ANTIBACTERIAL ACTIVITIES OF PHYTOCHEMICAL MIXTURE EXTRACTED FROM CLEOME GYNANDRA AND CLEOME VISCOSA STUDENTS MINI PROJECT Submitted to Tamil Nadu State Council for Higher Education By R. RAJAKUMARI, II M.Sc. DEPARTMENT OF ZOOLOGY QUEEN MARY’S COLLEGE (AUTONOMOUS) CHENNAI - 600 004 MARCH - 2018 ACKNOWLEDGEMENT I thank the Almighty for giving me the health, strength and courage, which enable me to successfully complete this research project. I am grateful to our principal Dr. (Mrs.) S. Santhi, Queen Mary’s College (Autonomous), Chennai-600 004, for providing the facilities to carry out my research work. My heartfelt thanks and gratitude to Dr. (Mrs.) Malathi, E. Associate Professor and Head, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004, for their valuable suggestions during the study period. I articulate my deep felt thanks to Dr. (Mrs.) S. Santhi, Assistant Professor, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004. She has been a constant source of inspiration with her valuable guidance, encouragement and critical suggestions throughout the course of the study. I am grateful to all the Staff Members of Department of Zoology, Queen Mary’s College, Chennai for their valuable inputs and encouragements. I wish to express my heartfelt gratitude to my father Mr. S. Ravichandiran, my mother Mrs. R Umaiyal, and especially all my family members for their moral support, blessings, encouragement and for being the source of inspiration who were the wind beneath my wings at every step in all endeavors. I also extend my heart full thank to my classmates and friends for their help and co- operation during the tenure of the work. I wish to thank the Lab Assistants, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004 for their timely assistance. I extend my sincere thanks to Mr A. Gnanavel, Lab. Technician, Kumaran Hospital, Chennai for providing the bacteria culture samples. With immense pleasure, I thank Mrs. D. Evangeline and Mrs. P. Nijanthi, Ph.D. Research Scholars for their generous help, support and cooperation during the research work. I wish to express my heartfelt gratitude to Mr. C. Palanisamy, SHV Energy Private Limited, Chennai – 600 017 for having given me the inputs and helping me in preparing the project report. I sincerely thank Tamil Nadu State Council for Higher Education, Lady Willington College Campus, Chennai for providing me the opportunity by funding this mini project. R. RAJAKUMARI Dr S. SANTHI, Department of Zoology, Research Supervisor Queen Mary’s College (Autonomous), Chennai - 600 004. CERTIFICATE This is to certify that the Students Mini Project entitled “Antibacterial Activities of Phytochemical Mixture Extracted from Cleome gynandra and Cleome viscosa” submitted to Tamil Nadu State Council for Higher Education is a bonafide record of research work done by R. RAJAKUMARI (Reg. No.1655006), ІІ M.Sc., Department of Zoology, Queen Mary’s College (Autonomous), Chennai during 2017 - 2018 and this work is carried out under my guidance and supervision. Research Supervisor Head of the Department (Dr. S. Santhi) DECLARATION R. RAJAKUMARI, B.Sc. Department of Zoology, Queen Mary’s College (Autonomous), Chennai- 600 004. I, hereby, declare that this Students Mini Project entitled “Antibacterial Activities of Phytochemical Mixture Extracted from Cleome gynandra and Cleome viscosa” submitted to Tamil Nadu State Council for Higher Education is a record of independent research work carried out by me under the guidance of Dr. (Mrs) S SANTHI, Assistant Professor, Department of Zoology, Queen Mary’s College (Autonomous), Chennai - 600 004, during the period 2017 - 2018 Place: Chennai (R. RAJAKUMARI) Date: 16- 03- 2018 CONTENTS PAGE S. No. TITLES No. LIST OF TABLES LIST OF FIGURES LIST OF PLATES 1.0 INTRODUCTION 1 1.1 Cleome gynandra (L.) 3 1.1.1. Scientific Classification 3 1.1.2. Medical Importance 4 1.2. Cleome viscosa (L.) 4 1.2.1. Scientific Classification 4 1.2.2. Medical Importance 5 1.3. Phytochemical and Antibacterial Activity 6 2.0 OBJECTIVES 7 3.0 REVIEW OF LITERATURE 8 4.0 MATERIALS AND METHODS 10 4.1 Collection of Plants 10 4.2 Preparation of Aqueous Leaf Extract 10 4.3 Phytochemical Screening of C. gynandra and C. viscosa 10 4.3.1 Detection of Alkaloids (Wagner’s Test) 12 4.3.2 Detection of Reducing Sugar (Fehling’sTest 12 4.3.3 Detection of Glycosides, (Borntrager’s Test) 12 4.3.4 Detection of Protein (Biuret’s Test) 12 4.3.5 Detection of Amino Acid (Ninhydrin Test) 12 4.3.6 Detection of Saponins (Foam Test) 13 4.3.7 Detection of Steroids (Salkowski Tests) 13 4.3.8 Detection of Tannins (Ferric chloride reagent test) 13 4.3.9 Detection of Flavonoids (Ferric chloride reagent test) 13 4.3.10 Detection of Anthraquinone (Borntrager’s reaction) 16 4.4 Selection of Microorganisms 16 4.5 Antibacterial Activity Assay 16 4.5.1 Preparation of Inoculum 16 4.5.2 Agar Disc Diffusion Method 16 4.6 Statistical Analysis 17 5.0 RESULTS AND DISCUSSION 18 Results of Phytochemical Screening of Cleome 5.1 gynandra and Cleome viscosa 18 5.2 Results of Antibacterial Activities of C. Gynandra and C. Viscosa 20 6.0 SUMMARY 25 7.0 CONCLUSION 27 8.0 REFERENCES 28 LIST OF TABLES TABLE PAGE TITLE No. No. Phytochemical Screening of Aqueous Leaf Extract of C. gynandra 1. 