of the data was classified into two categories by using the
median t-score analysis (Anwar, 2002):
t- score : 50 + 10 𝑥𝑖 − 𝑥
s
Description:
xi : total score of the respondents
x̅ : the average score
s : standard deviation score (1.7)
Basis for decision making:

If the t-score ≥ 50: favorable (strong / high / yes)
If the score-t ≤ 50: Unfavorable (weak / low / no)
All data collected will be compiled systematically by using a frequency distribution

was presented in the form of frequency tables and percentages. Frequency
distribution of group data in some classes and then counted the number of
86
observations that went into each class. The formula for the distribution of
frequencies used are:
Frequency Percentage = Frequency of class x 100%
Description
Frequency of class : The frequency response of Respondents
n : Total number of respondents answer
Attachment relative obtained from the preferences and the differentiation contained
in the table below :
Tabel 1 Attachment Differentiation
No Yes
Preference Strong Low attachment The highest
attachment
Weak The lowest attachment High attachment
Loyalty level was obtained by combining elements of repetition with the attachment
levels in the table below.
Tabel 2 Loyalitas Repeat Purchase

High Low
High Premium Loyalty Latent Loyalty
Attachment
Low Inertia Loyalty No Loyalty
RESULTS
In this paper we will discuss the result of research and analysis with the number
of respondents were 248 people who are patients at the Dental Polyclinic General
Hospital Cibabat Cimahi. The analysis will be presented in two parts, such as the
descriptive data analysis, and cross tabulation analysis.
A. Descriptive Analysis of Research Data.

Desriptive analysis of research data is directly related to the analysis of research
data. This analysis stems from the researchers distribute questionnaires to the
respondents as a patient at the Dental Polyclinic General Hospital Cibabat Cimahi.
The data have been collected from questionnaires completed by patients who had
Dental Polyclinic General Hospital Cimahi Cibabat further processed to obtain the
87
results of frequency analysis of respondents answers. By sex is almost the majority

of all respondents are respondents with female gender that is 193 respondents
(77,82%) and the rest is a respondent to the male sex as many as 55 respondents
(22,18%).
Table 3 Frequency Distribution of Gender Respondents

Gender F %
Male 55 22,18
Female 193 77,82
Total 248 100
Based on table 4 can be seen the frequency distribution of the highest levels of
patient satisfaction is the patient who states quite satisfied as much as 121
respondents (48,79%) and patients expressed at least as much as 6 respondents were
less satisfied (2,42%).
Table 4 Frequency Distribution Based on Level of Satisfaction

Level of Satisfaction f %
Very Satisfied 19 7,66
Satisfied 102 41,13
Quite Satisfied 121 48,79
Less Satisfied 6 2,42
Very Dissatisfied 0 0
Total 248 100
Based on table 5 that most of the frequency distributin of respondents was 169
(68,15%) agreed to recommend to others to perform dental work on General
Hospital Cibabat Cimahi and at least a respondent to answer as many as one
respondent disagreed (0,40%).
Table 5 Frequency Distribution of Respondents will Give Advice to Others to Perform Dental
Care
(Word of Mouth) in The Dental Polyclinic Hospital Cibabat Cimahi.
Word of Mouth f %
Very not Agree 0 0
Disagree 1 0,40
Less Agree 8 3,23
Agree 169 68,15
Strongly Agree 70 28,23
Total 248 100
88
B. Cross Table Analysis

The tables below explain about the picture of the differentiation of the preferences
of respondents, which was displayed in the form of cross-tabulations to describe the
proportion of respondents answer the item combination research, as follows:
Table 6 Cross Tabulation table Differentiation With Preferences

Respondents (Attachment / Attachment)
Differentiation
Total
Low High
f % f % f %
Strong 43 17,34 155 62,50 198 79,84
Preference
Weak 17 6,85 33 13,31 50 20,16
Total 60 24,19 188 75,81 248 100
The table can be seen that the respondents with the highest number of respondents were
with high attachment high by 188 respondents (75.81%), respondents with low
attachment of 60 respondents (24.19%).
Table 7 Cross tabulation table between Repetition With Attachments respondents

Repetition
Total
High Low
f % f % f %
High 159 64,11 39 15,73 198 79,84
Attachment
Low 37 14,92 13 5,24 50 20,16
Total 196 79,03 52 20,97 248 100
DISCUSSION
Based on the characteristics of the respondents, the author found out that
majority of respondents were female from the total of 193 respondents (77.82%). This
because Dental Polyclinic Cibabat General Hospital, Town of Cimahi open at 08.00 am
to 11.00 am on Monday to Thursday, and 8.00 am until 10.00 am on Friday and
Saturday. Based on the level of Patient Preferences in Dental Polyclinic Cibabat
General Hospital, the author obtained a frequency level of satisfaction with the answer
that most respondents were quite satisfied with as many as 121 respondents (48.79%),
in the mean time, the highest frequency respondents with 169 respondents (68.15%)
would recommend others to have their dental treatment at Dental Polyclinic Cibabat
General Hospital. Preference rate has shown how much confidence to the services
rendered by the hospital (Griffin, 2005). The results of data analysis above shows that
89
most respondents have a strong conviction of the need for services in the Dental
Polyclinic Cibabat General Hospital, Town of Cimahi. It also means that the patient
believes that service provided by the General Hospital, already according to the needs
of their health care required. Strong preference levels can give a positive impression for
the Dental Polyclinic Cibabat General Hospital, Town of Cimahi. Patients were satisfied
with the service provided, so that patients have confidence in the service, and willing to
come back if they need other dental treatments. With respect to patient satisfaction with
the service provided, it was consistent with theory proposed by Griffin (2005) that
patients who have high trust for a term of service willing to use other product or service
provided. The patient would happy to share their experience and knowledge with their
colleagues and family. Level of loyalty can be seen from two factors: The formation of
Attachment and Repetition. The results showed the level of patient loyalty in Dental
Polyclinic Cibabat General Hospital, Town of Cimahi were in the Premium Loyalty
(high loyalty) with 159 respondents (64.11%). Premium Loyalty rate include a high
level of loyalty, which means that patients who come in to Dental Polyclinic Cibabat
General Hospital not only have high levels of attachment, but also a high level of
repetition as well. Patient with a high level of loyalty was expected by the health
service in the hospital, because these type of patient can distinguish between good
services and satisfaction. High level of loyalty patient do not hesitate to recommend to
friends or family. As noted by Griffin (2005) that patients with the highest confidence
of a service and using health care services and are happy to share their knowledge with
colleagues and family. High loyalty patient will tend to buy more, so the hospital will
be able to maintain the loyalty of his patients, in the same time, the hospital will be able
to increase its revenue. Hospital are able to maintain positive cash flow. Positive cash
Flow means easier financing if management are planning to advance and stay superior
in the competition (Hurriyati, 2008).
CONCLUSION
Based on the results of research conducted by the author, the conclusion showed that
the patient at the Dental Polyclinic Cibabat General Hospital Town of Cimahi have high
levels of Premium loyalty.
90
REFERENCES
Budiman, R. T. 2003. Hubungan Kualitas Pelayanan di Balai Pengobatan Dengan
Loyalitas Pasien Terhadap Puskesmas Cisarua Kabupaten Sumedang. Bandung
: Tesis Unpad
Griffin, J. 2005. Customer Loyalty. (Sumiharti Y, Medya R, Kristiaji WC). Jakarta:

Gelora Aksara Pratama:5,18-24,31.
Hurriyati, R. 2008. Bauran Pemasaran dan Loyalitas Konsumen. Bandung: AlfaBeta,

