Volume 1 - Number 2 - August 2010

Volume 1 | Number 2 |August 2010
1
Asia Pacific Dental Students Journal
Asia Pacific Manusript of Papers presented at Scientific Research Competition,

37th Asia Pacific Dental Students Association Congress, Japan.
Dental Students
Journal Poster Presentation
3. PP1: THE AGE-DEPENDENT OSTEOGENIC DIFFERENTIATIONOF ADIPOSE-
DERIVED STEM CELLS
Tsai, Meng-Wen, Fu-Hung Tseng, Hen-Yu Liu
August 2010 Vol. 1 No.2
16. PP2: ORAL HEALTH STATUS AND ORAL HEALTH RELATED TO QUALITY
Editorial OF LIFE OF DENTAL STUDENTS IN UNIVERSITI SAINS ISLAM MALAYSIA
Nusaibah Mutmainnah Bt Mohamad Azmi, Mursyidatun Najihah Mohamad Nasar,
Editors-in-chief: Zulkarnain Sinor
Melissa Yap 28. PP3: THE EFFECT OF SOY MILK ON MANDIBLE BONE DENSITY IN RAT
Stanley Kamadjaja MODEL STUDY
Devi Gunawan, Diana S. Djohan, Tamara Gladysia E, William Adi Santoso, Lee Yan Ying
Publisher 39. PP4: TEST OF INHIBITORY POWER OF ESSENTIAL OIL EXTRACT

NUTMEG SEED (MYRISTICA FRAGRANS) ON GROWTH OF
Asia Pacific Dental Students STAPHYLOCOCCUS AUREUS IN ANGULAR CHEILITIS
Association (APDSA) Novita Eka Lestari, Aisyah Bella Azzanjani, Ita Purnama Alwi
53. PP5 : THE ANTI-INFLAMMATORY EFFECT OF CAHEW FRUIT EXTRACT ON
Executive Committee ARTIFICIAL EDEMA IN WISTAR RAT
Tiar Rennyka, Euis Mila Savista
APDSA 2009/2010
President: Rumi Sato 62. PP6: SYNTHESIZE AND CHARACTERIZE THE YTTRIUM STABILIZED
ZIRCONIA (YSZ) AS DENTAL RESTORATION MATERIAL
Secretary: Jun Ai Chong Arifialda. A
Treasurer: Karen Voon 71. PP7: THE SIZES OF MAXILLARY ANTERIOR TEETH IN ASIAN AND ITS
Editor: Stanley Kamadjaja CORRELATION WITH SELECTIVE CRANIOFACIAL ANTHROPOMETRIC
International Liaison Officer: MEASUREMENTS
Renette Gan Siok Lynn, Karthiravan Purmal, Ngeow Wei Cheong, Yeoh Oon Take
Jack Chao Ji
SRC Coordinator: Melissa Yap 88. PP8: INJURY TO THE ORAL CAVITY EXPERIENCED BY BRASS WIND
INSTRUMENT PLAYERS OF BANDUNG MARCHING BAND UNIT
President Elect: Peerapat Randita Diany Yordian, Sayed Mohamad Ridhwan, Puput Nurani
Kaweewongprasert 102. PP9:BACTERICIDAL AND CYTOTOXIC EFFECTS OF Erythrina fusca
LEAVES AQUADEST EXTRACT
Country Representatives: Timotius Andi Kadrianto, Nadya Saputri Halim, Melinia
Korea: Koo Seung Hwan 113. PP10: DENTAL SANTRI SCHOOL PROGRAM AS A SOLUTION FOR
INTEGRATED DENTAL HEALTH EDUCATION IN ISLAMIC BOARDING SCHOOL
Taiwan: Meng-Hsuan Tu Aditya Mukti Setyaji, Renna Maulana Yunus, Ira Willyanti, Dhea Adittya
Singapore: Syazwan Lim
124. PP11: PREVALENCE OF PERIODONTITIS IN DENTAL STUDENTS IN
Cambodia: Sok Chenh Chhean UNIVERSITY TECHNOLOGY MARA
Azwin Assilah bte Kamaruddin, Aiman Nadiah Ahmad Tajuddin, Farah Hidayah Mohd
The Asia Pacific Dental Students Journal is Fazli, Siti Sarah Nor Rizan, Maziahtul Zawani Munshi, Fouad Hussain M.H Al-Bayaty
the official scientific journal for the Asia 134. PP12: ORAL HEALTH STATUS OF CHINESE ELDERLY PEOPLE WITH
Pacific Dental Students Association DEMENTIA
(APDSA). Tam Hoy Suet Ailsa, Chan Yau Chuen, Cheung Wing Pan, Ho Tek Ka, Lau Chon Kit, Mak
Copyright: All rights reserved. No part of Ka Man, Ng Alice, Woo Cheuk Hang Timothy
this publication may be produced, stored in a
retrieval system or transmitted, in any form 165. PP13: THE EFFECT OF ETHANOLIC EXTRACT OF PINEAPPLE-STEM
( ANANAS COMOSUS (L.) MERR) ON INCREASING APOPTOSIS OF A HUMAN
or by any means (electronic, mechanical,
photocopying, recording or otherwise, ORAL TONGUE CANCER CELL ( SP – C1 ) IN VITRO
Agnes Bhakti Pratiwi, Muhammad Isa, Wisda Septiana Chandra Devi, Supriatno
without the written permission of the
publisher. 174. PP14: THE HEMOSTATIC EFFECT OF ARTEMISIA VULGARIS EXTRACT IN
TRAUMATIC BLEEDING ON MUS MUSCULUS
© 2010 Asia Pacific Dental Students Grace Angelina Samuel, Melisa Budipramana, Dian Lupita Sari, Astari Puteri,
Anisha Giantini, Jessica Theresia
Association
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PP1: THE AGE-DEPENDENT OSTEOGENIC DIFFERENTIATIONOF ADIPOSE-DERIVED

STEM CELLS
Tsai, Meng-Wen ,Fu-Hung Tseng, Hen-Yu Liu
Objectives: Adipose tissue is an ideal source of stem cells since we can obtain the adipose-
derived stem cells(ADSCs) from the tissue in a large quantity through simple surgery. In vivo
and in vitro studies indicate that a sub-population of ADSCs has potential to differentiate into
multiple cell types, including osteoblasts. Since the differentiation capacity of stem cells is
much affected by age, in this study, we want to compare the the growth kinetics and
differentiation potential on ADSCs in different ages. Methods: ADSCs were isolated from
adipose tissue of female SAMP8-1M and SAMP8-10M mice. First, we used flow cytometry to
examine the surface marker expression of CD45, CD34, Sca-1, CD44 and CD105 to
characterize the isolated cells. Furthermore, we compared the cell number and the gene
expression in PCR analysis between the older(ADSC-10M) and the younger(ADSC-1M) after
induction in osteogenic medium. Finally, we use Alizarin red staining to show the osteogenic
potential.Results: In cell number comparison, ADSC-1M grew faster than ADSC-10M. In PCR
analysis, ADSC-1M showed greater expression of osteoblast marker genes Runx2 and OPN
than ADSC-10M .In the meanwhile, the Alizarin red staining showed the same resalt.
Conclusions: Accordint to in vitro study, we can tell that the younger ADSCs have better
osteogenic capacity.
INTRODUCTION
Osteoporosis is a disorder characterized by compromised bone quality which predisposes

increased risks for fractures particularly in the hips, spine, and limbs, resulted from insignificant
trauma1. It is estimated over 200 million people worldwide have osteoporosis 2. The prevalence of
osteoporosis is continuing to escalate with the increasingly elderly population. The major complication
of osteoporosis is an increase in fragility fractures leading to morbidity, mortality, and decreased
quality of life. Hence osteoporosis is also known as ”the silence killer”.
The majority of current therapeutic protocols are focused on preventing excessive bone loss in
osteoporotic patients. For instance, bisphosphonates have been shown effective as antiresorptive agents
and approved by the FDA3. These agents exert their actions by inhibition of farnesyl diphosphate
synthase, a key enzyme of the mevaloneate pathway, leading to osteoclast apoptosis 4. However, these
therapies are not focused on the major factor in the pathogenesis of senile osteoporosis: the loss of
functional osteoblasts during aging5-8. In our study, we hypothesized that osteoblast dysfunction and the
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loss of osteoblasts leading to osteoporosis could potentially be prevented by administration of stem

cells that have been pre-differentiated to the osteoprogenitor phenotype.
Bone marrow is a common source of multipotent stem cells 9-11; however, there might be a better
choice. In previous studies indicates that a sub-population of adipose-derived stem cells(ADSCs) has
potential to differentiate into multiple types of cells, including osteocytes, adipocytes and
chondrocytes12. In the meanwhile, the proliferation rate of ADSCs is higher than bone marrow stem
cells(BMSCs), and the osteogenesis potential of ADSCs is better as well 13. Since there are more and
more people undergoing liposuction surgery, surplus adipose tissue has become common medical
waste. Therefore, adipose tissue is an ideal source of multipotent stem cells because of easy
obtainment. Since the differentiation capacity of stem cells is much affected by age 14and ADSCs have
better ability in osteogenesis than in adipocytogenesis, in this study, we compare the growth kinetics
and osteogenic potential on ADSCs in different ages. Thus, whether age affects the osteogenesis of
ADSCs will likely shed lights on the mechanism of osteoporosis and the development of its therapeutic
tools.
METHOD
1. Outline
First, we obtained ADSCs from adipose tissue of SAMP8-1M and SAMP8-10M mice. Since the
differentiation and self-renewal ability are significant characteristics of stem cells, we compared the
cell number/colony forming and the gene expression in PCR analysis between the older(ADSC-10M)
and the younger(ADSC-1M) after inducing the cells in osteogenic medium. Finally, we use Alizarin red
4
staining to observe the osteogenic potential.
2. ADSCs extraction
To obtain ADSCs, we have to prepare digest medium first. Standard digest medium contains
collagenase type IV 5mg/5c.c., hyaluornic acid 1.7mg/5c.c. and α-MEM(Minimum Essential
Medium Alpha Medium with 10% Fetal Bovine Serum and 1% PSA) 5cc filtrated through 0.22um
filter. Then we sacrificed the SAMP8 1M and 10M mice, and cut off the adipose tissue around the
abdominal region. Put the tissue in digest medium, cut it into small pieces, and shaking for 1 hour
in 37 ℃ incubator. Next, we centrifuged it of 1500 RPM for 10 minutes, seeding the cells on the
dish.
3. ADSCs cell culture

It usually takes 3~5 days to achieve 90% confluency in primary culture of ADSCs. We used 0.25%
trypsin-EDTA to "digest" the proteins that facilitate adhesion to the container and between cells, and
we could then seed the cells on a new dish. This process is done to permit subculture of the cells to a
new container, observation for experimentation, or reduction of the degree of confluency in the dish.
4. Cytochemistry Staining
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1.0 Alizarin Red S Staining

Alizarin Red ,may be used to identify calcium in tissue sections. Calcium forms an Alizarin Red
S-calcium complex in a chelation process, in which the end product would be red. First we used 10%
formaldehyde to fix the cells and applied 2% Alizarin Red staining for 15minutes. For quantification,
we added 10% cetylpyridnium chloride with 8mM Na 2HPO4 and 1.5mM KH2PO4 to separate out the
red color.
2.0 Oil Red O Staining

Oil Red O is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides
and lipids. First we used 10% formaldehyde to fix the cells. Then we mixed Oil Red O 0.4g and
isopropanol 80ml, diluting the stock with ddH2O. Finally we added the working solution to stain the
cell for 15 minutes so we could observe the results.
5. Osteogenic Medium Preparation

To prpare osteogenic medium, we had to prepare α-MEM with 10% FBS and 1% PSA first. Then
we added 0.1uM dexamethasone, β-glycerol phosphate and 50uM ascrobate into α-MEM.
6. mRNA Extraction
After we trypsinized the ADSCs, we centrifuged the cells over 1500 RPM for 5 minutes. Use 1ml
TRIzol to lysis the cells. Then we added 0.2ml chloroform and shook it for 15 seconds. 3 minutes later,
we centrifuged it over 4℃, 12000 RPM for 15 minutes. After centrifuged, the solution would be
6
separated into 3 layers. The upper layer contained RNA and should be removed into a new eppendorf.
Then we added Isopropanol of the same volume into the eppendorf, mixing well. We put the eppendorf
in -20℃ for 15 minutes, and centrifuged over 12000 RPM, 4℃ for 15 minutes to get the white pellet.
After removing the upper solution, we added 1 ml 75% ETOH. Then, we centrifuged it again and
removed the upper solution. Finally, we added 20 ul DEPC-ddH2O and mixed well.
7. Reverse transcription(RT)
The mRNA which we extracted would turn into complementary DNA (cDNA) through reverse
transcription. RT materials comprised SuperscripTM III, Oligo dT primer, 10mM dNTP mix, 10X RT
buffer, 25mM MgCl2, 0.1M DTT, RNase inhibitor and 4 ug mRNA. The total volume was 20 ul and
the reaction took place in PCR machine.
8. Polymerase Chain Reaction(PCR)

After RT, we did the PCR to enlarge the DNA fragment of our choosing. We added cDNA into
ddH2O, 2.5mM dNTP, 25mM MgCl2, upstream/downstream primer and Taq polymerase. The PCR
reaction would take place in PCR machine and go through 35 cycles of denaturation, annealing and
extension.
9. Agarose gel preparation

We added 0.5g Agarose powder into 50ml TAE buffer, heating with a microwave oven for 4
minutes. After mixed well, the gel was then poured into a mold. Finally, we inserted a comb and the gel
could be used after cooled.
RESULTS
I. The Cell Culture of ADSCs
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Fig.1
As Fig.1 shows above, ADSCs-1M were spindle-like and posses a more compact morphology. On
the other hand, ADSCs-10M were comparably larger and were more spread out.
II. Define the isolation cells on stem cell characterization

12. The Self-renewal ability of ADSCs
1.1. Colony forming Unit
Fig.2
We seeded ADSCs-1M and ADSCs-10M in two dishes. 14 days later, we used crystal violet
staining to assist in the observation of the number of cell colony. As Fig.2 shows above, the younger
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ADSCs formed about 150 colonies. In comparison, the older ADSCs formed only about 40 colonies
under the same condition. This result empirically demonstrates that the self-renewal ability gradually
gets worse as aging progresses.
1.2. Cell Proliferation
Fig.3
We counted the cell number on the 1 st,3rd,5th,7thand 9th day and drew the growth curves of
ADSCs-1M and ADSCs-10M. As Fig.3 shows above, the younger cells grew faster and the doubling
time was about 5 hours less than the older ones.
2. The Differentiation Potential of ADSCs
Fig.4
9
Mesenchymal stem cells have the potential to differentiate into multiple types of cells, including
osteocytes and adipocytes. We seeded the ADSCs-1M and ADSCs-10M in the induction medium to
compare their differentiation potential. As Fig.4 shows above, both 1M and 10M cells showed the
tendency towards osteogenesis and adipogenesis.
III. Semi-quantitative PCR Analysis
Fig.5
Fig.6
*P<0.05
10
Fig.7
Fig.5 is the result of PCR analysis. We used house-keeping genes(GAPDH) to semi-quantify the
Runx2 and OPN genes. Fig.6 and Fig.7 are bar charts after semi- quantification. Runx2, a key
transcription factor essential for early stage of osteoblast differentiation, is the most probable target
gene because its expression is demonstrated to be down-regulated during cellular senescence of
osteoblasts. OPN is a major gene essential for late stage of osteoblast differentiation. As Fig.6 shows
above, Runx2 gene expression of ADSCs-1M is more significant than ADSCs-10M on the 7 th day. In
the meanwhile, as Fig.7 shows above, OPN gene expression of ADSCs-1M is more significant than
ADSCs-10M on the 21th day.
IV. Alizarin red staining
Fig.8
11
Fig.9
Alizarin Red ,may be used to identify calcium in tissue sections. Calcium forms an Alizarin Red
S-calcium complex during a chelation process, in which the product would be red. After induction for
7, 14 and 21 days, we stained the cells with Alizarin red. As Fig.8 shows above, the color of the 21 days
observation was the darkest, and the younger cells was darker than the older ones.
DISCUSSION
In our study, we examined the various changes in the differentiation potential relative to age of
a stem cell population derived from adipose tissue of SAMP8 mice. Numerous studies in the past,
including those of our own group, have demonstrated the multi-lineage potential of these ADSCs 12,15.
Meanwhile, the proliferation rate of ADSCs is higher than that of bone marrow stem cells(BMSCs),
and the osteogenesis potential of ADSCs is better as well. However, recent studies have begun to
question the activity and potential of ADSCs with respect to mice age 16. To investigate this question,
this study presents data describing the relationship between ADSCs age, growth kinetics and
differentiation capacity.
We started our study by obtaining tissue from SAMP8-1M and SAMP8-10M mice. ADSCs
isolated from SAMP8-1M were spindle-like with a more compact morphology. Nevertheless, ADSCs-
10M were comparably larger and were more spread out (Fig.1). This could have been the result of the
condition of cells from the older mice was significantly worse. Previous study has demonstrated that
serially passaged long-term cultures would cause cellular aging and senescence17. Thus, we suggested
that we should attempt the experiment with cells of the first to the third passage to ensure the healthy
condition of the cells.
12
We then defined the isolation cells on stem cell characterization. Since the differentiation and self-
renewal ability are significant characteristics of stem cells, we compared the cell number/colony
forming and the gene expression in PCR analysis between ADSC-1M and ADSC-10M after inducing
the cells in osteogenic medium. In the colony forming unit (CFU) test, the younger ADSCs formed
about 150 colonies; in comparison, the older ADSCs formed only about 40 colonies under the same
condition (Fig.2). Furthermore, we compared the proliferation rate of ADSCs-1M and 10M. We
counted the cell number on the 1st,3rd,5th,7th and 9th day and drew the growth curves of ADSCs-1M and
ADSCs-10M(Fig.3). The younger cells grew faster and the doubling time was about 33.68 hours
compared to the 38.84hours of the older ones. These results empirically demonstrated that the self-
renewal ability gradually gets worse as aging progresses, which showed the same tendency toward the
study of age-related growth kinetics of ADSCs and BMSCs 18.19. Mesenchymal stem cells has the
potential to differentiate into multiple types of cells, including osteocytes and adipocytes. We seeded
the ADSCs-1M and ADSCs-10M in the osteogenic and adipogenic medium to compare their
differentiation potential. Both 1M and 10M cells showed the tendency towards osteogenesis and
adipogenesis (Fig.4).
Since the aim of our study is geared towards bone repair, we compared the osteogenic ability in
vitro. We used GAPDH as a standard to semi-quantify the Runx2 and OPN genes (Fig.5). Runx2, a key
transcription factor essential for early stage of osteoblast differentiation, is the most probable target
gene because its expression is demonstrated to be down-regulated during cellular senescence of
osteoblasts. OPN also happens to bemajor gene essential for late stage of osteoblast differentiation. We
extracted the RNA to see the gene expression of osteogenesis after induction for 7, 14 and 21 days.
Runx2 of ADSCs-1M had significant expression on the 7th day (Fig.6), and OPN of ADSCs-1M
expressed greatly on the 21th day(Fig.7) as well. As a result, we could tell that ADSCs-1M tends to
advance the time of osteogenesis after induction.
Furthermore, we use Alizarin red staining to compare the mineralization of ADSCs. After
induction for 21 days, the degree of mineralization of ADSCs-1M was higher than one of ADSCs-10M
(Fig.8). We could see the significant difference between 1M and 10M on the 21 th day through
quantification (Fig.9), which indicated that younger ADSCs might have better osteogenesis ability in
vitro. The same tendency could be seen in previous study of human BMSCs, in which the author
thought that stem cells would be affected by intrinsic factor and extrinsic somatic environment as aging
progresses.
13
In conclusion, we could sum up the osteogenic differentiation of ADSCs-1M and10M by the

following Fig.10. Runx2 was the major transcription factor during the early stage of the mineralization,
whereas OPN stood for the later stage instead. The lines in the following diagram represented ADSC-
1M and the dotted lines represented ADSCs-10M. The tendency towards osteogenesis of younger cells
was much stronger than the older ones.
osteogenic differentiation
5 5
1m/ODM(OPN)
10m/ODM(OPN)
1m/ODM(Runx2)
4 4
10m/ODM(Runx2)
1m/ODM(Alizarin red)
gene expression ratio
10m/ODM(Alizarin red)
Alizarin red O.D 405

3 3
2 2
1 1
0 0
day 7 day 14 day 21
Fig.10
CONCLUSION
ADSCs can be used to promote osteogenesis and bone regeneration as well. Moreover, age does
affect the differentiation of ADSCs. These findings provided important insights on emerging cell-based
therapeutic strategies, especially in the treatment of various bone disorders including osteoporosis.
14
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1. Consensus development conference: prophylaxis and treatment of osteoporosis. Am. J. Med. 90, 107-110
(1991).
2. Reginster,J.Y. & Burlet,N. Osteoporosis: a still increasing prevalence. Bone 38, S4-S9 (2006).
3. Rosen,C.J. Clinical practice. Postmenopausal osteoporosis. N. Engl. J. Med. 353, 595-6(2005).
4. Chapurlat,R.D. & Delmas,P.D. Drug insight: Bisphosphonates for postmenopausal osteoporosis. Nat. Clin.
Pract. Endocrinol. Metab 2, 211-219 (2006).
5. Cao,J.J. et al. Aging increases stromal/osteoblastic cell-induced osteoclastogenesis and alters the osteoclast
precursor pool in the mouse. J. Bone Miner. Res. 20, 1659-1668 (2005).
6. Chen,T.L. Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell
cultures by increased donor age and glucocorticoid treatment. Bone 35,83-95 (2004).
7. Kawaguchi,H. Molecular backgrounds of age-related osteoporosis from mouse genetics approaches. Rev.
Endocr. Metab Disord. 7, 17-22 (2006).
8. Labrie,J.E., III, Borghesi,L., & Gerstein,R.M. Bone marrow microenvironmental changes in aged mice
compromise V(D)J recombinase activity and B cell generation. Semin. Immunol.17, 347-355 (2005).
9. Caplan, A.I. Mesenchymal stem cells. J Orthop Res 9, 641-650 (1991).
10. Minguell, J.J., Erices, A. & Conget, P. Mesenchymal stem cells. Exp Biol Med (Maywood) 226, 507-520
(2001).
11. Pereira Lda, V. [The importance of the use of stem cells for public health]. Cien Saude Colet 13, 7-14 (2008)
12. Zuk, P.A., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue
Eng 7, 211-228 (2001).
13. Rider, D.A., et al. Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-
derived mesenchymal stem cells. Stem Cells 26, 1598-1608 (2008).
14. Zhou, S., et al. Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and
their differentiation to osteoblasts. Aging Cell 7, 335-343 (2008).
15. British Society for Matrix Biology meeting, Manchester, 2-3 April 2001. Abstracts. Int J Exp Pathol 82, A1-
25 (2001).
16. Shi, Y.Y., Nacamuli, R.P., Salim, A. & Longaker, M.T. The osteogenic potential of adipose-derived
mesenchymal cells is maintained with aging. Plast Reconstr Surg 116, 1686-1696 (2005).
17. Kassem, M., Ankersen, L., Eriksen, E.F., Clark, B.F. & Rattan, S.I. Demonstration of cellular aging and
senescence in serially passaged long-term cultures of human trabecular osteoblasts. Osteoporos Int 7, 514-
524 (1997).
18. Kassem, M. Stem cells: potential therapy for age-related diseases. Ann N Y Acad Sci 1067, 436-442 (2006).
19. Stenderup, K., Justesen, J., Clausen, C. & Kassem, M. Aging is associated with decreased maximal life span
and accelerated senescence of bone marrow stromal cells. Bone 33, 919-926 (2003)
15
PP2: ORAL HEALTH STATUS AND ORAL HEALTH RELATED TO QUALITY OF LIFE OF
DENTAL STUDENTS IN UNIVERSITI SAINS ISLAM MALAYSIA
Nusaibah Mutmainnah Bt Mohamad Azmi,Mursyidatun Najihah Mohamad Nasar,

Zulkarnain Sinor
Introduction: Two most common oral diseases affected world population were dental caries
and periodontal disease. Oral disease has significant influent on ones quality of life as shown
in many studies. The prevalence of dental caries and periodontal disease are high among
youth in Malaysia. Dental caries affect 60-90% of school children and adults in most
industrialized countries. Objectives: This study was conducted to assess oral health status of
dental students of Universiti Sains Islam Malaysia (USIM) and their oral health related to
quality of life (QOL). Method: This is a cross sectional study involving 62 Dental students of
USIM, aged 19-22 years old. The 12 items General Oral Hygiene Index (GOHAI) self guided
questionnaire was used to assess the impact of oral health condition on respondent’s quality
of life. Respondent’s oral health status was examined by single researcher using Debris
Index, Basic Periodontal Examination (BPE), and also DMFT index. Results: Prevalence of
dental caries was 53.2 %, and for periodontal disease was 91.9%. On analysis of the content
some items were felt to be relevant to the impact on quality of life, which consist of physical
function, pain or discomfort and psychosocial. Higher GOHAI score (3.58 s.d 0.71) was found
to be associated with higher number of teeth present and lower GOHAI score (0.58 s.d 0.82)
was associated with higher caries experiences. Conclusions: Poor oral health status
influences the quality of life among this study sample in varying degrees. Respondents with
poor oral health status have low quality of life with their main concern was poor dental
appearance.
INTRODUCTION
Two most common dental diseases of mankind and affected majority world population throughout
their life were dental caries and periodontal disease. Their unique cumulative in nature (Donald LP.,
2006) will become more and more complex over time. The diseases itself and their consequences posed
major burdens worldwide, which affecting health and well being of populations and the consequences
was not only involving physical limitation but also economic, social and psychological.
On top of that, according to Locker (1988) oral diseases seriously impair quality of life (QOL) and
can affect various aspect of life, including oral function, appearance, and interpersonal relationship.
The important of oral diseases on quality of life can be seen by looking at growing number of oral
health related to (QOL) instrument developed in dentistry and number of articles in oral health QOL
published.
16
Oral health is a fundamental factor for quality of life (WHO, 2005). A healthy mouth enables
people to speak, eat and socialize without pain, discomfort or embarrassment (Locker, 1988).
Moreover, the psychosocial impact of oral diseases often significantly diminishes quality of life
(Petersen, 2003). It influences eating, sleeping, working and social roles (WHO, 2005, MOH, 2002).
Oral health is defined as “the well being of the oral cavity, including the dentition and its supporting
structures and tissues, the absence of disease and the optimal functioning of the mouth and its tissues,
in a manner which preserves the highest level of self-esteem and interpersonal relationships” (WHO,
1999). Thus, dental health indices also contribute as indicators for healthy life (Centers for Disease
Control and Prevention, 2000).
OBJECTIVES AND RESEARCH QUESTIONS
General objectives
To assess oral health status of all dental students studying in Universiti Sains Islam Malaysia and their
oral health related to quality of life (QOL).
Specific objectives
To determine the prevalence of dental caries and periodontal disease of dental students in Universiti
Sains Islam Malaysia.
To assess oral health related quality of life (QOL) of dental students in Universiti Sains Islam
Malaysia.
To determine the relationship between dental caries and periodontal disease with oral health related
quality of life among respondents.
METHODOLOGY
Study design
This is a cross sectional study involving 62 dental students from Faculty of Dentistry Universiti Sains
Islam of Malaysia. This study was carried out from 2/1/2009 to 31/6/2009.
Reference population
The reference population for this study includes all students studying in University Sains Islam of
17
Malaysia (USIM) aged 19 years and above.
Source population
USIM students aged 19 years-old and above, who located in Pandan Indah Health Campus.
Sampling Frame
Dental students aged 19 years-old and above, registered in Faculty of Dentistry USIM from 1 st January
2009 to 31 June 2009, who fulfilled the inclusion and exclusion criteria and consented for the study.
Inclusion and Exclusion Criteria:

Inclusion criteria in this study was those aged 19 years and above. Since this study is also looking at the
association of Oral Health related Quality of Life with periodontal diseases therefore need to exclude
juvenile periodontitis and also to avoid pre-pubertal hormonal changes, adults age >19 years was
chosen. Other inclusion criteria were dentate which defined as having at least one natural root or tooth
in the mouth (MOH, 2004).
As for exclusion criteria, we exclude those who have been certified by medical doctor of having
systemic diseases. Pregnancy can play a role in periodontal disease development; hence, pregnant
mothers were also excluded. Patients on long term medication were also excluded to avoid influence on
saliva quality and also development of periodontal disease and dental caries. Patients who had
undergone invasive periodontal surgical procedure were also excluded as this may affect the
measurement for periodontal status.
Sample size calculation

Since all dental students available during this study period were taken, therefore no sample size was
calculated.
Data collection procedure

This study started with a session of giving self-administered GOHAI questionnaire to the respondents.
This procedure was followed by oral examination of the respondents to determine oral health status,
and was carried out in the dental treatment room. Oral health examinations were carried out on a dental
chair using mouth mirrors, probe number 9, CPI probe number 621 and under attached dental light. The
data were recorded into data form by a trained dental assistant.
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Research tools
Questionnaire
In this study, we used 12 item GOHAI questionnaires. Subjects were asked about three dimensions of
oral health related quality of life which are physical function including eating, speech and swallowing.
Secondly about psychosocial function including worry or concern about oral health, dissatisfaction
with appearance, self-consciousness about oral health and avoidance of social contacts because of oral
problems and thirdly is about pain or discomfort including the use of medication to relieve pain or
discomfort from the mouth. In addition, the questionnaire also included socio-demographic
characteristics such as age, sex, medical problem and orthodontic appliance.
Clinical examination
Caries measurement
The diagnosis of dental caries was based on the WHO criteria (WHO, 1998) using DMFT index.
Dental caries is defined as cavitated lesion found on visual observation. The examinations involve
inspection of the occlusal, facial, distal, lingual and mesial surfaces. A visual examination is carried out
using a mirror and is aided by an air syringe to dry a tooth surface. An explorer was used in caries
diagnosis as a tool to remove plaque and debris and check the surface characteristics of suspected
carious lesions. This explorer was handled with light pressure. The score was taken as the dependent or
outcome variable for the study where the score was range from 0 – 28 score. The exclusively visual
criteria of caries recorded as D, missing of tooth from arch recorded as M, and tooth surfaces fill with
any restorative material recorded as F. The total accumulated score of decayed, missing and filled was
taken as the dependent variable for analysis.
Periodontal disease measurement

Basic periodontal examination (BPE) index was used in this study. The standardized periodontal probe
with light pressure was used to examine the tissue for bleeding, plaque retentive factors and pocket
depth. The score was pen down according to standardized code, as follow:
Code
0 No bleeding or pocketing detected
1 Bleeding on probing - no pocketing > 3.5mm
19
2 Plaque retentive factors present - no pocketing > 3.5mm

3 Pockets > 3.5mm but <5.5mm in depth
4 Pockets > 5.5 mm in depth
Statistical analysis
Data was entered and analyzed using Statistical Package for Social Sciences (SPSS, version 17.0)
software. Descriptive statistic was presented in form of means and SD and for categorical variables
frequency and percentage. To test the relationship between variables, Pearson coefficient correlation
was used. Level of significant was set at 0.05.
RESULT
Table 1: Descriptive statistics variables understudy (n=62)*

Characteristics Mean (SD) Freq (%)
Sex
Male 18 (29.0)
Female 44 (71.0)
Age Overall 19.7 (0.85)
Male 19.4 (0.85)
Female 19.7 (0.84)
Past Medical History
No 57(91.9)
Yes 5 (8.1)
Wearing Orthodontic
appliance
No 60 (96.8)
Yes 2 (3.2)
BPE score
Healthy 5 (8.1)
BOD 12 (19.4)
Calculus 44 (71.0)
Deep Pocket 1 (1.6)
Oral Hygiene
Good 56 (90.3)
Fair 5 (8.1)
Poor 1 (1.6)
DMF Score Overall 3.7 (3.42)
Male 2.1 (2.07)
Female 4.4 (3.42)
*
all respondent are Malay
20
Table 2: Prevalence of Periodontal Disease and dental Caries among Respondents (n=62)
_______________________________________
Variable freq (%)_______________
Dental caries
Yes 33 (53.2)
No 29 (46.8)
Periodontal disease
Yes 17 (27.4)
No 45 (72.6)_______________
Table 3: Oral Health Quality of Life Score (n=62)

_________________________________________________
Variable mean (SD)__________
Total GOHAI Score (12 item) 11.1 (5.50)
GOHAI item 1 0.9 (1.11)
GOHAI item 2 0.8 (0.70)
GOHAI item 3 0.4 (0.64)
GOHAI item 4 0.4 (0.71)
GOHAI item 5 0.6 (0.88)
GOHAI item 6 0.4 (0.72)
GOHAI item 7 1.3 (1.07)
GOHAI item 8 0.9 (0.91)
GOHAI item 9 1.9 (0.98)
GOHAI item 10 1.2 (1.06)
GOHAI item 11 0.7 (0.83)
GOHAI item 12 1.5 (0.84)__________
Table 4: Relationship between oral health status and Oral Health related Quality of Life score
(GOHAI Score) (n=62)
________________________________________________________
Variables b1 coeff.(95% CI) p-value___
DMF 0.73 (0.35, 1.10) <0.001*
Periodontal Disease 0.39 (-1.56, 2.35) 0.688_____

Dependent Variable: total GOHAI Score; (Constant= 7.685): * significant at p-value < 0.05; 1 Pearson
correlation coefficient
21
Table 5: Relationship between Caries experience and item in Oral Health related Quality of Life score (GOHAI Score) (n=62)
________________________________________________________________________________________________
Variables b1 coeff.(95% CI) p-value___Adj.β2(95% CI) t-stat.(df) p-value
GOHAI item 1 -0.83 (-1.86, 0.20) 0.113
GOHAI item 2 1.50 (0.24, 2.76) 0.021*
GOHAI item 3 -0.55 (-2.03, 0.93) 0.460
GOHAI item 4 -1.63 (-2.96, -0.30) 0.018* -0.26 (-2.29, -0.17) -2.328 (60) 0.023*
GOHAI item 5 0.48 (-0.67, 1.64) 0.403
GOHAI item 6 0.31 (-1.18, 1.79) 0.681
GOHAI item 7 0.37 (-0.66, 1.40) 0.478
GOHAI item 8 -0.002(-0.92, 0.91) 0.996
GOHAI item 9 -0.60 (-1.60, 0.38) 0.224
GOHAI item 10 -0.92 (-2.16, 0.33) 0.145 -0.46 (-2.12, -0.76) -4.130 (60) <0.001*
GOHAI item 11 0.41 (-0.91, 1.72) 0.536
GOHAI item 12 -0.70 (-1.65, 0.24) 0.142__________

