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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 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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  • Download PDF (Inglês)

    Download PDF (Inglês)

    Revista Brasileira de Farmacognosia 28 (2018) 9–15 ww w.elsevier.com/locate/bjp Original Article Anatomy and microscopy of Piper caldense, a folk medicinal plant from Brazil a b c c Vera Lucia P. dos Santos , Vijayasankar Raman , Vanessa B. Bobek , Izabel P. Migacz , d b c,∗ Célia Regina C. Franco , Ikhlas A. Khan , Jane M. Budel a Escola Superior de Saúde, Biociência, Meio Ambiente e Humanidades, Centro Universitário Internacional Uninter, Curitiba, Paraná, Brazil b National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, Oxford, MS, USA c Programa de Pós-graduac¸ ão em Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brazil d Departamento de Biologia Celular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil a r a b s t r a c t t i c l e i n f o Article history: Piper caldense C. DC., Piperaceae, commonly known as “pimenta-d’água”, “pimenta-darda” or Received 27 September 2017 “paguarandy” in Brazil, is a shrub that grows mainly in humid and shaded habitats. The present study Accepted 25 November 2017 investigates the anatomy of the leaves and stems of P. caldense by light and scanning electron microscopy Available online 15 December 2017 in order to provide supporting data for correct identification of the species. The leaves are hypostomatic, have a 2-layered hypodermis, and posses pearl glands. The midrib shows a ‘U’-shaped stele comprised Keywords: of about ten collateral vascular bundles. The main anatomical marker of the stem is the presence of a Anatomy continuous sclerenchymatous sheath in the pith.
  • Rice Fermentation Starters in Cambodia: Cultural Importance and Traditional Methods of Production

    Rice Fermentation Starters in Cambodia: Cultural Importance and Traditional Methods of Production

    Southeast Asian Studies, Vol. 49, No. 2, September 2011 Rice Fermentation Starters in Cambodia: Cultural Importance and Traditional Methods of Production YAMAMOTO Sota* and MATSUMOTO Tetsuo** Abstract This paper focuses on the processes of producing fermentation starters in Cambodia, in order to explore the dispersal routes of starters in Southeast Asia. Spices, herbs, and a sweet ingredient are widely used to make starters in Cambodia, and many people put new starters on rice husks or straw. These widely distributed techniques may have originated in one place and later dispersed throughout Southeast Asia. Two different production processes are used in Cambodia: one based on a “rice wine culture”—character- ized by not using rice liquor, not using old starters, using leaves and branches to cover the starters, and not drying the starters; and the other based on a “rice liquor culture”—characterized by the use of rice liquor (blown over the starters), old starters (scattered over new starters and/or mixed with rice powder), and the addition of sugar without using plant materials. “Rice wine culture” seems to be the older type of process in Cambodia, and new techniques related to the “rice liquor culture” probably infiltrated the region later. The use of plants and the rituals related to starter production are very important in understanding the dispersal routes of starters in Southeast Asia. Keywords: amylolytic starter, capsicum (chili peppers), charcoal, dispersal routes, ethnobotany, rice liquor, rice wine, spices I Introduction Malted rice, or koji-cake—a fermentation starter in the form of a hard ball made from rice or other cereals—is thought to have originated in China, probably in the Yellow River basin [Hanai 1992: 62], 3,000–4,000 years ago [Ueda 1999: 89–90].