IDENTIFICATION OF ORAL BACTERIA FROM SUBGINGIVAL PLAQUE AND THE EFFECT OF Salvadora persica EXTRACTS ON THE GROWTH

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IDENTIFICATION OF ORAL BACTERIA FROM SUBGINGIVAL PLAQUE AND THE EFFECT OF Salvadora

persica EXTRACTS ON THE GROWTH

Nur Fatimah Zaharah binti Salehuddin

Thesis submitted in partial fulfilment for the degree of MASTER OF SCIENCE

UNIVERSITI SAINS ISLAM MALAYSIA

April 2023

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ii

AUTHOR DECLARATION

I hereby declare that the work in this thesis is my own except for quotations and summaries which have been duly acknowledged.

Date: 13^th April 2023 Signature:

Name: Nur Fatimah Zaharah Binti Salehuddin

Matric No: 3201478

Address: No 10B, Lot 3085, Kampung Padang Balang, Sentul, 51100 Kuala Lumpur

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iii

ACKNOWLEDGEMENTS

Upon completion of this research, I would like to express my gratitude to many parties.

My heartfelt thanks go to my supervisor, Dr. Rohazila Mohamad Hanafiah for giving me chance to get invaluable guidance, comments, and suggestions throughout this study.

I also would like to thank my co-supervisors, Prof. Madya Dr. Haslinda Ramli, Prof.

Madya Dr. Siti Aisyah Abd Ghafar and Prof. Madya Dr. Shahida Mohd Said for constantly motivating me to work harder and never give up. Without their guidance and persistent help, this project would not complete. This study is a collaboration with Universiti Kebangsaan Malaysia (UKM) and Universiti Sains Islam Malaysia (USIM) under MRUN-RAKAN RU-2019-002/2.

I also would like to thank Ms Wan Nor Asma Wan Razab, and Mr. Nik Muhammad Asyraf Mohd Azmi, as Science officers for their assistance when I am working on my research in the laboratory until it is done. Thank you to Mr. Muhammad Kamarulzaman Daud and Mrs. Nur Hana Mohd Rapiai, as laboratory staff for permitting me to borrow and use all the equipment and chemical required from starting until the end of the project.

I am taking the opportunity to sincerely thank Dr. Nor Zaihana Abd Rahman and Dr.

Nor Haliza Mat Baharin for becoming my panel and giving constructive comments during my proposal defence presentations. Those comments were very useful to be discussed. Thanks to Dr. Zulfahmi Said Student Coordinator for Postgraduate Students at the Faculty of Dentistry Universiti Sains Islam Malaysia, Pandan Indah Campus for guidance throughout my journey.

A great thanks to all my colleagues who are willing to share valuable information related to my project. They give me a lot of critical ideas for improving my methods to get good results, especially when I was performing the antibacterial activity technique of Salvadora persica extracts against selected oral bacteria. This thesis is dedicated to my family and friends who try their best to give me endless support by encouraging me to finish this master’s journey and supporting me financially to pay all the costs required during this project.