*significant at p<0.05: 1 crude regression coefficient in Simple linear regression; 2 Adjusted regression coefficient in Multiple linear regression (R2=0.324, The
model reasonably fits well; Model assumptions are met; there is no interaction between independent variables and no multicollinearity problem)
22
A total of 62 respondents (22 male and 40 female) agreed to participate and completed
the GOHAI questionnaire. Table 1 shows the distribution of finding related to variables
understudy. The mean age (SD) of respondent was 19.7 (0.85) years-old, with mean (SD)
for male was 19.4 (0.85) and female 19.7 (0.84) respectively. Most of the respondents (57
(91.9 %)) was noted without any medical problem, and only 2 (3.2 %) respondents
wearing orthodontic appliance.
On clinical examination finding, 57 (92.0%) of respondents experience some form
of periodontal problems with 12 (19.4%) of them having bleeding on probing, 44 (71.0
%) with gingival calculus and 1 (1.6 %) have deep pocket. Overall mean (SD) DMF
score among respondents was 3.7 (3.42) with female slightly higher (4.4 (3.42)) then
male (2.1 (2.07)). Overall oral hygiene status among respondents was fair/good (61
(98.4%)).
From table 2, it was noted that the prevalence of dental caries was 53.2%, while the
prevalence of periodontal disease was 27.4%. Total overall GOHAI mean (SD) score was
recorded at 11.1 (5.50), in which the highest score was for question number 9 (1.9 (0.98))
and the lowest score was for question number 3, 4 and 6 (0.4 (0.64)).
The relationship between oral health status and oral health related quality of life was
shown in table 4. There is significant association between DMF score and quality of life
among respondents (p < 0.001). However, periodontal disease have no immediate effect
on quality of life among respondents (p=0.688).
Further analysis of association between caries experience and GOHAI score reveled that
question number 2 and 4 shows significant association (p < 0.05). However, when
controlling the confounding factors during regression analysis, question 4 and 10 shows
significant association (Table 5).
DISCUSSION
Quality of life is increasingly acknowledged worldwide as a significant and appropriate

indicator of oral health burden and also accurate indicator for dental service needs
23
[Gherunpong S, Ng SK]. A lot of study had been conducted to assess the impact of oral
diseases on one quality of life. However due to cross-culture difference, the association
are varied from population to population [Koposova et al, Goursand et al, Oscarson et al].
Dental caries is one of a factor which has influence in quality of life. These diseases
could cause difficulty in chewing, swallowing and speech. It could also disrupt sleep and
productivity by compromising the ability to work. They could affect the way a person
socializes, with significant impact on self-esteem, employment and general well being.
Caries is the most prevalence affliction to world population. Despite the knowledge that
caries is preventable, dental caries still continuously become a major public health
problem. According to Thylstrup, dental caries is multifactorial disease and a lot of factor
involve in dental caries formation and progression. This statement further strengthens the
finding of this study regarding caries experience distribution (Table 2). These finding can
be due to the origin of the students, their background and socio-economic status and also
present of dental health services.
World Health Organization has defined health as “a complete state of physical, mental,
and social well-being, and not just the absence of infirmity.” Lot of studies had shows
that oral health influences one quality of life. Oral health affects people physically and
psychologically and also contributes to people daily activity such as enjoy life, look,
speak, chew, taste food and socialize, as well as feelings of social well-being. A part from
that, study in developing countries shows that poor oral health also contributes towards
loss of productivity, and not to forget million hour loss of schooling day. It was reported
that severe caries will cause pain, discomfort, acute and chronic infections, and eating
and sleep disruption as well as higher risk of hospitalization, high treatment costs and
loss of school days with the consequently diminished ability to learn. The association
between the impact of dental caries and low quality of life as shown in other studies is
also constant with this study finding.
24
Results from this study shows that low GOHAI score was a common characteristic of a
subjects with high caries experience. Even though the distributions of the score are varied
across the respondents, question number 2 and 4 are the most bothering issues among
respondents. Its shows that majority of respondents feel that they have problem with
physical function which are trouble biting or chewing and unable to speak clearly.
Finding from this study was further supported by other studies which reported that
irrespective of socioeconomic position, dental caries experience causes respondents
reporting dental pain and oral problems during the last 3 months were more likely than
those without such problems [Kida IA et al, Masalu and Astrøm, Astrøm and Okullo].
It was also shown that caries experience seems to have serious consequences for social,
functional and psychological performances [Kijakazi et al]. Kijakazi reported that, dental
caries can cause a problem in eating and cleaning but it was weakly associated with other
impairments. However, the characteristics of quality of life that an individual experience
are varied depending on different aspects of one perception [Kijakazi et al]. This
statement was consistent with finding from this study. This was further discussed by
Locker, which stated that the psychosocial impacts of oral disorders tend to vary from
individual to individual even though the severity of their clinical condition remains the
same [Locker].
Understanding dental need perceptions is important for the effective planning and
implementation of oral health care services; but this needs is not a significant finding
from this study. The result can be due to the fact that all dental students will be treated by
dental officer later on. This finding is obviously contradicted from other studies finding
where impaired OHRQoL was positively associated with perceived need for dental care,
indicating that of people's need for dental care [Astrøm and Kida, Jokovic and Locker].
However, result from this study shows that there is a significant association between
caries experience and appearance, which is strongly associated with perceived dental
25
treatment need among respondents. This finding was correlate by the finding by Locker .
Other finding from this study shows, student with orthodontic appliance have high
GOHAI score and high DMF score. Fixed appliances make oral hygiene practice difficult
even for the most motivated patients, and almost all of them experience some degree of
gingival inflammation. Study shows that gingival swelling and gingival recession are
common sequelae of orthodontic procedures. In addition to that, orthodontic appliances
also have the potential to damage the periodontal support of treated teeth. It is because
the alveolar bone loss occurs more often in orthodontic patients than others. The
orthodontic bands used in orthodontic treatment induce gingival inflammation is not
surprising, since bands are more plaque retentive and their margins are often placed
subgingivally. This finding had been published by Boyd and Baumrind, indicate that in
orthodontic treatment; plaque, bleeding tendency, and pocket depths were all significantly
greater in patient treated with banded teeth than using bonded ones.
CONCLUSION
Poor oral health status influences the quality of life among this study sample in varying
degrees. Respondents with poor oral health status have low quality of life with their main
concern was poor dental appearance and difficulty in function.
REFERENCES
1. Donald, L., Lee, R., Nucci, M., Grembowski, D., Jolles, C. Z. & Milgrom, P. (2006). Reducing oral health disparities: a
focus on social and cultural determinants. BMC Oral Health 6(Suppl 1), 6831-6.
2. Locker, D. (1988). Measuring oral health: A conceptual framework. Comm Dent Health, 5, 3-8.
3. Petersen, P. (2003a). Global framework convention on tobacco control: The implications for oral health. Comm Dent
Health, 20, 137–138.
4. Petersen, P. (2003b). The World Oral Health Report 2003: continuous improvement of oral health in the 21st century.
Comm Dent Oral Epidemiol, 31 (supp.1), 3-24.
5. WHO. (2005a). Caries Prevalence: DMFT and DMFS. [Online] [Accessed], Available from World Wide Web:
www.whocollab.od.mah.se/sicdata.html
6. MOH (2004) (Ed, MOH).
7. WHO. (1999a). Combating the tobacco epidemic. [Online] [Accessed 5 nov, 2006], Available from World Wide Web:
www.who.int/tobacco/resources/publications/tobaccocontrol_handbook.
8. WHO. (1999b, May/June). Global Tobacco Epidemic. [Online] [Accessed 5 nov, 2006], Available from World Wide Web:
26
www.who.int/whr/en
9. WHO. (1998). Oral Health Methods and Indices. [Online] [Accessed 5 nov, 2006], Available from World Wide Web:
http://whocollab.od.mah.se/expl/method.html.
10. Gherunpong S, Sheiham A, Tsakos G. A sociodental approach to assessing children’s oral health needs: integrating an oral
health-related quality of life (OHRQoL) measure into oral health service planning. Bull World Health Organ 2006; 84:36-
42.
11. Thylstrup A, Fejerskov O (1978). Clinical appearance of dental fluorosis in permanent teeth in relation to histologic
changes. Community Dent Oral Epidemiol6:315–328.
12. Ng SK, Leung WK. Oral health-related quality of life and periodontal status. Community Dent Oral Epidemiology
2006;34:114-22.
13. Kida IA, Astrøm AN, Strand GV, Masalu JR, Tsakos G: Psychometric properties and the prevalence, intensity and causes
of oral impacts on daily performances (OIDP) in a population of older Tanzanians. Health Quality of Life Outcomes 2006,
5:56.
14. Masalu JR, Astrøm AN: Applicability of an abbreviated version of the oral impacts on daily performances (OIDP) scale for
use among Tanzanian students. Community Dent Oral Epidemiology 2003, 31:7-14.
15. Astrøm AN, Okullo I: Validity and reliability of the Oral Impacts on Daily Performance (OIDP) frequency scale: A
16. Cross-sectional study of adolescents in Uganda. BMC Oral health 2003, 3:5.
17. Locker D: Concepts of oral health, disease and the quality of life In: Slade G, ed. Measuring oral health and quality of life.
In Dental Ecology Chapell Hill: University of North Carolina; 1997:11-23.
18. Åstrøm AN, Kida IA: Perceived dental treatment need among older Tanzanian adults- A cross-sectional study. BMC Oral
Health 2007, 7:9.
19. Jokovic A, Locker D: Dissatisfaction with oral health status in an older adult population. J Public Health Dent 1997, 57:40-
7.
27
PP3: THE EFFECT OF SOY MILK ON MANDIBLE BONE DENSITY IN RAT

MODEL STUDY
Devi Gunawan, Diana S. Djohan, Tamara Gladysia E, William Adi Santoso,

Lee Yan Ying
Objectives: By its intensity, exercise can be divided to light, moderate and high.
Light and moderate intensity exercise is categorized into aerobic exercise, while
high intensity exercise is categorized as anaerobic exercise. This activity can be
a long-term continuous light to moderate intensity exercise. Anaerobic activity is
high intensity exercise, which needs energy at once and cannot be done
continuously for a long duration of time. This activity needs break intervals.
Soybean milk is easy to obtain and has significant isoflavone content, which is
good for human body. Isoflavone can stimulate osteoblast functions in bones.
Isoflavone can be transformed into phytoestrogen, which stimulates the
osteoprotegrin receptors for activating the RANKL system in role of homeostasis
for bone remodeling process. Methods: 30 Wistar male rats divided into 3
groups: control, first treatment, second treatment with 10 rats each. In the control
group, rats got normal diet and no treatment. In the first and second treatment
groups, rats treated with 3-sets of anaerobic exercise in details: 1 set defined as
rats swam in the water with 75 cm height and laden with 13.5 grams loads,
followed with 3 minutes break. Before the treatments, rats were acclimated for
one week and trained twice before the real treatment, i.e. once in every two days
for eight weeks. Soybean milk was given to the first treatment group and water
for the second treatment group 3 ml for each rat. In the end of research, rats
were exterminated for soft tissue, mandible and femur bones removal for further
testing using densitometer. The data available in this research received from
analysis using ANOVA test. The test used for testing the significance of the
mandible and femur bone density data after they being swam and fed with
soybean milk. Result: There is a significant difference between the group treated
with soybean milk and with water (p < 0.05).Conclusion: The treatment using 3
ml soybean milk per day in Wistar rat shows an escalation in bone density and
proved to be preventive to further bone damaging.
INTRODUCTION
Generally exercise increases and maintains of bone mass. However, long duration
exercise and/or high intense exercise disturbs bone metabolism turnover, leading to bone
loss.1 The effect of pO2 on formation of osteoclasts, the cells responsible for bone
28
resorption.2 The exercise promptly induced metabolic acidosis around pH 7.2 with
increase of lactate about 10 fold as much as the base. These results suggest that a bout
intense anaerobic exercise induced metabolic acidosis, uncoupling of bone metabolism
with increased osteoclastic and unchanged osteoblastic activity and hypersecretion of
PBMC cytokines. Bone loss by prolonged severe anaerobic exercise might be caused by
hypersecretion of bone resorbing cytokines and metabolic uncoupling of bone.1
Blood pH is mainly buffered via the Formula system. Additional buffering is
contributed by the numerous histidine residues of hemoglobin and by plasma proteins.
Addition of CO2 to the system as a result of respiration causes an increase in H+
concentration (i.e., pH reduction) leaving the Formula concentration relatively unaltered.
If insufficient CO2 is expelled via the lungs, a respiratory acidosis results. Conversely,
addition of H+ to the system, for example as a result of the metabolism of sulfur-,
nitrogen-, and phosphorus-containing molecules, will decrease pH and reduce Formula
levels without altering the CO2 concentration much. Protons generated in this way,
together with associated waste anions, must be excreted via the kidneys to produce an
acidified urine; if insufficient H+ is eliminated, a metabolic acidosis results.3
A multitude of potential causes of systemic acidosis exist, in addition to renal and
respiratory disease. These include anaerobic exercise, gastroenteritis, excessive
consumption of protein or other acidifying substances, diabetes, anemia, AIDS, aging,
and the menopause. Acidosis can also arise locally as a result of growth factor or cytokine
stimulation of cell metabolism, vascular disease, ischemia, inflammation, infection,
tumors, wounds, and fractures. It should be borne in mind that although the pH of arterial
blood is normally close to 7.40, and that of venous blood ~7.36, the pH of the interstitial
fluid film bathing cells in tissues will generally be lower and subject to complex
gradients, depending on the metabolic activity of the cells, their distance from the nearest
capillary, and the quality of the microvasculature.3
Acidosis and osteoclast function. The negative impact of systemic acidosis on the
skeleton has long been known but was generally thought to result from physicochemical
dissolution of bone mineral, i.e., that the skeleton acted as a giant ion-exchange column
29
to buffer systemic acidosis in a passive manner. However, cell culture experiments

showed that protons exert a direct stimulatory effect on resorption pit formation by rat
osteoclasts cultured on bone slices. Similar acid-activation responses occur with avian
and human osteoclasts. Osteoclastic resorption in these simple in vitro systems involves
removal of both the mineral (mainly hydroxyapatite) and organic (mainly type 1
collagen) components of bone (or dentin) to create distinctive pits and trails similar to
those seen in vivo. These experiments show that osteoclasts are almost inactive at pH 7.4
and that bone resorption increases steeply as pH is reduced, reaching a plateau at about
pH 6.8. The sensitivity of osteoclasts to extracellular H+ is such that pH reductions of
≤0.1 unit can be sufficient to cause a doubling of resorption pit formation. This effect is
not subject to desensitization in longer-term cultures: acid-activated osteoclasts continue
to form resorption pits over periods of 7 d or more, amplifying the effects of modest pH
differences. Acidosis stimulates resorption in calvarial bone organ cultures similarly.
Furthermore, H+-stimulated Ca2+ release from calvaria is almost entirely osteoclast-
mediated, with only a small physicochemical component. These observations strongly
suggest that the effects of acidosis on bone loss in vivo are also osteoclast mediated.3
The responses of bone cells to pH changes constitute a homeostatic mechanism that helps
to maintain systemic acid-base balance. In acidosis, osteoclast resorptive activity is
increased, and the deposition of alkaline mineral in bone by osteoblasts is reduced, to
maximize the availability of hydroxyl ions in solution to buffer protons. Disruption of the
vascular supply to tissues is a key cause of local acidosis. This may occur, for example, in
tumors, inflammation, diabetes, wounds, and fractures and is accompanied by hypoxia.
Bone cells also show remarkable reciprocal responses to oxygen tension: in severe
hypoxia (PO2 < 1%), osteoclast formation and bone resorption are greatly increased,
whereas osteoblast growth, differentiation, and collagen production are curtailed. Diets or
drugs that shift acid-base balance in the alkaline direction may provide useful treatments
for bone loss disorders.3
One product that is considered to be useful in decreasing bone loss is
phytoesthrogen.4 The main consumable plant sources of phytoestrogens include
30
isoflavones and lignans found mainly in soybeans and flaxseed, respectively.5 Recent
discovery of ER-beta in osteoblast cells70 may explain the protective role of
phytoestrogens in bone loss, however, non-hormonal mechanisms have also been
suggested. It is well recognised that estrogens can down- or up-regulate the activity of
osteoclasts, thereby limiting the bone resorption8. However, the exact mechanism of
action is not clear. The phytoestrogens have a conservatory effect on calcium excretion.
Isoflavones have been proposed to inhibit activities of osteoclast-like cells by interfering
with tyrosine kinase activity of epidermal growth factor receptor protein. In vivo and in
vitro studies indicate that osteoclast formation and bone resorption are enhanced due to
the generation of free radicals. Since phytoestrogens have been reported to exert
antioxidant properties, they may reduce the rate of bone loss in postmenopausal women
partly by antioxidant effects. Considering animal and human data to date, phytoestrogens
appear to exert protective effect on bone. Ipriflavone (7-isopropoxy isoflavone) is a
synthetic isoflavone, having similar chemical structure to diadzein and genistein, and
former is one of its active metabolite. It has been reported to prevent bone loss in
postmenopausal women in doses of 600 mg/day and can be used as an alternative to HRT
in estrogen-deficient states.4
MATERIALS AND METHODS
This experiment was done in animal experimental unit of Airlangga University,

Indonesia. A total of 24 subjects were chosen based on the same species, gender, and age.
24 male Wistar rats around the age of twelve weeks and weighed around 150 gram were
chosen due to their hormonal balance compared to female Wistar rats. This experiment
needs 336 grams soymilk powder (here we used Melilea ®); 2240 ml aquadest; Ether
anaestheticus 10%; formalin 10%; scissors; 13.5 grams of load; two 90 centimeters
buckets; various hair dye; cotton; feeding tube size 8; 3 ml syringe; 30 cm sealed glass
box; surgical scissors; and DBM Sonic 1200 Ultrasound Digital Bone Measurement
31
product of IGEA S.p.A.

These 24 male Wistar rats divided into 3 groups: control, first treatment, second
treatment with 8 rats each. In the control group, rats got normal diet and no treatment. In
the first and second treatment groups, rats treated with 3-sets of anaerobic exercise in
details: 1 set defined as rats swam in the water with 75 cm height and laden with 13.5
grams load, followed with 3 minutes break. Before the treatments, rats were acclimated
for one week and trained twice before the real treatment, i.e. once in every two days for
eight weeks. Soybean milk was given to the first treatment group and water for the
second treatment group 3 ml for each rat. In the end of research, rats were anesthetized by
putting them one by one into a sealed glass box containing cotton with ether in it until
death. Then, the soft tissue, mandible and femur were removed by surgical scissors for
further testing using DBM Sonic 1200 Ultrasound Digital Bone Measurement. The data
available in this research received from analysis using a One Way ANOVA test. The test
used for testing the significance of the mandible and femur bone density data after they
being swam and fed with soybean milk.
RESULT
The results of the experiment are shown on Table 1.
Table 1. The experiment result of soymilk administration in rats bone density (in m/s).
Soft tissue Femur Mandible
Group Group Group Group Group Group Group Group Group
1 2 3 1 2 3 1 2 3
1 1400 1545 1797 1833 1431 1587 1479 1833 1833
2 1698 1525 1736 2044 1634 1985 1484 1876 1876
3 1672 1390 1708 1588 1320 1562 1472 1774 1774
4 1448 1511 1506 1544 1106 1520 1454 1602 1598
5 1457 1545 1710 1879 1431 1811 1151 1974 1974
6 1391 1516 1822 1588 1320 1891 1261 1700 1693

1733 1551 1755 1584 1320 1618 1443 1699 1699
32
8 1753 1625 1480 2045 1632 1325 1423 1839 1839
Table 2. The standard deviation and mean of the result of soymilk administration in rats
bone density
Standard Deviation Mean
Tissue
Group 1 Group 2 Group 3 Group 1 Group 2 Group 3
Soft Tissue 158.309 65.433 127.554 1569 1526 1689.25
Femur 213.17 175.716 217.211 1763.125 1399.25 1662.375
Mandible 122.451 117.969 199.622 1395.875 1787.125 1785.75
Which are group one is control rats, group two is rats with water treatment, and
group three is rats with soymilk treatment.
33
34
The interpretation for soft tissue is that One-Sample Kolmogorov-Smirnov Test is normal
in distribution, means the samples are in the same range. It can be detected from the score
for Asymp. Significance (2-tailed) above 0.05 (p=0.65, p=0.537, p=0.432). After then, we
can continue to the second test, ie. homogeneity test. Homogeneity test shows that the
data are homogenous, because the score is below 0.05 (p=0.003). The next test is
ANOVA, and the result is normal under 0.05 (p=0.04). Afterward, the LSD test can be
calculated and can be interpreted as: there is difference between control group and
swimming with milk group (p=0.065) and control group and swimming without milk
group (p=0.493). It means that the soya milk therapy has an efficacy in increasing the soft
tissue density.
The interpretation for bone tissue are divided into two, ie. Femur and Mandible. The
Femur testing shows that One-Sample Kolmogorov-Smirnov Test is normal in
distribution, means the samples are in the same range. It can be detected from the score
for Asymp. Significance (2-tailed) above 0.05 (p=0.492, p=0.887, p=0.993). After then,
we can continue to the second test, ie. homogeneity test. Homogeneity test shows that the
data are not homogenous, because the score is above 0.05 (p=0.078). However, some
35
statistics experts have suggested that even though the data are not homogenous, the
ANOVA testing still can be performed, and the result is not normal, above 0.05
(p=0.389). Because the ANOVA test result is not normal, the LSD test cannot be
performed.
The mandible testing shows that One-Sample Kolmogorov-Smirnov Test is normal in

distribution, means the samples are in the same range. It can be detected from the score
for Asymp. Significance (2-tailed) above 0.05 (p=0.903, p=0.321, p=0.992). After then,
we can continue to the second test, ie. homogeneity test. Homogeneity test shows that the
data are heterogenous, because the score is above 0.05 (p=0.563). However, some
statistics experts have suggested that even though the data are not homogenous, the
ANOVA testing still can be performed, thus the result is normal, below 0.05 (p=0.000).
Afterward, the LSD test can be calculated and can be interpreted as: there is difference
between control group and swimming with milk group (p=0.000), swimming with milk
group and swimming without milk group (p=0.000). It means that the soya milk therapy
has an efficacy in increasing the bone tissue density.
DISCUSSION
The present study examined the effectiveness of soymilk in preventing bone loss
thus promoting bone density. According to the data, there is a significant difference of
bone tissues between each groups especially from mandible. On the other hand, the soft
tissues differ only slightly.
Soymilk, which can be obtained through processing soybean, contains flavonoids,
in particular isoflavons (including genistin, genistein, daidzin, and daidzein). Recent
studies have shown that isoflavons, together with menaquinone-7 (an analogue of vitamin
K2 stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption, thereby
increasing bone mass. Genistein and daidzein also have anabolic effect on rat bone
36
metabolism.8,9 Another important mineral is Zinc, an essential trace element that also
potentially stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption.
Zinc stimulates protein synthesis in osteoblastic cells and bone tissue culture system in
vitro, activating aminoacyl-tRNA synthetase.8,9 Recent studies show that oral admission
of zinc may prevent bone loss in rats. The combination of nutritional factors has
undetermined effect on bone components (whether synergistic or additive), however
recently it has been established that combination of zinc and genistein can produce a
synergistic effect on bone components using femoral tissue from female rats. Recent
study also shows that soy milk-based diets were shown to increase calcium absorption in
rats and the isoflavones in soy protein isolate were shown to prevent femoral and
vertebral bone loss in rats.6
Exercise is an important to promote bone health. Immobilization induces severe
negative calcium balance as it increases urinary calcium excretion in humans, accelerates
bone resorption and progressive bone loss while suppressing serum PTH and 1,25-
(OH)2D3. Moderate endurance induces positive calcium balance and has a beneficial
effect on bone metabolism. In addition, a combination of moderate-impact exercise and
adequate calcium intake can increase bone strength during childhood. However,
strenuous exercise leads to detrimental effects on calcium metabolism. It increases serum
PTH concentration, thereby resulting in decreased BMD and low bone mass.7
Our study focused on bone density in which 24 rats were divided into three
groups with one group as the control with normal diet while the other two received same
exercise with different diet (soymilk and normal diet). The exercises were done once
every two days while soymilk intake was given daily. This lasted for 8 weeks.
Densitometer was used in this experiment to measure both soft tissues and bone tissues.
The rats were initially drugged and remained alive when the soft tissue was determined.
However to extract the bone tissue, the rats were killed and both mandible and femoral
bone of each rats were taken to measure. The high measurement of mandible bone
density from the first group (swimming, soymilk diet) suggests that soymilk boosts bone
health. The result was different in femoral bone and tissue bone. Presumably, the
37
strenuous swimming has more impact to mandible bone.
CONCLUSION
The treatment using 3 ml soybean milk per day in Wistar rat shows an escalation
in bone density and proved to be preventive to further bone damaging. This escalation of
bone density is probably due to isoflavones agent, which have been proposed to inhibit
activities of osteoclast-like cells by interfering with tyrosine kinase activity of epidermal
growth factor receptor protein.
REFERENCE
1. Kim CS, Maekawa Y, Fujita M, et al. 2001. Disturbance of Bone Metabolism Turnover Induced By A Single Bout
Anaerobic Exercise in Young Males. Medicine & Science in Sports & Exercise: Volume 33 - Issue 5 - p S39. Available at:
http://journals.lww.com/acsm-
msse/Fulltext/2001/05001/Disturbance_of_Bone_Metabolism_Turnover_Induced_By.227.aspx
2. Utting JC, Flanagan AM, Burch AB, et al. 2010. Hypoxia Stimulates Osteoclast Formation from Human Peripheral Blood.
Cell Biochemistry and Function Volume 28 Issue 5, pp. 374 – 380. Available at:
http://www3.interscience.wiley.com/journal/123502862/abstract?CRETRY=1&SRETRY=0
3. Arnett TR. 2008. Extracellular pH Regulates Bone Cell Function. American Society for Nutrition J. Nutr. 138:415S-418S.
Available at: http://jn.nutrition.org/cgi/content/full/138/2/415S
4. Rishi RK. 2002. Phytoestrogens In Health And Illness. Indian Journal of Pharmacology 2002; 34: 311-320
5. Arjmandi BH. 2001. The Role of Phytoestrogens in the Prevention and Treatment of Osteoporosis in Ovarian Hormone
Deficiency. Journal of the American College of Nutrition, Vol. 20, No. 90005, 398S-402S (2001). Available at:
http://www.jacn.org/cgi/content/full/20/suppl_5/398S
6. Choi, MJ. 2004. Effect of Soy Protein and Exercise on Bone Mineral Density and Bone Mineral Content in Growing Male
Rats. Journal Community Nutrition, Vol. 6, pp. 48-54. Available at: http://image.campushomepage.com/users/nutritionrp/6-
1-7.pdf
7. Charoenphandhu, N. Physical Activity Affect Intestinal Calcium Absorption: A Perspective Review. 2007. Journal of Sports
Science and Technology Volume 7, No. 1 and 2, pp. 171-181. Available at: http://www.narattsys.com/press/JSST2007.pdf
8. Zhong JM, Shimanuki S, Iraghasi A, et al. 2000. Preventive Effect of Dietary Fermented Soybean on Bone Loss in
Ovariectomized Rats: Enhancement with Isoflavone and Zinc Supplementation. Journal of Health Science Vol. 46(4), pp.
263-268. Available at: http://jhs.pharm.or.jp/data/46(4)/46(4)p263.pdf
9. Yamaguchi M, Iraghasi A, Sakai M, et al. Prolonged Intake of Dietary Fermented Isoflavone-Rich Soybean Reinforce with
Zinc Affects Circulating Bone Biochemical Markers in Aged Individuals. 2005. Journal of Health Science Vol. 51(2), pp.
191-196. Available at: http://jhs.pharm.or.jp/data/51(2)/51_191.pdf
38
PP4: TEST OF INHIBITORY POWER OF ESSENTIAL OIL EXTRACT NUTMEG

SEED (MYRISTICA FRAGRANS) ON GROWTH OF STAPHYLOCOCCUS
AUREUS IN ANGULAR CHEILITIS
Novita Eka Lestari, Aisyah Bella Azzanjani, Ita Purnama Alwi
Angular Cheilitis is acute or chronic inflammation at the corner of the mouth.

Characterized by fisurs, cracks on the corner of the lips, reddish, ulcerated
accompanied by burning sensation, pain, and dryness in the mouth corners.
Many factors predispose to the occurrence of angular cheilitis, both locally and
systemically, one secondary infection may occur due to candida albicans,
bacteria of Streptococcus and staphylococcus aureus. Based on the literature we
found nutmeg seed contains volatile oil, saponin, myristicin, elemisi, lipase,
pectin, acid and lemonena oleanolat and there proanthocyanidins, cyaniding,
flavonols, quercetin and kaempferol. Addition glyceride, proteins, fats and
glucose. Nutmeg seeds contain flavonoids that have antibacterial effects, and
therefore the availability of nutmeg seed is expected to be utilized as a valuable
natural resource, especially in the field of dentistry. The objectives this research
was conducted to determine the inhibitory power of nutmeg essential oil extract
(Myristica fragrans) on growth of Staphylococcus aureus in angular cheilitis. The
research is the experimental study quase experimental design, the location of
research at the Laboratory of Microbiology, Faculty of Dentistry Hasanuddin
University. The sample in this study is to staphylococcus aureus in 15 times
dilution, respectively - each 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,
25%, 50%, 75%, 100% with the test data analysis anova one-way. Overall, the
working procedures in this study consists of sterilization instruments, making the
medium, purified Staphylococcus aureus, Myristica fragrans dilutions of essential
oil, test the MIC (Minimum Inhibition Concentration) and inhibition zones.
Observations on the antibacterial activity of various concentrations of Myristica
fragrans performed after incubation for 24 hours at a temperature of 37ºC. This
research was conducted over 10 days and the results showed the lowest
concentration of essential oil from nutmeg seed extract can inhibit the growth of
staphylococcus aureus bacteria, known as the Minimum Inhibitory Concentration
(MIC), which aims to determine at what minimum concentration can inhibit
bacteria. The results obtained show that the concentration of volatile oil from
nutmeg seed extract is directly proportional to the resisting force against the
bacteria staphylococcus aureus in the angular cheilitis. The higher the volatile oil
of nutmeg Myristica fragrans contained, the larger of the inhibition of the
staphylococcus aureus bacteria. Test of LSD to see the magnitude of the
differences and determine the concentration has a significant difference between
some of the essential oil concentration of Myristica fragrans on the growth of
Staphylococcus aureus in angular cheilitis, so that the volatile oil from Myristica
fragrans can be combined as an alternative material to cure angular cheilitis.
39
Content of essential oil is effective for treating diseases Angular cheilitis is 50% -
100%. Because the results of studies on the concentration of essential oil on
Myristica fragrans is inhibiting the growth of Staphylococcus aureus Based on
the research that has been done, it can be concluded that essential oil contained
in seeds of nutmeg (Myristica fragrans) proved effective in inhibiting the growth of
Staphylococcus aureus in angular cheilitis with the minimum concentration of
50%.
INTRODUCTION
Advances in technology and science simply are not able to eliminate the sense
tradisional.1 Treatment plants use nutritious medicine has become part of the world
community, traditional medicine is effective, efficient, safe, and economical. This is in
line with the call of the world health organization (WHO) with the movement of "Back to
Nature".2
Therefore one alternative treatment is to increase the use of medicinal plants
among masyarakat.3 efficacious role of traditional medicine in order for public health
services can enhanced efforts are required recognition, research, testing and development
of the efficacy and safety of a plant medicine.4
Information about the chemical constituents contained in the tissue or organ of the
type plants in the genus Myristica fragrans not been widely publicized. Nutmeg seed
containing 9% water, 27% carbohydrate, 6.5% protein, 33% mixture of oil, 4.5%
essential oils. Sheathing seeds also contain 22.5% oil mixture and more than 10%
essential oils. Seeds containing 23-30% butter and, if separated consists of 73% and 13%
oil trimyristin essensial.5
Based on the results of a research study conducted the National Science and
Technology Authority in his book, Guidebook on the proper use of medicinal plants,
contain nutmeg seed that chemical compounds are beneficial to health. Nutmeg seed has
very high content of essential oils, saponins, myristicin, elemisi, lipase, pectin, acid and
40
oleanolat lamonena. Furthermore, nutmeg is not cyanogenic or alkaloids, iridoids acres

acres detected.There also note proanthocyanidins, cyaniding, flavonols, kaempferol and
quercetin.6
Previous research showed that the nutmeg seed (Myristica fragrans) has
analgesic and antiinflammatory properties. Addition glyceride, proteins, fats and glucose.
Nutmeg seed contains oil vapor (such as eugenol, iso-eugenol and alcohol) that has been
known to have antiseptic properties which can inhibit the growth bacteri.7
Staphylococcus aureus is a pathogenic species of the genus Staphylococcus,
which is round-shaped bacteria, gram-positive, usually arranged in irregular series, such
as grapes fruit.8
Several studies have reported that the region anterior nares are the main places
where Staphhylococcus aureus can be found. Suzuki, et al reported that the oral cavity
can be a reservoir for Staphhylococcus aureus. Knighton reported the existence of
coagulase-positive Staphhylococcus in the oral cavity, in saliva of 47, 5% of the samples
studied, while Piochi and Zelonte Staphhylococcus aureus detects 35% of the samples of
saliva and confirmed as the reservoir of the oral cavity Staphhylococcus phatogenic.9
Staphylococcus aureus was found on angular cheilitis, apart from that there are
various causes of angular cheilitis, but we discuss here especially that caused by the
bacterium Staphylococcus aureus.10 Angular cheilitis is generally known by the cut on the
corner of our mouth at the confluence of facial skin and lips. Inflammation, burning,
redness and ulceration or gap is a characteristic problem of this disease. Affected areas
are often very painful and itchy. In advanced cases, the cracks in the skin and bleeding
when open the mouth.11
After the study, it appears the content of nutmeg seeds (Myristica fragrans) can function
as an anti-bacterial and many other benefits that can be recommended as alternative
treatments on Angular Cheilitis. Based on Angular Cheilitis disease etiologies mentioned
before we can joint the of the nutmeg seed and its benefits on Angular Cheilitis infectious
diseases, such as saponins are useful as a source of anti-bacterial and anti-virus, boost
the immune system, increase vitality, and reduce blood clotting.12
41
Flavonoide are useful as anti-inflammatory, anti-oxidants, and helps reduce pain

in case of bleeding.13 Polyphenols useful as anti-histamine (allergy) and anti oksidant.14
We know that the use of antibiotics in infectious diseases in the oral cavity as in the
Angular Cheilitis caused a tendency of growing number of Staphylococcus aureus.15 This
easy Microorganisms resistant to antibiotics and can be caused more infection.16
Therefore nutmeg seed availability is expected to be utilized as a natural ingredient for
treatment in the field of dentistry. This research was conducted to determine the
minimum inhibitory power of essential oil extract of nutmeg seed (Myristica fragrans) on
the growth of Staphylococcus aureus.
METHOD
Type of Research: Experimental Study with Experimental Design Quase,

Location of research: Laboratory of Microbiology, Faculty of Medical and Pharmacy
Laboratory University of Hasanuddin. Research Time: 10 days.
The Population is Pure cultures of Staphylococcus aureus and The sample in this
study is to extract nutmeg seeds (Myristica fragrans) in dilutions of 15 times each 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15 %, 25%, 50%, 75%, 100%.
Type of Data this research is Primary Data, Processing of Data is Computers
SPSS version 16, Presentation of Data in Table Shape, Data analysis of one way Anova
test.
The tools used include petri dish, Erlenmeyer flask, measuring cups, glass Becker,
tips, mikropipet, Loop of Henle, Bunsen, analytical balance, test tubes, autoclave,
incubator, slide, tube racks, gloves, masks, tweezers, aluminum foil. The materials used
include pure cultures of Staphylococcus aureus, extract the essential oil (Myristica
fragrans), nutrient agar, rubbing alcohol,sterile swab / sterile cotton sticks, NaCl, distilled
water.
Research criteria is Minimum Inhibitory Concentration Test (Minimum Inhibator
42
Concertration) is the lowest concentration that can inhibit the growth of Staphylococcus
aureus, characterized by condensation in the tube which contains the volatile oil and
culturing the bacteria seem clear, whereas the inhibition is of the inhibition zone is seen
through a broad zone inhibition zones were marked by the bacteria in culture medium
were measured using a shove.
The working procedures in this study comprised as follows: Sterilization
equipment All equipment used in this study are sterilized in an autoclave at a temperature
of 121 º C for 15 minutes in the following manner:
a. Petri dish and the tip is wrapped with paper mikropipet
b. Diluent vial covered with aluminum foil
c. Pumpkin measure covered with parchment paper and tied with rope
d. Erlenmeyer pumpkin filled with 250 ml distilled water and then covered
with cotton that has been compacted
3. Making medium Medium NA
20. Composition
Peptone from meat 5gr
Meat Extract 3gr
Agar 12gr
21. How to Make: Nutrients to be weighed using an analytical balance as much as
10 grams, then add 500 ml distilled water into the Erlenmeyer, sterilize with
an autoclave at a temperature of 121 º C for 15 minutes. Then pour into a
sterile plate, where each plate containing 15-20 ml, let it solidify and it is then
ready to be used.
4. Purification of Staphylococcus aureus
Purification was carried out to obtain the bacterium Staphylococcus aureus from pure
culture. Purification of Staphylococcus aureus working stages are as follows:
V. The Loop of Henle is heated above the burning lamp of methylated spirit. Then
the loop is inserted into the pure culture of Staphylococcus aureus, but before
dipping in into the culture, the loop is allowed to cool to feel the temperature on
43
the tube wall.

