ANTI-QUORUM SENSING ACTIVITY OF MEDICINAL PLANTS [braheem Saheed Adebayo (Matric No. 3120280) Dissertation Submitted in Partial Fulfillment for the degree of MASTER OF SCIENCE (FOOD BIOTECHNOLOGY) Faculty of Science and Technology UNIVERSITI SAINS ISLAM MALAYSIA Nilai June 2014 II AUTHOR DECLARATION I 4i ý I hereby declare that the work in this dissertation is my own, except for the quotations and summaries which have been duly acknowledged. Date: June 2014 Signature: Name: lbraheem Saheed Adebayo Matric No: 3120280 Address: V3A-10-02A, Sri Palma villa, Jalan Persiaran UTL, Lagenda 71700 Mantin, Negeri Sembilan, Malaysia. iii AUTHOR'S BIODATA Saheed Adebayo Ibraheem (Matric No. 3120280 and Passport No. A04045946) is a Nigerian by nationality. He was born on the 5th January, 1981 at Kaduna in Northern part of Nigeria; though his parents were originally from South Western part. He was the senior prefect of Okere Secondary Grammar School (Saki West Local Government Area of Oyo state) in 1998 and he obtained his first degree; Bachelor of Science (Human Physiology) from University of llorin, Nigeria in 2003. With the aim of exploring knowledge and skills in the field of science, he proceeded to Universiti Sains Islam Malaysia in February 2013 to continue his Master Degree in Food Biotechnology. He is currently staying at V3A-10-02A, Sri Palma Villa, Jalan Persiaran UTL Lagenda, 71700 Mantin, Negeri Sembilan, Malaysia. iv ACKNOWLEDGEMENT All thanks, praise and adorations are due to all mighty Allah SWT who gave me all it requires (strength, knowledge and wisdom) towards the completion of my study. I affirm that completion of this study (MSc. ) is due to his mercy and I say Alhamdulilah for everything. I consider it a great privilege to have been given an opportunity to come and study at Universiti Sains Islam Malaysia, and for that, I'm grateful to the entire university community. I sincerely thank and appreciate the effort of my supervisor Prof. Abdul Jalil Bin Abdul Kader on the success of this research work. He was not just a supervisor but also a father with enough humility, wisdom, knowledge and patience to be emulated from. You will forever be remembered as the gentle approach and dedication i got from you will be put into practice anywhere i found find myself. Faculty of Science and Technology Universiti Sains Islam Malaysia is blessed with outstanding staffs that have large heart to accommodate people; so i appreciate the collective efforts of the staffs (teaching and non-teaching) as it was their approach that made it possible for me. Mr Fredy Kesnawan Shah bin Abdul Kahar and Ms Sarina Irma binti Saidon, I thank you both for you have always made yourselves available to us. I have this special appreciation from within to Assoc. Prof. Dr Zaiton binti Hassan and Assoc. Prof PM Chek Zaini. You both handled us (especially myself) like children. This exceptional relationship has indeed allowed us to gain lots of things from you; coupled with enormous knowledge you impacted in us while in class; thank you so much. Special thanks to Dr. Hanina binti Mohd Noor, the Head of the Programme; and every other lecturer in the Faculty of Science and Technology; Prof. Emeritus Jailani, Dr. Zazarina binti Ramly (my smart lecturer), Dr. Maryam binti Mohd Rehan and Dr. Nazmi to mention but few. Lots of people in my life contributed one way or the other to the success of this study progrmme. I appreciate