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iv ABSTRAK

Penyakit mulut biasanya ditemui pada populasi manusia disebabkan oleh pengumpulan plak bakteria. Kajian mengenai jenis bakteria mulut dalam populasi Malaysia masih berkurangan. Streptococcus mutans (S. mutans) adalah salah satu daripada streptokokus mulut yang memulakan penyakit periodontal sebagai penjajah awal. Lama-kelamaan, anaerob Gram-negatif menjadi lebih mantap, terutamanya Porphyromonas gingivalis (P. gingivalis). Walau bagaimanapun, bakteria boleh membina rintangan antibiotik terhadap ubat-ubatan dari semasa ke semasa. Jadi, Salvadora persica (S. Persica) atau dikenali sebagai miswak diakui kerana kehadiran sebatian bioaktif yang mengandungi aktiviti perencatan terhadap bakteria mulut. Objektif utama dalam kajian ini adalah untuk mengenal pasti bakteria mulut daripada plak subgingiva dan menentukan aktiviti antibakteria SPSE (ekstrak batang S. persica) terhadap bakteria mulut terpilih. Objektif khusus dalam kajian ini adalah untuk mengenalpasti jenis bakteria mulut dalam plak subgingiva menggunakan jujukan 16S rRNA, untuk menentukan aktiviti antibakteria SPSE terhadap bakteria mulut terpilih dan untuk menyiasat komposisi fitokimia SPSE menggunakan Spektrometri Jisim Kromatografi Gas (GCMS). Kajian ini telah berjaya menjumpai 176 bakteria mulut daripada 10 sampel plak subgingiva. Sebanyak 17 bakteria mulut telah dikenalpasti daripada semua sampel. 97 jenis bakteria Gram- negatif berjaya diasingkan daripada semua sampel. Majoriti adalah Gram-positif iaitu spesis Streptococcus dan lain-lain adalah bakteria Gram-negatif, termasuk P. gingivalis, A. actinomycetemcomitans dan K. penumoniae S. persica ekstrak telah dihasilkan dengan menggunakan pelarut n-heksana, diklorometana, aseton, etanol dan metanol.

Aktiviti antibakteria SPSE-n-heksana, SPSE-DCM, SPSE-aseton, SPSE-etanol, dan SPSE-metanol ditentukan dengan melakukan tiga teknik berbeza, seperti ujian resapan cakera (DDA), kepekatan perencatan minimum (MIC) dan minimum kepekatan bakteria mati (MBC) terhadap S. mutans dan P. gingivalis. Spektrometri jisim kromatografi gas (GCMS) telah dianalisis untuk menyiasat komposisi fitokimia dalam SPSE yang boleh menghalang pertumbuhan S. mutans dan P. gingivalis. Daripada dapatan, terdapat perbezaan yang signifikan antara jenis pelarut yang digunakan dalam SPSE dan diameter zon perencatan bagi S. mutans dan P. gingivalis (p < 0.001). Untuk keputusan MIC, S. mutans telah dihalang dengan SPSE-n-hexane pada 3.125 mg/mL.

Sementara itu, S. mutans dibunuh dengan 6.25 mg/mL SPSE-n-heksana Dalam analisis GCMS, fitokimia utama bagi semua ekstrak ialah asid benzoik, asid heksadeseniok dan asid oktadekanoik. Kesimpulannya, S. persica amat disyorkan sebagai salah satu alat kebersihan mulut untuk merawat penyakit mulut kerana keberkesanannya menghalang pertumbuhan bakteria mulut.

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v ABSTRACT

Oral diseases are commonly found in the human population caused by the accumulation of bacterial plaque. There is still lacking of study regarding types of oral bacteria in the Malaysian population. Streptococcus mutans (S. mutans) is one of oral streptococci which initiate periodontal disease as an early colonizer. Over time, Gram-negative anaerobes become more established, especially Porphyromonas gingivalis (P.

gingivalis). Nevertheless, bacteria can develop antibiotic resistance to medicinal drugs over time. However, Salvadora persica (S. persica) or known as miswak is acknowledged significantly due to the presence of bioactive compounds that contain inhibitory activity against oral bacteria. The main objective in this study is to identify oral bacteria from subgingival plaque and determine the antibacterial activities of Salvadora persica Stem Extracts (SPSE) against selected oral bacteria. The specific objective of this study is to identify the type of oral bacteria in subgingival plaque using 16S rRNA sequencing, to determine the antibacterial activities of SPSE against selected oral bacteria and to investigate the phytochemistry compositions of SPSE using Gas Chromatography Mass Spectrometry (GCMS). This study successfully isolated 176 oral bacteria from 10 subgingival plaque samples. A total of 17 oral bacteria had been identified from all samples. About 97 Gram-negative bacteria were successfully isolated from all samples. The majority of the bacteria are Gram-positive which is Streptococcus species and others are Gram-negative bacteria, including P. gingivalis, A. actinomycetemcomitans and K. pneumoniae. SPSE were produced using n-hexane, DCM, acetone, ethanol, and methanol. Antibacterial activities of SPSE-n-hexane, SPSE-DCM, SPSE-acetone, SPSE-ethanol, and SPSE-methanol were determined by performing three different techniques, such as disc diffusion assay (DDA), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. mutans and P. gingivalis. Gas chromatography mass spectrometry (GCMS) was analysed to investigate the phytochemistry composition in SPSE that can inhibit the growth of S. mutans and P. gingivalis. From the results, there is a significant difference between the type of solvents used in SPSE and the diameter of the inhibition zone for S. mutans and P. gingivalis (p < 0.001). For MIC results, S. mutans were inhibited with SPSE-n-hexane at 3.125 mg/mL. Meanwhile, S. mutans were killed with 6.25 mg/mL of SPSE-n-hexane. In GCMS analysis, the major phytochemical of all extracts was benzoic acid, hexadecanoic acid and octadecanoic acid. In conclusion, S. persica is highly recommended as one of the oral hygiene tools to treat oral diseases due to its effectiveness in inhibiting the growth of oral bacteria.