CV:28,128,141.
Notoatmodjo, S. 2005. Metodologi Penelitian Kesehatan. edisi 3. Jakarta: PT Rineka

Cipta.
Sudjana, M. 2005. Metoda Statistika. edisi 6. Bandung: PT Tarsito.
Sugiyono. 2007. Metode Penelitian Administrasi. Bandung: AlfaBeta:107.
Tjiptono, F. 2007. Pemasaran Jasa. edisi 3. (Wahyudi S, Basuki I). Malang: Bayumedia
Publishing:18-23,29,273,349,386,393-394,410-412.
91
DETERMINATION OF EFFECTIVE CONTACT

TIME OF SAGA LEAVES (Abrus precatorius L)
EXTRACT TOWARDS OF Candida albicans
(Preliminary Research)
Nuita, Warta Dewi, and Indrati
Padjadjaran University
ABSTRACT
Objective : Determining the effective contact time of 2 mg/ml minimum inhibitor
concentration (MIC) saga leaves ethanol extract towards of Candida albicans isolate
obtaining information that can be used as a reference to find alternative mouthwash
ingredients consisting herbal medicine.
Methods : Determination of contact time is conducted by making the saga leaves
ethanol extract on 2 mg/ml minimum inhibitor concentration. Samples used five isolates
of Candida albicans which were cultured on Chromogenic Candida Agar for diagnostic
medium and Sabouraud Glucose Agar for selective medium with two repetitions. 0.1
ml suspension of Candida albicans with a turbidity equivalent to 0.5 ml
Mc.Fahrland was inserted into tube and cultured in the Sabouraud Glucose Agar at 30
seconds ,60 seconds, 90 seconds, and 120 seconds contact time. The growth of
microorganisms in the Sabouraud Glucose Agar was observed after incubation with the
temperature 370C for 18-24 hours with facultative anaerob using the exicator. Every
single culturing in Sabouraud Glucose Agar showing minimum
colonies in the shortest contact time was chosen as the most effective contact time. Data
was analyzed using Anava.
Results : According to the method of dilusion and determination of contact time,
it can be obtained the effective contact time to decrease the number of Candida albicans
colonies is 30 seconds, because that contact time could reduce the Candida albicans
colonies more than 50% of population.
Conclusion : The Saga Leaves (Abrus precatorius L) ethanol extract which contains
with flavonoid, saponin, glisirizin, and phenol compounds that are effective to reduce
the Candida albicans colonies is 30 seconds.
Keyword: contact time, saga leaves extract, dan Candida albicans
92
INTRODUCTION
Candida albicans is a normal flora lived in the oral cavity that can cause an infection.
The important fungal infections of skin are those caused by dermatophytes and those
caused by Candida albicans (1).
Candida species can be isolated from the mouths of up to 70% of the normal
population, where it exits as a commensal organism. Candida albicans is
normally found on the surface of the body in small amounts in the skin, oral cavity,
gastrointestinal tract, and female genital tract. A variety of predisposing factor to the
development of candidal over growth and over clinical infection (2). Candida albicans
can become an opportunistic microorganisme in certain circumstances such as chronic
local iritants, ill-fitting appliances, inadequate care of appliances, disturbed oral ecology
or marked changes in the oral microbial flora by antibiotics, corticosteroids, xerostomia,
dietary factors, immunological and endocrine disorders, abnormal nutrition,
hospitalization, and heavy smoking (3).
The most common oral fungal infection is candidiasis (4). Oral candidiasis can
generally be dealt with antifungal drugs according to predisposing factor. A number of
effective antifungal agents are nystatin, amphotericin B, miconazole, fluconazole, and
itraconazole (5). That antifungal agents have proven the benefit clinically, but there are
sides effect occuring especially in long term use. The most common sides effect occur
are diarrhea, nausea or vomiting, burning, and erythema (6). In addition, antifungal
drugs expensively sold in the market.
WHO is recommended to use traditional medicines, including herbal to

maintanance the public health, prevention and treatment of disease, particularly for
chronic diseases, degenerative diseases, and cancer. WHO is also supporting efforts
increasing safety and efficacy of traditional medicine (7).
Saga leaves (Abrus precatorius L) is one of traditional medicine that potential

cultured to inhibit the growth of Candida albicans. Saga leaves contains antifungal
agents such as flavonoid, saponin, and phenol compounds (8,9,10) .
Paranto stated that the test fungal of saga leaves (Abrus precatorius L) ethanol
extract with concentration of 2 mg/ml could inhibited the growth of Candida albicans
(11)
.
Several studies have been conducted and showed that saga leaves ethanol extract
performed antifungal activity, but the study of effective contact time of saga leaves
ethanol extract towards of Candida albicans has not been investigated.
Determination of contact time is important because saga leaves (Abrus

precatorius L) ethanol extract is likely to use as alternative mouthwash ingredient
caused candidiasis after toxicity test using an animal and clinical test that can be used
93
everyday and more economical, because longer contact time could make more number
of dead fungal. Therefore, the contact time factor need to consider.

Population in this examination is the saga (Abrus precatorius L) which
growing in Bandung city with the criteria is small saga leaves and red-black seeds.
Sample which is used in this examination is saga leaves which growing in Ujung
Berung.
Cultivation of Candida albicans samples are used Chromogenic Candida Agar

for diagnostic and identification medium, also Sabouraud Glucose Agar (SGA) for
selective medium.
Examination procedures are performed as follows: cultivation the sample,

isolation and identification of Candida albicans, making of saga leaves ethanol extract,
making of Candida albicans suspension, making of minimum inhibitor concentration,
determination of contact time, and statistical analysis.
The isolation of Candida albicans was carried out by taking swabs from the
inner surface of mucosa. Swabs were cultured on Chromogenic Candida Agar for
medium for 18 to 24 hours at 370C with facultative anaerob and identified according to
culture characteristics, microscopic appearance, and germ tube formation.
The making of saga leaves ethanol extract is needed 70% ethanol. Extract is
processing with maceration during 3 x 24 hours and every 24 hours that macerate is
assembled and re-maceration again with the new 70% ethanol. The macerate is
evaporated using rotary evaporator after assembled. And then, that extract is poured into
the petri cup.
Determination of contact time is conducted by making the saga leaves ethanol

extract on 2 mg/ml minimum inhibitor concentration. 0.1 ml suspension
of Candida albicans with a turbidity equivalent to 0.5 ml Mc.Fahrland was inserted
into tube and subcultured in the Sabouraud Glucose Agar at 30 seconds ,60 seconds, 90
seconds, and 120 seconds contact time. The growth of microorganisms in the SGA was
observed after incubation with the temperature 370C for 18-24 hours with facultative
anaerob using the exicator. Every single culturing in Sabouraud Glucose Agar showing
minimum colonies in the shortest contact time was chosen as the most effective contact
time. Data was analyzed using Anava.
94
The following pathway of examination could be seen below.
Sample from saliva‟s student of dentistry

padjadjaran university
Cultivation on Candida Chromogenic

Agar medium
Incubated in 18 to 24 hours,
370C with facultative anaerob
Identification of C.albicans thorugh colony characteristic,

gram-staining, and microscopic appearance
C .albicans isolate
Incubated in 18 to 24 hours, Subcultured onto SGA

37oC with facultative anaerob
Making of saga leaves
ethanol extract Suspention of C.albicans
Mc.Fahrland 0,5 turbidity
/ Making Minimum Inhibitor
Concentration of 2 mg/ml
Determining of contact time 30 s, 60s, 90 s, 120 s
Incubated in 18 to 24 hours, Sector subcultured on SGA

