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ANTIBACTERIAL ACTIVITY of Persicaria Minor (Huds.) LEAF-EXTRACTS AGAINST BACTERIAL PATHOGENS

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ANTIBACTERIAL ACTIVITY of Persicaria Minor (Huds.) LEAF-EXTRACTS AGAINST BACTERIAL PATHOGENS ANTIBACTERIAL ACTIVITY OF Persicaria minor (Huds.) LEAF-EXTRACTS AGAINST BACTERIAL PATHOGENS MUSA AHMED ABUBAKAR UNIVERSITI TEKNOLOGI MALAYSIA ANTIBACTERIAL ACTIVITY OF Persicaria minor (Huds.) LEAF-EXTRACTS AGAINST BACTERIAL PATHOGENS MUSA AHMED ABUBAKAR A dissertation submitted in partial fulfillment of the Requirements for the award of Master of Science (Biotechnology) Faculty of Biosciences and Medical Engineering Universiti Teknologi Malaysia JANUARY 2015 iii DEDICATION To AR-RAZAQ The provider of assets and all Biotechnogists and Microbiologists who work assiduously towards ensuring the Nutritional values and Antimicrobial actions of naturally occurring plants & HIS EXCELLENCY ENGR. DR. RABIU MUSA KWANKWASO for providing the scholarship and may the blessings of Allah continue to follow him throughout his future endeavour- Amen. iv ACKNOWLEDGEMENT A research dissertation such as this, usually involves the efforts of many. I would like to start by expressing my profound gratitude to god Almighty Allah, the creator of plants, animals and tiny giants such as microbes and to whom all our praise is due, for making this journey up to the conclusion of my Masters degree, a relatively smooth and successful one. I also wish to express my sincere appreciation to my versatile supervisor, Dr. Razauden Bin Mohamed Zulkifli, for his encouragement, guidance, criticism and friendship without whose support, this research wouldn’t have been as presented here. I also admire and thank my respected parents, Alh. Modu Bukar and Haj. Rashidah Abubakar; without whom, I would not have the chance to understand the beauty of our universe and the tue meaning of love and patience. To this extent, I owe all the nice and valuable moments of my life to them. Leaving no stone unturned, I am so grateful to my darling wife, Hafsat Ahmad Maigida, for her kindness and never-ending motivations and encouragement; without her understanding and patience, I would’t have been able to dedicate my time towards the path of greater success in absolute tranquility. May god Almighty continue to bless her- Amen. May I also use this opportunity to recognize and appreciate the support of fellow postgraduate students at Nutritional Biochemistry Laboratory who in one way or the other assisted in the successful accomplishment of my research work and finally to all my family members especially Ismael Ibrahim and Fatima Moh’d Abubakar, for their moral and encouragement throughout my study in Malaysia. v ABSTRACT Persicaria minor (Huds.) Opiz known as Small water-pepper and well recognized locally in Malaysia as “daun kesum” is an edible vegetable with nutritional and medicinal benefits utilized generally by South-east Asians. The present study was conducted to evaluate the antibacterial activity of standardized aqueous-ethanolic and water extracts of P. minor leaves. The leaves of the plant undergone extraction based on Malaysian Standard Guideline which is 30% aqueous- ethanol and absolute water as normally used in traditional medicine to produce the respective extract concentrates. Both extracts were evaluated for total protein and polysaccharide contents in which aqueous-ethanolic extract was found to possess high contents of proteins (1713.67 µg/ml) while contents of polysaccharides were high in absolute water extract (17.6 µg/ml). These measurements were used as a standard for different batch extract. The extracts were then tested against four standard strains of bacteria which are Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 11229, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 at different concentrations using disc- diffusion (qualitative) and microplate dilution (quantitative) assays. For positive and negative control, penicillin and dimethylsulfoxide were used as controls, respectively. Both extracts showed antibacterial activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values in the range of 50 to 100 mg/mL against S. aureus, E. faecalis, and E. coli, respectively with aqueous-ethanolic extract being more potent. However, none of the extracts were active against P. aeruginosa. Results from this study truly illustrated high potential of P. minor leaves as natural antibacterial agent for the elimination of various bacterial disease and infections. vi ABSTRAK Sejumlah besar herba telah digunakan sebagai sumber pemakanan dan perubatan berasaskan tumbuhan dan dianggap berperanan dalam meningkatkan taraf kesihatan . Daun kesum dikenali sebagai Kecil waater-lada dan juga dikenali di Malaysia sebagai adalah sayuran yang membekalkan manfaat nutrisi dan perubatan yang diambil oleh kebanyakan penduduk Asia Tenggara. Kajian ini dijalankan bagi menganalisa aktiviti antibakteria daripada ekstrak daun P. minor iaitu ekstrak akues- etanol dan ekstrak. Untuk penghasilan ekstrak, daun diesktrak mengikut Garis Panduan Piawai Malaysia di mana 30% akeus-etanol digunakan dan juga teknik ekstrak air yang sering digunakan