you all and all your contributions are duly acknowledged but the whole of this is dedicated to my late parents; especially my angel mother. I pray you both continue to rest in peace. I'm still feeling your absence. V ABSTRAK Kemunailan bakteria rintangan pelbagai dadah telah membawa kepada pencarian pelbagai tumbuhan ubatan tempatan untuk aktiviti anti- penderiaan kuorum mereka. Sebatian anti- penderiaan kuorum telah dianggap sebagai novel yang bermakna untuk membendung tindakan kebisaan daripada bakteria rintangan pelbagai dadah kerana sebatian ini tidak mengenakan apa-apa tekanan terpilih pada bakteria . Spaciosa parkia, Centella asiatica dan Piper nigrum adalah tumbuhan yang terkenal di rantau tropika dengan banyak dilaporkan kepentingan perubatan. Oleh itu kajian ini dijalankan untuk menyaring tumbuh-tumbuhan untuk aktiviti anti- penderiaan kuorum menggunakan ekstrak metanol dan etanoli. Organisma pelapor dalam kuorum sensing Chromobacterium violaceum telah digunakan sebagai biomonitor organisma. Aktiviti anti- penderiaan kuorum tumbuh-tumbuhan terpilih diuji ke atas C. violaceum ATCC 12472 menggunakan asai agar difusi. Keputusan tiga eksperimen yang dilakukan pada tumbuhan menunjukkan zon boleh diperhatikan daripada perencatan cabutan dari kedua-dua pelarut kecuali ekstrak methanol P. nigrum yang tidak menunjukkan apa- apa perencatan walaupun pada kepekatan 800mg/ml. Kesan sensing Anti- kuorum telah dibezakan daripada kesan antibakteria dengan membandingkan zon jelas perencatan dihasilkan sebagai akibat daripada tindakan tetracycline dengan zon mendung perencatan yang dihasilkan oleh ekstrak dari tumbuh-tumbuhan yang terpilih . Walau bagaimanapun, ekstrak etanoli P. spaciosa menunjukkan aktiviti anti- bakteria pada kepekatan yang lebih tinggi sedikit (300mg/ml). Zon-zon diperhatikan perencatan adalah kepekatan bergantung dan minimum kepekatan perencatan QS telah ditubuhkan bagi setiap ekstrak tumbuhan. Ini menunjukkan bahawa jika diterokai dengan teliti; ekstrak tumbuh-tumbuhan yang terpilih boleh digunakan sebagai agen anti- kevirulenan. Kata kunci: Parkia spaciosa, Centella asiatica, Piper nigrum, Kuorum penderiaan, penderiaan Anti- kuorum. vi ABSTRACT The arising of multidrug resistance bacteria have led to the screening of various local medicinal plants for their anti-quorum sensing activity. Anti-quorum sensing compounds have been considered as a novel means to fight against these multidrug resistance bacteria as these compounds do not impose any selective pressure on the bacteria. Parkia spaciosa, Centella asiatica and Piper nigrum are well-known plants of tropical region with many reported medicinal importance. Therefore this study was carried out to screen the plants for anti-quorum sensing activity using methanolic and ethanolic extracts. A reporter organism in quorum sensing Chromobacterium violaceum was used as biomonitor organism. Anti-quorum sensing activities of the selected plants were tested on C. violaceum ATCC 12472 using agar well diffusion assay. The results showed observable zones of inhibition for both extracts except for the P. nigrum which showed no inhibition at the concentration of 800mg/ml of methanolic extract. Furthermore, comparison analysis between anti-quorum activity and antibacterial effects were carried out by comparing clear zone of inhibition produced as a result of tetracycline action with the cloudy zone of inhibition produced by the