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vi

صخلم

Streptococcus mutans (S.mutans)

ةثللا ضارمأ ببست تيلا ةيومفلا ةيدقعلا تاروكلما نم ةدحاو يه ًةصاخ ، اًخوسر رثكأ مارلجا ةبلاس تايئاوهلالا حبصت ،تقولا رورم عمو .ركبم تقو في رمعتسمك

ةيوثللا سناومويرفروبلا

( P. gingivalis⁽

اكيسيرب ارودافلس نإف ، كلذ عمو

)^{S. persica}

مسبا ةفورعلما وأ

⁾

م كاوسلما دض طبثم طاشن ىلع يوتتح تيلا ايجولويب ةطشنلا تابكرلما دوجو ببسب يربك لكشب ةفورع

.مفلا قيرط نع يايرتكبلا تتح تابسرت نم مفلا يايرتكب ىلع فرعتلا وه ةساردلا هذه نم يسيئرلا فدلها تاصلختسلم يايرتكبلل ةداضلما ةطشنلأا ديدتحو ةثللا

^{S. persica}

ةساردلا هذه .ة اقتنلما مفلا يايرتكب دض

لزع في تحنج

176

نم ةيومف يايرتكب

10

ةبجوم تناك يايرتكبلا ةيبلاغ .ةثللا تتح كلابلا نم تانيع

تاديحو كلذ في ابم ، مارلجا ةبلاس يايرتكبلا نم اهيرغو ةيدقعلا تاروكلما عاونأ نم يهو مارلجا ةيوثللا سناومويرفروبلا

( P. gingivalis

و

)^A

.

actinomycetemcomitans⁽

ةيوئرلا ةليسبلكلا و

^K.

pneumoniae

تابيذلما في اكيسيرب ارودافلس تاصلختسلم يايرتكبلل ةداضلما ةطشنلأا ديدتح تم .)

(

لوناثيم ،لوناثيإ ،نوتيسلأا ،ينليثيلما ديرولك ،يداعلا ناسكلها ، ةفلتمخ تاينقت ثلاث ذيفنت للاخ نم

)

ميثارلجا ديبم زيكترل نىدلأا دلحا و طبثلما زيكترلل نىدلأا دلحا ديدتح و ، صرقلا راشتنا صحف لثم

(MBC\MIC

ةيومفلا ةيدقعلا تاروكلما دض ةيويلحا ةيشغلأا صحفو )

^{S. mutans}

سناومويرفروبلا و ةيوثللا

( (P. gingivalis⁽

زاغلل نيوللا يلتكلا فيطلا سايقم ليلتح تم .

^GC-MS

ةبيكرت نم ققحتلل

⁾

تاصلختسم في ةيتابنلا ءايميكلا

( (S. persica⁽

ةيومفلا ةيدقعلا تاروكلما ونم عنتم نأ نكيم تيلا

^(S.

mutans

يوثللا سناومويرفروبلا و

^s⁽

ة

(P. gingivalis

ينب يربك قرف كانه نأ ينبت ، جئاتنلا للاخ نم . تاصلختسم في ةمدختسلما تابيذلما عون

( (S. persica

ةيدقعلا تاروكلما نم لكل طيبثت ةقطنم رطقو ةيومفلا

( (S. mutans⁽

ةيوثللا سناومويرفروبلا و

(P. gingivalis( p

<

^0.001

دحلل ةجيتنل ةبسنلبا .