37oC with facultative anaerob
Result
Statistical analysis
Figure 1. Pathway of examination
95
RESULTS
The cultivation of sample which isolate from saliva in the oral cavity to Chromogenic
Candida Agar medium that have been incubated for 18-24 hours at 370C by facultative
anaerobes showed a variety of microorganism colonies. Cultures grow on Candida
Choromogenic Agar medium as light green colonies, smooth surface, flat or
hemispherical in shape with a beer- like aroma. The Colony with a characteristic as
identified like above can be diagnosed as Candida albicans. That colony can be seen in
the image below.
Figure 2. Candida albicans colonies on Candida chromogenic Agar medium

The results obtained from Chromogenic Candida Agar medium then subcultured
on Sabouraud Glucose Agar medium then incubated by facultative anaerobes at 370C
for 18 to 24 hours. Candida albicans looks like a creamy white colonies, smooth
surface, flat or hemispherical in shape with a beer- like aroma. The result of cultivation
could be seen in the image below.
Figure 3. Candida albicans colonies on Sabouraud Glucose Agar medium

The results of microscopic examination of Candida albicans with gram-staining
showed round in shape, spherical to oval budding yeast cells 3-5 µm x 5-10 µm in size,
appear purple caused by gram-positive.
Figure 4. Results of microscopic examintaion with gram-staining

96
The result of determination of contact time on tube reaction towards to Candida

albicans suspension which equivalent with turbidity of McFarland 0,5 standard showed
the following results.
Figure 5. Effectivity test of contact time of saga leaves ethanol

extract towards of Candida albicans on tube.
The result of determination of contact time of saga leaves ethanol extract

towards of Candida albicans with time of 30 seconds, 60 seconds, 90 seconds, and 120
seconds by sector subcultured on Sabouraud Glucose Agar medium showed the
following results.
Figure 6. Sector subcultured of saga leaves (Abrus precatorius L)

ethanol extract towards of Candida albicans
on Sabouroud Glucose Agar medium
The result of various contact time of saga leaves ethanol extract (Abrus
precatorius L) towards of Candida albicans could be seen in the table below.
97
Table 1. The result of Candida albicans colonies on difference contact time of

saga leaves (Abrus precatorius L) ethanol extract
MIC 30 90 120 Control

60 seconds Control (+)
2mg/ml seconds seconds seconds (-)
I II I II I II I II I II I II
Sample 1 58 72 46 91 14 89 92 66 - - 224 253
Sample 2 51 8 224 39 101 80 89 159 - - 209 202
Sample 3 27 30 52 15 3 97 31 82 - - 201 237
Sample 4 109 60 102 40 64 116 41 1 - - 209 202
Sample 5 57 44 109 68 50 77 34 47 - - 201 237
Average 60,4 42,8 106,6 50,6 46,4 91,8 57,4 106,6 - - 211,33 230,67
Total
51,6 78,6 69,1 64,2 - 221
Average
(-) : negative control
(+) : positive control
I : repetition I
II : repetition II
The result of this table showed on positive control tube is turbid. The positive
control tube which containing of Candida albicans that equivalent with turbidity of
McFarland 0,5 standard that dissolved in glucose broth and untreated looked cloudy.
Solution which input into negative control tube looked clear because of containing only
a suspension of saga leaves ethanol extract in a glucose broth, there‟s no Candida
albicans is added. This indicated that negative control tube is not contaminated so it
could be used as controls in all repetitions.
According to Table 1, is notice that the number of Candida albicans colonies
appeared at least in 30 seconds (average of 51,6 colonies), then the number of colonies
is increasing in 60 seconds (average of 78,6 colonies). The Candida albicans colonies
have decreased in 90 seconds became 69,1 colonies and back to fall became 64,2 in 120
seconds.
Table 2. Kolomogorov Smirnov test for contact time of saga leaves
ethanol extract towards of Candida albicans isolate
Contact Time p-value* Data Distribution

30 seconds 0,900 Normal
Positive Control 0,941 Normal
*p-value is probability value.

98
Table 2. indicated that 5 group of data following the normal distribution which
showed by probability value (p-value) over 0,05. The data with normal distribution
value is used comparison analysis of parametric technique of One Way Anava test. The
result of anava test could be seen in this table below.
Table 3. The result of One Way Anava test of contact time of saga leaves
ethanol extract towards of Candida albicans isolate
F Ftable( 0,05;2;36) p-value* Conclusion

0,663 2,866 0,580 No significant difference exists
*p-value is probability value.
The result of one way anava test showed probability value of 0,580 that means
there are decreasing Candida albicans colonies in 4 time. According to One way anava
in a whole treatment group towards positive control, there were no statistical significant
difference exists among them that inhibited the growth of fungal on probability of 0.000
(p<0,05). The effective contact time could be determined from the shortest time with the
lowest number of colonies that growing.
H0 : the effective contact time of saga leaves (Abrus precatorius L) ethanol extract
towards to decrease Candida albicans colonies is 1 minute.
H1 : the effective contact time of saga leaves (Abrus precatorius L) ethanol extract
towards to decrease Candida albicans colonies is less than 1 minute.
Based on the result, the effective contact time of saga leaves (Abrus precatorius
L) ethanol extract towards to decrease Candida albicans colonies is 30 seconds, so it
could be stated that hypothesis1 (H1) is accepted and hypothesis0 (H0) is unaccepted.
DISCUSSION
Samples that use in this study are Candida albicans isolate from saliva in the
oral cavity with 5 samples twice repetition.
This study used Chromogenic Candida Agar medium for diagnostic and
identification Candida spp. At the medium, Candida albicans appeared as light green.
It could be happen because Chromogenic Candida Agar medium incorporates with two
chromogenic that indicated the presence of target enzymes such as X-NAG (5-bromo-
4-chloro-3-indolyl ß-D N-acetyl glucosaminide) which detected the activity of Candida
albicans hexosaminidase enzyme (12).
Based on table 1, there were differences in the contact time of saga leaves
(Abrus precatorius L) ethanol extract that affected number of Candida albicans
99
colonies isolate among 30 seconds, 60 seconds, 90 seconds, and 120 seconds but the
average difference is insignificant.
In 30 seconds, average Candida albicans colonies that grew was 51,6 colonies.
If it compared with the average of value positive control ,221 colonies, it will assume
that the contact time of 30 second is LD50 (Lethal dose) which means that saga leaves
ethanol extract assessed effective killed 50% or more of population of Candida albicans
sample.
Based on the results of hypothesis test, it could be concluded that H1 is accepted
and H0 is unaccepted that means the effective contact time of saga leaves (Abrus
precatorius L) ethanol extract towards to decrease Candida albicans colonies is 1
minute, is not suported. Optimum of contact time is determined from the shortest
contact time with the lowest number of colonies that growing, 30 seconds.
The effectiveness of contact time of saga leaves (Abrus precatorius L) ethanol
extract less than 1 minute were happened because the saga leaves is contained with
antifungal agents such as flavonoid, saponin, glisirhizin, and phenol compound (8,9,10).
Flavonoid compound are lipophilic, more lipophilic a flavonoid compound, then
it could be damaged the cell membranes and caused a lysis cell (13). Mechanism of
flavonoid is by forming a complex compound towards of extracellular protein which
could disrupt the membrane cell integrity (14). Flavonoid is binding with the lipid layer
of membrane cell and damaged the lipid structure of Candida albicans cell, so it could
interfered metabolism, food transport, and increased the permeability of cell wall (13).
Saponin could interacted with ergosterol in the cell wall Candida albicans to
form the complex compound, so resulted a pore in the cell membrane which disturbing
the electrolyte of transport membrane between natrium and kalium (Na-K) and calcium
and magnesium (Ca-Mg). Cell membrane leakage caused by activation of the enzyme
degradation and accumulation of free radical that damaging ion of hemostasis or
imbalancing metabolism so will lead a cell death. Saponin could also lower the surface
tension, because it could be interacted with protein and lipid in the cell membrane of
Candida (15).
Glisirizin glycoside are compound which could be hydrolyzed into sugar in
solution. The strong interaction between sugar molecules with water of molecule when
in solution, leaving little water to support the life of microorganism. Sugar content is
contained in this leave saga would causes around of fungal environment become
hypertonic. High osmotic pressure would lead the displacement of fluid in fungal cell
into environment, so that microorganism would plasmoptisis and caused a cell death (15).
Other substances that contained in saga leaves are phenol. Phenol compound
caused denaturation of protein in the membrane cell of Candida albicans. On protein
that have been denaturated, the hydrogen bond is damaged, resulting the changes of
protein structure that lead to change protein function (15).
Based on the mechanism of action, these active components is affected the lives
of fungal cells.
100
CONCLUSION
According to the result, it can be draw a conclusion that the effective contact time of
saga leaves (Abrus precatorius L) ethanol extract towards of Candida albicans colonies
reduction is 30 seconds.
REFERENCES
1. Bagg, J. 2002. Essentials of microbiology for dental students 2nd ed. New York.
Oxford University Press. 161,163,289-299 pp.
2. Chestnutt, I. G.andJ.Gibson. 2002. Churchill’s Pocketbook of Clinical Dentistry. 2nd