dalam perubatan tradisional. kandungan jumlah protin dan dan didapati ekstrak ekues-ethanol mempunyai kandungan protein yang tinggi (1713,67 μg / ml) manakala kandungan polisakarida yang tinggi dalam ekstrak air (17.6 μg / ml). Sukatan ini telah digunakan sebagai piawai untuk kumpulan ekstrak yang berbeza. Ekstrak kemudiannya diuji terhadap empat jenis bacteria piawai iaitu yang Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 11229, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 pada kepekatan yang berbeza menggunakan kaedah kualitatif (resapan cakera) dan kaedah kuantitatif (pencairan papan kecil) dengan penisilin digunakan sebagai kawalan positif dan dimethylsulfoxide (pembawa) sebagai kawalan negatif. Kedua- dua ekstrak menunjukkan aktiviti antibakteria dengan nilai kepekatan perencatan minimum (MIC) dan kepekatan bakteria minimum (MBC) masing-masing dalam lingkungan 50 hingga 100 mg/mL terhadap S. aureus, E. faecalis, dan E. Coli dengan ekstrak akueus-etanol menunjukkan kesan yang lebih ketara. Walau bagaimanapun, tiada ekstrak aktif terhadap P. aeruginosa. Oleh itu, keputusan yang diperolehi dalam kajian ini telah menunjukkan nilai-nilai nutrisi potensi tinggi daun P. minor untuk digunakan sebagai agen antibakteria semulajadi bagi menghapuskan kebanyakan penyakit dan jangkitan bawaan bakteria. vii TABLE OF CONTENTS CHAPTER TITLE PAGE DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv ABSTRACT v ABSTRAK vi TABLE OF CONTENTS vii LIST OF TABLES x LIST OF FIGURES xi LIST OF SYMBOLS/ABBREVIATIONS xii LIST OF APPENDICES xv 1 INTRODUCTION 1 1.1 Background of the Study 1 1.2 Statement of Research Problem 3 1.3 Significance of the Study 4 1.4 Objectives 4 1.5 Scope of the Study 4 2 LITERATURE REVIEW 5 2.0 General Aspects about Medicinal Plants 5 2.1 Traditional Medicine 8 2.2 Problem and Prospect of Traditional Medicine 9 2.3 Biology of Target Plant: Persicaria minor 10 2.3.1 Scientific Classification 10 viii 2.3.2 Characteristics and Distribution of Persicaria minor 10 2.3.3 Phytochemicals of Persicaria minor 12 2.3.4 Pharmacological Properties of Persicaria minor 13 2.4 General Applications of Persicaria minor (Huds.) 14 2.4.1 Uses of Persicaria minor in Cooking 14 2.4.2 Traditional Uses and other Applications of Persicaria minor 15 2.5 Extraction Techniques and Standardization of Plant Extracts 16 2.6 Pathogenicity and Lab Features of the Research Bacteria 19 2.6.1 Staphylococcus aureus 19 2.6.2 Enterococcus faecalis 20 2.6.3 Escherichia coli 21 2.6.4 Pseudomonas aeruginosa 22 3 MATERIALS AND METHODS 24 3.1 Collection of Plant Material and Preparation 24 3.2 Plant Extraction Technique 24 3.3 Standardization of the Extracts 25 3.3.1 Total Protein Estimation 25 3.2.2 Total Polysaccharide Estimation 26 3.4 Preparation of Different Concentrations of the Extracts and Standard agents Used for Antibacterial Assay 27 3.5 Paper Disc Preparation 27 3.6 Bacterial Organism and Culture Media 27 3.7 Preparation of McFarland Standard and Saline Solutions 28 3.8 Standardization of Bacterial Stock Culture 28 3.9 Disc Diffusion Susceptibility Test 29 3.10 Determination of Minimum Inhibitory ix Concentration 30 3.11 Determination of Minimum Bactericidal Concentration 30 3.12 Data Analysis 31 3.13 Methodology Work-flow Chart 31 4 RESULTS AND DISCUSSION 32 4.0 Introduction 32 4.1 Extraction Outcome of Persicaria minor Leaves 33 4.2 Standardization for Total Protein and Polysaccha- ride Estimation 33 4.3 Antibacterial Activity of Persicaria minor Leaf-extracts 36 4.3.1 Minimum Inhibitory Concentration Test (MIC) 41 4.3.2 Minimum Bactericidal Concentration Test (MBC) 41 4.3.3 Consequence of Using Different Concen- tration of the Extracts 44 4.3.4 Effects of Using Different Extraction Solvents 44 4.3.5 Antimicrobial Susceptibility of Bacterial Isolates 45 5 CONCLUSION AND RECOMMENDATION 47 5.1 Conclusion 47 5.2 Recommendations 48 LIST OF REFERENCES 49 APPENDICES 59-66 x LIST OF TABLES TABLE NO. TITLE PAGE 1.1 Persicaria minor Description 2 2.1 Solvents for Active Components Extraction 18 4.1 Comparison of Total Contents of Protein and Polysaccharide of 30% aqueous-ethanolic and 100% aqueous extracts of P. minor leaves 34 4.2 Antibacterial Evaluation of Persicaria minor Leaf-extracts against Pathogenic Bacterial Isolates 37 4.3 Results of Minimum Inhibitory Concentration (mg/ml) of P. minor leaf-extracts 41 xi LIST OF FIGURES FIGURE NO. TITLE PAGE 2.1 Fresh Leaves of P. minor immediately after purchase 11 2.2 Geographical regions of Southeast Asia 12 4.1 Standard Calibration Curve of BSA from which Total Protein Contents was Estimated 34 4.2 Standard calibration curve of glucose from which Total Protein Contents was Estimated 35 4.3 Zones of inhibition produced by S. aureus (A), E. coli (B) and E. faecalis (C) Against aqueous-ethanolic extract of P. minor 39 4.4 Zones of inhibition produced by S. aureus (A), E. coli (B) and E. faecalis (C) Against water-extract of P. minor 40 4.5 MBC results for aqueous-ethanolic Extract Against S. aureus (A), E. coli (B) E. faecalis (C) 42 4.6 MBC results for water-extract against S. aureus (A), E. coli (B) and E. faecalis (C) 43 4.7 MBC results for Penicillin Antibiotic agent against S.
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