⁾

طيبثتلل نىدلأا

( MIC⁽

ةيومفلا ةيدقعلا تاروكلما طيبث ت تم ، )

^{S. mutans}

يداعلا ناسكلها تاصلختسبم

⁾

نم

( S. persica

( ميثارجلل ديبم نىدلأا دلحا ،هسفن تقولا فيو .لم / ممج

^3.125

دنع

)^MBC)

تم يأ ةيومفلا ةيدقعلا تاروكلما لتق

( S. mutans

.يداعلا ناسكلها تاصلختسم نم لم / غلم

^6.25

دنع

)

ةبسنلباو

زاغلل نيوللا يلتكلا فيطلا سايقم ليلتح جئاتنل

^GCMS

ةيسيئرلا ةيتابنلا ةيئايميكلا ةدالما تناك ،

، ماتلخا في .كيوناكيداتكولأا ضحمو كيونيسيداسكيه ضحمو كيوزنبلا ضحم يه تاصلختسلما عيملج

مادختسبا ةدشب ىصوي

^{S. persica}

طيبثت في اهتيلاعفل اًرظن مفلا ضارمأ جلاعل مفلا ةفاظن تاودأ دحأك

مفلا يايرتكب ونم

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vii

TABLE OF CONTENTS

CONTENT PAGE

AUTHOR DECLARATION ii

ACKNOWLEDGEMENTS iii

ABSTRAK iv

ABSTRACT v

AL-MULAKHKHAS vi

TABLE OF CONTENTS vii

LIST OF TABLES ix

LIST OF FIGURES x

LIST OF APPENDICES xiv

LIST OF UNITS OF MEASUREMENTS xv

LIST OF SYMBOLS xvi

LIST OF ABBREVIATION xvii

CHAPTER 1: INTRODUCTION 1

1.1 Introduction 1

1.2 Background of the Study 2

1.3 Statement of the Problem 3

1.4 Objectives of the Study 3

1.4.1 Specific Objectives 4

CHAPTER 2: LITERATURE REVIEW 5

2.1 Oral Health Disease 5

2.1.1 Dental Caries 6

2.1.2 Periodontitis 6

2.2 Oral Pathogens 9

2.2.1 Streptococcus mutans 10

2.2.2 Porphyromonas gingivalis 11

2.3 Salvadora persica as a Therapeutic Agent 13 2.3.1 Composition in Salvadora persica Extracts 15 2.3.2 Antibacterial Actions of Salvadora persica Extracts

Against Oral Pathogens

16

CHAPTER 3: RESEARCH METHODOLOGY 18

3.1 Sample Processing 18

3.2 Colony Forming Unit for Each Sample 19

3.3 Isolation and Identification of Oral Bacteria from Subgingival Plaque

20

3.3.1 Morphology of Oral Bacteria 20

3.3.1.1 Gram-staining 20

3.4 Polymerase Chain Reaction (PCR) Analysis 20

3.4.1 DNA Extraction 20

3.4.2 Polymerase Chain Reaction (PCR) 21

3.4.3 DNA Purification 22

3.4.4 Gel Electrophoresis 23

3.5 Sequencing Analysis 24

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viii

3.5.1 16S rRNA Sequencing 24

3.5.2 Basic Local Alignment Search Tool (BLAST) Program

24

3.6 Salvadora persica Stem Extraction 24

3.7 Antibacterial Activities Against Streptococcus mutans and Porphyromonas gingivalis from Subgingival Plaque

25 3.7.1 Bacterial Cultures for Selected Oral Bacteria

(Porphyromonas gingivalis and Streptococcus mutans) 25

3.7.2 Disc Diffusion Assay (DDA) 26

3.7.3 Minimum Inhibitory Concentration (MIC) and

Minimum Bactericidal Concentration (MBC) 27 3.8 Phytochemistry of Salvadora persica Extracts (SPSE) 28 3.8.1 Gas Chromatography Mass Spectrometry (GCMS) 28