ed. Churchill Livingstone: London. 442 pp
3. Samaranayake. 2002. Essential Microbiology for Dentistry. 2nd Edition. Churchill

Livingstone. Edinburgh. 142-147; 239-243 pp.
4. Lamont, R.J. 2006. Oral Microbiology and Immunology 1st ed. Washington. American
Society for Microbiology (ASM) Press. 333-340 pp.
5. McCullough, M.J.and Savage, N.W. 2005. Oral candidosis and the therapeutic use
of antifungal agents in dentistry. Australia Dent J Medication Suplement 50: S36-
S39. Available online at: www.ada.org.au(diakses 25 November 2011).
6. Lubis, R.D. 2008. Pengobatan Dermatomikosis. Fakultas Kedokteran USU. USU e-

Repository. Melalui:www.repository.usu.ac.id (diakses 25 Oktober 2011).
7. WHO. 2003. Traditional medicine. Available online at : http://www.who.

int/mediacentre/ factsheets/fs134/en. (diakses November 2011).
8. Hariana, A. 2011. Tumbuhan Obat dan Khasiatnya Seri 3. Penebar Swadaya.

Jakarta. hal17-22.
9. Indrati, G. dan Khaerati. 2009. Potensi tanaman saga sebagai pestisida nabati
dalam WartaPenelitian dan Pengembangan Tanaman Indonesia. Badan Penelitian
dan Pengembangan Pertanian Pusat Penelitian dan Pengembangan Perkebunan.
Vol. 15 No. 1. Hal. 21-23. Melalui: www.perkebunan.litbang.deptan.go.id (diakses
20 Maret 2012).
10. Gunawan, D. dan Purwantini, I. 2010. Abrus precatorius L.hal. 1-10.Melalui :

www.fleppc.org (diakses 16 Oktober 2011).
101
11. Paranto, M. 2007. Uji daya antijamur ekstrak daun saga (Abrus precatorius L)
terhadap Candida albicans. Bandung. Fakultas Kedokteran Gigi Universitas
Padjadjaran. hal 51.
12. Toku. 2010. Candida selective supplement. Toku E-application data sheet. hal 1-4.
Available online at : www.toku-e.com. (diakses 24 Maret 2012).
13. Jawetz, E., Melnick, J. L., Adelberg, E. A. 1996. Mikrobiologi Kedokteran. Edisi
20. Jakarta : EGC. hal 167-202.
14. Juliantina, F., Citra, D.A., Nurmasitoh, T., dkk. 2009. Manfaat sirih merah (Piper
croatum) sebagai agen antibakteri terhadap bakteri gram positif dan gram negatif.
JKKI–Jurnal Kedokteran dan Kesehatan Indonesia. Melalui:
www.journal.uii.ad.idhal 1-10 (diakses 20 Maret 2012).
15. Herawati, E. 2011. Uji efek anti jamur fraksi n-heksana dan etil asetat daun sirih
(Piper betle L.) terhadap Candida albicans (isolat gigi tiruan lengkap akrilik
rahang atas). Universitas Padjadjaran. Bandung. hal 24-26.
102
EFFECTS OF PROBIOTIC DRINK ON TOOTH

DISCOLORATION -- AN IN-VITRO STUDY
Andita W. Maharani1, Mohammad Yogiartono2
ABSTRACT
Background: Yakult is a leading brand of probiotic drink all over the world. It is made
by fermenting mixtures of skimmed milk with special strain of bacterium called
Lactobacillus casei Shirota strain. This Lactobacillus belongs to the lactic acid
producer bacteria, which also produce hydrogen peroxide (H2O2). Hydrogen peroxide
has the ability to penetrate stains on enamel and dentin layers, which accumulated for a
long term. It is also one of the oxidizer which is commonly used in tooth bleaching
procedures.
Objective: The aim of this study was to know the influence of probiotic drinks on tooth
discoloration.
Methods: This research was an experimental in-vitro study of extracted tooth examined
under controlled post-test group design. The sample consists of 24 teeth divided equally
into four groups. Sample was soaked in distilled water and probiotic drinks (Yakult) for
3 to 6 hours. An optical photodiode sensor was in observing the discolouration
occurred. The data were tested using a non-parametric statistical test, Kruskal – Wallis,
with significancy of 95% (p < 0,05) .
Result: There were significant difference of tooth colour between four groups (p = 0,00
< 0,05).
Conclusion: Probiotic drinks can affect the tooth discoloration.
Key Words: Tooth discoloration, Lactobacillus Casei Shirota strain, Hydrogen
peroxide.
INTRODUCTION
Tooth discoloration presents two major challenges to dentistry. The first
challenge is to ascertain the cause of the stain and the second is its management.
Correction of these types of dental problems can produce dramatic changes in
appearance, which often result in improved confidence, personality and social life.1
Many factors can cause tooth discoloration. These factors are classified in four major
103
groups: genetics,2-3 congenital factors,4 acquired or environmental factors2 and

iatrogenic factors.5
Over the past two decades, tooth whitening or bleaching has become one of the
most popular esthetic dental treatments. Current tooth bleaching materials are based
primarily on either hydrogen peroxide or carbamide peroxide. Both may change the
inherent color of the teeth, but have different considerations for safety and efficacy. In
general, most in-office and dentist-prescribed, at-home bleaching techniques have been
shown to be effective, although results may vary depending on such factors as type of
stain, age of patient, concentration of the active agent, and treatment time and
frequency. However, concerns have remained about the long-term safety of
unsupervised bleaching procedures, due to abuse and possible undiagnosed or
underlying oral health problems.6
Probiotics are defined as „live microorganisms which when administered in
adequate amounts confer a health benefit on the host‟.7 Probiotics are living, health-
promoting microorganisms that are incorporated into various kinds of foods. The ability
of probiotics to withstand the normal acidic conditions of the gastric juices and the
bactericidal activity of the bile salts, as well as the production of lactic acid that inhibits
the growth of other microorganisms, allow them to be established in the intestinal tract.8
The concept of probiotics progressed around 1900, when Elie Metchnikoff hypothesized
that the long and healthy lives of Bulgarian peasants were the outcome of their
consumption of fermented milk and milk products.9 Members of the genera
Lactobacillus, Bifidobacterium and Streptococcus are the most common probiotics used
in commercial fermented and non-fermented dairy products today.10
Yakult is a leading brand of probiotic drink all over the world. Yakult is a
fermented milk drink containing Lactobacillus casei Shirota (LcS), a unique probiotic
strain discovered in 1930.11 Lactobacillus sp. is bacteria that belong to a group generally
referred to as lactic acid bacteria.12 The ability of lactic acid bacteria to produce enough
hydrogen peroxide (H2O2) without growing at refrigeration temperature should enable
them to extend the shelf-life of some refrigerated foods without altering the acidity of
the food.13 Hydrogen peroxide has the ability to penetrate stains on enamel and dentin
layers, which accumulated for a long term. It is also one of the oxidizer which is
commonly used in tooth bleaching procedures.
The purpose of this study was to determine the effect of probiotic drinks on
tooth discoloration