VI. Next, the loop is scrawled on pure cultures of microbes, then inserted into the dish
disk containing NA medium that had been prepared beforehand.
VII. Test tube containing the bacteria Staphylococcus aureus were incubated for 1 x 24
hours at a temperature of 37 º C.
5. Myristica fragrans dilution of essential oil

Dilution study aimed to produce some of the concentration of essential oil extracts of
Myristica fragrans that will be used to test the minimum inhibitory concentration or
MIC of Myristica fragrans extract which can inhibit the growth of Staphylococcus
aureus and inhibitory zone (zone of inhibition).
In this study, dilutions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 25%,
50%, 75%, 100% is used.
6. Test MIC (Minimum Inhibition Concentration) MIC test was conducted to
determine the minimum concentration that still can inhibit microbial growth.
Test Procedure:
1. Prepare 16 petri dish that has been sterilized in an autoclave for 15
minutes at a temperature of 121 º C, 15 dish is used to test the
concentration of extract of Myristica fragrans and one dish as a
control dish.
2. Each filled with 1 ml tubes cultured bacteria except the control tube,
and mix until homogeny with the medium. Then, the tube is inserted
with 1 ml of cinnamomun burmani solution that has been diluted.
Each concentration dilution tube is inserted into a bacterial culture.
3. All tubes are then kept in an autoclave at a temperature of 37 º C for
24 hours.
4. Minimum inhibitory concentration is determined based on the lowest
concentration of Myristica fragrans essential oil that can inhibit
bacterial growth marked by a clear and visible solution in the tube.
44
Turbidity indicates the existence of bacterial growth.

7. Inibisi Test Zone
3.0 Make dilutions of extracts of Myristica fragrans in various dilutions (1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 25%, 50%, 75% , 100%)
4.0 Create suspense Staphylococcus aureus in physiological NaCl solution
(0.9%) using a standard Mac Farland
5.0 Enter suspense NA bacteria in medium plate that previously had to be given
the drug disc
6.0 Add the extract of Myristica fragrans have been made into discs drugs on NA
medium
7.0 Incubation at 37 º C for 18-24 hours
RESULTS
The results of inhibition zones and MIC tests performed in the laboratory of
Microbiology, Faculty of Medicine University of Hasanuddin shows Myristica fragrans
is used to inhibit the growth of Staphylococcus aureus.
Observations on the antibacterial activity of various concentrations of Myristica
fragrans performed after incubation for 24 hours at a temperature of 37 º C. The MIC
tests and observations of the inhibition after 24 hours incubation period can be viewed at
the following table.
45
Figure 6. Power Test Results Myristica fragrans Inhibitory concentration of 10% -100%
Source: Primary data
13. Minimal Test Consentrasion inhibitors (MIC)
Table 1. Test observations from Myristica fragrans MIC after 24 hours incubation.
No Concentration Myristica Trial 1 Trial 2 Trial 3

fragrans essential oil (mm) (mm) (mm)
1 2 3
1 1% 0 0 0
2 2% 0 0 0
3 3% 0 0 0
4 4% 0 0 0
5 5% 0 0 0
6 6% 0 0 0
7 7% 0 0 0
8 8% 0 0 0
9 9% 0 0 0
10 10% 10 10 10
11 15% 12 12 12
12 25% 14 14 14
13 50% 15 15 15
14 75% 16 16 16
15 100% 18 18 18
Source: Primary Data
Table 1 shows that the concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%
and 9% in treatment 1, 2, and 3 there is no meaningful results were 0 mm, whereas at
a concentration of 10% is 10 mm, a concentration of 15% is 12 mm, a concentration
46
of 25% is 14 mm, a concentration of 50% is 15 mm, a concentration of 75% is 16

mm, and 100% concentration is 18 mm. The results in the table can also be known
Consentration Minimum inhibitory (MIC) of Myristica fragrans extract at
concentration of 10%.
14. Inhibitory zone / Zone Inhibition Myristica fragrans

Table 2. Test observations MBC / MIC Myristica fragrans for Staphylococcus aureus
after 24 hour incubation period.
No Concentration of Trial 1 Trial 2 Trial 3

Myristica fragrans (mm) (mm) (mm)
essential oil 1 2 3
1 1% Ineffective Ineffective Ineffective
10 10% Effective Effective Effective
Source: Primary Data
Description: 0 mm = does not inhibit bacterial growth
The table shows that the concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%
47
and 9% in treatment 1, 2, and 3 there is no meaningful results are not effective in

inhibiting Staphylococcus aureus bacteria growth, while at a concentration of 10%,
15%, 25%, 50%, 75%, 100% in treatment 1, 2, and 3 is effective in inhibiting the
growth of Staphylococcus aureus. From the results in the table can also note the
Minimum Inhibitory Concentration (MIC) of Myristica fragrans extract at
concentration of 10%. Overview of the results of this study can be seen in the
following chart:
Concentration of Extract Nutmeg Seed
(Myristica fragrans)
Inhibition of zone area
Figure 2. Relationship Between the Concentration of Extract Nutmeg Seed (Myristica fragrans) and
Inhibition of Zone Area.
Source : Primary Data
Table 3. One Way ANOVA Test Results for MBC / MIC
Concentration Mean Error Standart F Sig
Of Myristica
fragrans
0.1 5.00000 1.32453 1.317 33 0.000
0.15 10.00000 1.32453
0.25 20.00000 1.32453
0.5 45.00000 1.32453
0.75 70.00000 1.32453
1.00 95.00000 1.32453
48
In table 3 shows the value of F at a concentration of Myristica fragrans 1.31733

with significance of 0000 (sig. <0.1) means that there are significant differences between
various concentrations of Myristica fragrans in inhibiting the growth of Staphylococcus
aureus. Further test LSD (Least Significant Different) to see the magnitude of the
differences of various concentration. Results of LSD (Least Significant Different) to see
the magnitude of the differences of various concentration. Results of LSD (Least
Significant Different) can be seen in table 4.
Table 4. Test LSD (Least Significant Different) to TEST MBC / MIC
Trial 0.1 0.15 0.25 0.5 0.75 1.00
0.1 - 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽
0.15 0.000 ̽ - 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽
0.25 0.000 ̽ 0.000 ̽ - 0.000 ̽ 0.000 ̽ 0.000 ̽
0.5 0.000 ̽ 0.000 ̽ 0.000 ̽ - 0.000 ̽ 0.000 ̽
0.75 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽ - 0.000 ̽
1.00 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽ 0.000 ̽ -
̽ ignificant at P <0.05
De s cription: :S
In table 4, the results of LSD (Least Significant Different) test for zones of inhibition
showed significant differences in inhibiting the growth of Staphylococcus aureus, these
concentrations have a tendency to the same effect in inhibiting bacterial growth.
DISCUSSION
Research had been conducted in the laboratory of Microbiology Faculty of

Medical Hasanuddin University which has a goal to know the minimum inhibitory
essential oil extract of Myristica fragrans nutmeg on the growth of Staphylococcus
aureus in the Angular Cheilitis. Therefore, the results can be accounted for scientifically
based hypothesis on existing research previously that extracts essential oil can inhibit the
growth of Staphylococcus aureus.
This research was conducted over 10 days and the results showed the lowest
concentration of the extract of nutmeg essential oil can inhibit the growth of
49
Staphylococcus aureus, known as the Minimum Inhibitory Concentration (MIC), which

aims to determine at what minimum concentration can inhibit bacteria. Before conducting
the tests of the inhibition of essential oils of nutmeg seed (Myristica fragrans), the first
step is to test the MIC (Minimum Concentration inhibition) to determine the minimum
concentration of Myristica fragrans which can inhibit the bacteria Staphylococcus
aureus.
Based on the results from table 1 in the MIC tests (Minimum Concentration
Inhibition) we can see that the essential oil extract of Myristica fragrans has the ability
to inhibit the growth of Staphylococcus aureus, at concentrations starting from 10%,
15%, 25%, 50%, 75%, and 100% which inhibits the growth of Staphylococcus aureus.
Based on the results in table 2, on the inhibition zone test and inhibition zone
Myristica fragrans against Staphylococcus aureus after 24 hour incubation period, it can
be viewed that the average inhibitory region of Myristica fragrans essential oil extract in
a concentration of 10% is 10 mm, a concentration of 15% is 12 mm, a concentration
of 25% is 14 mm, 50% is 15 mm, 75% is 16 mm and 100% is 18 mm, whereas at a
concentration of 1% - 9% did not inhibit the bacterial growth.
The results obtained show that the concentration of volatile oil from nutmeg seed
extract is directly proportional to the resisting force against Staphylococcus aureus in the
angular cheilitis. The higher the volatile oil of nutmeg seed (Myristica fragrans)
contained, the larger the inhibition on the growth of Staphylococcus aureus.
LSD test is done to see the magnitude of the differences and determine the
concentrations that have significant difference. The result can be seen significant
differences between some of the essential oil concentration of Myristica fragrans on the
growth of Staphylococcus aureus in angular cheilitis, so that the volatile oil from
Myristica fragrans can be combined as an alternative material to cure angular cheilitis.
Nutmeg essential oil content is effective to treat angular cheilitis is 10% - 100%, because
the research results on the concentration of essential oils on Myristica fragrans is
inhibiting the growth of Staphylococcus aureus.
Based on the results of the inhibition test performed at least suggest that the
50
successful use of anti-microbial treatment depends activities to achieve inhibition or

destruction at the site of infection without causing significant toxic effects on hospes.
Minimal drug concentration achieved in the place of infection must be balanced with
minimum inhibitory concentration for the microorganisms that cause infections such as
Staphylococcus aureus.17
CONCLUSION
Based on the results of research conducted at the Laboratory of Medical
Microbiology, Hasanuddin University, it can be concluded that the essential oil
(Myristica fragrans) proved effective in inhibiting the growth of Staphylococcus aureus
in angular cheilitis disease with 10% concentration. This research also suggest to conduct
further research about the use of appropriate concentration and whether it is safe to use
essential miyak nutmeg seed (Myristica fragrans) in the field of dentistry and to consider
the use of extract of nutmeg seed (Myristica fragrans) against infectious diseases that one
factor causes the bacterium Staphylococcus aureus, especially on Angular Cheilitis.
REFERENCE
1. Biomedical and Pharmaceutical Research and Development Center. Native medicinal plants belong to the community and
the Nation State in 2007. Available from http://www.bmf.litbang.depkes.go.id [accessed on May 15, 2010]
2. [WHO] Word Health Organization.2004. Guidelines for Drinking-water Quality 3rd ed. Geneva: World Health
Organization.
3. AP Dharma. Traditional Medicinal Plants of Indonesia. Jakarta: PN Balai Pustaka, 1985. P 2-13.
4. Santoso, Sarjono O. Article: Use of Traditional Medicine in a Rational World in the Mirror of Medicine. Published by
Research and Development Centre Kalbe Farma. 1989.
5. Purwadakasi. Utilizing the yard for family medicine. Jakarta: PT. Conversely, in 2007. P 55.
6. Wijayakusuma. Drugs Indonesia nutritious plants: herbs, rhizomes and tubers. Sydney: Millennium popular.2001.p.13
7. Pelawi J. Franta 2009. Isolation of alkaloid compounds Nutmeg Seeds (Myristica fragrans Houtt), Thesis. USU. Medan.
8. Jawetz, E., Melnick, and Adelberg J., E. Medical Microbiology. Translation languages: Edi Nugroho, Maulany, Ed. 20.
Jakarta: EGC; 1996. P 211-12
9. Clelia A.P.M., C. Y. Koga-Ito, Sntonio Olavo Cardoso Jorge. Presence of Staphylococcus spp and Candida spp. In the
Human Oral Cavity. Branz. J. Microbiol. July / Sept. Vol.35 No 1-2 Sao Paulo.2002
10. Brooks, G.F., Butel, J.S., and Morse, S.A. Medical Microbiology. Jakarta: Salemba Medika; 2005. P 317, 318-2-0.
11. James, William D.; Berger, Timothy G., et al. (2006). Andrews' Diseases of the Skin: Clinical Dermatology. Saunders
Elsevier.
12. Pratama, Hadi Azis.2007. Studying with How to Determine Drying Characteristics of Water Balance and Fruit Drying
Constant (Phaleria macrocarpa [Scheff] Boerl), Thesis. IPB. Bogor
13. K. Hahlbrock 1981. Flavonoids in the Biochemisry of Plants, vol. 7: Secondary Plant Products. New York: Academic
51
Press. Page: 425-456

14. Arts, I. C. And P.C. Hollman, polyphenols and desease risk in epidemiologic studies. Am J Clin Nutr, 2005. 81 (1suppl):
p.3175-3255
15. Allen L. Honeyman, Herman Friedman, Mauro Bendinelli. Staphylococcus aureus: Infection Agents and Phatogenesis.
Kluwer Academic Publishers
16. Levinson, W. and Jawetz, E. Medical Microbiology and immunology Ed. 7th.Boston: McGrawHill, 2003. P 91.
17. Honeyman AL, Friedman H. Bendinelli M.2001. Staphlycoccus aureus Infection and Disease. New York: Plenum
Publishers.
52
PP5 : THE ANTI-INFLAMMATORY EFFECT OF CAHEW FRUIT EXTRACT ON

ARTIFICIAL EDEMA IN WISTAR RAT
Tiar Rennyka, Euis Mila Savista
Objective: To determine the anti-inflammatory effect of cashew fruit extract on

artificial edema in Wistar rat.Method: Fifteen male Wistar rats divided into five
groups, each consisting of three rats. In the treatment group, extract of cashew
fruit was per oral administered consecutively by various concentrations, which
were 2.5%, 5%, and 7.5%. In the negative control group, the aquadest was
administered in dose of 10 ml / kg BW and the 10 mg / kg BW sodium diclofenac
was administered in positive control. In order to induce the edema, one hour after
administration, in each group, hind paw of the rats were injected subcutaneously
with 1% carrageenin. The volume measurement performed with one hour interval
for 7 hours assessing with pletysmometer.Result: Rats treated with sodium
diclofenac (10 ml / kg BW) and cashew fruit extract (2,5% and 7.5%) showed no
significant reduction in percentage of paw edema volume. The area under curve
(AUC) of 2.5%, 5%, and 7.5% cashew fruit extract were equivalent to sodium
diclofenac 10 ml/kg BW. The % inhibition of paw edema of 2.5%, 5%, 7.5%
cashew fruit extract were respectively 61.3%, 40.53% and 64.78%. Conclusion:
2.5% and 7.5% cashew fruit extract have anti-inflammatory effect in Wistar rats.
INTRODUCTION
Anacardium occidentale L. of the family anacardiaceae is a multipurpose tree.

The leaves, stem bark, stem and fruits of cashew have been used as traditional medicine
for a long time in some countries. The plant is employed for various inflammatory
conditions such as arthritis. It is also used in fever, aches and pain, and asthma.1 In the
traditional Nigerian and Brazilian pharmacopoeia, stem bark of cashew is known for its
anti-inflammatory effect.2 The anti-inflammatory effect from the extract of the bark of
cashew is attributed to the presence of tannin, which has been shown to have an effect in
cases of acute and chronic inflammation.3
Tannin is also found in the fruit of cashew. The other compounds of cashew fruit
are flavonoid, anacardonic acid, vitamin C and vitamin A.4 Syawalia et al. (2009)
reported the ability of 2% cashew fruit extract to cure stomatitis on day 7 in clinical
53
applications. Cashew extract 2% is expected to impede or to stop the inflammatory

process that occurs in the oral cavity mucosa.5 Based on previous study, this study was
made to further provide evidence for the anti-inflammatory effect of cashew by using the
carrageenan-induced edema test.
Plant material – The specimens of the plant were submitted to the department of
pharmacy biology, University of Gadjah Mada and taxonomically identified and
authenticated by the experts (Voucher number: FA/BF/49/Ident/III/09).
Preparation of extract – The cashew fruits were dried at 450C for 24 hours, and coarsely
powdered. The coarse powder was macerated with 80% ethanol and stirred using
magnetic stirrer. The extract was filtered using a Buchner funnel. The filtrate thus
obtained was concentrated in rotary vacuum evaporator at 70 0C to obtain the extract. The
extract was made at concentration 2.5%, 5%, and 7.5% by adding aquadest as much as
needed.
Animals – The experimental animals used in this study were fifteen male Wistar albino
rats each weighing 130-200g and aged 2-3 months. Twenty-four hours before the
experiments they were fasting and were maintained only with water ad libitum. Ethical
approval for this study was obtained from the Research Ethics Committee of the dentistry
faculty, Gadjah Mada University.
Anti-inflammatory test
Screening for anti-inflammatory activity of cashew extract was done with a
carrageenan-induced paw edema model. The carrageenan-induced paw edema is a
suitable model to study acute local inflammation and widely considered to be one of the
most useful models in the evaluation of anti-inflammatory activity of investigational
compounds.
Fifteen experimental animals were randomly selected and divide into five groups
denoted as Group I, Group II, Group III, Group IV, and Group V, consisting of 3 rats in
each group. Each group received a particular treatment i.e. control, positive control, and
the extract (2.5%; 5%; 7,5%). Prior to any treatment, each rat was weighed properly and
54
the doses of the test samples and control materials were adjusted accordingly. Group III,
IV, and V received the crude extract orally at the concentration of, 2.5%; 5%; and 7.5%
respectively. Group II received oral administration of diclofenac sodium as standard anti-
inflammatory drug at a dose 40 mg/kg WB while Group I was kept as control giving
aquadest. All treatment were given orally at volume 10 ml/kg WB. Each animal received
an injection (0,1 ml) of carrageenan 1% in the sub-plantar region of the right hind paw 1
hour later. The paw volume measured before carrageenan injection and in every hour for
7 hours after carrageenan injection using a plethysmometer (ml). Each measurement was
repeated three times and then averaged. The edema volume was quantified by measuring
the difference in paw volume before the injection of carrageenan (V 0) and at the various
time points (Vt). The percentage of edema volume is calculated in accordance to
Mansjoer (1997)6,7 as follows:
% volume edema = 100%
Total edema volume for each treatment was calculated in arbitrary unit as the area under
the curve (AUC) and to determine the percentage inhibition for each treatment. The AUC
for each rat was calculated using the formula:
(Vtn – V0) + (V(tn+1) – V0)
AUCtn = x [ (tn+1) - tn]
tn+1
2
Where, tn = at time - n
AUCcontrol – AUCtreatment
% inhibition of inflammation = x 100%
AUCcontrol
Chemicals – Carrageenan (St.Louis, MO, USA) and diclofenac sodium.

Statistical analysis – The values are reported as mean ± standard deviation (SD). For
normally distributed data, difference in mean values between groups were analyzed by
55
one-way analysis of variance (ANOVA) followed by post-hoc Least Significance

Difference (LSD) tests for multiple comparisons. For data which was not normally
distributed (percentage of edema volume data), analysis data by Kruskal-Wallis tests
(non-parametric ANOVA) followed by the Mann-Whitney test for comparison between
two groups of subjects. P < 0.05 was considered statistically significant.
RESULTS
The percentage of edema volume values, calculated for each group of cashew
extract, diclofenac sodium, and control, are presented in Table I. The subcutaneous
injection of carrageenan produced a local edema that increased progressively to reach a
maximal 3 hours after the injection, except the group of diclofenac sodium and cashew
extract at concentration. The percentage of edema volume for all groups showed
reduction after 5 hours carrageenan injection (Fig. I).
Table I. Changes in edema volume (%) from 1-7 hours after carrageenan injection
following oral administration of aquadest, various concentration of cashew
extract (2.5%; 5%; 7.5%), and diclofenac sodium
Percentage of edema volume

Dose/ (Mean ± SD)
Treatment concentration
1 hr 2 hr 3 hr 4 hr 5 hr 6 hr 7 hr
10 ml/kg 17,12 ± 19,43 ± 32,64 ± 31,71 ± 21,67 ± 18,63 ± 14,80 ±
Control
WB 5,54 5,66 9,44 7,69 3,71 4,84 5,43
10,59 ± 10,61 ± 7,63 ± 9,87 ± 9,85 ± 8,28 ± 3,03 ±
Extract 2.5%
1,12 1,34* 3,66* 3,52* 5,25* 3,30* 1,31*
12,28 ± 14,59 ± 19,79 ± 15,24 ± 11,55 ± 12,21 ± 5,41 ±
Extract 5%
5,87 6,42 3,50 3,31* 6,62 3,06 4,79
8,38 ± 10,56 ± 10,63 ± 9,69 ± 6,86 ± 5,28 ± 1,52 ±
Extract 7.5%
6,07 3,23 5,34* 7,61* 6,16* 4,63* 1,32*
Diclofenac 40 mg/kg 5,85 ± 5,14 ± 7,29 ± 8,78 ± 11,69 ± 7,32 ± 5,12 ±
sodium WB 1,30* 3,43* 1,21* 3,84* 2,60* 4,62* 1,34*
Values are mean ± SD; n = 3; significantly difference from control group *p < 0.05
Kruskal-Wallis followed by Mann-Whitney tests
56
Figure 1. Percentage of paw edema volume exhibited by extract of cashew at various

time interval
The average AUC of edema volume in the extract group showed a significant
difference from control group, but was equivalent to the standard reference drug
diclofenac sodium (p > 0.05). The percentage inhibition of inflammation will be higher
when the AUC showed a low value (Table II). Extract of cashew at concentration 2.5%
and 7.5% inhibited the inflammation by 61.3% and 64.78% (Figure II). Extract of cashew
at concentration 5% also exhibited the inflammation inhibition although less than 50%.
Table II. The average AUC of edema volume after 7 hours and the percentage inhibition
of inflammation for each group
Dose/ AUC1-7 % Inhibition of

Treatment
Concentration ( Mean ± SD) Score Inflammation
Control 10 ml/kg WB 0,602 ± 0,123 -
Extract of cashew 2.5% 0,233 ± 0,036* 61,3
Extract of cashew 5% 0,358 ± 0,099* 40,53
Extract of cashew 7.5% 0,212 ± 0,102* 64,78
Diclofenac sodium 40 mg/kg WB 0,208 ± 0,058* 65,45
Values are mean ± SD; n = 3; significantly difference from control group *p < 0.01
One-way ANOVA followed by LSD tests
57
Figure II. Anti-inflammatory effect of the extract of cashew fruits on carrageenan-

induced rat paw edema in rats
DISCUSSION
Carrageenan-induced paw edema is a suitable experimental animal model for
evaluating an antiedematous effect. Edema developed following injection of carrageenan
serves as an index of acute inflammatory changes, was and can be determined from
differences in the paw volume measured immediately after carrageenan injection and then
every hour for 7 hours.8 Carrageenan is the phlogistic agent of choice for testing anti-
inflammatory drugs as it is not known to be antigenic and is devoid of apparent systemic
effects.9
In the carrageenan-induced paw edema test the development of edema
(inflammatory response) is a biphasic event with a maintence phase in between (2-3
hours): initial non phagocytic exudative inflammatory phase lasting up to 2 hours and a
delayed phagocytic inflammatory phase from 3-5 hours. The initial phase is primarily
mediated by histamine, serotonin and increase in prostaglandin synthesis in the
surroundings of the damaged tissue while the late phase is mediated by leukotrienes,
mobilized phagocytic cells, polymorphonuclear cells, monocytes, macrophages,
prostaglandins produced by tissue macrophages, oxygen free radicals, nitric oxide,
proteolytic enzymes and platelet activating factor.10 In this study, the extracts did not
58
show a significant effect in edema formation at the early phase but showed significant
effect at the later phases after 3 hours.
The inhibitory activity shown by the extract of cashew (2.5% and 7.5%) over a
period of 7 hr in carrageenan-induced paw inflammation was quite similar to that
observed in the group treated with diclofenac sodium. This anti-inflammatory effect of
the extract observed might be due to the presence of tannin and flavonoid in the plant.
According to Jeffers (2006)11, the role of tannin as anti-inflammatory is to inhibit the
production of prostaglandin E2 (PGE2), whereas flavonoids inhibit the lipoxygenase and
cyclooxygenase pathways of arachidonate metabolism.12 Another anti-inflammatory
property of flavonoid was also demonstrated by its ability to inhibit neutrophils
degranulation. This is a direct way to diminish the release arachidonic by neutrophils and
other immune cells.13 Therefore, it can be assumed that the inhibitory effect of the extract
of cashew on carrageenan-induced inflammation could be due to the inhibition of the
enzyme cyclooxygenase, leading to the inhibition of prostaglandin synthesis by tannin
and flavonoids.
Cyclooxygenase (COX) is an enzyme needed for the conversion of arachidonic
acid into prostaglandin. This enzyme has two isoforms of COX-I and COX-II. COX-I
represents a key enzyme in many cells and tissues, whereas COX-II was induced by
inflammatory stimuli so that the enzyme is not found in normal cells.14
Extract of cashew at concentration 2.5% and 7.5% exhibited the inhibitory
activity more than 50%, whereas at concentration 5% less than 50%. The difference in
the concentration of the extract is suspected in affecting the effectiveness of each anti-
inflammatory compounds that also indirectly affect the anti-inflammatory effect of
cashew extract. The highest percentage inhibition of inflammation was exhibited by
cashew extract at concentration 7.5%. This might be due to the higher concentration of an
extract, the higher active substances contained therein, thus more anti-inflammatory
substances and resulted better anti-inflammatory effect. In contrast, the inhibitory activity
extract of cashew at concentration 2.5% was better compared to concentration 5%. This
might be because of substance or compound that only produce a response at low
59
concentrations, so that if the concentration is increased, the compound will produce a

constant response or opposite response (aggravate inflammation).
Variance response mechanism of the body (biological variation) experimental
animals also suspected of affecting the result. According to Katzung (1997),15 the
relationship between drug dose (concentration of the extract) and the clinical response
can be quite complex, because the response of each individual/animal to a drug can vary
widely. These responses can be caused by genetic differences in drug metabolism or
immunological mechanisms.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. drg. Juni Handajani, M. Kes and drg. Heni
Susilowati, M. Kes, Ph.D from department of oral biology of Gadjah Mada University for
their advice and input to this study.
CONCLUSION
This study concluded that extract of cashew at concentration 2.5% and 7.5% has
anti-inflammatory properties on carrageenan-induced rat paw edema model.
The presence of one or more phytochemical constituents present in the extract
further study is warranted, for isolation of the constituents responsible for the activity and
also to explore the exact mechanism of action of the activity.
REFERENCES
1. Olajide OA, Aderogba MA, Adedapo ADA, Makinde JM. Effects of Anacardium occidentale stem bark extract on in vivo
inflammatory models. J Ethno pharmacol 2004; 95, 139-142
2. Tedong L, Dzeufiet PDD, Dimo T, Asongalem EA, Sokeng SN, Flejou J, Callard P, Kamtchouing P. Acute And Subchronic
Toxicity Of Anacardium Occidentale Linn (Anacardiaceae) Leaves Hexane Extract In Mice, Afr. J.
Traditional 2007; 4 (2): 140-147. Available at:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816447/pdf/AJT0402-0140.pdf. Accessed Aug 2, 2010
60
3. Pereira JV, Sampaio FC, Pereira M, Melo AFM, Higino JS, Carvalho A. In vitro Antimicrobial Activity of an Extract from
Anacardium occidentale Linn. On Streptococcus mitis, Streptococcus mutans and Streptococcus sanguis. Odontolgia. Clin.
Cientif 2006; 5 (2): 137-141
4. Raharni, Mutiatikum D, Saroni. Uji Antiiflamsi Ekstrak Buah Semu Jambu Mede
(Anacardium occidentale L) (Abstr.), Available at:http://www.litbang.depkes.go.id/risbinkes/Buku
%20laporan%20penelitian%201997-2006/26-uji_antiiflamsi_ekstrak_buah_sem.htm. Accessed May 6, 2009.
5. Syawalia ZA, Fadlilah R, Rennyka T, Werdiningsih NE, Ningsih JR. Pemberian Topikal Ekstrak Buah Jambu mete
(Anacardium occidentale L) Konsentrasi 2% pada Epitel mukosa Rongga Mulut Wanita Penderita Recurrent Apthous
Stomatitis, Laporan Penelitian Program Kreativitas Mahasiswa, Universitas Gadjah Mada, Yogyakarta, 2009. [Indonesian]
6. Mansjoer S. Efek Antiradang Minyak Atsiri Temu Putih (Curcuma Zedoria Rosc.) Terhadap Udem Buatan Pada Tikus
Putih Betina Galur Wistar. Majalah Farmasi Indonesia 1997; 8: 35-41. [Indonesian]
7. Hapsari HD, Handajani J, Tandelilin RTC. Efektivitas Ekstrak Etanol Buah Mengkudu sebagai Bahan Antiinflamasi pada
Tikus Wistar. Majalah Ilmiah Kedokteran Gigi 2006; 21(2): 60-68. [Indonesian]
8. Buadonpri W, Wichitnithad W, Rojsitthisak P, and Towiwat P. Synthetic Curcumin Inhibits Carrageenan-Induced Paw
Edema in Rats. J Health Res 2009; 23(1): 11-16.
9. Sudjarwo SA. The Potency of Piperine as Antiinflammatory and Analgesic, Folia Medica Indonesiana 2005; 41(3): 190-
194.
10. Ratnasooriya WD and Fernando TSP. Anti-inflammatory Activity of Sri Lankan Black Tea (Camellia sinensis L.) in rats,
Phcog Res [serial online] 2009; 1:11-20. Available at: http://www.phcogres.com/text.asp?2009/1/1/11/58142. Accessed
Aug 2, 2010.
11. Jeffers MD. Tannins As Anti-Innflammatory Agents. Thesis. Miami University Oxford: Ohio, 2006.
12. Ebadi M. Pharmacodynamic Basis of Herbal Medicine. CRC Press LLC: Florida, 2002: 393-395.
13. Nijveldt RJ, Nood EV, Hoorn DECV, Boelens PG, Norren KV, Leeuwen PAMV. Flavonoids: a review of probable
mechanisms of action and potential applications [online]. Available at: http://www.39kf.com/cooperate/qk/American-
Society-for Nutrition/017404/2008-12-28-550119.shtml. Accessed March 5, 2010.
14. Cheng Z, Nolan AM, and Mc Kellar QA. Measurement of Cyclooxygenase Inhibition in vivo: A Study of Two Non-
Steroidal Anti-Inflammatory Drugs in Sheep. Inflammation 1998; 22(4): 353-366.
15. Katzung BG. Farmakologi Dasar dan Klinik. 6th ed. EGC: Jakarta, 1997: 564-565.
61
PP6: SYNTHESIZE AND CHARACTERIZE THE YTTRIUM STABILIZED

