1 Amadi, Emenike Benjamin PG/M.Sc/12/61609 METHANOL EXTRACT OF ACALYPHA CILIATA LEAVES HAS ANTIBACTERIAL AND ANTI-DIABETIC ACTIVITY Department of Biochemistry Faculty of Biological Sciences Digitally Signed by : Content manager’s Name Nwamarah Uche DN : CN = Weabmaster’s name O= University of Nigeri a, Nsukka OU = Innovation Centre 2 CERTIFICATION Amadi, Emenike Benjamin, a postgraduate student with Registration Number PG/M.Sc/12/61609 of the Department of Biochemistry has satisfactorily completed the requirements for course work and research for the degree of Master of Science in pharmacological Biochemistry. The work embedded in this report is original and has not been submitted in part or in full for any other diploma or degree of this or any other university. …………………………. ………………………… Prof. O F C Nwodo Dr. C.A Anosike Supervisor Supervisor …………………………. …………………………. Prof. O F C Nwodo External Examiner Head of Department 3 DEDICATION This work is dedicated to the God Almighty. 4 ACKNOWLEDGEMENT My special appreciation goes to God, the creator of heaven and earth. I also thank my supervisors, Prof. O F C Nwodo and Dr. C .A Anosike for their supports and efforts throughout the execution of this work. My special thanks also goes to my brothers and sisters; William, Christopher, Josephine, Tessy, Gloria, Anthony Chidozie(junior), Collins Nnanna, Chinazom, Ngozi and Kingsley Obiora Amadi. I also thank my in-laws, Mr. Ugbor Christopher and Mr. Agu Clement for their constant supports in this degree programme. I also remain indebted to my mum, Mama Ike and my aunty, Mrs. Amadi Kate. I thank the Head of Department, Prof. O F C Nwodo and all the academic and non-academic staff in the Department of Biochemistry University of Nigeria, Nsukka for their good work. I also thank the families of Dr. C D Nwani Department of Zoology University of Nigeria Nsukka, Mr. Diyoke Victor Chinero and Mr. Diyoke Innocent for their kindness and generosity. Special thanks also go to Mr. Ebuka Nedu for putting me through in the laboratory practical work. I also thank my colleagues; all the post graduate students, 2012 academic session. Finally, I thank my other friends; Anwana Ugbor, Chukwuemeka Ugbor, Chiamaka Agu, Chukwuemeka Ugwu (“badest son”), Ebere Aniaku, Helen Aniaku, Chukwuma Obodo, Ugwu Festus, Chizoba Anosike, Diyoke Christopher (Okotie), Chidubem Diyoke, Dike Adaeze, Onyedika Diyoke, Onyinyechi Diyoke and Chidimma Diyoke for their care and concern. 5 ABSTRACT Microorganisms have developed resistance to several antimicrobial drugs. This resistance may be due to long term over use or under use of these drugs or due to drug abuse. For this reason, there is an increase in the rate of microbial infection which threatens human population and health status of individuals. Microbial infections are treated with antibiotics, but these treatments are often associated with harmful side effects. Therefore, there is need to develop a new effective antimicrobial drugs of plant origin that is not associated with much harmful side effects. Acalypha ciliata is a plant from the family of euphobiaceae. This research examined the antibiotic and anti-diabetic effects of aqueous and methanol extracts of Acalypha ciliata leaves. Quantitative and qualitative phytochemical results in both extracts reveal the presence of tannins, hydrogen cyanide, soluble carbohydrates, steroids, saponin, flavonoid, alkaloids, glycosides, reducing sugar, and phenol. Hydrogen cyanide had the least composition in aqueous extract (0.64 Ə 0.01 mg/100g) while reducing sugar has largest composition in methanol extract (792.54 Ə0.03 mg/100g).The sensitivity test of the aqueous extract of Acalypha ciliata showed antibacterial activity with the range from 8.00 - 17.00 Ə 2.00 mm, while methanol extract was from 9.00 - 20.00 Ə4.00 mm. The duration of antibacterial activity observed in aqueous extract ranged from 5.00 – 11.00 Ə 2.00 