This research was done in the Physical Optic and Laser Applications Laboratory,
Faculty of Science and Technology, Airlangga University, Indonesia. This experimental
research was using an experimental in-vitro study of extracted tooth examined under
controlled post-test group design. Sample for post extraction of premolar teeth derived
from clinic in Dharmahusada 1, Surabaya, Indonesia. The sample group was divided
104
into group A (control): post extraction of premolar teeth soaked in distilled water for 3
hours. Group B: post-extraction of premolar teeth soaked in probiotic drinks (Yakult)
for 3 hours. Group C (control): post extraction of premolar teeth soaked in distilled
water for 6 hours. Group D: post extraction of premolar teeth soaked in probiotic drinks
(Yakult) for 6 hours. The sample size of each group are 6 pieces, so the total sample
with four treatment groups were 24 samples. This research was conducted from 15 to 16
March 2012. Criteria of materials that used were 24 premolars post extraction without
anomalies and do not have tetracycline staining or other staining.
Figure 1. Sample was soaked in distilled water

and probiotic drinks (Yakult) for 3 to 6 hours
Yakult is a fermented milk drink containing Lactobacillus casei Shirota (LcS), a
unique probiotic strain backed by over 70 years of scientific research. Water, skimmed
milk (reconstituted), glucose-fructose syrup, sugar, maltodextrin, flavouring are the
ingredients of Yakult. And it also contains 1010 Lactobacillus casei Shirota per 100ml
when refrigerated (6.5 billion per bottle). 11
The research was started by washing 24 premolars post extraction with flowing
water, then dried with tissue. Each group in accordance to the sample soaked in a tube
for 3 and 6 hours. Before the measurement with optical photodiode, the sample washed
by flowing water and then dried with tissue.
Optical Photodiode was used to measure the tooth discoloration. First, He-Ne
laser light, photo detector OPT 101, and digital microvolt was placed on desk research.
Then, He-Ne laser and photo detector OPT 101 position was placed in a straight line.
Photo detector OPT 101 was connected with digital microvolt that functions to read the
voltage value derived from the photo detector OPT 101. Photo detector OPT 101
changed the intensity of voltage value.
105
Figure 2.Optical Photodiode

The data were tested using a non-parametric statistical test, Kruskal – Wallis,
with significancy of 95% (p < 0,05)
RESULTS
The data obtained from this research shows that there were a significant
difference of tooth discoloration between four groups. The measurement results of
premolar post extraction discoloration in distilled water and probiotic drinks (Yakult)
immersion for 3 and 6 hours was analyzed with a non-parametric test Kruskal – Wallis,
with significancy of 95% (p < 0,05), obtained asymp. Sig value = 0,000 which means
that there was a significant difference of tooth discoloration between four groups (Table
1).
Tabel 1. Kruskal-Wallis Test

Test Statisticsa,b
milivolt
Chi-Square 17.875
df 3
Asymp. Sig. .000
a. Kruskal Wallis Test
b. Grouping Variable: group
106
DISCUSSION
In our study, the purpose is to know the affect of probiotic drinks on tooth
discoloration. To find the comparison of the discoloration that occurred in distilled
water and probiotic drinks (Yakult) immersion the research was using an experimental
in-vitro study of extracted tooth examined under controlled post-test group design, by
soaked the sample for 3 and 6 hours. The duration of immersion performed for 3 and 6
hours due to estimates of person drink Yakult package (65 ml) is 30 seconds and
consumes two packs a day so if it consumed for 6 months and 1 year the results that
obtained for laboratory testing are 3 and 6 hours.
By using a non-parametric statistical test, Kruskal-Wallis, there were significant
differences between four groups. But there were no significant discoloration from
distilled water and probiotic drinks (Yakult) immersion by view the sample directly.
Yakult contains Lactobacillus casei Shirota contain which is bacteria that belong
to a group generally referred to as lactic acid bacteria.The ability of lactic acid bacteria
to produce enough hydrogen peroxide (H2O2)12,13. We concluded that probiotic drinks
can affect the tooth discoloration. Further studies are required to clarify the mechanism
of tooth discoloration by Lactobacillus casei Shirota, which belongs to the lactic acid
producer bacteria, which also produce hydrogen peroxide (H2O2)
CONCLUSION
This research showed that probiotic drinks can affect tooth discoloration.
REFERENCES
1. Hattab FN, Qudeimat MA, al-Rimawi HS. 1999. Dental discoloration: an
overview. J Esthet Dent; 11(6): 291-310.
2. Regezi JA, Sciubba JJ, Jordan RCK. 2003. Oral pathology: clinical pathologic
correlations. 4th ed. St. Louis: Sunders.
3. Greenberg MS, Glick M. 2003. Burket's oral medicine diagnosis and treatment.
10th ed. Hamilton: BC Decker Inc.
4. Pindborg JJ. 1982. Aetiology of developmental enamel defects not related to
fluorosis. Int Dent J; 32(2): 123-34.
5. Van der Burgt TP, Mullaney TP, Plasschaert AJ. 1986. Tooth discoloration
induced by endodontic sealers. Oral Surg Oral Med Oral Pathol; 61: 84-9.
6. ADA. 2009. Tooth Whitening/Bleaching: Treatment Considerations for Dentists
and Their Patients
7. WHO/FAO Joint Working Group. 2002. Guidelines for the evaluation of
probiotics in food.
107
8. Catanzaro J, Green L. 1997. Microbial ecology and probiotics in human

medicine (Part II). Altern Med Rev; 2(4):296-305.
9. Kopp-Hoolihan, L. 2001. Prophylactic and therapeutic use of probiotics: A
review. J Am Diet Assoc. 101(2): 229-241.
10. Heller, K. 2001. Probiotic bacteria in fermented foods: Product characteristics
and starter organisms. Am J Clin Nutr; 73(2):374S-379S.
11. Yakult. Available from http://hcp.yakult.co.uk. Accessed at 2012,28th April
12. Gilliland SE. 1990. Health and nutritional benefits from lactic acid
bacteria.FEMS Microbiol Rev; 87: 175 – 8.
13. P.S. Yap, S.E. Gilliland. 2000. Comparison of newly isolated strains of
Lactobacillus delbrueckii subsp. lactis for hydrogen peroxide production at 5
°C, J. Dairy Sci. 83 628– 632.
108
INFLUENCE OF PLAYING VIOLIN AND VIOLA TO