ZIRCONIA (YSZ) AS DENTAL RESTORATION MATERIAL
Arifialda. A
Objectives:To get the nanoparticle size from Yttrium Stabilized Zirconia (YSZ)
on sol-gel method as new bioceramics in dental restoration
material.Methods:The precursor concentration (Zirconium Chloride/ZrCl 4) that
used in this research was 0.05 M and 0.01 M which allow the retention of
tetragonal structure and efficiently arrest crack propagation. 3% and 8% of
Yttrium Nitrate/ Y(NO3) added as stabilizer despite use pulp acasia as template.
This research involved the using of Scanning Electron Microscope (S.E.M) type
JSM – 6360 to characterize the visual grain size (particle size) also the
morphology of particle and X-ray Diffraction (XRD) to characterize the cristal
structure (structure phase) of Zirconia. Results:There are differences on visual
grain size between samples with precursor concentration 0.05 M and 0.01 M,
which grain 0,05 M has smaller particle size (14.2 nanometer) than 0,1 M (17.7
nanometer). The crystal structure that shaped are tetragonal and monoclinic. The
colour result is white opaque.Conclusion:It could be concluded from S.E.M
result table that the particle size depends on precursor concentration. As small as
precursor concentration, the particle size becomes smaller. The YSZ has greatly
stable tetragonal structure because of small particle size which is able to reduct
the free energy where can be forming. The good characteristic of YSZ is shape
memory ceramics where can be used to apply in posterior teeth with good
mechanical characteristic.
INTRODUCTION
As a current Indonesian mineral resource, zircon is very potential to be a basic

material of synthesis of partially stabilized zirconia (PSZ) that constitutes an important
component in advanced ceramics. In manufacture industry, we can find zirconia
extensively as a basic material in tile production and refractory material to be a layer of
melting furnace, kiln furnace, nozzles, crucible, as a component of sensory ceramics and
SOFC, healthy application (especially as heads for hydroxyapatite), as well as jewelries.
The variousness of the application of zirconia (ZrO2) is absolutely related to its specific
62
characteristics and its ability; for instance, it has a very high refractoriness, namely, about
2750° C for pure zirconia, its easiness to phase transform to result in mechanical
characteristics intended, its good ionic conductance as well as its easiness to be stabilized
by the other metal oxide to modify the physical, mechanical and chemical characteristics.
The existence of ZrSiO4 in Indonesia has been recognized since many years ago.
It can be found in water logged area Bangka-Belitung as alluvial sediment a long with tin
sand and other mineral resources. Furthermore, zircon is also existed along the watershed
of rural region in Central Kalimantan along with alluvial sediment of gold. Until the
beginning of year 2000, zircon from Bangka-Belitung was still considered as residue of
ore that did not have much attention but had given the good result, until now the use of
zirconia with high quality still depends on import products, where as the existence of
zircon (ZrSiO4) as a main source of zirconia has a big reserve potency. Zircon sand
(ZrSiO4) that is existed in an abundant amount in South Kalimantan presently has not
been used optimally as we can see in the mineral map distribution below.
This big potency of natural resource has not been used optimally to produce the products
that have value added and high utility and therefore have a certain up sell.
Because the abundance of zircon sand in Indonesia, it is important to develop the
63
function of zirconia in order to increase the value added of Indonesian mineral resources
both in bioceramics and especially in dental material. This condition is appropriate with
the research of the success of zirconia applications in orthopedic or implant that
encourages the practioners of dentistry to explore the possibility of the use of material
fulfill aesthetic aspects as support material (alloy substitute) or, in other words, if it is
viewed from its mechanical aspect, it has resemblance to metal alloy.
Nowadays, the need of the use of zirconia is significantly increasing along with
global era that emphasizes the aesthetic aspects in one side and has superiority in
mechanical aspects in another side. Traditionally, alumina has been used as implant or as
addition into dentifrice. Nevertheless, the experts of dentistry prefer to develop zirconia
compared to alumina because zirconia is stronger than alumina and the other bioceramics
besides it has a good resistance to fracture. Zirconia substitutes gold or stainless steel that
has traditionally been used in dentistry as a tooth mantle because it is more translucent
and transparent to x-ray. In recent years, zirconia has been used in cosmetics dentistry for
the purpose of aesthetic application. In addition, it is extensively selected as the
application of dental material. There are many researches to renew zirconia since the
invention of transformation toughening capabilities of zirconia in the middle of 1970.
Zirconia is one of bioceramics that has been introduced intensively in prosthetics to be
used as the material of crown, bridge or fixed partial denture because of its characteristic
that is inert to body liquid and aesthetic. Bioceramics as dental material must fulfill the
following requirements (1) has compression strength (2) wear and corrosion resistance
(3) can be polished (4) bioactive or inert, appropriate with the indication (5) fatigue
resistance (6) easy to sterilize (7) easy to fabrication, and (8) shape memory where the
material is able to return to the initial form after getting the pressure. From biomaterial
aspect zirconia constitutes a selected material because it fulfills the requirements of
protheses, namely, it must be bioactive, bionert and biocompatible where it is able to
adopt and integrate well with minimal adverse reaction.
There are some types of zirconia, that is; Tetragonal Zirconia Polycrystals (TZP),
Fully Stabilized Zirconia (FSZ) and Partially Stabilized Zirconia (PSZ) that constitutes a
64
type of zirconia used as dental material. As dental material, zirconia has good
characteristics with physical, mechanical, chemical and biological characteristics. From
the literature aspect, zirconia described as bioceramics material partially stabilized.
Accordingly, it needs the addition of single oxide in order to be absolutely stable in room
temperature by adding Yttrium Oxide (Y2O3), Calcium Oxide (CaO) or Magnesium
Oxide (MgO). The selected oxide in this research is Yttrium Oxide (Y 2O3), that is able to
retain the tetragonal structure and retain it, in order not to be monoclinic structure. In the
field of dental material, Yttrium Stabilized Zirconia (YSZ) is very potential to develop,
because it is stable both chemically and biologically besides it is very strong
mechanically with the toughness that reaches 1000 Mpa.
METHODS
The method of synthesis of Yttrium stabilized zirconia (YS2) that will be used in
this research is the method of sol gel with the purpose of obtaining nonparticles of YSZ.
It is important to process this material to be nanoparticles because it is smaller size of the
particle, the more active it is, then the genuine characteristics will be emerged, namely,
electrical, magnetical and mechanical characteristics. In addition, the process will be
more efficient because as smaller as particles, the process of temperature used can be
reduced. The method of sol gel is a process of producing the advanced ceramics
constituing bottom-up process that involves the formation of colloid (sol) and the
formation of polymer chains (gelation) from the sol in the medium (gel). In the process of
producing zirconia, precursor used is zirconium chloride (ZrCl 4). In this research, the
process of gelation will be substituted by the pulp of paper as template.
Process of Producing
Process of producing zirconia involves two steps, as follow:
8.0 Hydrolysis
The reaction of cutting metal tie by water that, then, forms a compound of hydroxide
65
metal.
Si(OR)4 + H2O  HO-Si(OR)3 + ROH
9.0 Condensation
The reaction polymerization hydroxide metal to form the bigger particle or molecule.
(OR)3Si-OH + HO-Si(OR)3 (OR)3Si-O-Si(OR)3 + H2O
or
(OR)3Si-OH + HO-Si(OR)3 (OR)3Si-O-Si(OR)3 + ROH
The Processing Methods of The Yttrium Stabilized Zirconia Grain
Precursor AQUA
BIDESTILATA
ZrCl4
Mixing and Aging for 2

days
Adding and mixing with pulp

acasia
Stirring continuing methods
Y(NO3)3.6H2O
Heating on temperatures 90-100oC

(1 days)
Heating on temperatures 1652oF/900oC in

order to get nanoparticles YSZ form
66
Get specimen with 3 point bending Characterizing

methods (XRD, SEM)
Bending test
The precursor concentration (Zirconium Chloride/ ZrCl4) that used in this research
was 0.05 M and 0.01 M which allow the retention of tetragonal structure and efficiently
arrest crack propagation. 3% and 8% of Yttrium Nitrate/ Y(NO 3) added as stabilizer
despite use pulp acasia as template. This research involved the using of Scanning
Electron Microscope (S.E.M) type JSM – 6360 to characterize the visual grain size
(particle size) also the morphology of particle and X-ray Diffraction (XRD) to
characterize the cristal structure (structure phase) of Zirconia.
67
Picture 1 SEM Result 20000X ZrCl4 0,1 M (17,7 nanometer)
Picture 2 XRD Result of ZrCl4 0,1 M
68
Picture 3 SEM Result 20000X ZrCl4 0,05 M (14,2 nanometer)
Picture 4 XRD Result of ZrCl4 0,05 M
69
RESULTS
There are differences on visual grain size between samples with precursor concentration
0.05 M and 0.1 M, which grain 0,05 M has smaller particle size (14.2 nanometer) than
0,1 M (17.7 nanometer). The crystal structure that shaped are tetragonal and monoclinic.
The colour result is white opaque.
CONCLUSION
It could be concluded from S.E.M result table that the particle size depends on precursor
concentration. As small as precursor concentration, the particle size becomes smaller. The
YSZ has greatly stable tetragonal structure because of small particle size which is able to
reduct the free energy where can be forming. The good characteristic of YSZ is shape
memory ceramics where can be used to apply in posterior teeth with good mechanical
characteristic.
REFERENCE
1. Park, J.B., Bronzino, J.D. 2003. Biomaterials: Principles and Applications. New York: CRC Press. 38, 41-42
2. Black, J., Hastings, G. 1998. Handbook of Biomaterial Properties. London:

Chapmann & Hall. 340, 343.
70
PP7: THE SIZES OF MAXILLARY ANTERIOR TEETH IN ASIAN AND ITS

CORRELATION WITH SELECTIVE CRANIOFACIAL ANTHROPOMETRIC
MEASUREMENTS
Renette Gan Siok Lynn, Karthiravan Purmal, Ngeow Wei Cheong, Yeoh Oon
Take
Selecting appropriate anterior teeth size becomes difficult when there are no pre-
extraction records of the missing natural teeth. Objective: This study determines
the sizes of maxillary anterior teeth in Asian, and investigates if selective
craniofacial anthropometric measurements could be used to predict anterior teeth
sizes. Material & Methods: This study was conducted on a group of Asian
subjects (mean age: 22.3 + 1.7 years; range 19-25 years) with Class I occlusion,
where direct measurements were made of the anterior maxillary teeth on stone
casts. Selective craniofacial anthropometric measurements were correlated to
the sizes of their anterior teeth. Results: The mesiodistal diameter of the
maxillary central incisor, lateral incisor and canine were 8.67 + 0.64 mm, 7.00 +
0.65 mm and 7.91 + 0.71 mm respectively. The anterior arch width, as
represented by the inter-canine cusp tip distance was 34.87 + 2.19 mm. Sex
differences in tooth sizes were seen in all anterior teeth. However, there were no
racial differences between 3 major races (Malay, Chinese and Indian) recruited.
The intercanine distance was similar to that reported for Singaporean Chinese,
and this was larger than Caucasian’s. The height, and facial, mandibular and
mouth widths of the subjects were significantly greater in males. The mandibular
width showed significant correlation with all anterior teeth concerned.
Conclusion: The maxillary anterior teeth of men were greater than those of
women in the Asian population studied. The lateral incisor and canine of Asian
males were larger than that reported for other races. The anterior arch width was
similar to that reported for Singaporean Chinese.
INTRODUCTION
Ideal selection and arrangement of anterior teeth in prosthodontics can be challenging in
the absence of pre-extraction records. In this aspect, the mesio distal width of the
anterior teeth is harder to determine compared to its height (also called tooth length). 1-3
The height of the anterior teeth can usually be determined according to the length of lip
at smiling and rest position.4
71
Although several authors have attempted to use biometric guide as an aid in the
positioning of the anterior teeth, there is still a lack of agreement in relating successive
width of the anterior teeth.3 5 6
This can be attributed to the fact that tooth size and
antropometric measure are race and gender specific.7 Table 1 provides a summary of the
size of the central incisor in various races.
Lombardi8 indicated that there is a recurring ratio in the width of successive teeth from
the central incisor. Lombardi also mentioned that central incisor should be larger than the
lateral incisor to dominate the composition and bring order and coherence. Levin 9 and
others10 11
expanded this concept to come up with proportions that was deemed
aesthetically pleasing in the arrangement of teeth. They used the Fibonacci Number of
1.618 (simplified in literatures as ‘the golden proportions’) as used in the ancient Greek
architecture and adapted it to suit the use in dentistry. If the same ratio between the width
of the central incisor and lateral incisor is repeated between the lateral incisor and the
amount of cuspid shown, and between the cuspid and bicuspid, each tooth size will be
different but related because of the repetition of the same ratio. This is bringing order by
organizing the elements according to a principle. 8 However there also has been
conflicting reports that pleasing arrangement of teeth does not necessarily follow the
golden proportions.12 13 For example Ward 12
believed that when the golden proportion is
used, the lateral incisor appears too narrow, and the resulting canine is not prevalent
enough. Preston14 reported that the golden proportion was found in the relationship
between the maxillary central and lateral incisors in only 17% of the casts of patients he
studied when viewed from the frontal.
Therefore the purpose of this study was to determine the width of maxillary anterior teeth
and maxillary intercanine width of adult Asians residing in Malaysia, and investigate if
selected craniofacial antropometric measurents could be used to predict the sizes of
anterior teeth. The existence of the golden proportion in the maxillary anterior teeth were
also explored.
72
Size of
Intercanine Findings
Author Year Sample Size Race central
width
incisor
The widths of the anterior teeth can be predicted
22 (male) 46 Malay by a combination of the interpupillary width,
Zakiah et al 2010 8.50 ± 0.50 NA
38 (female) 14 Chinese inner canthal distance, and inter alar distance.
The moulds available for artificial teeth were

Woodhead
1977 284 Caucasian 9.00±0.40 NA narrower then the width of incisor measured
C.M
Tooth size is highly correlated in Negroids as

Lavelle, 60 (male) 40 compared to Caucasians. Tooth size may be a
1972 8.79 ± 0.45 NA
C.L.B 60 (female) Caucasian factor in malocclusions.
40 Negroid 9.33±0.59 NA
40
8.67±0.42 NA
Mongoloids
Maxillary Arch dimensions and central incisor

100 widths of patients of Nigerian birth are in general
Mack P.J 1981 200 9.9 ± 0.74 37.32 ±3
Nigerians greater than those of British origin.
100 British 8.8 ± 0.66 34.68±2.49
Keng S.B 42.8 percent of the studied sample had maxillary

and Fong 1996 64 (male) Chinese 8.85±0.59 35.74±2.17 central mesiodistal diameters greater than 9 mm.
K.W.C 73
Table 1: The size of the central incisor in various studies.
MATERIAL AND METHODS
Sample
Selection criteria
The subjects consisted of convenient samples of students of the University of Malaya and
another nearby learning institution. They were recruited on a voluntary basis. The
subjects were generally healthy and exhibited no craniofacial abnormalities. Subjects of
mixed parentage were excluded from this study. Ninety four students volunteered for the
project, but 4 were excluded because of their prior orthodontic treatment. In addition,
damages to study cast resulted in exclusion of another 6 subjects. Therefore only 84
subjects were recruited into this study.
The inclusion criteria were:

1. Healthy state of gingival and periodontium
2. Full complement of teeth which is caries free from second molar to second molar
in both arches
3. No supernumerary teeth present
4. Normal molar and canine relationship (Angle Class I relationship)
5. Normal overjet (< 3 mm) and overbite (<4 mm)
6. No crossbite
7. Absence of spacing in the anterior teeth or spacing (<3 mm)
8. Minimal total arch crowding (<3 mm)
9. Minimal rotations
10. Minimal attrition
11. No prior orthodontic treatment
The exclusion criteria were:
74
1. Obvious diastema
2. Reverse curve of spee
3. Abnormal buccal or lingual tipping of teeth
4. Crossbite relationship
5. Peg-shaped lateral incisors or other anomalies
Collection of data
Ethical approval for the study was obtained from the Ethics Committee, Faculty of
Dentistry of the University of Malaya. Written consent was obtained from all subjects
who underwent examination and/or impression taking. Measurements were taken on each
subject on two areas:
i. On dental casts to obtain measurements of the width and coronal length of the
maxillary central incisors, lateral incisors and canine. In addition, intercanine distance
was obtained by measuring the distance between the cuspal tip of the left maxillary
canine and the right maxillary canine.
ii) Using the craniofacial landmarks to obtain selective anthropometric norms, i.e.
measurements of the facial framework of each subject.
All measurements were under taken by a single examiner.
i) Measurement of anterior teeth sizes

The maxillary central incisors, lateral incisors and canine sizes and intercanine distance
were obtained from a study cast made from each subjects. A dental impression was taken
from the subjects using alginate on a stock tray, and a cast is the poured using hard dental
stone. A Vernier Calliper (Least Count-0.02mms) was used for obtaining measurements.
ii) Measurement of the facial framework (Craniofacial anthropometric

measurements)
The measurements of the face and the mouth were carried out according to standard
methods of physical anthropometry developed by Farkas. Three measurements were
75
taken from the craniofacial region, namely:

22. Face width (zy-zy)
23. Mandible width (go-go)
24. Mouth width (ch-ch)
In addition, the height of the subjects was also obtained.
Error of method
A test-retest exercise was undertaken to ensure that the data collected were consistent and
accurate. Twenty random subjects and dental casts were re-measured 2 weeks following
the first measurement. The reproducibility of measurements was analysed using intraclass
coefficient correlation (SPSS, Chicago, USA) and the Bland and Altman plot for mean
percentage of difference (MedCal, Belgium). These findings indicate that the errors were
minimal and unlikely to bias the results.
Statistic Analysis
Data collected were entered into Statistical Package for Social Science statistical software
(Version 16.0; SPSS Inc, Chicago, Illinois, USA). The anterior teeth sizes and anterior
arch width, and craniofacial anthropometric measurements were recorded for each
subject to the nearest 0.01 and 0.1 mm respectively, and described in terms of average
values and standard deviations.
A t-test for independent samples was used to determine whether there was a significant
difference in tooth sizes and arch width values for males and females. Analyses of
variance (ANOVA) were used to compare the differences between the three races. Post-
Hoc tests with Tukey’s adjustment were undertaken to determine the races involved.
Correlations analysis was made between the anterior teeth widths and various
craniofacial anthropometric measurements. The significance value was set at 95%
(p<0.05).
76
1.8
1.6
1.4
1.2
1
Male
Ratio
Female
Ideal
0.8
0.6
0.4
0.2
0
Central to lateral ratio Lateral to canine ratio
Comparisons were made between the calculated ratios of central incisors and canines to
lateral incisors, with anticipated golden ratio for central incisor (1.618) and canine
(0.618) teeth using paired t-test. Similarly, comparison was also made between the incisor
to face width ratio and the ideal 1:16 for golden proportion.
16.2
16
15.8
15.6
15.4
Male
Ratio
Female
Ideal
15.2
15
14.8
14.6
14.4
Incisor to face width ratio
77
RESULTS
The subjects included consisted of 84 university students who volunteered for the study.
Their age ranged from 19 to 25 years, with a mean age of 22.3 + 1.7 years. Forty three
(n=43) of the subjects were male, with the remaining 41 female. Their ethnic/racial
distribution was 32 Malay, 28 Chinese and 24 Indian (Figure 1).
Figure1: Racial and gender distribution of subjects

18
16
14
12
10
Number
Male
Female
8
0
Malay Chinese Indian
Race
Sex differences in tooth sizes were seen in all anterior teeth with male exhibiting
significantly larger anterior teeth (Figure 2 and Figure 3). However, there was no racial
influence from the 3 major races recruited.
78
Figure 2: Mesio-distal width of anterior teeth according to gender

10
7
Mesio-distal width
Male
5
Female
0
Central incisor Lateral incisor Canine
Teeth
The mesio-distal widths of the anterior teeth are shown in Table 2. The average
mesiodistal diameter of the maxillary central incisor, lateral incisor and canine were 8.67
+ 0.64 mm, 7.00 + 0.65 mm and 7.91 + 0.71 mm respectively. In addition, the combined
incisor widths of male (32.21 + 1.97) was significantly greater than female (30.46 + 2.10)
(Independent t-test; P = 0.00).
Table 2: Mesio-distal width of anterior teeth
Tooth width (mm) Mean (SD) mm* Range (mm)
Central incisor Male 8.87 + 0.53 7.70 -- 10.10

Female 8.47 + 0.68 6.00 – 10.10
Combined 8.67 + 0.64 6.00 – 10.10
Lateral incisor Male 7.24 + 0.64 5.20 – 8.60

Female 6.76 + 0.58 5.50 – 7.80
Combined 7.00 + 0.65 5.20 – 8.60
Canine Male 8.21 + 0.54 7.00 – 9.60

Female 7.60 + 0.73 4.50** – 8.90
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Combined 7.91 + 0.71 4.50** – 9.60
* Independent t-test; P<0.05

The anterior arch width as represented by the inter-canine cusp tip distance was 34.87 +
2.19 mm (range = 27.4 to 39.7 mm). However, as there was gender dimorphism on this
distance, this was significantly larger in male (35.61 + 1.65 mm) than female (34.10 +
2.42 mm)(Independent t-test: P = 0.002)
The height, and facial, mandibular and mouth widths of the subjects were significantly
greater in males (Table 3).
Table 3: Selective anthropometric measurements

Anthropometric measurments Mean (SD)* Range
Height Male 170.58 + 8.71 cm 145.00-183.00 cm

Female 160.49 + 7.07 cm 145.00-176.00 cm
Combined 165.65 + 9.39 cm 145.00-183.00 cm
Face width Male 132.79 + 10.61mm 105.00-150.00 mm

Female 129.27+ 8.87 mm 98.00 -145.00 mm
Combined 131.07 + 9.90 mm 98.00 -150.00 mm
Mandibular width Male 108.58 + 8.51 mm 95.00-130.00 mm

Female 103.05 + 7.17 mm 90.00-120.00 mm
Combined 105.88 + 8.32 mm 90.00-130.00 mm
Mouth width Male 49.78 + 4.24 mm 37.69-56.00 mm

Female 46.55 + 3.33 mm 39.00-55.00 mm
Combined 48.20 + 4.13 mm 37.69-56.00 mm
* Independent t-test; P<0.05
There was a weak significant correlation between the canine width to all these
80
anthropometric measurements (Pearson correlation coefficient; P<0.05)(Table 4). The

mandibular width showed significant correlation with all anterior teeth concerned.
Further analysis using general linear measure however, showed no influence of

craniofacial anthropometric measurements on tooth sizes.
Table 4: Correlation between antropometric measurement, width of anterior teeth and
intercanice distance.
Width Central Lateral Canine Intercanine
Height Pearson 0.291 0.245 0.305 0.196

correlation
P value 0.007* 0.024* 0.005* 0.074
Facial width Pearson 0.187 0.200 0.418 0.261

correlation
P value 0.088 0.067 0.000* 0.017*
Mandibular Pearson 0.288 0.276 0.289 0.348

width correlation
P value 0.008* 0.011* 0.008* 0.001*
Mouth Pearson 0.123 0.185 0.310 0.238

width correlation
P value 0.266 0.093 0.004* 0.029*
Pearson correlation coefficient; *P<0.05
The existence of the ‘‘golden proportion’’ for the maxillary anterior teeth as a whole was
not found. Significant differences emerged when comparing the calculated ratios of
central incisors and canines to lateral incisors (1.242 and 0.889 respectively), with
81
anticipated golden ratio for central incisor (1.618) and canine (0.618) teeth (P<0.05).
The central incisors to lateral incisors ratio of Asian was significantly lower than the
proposed ideal golden proportion (Paired sample t-test; df= 83, P = 0.00), while their
lateral incisors to canines ratio was significantly higher than the proposed ideal golden
proportion (Paired sample t-test; df= 83, P = 0.00)(Figure 3).
Figure 3: The central incisors to lateral incisors ratio and lateral incisors to canines ratio
in comparison to the ideal golden proportion.
The central incisor to face width ratio of 1:15.2 was significantly lower than the ideal
1:16 for golden proportion (Paired sample t-test; df= 83, P = 0.00)(Figure 4).
Figure 4: The central incisor to face width ratio of subjects in comparison to the ideal
proportion.
Table 5: Fitted regression model and correlation (r) between width of anterior teeth,
intercanine distance and mandibular width.
Tooth Predicted width Correlation (r) P value
Right central incisor 6.54+0.02(MW) 0.27 0.01*
Left central incisor 6.21+0.02(MW) 0.29 0.01*
Right lateral incisor 5.14+0.02(MW) 0.22 0.05
Left lateral incisor 4.59+0.02(MW) 0.30 0.01*
Right canine 5.37+0.02(MW) 0.30 0.01*
Left canine 5.56+0.02(MW) 0.25 0.02*
Intercanine distance 25.15+0.10(MW) 0.35 0.01*
82
MW = Mandibular width
*= P < 0.05
There is a significant correlation between the upper anterior teeth, intercanine distance of
the upper arch and mandibular width (Table 5).
DISCUSSION
The Malaysian population consists of three main Asian races (Malay, Chinese and Indian)
with diverse craniofacial features.15 Although variation in tooth size is known to exist
between different racial groups,16 such a difference was not found to be significant among
the samples recruited, suggesting that Malaysians were more homogenous in relation to
tooth size than anticipated. This finding is consistent with that reported on Malaysian
Chinese and Malays.17 As a matter of fact, the difference in the width of central incisor
17
between our study and Isa et al is only 0.13 mm for central incisor, 0.09 for lateral
incisor and 0.03 for the canines respectively.
However, significant sexual dimorphism was noted to be present in the measurement of

tooth width. The males had significant larger width of incisors and canines compared to
the females. This is in agreement with all studies that measured tooth size. 18-21 The
intercanine width was significantly larger in the males, similar to that reported on
Singaporeans by Keng22 but in contrast with study by Hasanreisoglu et al 5. The
difference could be due to the diverse population that was studied by Hasanreisogulu et
al.
Malaysians were found to have wider anterior arch. This would have a tendency to result
in bimaxillary protrusion, just like the Singaporeans as reported by Lew and Keng. 23The
contributing factor to the wide anterior arch could be the lateral incisors and canines. This
83
is because, when using the Caucasian teeth sizes as standards, the Malaysian was found
to have smaller central incisor, wider laterals and canines. 24 As a result, this fact would
require us too, to rethink in the usage of the teeth straight from the standard mould. Asian
dentists treating Malay, Chinese or Indian patients can consider selecting teeth from
various group of moulds or establish moulds specially for their population, as suggested
by by Lew and Keng.23
The size, shape and arrangement of anterior teeth in particular the central incisor would
determine the harmony and aesthetic of a denture replacing the anterior teeth. 10 11 16 To
appear attractive the anterior teeth must be in harmony and proportion to the facial
morphology.11 Various guides have been used to select the size of the anterior teeth based
on anatomical landmarks. Example of some of the landmarks commonly used are
intercommisurral width,17 interpupilary width,17 interalar width,22 incanthal distance,25
bizygomatic (face) width,18 width of mouth26 and incisive papilla.27 Scandrett et al28 have
suggested that more than one anatomic reference was needed to predict the width of
anterior teeth. Because of that, this study investigated a few additional anatomic
landmarks that may be useful to help on the positioning of the maxillary teeth, by the use
of craniofacial anthropometry.
The anthropometric measurements of the male were found to be significantly higher than
the female because their stature is bigger as confirmed by the height. This is consistent to
that reported by Farkas.29
The correlations between three craniofacial anthropometric measurements, namely

height, facial width and mouth width with the sizes of anterior teeth were variable and
inconsistent. Only the mandibular width was found to consistently provide a significant
correlation with all the anterior teeth sizes and anterior arch size. The mandibular width is
a distance measured from goinon on the left to the gonion on the right according to
Farkas.29 This is a stable point and can be easily measured. From our search of literature
84
this is the first report of significant correlation of mandibular width to the anterior
incisors and intercanine width. Regression analysis shows the upper anterior teeth and
upper intercanine width is significantly correlated with the mandibular width. (Table 5).
Levin9 and others10 11 have mentioned that the golden proportion can be applied for the
arrangement of anterior teeth. The golden proportion shows that the perceived width of
the next anterior teeth is 62% smaller than the first anterior teeth. For example the lateral
incisor should be 62% smaller than the central incisor and the canine should be 62%
smaller than the lateral incisor. The finding of this study, however, indicated that the
golden proportion was not suitable for the arrangement of anterior teeth of Asians.
Significant differences emerged when comparing the calculated ratios of central incisors
and canines to lateral incisors (1.242 and 0.889 respectively), with anticipated golden
ratio for central incisor (1.618) and canine (0.618) teeth (p<0.05).
Golden proportion is related to aeshthetic pleasing smile rather than random selection of
Class I incisor as done in this study. In an aesthetic smile, the perceived dimension of the
anterior teeth when viewed from the front may follow the golden proportion. This is
because of the curvature of the arch and angulation of the anterior teeth which shows less
teeth distally. In this respect, the central incisor is the dominant teeth because it can be
seen in full size in frontal view. Because of this, this study evaluated if the width of this
central incisor was in golden proportion the face width. As with the evaluation for the
golden proportion of teeth, the result obtained did not provide the proportion advocated.
The central incisor to face width ratio of 1: 15.2 was significantly lower than the ideal 1:
16. One possible explanation of this finding could be because beauty and pleasing smile
is subjective. Asians with wider and proclined incisors with different skeletal structure
would not fit in the ideal 1:16 for golden proportions.
CONCLUSION
Within the limitation of this study, the following conclusions can be made.
85
1. The anterior teeth of male were wider compared to females in our population.
2. The mandibular width showed significant correlation with all anterior teeth
concerned.
3. The arrangement of the anterior teeth do not follow the golden proportion.
4. The central incisor width do not follow the golden proportion with the facial
width.
REFERENCES
1. McArthur DR. Determining approximate size of maxillary anterior artificial teeth when mandibular anterior teeth are present. Part I:
Size relationship. J Prosthet Dent 1985;53(2):216-8.
2. McArthur DR. Determination of approximate size of maxillary anterior denture teeth when mandibular anterior teeth are present.
Part II: Mold selection. J Prosthet Dent 1985;53(3):369-73.
3. Abdullah MA. Inner canthal distance and geometric progression as a predictor of maxillary central incisor width. J Prosthet Dent
2002;88(1):16-20.
4. Goncalves LC, Gomes VL, De Lima Lucas B, Monteiro SB. Correlation between the individual and the combined width of the six
maxillary anterior teeth. J Esthet Restor Dent 2009;21(3):182-91; discussion 192.
5. Hasanreisoglu U, Berksun S, Aras K, Arslan I. An analysis of maxillary anterior teeth: facial and dental proportions. J Prosthet
Dent 2005;94(6):530-8.
6. Gomes VL, Goncalves LC, do Prado CJ, Junior IL, de Lima Lucas B. Correlation between facial measurements and the mesiodistal
width of the maxillary anterior teeth. J Esthet Restor Dent 2006;18(4):196-205; discussion 205.
7. Johnson PF. Racial norms: esthetic and prosthodontic implications. J Prosthet Dent 1992;67(4):502-8.
8. Lombardi RE. The principles of visual perception and their clinical application to denture esthetics. J Prosthet Dent
1973;29(4):358-82.
9. Levin EI. Dental esthetics and the golden proportion. J Prosthet Dent 1978;40(3):244-52.
10. Ricketts RM. The biologic significance of the divine proportion and Fibonacci series. Am J Orthod 1982;81(5):351-70.
11. Marquardt SR. Dr. Stephen R. Marquardt on the Golden Decagon and human facial beauty. Interview by Dr. Gottlieb. J Clin
Orthod 2002;36(6):339-47.
12. Ward DH. Proportional smile design using the recurring esthetic dental (red) proportion. Dent Clin North Am 2001;45(1):143-54.
13. Rosenstiel SF, Ward DH, Rashid RG. Dentists' preferences of anterior tooth proportion--a web-based study. J Prosthodont
2000;9(3):123-36.
14. Preston JD. The golden proportion revisited. J Esthet Dent 1993;5(6):247-51.
15. Al-Junid ST. Craniofacial anthropometric norms of the Malaysian. University Malaya, 2005.
16. Brisman AS. Esthetics: a comparison of dentists' and patients' concepts. J Am Dent Assoc 1980;100(3):345-52.
17. Isa ZM, Tawfiq OF, Noor NM, Shamsudheen MI, Rijal OM. Regression methods to investigate the relationship between facial
measurements and widths of the maxillary anterior teeth. J Prosthet Dent 2010;103(3):182-8.
18. Gillen RJ, Schwartz RS, Hilton TJ, Evans DB. An analysis of selected normative tooth proportions. Int J Prosthodont
1994;7(5):410-7.
19. Sterrett JD, Oliver T, Robinson F, Fortson W, Knaak B, Russell CM. Width/length ratios of normal clinical crowns of the maxillary
anterior dentition in man. J Clin Periodontol 1999;26(3):153-7.
20. Sherfudhin H, Abdullah MA, Khan N. A cross-sectional study of canine dimorphism in establishing sex identity: comparison of
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two statistical methods. J Oral Rehabil 1996;23(9):627-31.

21. Owens EG, Goodacre CJ, Loh PL, Hanke G, Okamura M, Jo KH, et al. A multicenter interracial study of facial appearance. Part 2:
A comparison of intraoral parameters. Int J Prosthodont 2002;15(3):283-8.
22. Keng SB. Nasal width dimensions and anterior teeth in prosthodontics. Ann Acad Med Singapore 1986;15(3):311-4.
23. Lew KK, Keng SB. Anterior crown dimensions and relationship in an ethnic Chinese population with normal occlusions. Aust
Orthod J 1991;12(2):105-9.
24. Lysell L, Myrberg N. Mesiodistal tooth size in the deciduous and permanent dentitions. Eur J Orthod 1982;4(2):113-22.
25. Al Wazzan KA. The relationship between intercanthal dimension and the widths of maxillary anterior teeth. J Prosthet Dent
2001;86(6):608-12.
26. Latta GH, Jr., Weaver JR, Conkin JE. The relationship between the width of the mouth, interalar width, bizygomatic width, and
interpupillary distance in edentulous patients. J Prosthet Dent 1991;65(2):250-4.
27. Varjao FM, Nogueira SS, Filho JN. The center of the incisive papilla for the selection of complete denture maxillary anterior teeth
in 4 racial groups. Quintessence Int 2008;39(10):841-5.
28. Scandrett FR, Kerber PE, Umrigar ZR. A clinical evaluation of techniques to determine the combined width of the maxillary
anterior teeth and the maxillary central incisor. J Prosthet Dent 1982;48(1):15-22.
29. Farkas LG, editor. Antropometry of the Head and Face. 2 ed. New York: Raven Press, 1994.
87
PP8: INJURY TO THE ORAL CAVITY EXPERIENCED BY BRASS WIND

INSTRUMENT PLAYERS OF BANDUNG MARCHING BAND UNIT
Randita Diany Yordian, Sayed Mohamad Ridhwan, Puput Nurani

Objective : this research was conducted to gather subjective data on injury to
the oral cavity of brass wind instruments player who have been playing routinely
for more than a year based on etiology of injuries that has profound effect on
anatomy, vascular, and nervous system. Methods: This research is descriptive
retrospective base study and surveyed subjectively. Study was conducted on
Marching Band Gema Wibawa Mukti Unit, Sadaluhung Padjadjaran, and Waditra
Ganesha with random sampling technique. The data was collected using
purpose sampling technique through questionnaire of 132 brass wind instrument
player, age range 17–30 years, and have been playing routinely for more than a
year. Results : As per attached below are the result of the study on etiology of
injury. Injury caused by improper blowing or over blowing technique marked the
number of (58,33 %). On anatomical point of view, the injury is marked on the
lips fissure as much as (37,12 %). Injury to the vascular system show the present
of lips swelling (25,76 %). Injury on innervation system was marked by quivering
lips as high as (40,15 %). Conclusion: The verdict of this study has shown that
there are marked effect and produced injury to the oral cavity and oral mucosa of
brass wind instrument players who have been playing routinely for more than a
year with over blowing act and improper blowing technique.
INTRODUCTION
Brass wind instrument is a music instrument which made of metal elements (Kirnadi,
2004). Optimum practice is required to develop the ability to play brass. Optimum
practice includes the correct blowing technique, body position while blowing, song
material comprehensive, and blowing activity (Lewis, 2008). Injury in the oral cavity on
brass wind instrument players caused by excessive blowing activity (overuse) and bad
blowing technique (bad habits) (Lewis, 2008).
Brass wind instrument related to the oral cavity through an instrument part called
mouthpieces which contacted with the lips (Kirnadi, 2004). Placement of the lips, tongue,
and muscles around the mouth of the mouthpieces, causing a gap in the lip to drain the air
named embouchure (Harnum, 2008). Embouchure is an important factor in sound
production. It formed by the contraction of muscle expression of lip region, namely
88
expression of smiling (orbicularis oris muscle) and an expression of pucker (buccinators

muscle) (Campos, 2005). Playing wind instrument activities is an abnormal respiration
function. This condition has some similarities with mouth breathing activity.
Oral cavity injuries on brass wind instrument players are due to excessive blowing
activity of normal duration (overuse), and the technique of blowing the wrong habits (bad
habits). Bad habits in blowing activities include wrong mouthpieces placement, excessive
pressure of the mouthpieces on the lips, the wrong body position when blowing, and
cheek bulging activity (Campos, 2005; Lewis, 2008).
Injury to the oral cavity called embouchures overuse syndrome. Embouchures overuse
syndrome is a chronic injury of the forming embouchures muscle which experienced after
blowing excessive activity (overuse) and incorrect blowing habits (bad habits) (Lewis,
2008).
Initial clinical picture of the embouchure overuse syndrome in the oral cavity, consisting
of forming embouchure muscle fatigue, and discomfort when in contact with
mouthpieces. Clinical embouchure overuse syndromes in the oral cavity are classified
based on anatomical injuries, vascular injuries, and innervations injuries (Lewis, 2008).
Anatomical injuries are including the fissure lips, abrasion on the lips because the
mouthpieces, air flow, excessive pressure of the mouthpieces, and small cracks on the
lips. Vascular system injuries include bruises, swelling, and contact dermatitis.
Innervations injuries include muscle pain, numbness of the lips, spasm (stiffness), tremor,
and muscle cramps (Yeo, et al., 2002; Lewis, 2008; Ghoussoub, 2008).