extract from the selected plants. However, ethanolic extract of P. spaciosa showed antibacterial activity at a slightly higher concentration (300mg/ml). The observed zones of inhibition were concentration dependent and minimum of QS inhibition concentration were established for each plant extract. This result suggests that the extract of the selected plants can be used as anti-virulence agents. Keywords: Parkia spaciosa, Centella asiatica, Piper nigrum Quorum sensing, Anti- quorum sensing. vii ýýA äJýl äulzll ýI; LJI ýý ýý ýl öý_Lý11 `ý9ýý äA9Li1l L, ÄS. JI vyLr ýýl Ln1 ; -L-L, ä_9 yLQ11 öýLý ä. sLl ýLS, JI a*-'9 Anti-quorum v, oý. a, y ýLS, 11 oW i) Y öý a+c ýl Ä'9ýý äP9Lä1l ýLý.:, ý., il ýý äýLJI ÄýL.. JI äJl*cll äýlsL 9 LýLM I ýLL.: ýS ý P. spaciosa 0 Ä99,, ý. Le Ia, j, .i ýrl ýýL'LJI `ýcJ äe 111 o LA `, ýal ý11 L19 . ä_. bJl Lg: ýl ý. l, Lýe11 ý ä; ýyl ääm: 11 -L"9 äJ9; lxýyl9 äJýLJ l ýl ý. J lldý ä oý: ,P anti-quorum L) L5ý I L. r Jl anti-quorum L3 Ä,.; JI C :, L, 'LJI 5., 11 biomonitor I.. L. r La1.:,; . u, pTI 12472 jc ý,,.. mg/m1800 _L=:, si e4.0.; lc -Lttf änl. ý- v. lr I j,: l lJ. o. c , gJýs 1 ji P. nlgr-um `,.. ýýI äýlo 91 äýal9 ý. ; ýtä1z . ä; ýLä o JI& anti-quorum , u: I. -ý , lý. LJi P. spaciosa tji mg/m1300 SL, aA Z; u `}.;:. t; i QS P. spacrosa ý,; ýI L.., =J. I "v. ý LG ý ýýyý viii TABLE OF CONTENTS CONTENTS TITLE PAGE AUTHOR DECLARATION AUTHOR'S BIODATA ACKNOWLEDGEMENT ABSTRAK ABSTRACT MULAKHAS TABLE OF CONTENT LIST OF FIGURE LIST OF TABLES ABBREVIATIONS APPENDICES CHAPTER 1: INTRODUCTION 1.1 Problem statement 1.2 Objectives of the study CHAPTER II: LITERATURE REVIE 2.1 Parkia spaciosa 2.1.1 Physical properties of Parkia spaciosa 2.1.2 Chemical composition of Parkia spaciosa 2.1.3 Nutritional value of Parkia spaciosa 2.1.4 Medicinal importance of Parkia . spaciosa 2.1.4.1 Antioxidant activity of P. spaciosa 2.1.4.2 Antiangiogenic property of P. spaciosa 2.1.4.3 Hypoglycemic effect of P.. spaciosa 2.1.4.4 Antimicrobial activity of P. spaciosa 2.1.5 Excretion and toxicity of Parkia spaciosa Page i ii 111 iv V vi V11 viii xi Xll X111 XIV I 5 5 6 6 7 8 9 9 9 10 I1 13 13 ix 2.2 Ceniella asialica 2.2.1 Physical properties of Centella asiatica 2.2.2 Chemical composition of Centella asiatica 2.2.3 Nutritional composition of Centella asiatica 2.2.4 Medicinal importance of Centella asialica 2.2.4.1 Antimicrobial effect of C. asialica 2.2.4.2 Stimulating effect on the brain 2.2.4.3 Effect on wound healing 2.2.4.4 Anticancer effect 2.2.4.5 Preventive effect on gastric ulcer 2-2.5 Toxicity of Centella asiatica 2.3 Piper nigrum 2.3.1 Physical properties of Piper nigrum 2.3.2 Chemical composition of Piper nigrum 2.3.3 Nutritional value of Piper nigrum 14 15 16 17 17 17 18 19 19 20 21 21 23 23 24 2.3.4 Medicinal importance of Piper nigrum 25 2.3.4.1 Anti-inflammatory function of P. nigrum 25 2.3.4.2 Antimicrobial activity of P. nigrum 25 2.3.4.3 Antimutagenic and antitumor activity of P. nigrum 26 2.3.4.4 Antioxidant activity of P. nigrum 27 2.4 Quorum sensing 28 2.4.1 General mechanism of quorum sensing 29 2.4.2 Classification of autoinducers 29 2.4.2.1 Acylated Homoserine Lactone 30 2.4.2.2 Peptides 30 2.4.2.3 Furanosyl borate diester 31 2.4.2.4 Autoinducer type 3 32 2.4.3 Quorum sensing in Gram negative bacteria 33 2.4.4 Quorum sensing in Gram positive bacteria 35 s 2.5 Anti-quorum sensing 2.5.1 Mechanism of anti-quorum sensing 2.6 Chromobacterium violaceum 2.6.1 Quorum sensing