SEVERITY OF TEMPOROMANDIBULAR JOINT
DISORDER
Gempita, Ade Sri Nengsih, Hutami Fitri Widhiyanti, Rasmi Rikmasari
Universitas Padjadjaran
ABSTRACT
Introduction: Temporomandibular joint disorders (TMD) are often found on violinists
and violists. When they play violin or viola, neck, shoulders, and jaw are in the same
position within a period of time, this condition increases stress of jaw. In addition, the
body is also in imbalance position when they play violin and viola. These factors can
accumulate the occurrences of TMD. By its severity, TMD can be divided into mild,
moderate, and severe.
Objectives: The purpose of this study was to prove whether there were correlations
between long period of time, frequency, and routinity on practice with severity of TMD
on violinists and violists.
Method: This study was conducted on 30 of 50 violinists and violists, age range 18-31
years old, of ISO (Institute Technology of Bandung Students Orchestra) taken by
random sampling technique. The participants were given questionnaires and their
temporomandibular joints (TMJ) were examined objectively.
Result: All participants showed 100% prevalence of TMD during TMJ examination.
Prevalence of severity among participants were 23.3% mild, 60% moderate, and 16.7%
severe respectively. Analysis by using Chi-Square method showed that there were non
significant correlations between long period of time, frequency, and routinity on
practice with severity of TMD on violinists and violists (p>0.05).
Conclussion: This study showed that long period of time, frequency, and routinity on
practice did not influence the severity of temporomandibular joint disorder (TMD) on
violinists and violists. However, violinists and violists have great risks of TMD and
most of them have moderate severity of TMD.
Keywords: Temporomandibular Joint Disorder, violin and viola, long period of
time, frequency, routinity.
109
INTRODUCTION
Temporomandibular joints (TMJs) are the small joints in front of each ear that
attach the lower jaw (mandible) to the skull. Temporomandibular joints consist of the
condyle, articular disc, articular eminence, glenoid fossa, muscles, tendons, nerves,
bones, and connective tissues. They are the most complex joints in the human body.
These joints have rotation and translation movement in mastication system, such as
swallowing and speaking.1,2,3
Temporomandibular joints can have several abnormalities or disturbances which
is called temporomandibular joint disorders (TMDs). Temporomandibular joint
disorders (TMDs) are disturbance of the jaw and joint relation in orofacial region due to
imbalance of relation between the jaw and skull with the attached muscles while the jaw
is moving.2 The symptoms of TMD are pain in masticatory muscles and
temporomandibular joint, stiffness of jaw muscles, limited jaw movement or locked jaw
opening, joint sounds (clicking), and changes in occlusion. Other symptoms that
accompany the occurrence of TMD are headache, pain in the ears, dizziness, and
hearing disturbances.2,4 Temporomandibular joint disorders are caused by malocclusion
or overbite of the teeth, poor mechanical relation (inflammation, subluxation of the disc,
and asymmetrical joints), muscles spasm, postural imbalance, bad habit (grinding,
clenching, and chewing gum), and trauma.4 Postural imbalance can also cause TMD,
such as in the violinist and violist.5,6,7
The violinists and violists experience much pain in the neck, jaw, and head
compared with the normal population. The violinists and violist often complain pain and
noise in TMJ (clicking) when the jaw is moving, locked mouth opening, and tilt
movement of jaw to the right side.7,8 Kovero7 added the pain occurs in mastication
muscles of violinists and violists at maximum of mouth opening. This condition is
caused by continuous pressure in lower jaw (mandible) by the instrument and clenching
or crossbite relation of maxillar and mandibular teeth while playing violin or viola.9
Temporomandibular joint disorder of violinists and violists can also be caused by
tension on neck, shoulders, and jaw muscles.10 The cervical spine can also be affected
due to the prolonged head and neck position to hold the instrument (violin or viola).
This position can cause muscle spasms and nerve compression.11,12

This type of research was a descriptive study with random sampling method.
The research was conducted in March 2012 at the Institute Technology of Bandung
(ITB), on Ganeca Street number 10, Bandung. The materials that were used in the study
were handscoons, dentist mouth mirror, stationeries, and questionnaires.
The population in this study were violinist and violist members of the ITB
Student Orchestra. There were 30 samples taken randomly from 50 population, age
between 18-31 years old. Avoided biases, there were some criteria to select samples, as
follows: not being in the care of a physician; not seeing a health professional; currently
110
not suffering from inflammation in the ears, nose and throat; not having a toothache;
never been suffered from pain in bone and knee; currently not in orthodontics treatment.
Participants who have met the criteria were asked to complete informed consent and
then were given questionnaires. The first questionnaire contained specific questions about
their activities playing the violin and viola : how many years they played violin; the
routinity; how many hours a day; how many times a week; whether there was a pause
while playing.
Next step was filling a questionnaire on signs and symptoms of
temporomandibular joint disorders. On a questionnaire on symptoms of TMD, there
were 12 questions. The questions included whether there were ever difficulties or sound
when opening mouth and pain around the face, ears and neck. The next step was
examined the signs of temporomandibular joint disorders. These consisted of 15
examinations: the examinations of jaw movement (smooth or rough), opening of mouth
(zig-zag, swing or not), pain and difficulties on opening the mouth, palpation on
masticatory system muscles (Masseter and temporal) and joint (intra meatal and pre
auricular), joint sounds, occlusal wear, the impression of the bite, facial and dental arch
asymmetries, body posture, and occlusal curve. One of functional disorders of the
temporomandibular joint can be seen from a limited mouth opening and deviation from
midline mandibular movement (or a zig-zag motion).13
Furthermore, to validate the severity of TMJ disorders required score to
determine the occurance of severity of TMD. At the questionnaire of TMD symptoms,
there were 3 answer options, which were not, sometimes and often, with a points
assessment were 0, 5 and 10. While the examination of the signs of TMD, the value
range of were 0-10, 0 if there was no sign of abnormality; 5 if there was one sign at
each examination or interference on only one side of the jaw (unilateral); 10 if there
were to 2 marks when the examination or interference on both sides of the jaw
(bilateral).
The values of test results of signs and symptoms of TMD were summed. There
were 3 obtained on the severity of TMD, which were: when the value 0 there was no
disorder; mild TMD, if the value ranging between 5-50; moderate TMD, if the value
ranging from 55-100; and severe TMD, if the value ranging from 105-150.
Then the results were analyzed by using Chi-Square statistical analysis to test
whether there were relation between long periods of time, routinity and the frequency of
playing the violin and viola to the severity of TMD. Chi-square analysis has a
significance value ( = 0.05). After that, all datas were analyzed by Statistical Software
to obtain p values, then it was compared with  = 0.05. If the p value less than  =
0.05, there was relation between the two variables and otherwise.14 Hypothesis of this
study were H0 = there is no significant relationship between long period of time,
frequency, and routinity on practice with severity of TMD on violinists and violists and
H1 = there is significant relationship between long period of time, frequency, and
routinity on practice with severity of TMD on violinists and violists.
111
RESULTS
Description of research data regarding distribution of gender in violinist and
violist to severity can be seen in Table 1.
Table 1. Distribution data of gender in violinist and violist to severity of TMD
Gender Score Total
Mild Moderate Severe
Male 4 (25%) 12 (75%) 0 (0%) 16 (100%)
Female 3 (21.4%) 6 (42.9%) 5 (35.7%) 14 (100%)
Total 7 (23.3%) 18 (60%) 5 (16.7%) 30 (100%)
Description of research data regarding distribution long of period time playing