MATERIALS:
1. Questioners forms (170 plies)
2. Stationery
3. Digital camera
METHODS:
89
This research is classified as descriptive retrospective research with subjective scope

survey techniques. On descriptive retrospective research with survey technique, the
research is directed to describe or analyze some condition in a commnunity or society by
trying to look back in the past (backward looking), which means the data gathering or
survey started from effect or result that has been happening. Afterward, from that effect
we can browse the cause or variable that affect the results. Commonly, research on
descriptive survey only answers "how" question (Notoatmodjo, 2005).
This research provides subjective description about oral cavity injuries on brass wind
instrument player who had undergone activity of routinely playing wind instrument more
than one year in Marching Band Gema Wibawa Mukti Unit, Sadaluhung Padjadjaran, and
Waditra Ganesha in Bandung City. The research population is brass wind
instrument players participated in Bandung Marching Band Units.
The criteria of the population is :
1. Male or Female brass wind instrument players.
2. Age between 17 – 30 year.
3. Routinely playing wind instrumet more than a year.
4. Have good health condition.
5. Didn't wear protesa atau orthodontic device.
The sample picking tehcnique that used for picking Marching Band activity unit in
Bandung City is simple random sampling technique. In simple random
sampling technique we have to determine first the amount of population that will be
getting sampled. Then take some part of the population by using random numbers table
(Sastroasmoro, dkk., 1995).
Sampling was done to determine 3 Marching Band activity unit from 10 Marching
Band activity unit that exist in Bandung regional by using random numbers table.
The Marching Band activity unit that selected are:

1. Sadaluhung Padjadjaran De Corps Padjadjaran University.
2. Marching Band Waditra Ganesha Unit in Bandung.
90
3. Marching Band Gema Wibawa Mukti Pemerintah Kota Bandung.
Research sample taken from population of Marching Band activity unit that selected
with purpose sampling technique. Purpose sampling method is sampling by including
all brass wind instrument player that fulfill the population criteria into the research at a
certain time range, so that the amount required for brass wind instrument player sample
can be fulfilled (Sastroasmoro, 1995).
The data was collected using a questionnaire form to be filled solely by the respondents
guided by the researcher. From the results of questionnaires interviews was being
conducted to respondents who had suffered soft tissue injuries.
All data obtained from the research are collected, recorded, processed and analyzed in a
simple, modified in percent and presented in tabular format to determine the size
frequency distribution frequency of the research respondents’ answers.
RESULT
Research on subjective description of the oral cavity injury in brass wind instrument
players in the Marching Band Gema Wibawa Mukti unit, Sadaluhung Padjadjaran, and
Waditra Ganesha was conducted in February-March 2010.
The study was conducted in 132 players brass instrument which meet the criteria of
population. Based on the total number there are 51 women (38.64 %) and 81 men
(61.36%). The sample is classified by type of brass instrument. The classification are
high brass 47 people (35.60%), middle brass 32 people (24.24%), and low brass 53
people (40.15%).
Table 1 shows the number and percentage of brass player, which is still in a routine and
non routine activity blowing brass instrument. Blowing routine activities with the criteria
practice more than one years, 2-4 days a week, daily exercise for 2,5-4 hours with a break
of 45 minutes–1 hour.
Table 1 Number and percentage blow brass instrument players who doing routine
activity and not doing routine activities
91
Information Amount of people Percentage (%)

Doing routine activity. 132 81.48
Not doing routine activities. 30 18.52
Amount 162 100
Aetiology Distribution Embouchure Overuse Syndrome Based Activities and

Habits Overstated Blowing Blowing Technique Wrong
In Table 2 below shows the distribution of embouchure overuse syndrome etiology in the
activities of Marching Band Gema Wibawa Mukti unit, Sadaluhung Padjadjaran and
Waditra Ganesha. Distribution of embouchure overuse syndrome etiology are classified
by only have overuse blowing activities, only have bad habits blowing activities, have
overuse blowing activities and bad habits blowing activities, and have no overuse
blowing activities and have no bad habits blowing activities. Results of research on the
number and percentage of brass wind instrument players who had experienced excessive
blowing activities with minimal or activity break blew exceeding the normal duration
with a never experienced excessive blowing activity can be seen in Table 2.
Table 2 Number and Percentage Players Musical Instrument Brass Blow By

Blowing who experience excessive activity (Overuse)
Have experienced excessive
94 71.21
blowing activities.
Never experienced excessive
38 28.79
blowing activities.
Amount 132 100
Excessive blowing activities mostly done by wind instrument players when it will be seen
in public (60%), when it wants to master the game of brass wind instruments (20.80%)
and when it wants to master the song material (19.20%) . (Table 3)
Table 3 Number and Percentage of etiology Players Musical Instrument Brass Blow
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By Blow who experience excessive activity (Overuse)

When we want to master the brass
26 * 20,80
brass playing techniques.
As will be seen in public. 75 * 60
When we want to master the song
24 * 19,20
material.
Amount 125 ** 100
* Total represents the total number of answers for each answer choice. A total of 94
person brass wind instrument players who had experienced excessive blowing
activities select more than one answer choice.
** The answer is not equal to the number of respondents.
Results of research on the number and percentage of brass wind instrument players who
have a habit of blowing the wrong technique (bad habits) and didnt have a habit of
blowing the wrong technique (bad habits) when blowing activity can be seen in Table 4
below.
Table 4 Number and Percentage Players Musical Instrument Brass Blow By

Blowing Technique's Wrong Habit (Bad Habits)
Have bad habits when
105 79.55
blowing activities.
Did not have bad habits
27 20.45
when blowing activities.
Amount 132 100
Technique of blowing the wrong habits (bad habits) on brass wind instrument players of
the most widely performed is always blowing activities with mouthpieces that are too
pressing lips (33.68%), then the wrong body position while blowing activity (26.94%),
93
put the mouthpieces are not in the middle of the lips (14.51%), using breathing chest
when blowing activity (13.99%), habit of blowing with his cheeks bulging (6.22%), and
the smallest is a form of cleft lip toogreater when in contact with mouthpieces
(4.66%). (Table 5)
Table 5 Number and Percentage Players Musical Instrument Brass Blow Based on
Various Kinds of False Blowing Technique Habit (Bad Habits)
Mouthpieces do not put the
28* 14.51
middle lip
Always blow with mouthpiece
65* 33.68
that are too pressing her lips
Using respiratory chest.
Form a gap of lip too large when 27* 13.99
contact with the mouthpieces.
9* 4.66
Habit of blowing with his cheeks
12* 6.22
bulging.
The wrong body while blowing
52* 26.94
activity.
Amount 193** 100
* Total represents the total number of answers for each answer choice. A total of
105 people brass horn players who never have a habit of blowing the wrong (bad
habits) choose more than one answer choice.
** The answer is not equal to the number of respondents.
Table 6 shows the distribution of etiologies of embouchure overuse syndrome in the

Echoes of activity units Marching Band Wibawa Mukti, Sadaluhung Padjadjaran and
Waditra Ganesha.
Distribution of embouchure overuse syndrome etiology are classified based only ever
94
experienced excessive blowing activity (overuse), just have a habit of blowing the wrong
(bad habits), had experienced excessive blowing activity (overuse) and has a habit of
blowing the wrong technique (bad habits), and never experienced excessive blowing
activity (overuse) and do not have a habit of blowing the wrong technique (bad habits).
Table 6 Distribution of embouchure overuse syndrome etiology in the activities of Gema

Wibawa Mukti Marching Band, Sadaluhung Padjadjaran and Waditra
Ganesha
Only ever experienced excessive
17 12.88
blowing activities.
Only have bad blowing habit. 28 21.21
Had experienced excessive
blowing activity and has bad 77 58.33
blowing habit.
Never experienced excessive
blowing activity or bad blowing 10 7.58
habits.
Amount 132 100
Table 7 Distribution table of Anatomical Injuries in Oral Cavity

Information A (people) B (people) C (people) D (people)
Split lip. 9 11 49 3
Lip abrations. 2 9 43 –
Pressure point
6 11 35 –
abrations.
Air induced abrations. 1 3 16 –
Lip divots. – – 1 –
Table 8 Distribution table of vascular injuries in oral cavity

Keterangan A (people) B (people) C (people) D (people)
Lip swelling. 1 1 34 –
Blue bruise spots on a 1 – 21 –
95
lip.
Contact dermatitis. 3 2 15 2
Table 9 Distribution table of Innervations injuries in oral cavity

Keterangan A (people) B (people) C (people) D (people)
Quivering lip. 11 12 53 5
Oral cavity muscles
9 9 51 –
spasms.
Thick rim sensation. 7 15 45 3
“Pin prick”
5 10 48 –
sensation.
Oral cavity muscles
5 3 32 –
cramp.
INFORMATION
A = Rarely experiencing excessive blowing activities.
B = Having bad habit on using the wrong blowing technique to play wind instrument
C = Rarely experiencing excessive blowing activities, and Having bad habit on using
the wrong blowing technique to play wind instrument
D = Never experiencing excessive blowing activities, and didnt have bad habit on
using the wrong blowing technique to play wind instrument
DISCUSSION
A descriptive study was conducted to examine the subjective description of the oral
cavity injured players brass wind instrument in Marching Band activities Reverberation
unit Wibawa Mukti, Sadaluhung Padjadjaran, and Waditra Ganesh who has been blowing
the routine for more than a year. Research was done by giving a questionnaire on brass
wind instrument players followed by an interview with the brass wind instrument players
who have had experienced of oral cavity injuries.
Reason for selecting the units of activity as the Marching Band research site because it is
96
an activity that combines a balance between the elements of art in the game of musical
instruments and sporting elements indicated by the movement, while in place and running
(Banoe, 1987). In addition, they are doing an exercise routinely, participating in the
championship Marching Band and other performances in public. Therefore, a brass wind
instrument players should be able to combine walking with movements in place while
accompanied by a brass wind instrument playing. To combine the two elements necessary
optimal exercise. Optimal exercise include the correct blowing technique, good body
position when the blow, over matter song, blowing a routine activity, and exercise
combines walking movements and movements in place, accompanied by the blowing
activities (Lewis, 2008). Excessive blowing activity (overuse) exceeds the normal
duration is not recommended because it is not useful and the possibility of things that are
not desirable, especially injuries will increase significantly (Giam and Tea, 1993).
Based on research results, shows that the most common etiology of oral cavity injuries on
brass wind instrument players excessive blowing activity (overuse) and bad blowing
habit (58.33%). Lewis (2008) said that the injury in the oral cavity occurs because the
players practiced excessively (overuse) and bad habits. Injury to the blower was named
embouchures overuse syndrome. Embouchures overuse syndrome is a chronic injuries
experienced by the muscle-forming activity embouchures after blowing excessively and
bad blowing habits (Lewis, 2008).
Excessive blowing activity happen to players when they about perform on public (60%)
and when they want to master the technique (20.21%), and want to master the song
(19.20%). The results are consistent with research conducted by Schuele and Lederman
(2004) towards 264.000 United States musicians who have attended the orchestra and
haven’t been in orchestra. Results showed that injuries happen to orchestra musician that
is equal to 76% (Heinan, 2008). Lewis (2008) suggested that the desire to perform
perfectly with brass instruments will make players practicing nonstop with minimal
breaks. the most common bad blowing habit that occurs in brass player are pressing the
lips too much (33.68%), wrong position (26.94%), using chest breathing
(13.99%),bulging cheecks (6.22%), and opene the lips too much when contact with the
97
mouthpieces (4,66%). This is consistent with that suggested by Campos (2005) that the
bad habits most often done by players is a brass wind instrument mouthpieces excessive
pressure on the lips, the wrong body position while blowing activity, and breathing
techniques using chest breathing. Mouthpieces pressure is one important thing in play.
Pressing mouthpieces to much to the lips to make lips opening become smaller, so it can
play high notes with ease, can cause embouchure overuse syndrome (Campos, 2005).
Anatomical injuries that most happend to players who had experienced excessive
blowing activity (overuse) and have bad blowing technique (bad habits) include the
fissures of the lips (split lip) 49 (37.12%), lips abrasion 43 people (32.57%), abrasion by
the pressure (pressure point abrasions) 35 people (26.51%) , abrasion by the air flow (air
induced abrasions) (12.12%) and small cracks on the lips while closing the mouth (lip
divots) that consisted of one person (0.75%).
Lewis (2008) suggested lips fissure has a side effect of decreasing the control of
embouchure muscle. Eembouchure muscle fatigue is caused by excessive embouchure
blowing activity and excessive pressure on the lips mouthpieces (bad habits), causing the
skin becomes dry and peeling lips (Lewis, 2008).
Lip abrasion is a mild symptom that occurs in brass player. This abrasion may soon
disappear, when the embouchure muscles have to function optimally (Lewis, 2008). Ellis
(2003) suggested that these injuries occur because of friction on the skin or mucosa of the
soft tissues oral cavity with instrument’s body (brass). Based on Ghoussoub’s research in
2008 towards 340 male wooden-brass player and brass in Lebanon, lip muscles abrasion
injuries occur as 7.8%. According to Lewis (2008), when a wind instrument played
continuously with mouthpieces that pressing lips too much will be formed thickening and
hardening. This is called abrasion by the pressure (pressure point abrasions). Lewis
(2008) suggested that abrasion by the flow of air (air induced abrasions) occurred on the
upper lip and lower lip which is split to remove the air. This happens when the blower
was playing high notes with the embouchure muscles fatigue so that the aperture as an
opening for air flow will be inadequate to contract. This contraction causes minimal lip
trembled so that when the blower must issue an air flow from the aperture into the
98
mouthpieces with high speed, can cause abrasion. A lip divot is a small cleft lip when his
mouth shut. Lip divots occur because of loss of control embouchure muscles due to
excessive activity and blowing mouthpieces excessive pressure on the lips (Lewis, 2008).
The most common injuries that happend to brass player are lip swelling ,34 people
(25.76%), bruising of the lips (blue bruise spots on lip), 21 people (15.90%), and contact
dermatitis, 15 people (11.36%). Etiology lip muscles caused by stress due to excessive
blowing activities (Lewis, 2008). Bruises caused by impact or blunt (brass) are directly
related to the area, causing skin bluish or blackish (Giam and Tea, 1993). Based on
research conducted by Gambichler, et al. (2004) towards 97 orchestra musicians, there
are three people who experience allergic to metal instruments. This is called allergic
contact dermatitis. The subject of research in the Marching Band activity unit that
experienced allergic to metal was changing the substance of mouthpieces in brass with
plastic (interview with brass wind instrument players, 2010).
The most common innervation injuries on brass player who had experienced excessive
blowing activity (overuse) and have bad blowing technique (bad habits) are ttremor
(quiver) in the lip muscle as much as 53 people (40.15%), muscle spasm of the oral
cavity of 51 people (38.64%), sharp pain sensation ("pin prick" sensation) as many as 48
people (36.36 %), sensation of a thickened rim mouthpieces (thick ream of sensation) as
many as 45 people (34.09%), and muscle cramps were 32 people (24.24%). Based on the
results of research conducted by Ghoussoub, et al (2008) towards 340 male’s wind
instrument musicians and brass timber players in Lebanon, injury tremors, cramps,
spasms in the muscles of the lips and oral cavity happend as much as 34.5%. Lewis
(2008) suggested that the thickened rim mouthpieces are effects of the pain and swelling
of the lips, so that the lips become numb. According to Lewis (2008), the sensation of
sharp pain felt by the upper and lower lips are in contact with mouthpieces that occurred
due to excessive blowing activities so that the embouchure muscle fatigue.
Thus there is a subjective description in the oral cavity brass wind instrument players
who had undergone a routine activity for more than a year blow to the etiology of brass
wind instrument players who have done blowing excessive activity (overuse) and has bad
99
blowing habit.
CONCLUSION
From the research of the oral cavity injuries on brass wind instrument players in
Marching Band Gema Wibawa Mukti, Sadaluhung Padjadjaran, and Waditra Ganesha, it
can be concluded as follows:
1. Aetiology of oral cavity injuries on brass wind instrument players is happening
commonly on players who had experienced excessive blowing activity (overuse)
and had a habit of wrong technique blowing (bad habits) (58.33%).
2. Excessive blowing activities mostly done by wind instrument players when they
are preparing shows and performing in public (59.84%).
3. The wrong habits blowing technique (bad habits) on brass wind instrument
players that most widely occured are always blowing activities with mouthpieces
that are too pressing his/her lips too much as 33.68%.
4. The common type of injuries related to the anatomy of the oral cavity is lip fissure
(split lip) of 37.12%. The common type of injuries related to the oral cavity
vascularities is lip swelling of 25.76%. The common type of injuries related to the
oral cavity is the neural tremor (quiver) in the muscles of the lips as much as
40.15%.
SUGGESTIONS
Advices from the research of the oral cavity injury on brass wind instrument players in
the Marching Band Gema Wibawa Mukti unit, Sadaluhung Padjadjaran, and Waditra
Ganesha, are as follows:
1. Each unit in Marching Band activities, brass players, coaches, and administrators
is expected to do injuries prevention efforts that may occur while doing activities
in the Marching Band, that can be done by doing warming up blowing exercise
with brass instrument properly, and if embouchure muscle fatigue happens, then it
is advised to take a break from blowing activities so that the risk of injury to the
oral cavity can be reduced.
100
2. Further research is needed about the side effects from excessive blow activity
(overuse) and the habit of using wrong blowing technique (bad habits) in order to
conduct long-term prevention of oral cavity injuries seriously.
3. Preventive measures to prevent adverse effects of the oral cavity on blower
performer at an early stage brass musical instrument lessons. Preventive measures
include: proper blowing technique, did not do over-blowing activities, and
designing optimal training according to players/performers ability.
REFERENCES
1. Banoe, P. 1987. Marching Band Indonesia. Jakarta: Lembaga Pendidikan Umum Suling Bambu. 7, 9-10, 21-22, 46-47, 58.
2. Campos, F. G. 2005. Trumpet Technique. New York: Oxford University Press, Inc. 30-135.
3. Ellis, E. 2003. Soft Tissue and Dentoalveolar Injuries. In: Peterson L. J.; E. Ellis; J. R. Hupp; et al. Contemporary Oral and
Maxillofacial Surgery. Fourth Edition. Missouri: Mosby, Inc. An Affiliate of Elsevier Science. 504-509.
4. Gambichler, T; S. Boms; and M. Freitag. 2004. Contact dermatitis and other skin conditions in instrumental musicians.
BioMed Central Dermatol 4(1):3.
5. Giam, C. K.; and K. C. Teh. 1993. Ilmu Kedokteran Olahraga Pedoman untuk Semua Orang. Translated by H. Satmoko.
Jakarta: Binarupa Aksara. 17, 187, 191-193.
6. Ghoussoub, M. S.; K. Ghoussoub; A. Chaaya; et al. 2008. Orofacial and hearing specific problems among 340 wind
instrumentalists in Lebanon. Le Journal Mèdical Libains (J. Med Liban) 56(3):159-167.
7. Harnum, J. 2008. All About Trumpet: A Fun and Simple Guide to Playing Trumpet. Wisconsin: Hal Leonard Corporation.
5, 8-9, 11-15, 18-28, 31-37, 48-55, 57-59, 85-97, 100-101.
8. Heinan, M. 2008. A review of the unique injuries sustained by musicians. Journal of American Academy of Physician
Assistants (JAAPA) 21(4):45-52. Available at http://media.haymarketmedia.com/documents/2/musician 0408_1280.pdf
(15 November 2009, 20.15).
9. Kirnadi. 2004. Pengetahuan Dasar Marching Band: Sumbangan untuk Komunitas Marching Band di Indonesia. Jakarta:
PT Citra Intirama. 1-7, 21-22, 43-48.
10. Lewis, L. 2008. Broken Embouchures: An Embouchure Handbook and Repair Guide for Brass Players Suffering from
Embouchure Problems Caused by Overuse, Injury, Medical/Dental Conditions, or Damaged Mechanics. Revised Edition.
New York: Oscar’s House Press. 2-123.
11. Notoatmodjo, S. 2005. Metodologi Penelitian Kesehatan. Edisi Revisi. Cetakan Ketiga. Jakarta: PT. Rineka Cipta. 26-27,
85, 88.
12. Sastroasmoro, S.; S. Ismael; A. R. Tumbelaka; dkk. 1995. Dasar-Dasar Metodologi Penelitian Klinis. Jakarta: Binarupa
Aksara. 45-46, 49, 55.
13. Yeo, D.K.L; T.P. Pham; and S. A. T. Porter. 2002. Specific orofacial problems experienced by musicians. Australian Dental
Journal 47(1):2-11. Available online at http://ada.org.au/App.../M28761_v1_632973754834191250.pdf (10 November
2009, 23.17).
101
PP9:BACTERICIDAL AND CYTOTOXIC EFFECTS OF Erythrina fusca

LEAVES AQUADEST EXTRACT
Timotius Andi Kadrianto, Nadya Saputri Halim, Melinia
Objectives: To investigate the bactericidal effect on Porphyromonas gingivalis

(P. gingivalis) and cytotoxic effect on fibroblast with various concentrations of
Erythrina fusca Leaves Aquadest Extract (EFLAE) in vitro. Methods: Pure P.
gingivalis was cultured in Brain Heart Infusion (BHI) medium for 24 hours
with/without various concentrations treatment of EFLAE. Observation, calculation
and statistical analysis of remaining bacteria were performed by Inhibitory Zone
method to evaluate EFLAE bactericidal effect and compared to chlorhexidine as
positive control. To evaluate the cytotoxic effect, NIH 3T3 cells (fibroblast) were
cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) containing 10%
Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin, pH 7.2, in 5% CO2,
37OC humidified incubator. Cells were treated with/without various concentrations
of EFLAE for 48 hours. The viable cells were then counted by 3-(4,5-
Dimethylthiazol-2-yl)-2,5 diphenyl tetrazodium bromide (M.T.T.) method. Results:
EFLAE have bactericidal effect on P. gingivalis in a concentration dependent
manner starting from 78%. The concentration of 90% EFLAE had stronger
bactericidal effect (35.004+1.546) than those in chlorhexidine as positive control
(32.313+1.619). One-way ANOVA showed significant bactericidal effect
differences among concentrations of EFLAE and Chlorhexidine (p < 0.05) while
Tuckey HSD test showed significant difference only between lower concentration
of EFLAE (78%, 79%) and chlorhexidine. With the highest concentration of
EFLAE (100%) applied in the bactericidal test, no cytotoxic effect of EFLAE on
NIH 3T3 cells was detected. Conclusion: EFLAE could inhibit the growth of P.
gingivalis in a concentration dependent manner, starting from 78%. There was no
evidence of EFLAE’s cytotoxic effect on fibroblast.
INTRODUCTION
Erythrina fusca (E. Fusca) is the most widespread species in the genus occurring
wild in both the Old and New World tropics. In Asia and Oceania it occurs along coats
and rivers from India to the Philippines, New Guinea, and Polynesia; in Africa it occurs
in Madagascar, The Mascarene Islands, The Comoro Island, and Pemba Island, but not in
Continental Africa. Furthermore in Central and Amazon basin and along coast of Brazil,
102
Colombia, up to Honduras and Guatemala; planted throughout the humid tropic. 1,2 E.
fusca is found from sea level up to 200 m altitude, within a wide range of rainfall pattern,
from 1,200 mm to over 3,000 mm annually, with or without seasonal distribution.
Average daily temperatures range from 16-24°C at the higher elevations to over 26°C in
the lowland. It seems to prefer littoral locations with badly drained soils like swamps and
stream banks and upland riverine marshes. In low-lying freshwater swamps E. fusca
attains huge dimensions and sometimes develops almost pure stands.1
E. fusca has many functions and been used by several countries; as in Indonesia,
the scraped inner bark is used for poulticing fresh wounds, and bark or root decoctions
are applied against beriberi. The grated wood is used to treat haematuria; the root is used
for rheumatism; bark and leaves serve as vermifuge. In Thailand, root, bark, and leaves
are used as an antipyretic. In Vietnam, the bark is used to treat toothache. The young
leaves are eaten as a vegetable in Java and Bali, as are the followers in Guatemala. In
Central America, the leaves are a source of animal fodder.3
The first compounds isolated from Erythrina were alkaloids. Subsequently,
homoerythrina alkaloids were investigated for their anti-cancer activity.4 Recently,
research involving Erythrina has focused on other chemical effects, primarily the
antimicrobial action of Erythrina lectins and the enzymology of proteinase inhibitors
isolated from Erythrina.5
Phytochemistry test on E. fusca leaves aquadest extract (EFLAE) in Balai
Penelitian Tanaman Obat dan Aromatik (BALITTRO), Bogor (2010), showed that
EFLAE contains alkaloid, glycoside, saponin, tanin, triterphenoid and steroid. Two
strongest compounds found in EFLAE were alkaloid and glycoside. Alkaloids have the
ability as anti-bacterial agent by disrupting the compiler’s components of peptidoglycan
bacterial cell. This cause the cell wall layer is not fully formed, then resulting apoptosis
of the cell.6 Tanin has also shown potential as antibacterial agent7,8 and other previous
research concluded that triterphenoid and saponin worked as antibacterial agent.9
The incidence of periodontal disease reached 70% in entire population of the
world, including Indonesia, especially in elderly. 10 Periodontal disease is an infectious-
103
typed disease which can be caused by local factor as well as systemic factor. Commonly,
the main cause of periodontal disease is local factor, which is caused by bacteria and
afterwards is aggravated with the existence of systemic factor. The main pathogenic
bacteria which cause the periodontal disease is Pophyromonas gingivalis (P. gingivalis),
This bacteria has the ability to infect the periodontal ligament; which in early stage starts
with infection of the gum (gingivitis) and continue to chronic infection which involve all
the periodontal ligament (periodontitis).11
These knowledge that afterward become the foundation for the implementation of
a scientific research about the bactericidal effect of EFLAE on P. gingivalis. After the
bactericidal test was conducted, to evaluate the cytotoxic effect, NIH3T3 cells (fibroblast)
were cultured; which fibroblast are one of the component in oral mucosa.12
The objectives of this research were to investigate the bactericidal effect on P.
gingivalis and cytotoxic effect on fibroblast with various concentrations of EFLAE in
vitro.
In this study, extraction of E. fusca leaves were performed by maseration

tehnique. E. fusca leaves (50mg) were dried for 5 days, grinded, diluted in Aquadest for
24 hours, refined, then evaporated with rotary evaporator 40OC.
Pure P.gingivalis was cultured in Brain Heart Infusion (BHI) medium for 24 hours
in 37OC humidified incubator, with/without various concentrations treatment.
Observation and calculation of remaining bacteria were performed by Inhibitory Zone
method to evaluate EFLAE bactericidal effect and compare to Chlorhexidine as positive
control. The results were then analyzed using One Way Anova with  0.05.
To evaluate the cytotoxic effect, NIH3T3 cells (fibroblast) were cultured in
100 L Dulbecco’s Modification of Eagle’s Medium (DMEM) containing 10% Fetal
Bovine Serum (FBS) and 1% Penicillin-Streptomycin using the 96 wells-plate, pH 7.2, in
5% CO2 37oC humidified incubator.13,14,15 Cells were treated with/without various
104
concentrations of EFLAE (0%, 10%, 100%) for 48 hours. The viable cells were then
counted by 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazodium bromide (M.T.T.)
method. This assay based on the changes of tetrazodium salt. M.T.T. will transmute
formazan in mitochondria. The formazan’s concentration, purple in colour, can be
determined by spectrophotometry. Formazan crystal which was formed, will dissolve
with the addition of acid isopropanol. The absorbance was then evaluated using Elisa
plate reader with   570nm. In this study, the cells absorbance was in liniar with
viability 16,17,18,19
RESULTS
In this study, EFLAE were obtained in gel form, brownish-green in colour and
solid consistency. P. gingivalis, which was cultured in BHI medium, after treated with
various concentrations of EFLAE showed bactericidal effect of EFLAE starting from
78%, having tendencies to increase and reached its peak on 80% (Fig. 1 and Fig. 2)
a. b. c.
105
d. e. f.
g.
Figure 1. Diameter bactericidal effect represented in Inhibitory Zone. Pure P.

gingivalis were cultured in BHI medium for 24 h with treatment of 78% (a), 79% (b),
80% (c), 90% (d), 100% (e), Chlorhexidine (f), Aquadest (g).
106
Figure 2. Diameter bactericidal effect of EFLAE in various concentrations;

Aquadest; Chlorhexidine
Aquadest as negative control did not show any bactericidal effect. EFLAE has
bactericidal effect on P. gingivalis in a concentration dependent manner starting from
78%. The concentration of 90% EFLAE had stronger bactericidal effect (35.004+1.546)
than those in chlorhexidine as positive control (32.313+1.619). The concentration of
100% and 80% EFLAE showed no significant difference with chlorhexidine as positive
control. One-way ANOVA showed significant bactericidal effect differences among
concentrations of EFLAE and Chlorhexidine (p < 0.05) while Tuckey HSD test showed
significant difference only between low concentration of EFLAE (78%, 79%) and
chlorhexidine.
In cytotoxic test, NIH3T3 cells were cultured in DMEM using 96 wells-plate for
24 hours in various quantity of cells. The result showed the absorbance of the cells; in
which a formula acquired (Fig 3.). The test was conducted triplo using ELISA plate
reader, with   570nm.
107
Figure 3. The formula which acquired from various quantity of cells (500, 1,000,
5,000, 10,000, 30,000).
NIH3T3 with the same quantity of cells (2000 cells) were followed by treatment
with EFLAE in various concentrations (0%, 10%, 100%) for 48 hours, 24 hours after the
first seeding. The result of this treatment, the absorbance of NIH3T3 cells were acquired
(Table 1.).
0% 10% 100%
Test 1 0.195 0.2253 0.2069
Test 2 0.2707 0.3317 0.2853

Test 3 0.2126 0.2438 0.2308
Table 1. The absorbance of NIH3T3 cells which were cultured in DMEM in 96

wells-plate for 24 h, followed by treatment after 48 h.
Then, the result of the cell’s absorbance was substituted to the formula to calculate
the number of viable cells (Table 2).
108
0% 10% 100%
Test 1 25371 29700 27457
Test 2 23856 30633 25478
Test 3 26467 31667 29500
Mean 25231 30667 27478
Standard Deviation 1311.19 983.757 2011.195
4
Mean / 10 2.5231 3.0667 2.7478
Standard Deviation/104 0.13112 0.09838 0.20112
Table 2. Viable cells with various concentrations of EFLAE after 3 days.
Number of NIH3T3 cells which were cultured in DMEM with/without various

concentrations of EFLAE showed that EFLAE did not induce cytotoxicity on NIH3T3
cells (Fig 4.). The number of NIH3T3 cells with 10% and 100% of EFLAE were slightly
higher than in control (0%) between the first day seeding and after three days (Fig 5.)
a. b. c.
Figure 4. EFLAE did not induce cytotoxicity on NIH3T3 cells. NIH3T3 cells were
cultured in 96 wells-plate for 24 h, followed by treatment of 0% (a), 10% (b) and 100%
(c) EFLAE for 48 h. Pictures were captured under light microscope. Black bar = 100mm.
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Figure 5. Number of NIH3T3 cells with various concentrations of EFLAE after 3

days.
DISCUSSION
The result of this study showed that 100% concentration of EFLAE has
bactericidal effect which is almost equal strength to those of Chlorhexidine moreover the
90% concentration of EFLAE had stronger bactericidal effect (35.004+1.546) than those
of chlorhexidine as positive control (32.313+1.619).
One-way ANOVA showed no significant bactericidal effect differences among
concentrations of EFLAE and Chlorhexidine (p < 0.05) while Tuckey HSD test showed
significant difference only between lower concentration of EFLAE (78%, 79%) and
chlorhexidine. It means that 80%, 90% and 100% of EFLAE has no differences or almost
equal bactericidal effect with those of Chlorhexidine. From this study, it can be concluded
that 80% of EFLAE is the optimum concentrations, which in the future can be used as
medication. This is because after 80% of EFLAE, there was no significant increase in
bactericidal effect.
Bactericidal effect of EFLAE, were suspected from some of the components
found in EFLAE based on the phytochemistry test of EFLAE, such as Alkaloid, Tannin,
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Saponin.7,8,9 Previous research by Robinson, 1991 stated that Alkaloid act as antibacterial
agent, by means of disrupting the compiler’s component of peptidoglycan bacterial cell
therefore the cell wall is not fully formed, resulting in apoptosis of the cell. The result of
this study can be concluded that Alkaloid which was found in EFLAE is suspected to
play a role in resulting the bactericidal effect on P. gingivalis. In order to ensure this
mechanism, further research need to be conducted.
After EFLAE’s bactericidal effect was found, cytotoxic test was conducted to test
EFLAE’s cytotoxic effect on human oral mucosa. The result of this test showed that with
the highest concentration of EFLAE that was tested for bactericidal effect, EFLAE did
not induce the growth of NIH3T3 cells (fibroblast). From this result, it can be concluded
that developing 80% EFLAE to a traditional herbs in gel state for periodontal disease,
will have minimal cytotoxic effect and urge the growth of the cells, resulting in maximum
recuperation and regeneration of cells in human oral mucosa.
CONCLUSION
EFLAE could inhibit the growth of P. gingivalis in a concentration dependent
manner, starting from 78%. There was no evidence of EFLAE’s cytotoxic effect on
fibroblast.
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1. Hanum F I and L.J.G van der Maesen. Plant Resources of South-East Asia : Auxiliary plants. Backhuys Publishers,
Leiden. 1997;11:121-23.
2. Marin, N B. Erythrina fusca Lour. Tropical Tree Seed Manual. Reforestation, Nurseries & Genetics Resources.
2003.- Online http://www.rngr.net/Publications/ttsm/Folder.2003-07-11.4726/PDF.2004-03-03.5508/file
3. Valkenburg JLCH and Bunyapraphatsara, N. Plant Resources of South-East Asia : Medicinal and Poisonous Plants 2.
Backhuys Publishers, Leiden. 2001; 12(2):252-53.
4. Kass D L. Erytrina Species – Pantropical Multipurpose Tree Legumes. Forage Tree Legumes in Tropical Agriculture.
Food and Agriculture Organization.- Online http://www.fao.org/ag/AGP/AGPC/doc/Publicat/Guttshel/x5556e0b.htm
5. Payne, L. The Alkaloids of Erythrina: Clonal Evaluation and Metabolic Fate. PhD Thesis, Department of Chemistry,
Louisiana State University, 1991; 160 pp.
6. Robinson, T. Kandungan Organic Tumbuhan Tingkat Tinggi (Organic Content of Plant). ITB, Bandung. 1991; 132-6.
7. Akiyama H, Fujii K, Yamasaki O, Oono T, Iwatsuki K. Antibacterial Action of Several Tannins Against
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Staphylococcus aureus. J. Antimicrob. Chemother. 2001;48(4): 487–91.
8. Funatogawa K, Hayashi S, Shimomura H, et al. Antibacterial Activity of Hydrolysable Tannins Derived From
Medicinal Plants Against Helicobacter pylori. Microbiol. Immunol. 2004;48(4): 251–61.
9. Yadava, RN, Jharbade J. New Antibacterial Triterpenoid Saponin from Lactuca scariola. Fitoterapia . 2008;
79(4):245-9.
10. Situmorang, N. Profil Penyakit Periodontal (Periodontal Disease Profile). Dentika Dental Journal. 2004; 9:71-77.
11. Agtini MD. Epidemiologi dan Etiologi Penyakit Periodontal (Epidemiology and Etiology of Periodontal Disease).
Cermin Dunia Kedokt (Medical World Image). 1991;72: 42-6
12. Oie Y, Hayashi R, Takagi R. A Novel Method of Culturing Human Oral Mucosal Epithelial Cell Sheet Using Post-
mitotic Human Dermal Fibroblast Feeder Cells and Modified Keratinocyte Culture Medium for Ocular Surface
Reconstruction. Br.J Ophthalmol. 2010 - Online
http://bjo.bmj.com/content/early/2010/06/09/bjo.2009.175042.abstract
13. Todaro GJ and Green H . Quantitative Studies of the Growth of Mouse Embryo Cells in Culture and Their
Development into Established Lines. J. Cell Biol. 1963; 17: 299-313.
14. Phelan M C. Basic Techniques for Mamalian Cell Tissue Culture. Current Protocols in Cell Biology. 1998; (1.1):1-
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15. Takashima A. Establishment of Fibroblast Cultures. Current Protocols in Cell Biology. 1998; (2.1): 1-12.
16. Campling B, Pym J, Galbraith PR, Cole SPC. Use of MTT Assay for rapid determination of chemo sensitivity of
human leukemic blast cells. Leukemia Research. 1988; 12:823-31.
17. Ciapetti G, Cenni R, Pratelli L, Pizzoferrato. In Vitro Evaluation of cell/biomaterial interaction by MTT Assay.
Biomaterials. 1992; 14:359-64.
18. Mosmann T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and
Cytotoxicity Assays. Journal of Immunological Methods. 1983; 65(1-2): 55–63
19. Shi Y, Kornovski BS, Savani R, Turley EA. A Rapid, Multiwall Colorimetric Assay for Chemotaxis. J Immun
Methods. 1993; 164:149-54.
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PP10: DENTAL SANTRI SCHOOL PROGRAM AS A SOLUTION FOR