19 and C. viscosa 2. Antibacterial Activity of Leaf Extract of C. gynandra and 21 C. viscosa LIST OF FIGURES FIGURE PAGE TITLE No. No. Zone of Inhibition (Mean) of C. gynandra and C. Viscosa 1. Against Gram Negative Bacteria Klebsiella pneumonia and 23 Gram Positive Bacteria Staphylococcus aureus LIST OF PLATES PLATE PAGE TITLE No. No. 11 1. Cleome gynandra 11 2. Cleome viscosa Phytochemical Analysis of C. gynandra and C. viscosa 14 3. Aqueous Leaf Extract Phytochemical Analysis of C. gynandra and C. viscosa 15 4. Aqueous Leaf Extract Zone of Inhibition of C. gynandra and C. Viscosa 22 5. Against Gram Negative Bacteria Klebsiella pnumonia Zone of Inhibition of C. Gynandra and C. Viscosa Against 22 6. Gram Positive Bacteria Staphylococcus aereus 1. INTRODUCTION India has a flora that is widely distributed throughout the country which is one of the richest with enormous medicinal and aromatic plants. It constitutes 11% of total known flora having medical property (Sati et al., 2010). Herbal drugs have been the basis of treatment and cure for innumerable diseases and functional condition in traditional systems practiced as Ayurveda, Unani and Siddha. Currently, multiple drug resistance microorganisms have been developed due to indiscriminate use of commercial antimicrobial drugs generally used in the treatment of infectious disease (Bhaskarwar et al., 2008) thus additional therapy is needed for the future. At present, there has been a lot of attention focused on producing medicines and products that are natural. Plants are good sources for new, safe, eco friendly and renewable drugs. Plants are used medically in several countries and are a basis for many effective and prevailing drugs (Srivastava et al., 2008). A number of plants with substantial antimicrobial activity have been stated in different traditional works (Jones et al., 1996 and Satish et al., 1999). Numerous medicinal plants are used daily in Ayurvedic practices. In India more than 7000 medicinal plants are recognized. According to a report of World Health Organization, more than 80% of world’s populations depend on traditional medication for the primary healthcare requirements (Umamaheswari et al., 2008). The fewer availability and high rate of new generation antibiotics needs substitute medicines with antimicrobial activity (Poovendran et al., 2011). Therapeutic plants represent a ridiculous source of antimicrobial mediators. Green plants represent a reservoir of effective chemotherapeutic agent and can provide valuable sources of natural pesticides. Bio pesticides have been suggested as an effective 1 substitute for chemicals. Majority of medicinal plant species are rich in bio-molecule contents which can cope up with health hazard. Recently, antibacterial activities of many plants species have been reported by Pandey and Mishra (2010). The vast majority of modern medications were derived originally from ancient herbal traditions. There are numerous natural products of plants which shows an antifungal, antibacterial and antiprotozoal activities that could be used either systemically or locally (Cowman, M.M. 1999). Many of the medicinal plants used in traditional medicine are readily available in rural areas which are relatively cheaper than modern medicine. Nowadays many of the medicines are acquired from natural products or partial synthetic byproducts of natural sources and used in the customary systems of medicine (Sukanya et al., 2009). Most of these published works come from many countries that are still in practice and the herbal medicines are used for treatment of various diseases for practical and economic reasons (Nascimento et al., 2000). Many plants are used by native groups to satisfy their food needs, nutrition and health and found a source of extra income and employment (Sandhya et al., 2006; FAO, 2012 and Visweswari et al., 2013). Usually, the plants consist of metabolites that deliberates to them, have more medicinal advantages that are at the basis of their consumption by people (Zwenger and Basu, 2008; Visweswari et al., 2013 and Adjatin et al., 2013). The various parts of the plants are conventionally used for the treatment of several complaints and as an antidote for snake bites and scorpion stings (Uma Devi, 2001). The secondary metabolites are the bioactive substances in plants. Most promising targets in search for such biologically active compounds are plants used 2 in folk medicines. Medicinal plants, with their wide variety of chemical constituents, offer a promising source of new antimicrobial principle (Cooper, 2004 and Patwardhan et al., 2005). Several leaves extract have been found to have antimicrobial activity against various microorganisms (Kim et al., 2004).