violin and viola to severity of TMD can be seen in Table 2.
Table 2. Distribution long period of time playing violin and viola to severity of TMD
Long Period of Time (years) Score Total
1-3 4 (33.3%) 6 (50%) 2 (16.7%) 12 (100%)
4-6 1 (9.1%) 8 (72.7%) 2 (18.2%) 11 (100%)
7-10 1 (33.3%) 2 (66.7%) 0 (0%) 3 (100%)
11-13 0 (0%) 3 (75%) 1 (25%) 4 (100%)
Total 6 (20%) 19 (63.3%) 5 (16.7%) 30 (100%)
Description of research data regarding distribution frequency playing violin and

viola in a week to severity can be seen in Table 3.
Table 3. Distribution frequency playing violin and viola in a week to severity of TMD
Frequency Playing in a Week Score Total
1-2 3 (30%) 5 (50%) 2 (20%) 10 (100%)
3-4 2 (33.3%) 2 (33.3%) 2 (33.3%) 6 (100%)
112
5-6 0 (0%) 5 (3.3%) 1 (16.7%) 6 (100%)
7-8 1 (12.5%) 5 (87.5%) 1 (16.7%) 6 (100%)
Total 6 (20%) 19 (63.3%) 5 (16.7%) 30 (100%)
Description of research data regarding distribution routinity playing violin and

viola to severity can be seen in Table 4.
Table 4. Distribution routinity playing violin and viola to severity of TMD
Routinity Score Total
Yes 3 (14.3%) 13 (61.6%) 5 (23.8%) 21 (100%)
No 3 (33.3%) 6 (61.9%) 0 (0%) 9 (100%)
Total 6 (20%) 19 (63.3%) 5 (16.7%) 30 (100%)
Description of analysis data by using Chi Square method of gender, long period
of time, frequency, and routinity to severity of TMD can be seen in Table 5.
Table 5. Data by using Chi Square method of gender, long period of time, frequency,
and routinity to severity of TMD
Distribution of Severity P-Value Result
Gender 7 (23.3%) 18 (60%) 5 (16.7%) 0.030 p-value < 
Long period of time 6 (20%) 19 (63.3%) 5 (16.7%) 0.670
Frequency in a week 6 (20%) 19 (63.3%) 5 (16.7%) 0.311 p-value > 
Routinity 6 (20%) 19 (63.3%) 5 (16.7%) 0.191
* = 0.05
An analysis with Chi-Square method was used to determine whether there were
a relation between long period of time, frequency, and routinity on practice with
severity of TMD on violinists and violists. As we could see on the Tabel 5, p-value each
of them were 0.670, 0.311, dan 0.191 respectivley, p-value >  ( = 0.05). It meant H0
was accepted and H1 was rejected, so there were no significant relation between long
period of time, frequency, and routinity on practice with severity of TMD on violinists
and violists. But, as we could see too on the Tabel 5 that showed p-value of gender is
113
0,030 with  = 0.05, so p-value < . It meant there was significant relation between
gender of violinist and violist to severity of TMD.
DISCUSSION
Based on the result in this study, all the participants were suffered from
temporomandibular joint disorder (TMD). Lozano5 and Kovero7 said that playing violin
and viola appears to be a factor associated with TMD-related findings. Violin and viola
players often report signs and symptoms identical to those of TMD.5 The violinists and
violist often subject to prolonged static use of the neck and shoulders, monotonous
repetitive use of temporomandibular joints, asymmetric body postures, or even
combination of all of them.15
A study which done by Moraes17 investigated temporomandibular dysfunction in
92 musicians, including 22 violists and violinists, found that 18 string instrumentalists
musicians had a history of temporomandibular pain. Neck and jaw positioning while
playing, excessive pressure applied to hold the instrument and occlusion with excessive
force, were the primary causes of temporomandibular dysfunction. More problems were
found in the violists, such as pain may seem to be a headache or involve the face and
neck, and is usually related to either excessive muscle tension (for example, teeth
clenching) or degradation of the joint itself. Symptomatic violinists and violists can
change their technique to reduce the force caused by the instrument on the jaw, reducing
pain and dysfunction.16,17
Prevalence of severity among participants were 23.3% mild, 60% moderate, and
16.7% severe respectively. In this study, the most common severity among violinist and
violist was moderate. In a previous study by Nomura18 on dental students in
Brazil with the results obtained using the Fonseca questionnaire, the participants
were 46.79% TMD-free and 53.21% were suffered from TMD with
severity percentage 35.78% mild, 11.93% moderate and 5.5% severe respectively. This
adds evidence to the importance of the clinical examination of the TMD. Approximately
¾ of the population may have some sign and symptom of TMD. Dental occlusion
studies have demonstrated a low percentage of individuals who are completely TMD–
free. Violinist and violist have greater risk of severity in TMD than normal population
and they are unaware that they have TMD. Using a simplified questionnaire, they were
able to recognize unnoticed symptoms that could lead to a greater TMD. The
individuals presenting signs and symptoms followed preventive measures, making
treatment easier.5
Based on the results, we can see that there were no significant relation between
long period of time, frequency, and routinity on practice with severity of TMD on
violinists and violists. In this study showed that long period of time didn‟t relate
significantly with severity of TMD. According to Kovero‟s studies7 say that long
period of time playing the violin and viola is associated with the occurrence of TMD.
Allsop19 studied about pianist said that there is significant relation between long period
114
of time playing piano and reported of playing‐related musculoskeletal disorders