INTEGRATED DENTAL HEALTH EDUCATION IN ISLAMIC BOARDING
SCHOOL
Aditya Mukti Setyaji, Renna Maulana Yunus, Ira Willyanti, Dhea Adittya
This research aimed to give awareness about dental health education by

innovative learning method and also create health cadres to be responsible to
the dental health of boarding school society. Boarding school has an important
role for national progress and development, especially for Muslims to be the next
generation leaders with moral integrity. Unfortunately, a boarding school is often
seen as a slum educational environment, poorly maintained, and far from the
health values, particularly on dental and oral health. Can be seen from our
sample, 18 of 21 santri had decays and only 2 of them have treated. Thus, is
needed an integrated educational programs between educational boarding
school and the dental health education that can improve the quality of dental
health in boarding school in order to create a caring environment and to respond
to oral and dental health. As a solution, we created Dental Santri School Program
(DENTSPRO) which applied in the boarding school. DENTSPRO contains three
interrelated methods to measure student ability to capture resources. There are
DENTS-Classical method as introduction, DENTS-Group (an interactive
discussion method which demonstrates the learning that is integrated), and
DENTS-Skills. DENTSPRO measure student abilities in three aspects. The
assessment is involved the cognitive which examined by giving pre-test to
DENTSPRO cadres. The affective is observed by the cadre’s motivation DENTS-
Group and DENT-Skills and the psychomotor are observed by the awareness of
dental and oral health of boarding school. After this program, their ability to know
about dental health is increasing rapidly, we can look their pre-mark and post-
mark knowledge, from 40,93% increases into 83,2%. It can be concluded that
DENTSPRO is succeeded in providing interpersonal communication learning
with the result the boarding school student paradigm about dental health
education is changed after they take part on DENTSPRO.
INTRODUCTION
Background of Problem
Boarding school is one institution that has an important role for national
development and progress, especially for Muslims. Create a cadre of boarding schools to
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participate - a nation that has the capability of cadres in the field of science and
technology and religious education to become a caliph or leader of the future later1.
But in fact, always seen as a boarding school educational environment slums,
poorly maintained, and far from the values of health. This was shown by the pattern of
daily life that ignores the students health, especially dental health. For example, in
general, they use toothbrushes together. This can lead to easy transfer between individual
bacteria and germs to each other individual that could result in ease of transmission of
various diseases. In addition the students rarely to clean his teeth twice a day as
recommended by your dentist causes plaque buildup, which can be of various diseases
resulted in the oral cavity.
Picture 1. Dental health condition in Islamic boarding school

Oral cavity is a gateway for entry of germs, so that if the oral cavity are not kept
clean will affect or aggravate health problems in other body parts. Such occurrence or the
severity of heart disease caused by the entry of germs go into the bloodstream.
The lack of knowledge about the importance of maintaining healthy teeth and
mouth as the major factor of health problems among dental, in students boarding school.
Religious education which has been in the boarding school curriculum has not been able
to form healthy lifestyle, so we need an integrated learning between religious education
and health sciences in general, especially the science of dental and oral health.
Objective of Program
This program provide to create a cadre of health, especially for dental health in
Islamic boarding school and local community as well. Also, to discover an innovative
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extension of media thet will be received well in boarding school beside as an additional
learning which integrates between dental and oral health sciences and religious sciences.
Required Output
Dental Santri School Program (DENTSPRO) is a study group with a device that
integrates learning of oral and dental health lessons with religious lessons in islamic
boarding school. In practice, DENTSPRO will be equipped with a guide which contains :
the basic curriculum, teaching methods, and reference knowledge about dental health.
Integrated system that will be developed in this DENTSPRO learning format that will
improve aspects of cognitive, psychomotor, and affective from the participants/students
/santri. Tutorial with a brief introduction to some references and an explanation of dental
and oral health in brief and general, which is an increase in cognitive devices. Next
tutorial will be followed by group discussion of learning who was accompanied by a
supervising tutor a student in each group to solve a common problem in the field of
dental and oral health are common among the people as the device increase in the
psychomotor and affective. Then proceed with the learning with proceed the their
application skills, which is also an increase in the psychmotor.
Expected output from DENTSPRO is created health cadres, particularly dental
and oral health are able to apply it in life in boarding school by integrating it with the
religious sciences.
Benefits of Programs
1. As an innovative new breakthrough in dental sciences, especially science
teaching oral and dental health in Islamic boarding school,
2. Creating a boarding school with a healthy and ideal to improve dental
health,
3. Removing the general paradigm regarding Islamic students (santri) living
patterns.
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General Description of Target Society

Boarding school is an institution of Islamic education which was introduced in
Java since 500 years ago. Islamic boarding school is a boarding school (Islamic boarding
school). The term itself is derived from Pondok Arabic (‫فندوق‬, funduuq), while the term is
derived from a boarding school-the students. So, students boarding school (santri) not
only learn, but also lived in a dormitory that had been provided. Boarding school led by a
kyai as a regulator of boarding school life, then kyai appoint a senior students to organize
their class brothers, they are usually called the village elder cottage or cabin management.
Pesantren education aims to deepen knowledge about the Qur'an and Sunnah, by learning
Arabic and the rules of Arabic grammar3,4,5.
Boarding School is the percentage of general education school subjects more
sciences of Islamic religious education than the general knowledge such as science, social
science, etc. Boarding school that teaches only Islamic religious knowledge only
boarding school, commonly known as salafi, as for a modern boarding school, a boarding
school which integrates between religious knowledge and science.
As a religious educational institution, the boarding school experience many
changes and plays various roles in Indonesian society, such as forming the paradigm of
religious life and social life. Boarding schools emphasize the values of simplicity,
sincerity, independence, and self-control. The students were separated from their parents
and families with the aim of forging themselves to be able to fulfill their duty as a Caliph
on earth4,5.
The students generally spend up to 20 hours a day with full of activities, starting
from tahajud prayers at 3 am until they go back to sleep at night. By day, the students
were learning science, which can be obtained from public schools, while in the evening,
they attended a recitation of the Kyai or their religious teacher, to further his religious
studies and the Qur'an.
Boarding school should be equipped with adequate health facilities. But in fact
there are many boarding schools that do not have health facilities. In order to avoid the
disease, one way that can be done is to maintain and preserve the environment and
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cleanliness of the boarding school students each so as not to disturb the teaching and
learning activities as well as other religious activities.
While the problem with the boarding school sanitation is the density of occupants
and ventilation in the bedroom area students, area ventilation and natural lighting in
classrooms, Waste Water Treatment, waste management, environmental sanitation
boarding school, and the lack of attention to the students as well as some party involved
in the importance of maintaining health have been a factor in the spread of various
diseases among students, including dental and oral diseases.
Using a toothbrush together is one example of the indifference / ignorance of the
students on the health of teeth and mouth. This can lead to easy transfer between
individual bacteria and germs to each other individuals, causing various diseases spread
easily. Not only that, the behavior before bed without brushing your teeth play an
important role in the occurrence of caries in the majority of students. Nutrition also plays
an important role in maintaining the resilience and strength of teeth. Provision of food at
several boarding schools in Indonesia are still not meet health standards. Nutrition has
also become an important part in forming and immune defense. So when someone
nutrients are lacking, then the disease germs and bacteria will be easy to attack a person's
body. In addition, the habit of students do not wash their hands before meals provide an
opportunity for germs and bacteria easy entry and lodged in the oral cavity.
The lack of health education in boarding schools became one of the many factors
of dental care problems, especially problems of caries in the boarding school. In some
boarding schools, the students still do not care about the lessons or general knowledge,
especially health. Fun and compliance with the students in the religious sciences is very
high. So sometimes they neglect the things that no religious overtones. Though
maintaining hygiene and dental health is one of the application of Islamic teachings 1.
With the existence of integrated learning in some students, which links between
religious education and particularly health education dental health will be formed cadre
of dental health and support healthy lifestyles in the boarding school with the basics of
the Qur'an and Hadith. In oral and dental health study is expected the students can
117
mengamalkannya as a form of healthy lifestyles and worship to Allah SWT 1.
METHODS
An intergrated program design in making concept of integrated learning raises
three learning and valuation methods to produce an optimal achievement. The
DENTSPRO has three kinds of interrelated methods and different assessment standards
to measure students ability to understand and feasibility of this learning program. The
first methods is called DENTS-Classical method, the method created a dynamic learning
concepts and the creation of two-way communication of introductory learning which help
students to learn about dental and oral education. The second method is DENTS-Group
discussion, the learning method that is managed by the facilitator in it and also managed
peers learning as a form of interaction and authentic assessment to demonstrate the
intergrated learning. The last methods is DENTS Skills fields method as the application
from the first and second methods.
Picture2. DENTSPRO’s method
In this methods can be used for the development of psychomotor skills and the
imagination of students. The cognitive assessment is the monitoring and evaluation of the
program which is done on a pre-test and post-test as cognitive reference students. And
affective assessment is the attitudes and behaviors that occur when DENTSPRO program
118
was held at their boarding school. While psychomotor assessment was taken by the
number or the accumulation of debris on the surface of dental students through
observation of the pre and post program.
RESULTS
Table 1. program’s result

Group I (control) Group II (DENTSPRO)
No
Cognitive Cognitive
Sample Pre Afectiv Psychomo Afectiv Psychomoto
Post Pre Post
e tor e r
test test test test
1 5 7 C 2,5 4 10 B 0,66
2 6 8 C 2,33 7 14 B 2,5
3 7 8 C 3,16 6 13 A 2,33
4 7 9 C 5 7 14 B 3,16
5 7 8 C 2,33 6 12 B 3
6 4 8 C 2 6 13 B 2,33
7 7 9 C 5,83 5 13 B 3
8 7 7 C 3,16 6 14 B 1,3
9 8 9 C 5 5 12 B 3
10 7 7 C 2,33 5 15 B 0,16
11 3 7 C 4,66 5 12 B 1,33
12 6 7 C 1,83 6 12 B 2
13 7 7 B 2,33 5 13 A 1,66
14 6 7 C 3,16 6 14 B 0,83
15 5 6 B 5 8 13 A 0,5
16 7 7 C 4,33 5 13 B 1,5
17 6 7 C 3,16 7 14 B 1,66
18 7 7 C 3 5 13 B 1,83
19 7 7 C 2,33 6 14 B 1,83
20 4 5 C 5 6 14 B 1,83
21 4 5 C 4,66 7 14 A 0,33
22 5 5 C 5,83 6 12 B 2
23 5 5 C 4,66 5 13 B 1,66
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24 4 4 C 5 5 12 B 0,83
25 5 5 C 2 6 11 B 0,5
26 6 4 C 4,16 5 11 B 1,5
27 6 5 C 2,5 6 12 B 1,66
28 3 4 C 5 6 13 AB 1,83
29 4 4 C 5,33 7 10 B 1,83
30 6 5 B 4,66 5 13 A 1,83
171 193 - 174 383 -
DISCUSSION
According to Green (1999), Health education process put the heath information in
contact with heath practice. It’s affected by three factors; there are predisposing,
reinforcing and enabling construct8. The reinforcing factor become the basis of our
project, due to it correlation with health education and we has designed it in our project :
Dental Santri School Program (DENTSPRO) as an integrated learning of dental and oral
health in Islamic boarding school. This program consists of three aspects in order to
developing of education goals based on Bloom taxonomy’s theory (1956) 7. The three
aspects are cognitive which related with acknowledgment, affective which related with
awareness and willingness and also psychomotor which have correlation with motorive
activities.
Dental Santri School Program (DENTSPRO) as an integrated learning of dental
and oral health in Islamic boarding school is finished by developing the concept of
education that integrate dental and oral health education with an understanding of health
concern in daily activities of islamic boarding school students. The learning concept
prepares the santri to be health cadres in DENTSPRO programs. Later on, they drive the
society to concern with their oral hygiene and dental health. The learning concept is using
subject programs that preparing students as the health cadres to attend several
DENTSPRO's programs. As a result, these health cadres can force the society to care
120
with their dental and oral health.
Table 2. Independent samples test
Independent Samples Test

Levene's Test
for Equality t-test for Equality of Means
of Variances
Sig. Std. 95% Confidence
Mean
(2- Error Interval of the
F Sig. T df Differen
tailed Differenc Difference
ce
) e Lower Upper
Equal
-,33
variances 8,220 ,006 58 ,738 -,10000 ,29769 -,69590 ,49590
6
assumed
pre_t
Equal
est
variances -,33 49,7
,738 -,10000 ,29769 -,69802 ,49802
not 6 03
assumed
Equal -
- -
variances 3,950 ,052 17,4 58 ,000 -6,33333 ,36315
7,06026 5,60641
assumed 40
post_
Equal
test -
variances 54,7 - -
17,4 ,000 -6,33333 ,36315
not 40 7,06118 5,60549
40
assumed
From the table above shows that there are significant differences between
following DENTSPRO program and didn’t follow this program. Showed by sig. (2-
tailed) 0,000. These results proves that DENTSPRO's program increased the cognitive
121
abilities of dental health cadres. Provision of general education about dental health and
groups discussion to increase knowledge, also followed by doing counseling exercises to
younger students about Dental Health Education and stadium general counseling by
profesional dental care from Faculty of Dentistry in Airlangga University. Cognitive
domains of DENTSPRO program has reached the third level in Bloom's theory category,
namely Application, that is the ability to apply ideas, procedures, methods which have
been studied on a new and concrete situation. on the theory of Bloom7.
The result of pre-test and post-test was obtained by granting a questionnaire
about general knowledge of dental health. The assessment of affective aspects of the
participants was measured the enthusiasm, attitude and behavior during the
DENTSPRO's counselling. It assessed by the tutor with the value of A, AB, and B. The
criteria for an A if the enthusiasm, attitude and behavior of the participants is very well.
The criteria of AB if the enthusiasm, attitude and behavior is good. and the value of B if
the enthusiasm, attitude and behavior of participants is enough.
The psychomotor aspects of the participants was measured by the ability to
deliver information about brushing teeth, included the value of communication skills.
While, mean of psychomotor mark 0,433 showed in group II (DENTSPRO), it means
their plaque accumulation is in good condition. In general, the measurement of the
affective and psychomotor aspects gave the good results. The participants showed the
interest by asking question or discuss and also convey the information to other
participants in the next-day counseling. The assessment of psychomotor and affective
aspects is based on the evaluation by the tutors during DENTSPRO program activities.
CONCLUSION
Dental Santri School Program (DENTSPRO) can change the mindset and the
knowledge of students about dental health education as their foundation to support their
life in their boarding school. This program also provide the learning of interpersonal
communication in conducting the follow-up activities DENTSPRO cadre after they
learning this integrated program to improve their quality health of care in their Islamic
122
boarding school environment.
REFERENCES
1. Bambang, Aris. 2005. Pembelajaran Terintegrasi. www.arisbambang.wordpress.com, access on 30 Maret 2009
2. Anonim. 2005. Kehidupan Pendidikan Pondok Pesantren. http/www.almukhlisin.com, access on 30 Maret 2009
3. Fatah HRA, Taufik MT, Bisri AM. Rekontruksi Pesantren Masa Depan. Jakarta Utara: PT. Listafariska Putra.
2005. p:11
4. HS Mastuki, El-sha MI. Intelektualisme Pesantren. Jakarta: Diva Pustaka. 2006. p:1
5. Haedari, H. Amin. Transformasi Pesantren. Jakarta: Media Nusantara. 2007. p:3
6. Wahab, Rochidin. Sejarah Pendidikan Islam di Indonesia. Bandung: Alfabeta,CV. 2004. pp:153-154
7. Bloom, Benjamin S. Taxonomy of Educational Objectives (1956). Published by Allyn and Bacon, Boston, MA.
Copyright (c) 1984 by Pearson Education.
8. Green, J. and Tones, K. (1999) Towards a secure evidence base for health promotion. Journal of Public Health
Medicine, 21, 133–139.
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PP11: PREVALENCE OF PERIODONTITIS IN DENTAL STUDENTS IN

UNIVERSITY TECHNOLOGY MARA
Azwin Assilah bte Kamaruddin, Aiman Nadiah Ahmad Tajuddin, Farah
Hidayah Mohd Fazli, Siti Sarah Nor Rizan, Maziahtul Zawani Munshi, Fouad
Hussain M.H Al-Bayaty
Objectives: To evaluate and compare the prevalence of periodontitis in both

gender of dental students in Universiti Teknologi MARA. Material and methods:
One hundred and eighteen bite wing radiographs of 59 dental students of year 2
and 3 were collected from their previous clinical projects. Eighteen radiographs
were excluded due to current active orthodontic treatment. All radiographs were
scanned and uploaded to specific computer software to measure the amount of
bone loss in the radiographs. The distance from cementoenamel junction to
alveolar crest were measured in millimeters of each mesial and distal sites of
upper and lower bicuspids, 1st molar and mesial site of 2nd molar. Measurements
of 1322 sites were analyzed using students T-test to compare gender differences.
Results: All the students showed 100% prevalence of periodontitis due to more
than 1mm bone loss at least in one site. Females showed higher number of sites
involved with bone loss than males which equals to 38.8% and 19% respectively.
The results showed total number of sites of males (± 8.333) and females (±
7.952) statistically non significant differences (p>0.05). Non significant
differences were found in total number of upper teeth between males(± 4.100)
and females (± 3.671).however, significant differences between male(± 4.233)
and female (± 3.686) were found in total number of lower teeth (p<0.05). The
mesial and distal sites of second premolar showed high prevalence of alveolar
bone loss equals to 41.5%. Conclusion: High prevalence of periodontitis was
found among all dental students. Females showed higher prevalence than males.
Future planning management programs should be scheduled to prevent
progression of bone loss among dental students.
INTRODUCTION
In this study, CEJ-AC distance was taken to access the interproximal bone loss
considering 1mm as normal alveolar bone loss (Eliasson,1998)
Periodontitis is defined as an inflammatory disease of the supporting tissues of the teeth
resulting in progressive destruction of the periodontal ligament and alveolar bone with
pocket formation, recession, or both. (Carranza, 2002).
124
It is possible to measure the attachment and alveolar bone loss by measuring the height
and outline of alveolar bone on clinical radiographs due to the destructive nature of
periodontitis (Hugoson and Norderyd, 2008)
The height of alveolar bone may be evaluated by intrasurgical inspection or by
radiographic examination (Mehdizadeh, 2006)
However radiographic assessment tends to underestimate the amount of bone loss
(Theilade 1960, Suomi et al. 1968, Shrout et al. 1991, Akesson et al. 1992, Tonetti et al.
1993, Eickholz et al. 1996, 1998a, 1999b, Eickholz & Hausmann 2000)
On the other hand, other studies have shown that underestimation of bone loss is common
on both intra oral and panoramic radiographs when compared with direct measurements
during surgery (Hammerle et al 1991).
In addition digital processing and manipulation of radiographic images may improve
diagnostic interpretation of radiographs in terms of reproducibility and validity (Wolf et
al, 2001). In order to optimize diagnostic information from routine radiographic
examination, appropriate radiographs of the highest quality should be used (Mehdizadeh,
2006)
This matter of fact that radiographs may be useful to detect minute changes within the
alveolar bone, has led to studies on radiographic techniques such as subtraction
radiography and radiometric analysis, however, most practitioners still depend heavily on
routine intraoral radiographic technique for periodontal assessment. One of the most
useful techniques in evaluation of periodontal disease is bitewing technique.
(Mehdizadeh, 2006).
Many epidemiological studies have been done to asses the status of periodontal health in
various parts of the world. The interpretation of epidemiological data of periodontal
disease is difficult, due to inconsistencies in the methodology used. It is not possible,
therefore, to accurately assess if the prevalence of the periodontal diseases shows a
world-wide decline. As long as the disease is assessed through accumulated clinical
attachment loss, retention of the natural dentition in older ages entails increased
prevalence in these cohorts (Papapanou, 1996).
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The amount of pathological bone loss was interpreted as the distance from CEJ to AC
subtracted with normal alveolar bone loss (1mm) (Eliasson,1998)
The aim of this study was to evaluate and compare the prevalence of periodontitis in both
genders of dental students in Universiti Teknologi MARA by measuring the alveolar bone
loss in available bitewing radiographs
A descriptive cross sectional study was carried out among the Year 2 and year 3
dental students in Faculty of Dentistry, University Technology MARA. Bitewing
radiographs were optioned from Year 2 and year 3 dental students radiography clinical
training in the Faculty of Dentistry University Technology MARA, Malaysia. The
radiographs were taken under supervision of a qualified radiographer. Available
radiographs were chosen to limit unnecessary exposure of radiation to dental students.
One hundred and eighteen bite wing radiographs of 59 dental students of year 2
and 3 were collected. All radiographs were taken by paralleling technique. Eighteen
radiographs were excluded due to active orthodontic treatment. Overall one hundred
bitewing radiographs were collected, scanned and uploaded into Microtek Scanwizard
Pro V7.041.
Planmeca Romexis 2.1.1.R computer software was used to measure the
radiographic alveolar bone loss which is defined as the distance from cementoenamel
junction (CEJ) to alveolar crest (AC) up to a fraction of millimeters (mm). Bone loss was
considered to be present when the distance between CEJ-AC exceeds 1 mm. The sites
measured included the mesial and distal sites of upper and lower teeth first premolars,
second premolars , first molars and the mesial site of upper and lower second molars .
1322 sites were measured. All data were analyzed by using the Statistical Package for
Social Science (SPSS) Version 17.0. Statistical significance of differences between means
was tested with the Student T-Test. Significance was accepted at the probability level P <
0.005.
126
RESULTS
Out of 1322 sites measured 764 sites were found to have bone loss. All the students
showed at least one site with more the 1 mm of alveolar bone loss. About 38% of the sites
in females and 19% in males had more than 1 mm of alveolar bone loss (Table 1). Male
have significantly higher mean pathological alveolar bone loss as compare to females
(figure 1). Table 2 and Table 3 shows the mean pathological bone loss in different
locations in male and female students. Meanwhile, figure 2 shows percentage of sites
with amount of pathological alveolar bone loss (mm).
Table 1; represent the comparison of mean alveolar bone loss (mm) in the upper teeth in
female and male for each tooth statistical analyses revealed non significant differences.
Table 2 showed the comparison of mean alveolar bone loss (mm) in the upper teeth in
female and male for each tooth. Statistical analyses revealed that there is a significant
difference for the 1st upper molar between male and female. In the lower teeth, the 1st
lower premolar and 2nd lower premolar shows significant difference between male and
female.
Figure 2.demonstrated the percentage of alveolar bone loss according to the amount of
bone loss measured by the software.
Table 1: Percentage of sites with pathological bone resorption in both female and
male
Gender Total No Of Sites Total No Of Sites Percentage(%)
Available With Pathological
Bone Resorption
Male 388 251 19
Female 934 513 38
Total 1322 764 57
127
Figure 1: The mean pathological alveolar bone loss in female and male
Pathological
Alveolar Bone
Tooth Gender Loss (Mean, Sd) Sd P Value
1st Upper
Premolar Male 0.250 0.286
Female 0.251 0.300 0.987
2nd Upper
Premolar Male 0.866 0.444
Female 0.695 0.456 0.087
1st Upper Molar Male 0.713 0.522
Female 0.430 0.437 0.006*
2nd Upper Molar Male 0.201 0.359
Female 0.149 0.283 0.436
Table 2: Comparison Of Mean Pathological Alveolar Bone Loss(mm) In The Upper
Teeth in Female And Male For Each Tooth
*The result shows there are significance different between male and female in each tooth.
128
Pathological Alveolar
Tooth No. Gender Bone Loss (Mean, Sd) Sd P Value
1st Lower Premolar Male 0.403 0.431
Female 0.256 0.275 0.043*
2nd Lower Premolar Male 0.590 0.361
Female 0.358 0.328 0.020*
st
1 Lower Molar Male 0.292 0.240
Female 0.207 0.224 0.094
2nd Lower Molar Male 0.100 0.220
Female 0.020 0.073 0.060
Table 3: Comparison Of Mean Pathological Alveolar Bone Loss(mm) In The Lower
Teeth in Female And Male For Each Tooth
*The result shows there are significance different between male and female in each tooth.
Figure 2: Percentage of sites with amount of pathological alveolar bone loss (mm)
129
DISCUSSION
The present study was carried out to evaluate and compare the prevalence of
periodontitis in both genders of dental students in University Technology MARA.
Participation rate was 84.7 % whereby students who are undergoing active orthodontic
treatment are excluded as bone resorption may not be a justified representative of an
underlying periodontitis.
In order to obtain a reproducible epidemiological data suitable for the prevalence
of periodontitis, it is necessary to establish a set of criteria for the clinical and
radiological diagnosis of gingivitis and periodontitis. Thus, essentially, diagnosis of
periodontal disease is still primarily based on the diagnosis of periodontal pocket, loss of
connective tissue attachment and/or assessment of alveolar bone loss performed on
radiographs (Albandar, 2000).
In this study, intraoral bitewing radiographs were used to provide information
about accumulated severity of periodontal disease. While clinical measures of
inflammation, including assessments of gingival index and bleeding, probing depths, and
clinical attachment levels are commonly used in clinical practice, such assessments only
provide information of the condition at the time of examination (Persson et al. 1998)
However, these radiographs provide only 2 dimensional images of 3-dimensional
structures. Hence, the radiographic image of interproximal bone loss may change with
changing projection geometry. Additionally, evaluation of radiographs tends to
underestimate the extent of alveolar bone loss as compared to the gold standard of
intrasurgical measurements (Theilade 1960, Suomi et al. 1968, Shrout et al. 1991,
Åkesson et al. 1992, Tonetti et al. 1993, Eickholz et al. 1996, 1998a, 1999b, Eickholz &
Hausmann 2000).
Radiographic methods are frequently used in epidemiologic studies on
periodontitis. In this study, the distance from the cementoenamel junction (CEJ) to the
alveolar crest (AC) has been assessed on bite wing radiographs. The diagnostic parameter
of amount of bone loss which indicates periodontitis, however, varies. Periodontitis is
130
diagnosed when the normal radiographic distance between CEJ and AC .According to
(Lindhe et al. 2003, Paulander et al. 2004), the ideal distance in health should be 1mm.
In the present study, we consider the normal distance between the CEJ and AC is 1
mm and distance that exceeds that parameter is considered as bone loss which is one of
the most important diagnostic criteria of periodontitis.
The prevalence of periodontitis in which the amount of bone resorption is more
than 1 millimeter from cementoenamel junction of at least one site among dental students
of University Technology MARA was 100%. This is not consistent with the authors’
expectations and hypothesis. Dental students are representative of the educated,
urbanized, influential, and motivated class of individuals. However, an early study has
indicated that the effect of joining a dental profession and its relationship with the
personal level of oral health has not shown any correlation (Maatouk, 2006).
It is clearly demonstrated from the current sesults that male have a higher mean
alveolar bone loss of 0.427mm (p>0.05) compared to females with a mean alveolar bone
loss of 0.296mm (p>0.05). There is a significant difference between males and females
(p>0.05). These findings were in consistent with other studies conducted by several
researchers (Albandar et al. 1999, Kelly et al. 2000, Krustrup & Erik Petersen 2006,
Bourgeois et al. 2007, Suominen-Taipale et al. 2008). The fact that women had
substantially less documented periodontal disease might be due to differences in
periodontal risk factors, sociocultural determinants, or differences in dental and general
health behaviour. Smoking patterns, for example, were different across genders, favoring
males (ever smokers: 59.5% versus 52.5% in adults and 61.4% versus 35.9% in seniors).(
Paulander et al.2004).
Our results revealed that the percentage of alveolar bone loss of 0.1 mm to 1 mm
was 91.9 % and alveolar bone loss of 1.1 mm to 2 mm was 7.6 %. While the percentage
of alveolar bone loss for 2.1 mm to 4 mm is 0.4%. This suggests that despite the high
prevalence of periodontitis in the dental students, the amount of alveolar bone loss is still
minimal. Future management programs should be scheduled to prevent progression of
bone loss among dental students..
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CONCLUSION
High prevalence of periodontitis was found among all dental students. Males showed
higher prevalence than females. Future management programs should be scheduled to
prevent progression of bone loss among dental students.
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133
PP12: ORAL HEALTH STATUS OF CHINESE ELDERLY PEOPLE WITH

DEMENTIA
Tam Hoy Suet Ailsa, Chan Yau Chuen, Cheung Wing Pan, Ho Tek Ka, Lau
Chon Kit, Mak Ka Man, Ng Alice, Woo Cheuk Hang Timothy
Aims: To compare oral hygiene habits and oral health status of Chinese elderly
people with and without dementia and to promote the oral health to elderly
people with dementia. Methods: Chinese elderly people with dementia attending
day-care centers were invited for this study. Age and gender matched people
without dementia were recruited as control. All participants and their care-takers
were informed of the study purposes and procedures, and consent was sought.
The study comprised of questionnaire survey, saliva collection and clinical
examination. Oral hygiene habits, use of dental aids, dental service utilization
were explored in the questionnaire survey. Unstimulated salivary flow rate was
measured. Caries experience, oral hygiene status, periodontal status, were
measured by DMFT index, Visible Plaque Index (VPI) and Community
Periodontal Index (CPI). Results: Fifty-nine Chinese elderly people with
dementia (47 female) were recruited, and their mean age was 80±7. Compare
with the control, there were more people with dementia who received assistance
on tooth-brushing (31% vs. 5%; p<0.001) and brushed less than twice a day
(33% vs. 17%; p=0.045). Their unstimulated salivary flow rate was lower than
that of the control (0.30ml/min vs. 0.41ml/min; p=0.043). Their mean DMFT score
was similar to control (22.3±8.2 vs. 21.5±8.2). They had a higher VPI score (0.78
vs. 0.55; p<0.001) and a similar percentage of people with CPI 3 or larger when
compared to control (78% vs. 74%). Conclusion: Compared with people without
dementia, Chinese elderly people with dementia had poorer oral hygiene and
less unstimulated salivary secretion, but their caries experience and periodontal
conditions were found similar to the people without dementia.
INTRODUCTION
Definition of dementia
Dementia is a clinical syndrome characterized by the loss of intellectual capability

(memory impairment, aphasia, apraxia, agnosia, or a disturbance in executive
functioning) of sufficient severity that social or occupational functionings are interfered
(American Psychiatric Association, 1994).
134
The classical presentation of dementia is the development of multiple cognitive

deficits, like memory impairment, deterioration of language function, disturbances in
executive functioning, failure to recognize or identify objects despite intact sensory
function. In addition, there are psychiatric and behavioral changes such as personality
changes, depression and agitation (Chiu et al., 2002).
Dementia can be classified into different subtypes according to the underlying

brain pathologies. Alzheimer’s disease (AD), vascular dementia (VD), dementia with
Lewy bodies (DLB) and frontotemporal dementia (FTD) are the most common subtypes.
According to the World Alzheimer Report (2009), AD is the most common

subtype of dementia among the elderly and accounts for 50-75% of all dementia cases.
Characteristics of the common dementia subtypes are summarized in Table 1 (World
Alzheimer Report 2009).
Table 1 Common subtypes of dementia
Subtypes Early characteristic Neuropathology Proportion

symptoms
Alzheimer’s • Impaired memory, apathy Accumulation of cortical 50-75%

Disease (AD) and depression amyloid plaques,
• Gradual onset formation of
neurofibrillary tangles
Vascular • Similar to AD, but memory Single infarcts (Stroke) 20-30%