in Chromobacterium violaceum CHAPTER III: MATERIALS AND METHODS 3.1 Collection of plant extracts 3.2 Extract preparation 3.2.1 Drying 3.2.2 Extraction 3.3 Biomonitor organism 3.4 Anti-quorum sensing test 3.5 Test for minimum of QS inhibitory concentration 3.6 Anti-quorum sensing activity and antibacterial effect 3.7 Statistical analysis CHAPTER IV: RESULTS AND DISCUSSION 4.1 Anti-quorum sensing activity of Parkia spaciosa 4.2 Anti-quorum sensing activity of Centella asiatica 4.3 Anti-quorum sensing activity of Piper nigrum 4.4 Minimum of QS inhibitory concentration 4.5 Anti-quorum sensing activity and antibacterial effect 4.6 Discussion CHAPTER V: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusion 5.2 Recommendation References Appendices 36 37 39 40 42 42 42 42 43 43 44 46 47 47 48 48 51 54 56 57 59 66 66 67 69 88 xi LIST OF FIGURES Page Figure 2.1: P. spaciosa attached to the stalk and P. sapciosa seeds 7 Figure 2.2: C. asiatica leaves with its petioles 16 Figure 2.3: Ripping Piper nigrum 22 Figure 3.1: Schematic illustration of extract preparation 45 Figure 3.2: Schematic illustration of experimental procedure 47 Figure 4.1: Bioassay of the methanolic extract of P. spaciosa for anti-quorum sensing activity with C. violaceum ATCC 12472 50 Figure 4.2: Bioassay of the ethanolic extract of P.. spaciosa for anti-quorum sensing activity on C. violaceum ATCC 12472 50 Figure 4.3: Anti-quorum sensing activity of P. spaciosa 51 Figure 4.4: Bioassay of the methanolic extract of C. asiatica for anti-quorum sensing activity with C. violaceum ATCC 12472.52 Figure 4.5: Bioassay of the ethanolic extract of C. asiatica for anti-quorum sensing activity with C. violaceum ATCC 12472.53 Figure 4.6: Anti-quorum sensing activity of C. asiatica 54 Figure 4.7: Bioassay of the methanolic and ethanolic extract of P. nigrum for anti-quorum sensing activity with C. violaceum ATCC 12472.55 Figure 4.8: Anti-quorum sensing activity of P. nigrum 56 Figure 4.9: Anti-quorum sensing activity and antibacterial effect 58 Figure 4.10: Minimum of QS inhibitory concentration of the selected medicinal plants 59 xii LIST OF TABLES Page Table 4.1: Zones of inhibition produced by methanolic and ethanolic extracts on P. spaciosa on C. violaceuni ATCC 12472 51 Table 4.2: Zones of inhibition produced by methanolic and ethanolic extracts on C. asiatica on C. violaceum ATCC 12472 53 Table 4.3: Zones of inhibition produced by methanolic and ethanolic extracts on P. nigrum on C. violaceum ATCC 12472 55 Table 4.4: Minimum of QS inhibitory concentration (mg/ml) value for the selected plants extracts 57 xiii APPENDICES Pages Appendix 1: Analysis of extracts of P. sjpaciosa by One-Sample t-test 88 Appendix 2: Analysis of extracts of C. asiatica by One-Sample t-test 89 Appendix 3: Analysis of extracts of P. nigrum by One-Sample t-test 90 xiv ABBREVIATION QS Quorum sensing FRAP Ferric iron antioxidant potential HUVEC Human umbilical vein endothelial cell GC-MS Gas chromatography mass spectrometer CNS Central nervous system LPS Lipopolysaccharide 1L6 Interleukin 6 PGE2 Prostaglandin E2 DPPH 2,2-diphenyl- I -picrylhydrazyl ABTS 2,2-azinobis-3-ethylbenzothiazolina-6-sulfonic acid AHL Acylhornoserine lactone DNA Deoxyribonucleic acid AIP Autoinducer peptide CSP Competent stimulating peptide QSI Quorum sensing inhibitor LB Luria bertani STA Soft top agar DMSO Dirnethyl sulfoxide SAM S- adenosyl methionine ml milliliter µl microliter