3.9 Statistical Analysis 28

CHAPTER 4: RESULTS 30

4.1 Population of Samples 30

4.2 Colony Forming Unit 30

4.3 Morphology of Oral Bacteria 34

4.4 Isolation and Identification Type of Oral Bacteria from

Subgingival Plaque Samples 39

4.5 Antibacterial Activities of Salvadora persica Stem Extracts (SPSE) Against Selected Oral Bacteria from Subgingival Plaque

58 4.5.1 Extraction of Salvadora persica 58

4.5.2 Selected Oral Bacteria 59

4.5.2.1 Disc Diffusion Assay (DDA) 59 4.5.2.2 Minimum Inhibitory Concentration (MIC)

and Minimum Bactericidal Concentration (MBC)

61 4.6 Gas Chromatography Mass Spectrometry (GCMS) 63 CHAPTER 5: DISCUSSION, RECOMMENDATIONS AND

CONCLUSION

71

5.1 Introduction 71

5.2 Summary and Discussion of Results 71

5.2.1 Microbial Composition in Subgingival Plaques 71 5.2.2 Antibacterial Activity of Salvadora persica Stem

Extracts (SPSE) Against Streptococcus mutans and Porphyromonas gingivalis

74 5.2.3 Chemical Compositions in Salvadora persica Stem

Extracts (SPSE)

75

5.3 Recommendations of the Study 76

5.4 Conclusions 77

REFERENCES 78

APPENDICES 87

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ix

LIST OF TABLES

Tables Page

Table 2.1 : Important Bioactive Constituents Found in S. persica

Extracts 16

Table 3.1 Selection Criteria of The Sample 19

Table 3.2 : Details of Primer Used in PCR 21

Table 3.3 : PCR Components Used in the Amplification of Specific

DNA Target with a Total of 25 µL for One Reaction 22

Table 3.4 : Details of PCR Amplification Cycles by Using a Positive,

Negative, and Universal Primer in T100 Thermal Cycler 22

Table 4.1 : Number of Viable Colonies for Each Subgingival Plaque

Sample 34

Table 4.2 : The Total Number of Gram-positive and Gram-negative

Oral Bacteria Isolated from Subgingival Plaque Samples 39

Table 4.3 : Morphology of 176 Isolated Oral Bacteria on Media and

Under Microscope 41

Table 4.4 : Oral Bacteria Species Which Are Mostly Found in the

Subgingival Plaque Samples 56

Table 4.5 : Total Yield of Five Types of Different Types of SPSE 58

Table 4.6 : Inhibition Zone of DDA for SPSE Against S. mutans 59

Table 4.7 : Inhibition Zone of DDA for SPSE Against P. gingivalis 60

Table 4.8 : MIC and MBC of SPSE Against S. mutans and P. gingivalis 61

Table 4.9 : Phytochemical Compounds Found in SPSE Using GCMS

Analysis 67

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x

LIST OF FIGURES

Figures Page

Figure 2.1 : The Stages of Periodontal Disease from Healthy Gums

Until Chronic Periodontitis 8

Figure 2.2 : The Morphology of P. gingivalis on Blood Agar Was

Black-Pigmented 12

Figure 2.3 : Chewing Stick of S. persica 17

Figure 3.1 : Flowchart of Research Methodology 29

Figure 4.1 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Rod-Shaped) from Subgingival Plaque Sample 1

31

Figure 4.2 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped and Aggregates in

Chains) from Subgingival Plaque Sample 2 31

Figure 4.3 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped) from Subgingival Plaque Sample 3

31

Figure 4.4 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped and Aggregates in Chains) from Subgingival Plaque Sample 4

32

Figure 4.6 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Rod-Shaped and Aggregates in Chains) from Subgingival Plaque Sample 6