Dementia less affected, more mood in critical regions, or
(VD) fluctuations more diffuse multi-
• Physical frailty infarct disease
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• Stepwise onset
Dementia 1) Marked fluctuation in Cortical Lewy bodies <5%

with Lewy cognitive ability (alpha-synuclein)
Bodies 2) Visual hallucinations
(DLB) 3) Parkinsonism (tremor
and rigidity)
Fronto- • Personality changes No single pathology, 5-10%

temporal • Mood changes limited to frontal and
Dementia • Disinhibition temporal lobes
(FTD) • Language difficulties
Etiology, pathogenesis and risk factors
Common causes of dementia can be classified into two main groups, reversible
(treatable) and irreversible (Frenkel, 2004). The reversible causes that lead to dementia
can be arranged into a useful mnemonic “DEMENTIA”. D for drugs and alcohol, E for
emotional illness like depression, M for metabolic disorders like pernicious anaemia, E
for endocrine disorders like hypo- or hyper-thyroidism, N for nutritional deficiencies like
vitamins B deficiency, T for brain trauma or tumors, I for infections like tuberculosis and
AIDS, and A for cerebral arteriosclerosis. Common irreversible causes of dementia
include degenerative disorder of brain as observed in AD, cerebral infarct as observed in
VD. Rarer causes include Parkinson’s disease, DLB, FTD (Ettinger, 2000).
In AD, the patients suffered from accelerated neuron death leading to gross atrophy
of affected area of the brain. The neuron death is caused by the localized accumulation of
beta-amyloid protein and formation of neurofibrillary tangles adjacent to nucleus of nerve
cells (Frenkel, 2004). In VD, vascular changes such as thrombotic or embolic vascular
136
occlusion produce either focal or diffused damage in the brain. In DLB, abnormal
proteinaceous cytoplasmic inclusion, called Lewy bodies, are developed in the neocortex.
The function in information processing and in relaying data is probably impaired. Also,
there is an altered level of neurotransmitter which affects the function of neuronal
circuits. In FTD, the temporal and frontal lobes of the brain undergo progressive
degeneration. Changes are observed in the tau proteins, which make up the skeletal
support of neurons; TAR-DNA binding protein (TDP43) that regulates gene expression;
and the progranulin which stimulates cell growth and inflammation. These cause cell
dysfunction and death in frontal and temporal lobes.
The main risk factor for most forms of dementia is advanced age, with prevalence
approximately doubling every five years over the age of 65 (World Alzheimer Report,
2009). People having family history of dementia, apolipoprotein E genotype, limited
education or head injury are also at a higher risk. Cardiovascular disease and
corresponding risk factors also play a causal role in dementia. Therefore, medical
conditions such as hypertension, diabetes mellitus, stroke, hypercholesterolemia, and
lifestyle such as high fat diet, smoking, and physical inactivity have an increased risk of
dementia incidence (Ng et al., 2009).
Diagnosis and assessment
One of the most commonly used diagnostic criteria for dementia is developed by the
American Psychiatric Association (APS) (APS, 1994). This diagnostic criteria includes
impairment of memory and at least one of the following domains: language, praxis,
gnosis and executive functioning. The cognitive deficits must be sufficiently severe to
cause impairment of social or occupational life compared to the previous level of
functioning, and the decline does not occur only during delirium. A comprehensive
assessment includes history taking, physical examination, cognitive assessment; and is
supplemented by appropriate blood and neuroimaging investigation (Small, 2006).
Several screening tests are advocated for diagnosing and assessing dementia with
137
reasonable reliability. Mini-Mental State Examination is also widely used. It covers the
subject’s orientation to time and place, recall ability, short-term memory and arithmetic
ability (Folstein et al., 1975). Apolipoprotein E genotype was shown to be a major
susceptibility factor for AD, and this association was also found in Chinese in Hong
Kong and Taiwan (Mak et al., 1996; Hong et al., 1996).
Prevalence
The crude prevalence of dementia in Europe varies from 5.9 to 9.4% for elderly people
aged over 65 (Berr et al., 2005); In Canada, the prevalence of dementia of elderly people
aged over 65 is about 8% (Canadian Study of Health and Ageing Working Group, 1994).
In Hong Kong, the prevalence of dementia in Chinese elderly people aged 70 years and
older is 6% (Chiu et al., 1998). AD and VD are accounted for 65% and 29% of all
dementia patients, respectively.
Dental and oral features of people with dementia
With the progression of severity in dementia, the ability for patients to perform self-care,
including oral hygiene practice, deteriorates gradually. Salivary dysfunction may also be
present due to the pharmaceutical treatment of the disease, such as the side effects from
cholinesterase inhibitors. In particular to patients with AD, the submandibular salivary
output can be impaired (Ship et al., 1990). All these were possible factors contributing to
the significantly higher number of coronal and root surface caries (Warren et al., 1997;
Ellefsen et al., 2008).
The salivary dysfunction may also increase the risk of burning mouth syndrome and
opportunistic infection such as oral candidiasis. People with dementia had decreased use
of dentures, increased prevalence of denture related mucosal lesions, increased plaque
accumulation, increased number of decayed retained roots (Chalmers et al., 2003).
Higher risks in oral disease were related to the severity of dementia, but not to specific
dementia subtypes. So far there was no study reporting the oral health conditions of
138
people with dementia in Chinese population. This community health project aimed to
study the oral health status of Chinese elderly people with dementia.
Aims and Objectives
There was no study published in English reporting the oral health conditions of Chinese
people with dementia in Chinese population.
Aims
This community health project aimed to compare oral health status of Hong Kong
Chinese elderly people with and without dementia and to promote oral health to elderly
people with dementia.
Objectives
The objectives of this community health project were
1. To compare oral hygiene of Chinese elderly people with and without dementia,
2. To compare caries experience of Chinese elderly people with and without dementia,
3. To compare periodontal status of Chinese elderly people with and without dementia,
4. To apply topical fluoride to elderly people with dementia, and
5. To deliver oral hygiene instructions to Chinese elderly people with dementia and
their caregivers.
Study Sample
The target population selected for this project was elderly people who were 1) aged 60 or
139
above, 2) fit for periodontal probing (people who need antibiotics prophylaxis for dental
treatment were excluded), and 3) diagnosed with dementia. Recruitment of participants
was carried out in the day-care centres of the Hong Kong Alzheimer’s Disease
Association and St. James’ Settlement Kin Chi Dementia Care Support Service Center in
March 2010. All eligible elderly people were invited to the study through the day-care
centres by emails and invitation letters. The invitation letter described and explained the
purposes and procedures of the study. They were registered by phone with their age and
gender recorded. A group of elderly people without dementia with corresponding age and
gender was recruited from people who attended the Prince Philip Dental Hospital
(PPDH). The examination of the elderly people in control group was carried out at
PPDH. The progress summary of this community health project can be found in
Appendix 1.
Pilot Study
Data and Information for the study were obtained from two measures: a questionnaire
and an oral health assessment encompassing an oral examination and unstimulated
salivary flow rate measurement. Prior to the study, a pilot study was conducted in PPDH
to 1) evaluate the questions set in the questionnaire, 2) study the feasibility of
unstimulated salivary flow rate measurement, and 3) calibrate inter and intra-examiner
agreement on oral examination.
The pilot study was conducted in the Reception Patient Clinic at PPDH in January,
2010. Ten elderly people attending PPDH were recruited for pilot test. They were not
participants of the main study. The purpose of the pilot study was explained and consent
was sought. Firstly, they were asked to answer a questionnaire. This was to evaluate and
to check the comprehensiveness of responses to the questions. Secondly, they were asked
to expectorate their saliva in a specimen container for 5 minutes. This was to test the
feasibility and precision of measurement of the volume of unstimulated saliva. Finally,
they were examined by two examiners for calibration of the examination criteria,
140
employing the Oral Health Assessment Form (Appendix 9).
Registration and Questionnaire Survey
All participants and their care takers in the main study were informed of the study
purpose and procedures. They were required to make consent before the study
(Appendices 2, 3 & 4). Participants who had given their consent registered in their
respective centers of the Hong Kong Alzheimer’s Disease Association and the St. James’
Settlement Kin Chi Dementia Care Support Service Center for the study. Medical history
of all participants was taken prior to the oral examination. The date of diagnosis and the
stage of dementia were recorded.
A questionnaire was used to study participants’ oral hygiene habits, use of dental
aids, service utilization and personal data. All participants were requested to complete the
questionnaire (Appendices 7 & 8) prior to the day of the examination. Participants of the
control group had to fill in the questionnaire on the day of their visits to PPDH. The
answers of questionnaires were checked during registration and any missing or unclear
responses were identified and corrected before the oral examination.
Sialometric Assessment
The purpose of the sialometric assessment was to study the unstimulated salivary flow
rate and salivary acidity (pH value) of elderly participants. Stimulated salivary flow rate
test was not carried out because of the risk of swallowing the salivary stimulator (rubber
tubing) during chewing by elderly participants, particularly those with dementia.
Participants were instructed not to eat, drink, and smoke for an hour before the
sialometric assessment. During the salivary flow rate test, they were asked to expectorate
resting saliva in a 50ml disposable specimen container for 5 minutes. All salivary
measurements were made on the day in the laboratory after the specimens were collected.
The volume was measured in ml/min according to the guidelines described by Speight et
al. (1992) using a pipette (Drummond Pipet Aid XP, Golden Valley, Minnesota, USA).
141
The pH of the specimens was then measured with a pH meter (Shindengen mini Lab pH
meter, Camarillo, California, USA). Unstimulated salivary flow rate less than 0.1ml/min
is considered as hyposalivation (Rantonen, 2003).
Clinical Examination
Clinical examination was conducted according to the guidelines by World Health

Organization (WHO, 1997). The information collected was recorded in an Oral Health
Assessment Form (Appendix 9). The oral examination included evaluation of the oral
mucosal condition and location, the prosthetic status, the dentition status and caries
experience using DMFT index, oral hygiene status using visible plaque index (VPI),
periodontal status using community periodontal index (CPI), and lastly the loss of
attachment. Two examiners used dental mirrors with LED lights and WHO probes for the
oral examination. Cotton rolls were used for moisture control and visualization of tooth
surfaces. Mirrors and cheek retractors were used for photo-taking and documentation of
abnormal lesions.
Oral mucosal assessment including the conditions of lesions, locations of lesions,

prosthetic status and dentition status were performed and recorded using codes according
to the guidelines suggested by WHO (1997) in Tables 2, 3, 4 and 5, respectively.
Table 2 Codes used in condition of oral Table 3 Codes used in location of oral
mucosal condition assessment mucosal condition assessment
Code Condition Code Condition
0 No abnormal condition 1 Vermillion border
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1 Malignant tumour (oral cancer) 2 Commissures

2 Lichen planus 3 Lips
3 Ulceration (aphthous, traumatic) 4 Buccal mucosa
4 Acute necrotizing gingivitis 5 Floor of mouth
5 Candidiasis 6 Tongue
6 Abscess 7 Hard and / or soft palate
7 Other condition (specify) 8 Alveolar ridge / gingiva
8 Not recorded 9 Not recorded
Table 4 Codes used in prosthetic status Table 5 Codes used in caries status
Code Condition Code Condition
0 No prosthesis 0 Sound
1 Bridge 1 Decayed
2 More than 1 bridge 2 Filled, with decay
3 Partial denture 3 Filled, without decay
4 Both bridge(s) and partial denture(s) 4 Missing, as a result of caries
5 Full removable denture 5 Missing, any other reason
9 Not recorded 6 Fissure sealant
7 Bridge
abutment/crown/veneer/implant
8 Unerupted, unexposed
9 Not recorded
143
Visible plaque index (VPI) assessing oral hygiene status and Community periodontal
index (CPI) assessing periodontal status were measured according to the guidelines
suggested by WHO (1997) on six sextants. Six index teeth, defined according to the FDI
tooth number system, using 16/17, 11, 26/27, 46/47, 31, 36/37 were examined. If the
index tooth in a particular sextant was missing, the highest score among all teeth
remained in the sextant was taken. VPI was measured as present (Code 1) or absent
(Code 0) on the buccal and lingual surfaces of the index teeth. The periodontal status was
assessed using a WHO probe measuring in millimeters. (Table 6) The loss of attachment
was measured according to the guidelines suggested by WHO (1997) using WHO probe
measuring in millimeters. (Table 7)
Table 6 Codes used in CPI
Code Condition
0 Healthy
1 Bleeding after probing
2 Calculus, all black band visible
3 Pocket 4 – 5 mm, gingival margin with black band
4 Pocket > 6mm, black band not visible
5 Excluded sextant (<2 teeth present)
9 Not recorded
Table 7 Codes used in assessment in loss of attachment
144
Code Condition
0 0-3mm
1 4-5mm (CEJ within black band)
2 6-8mm (CEJ between upper limit of black band and 8.5mm ring)
3 9-11mm (CEJ between 8.5mmand 11.5mm rings)
4 12mm or more (CEJ beyond 11.5 mm rings)
X Excluded sextant (<2 teeth present)
9 Not recorded
Report of Assessment and Follow Up
A report on oral examination (Appendix 10) and a souvenir were given to each
participant after the assessment. Participant was individually informed about their oral
health conditions accordingly. They were given fluoride varnish treatment and advised to
seek dental treatment immediately if necessary. For participants with unsatisfactory oral
hygiene, they were advised to reinforce their oral health care and visit a dentist for follow
up.
Oral Health Promotion
Oral health promotion talks were conducted to increase the oral health awareness,
introduce and demonstrate oral hygiene instruction to participants, caregivers and staff.
Oral healthcare pamphlets (Appendix 11) were tailor-made for elderly people with
dementia. They were distributed during the talks. Dental knowledge education was given
via interactive talks (Appendix 12), experience sharing, and problem discussion. Dental
information was revised at the end of the talks. In addition, an oral health care article was
published in the St. James’ Settlement monthly publication (Appendix 13).
145
Data Entry and Statistical Analysis
The data from the questionnaires and charting sheets were entered into Microsoft Excel
2007 and proof-read by all members together. The data were then analyzed using SPSS
17-0 (SPSS Inc., Chicago, USA). Parametric t-test and Chi-square test were used to study
various independent variables. The cut-off point for statistical significance was set at
0.05.
RESULTS
Sample size
For this study, 82 elderly people with dementia were invited in total. 59 elderly people
(47 female, 12 male) in the dementia group responded. The response rate was 72%. There
were 59 elderly people without dementia (47 female, 12 male) in the control group. The
mean age of dementia group was 79.8 ± 7.4 while the mean age of the control group was
79.4 ± 6.8 (p=0.707). The difference in mean age was due to the lack of exact age-
matching pair for three of the following ages, 92, 94 and 98. Three control people of age
84, 85 and 89 were used to pair up with them.
Oral hygiene habits
For the dementia group, 67% brushed at least twice a day and 33% brushed less than
twice a day. For the control group, 83% brushed at least twice a day and 17% brushed
less than twice a day. There were more people from the dementia group who brushed less
than twice a day when compared to the control group (p=0.045). For the dementia group,
31% received assistance on brushing, while only 5% of the control group received
assistance on brushing (p<0.001).
There were 37 people (63%) from the dementia group and three people (5%) from
the control group who reported difficulties during oral hygiene practice. The problem of
146
forgetfulness included forgetting to brush and forgetting that he/she had already brushed.
Inability to brush included the lack of dexterity of the elderly people and the lack of
assistant to help them perform oral hygiene practice. Table 8 showed the problems
encountered during oral hygiene practice in both groups.
Table 8 Problems encountered during oral hygiene practice
Forgetfulness Unwillingness Inability to Caregiver lacks the
to brush brush skills to assist
Dementia
27 (73%) 13 (35%) 8 (22%) 6 (16%)
(N=37)
Control (N=3) 0 (0%) 0 (0%) 3 (100%) 0 (0%)
Dental service utilization
The time elapsed since the last dental visit was similar within the two groups. About 50%
of the dementia group and 49% of the control group had not visited a dentist for the past
one year. There were three people from the dementia group and two people from the
control group who were not sure when was the last time the elderly visited a dentist.
Time of last dental visit
Time of last dental visit Dementia (N=56) Control (N=57)
Less than one year ago 28 (50%) 29 (51%)

One to three years ago 18 (32%) 11 (19%)
More than three years ago 10 (18%) 17 (30%)
There were 10 people from the dementia group and 17 people from the control
group who had not visited a dentist for the past three years. The reasons why they did not
see a dentist were shown in Table 10.
147
Table 10 Reasons for not visiting a dentist
Dementia (N=10) Control (N=17)
Perceived no problems 8 (80%) 7 (41%)
Cost 4 (40%) 8 (47%)
Unwilling to go 4 (40%) 15 (88%)
Dental fear 2 (20%) 1 (6%)
There were 66% of the dementia group who perceived a need to visit a dentist, while
only 54% of the control group perceived a need (p=0.009). Table 11 showed the reasons
for visiting a dentist.
Table 11 Reasons for visiting a dentist

Caries 30 (56%) 36 (64%)
Periodontal problems 28 (52%) 31 (55%)
Ill fitting dentures 19 (35%) 21 (38%)
Chewing difficulties 11 (20%) 23 (41%)
Halitosis 8 (15%) 1 (2%)
Check up 6 (11%) 5 (9%)
Food trapping 2 (4%) 12 (21%)
Ulceration 1 (2%) 7 (13%)
Dry mouth 0 (0%) 13 (23%)
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1. Unstimulated salivary flow rate

There were 11 people from the dementia group and one from the control group who were
unable to complete the unstimulated salivary flow rate test (p=0.010). The unstimulated
salivary flow rate of the dementia group was lower than that of the control group
(p=0.043) as shown in Table 12.
Table 12 Mean unstimulated salivary flow rate
Mean flow rate (ml/min) P value

Dementia (N=48) 0.30 ± 0.17
0.043
Control (N=58 ) 0.41 ± 0.28
For the dementia group, the mean unstimulated salivary flow rate of the people
under medication associated with xerostomia (N=8) was 0.23 ± 0.17 ml/min and for those
who were not under medication associated with xerostomia (N=40) was 0.31 ± 0.16
ml/min (p=0.190).
For the control group, the mean unstimulated salivary flow rate of the people under
medication associated with xerostomia (N=9) was 0.30 ± 0.41 ml/min and for those who
were not under medication associated with xerostomia (N=49) was 0.43 ± 0.37 ml/min
(p=0.354).
Oral mucosal status
For the dementia group, one person presented with lichen planus on the buccal mucosa
and two people from the dementia group and two people from the control group
presented with candidiasis on the palate and labial commissures.
4.5 Prosthesis
There were more bridges found in subjects with dementia than those without (p=0.018)
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as shown in Table 13.
Table 13 Types of prosthesis used
Dementia (N=59) Control (N=59) P value

Yes 14 (24%) 13 (22%)
Complete denture 0.827
No 45 (76%) 46 (78%)
Yes 24 (41%) 33 (56%)
Removable Partial denture 0.097
No 35 (59%) 26 (44%)
Yes 16 (27%) 6 (10%)
Fixed Bridge 0.018
No 43 (73%) 53 (90%)
Caries experience
DMFT score
The mean DMFT score was 22.3 ± 8.2 for the dementia group, and 21.5 ±8.2 for the
control group (p=0.585). Table 14 below compared the DMFT, DT, MT and FT between
the two groups.
Table 14 DMFT, DT, MT and FT in both groups
Mean value Mean value P value

DMFT 22.3 ± 8.2 21.5 ± 8.2 0.585
DT 1.2 ± 1.9 0.8 ± 1.4 0.282
MT 18.9 ± 9.4 18.3 ± 8.9 0.748
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FT 2.5 ± 3.3 2.4 ± 2.5 0.875
For the dementia group, no association was found between the DMFT score and
brushing frequencies, recent dental visit, salivary flow rate, assistance on brushing,
perceived dental need as shown in Table 15.
Table 15 Brushing frequencies, recent dental visit, salivary flow rate, assistance on
brushing, perceived dental need and DMFT in dementia group
No. of people Mean DMFT P value

Yes 38 22.2 ± 7.4
Brush at least twice a day (N=57) 0.944
No 19 22.4 ± 9.9
Yes 46 22.4 ± 7.9
Recent dental visit (N=56) 0.622
No 10 21.0 ± 9.4
Hyposalivation (<0.1ml/min) Yes 4 23.0 ± 7.3
0.802
(N=48) No 44 22.0 ± 8.3
Yes 18 22.4 ± 8.2
Assistance on brushing (N=57) 0.801
No 39 21.8 ± 8.2
Yes 39 22.0 ± 8.0
Perceived dental need (N=47) 0.214
No 8 26.0 ± 9.3
For the control group, there was an association found between DMFT score and
perceived dental need as shown in Table 16.
Table 16 Brushing frequencies, recent dental visit, dental attendance, salivary flow
rate, assistance on brushing, perceived dental need and DMFT in control
group
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No. of people Mean DMFT P value

Yes 49 21.3 ± 8.3
No 10 22.4 ± 8.0
Yes 40 21.0 ± 7.8
No 17 21.5 ± 9.1
0.346
(N=58) No 49 21.0 ± 8.6
Yes 3 18.7 ± 2.9
No 55 21.9 ± 8.4
Yes 32 23.8 ± 7.1
No 22 18.5 ± 8.8
Untreated root caries
The mean number of untreated root caries was 0.6 ± 1.3 for the dementia group, and 0.4
± 0.9 for the control group (p=0.360). No association was found between the number of
untreated root caries and brushing frequencies, recent dental visit, salivary flow rate,
assistance on brushing. There was an association found between the number of untreated
root caries and perceived dental need (p=0.049) as shown in Table 17.
brushing, perceived dental need and untreated root caries in dementia
group
No. of Mean no. of

P value
people untreated root
Yes 38 caries
0.3 ± 1.0
No 19 1.1 ± 1.7
Recent dental visit (N=56) Yes 46 0.5 ± 1.3 0.863
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No 10 0.6 ± 1.4
0.971
(N=48) No 44 0.5 ± 1.2
Yes 18 0.3 ± 0.7
No 39 0.7 ± 1.5
Yes 39 0.7 ± 1.5
No 8 0.1 ± 0.4
No significant association was found between the number of untreated root caries
and brushing frequencies, recent dental visit, salivary flow rate, assistance on brushing,
perceived need for the control group as shown in Table 18.
brushing, perceived dental need and untreated root caries in control group
Mean no. of
No. of
untreated root P value
people
caries
Yes 49 0.2 ± 0.6
No 10 1.1 ± 1.6
Yes 40 0.2 ± 0.6
No 17 0.8 ± 1.3
0.568
(N=58) No 49 0.4 ± 0.8
Yes 3 0.3 ± 0.6
No 55 0.4 ± 0.9
Yes 32 0.5 ± 1.0
No 22 0.3 ± 1.0
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Oral hygiene status
The dementia group had a higher mean VPI score of 0.78 ± 0.26 compared to the control
group 0.55 ± 0.32 (p<0.001). No significant association was found between the VPI score
and the brushing frequencies in both the dementia group and the control group.
Community Periodontal Index and Attachment Loss Code
There were 10 edentulous participants from the dementia group and eight from the
control group who were excluded from the periodontal status examination. Two
participants from the dementia group and one from the control group were unable to
complete this assessment.
The need of advanced periodontal treatment (CPI ≥ 3) was 78% in the dementia
group and was 74% in the control group. Probing attachment level greater than 3mm
(Attachment Loss Code > 0) was 85% in the dementia group and 94% in the control
group. Table 19 showed the highest CPI and Table 20 showed the highest attachment loss
between the dementia and control group.
Table 19 CPI in both groups
Highest CPI Dementia (N=47) Control (N=50)

0 0 (0%) 1 (2%)
1 5 (11%) 7 (14%)
2 5 (11%) 5 (10%)
3 24 (51%) 26 (52%)
4 13 (27%) 11 (22%)
Table 20 Attachment loss code in both groups
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Highest Attachment Loss code Dementia (N=47) Control (N=50)

0 7 (15%) 3 (6%)
1 22 (47%) 17 (34%)
2 11 (23%) 20 (40%)
3 3 (6%) 7 (14%)
4 4 (9%) 3 (6%)
The relationship between CPI and brushing frequency (p=0.008) and between CPI
and recent dental visit (<3 years) (p=0.034) in the dementia group were significant. No
association was found between CPI and assistance on brushing, perceived dental need, as
shown in Table 21.
Table 21 Brushing frequency, recent dental visit, assistance on brushing, perceived

dental need and CPI in the dementia group
CPI < 3 CPI ≥ 3 P value

Yes 3 28
No 7 8
Yes 6 32
No 4 3
Yes 1 13
No 9 24
Yes 9 25
No 1 3
DISCUSSION
Study sample
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In Hong Kong, the prevalence of dementia in Chinese elderly people aged 70 years and
older was 6.1% (Chiu et al., 1998). We targeted daycare centers for elderly people with
dementia which the members were all diagnosed with dementia by medical doctors. We
sent out invitation letters to several centres and three of them showed interest in our
project. We designated some dates for this study and the centres helped to distribute
invitation letters and consent to the corresponding participants. Despite our project
schedule was restricted by the university’s curriculum and confined resources, we
managed to examine 59 people with dementia. The response rate was considered
satisfactory. The participants of control group were generated by the computer system
from people registered in PPDH. They were invited through telephones. The ages of 3
pairs of participant (92, 94 and 98 years old) could not be matched due to difficulties of
finding corresponding controls. Many of them in this age group had either passed away
or refused to come due to health reasons.
Due to the lack of cooperation of elderly people with dementia at the severe
stage, we were only able to recruit and carry out examinations on those with mild to
moderate dementia. Also, some of the elderly people in the daycare centres were having
regular dental checkups and perceived no need in joining our study. Hospitalized and
institutionalized subjects were not included in this study. Therefore the participants were
by no means a full representation of the population of people with dementia in Hong
Kong. However, this study still provided useful information and allowed a rational
understanding of the oral health status of people with dementia.
Questionnaire survey and clinical assessment
Both the clinical examinations and the questionnaire surveys were conducted smoothly
and efficiently. This could be attributed to the pilot study which allowed an evaluation of
trial run before the main study. The questionnaires were found to be easily understood.
The close-ended questions were quick to complete. Two examiners were involved in the
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clinical examinations. The level of inter-examiner agreement was good (Kappa

value=0.95, 0.85 and 0.81 in DMFT score, VPI and CPI charting respectively). Well-
defined diagnostic criteria, calibration exercises and the pilot study all contributed to the
good level of inter-examiner agreement. The level of cooperation of the participants was
satisfactory. For those people with dementia who were unable to follow instructions of
the examination, great patience and assistance from the staff of the centres were needed.
A few participants refused to cooperate and even showed aggressive behaviour. Therefore
examinations on them could not be completed.
Oral hygiene behaviour
There were significantly more elderly people from the dementia group who brushed less
than twice a day compared to the control group. The reasons of brushing less could be
attributed to their cognitive declination and the difficulty in performing oral hygiene
practice on them. In our survey, caretakers reported several problems when performing
oral hygiene practice on elderly people with dementia. The elderly might not understand
the need therefore refused to brush. They might refuse to open their mouths and showed
aggressive behaviour towards the caretakers. The lack of knowledge in correct oral
hygiene practice was also a factor. Lack of dexterity, lack of assistance and forgetfulness
were the major problems encountered by elderly people with dementia who brushed by
themselves. We encouraged all caretakers to supervise the elderly when they were
brushing. Therefore if there was any lack of dexterity or areas that was not cleaned well,
the caregiver could give assistance immediately. The caregivers should also remind the
elderly to brush and be familiarised with the proper method of brushing. In uncooperative
cases, other methods such as mouth cleaning swabs might be a choice, though swabs
could not remove as much plaque and debris as toothbrushes (Chalmers et al., 2005).
We have found that there were significantly more elderly people in the
dementia group needed assistance on brushing than that in the control group. At the time
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of this study, there was no oral health promotion in Hong Kong targeted for elderly
people with dementia. We organized two talks and made pamphlets to improve the
efficacy of oral hygiene practice performed on elderly people with dementia. The
audiences consisted of caretakers, family members of the elderly and the staff of the
centres. They showed interest particularly in the provision of oral hygiene instructions
and replacement of missing teeth. Three hundred copies of the pamphlet written in
Chinese were made and given to the centres. Those who could not join our talks were
able to access the invaluable knowledge through the pamphlets. Soft copy of the
pamphlet was also given to the centers for continuous printouts. An oral health care
article was also published in the St. James’ Settlement monthly publication. Therefore the
promotion of oral health to the elderly would be more effective and sustainable.
Dental service utilization
There was no significance difference in the frequency of dental visits comparing the
dementia group to the control group. In our sample, half of the elderly people in the
dementia group had not visited a dentist for more than a year. In those who had visited a
dentist within a year, most were due to dental problems but not for regular check-ups.
However, the perceived need of dental treatment in the dementia group was found to be
significantly higher than the control group. This might be because the caretakers of the
elderly were aware of the possible increased need of dental treatment, but its relationship
with regular dental visits was not understood. There was a need to emphasize the
importance in frequent regular dental check-ups. Future oral conditions of the elderly
should be assessed by a dentist and should be monitored regularly. Complex treatment
procedures should be carried out in the early stage of dementia than at the later stage
during which patient management was compromised (Frenkel, 2004). Prevention could
help elderly people with dementia to retain their natural µ dentition.
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Sialometric Assessment
There were some difficulties in performing the assessment. We could not carry out the
analysis of pH value of the saliva and there were two reasons. First, we could not alter the
mealtime of the elderly with dementia since they had fixed schedules set by the centres.
Therefore the pH value of saliva might be influenced by their meal not long before the
collection. Second, they might had the risk of swallowing the buffer solution as some of
them were unable to follow instructions. There were significantly more elderly people in
the dementia group who were unable to complete the test compared to the control group.
Using the remaining data for analysis, a significant difference in unstimulated salivary
flow rate was found. One other study also found that people with AD had significantly
lower unstimulated submandibular salivary flow when compared to the control (Ship et
al., 1990). If we took the drugs that would induce xerostomia into account, no significant
difference in unstimulated salivary flow rate was found. The result might be explained by
that elderly people with dementia had organic reasons in reduction of unstimulated saliva
flow such as neurological changes due to underlying brain pathology. Another reason
might be because the dementia patients could not understand the instruction of the test
and did not expectorate all the saliva out for sampling.
Prosthetic status
There were significantly more bridges found in the dementia group than in the control
group. For the elderly people with dementia, fixed prosthesis required less compliance
and it was advantageous in the use of them over removable prosthesis. Moreover, fixed
prosthesis did not have the problems brought by removable prosthesis such as denture
stomatitis. In other studies, denture stomatitis was found to be prevalent in elderly people
with dementia (Chalmers et al., 2003). However, bridges might be more difficult to clean,
which may lead to development of caries and periodontal disease around the abutment
teeth. Therefore the need of cleaning the prosthesis should be emphasized and their
159
caretakers should know how to assist on cleaning. Adaptation to new removable

prosthesis might be difficult for these patients. Whenever possible, relines for ill-fitting
denture was preferable (Ghezzi et al., 2000).
Caries experience
Although in our study there was more decayed teeth, missing teeth and filled teeth found
in elderly people with dementia than the control group, statistically there was no
significance between them. However, in other studies, there were significantly more
coronal and root caries, filled surface found in elderly people with dementia than those
without it (Chalmers et al., 2003; Ellefsen et al., 2008).
Elderly people with dementia on average had more than one decayed tooth.
Caretakers should be noted that caries should not be left untreated. The complexity of
restoration increased and restorability of the decayed tooth would be reduced over time.
In later stage of dementia, the elderly might not cooperate in more complex treatments or
extraction of hopeless tooth. Hence regular dental check-up for diagnosing of carious
lesion as early as possible was necessary. Comprehensive preventive measures might be
necessary for high risk group.
Oral hygiene and periodontal status
Elderly people with dementia had significantly poorer oral hygiene than the control
group. However they did not show less tooth brushing frequency in this study. Therefore
it might be the problem in brushing technique that led to the increased plaque
accumulation in the elderly people with dementia. Some similar findings could be found
in other studies. Significantly higher gingival index (Warren et al., 1997) and plaque
index (Chalmers et al., 2003) were reported in elderly people with dementia when
compared to those without. There was no significant difference in the periodontal
160
condition between the elderly people with and without dementia. Although the majority
of the elderly brushed twice a day, the periodontal condition was still unsatisfactory.
It was of noteworthy that the majority (78%) of the elderly people with
dementia group had periodontal pockets and required advanced periodontal treatment.
This indicated that there could be problems in brushing techniques or presence of plaque
retentive factors, which affect efficient plaque removal. Also 85% of the elderly with
dementia had an experience in periodontitis according to the attachment loss level. The
importance of periodontal care of the elderly should be emphasized especially those with
dementia. It was important to give adequate plaque control to the best ability of the
elderly and caretakers in order to limit the progression of periodontal diseases. Further
tooth loss due to periodontal diseases would lead to several problems, including a
decrease in masticatory function and ill-fitting dentures. Problems such as inability to
cooperate with dental procedures, inability in accepting new changes might occur in
remaking a new denture. Even if remaking of the denture was successful, they might still
refuse to use the new one.
Dental management of elderly people with dementia
Hong Kong was facing an aging population phenomenon. The proportion of people aged
65 and above in Hong Kong in 2008 was 13% and it was estimated to reach 24% by the
year 2025. As the risk of dementia was related to aging, there would be more people with
dementia in the future. Dentists in Hong Kong would face a greater challenge in
maintaining the oral health of the elderly with dementia. Dentists should pay attention in
the treatment planning and behavioral management for people with dementia. Treatment
planning must be designed with consideration of the severity of the condition and involve
caretakers and guardians (Ghezzi et al., 2000). They should try to anticipate the future
oral condition of the elderly (Kocaelli et al., 2002).
161
The prevention of deterioration of oral health was very important in elderly

people with dementia despite they might not be have a higher risk in oral conditions
according to the study. As the cognitive impairment progressed, the elderly would be
more uncooperative towards dental treatment, and find it more difficult adapting to new
dentures (Kocaelli et al., 2002). Proper oral hygiene instructions should be taught to the
people with dementia and they should be encouraged to do so independently in early
stage (Frenkel, 2004). An electric toothbrush or modified handle might be easier to grip if
manual dexterity was impaired. Caretakers should also learn the proper way of oral
hygiene instructions. As in later stage of dementia, the elderly frequently needed
assistance (Frankel, 2004). Comprehensive preventive measures might be necessary.
Frequent dental recall visits should be emphasized to reduce changes in oral conditions
and to prevent the further need of complex treatments. If major treatments were
indicated, it should be carried out in early stages of dementia (Frenkel, 2004). As the
cognitive declination proceeded, shorter, less stressful appointments and the use of the
least traumatic interventions should be arranged (Ettinger, 2000).
Hand scaling might be less frightening than ultrasonic scalers. It was also
possible to consider chemical caries removal techniques (Frenkel, 2004). The aim in
dental treatment for elderly people with dementia was to maintain existing dentition with
minimal changes (Ettinger, 2000). Dentists can speak with calm voice and physical
reassurance and perform only the necessary treatments. Oral sedatives or anxiolytics,
such as short-acting benzodiazepines before treatment could be considered to manage
behavioural problems (Kocaelli et al., 2002). Intravenous sedation or general
anesaethesia could also be considered. Complex and time-consuming dental treatment
should be avoided in people with severe dementia (Ghezzi et al., 2000).
CONCLUSION AND RECOMMENDATIONS
Conclusion
162
In this community health project, we concluded that

1. Hong Kong Chinese elderly people with dementia had a poorer oral hygiene than
those elderly without dementia.
2. Hong Kong Chinese elderly people with dementia had a similar caries experience
liked those elderly without dementia.
3. Hong Kong Chinese elderly people with dementia had a similar periodontal status
liked those elderly without dementia.
4. This community health project successfully delivered topical fluoride varnish to the
exposed root surfaces of teeth in elderly people with dementia.
5. This community health project successfully delivered oral hygiene instructions to
Chinese elderly people with dementia and their caregivers.
Recommendations
From the findings of this study, we recommend:
1. Oral health education should be provided to caretakers on how to perform daily oral
hygiene care for the elderly. Assistance or supervision on tooth-brushing is
recommended.
2. Comprehensive treatment should be provided as soon as the elderly is diagnosed
with dementia. Because later complex treatment would be difficult to ensue during
late stage of the disease.
3. Regular dental check-up should be emphasized for this group of patient to provide
prevention and intervention of oral disease.
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PP13: THE EFFECT OF ETHANOLIC EXTRACT OF PINEAPPLE-STEM (

ANANAS COMOSUS (L.) MERR) ON INCREASING APOPTOSIS OF A
HUMAN ORAL TONGUE CANCER CELL ( SP – C1 ) IN VITRO
Agnes Bhakti Pratiwi, Muhammad Isa, Wisda Septiana Chandra Devi,

Supriatno
Background: Human oral tongue cancer cells are one of the most common
malignancies in the oral cavity. At the present, the new target for cancer therapy
is apoptosis induced by herbal medicines. Pineapple stem is one of a potential
herbal medicine because of its bromelain enzyme content. Bromelain is one of
an anticancer agents that can induce apoptosis. The aim of the study was to
evaluate apoptosis induction by various concentrations of pineapple-stem
ethanolic extract in a human oral tongue cancer cell Supri’s Clone-1 (SP – C1).
Methods: SP – C1 cells were treated with three concentrations under IC50 of
pineapple stem ethanolic extract, 5.000, 5.500 and 6.000 μg/ml. Apoptosis
examination were delivered by cells stained with fluorochrome ethidium bromide
and acridine orange after 24 hours incubation. Fluorescence microscope was
used for counting the cells. Viable cells would be stained green and apoptotic
cells would be stained yellow to orange. Data was analyzed by One way ANOVA
and post hoc LSD test with significance value 95 % (p<0,05) Results: SP – C1
cells treated with 5.000, 5.500 and 6.000 μg/ml pineapple stem ethanolic extract
showed the percentage of apoptosis 65,18%; 82,86% and 98,52% respectively,
one-way ANOVA test revealed a statistically significant difference between the
group treated with different concentration (p<0,05). Moreover post hoc LSD test
showed a statistically significant difference between each group. Conclusion:
Pineapple stem ethanolic extract concentration may induce apoptosis of human
oral tongue cancer cell (SP – C1).
INTRODUCTION
Cancer is defined as uncontrolled tissue growth in susceptible patients caused by

imbalance of cells division and programmed cell death or apoptosis (Ponder, 2001). Head
and neck cancers are primary malignant neoplasms that occur in several anatomical sites
in the head and neck region such as oral cavity, ear, nasal cavity, paranasal sinuses,
nasopharynx, hypopharynx, oropharynx and salivary glands (Cassidy, et al., 2002). Head
and neck cancers are the 10th most common cancer in the world (Adeyemi et al., 2008).
165
Over 90% of oral and pharyngeal cancers are squamous cell carcinoma (Scully, 2003;
Bsoul et al, 2005).
The tumor growth is characterised by an imbalance between cell proliferation and
programmed cell death or apoptosis (Baltaziak, 2004). Apoptosis can be induced by
radiation and chemotherapy treatment (Susworo and Putra, 2006). Cancer therapy by
inducing apoptosis has become a new strategy since the objective of cancer therapy was
to inhibit cancer cells growth without damaging normal cells. (Gerl and Vaux, 2005).
The medicinal properties of pineapple (Ananas comosus (L.) Merr) have been
recognized for a long time (Kelly, 1996). Pineapple has 90% water content, high in
potassium, calcium, iodine, sulfur and chlorine. It is also rich in acid, biotin, vitamin B12,
vitamin E, and bromelain enzyme (Kurniawan, 2008). Bromelain was found in high
concentrations in pineapple stem and introduced as therapeutic agent. Bromelain actions
include : inhibition of platelet aggregation; fibrinolytic activity ; anti-inflammatory
action; anti-tumor action; modulation of immunity; skin debridement properties;
enhanced absorption of other drugs; mucolytic properties; digestive assistance and
enhanced wound healing (Kelly, 1996).
In cancer treatment, bromelain was reported to inhibit tumor cell proliferation and
differentiation (Mynott et al., 1999; Maurer, 2001). Treatment with bromelain decreased
viability of mouse melanoma cells in vitro (Guimaraes-Ferreira et al., 2007). Other in
vitro studies showed the ability of bromelain to reduce cells migration and invasion in
glioma cells (Tysnes et al, 2001). Bromelain which was given per-oral, functioned as an
immunomodulatory of monocytes immunocytotoxicity repairs to fight cancer cells.
Bromelain combined with chemotherapeutic agent such as 5-fluorouracil and vincristine,
has been reported to result in tumor regression (Gerard, 1974 cit Kelly. 1996). So far,
research on the effects of pineapple-stem ethanol extract towards apoptosis in human oral
tongue squamous cell carcinoma has not been reported.