(PRMDs). Zetterberg20 said that the longer time of playing piano (years) have
correlation with high risk of musculoskeletal disorder. Respondents who began playing
piano at teenager age have greater risk of musculoskeletal disorder than respondents
who started in adulthood. So, as we can say that long period of time is correlated with
occurance of TMD, but it doesn‟t determine its severity.
The results also say that there is no significant relation between frequency of
playing in a week violin and viola to severity of TMD. In the Allsop 19 studies
suggested that PRMDs were associated with frequency and duration of practice. But, it
was not correlated with occurrance and severity of TMD.
About the relation between routinity to severity of TMD, it also showed non
significant result. The severity of TMD in participants who played the violin on a
regular basis is not much different from the violinist is not routine. Playing violin and
viola will cause the body to be in an imbalanced position and the jaw will be depressed.
The more routine playing the violin means that the situation will become increasingly
common. The profesional violinist or violist have been played more routine than the
amateur. So, it is needed to do further studies to compare the severity of TMD in
profesional and amateur violinists or violists.
The results showed that, there is significant relation between gender of violinist
and violist to severity of TMD. The results showed that female violinists and violists
were the most suffered from severe TMD. In previous studies said that women have a
higher risk to suffer from TMD than men.21,22 Considering only severe of TMD, women
were approximately 9 times more affected than men. This is same as musculoskleletal
disorder (PRMDs) of musicians. Zaza and Farewell23 who studied about cello and
double bass players said that female players have greater risk of musculoskeletal
disorder (PRMDs), such as upper limb, hands, asymmetrical extension, muscles, and
nerves disorder than male players. The high prevalence of TMD/MSD in women may
be related to their different physiological characteristics, such as regular hormonal
variations, muscle or cartilage structures and different characteristics of the connective
tissue.18,24
So as we can see that both males and females can suffer from TMD. Many of the
musicians, especially the violinist and violists don‟t know that they have high risk to
suffer from TMD. If this condition is left for a long time, it will become more severe
and will affect their performance playing the violin and viola. Hence, early prevention
and treatment are so important in order to prevent further TMJ disorder.
CONCLUSSION
This study showed that long period of time, frequency, and routinity on practice
did not influence the severity of temporomandibular joint disorder (TMD) on violinists
and violists. However, violinists and violists have great risks of TMD and most of them
have moderate severity of TMD.
115
REFFERENCES
1. Okeson JP. Management of temporomandibular disorders and occlusion, 4th ed. St.
Louis: Mosby; 1998. p. 78, 93, 148,180.
2. Plain. Professional musicians with craniomandibular dysfunctions treated with oral
splints. The Patient Education Institute; 2008
3. Nardini LG, Manfredini D, Ferronato G. Temporomandibular joint total
replacement prosthesis : current knowledge and conciderations for the future. Int J
Oral Maxillofac Surg 2008;37:103-10.
4. Wright EF. Manual of temporomandibular disorders. Blackwell Munksgaard. USA:
Blackwell Publishing Company; 2005. p. 29-31.
5. Lozano R, Sáez-Yuguero MR, Bermejo-Fenoll A. Prevalence of
temporomandibular disorder–related findings in violinists compared with control
subjects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109(1):15-9.
6. Yeo DKL, Pham TP, Baker J, Porter SAT. Specific orofacial problems experienced
by musicians. Aust Dent J 2002;47(1):2-11.
7. Kovero O, Kononen M, Pirinen S. The effect of violin playing on the bony facial
structures in adolescents. Europe J Orthod 1997;19:369-75.
8. Kadmin, S. When the TMJ is Affected; 2011 [cited 2012 Februari 14]. Available
from: http://www.1800dentist.com/women-account-for-90-of-tmj-sufferers/
9. Ciancaglini R, Gherlone EF, Radaelli G. Unilateral temporomandibular disorderand
asymmetry of occlusal contacts. J Prost Dent 2003;89:180-4.
10. Stechman N J, Almeida C, Bradasch ER, Corteletti LCBJ, Silvério KC, Pontes
MMA, et al. Ocorrência de sinais e sintomas de disfunção temporomandibular em
músicos. Rev Soc Bras Fonoaudiol 2009;14:362-6.
11. Steinmetz A, Ridder PH, Methfessel G, Muche B. Professional musicians with
craniomandibular dysfunctions treated with oral splints. J Craniomand Pract 2009
[cited 2012 Februari 2]. Available from:
http://www.freepatentsonline.com/article/CRANIO-Journal-Craniomandibular-
Practice/210722709.html
12. Lederman RJ. Neuromuscular and musculoskeletal problems in instrumental
musicians. Muscle Nerve 2003;27:549-61.
13. Lin YC, Hsu ML, Yang JS, Liang TH, Chou SL, Lin HY. Temporomandibular joint
disorders in patients with rheumatoid arthritis. J Chin Med Assoc 2007;70(12):527–
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14. Rahmawati A. Faktor yang berhubungan dengan pemberian ASI eksklusif di
wilayah Kerja Puskesmas Bonto Perak Kabupaten Pangkep [cited 2012 March 21].
Available from: http://www.damandiri.or.id/file/rahmawatiunhasbab3.pdf
15. Paarup HM, Baelum J, Holm JW, Manniche C, Wedderkopp N. Prevalence and
consequences of musculoskeletal symptoms in symphony orchestra musicians vary.
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16. Safety and Health in Arts Production and Entertainment (SHAPE). Musicians and
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17. Moraes GFS, Papini AA. Musculoskeletal disorders in professional violinists and
violists. systematic review. Acta Ortop Bras 2012;20(1):43-7.
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prevalence and severity of temporomandibular disorders in brazilian dental
undergraduates. Brazilian Dent J 2007;18(2):827-42.
19. Allsop L, Ackland T. The prevalence of playing‐related musculoskeletal disorders
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Issue Music and Health 2010;3(1):61‐78.
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problems among male and female music students. Med Probl Perform Art
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21. Bursky P. Performance related musculoskeletal disorders in bassoon players.
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22. Kitsoulis P, Marini A, Iliou K, Galani V, Zimpis A, Kanavaros P, et. al.
Signs and Symptoms of Temporomandibular Joint Disorders Related to the Degree
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117

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Asia Pacific Dental Students Journal | Volume 3| Number 2| June 2012

EFFECTIVENESS OF ELLAGIC ACID THAT

Airlangga University, Indonesia

Asia Pacific Dental Students Journal 2012

MATERIAL AND METHODS

Distilled Water Strawberry

First, the result is tested by Kolmogorov-Smirnov test to analysis normality of

Sum of Squares df Mean Square F Sig.

Between Groups 3.203 1 3.203 12.779 .005

Within Groups 2.507 10 .251

DLINGO (Acorus calamus L) EXTRACT AGAINST

Asia Pacific Dental Students Journal 2012

Conclusion : The usage of dlingo extract (ethanol) to Candida albicans more

Key words: Candida albicans, rhizome of dlingo, MIC.

Prevalance of denture stomatitis among denture used is varieties, but in population

One of the potential plants to develop as traditional medicine is dlingo (Acorus

MATERIAL AND METHODS

Minimum inhibitory concentration is the smallest concentration of antimicrobial

Figure 1. The equipment

A : microscope H: absorbent pipette

B : reaction tube I : erlenmeyer 100 ml

C : tub shelf J : aquades

D : bulyon glucose K: filter paper

E : spiritus lamp L : object glass

G : sabouraud dextro agar

The steps that are going to do in this experiment are:

1. Taken and cultivate the examination material

2. Isolation and identification of Candida albicans

Characteristic examination of the colony in Candida Chromogenic Agar is going to

3. Make a suspension of Candida albicans

Suspension of Candida albicans is made equivalent with Mc Fahrland 0,5 turbidity

4. Make an extract of dlingo rhizome (Acorus calamus L)

Making extract of dlingo rhizome (Acorus calamus L) using ethanol 70 % by

5. Examination of inhibition power of dlingo extract (Acorus calamus L) according to

The determining of antifungal activity by diffuse method is a method to see the

6. Diluting test of testing substance and the determination of minimum inhibitory

Preparation of standard solution by diluting the extract of the rhizome dlingo in

Determination of minimum inhibitory concentration dlingo rhizoma extract toward

Table 1. Preparation of the rhizome extract dlingo

Tube Bulyon The suspension of Concentration Suspensition

1 - 4 ml from standard solution 80 mg/ml 0,1 ml

2 2 ml 2 ml from tube 1 40 mg/ml 0,1 ml

Turbidity of each tube is recorded and taken an oese from each

Keys: N= Number of all data

nj = Number of j treatment of data

𝑅𝑗 = average ranking of the j treatment

T i= Number of points equal to-i

If the two treatments revealed different (significantly), then further tested by

𝑅𝑢 − 𝑅𝑣 = Difference in average ranking of the two treatment

Figure 2. The determining of minimum inhibitory concentration of Candida albicans

The total colony of Candida albicans in every tube

The concentration of ethanol extract of dlingo rhizome in tube :

1 : 80 mg/ml 4 : 10 mg/ml 7 : 1,25 mg/ml

2 : 40 mg/ml 5 : 5 mg/ml 8 : 0,625 mg/ml

3 : 20 mg/ml 6 : 2,5 mg/ml K (-) : negative control

K (+): positive control

The total colony of Candida albicans in every tube

1 0 7,5 5,5 1,5 3 1,5 3,5 8 0 105

7 0 221,5 3,3.104 3,3.104 3,3.104 3,3.104 105 105 0 105

The concentration of ethanol extract of dlingo rhizome in tube :

1 : 80 mg/ml 4 : 10 mg/ml 7 : 1,25 mg/ml

2 : 40 mg/ml 5 : 5 mg/ml 8 : 0,625 mg/ml

3 : 20 mg/ml 6 : 2,5 mg/ml K (-) : negative control

K (+): positive control

OSTEOCYTE 0.097 0.186 0.027* 0.010 0.090