32

Figure 4.7 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped) from

Subgingival Plaque Sample 7 33

Figure 4.8 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped) from

Subgingival Plaque Sample 8 33

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xi

Figure 4.9 : Bacteria Colonies on Blood Sheep Agar and Microscopic Analysis (Cocci Shaped) from Subgingival Plaque Sample 9

33

34

Figure 4.11 : Bacteria Colonies on WCA Agar and Microscopic Analysis (Rod-Shaped) from Subgingival Plaque

Sample 1 35

Figure 4.12 : Bacteria Colonies on BHI Agar and Microscopic Analysis (Cocci Shaped) from Subgingival Plaque Sample 2

36

Figure 4.13 : Bacteria Colonies on WCA Agar and Microscopic Analysis (Rod-Shaped) from Subgingival Plaque Sample 3

36

Figure 4.15 : Bacteria Colonies on WCA Agar and Microscopic Analysis (Cocci Shaped) from Subgingival Plaque

Sample 5 37

Figure 4.16 : Bacteria Colonies on WCA Agar and Microscopic Analysis (Rod Shaped) From Subgingival Plaque

Sample 6 37

37

Figure 4.18 : Bacteria Colonies on WCA Agar and Microscopic Analysis (Cocci Shaped) from Subgingival Plaque Sample 8

38 Figure 4.19 : Bacteria Colonies on BHI Agar and Microscopic

Analysis (Cocci Shaped) from Subgingival Plaque Sample 9

38

Figure 4.20 : Bacteria Colonies on BHI Agar and Microscopic Analysis (Rod-Shaped) from Subgingival Plaque Sample 10

38

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xii

Figure 4.21 : 1.5% Agarose Gel electrophoresis of PCR Using PCR Products After DNA Purification Obtained from Sample 1, 2, 3 and 4 in 100 V For 60 Minutes

54

Figure 4.22 1.5% Agarose Gel electrophoresis of PCR Using PCR Products After DNA Purification Obtained from Sample 4,6, and 7 in 100 V For 60 Minutes

54

Figure 4.23 : 1.5% Agarose Gel electrophoresis of PCR Using PCR Products After DNA Purification Obtained from

Sample 5 in 100 V For 60 Minutes 55

Figure 4.24 : 1.5% Agarose Gel electrophoresis of PCR Using PCR Products After DNA Purification Obtained from Sample 8 in 100 V For 60 Minutes

55

Figure 4.25 : 1.5% Agarose Gel electrophoresis of PCR Using PCR Products After DNA Purification Obtained from Sample 10 in 100 V For 60 Minutes

56

Figure 4.26 : Alignments of The Nucleotide Sequence for Sample 4

After The BLAST Program in NCBI Data for S. mutans 57

Figure 4.27 : Alignments of The Nucleotide Sequence for Sample 5 after The BLAST Program in NCBI Data for P.

gingivalis

57

Figure 4.28 : Phylogenetic Tree of Oral Bacteria Species from

Subgingival Plaque 58

Figure 4.29 : Inhibition Zone of S. mutans When Exposed to Different Concentrations of SPSE-Methanol After 24 Hours

60

Figure 4.30 : Inhibition Zone of P. gingivalis When Exposed to Different Concentrations of SPSE-Methanol After 24

Hours 60

Figure 4.31 : The Colour of MTT Was Change After S. Mutans

Treated with SPSE (MIC) 62

Figure 4.32 : The growth colonies of S. mutans treated with SPSE at (1) 25.00 mg/mL; (2) 12.50 mg/mL; (3) 6.25 mg/mL;

(4) 3.13 mg/mL; (5) 1.56 mg/mL; (6) 0.78 mg/mL; (7) 0.39 mg/mL; (8) 0.20 mg/mL on BHIA agar (MBC)

62

Figure 4.33 : The growth colonies of P. gingivalis treated with SPSE

at (1) 25.00 mg/mL; (2) 12.50 mg/mL; (3) 6.25 mg/mL; 63

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xiii

(4) 3.13 mg/mL; (5) 1.56 mg/mL; (6) 0.78 mg/mL; (7) 0.39 mg/mL; (8) 0.20 mg/mL on WCA agar (MBC)