Pineapple stem ethanol extract
166
Pineapple stem was dried in a drying chamber at a temperature of 45 ° C. Dried

pineapple stem was blended into powder until smooth and fluffy. This extract preparation
used maceration, by immersing the pineapple-stem powder in 96% ethanol for 24 hours
and filtered. This process was repeated 3 times. Obtained filtrate was evaporated with
rotary vacuum evaporator with temperature of 70 ° C . This process was to evaporate
ethanol in order to obtain viscous extract. Pineapple stem ethanol extract 100 mg
dissolved in DMSO into 100 μl, was then added medium (DMEM) until the
concentration of 1 ml (stock solution contained pineapple stem ethanol extract 100 mg /
ml).
Human oral tongue cancer cells (SP-C1)
Cancer cells used in this study were human oral tongue squamous cell carcinoma
cell lines cloned by Supriatno, DD.S, MDSc., Ph. D. stored at the Center for Integrated
Research and Testing Laboratory (LPPT), Gadjah Mada University, Yogyakarta. Cancer
cells were cultured in tissue culture flask and incubated at 37 ° C and 5% CO2.
Apoptosis
Ethidium bromide-acridine orange stock solution was made, consisting of
ethidium bromide 50 mg and acridine orange 15 mg dissolved in 1ml ethanol 95%, then
added 49 ml of distilled water. From the stock solution, 1 ml was taken and diluted with
PBS (1:100).
A microplate was filled with the following conditions: (a) In each well, a
coverslip was placed in the bottom of wells, (b) Row A, columns 1-3 were filled with
cancer cells without treatment (negative control), (c) Row B columns 2-4 were filled with
pineapple stem ethanol extract concentration 5000, 5500 and 6000 µg / ml and tongue
cancer cells.
A microplate was placed in an incubator with a temperature of 37 º C and 5%
CO2. After 24 hours, the coverslips were removed and placed on glass objects. The
ethidium bromide-acridine orange solution 50-10 μl was dropped on the coverslip.
Preparations were observed under fluorescent microscope with a magnification of 100-
400x. Whole cell nucleus was colored light green and cells undergoing apoptosis were
167
colored orange. The number of cells that undergo apoptosis are calculated on four fields
of view.
Statistics
Data was analyzed using one-way ANOVA and post-hoc test with level of significance
95% (p<0,05).
RESULTS
Apoptosis was performed on human oral tongue cancer cell (SP – C1) treated by
pineapple stem ethanolic extract. After 24 hours of incubation by staining with
fluorochrome ethidium bromide and acridine orange, samples of incubated cells were put
in an object glass, dropped with fluorochrome and observed with fluorescence
microscope.
.
(a) (b)
(c) (d)
Figure 1. Figure of human oral tongue squamous cell carcinoma (Sp – C1) after 24
168
hours incubated with pineapple stem ethanol extract and stained with
fluorochrome ethidium bromide – acridine orange (a) negative control, (b)
pineapple stem ethanol extract concentration 5000  g / ml , (c) 5500  g /
ml, and (d) 6000  g / ml, (fluorescence microscope, 40x magnification).
Observation was carried out on four different fields of view. Viable cells presented
a green color ( red arrows in Fig. 1), whereas apoptotic cells presented a yellowish orange
in color ( black arrow in Fig. 1).
Table 1. Results of Mean, Standard Deviation, Degrees of Freedom, and Significance of
One-Way ANOVA in Apoptosis of Human Oral Tongue Squamous Cell
Carcinoma (Sp – C1) after 24 hours Incubation in Pineapple Stem Ethanol
Extract
Concentration of
Pineapple Stem Ethanol x± sd (%) df P
Extract ( g/ml)
Negative Control 0
5000 65,18 ± 2,29

3058, 480 0,000
5500 82,86 ± 1,71
6000 98,52 ± 1,29
x : Mean
sd : Standard Deviation
df : Degrees of Freedom
p : Significance value of One-Way ANOVA test
169
100
90
80
70
apoptosis(%)
60
50
40
30
20
10
0
0 5000 5500 6000
concentration of pineapple stem ethanolic extract ( g/ml)
Figure 2. Mean of Apoptotic Persentage of Human Oral Tongue Cancer Cell (SP – C1)
After 24 hours Incubation in Pineapple Stem Ethanolic Extract Concentration
5000, 5500 and 6000  g/ml
** P < 0,01
As shown in fig.2, percentage of apoptotic human oral tongue cancer cell (SP –
C1) increased significantly with the increase of concentration of pineapple stem
ethanolic extract. The concentration of pineapple stem ethanolic extract 6000  g/ml
caused 98.52% apoptotic cells, concentration of 5500  g/ml showed 82.86% apoptotic
cells, whereas concentration of 5000 g/ml revealed 65.18% apoptotic cells (Table 1).
DISCUSSION
Tongue cancer has general characteristics like most common cancer (Revianti and
Parisihni, 2005), has its own growth signals, is insensitive to anti-growth signals, is able
to avoid apoptosis, has unlimited replication abilities, has a mechanism of angiogenesis,
invasive and metastasis (Hanahan and Weinberg , 2000). Human oral tongue cancer cell
is more aggressive than other cancer cell of the head and neck (Ye et al., 2008).
Human oral tongue cancer cell (SP-C1) was cultured from the tongue squamous
cell carcinoma medium differentiation, without invasion to muscle tissues. SP-C1 cell
170
cultures have a rapid cell growth, invasion and metastasis to regional cervical lymph
nodes and often cause recurrence despite radical surgery was performed. This occurs
because the micro-invasion of cells and metastasis of the primary cancer site (Supriatno,
2008).
Treatment for cancer is definite and alternative treatments using herbal medicine
is needed. Pineapple is one of the herbal medicine that can be used as an anti-cancer
agent. Studies in Europe have been investigated the efficacy of pineapple extract in the
treatment of breast, colorectal, and plasmacytoma cancer patients (Beuth, 2008). IC50
value of pineapple stem ethanolic extract was detected at 6324.49  g/ml. Based on the
IC50 value, we used concentration of 5000, 5500 and 6000  g / ml.
The results of this study showed that pineapple stem ethanolic extract inhibit
cancer cells through induction of apoptosis. Previous studies reported that bromelain
from pineapple stem ethanol extract could inhibit tumor growth by inhibiting tumor cell
proliferation (Mynott et al, 1999; Maurer, 2001) and apoptosis (Kalra et al., 2008;
Chobotova et al, 2009.)
This study indicated that pineapple stem ethanol extract caused apoptosis of
human oral tongue cancer cell. Cells treated by pineapple stem ethanolic extract were
stained with fluorochrome ethidium bromide and acridine orange, and observed with
fluoresence microscope to confirm the occurrence of apoptosis. Acridine orange-ethidium
bromide has the ability to stain the nuclei of cells in a late phase of apoptosis yellow and
to stain the nuclei of necrotic cells orange-red. This is a useful tool to discriminate normal
from apoptotic and apoptotic from necrotic cells (Buomino, 1999). An apoptotic cell
usually has morphological changes such as: DNA fragmentation, cell volume decrease,
mitochondrial function loss, membrane blebbing, compaction of chromatin into dense
masses that lie at the periphery of the nucleus or, in other cases, condensation of the entire
nucleus into a dense ball with the chromatin distributed evenly throughout the nucleus
(Ross et al., 2003). In this study, human oral tongue cancer cell pointed apoptotic
morphological changes as incomplete nucleus formed and stained as yellow to orange
cells by fluorochrome ethidium bromide when the cell was observed using a fluoresence
171
microscope.
Apoptosis, a type of a programmed cell death, is an active process. It is a normal
component of the development and health of multicellular organisms. Apoptosis can
occur by internal and external pathway (Elrod and Sun, 2008). In this study, the cause of
the apoptotic is a signal from outside the cell by treatment using various concentrations of
pineapple stem ethanol extract. This apoptosis induction ability is due to bromelain
content in pineapple stem ethanol extract (Kurniawan, 2008; Tochi, 2008). The results of
this study correspond to Maurer (2001) which states that bromelain can inhibit tumor cell
proliferation and differentiation of leukemia cells in vitro through apoptotic mechanisms.
According to Kalra (2008), apoptosis of a human oral tongue cancer cell caused by
bromelain may occur through the intrinsic or extrinsic pathway to improve regulation
p53, Bax, caspase 3 and caspase 9 and decrease anti-apoptotic protein bcl2.
CONCLUSION
Pineapple stem ethanol extract may induce apoptosis of human oral tongue cancer
cells (SP–C1).
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1. Adeyemi, B. F., Adekunle, L. V., Kolude, B. M., Akang, E. E. U., dan Lawoyin, J. O., 2008, Head and Neck Cancer-
A Clinicopathological Study in a Tertiary Care Center, J Natl Med Assoc, 100 (6) : 690-697.
2. Baltaziak, M., Koda, M., Barwijuk-Machala, M., Musiatowicz, B., Duraj, E., Kanczuga-Koda, L., Musiatowicz, M., dan
Reszee J., 2004, The Role of Bak Expression in Apoptosis of the Oral Squamous Cell Carcinoma (OSCC) and Metastases
to Lymp Nodes (LNMs), Annal Acad Med Biol, 49 (Suppl. 1): 14-15.
3. Batkin, S., Taussig, S., dan Szeckerczes, J., 1988, Antimetastatic Effect of Bromelain with or without Its Proteolytic and
Anticoagulant Activity, J. Cancer Res. Clin. Oncol., 114 : 507-508.
4. Beuth, J., 2008. Proteolytic enzyme therapy in evidence-based complementary oncology: fact of fiction, Integr Cancer
Ther. 7:311-316.
5. Bsoul, S. A., Huber, M. A, dan Terenzhalmy, G. T., 2005, Squamous Cell Carcinoma of the Oral Tissues : A
Comperehensive Review for Oral Health Care Providers, J. Contempt. Dent. Practice, 6(4) : 001-016.
6. Buommino, E., Morelli, F., Metafora, S., Rossano, F., Perfetto, B., Baroni, A., Tufano, M., 1999. Porin from Pseudomonas
aeruginosa Induces Apoptosis in an Epithelial Cell Line Derived from Rat Seminal Vesicles, American Society for
Microbiology Vol. 67, No. 9
7. Cassidy J., Bissett D., dan Spence R. A. J., 2002, Oxford Handbook of Oncology, Oxford University Press: New York.
172
8. Chobotova, K., Vernallis, A. B., Majid, F. A. A., 2009, Bromelain’s Activity and Potential As An Anti-Cancer Agent:
Current Evidence and Perspectives, Cancer Lett., doi:10.1016/j.canlet.2009.08.001
9. Elrod, H. A. dan Sun S. Y., 2008, PPARγ and Apoptosis in Cancer, PPAR Research,1-7.
10. Gerl, R. dan Vaux, D. L., 2005, Apoptosis in the Development and Treatment of Cancer, Carcinogenesis, 26(2): 263-270.
11. Guimaraes-Ferreira, C. A., Rodrigues, E. G., Mortara, R. A., Cabral, H., Serrano, F. A., Ribeiro-dos-Santos, R., dan
Travassos, R. L., 2007, Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-
Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa, Neoplasia, 9 (9): 723-733.
12. Hanahan, D. dan Weinberg, R. A., 2000, The Hallmarks of Cancer, Cell, 100: 57-70.
13. Kalra, N., Bhui, K., Roy, P., Srivastava, S., George, J., Prasad, S., dan Shukla, Y., 2008, Regulation of p53, Nuclear Factor
κB and Cyclooxygenase-2 Expression by Bromelain Through Targeting Mitogen-Activated Protein Kinase Pathway in
Mouse Skin, Toxi Appl Pharmacol, 226: 30-37, www.elsevier.com, 12/07/09. 62
14. Kelly, G. S., 1996, Bromelain : A Literature Review and Discussion of Its Therapeutic Application, Alternative Medicine
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15. Kurniawan, F., 2008, Sari Buah Nanas Kaya Manfaat : Alternatif Meningkatkan Nilai Ekonomis Hasil Panen, Sinar Tani
edisi 13-19 Agustus 2008.
16. Maurer, H. R., 2001, Bromelain : Biochemistry, Pharmacology and Medical Use, Cell. Mol. Life Sci., 58: 1234-1245.
17. Mynott, T. L., Ladhams, A., Scarmato, dan P., Engwerda, C. R., 1999, Bromelain, from Pineapple Stems, Proteolytically
Blocks Activation of Extracellular Regulated Kinase-2 in T Cells, J Immun, 163: 2568-2575.
18. Ponder, B.A. J., 2001, Cancer Genetics, Nature, 411: 336-341, www.nature.com, 10/07/2009.
19. Revianti, S. dan Parisihni, K., 2005, Peran MMP pada Metastasis Karsinoma Sel Skuamosa Rongga Mulut, Jurnal PDGI,
Edisi khusus tahun ke-55, h. 232-236.
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10 (1) : 75-78.
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Review, Pakistan J Nutr, 7 (4) : 513-520
25. Tysnes, B. B., Maurer, H.R., Porwol, T., Probst, B., Bjerkvig, R., dan Hoover, F., 2001, Bromelain Reversibly Inhibits
Invasive Properties of Glioma Cells, Neoplasia, 3(6): 469–479.
26. Ye, H., Yu, T., Stephane, T., Barry, L. Z., Wang, J. G., Joel, L. S., Mao, L., David, T. W., dan Xiaofeng, Z., 2008,
Transcriptomic Dissesction of Tongue Squamous Cell Carcinoma, BMC Genomic, 9 (96) : 1-11,
http://www.biomedcentral.com, 08/02/2009.
173
PP14: THE HEMOSTATIC EFFECT OF ARTEMISIA VULGARIS EXTRACT IN

TRAUMATIC BLEEDING ON MUS MUSCULUS
Grace Angelina Samuel, Melisa Budipramana, Dian Lupita Sari, Astari

Puteri,
Anisha Giantini, Jessica Theresia
Background: Artemisia vulgaris is a herbal plant that can be found nearly all
over the world. In Ayurveda (Indian traditional medicine), it is used to ease
cardiac complaints and to relieve feelings of unease, unwellness and general
malaise. Moxibution of mugwort is also done in the practice of traditional Chinese
medicine. Different parts of A. vulgaris are also used for a multitude of other
medicinal purposes including as antibacterial, anti-inflammatory, antiseptic,
diaphoretic, emmenagogic, and stimulatory agents. It can also be used as a
traditional medicine to stop bleeding and regularize menstrual disorders.
Objective: This study is intended to prove that this herb can be used as a
hemostatic medication in traumatic bleeding. Method: This study was a
randomized controlled trial done in Mus musculus. Before the experiment is
conducted, subjects were acclimatized for 1 week. Subjects were divided into 4
groups, 10 M. musculus in each group. Group 1 using 25% A. vulgaris extract,
group 2 50%, group 3 100%, and group 4 as the control group that uses
aquadest. The tails of M. musculus were cut at certain part with 0.3 cm in
diameter. The bleeding part of the remaining tails were put into the extract for 3
seconds by group 1,2, and 3 as well as into aquadest by group 4. The duration of
bleeding in second was recorded. Result: One way ANOVA continued by LSD
revealed that there were significant differences between group 1 (25% A. vulgaris
extract) and control group (p=0.011), group 2 (50% extract) and control group
(p=0.004), group 3 (100% extract) and control group (p=0.000). Conclusion:
This study demonstrated that A. vulgaris extract can stop the bleeding in a
shorter time than that by control group. The higher the concentration of A.
vulgaris leaf extract, the greater the hemostatic effect.
INTRODUCTION
Bleeding, scientifically known as hemorrhage, refers to the loss of blood. 1 Unspe-

cific hemorrhage patients are usually prescribed to epinephrine as vasoconstrictor, or
tranexamic acid as antifibrinolytic agent.2,3 Bleeding is also unavoidable in oral surgery
practice, even after a routine dental extraction as it is also categorized as traumatic bleed-
174
ing. It usually stops no more than 10 minutes after the extraction. If bleeding continues,
the area should be inspected for signs of mucosal tearing or other evident cause for con-
tinued hemorrhage. In the absence of any such sign, a further period of 10 minutes with
firm pressure on the wound should be tried. 4 Occlusal pressing with tampon is the best
way to stop the bleeding and can induce the formation of stable thrombus. 5 Besides, a re-
sorbable hemostatic agent (e.g. oxidized cellulose, fibrin foam, gelatin foam, collagen
granules, alginate fibres) can also be given into the socket after dental extraction.2
However, those hemostatic medications have several disadvantages. Epinephrine
can influence systemic circulation and cause allergic reactions in a few cases 6 while
tranexamic acid can cause vascular occlusive events (myocardial infarction, stroke, pul-
monary embolism, deep vein thrombosis).7 Besides, the price of the drugs is relatively
high. They are also difficult to be obtained in remote places. On the other hand, several
plants are known as traditional hemostatic medications, but yet they need to be proven.
One of them is Artemisia vulgaris (A. vulgaris) that can be found nearly all over the
world.8,9 It is expected that it can be used as a cheaper and safer hemostatic medication.
A. vulgaris, widely known as mugwort, has been used as traditional medicine in
several countries. In Ayurveda (Indian traditional medicine), it is used to ease cardiac
complaints and to relieve feelings of unease, unwellness and general malaise. Moxibution
of mugwort is also done in the practice of traditional Chinese medicine. 10 A. vulgaris are
also used for a multitude of other medicinal purposes including as antibacterial, anti-
inflammatory, antiseptic, diaphoretic, and emmenagogic.11,12 It can also be used as a
traditional medicine to stop bleeding12, and regularize menstrual disorders.11 This herb
contains tannin that is also found in other hemostatic herbs, such as: Ageratum
conyzoides L., Alchornea cordifolia, Aspilia africana Pers., Baphia nitida, Chromolaena
odorata L., Landophia owariensis, Ageratum conyzoides L., Piper betle L., and Psidium
guajava.13,14,15,16
The purpose of this study was to examine whether it could be used as a local
hemostatic agent on traumatic bleeding, especially post-dental-extraction bleeding, due to
the ability of A. vulgaris to stop bleeding in traditional medicine as it contains tannin,
175
Although post-extraction bleeding is usually not dangerous, patient’s feeling of

discomfort will be minimized if the bleeding time can be reduced. This preliminary study
was a randomized controlled trial done in Mus musculus (M. musculus) or mice. A certain
part of the tails were cut and then dipped into 25%, 50%, and 100% of A. vulgaris extract
and aquadest as control group. The bleeding time of the tail was recorded to know the
hemostatic effect of the extract.
MATERIALS AND METHOD
Extract of Artemisia vulgaris preparation
A. vulgaris extract was made by simple extraction. One kilogram fresh A. vulgaris leaves
were needed to make 200 ml pure extract. To make pure extract, the leaves were washed
with water at the temperature of 60o-70o C. Then, the clean leaves were put into sterilized
blender. The blendered leaves were filtered with sterilized rough filtrate paper. Finally,
pure extract was ready and then put into a sterilized flask. To make 50% extract, pure
extract was diluted into the same proportion of aquabidest. To make 25% extract, 50%
extract was diluted into the same proportion of aquabidest. Aquabidest was chosen as
solvent because it is considered as neutral, sterile, and pure solvent that does not affect
the characteristic of the extract. Effective concentration of A. vulgaris extract as local
hemostatic agent on traumatic bleeding is for further study.
Hemostatic test
This randomized controlled trial was done in animal experimental unit of University of
Airlangga, Surabaya Indonesia. A total of 40 Mus musculus were chosen based on the
same gender, age, and species. Male mice around the age of 3 months and weighed 20-25
grams were chosen due to their hormonal balance compared to female mice.17,18 This
experiment was done using vernier calliper, scissors, anesthetics agent (ether 10%), A.
vulgaris extract, aquadest, and filter paper on which the blood droplets drip. Subjects
were divided into 4 groups, ten M. musculus in each group. Group I as the control group
that using aquadest; group II using 25% A.vulgaris extract; group III 50%; and group IV
100%.
The mice were anesthesized by putting them one by one into a sealed flask
176
containing cotton with ether in it. Soon after they were unconscious, the mice were then
taken out. Then, their tails were cut off by a pair of scissors at a certain part with 3 mm in
diameter. The remaining tails were put into A. vulgaris extract for the treatment groups, or
aquadest for the control group. The bleeding time was recorded right after the tail was cut
until the bleeding completely stop. The data were then analyzed using a One Way
ANOVA continued by LSD for Windows 2007. These tests were performed to find any
correlation between the effect of A. vulgaris extract and the length of bleeding time.
RESULTS
The mean and standard deviation of the hemostatic effect in traumatic bleeding on
M. musculus with 25%, 50%, and 100% concentration of A. vulgaris extract and control
group, are shown on Table 1. The results showed that the hemostatic effect tends to
increase by the higher concentration. Test of Normality (Kolmogorov-Smirnov Test)
showed that the probability was greater than 0.05 (p = 0.195). Test of Homogenicity of
Variances showed that the probability was also greater than 0.05 (p = 0.108). The result
of the One-Way ANOVA showed that the probability was lower than 0.05 in all
concentration of A. vulgaris extract. It means that different concentration of A. vulgaris
extract significantly influences the reduction of the bleeding time on the M. musculus.
The results is shown on figure 1.
Table 1. Mean and standard deviation of the hemostatic effect in control group
and extract groups in traumatic bleeding on M. musculus
Mean of Bleeding Time +

Group Number
Standard Deviation (seconds)
I
10 210,50 + 37,610
(Control)
II
10 169,00 + 33,423
(25% A. vulgaris extract)
177
III
10 163,00 + 42,361
(50% A. vulgaris extract )
IV
10 126,70 + 19,995
(100% A. vulgaris extract)
The 95% confidence interval for mean; mean; maximum and minimum value for
each group are shown on Figure 1. The lower bound for group 1 is 183.60 and the upper
bound is 237,40 while the minimum data is 158 and the maximum is 272. The lower
bound for group 2 is 145.09 and the upper bound is 192.91 while the minimum data is
116 and the maximum is 216. The lower bound for group 3 is 132.70 and the upper
bound is 193.30 while the minimum data is 118 and the maximum is 245. The lower
bound for group 4 is 112.40 and the upper bound is 141,00 while the minimum data is
105 and the maximum is 117.
Figure 1. Diagram of mean the hemostatic effect in control group and extract
groups in traumatic bleeding on M. musculus
= 95%
confidence
Bleeding Time (seconds)
250
interval
for mean
200
150
0
I II III IV
Group
178
The LSD tests showed significant differences between the control group and
treatment group, but the difference of the length of bleeding time between 25% and 50%
A. vulgaris extract is not significant (p = 0.699). It is shown on Table 2.
Table 2. Statistical results of LSD test from the hemostatic effect in control group and
extract groups in traumatic bleeding on M. musculus
II III IV
I (25% A. (50% A. (100% A.
Group
(Control) vulgaris vulgaris vulgaris
extract) extract) extract)
I
-
(Control)
II
(25% A. p = 0.011 -
vulgaris extract)
III
(50% A. p = 0.004 p = 0.699* -
vulgaris extract)
IV
(100% A. p = 0.000 p = 0.009 p = 0.024 -
vulgaris extract)
*The mean difference is not considered significant because p > 0.05.
DISCUSSION
From the data, it can be seen that there is significant difference of the length of the
bleeding time between control group and treatment groups. However, the difference
between 25% and 50% A. vulgaris extract is not significant. However, the greater
concentrations of A. vulgaris extract shows the tendency to shorten the bleeding time on
the M. musculus.
A. vulgaris is well known to have the ability to stop bleeding in traditional
179
medicine. The shortening of bleeding time is also shown on experimental mice which
given 15 g/kg A. vulgaris extract for 5 days.19 A. vulgaris leaves contain several chemical
substances. Its essential oil consists of phellandrene, cadinene, and  –thujone. The other
substances are  –amirin, fernenol, dihydromotriacaria ester, cineole, 1- -terpineol,
 -kariophilene, 1-quebrachitol, and tannin.20 The active ingredient that act as hemostatic
agent is probably tannin, as several other plants which contain tannin are also have
hemostatic effect.14,15,16
The mechanism of tannin in blood coagulation is presumed as follows. Tannic
acid – a derivate of tannin – acts as astringent. As it shrinks the epithelial surface, tannin
can stop capillary bleeding.21,22 Besides, tannin can also precipitate blood protein, such as
albumin. Serine protease inhibitors (serpins) which bind to albumin act as blood
anticoagulant by inhibiting the activation of serine proteases. Serpins inhibit the action of
their respective serine protease by mimicking the three-dimensional structure of the
normal substrate of the protease. Serine proteases play an important role in activating
blood coagulation factors: factor X, XI, thrombin, and plasmin. While tannin precipitates
albumin, serpins as inhibitor of serine proteases are also inactive. Due to that reason,
serine proteases are activated and stimulate blood coagulation cascade.23,24,25
In this experiment, the bleeding time of the animals’ tails was recorded in
seconds. This preliminary study on mice’s tail is expected to be applied on post-
extraction traumatic bleeding. The use of M. musculus as sample is based on the
presumption that the size of capillary blood vessels on its tail is about the same size of
those on teeth.14 This was done to see the possibility of using A. vulgaris as a hemostatic
agent on more severe bleeding after dental extraction. After being cut, the remaining tails
were dipped into A. vulgaris for 3 seconds in treatment groups and aquadest in control
group. We would like to examine whether the extract can act as a hemostatic agent
without other interfering factors, such as pressure factor and blood coagulation factors in
saliva.
It is expected that the result of this preliminary study can be applied in clinical
practice, especially in post-extraction bleeding. It is suggested to make A. vulgaris gel so
180
that the extract can be used and produced widely. The gel is then applied on tampon
which is usually used after dental extraction. Finally the bleeding can stop faster.
CONCLUSION
A. vulgaris can function as a hemostatic agent on traumatic bleeding on mice (M.
musculus). The higher the concentration of A. vulgaris extract, the better the hemostatic
effect. Time needed for 25% A. vulgaris extract to stop bleeding was 169.00 seconds (p =
0.011), while 50% extract was 163.00 seconds (p = 0.04) and 100% extract was 126.70
seconds (p = 0.00). The time for each group was shorter than control group, which is
210.50 seconds. The effect of hemostatic is possibly due to tannin, as it acts both as
astringent and as precipitated serine protease inhibitor in albumin.
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Volume 1 | Number 2 |August 2010

Asia Pacific Manusript of Papers presented at Scientific Research Competition,

Publisher 39. PP4: TEST OF INHIBITORY POWER OF ESSENTIAL OIL EXTRACT

PP1: THE AGE-DEPENDENT OSTEOGENIC DIFFERENTIATIONOF ADIPOSE-DERIVED

Tsai, Meng-Wen ,Fu-Hung Tseng, Hen-Yu Liu

Osteoporosis is a disorder characterized by compromised bone quality which predisposes

loss of osteoblasts leading to osteoporosis could potentially be prevented by administration of stem

staining to observe the osteogenic potential.

3. ADSCs cell culture

1.0 Alizarin Red S Staining

2.0 Oil Red O Staining

5. Osteogenic Medium Preparation

8. Polymerase Chain Reaction(PCR)

9. Agarose gel preparation

I. The Cell Culture of ADSCs

II. Define the isolation cells on stem cell characterization

1.2. Cell Proliferation

2. The Differentiation Potential of ADSCs

III. Semi-quantitative PCR Analysis

IV. Alizarin red staining

In conclusion, we could sum up the osteogenic differentiation of ADSCs-1M and10M by the

Alizarin red O.D 405

Nusaibah Mutmainnah Bt Mohamad Azmi,Mursyidatun Najihah Mohamad Nasar,

OBJECTIVES AND RESEARCH QUESTIONS

Malaysia (USIM) aged 19 years and above.

Inclusion and Exclusion Criteria:

Sample size calculation

Data collection procedure

Periodontal disease measurement

2 Plaque retentive factors present - no pocketing > 3.5mm

Table 1: Descriptive statistics variables understudy (n=62)*

Table 3: Oral Health Quality of Life Score (n=62)

DMF 0.73 (0.35, 1.10) <0.001*

Periodontal Disease 0.39 (-1.56, 2.35) 0.688_____

GOHAI item 1 -0.83 (-1.86, 0.20) 0.113

GOHAI item 2 1.50 (0.24, 2.76) 0.021*

GOHAI item 3 -0.55 (-2.03, 0.93) 0.460

GOHAI item 5 0.48 (-0.67, 1.64) 0.403

GOHAI item 6 0.31 (-1.18, 1.79) 0.681

GOHAI item 7 0.37 (-0.66, 1.40) 0.478

GOHAI item 8 -0.002(-0.92, 0.91) 0.996

GOHAI item 9 -0.60 (-1.60, 0.38) 0.224

GOHAI item 11 0.41 (-0.91, 1.72) 0.536

GOHAI item 12 -0.70 (-1.65, 0.24) 0.142__________

Quality of life is increasingly acknowledged worldwide as a significant and appropriate

PP3: THE EFFECT OF SOY MILK ON MANDIBLE BONE DENSITY IN RAT

Devi Gunawan, Diana S. Djohan, Tamara Gladysia E, William Adi Santoso,

to buffer systemic acidosis in a passive manner. However, cell culture experiments

MATERIALS AND METHODS

This experiment was done in animal experimental unit of Airlangga University,

product of IGEA S.p.A.

1 1400 1545 1797 1833 1431 1587 1479 1833 1833

2 1698 1525 1736 2044 1634 1985 1484 1876 1876

3 1672 1390 1708 1588 1320 1562 1472 1774 1774

4 1448 1511 1506 1544 1106 1520 1454 1602 1598

5 1457 1545 1710 1879 1431 1811 1151 1974 1974

6 1391 1516 1822 1588 1320 1891 1261 1700 1693

8 1753 1625 1480 2045 1632 1325 1423 1839 1839

The mandible testing shows that One-Sample Kolmogorov-Smirnov Test is normal in

strenuous swimming has more impact to mandible bone.

PP4: TEST OF INHIBITORY POWER OF ESSENTIAL OIL EXTRACT NUTMEG

Novita Eka Lestari, Aisyah Bella Azzanjani, Ita Purnama Alwi

Angular Cheilitis is acute or chronic inflammation at the corner of the mouth.