Figure 4.34 : Chromatograms for SPSE-n-hexane from GCMS

Analysis 64

Figure 4.35 : Chromatograms for SPSE-DCM from GCMS Analysis 65

Figure 4.36 : Chromatograms for SPSE-Acetone from GCMS

Analysis 65

Figure 4.37 : Chromatograms for SPSE-Ethanol from GCMS

Analysis 66

Figure 4.38 : Chromatograms for SPSE-Methanol from GCMS

Analysis 66

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xiv

LIST OF APPENDICES

Appendices Page

Appendix 1 : Alignment of Nucleotide Sequence from All Samples

After BLAST Program in NCBI Data 87

Appendix 2 : Inhibition Zone of SPSE Against S. mutans and P.

gingivalis 111

Appendix 3 : Colour Changes for MIC After Added MTT Solution

Into 96-Well Plate 112

Appendix 4 : Growth Colonies of S. mutans and P. gingivalis on

BHI and WCA Agar for MBC 114

Appendix 5 : Conference 121

Appendix 6 : Publication 121

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xv

LIST OF UNITS OF MEASUREMENTS

Colony forming unit per millilitre CFU/mL

Gram G

Mass-to-charge ratio m/z

Micro litre µL

Milli gram Mg

Milli gram per millilitre mg/mL

Millilitre mL

Milli molar mM

Nanometre Nm

Svedberg S

Volt V

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xvi

LIST OF SYMBOLS

°C Celsius

= Equal

< Less than X Multiply

% Percentage

: Ratio

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xvii

LIST OF ABBREVIATIONS

A. actinomycetemcomitans Aggregatibacter actinomycetemcomitans

ADA american diabetes association

ANOVA analysis of variance

BHI brain heart infusion

BLAST basic local alignment search tool

Bp base pair

CHX Chlorohexidine

C. rectus Camphylobacter rectus

CXCL8 c-x-c motif chemokine ligand 8

DCM dichloromethane

DDA disc diffusion assay

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

E. clocae Enterobacter clocae

E. corrodens Eikenella corrodens

E. ludwigii Enterobacter ludwigii

E. mori Enterobacter mori

E. nodatum Eubacterium nodatum

E. roggen kampii Enterobacter roggen kampii

E. tabacii Enterobacter tabacii

F F-value

F. nucleatum Fusobacterium nucleatum

Gbp glucan binding protein

GCMS gas chromatography mass spectrometry

Gtf glycosyltransferase

IL interleukin

Kgp lyseine-specific

K. pneumoniae Klebsiella pneumoniae

MBC minimum bactericidal concentration

MIC minimum inhibitory concentration

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl tetrazolium bromide

NA not available

NCBI national centre for biotechnology

information

OD absorbance

P p-value

PBS phosphate-buffered saline

PCR polymerase chain reaction

P. gingivalis P. gingivalis

pH potential of hydrogen

P. intermedia Prevotella intermedia

P. micros Peptostreptococcus micros

QS quorum sensing

Rgp arginine-specific

Rpm revolutions per minute

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xviii

rRNA ribosomal ribonucleic acid

S. agalactiae Streptococcus agalactiae

S. anginosus Streptococcus anginosus

SD standard deviation

S. downei Streptococcus downei

S. gordonii Streptococcus gordonii

S. lutetiensis Streptococcus lutetiensis

S. mutans Streptococcus mutans

S. oralis Streptococcus oralis

S. persica Salvadora persica

spp. several species

SPSE Salvadora persica stem extract

SPSS statistical package for the social sciences

S. salivarius Streptococcus salivarius

S. sanguinis Streptococcus sanguinis

TAE tris-acetate-EDTA

T. denticola Treponema denticola

T. forsythia Tannerella forsythia

TGF-β transforming growth factor beta

Tm temperature

TNF-α tumour necrosis factor alpha

TNTC too-numerous-to-count

USIM university sains islam malaysia

WCA wilkins chalgren anerobic

WHO world health organization