days, while methanol extract ranged from 5.00 – 12.00 Ə 1.00 days. The result of colony forming unit shows that Klebsiella pnumonia has the highest CFU value of 4.15 Ə0.04 x 10 8cfu/ml while Proteus mirabilis has the least colony forming unit of 1.27 Ə 0.06 x 10 8cfu/ml. The minimum inhibitory concentrations (MIC) of the test bacteria in both aqueous and methanol extracts were within 6.25 – 12.50 Ə 0.00 mg/ml. However, the minimum bactericidal concentration (MBC) of the test bacteria in both aqueous and methanol extracts ranged from 9.38 – 25.00 Ə 0.00 mg/ml. The acute toxicity tests of the methanol extract of Acalypha ciliata was greater than 5000mg/kg body weight. The blood of the glucose levels of the whistar albino rats were measured and recorded. On day 6, a non-significant (p>0.05) increase in blood glucose level was observed in all the test groups when compared to the diabetic untreated control (group 6). On the final day (day 14), a significant (p<0.05) decrease in glucose level in all the groups treated with extract was observed when compared to the diabetic untreated control (group 6). The decrease levels observed in the liver enzymes (AST, ALT) and lipid profile (LDL) of rats treated with methanol extract is an indication of hepatoprotective activity and ability of the extract to prevent cardiovascular diseases. 6 TABLE OF CONTENTS Certification i Dedication ii Acknowledgement iii Abstract iv Table of Contents v List of Figures x List of Tables xii List of Abbreviations xiii CHAPTER ONE: INTRODUCTION 1.1 Indian nettle (Acalypha ciliata) 1 1.1.2 Origin and geographic distribution 2 1.1.3 Ethnomedicinal use of Acalypha ciliata 3 1.2.1 Plant phytochemical constituents 4 1.2.2 Alkaloids 4 1.2.3 Polyphenols (Phenolics) 4 1.2.4 Flavonoids 5 1.2.5 Tannin 5 1.2.6 Glycosides 6 1.2.7 Saponins 6 1.2.8. Steroids 7 1.3.1 Infection 7 1.4.1 Bacteria 7 1.4.2 Classification of bacteria based on cell wall 8 1.4.3 Gram-positive bacteria 9 1.4.4 Gram- negative bacteria 9 1.4.5 Bacterial interaction with other organisms 10 1.5.1 Antibiotics 11 1.5.2 Pharmacodynamics 12 1.5.3 Classes of antibiotics 12 1.5.4 Routes of administration 12 1.5.5 Side effects of antibiotics 12 1.6.1 Diabetes mellitus 13 7 1.6.2 Causes of diabetes 14 1.6.3 Types of diabetes 14 1.6.4 Diabetic complications 16 1.6.5 Management of diabetes mellitus 17 1.6.6 Drugs used in the treatment of diabetes mellitus 17 1.6.7 Herbal anti-diabetic agents 18 1.7.1 Liver 18 1.7.2 Hepatotoxicity 19 1.7.3 Drug metabolism in liver 19 1.7.4 Liver function tests 20 1.7.4.1 Aspartate amino transferase (AST) 21 1.7.4.2 Alanine aminotransferase (ALT) 21 1.7.4.3 Alkaline phosphatase (ALP) 22 1.7.5 Clinical significance of liver function tests 22 1.8.1 The kidney 22 1.8.2 Renal function 22 1.8.2.1 The urea 23 1.9.1 Lipid profile 23 1.9.2 Cholesterol 23 1.9.3 Triacylglycerol (TAG) 24 1.9.4 High density lipoprotein (HDL) 24 1.9.5 Low density lipoprotein (LDL) 24 1.10.1 Aim and objectives 25 1.10.2 Aim of the study 25 1.10.3 Specific objectives 25 CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials 26 2.1.1 Plant materials 26 2.1.2 Animals 26 2.1.3 Micro organism 26 2.1.4 Chemical reagents 26 2.1.5 Instruments and equipment 27 2.1.6 Preparation of reagents 28 2.2.1 Methodology 29 8 2.2.2 Preparation of plant material 29 2.2.3 Extraction of plant material 30 2.2.4 Bacterial species used for the study 30 2.2.5 Preparation of test organisms 30 2.2.6 Preparation of stock from concentrated extract 30 2.2.7 Preparation of nutrient agar 30 2.2.8 Preparation of normal saline 30 2.2.9 Tests for identification of bacteria 31 2.3.1 Test for determination of colony forming unit (CFU) 31 2.3.2 Antibacterial activity test (sensitivity) 31 2.3.3 Determination of the minimum inhibitory concentration (MIC) 32 2.3.4 Determination of the minimum bactericidal concentration (MBC) 33 2.3.5 Test for duration of antibiotic activity 33 2.4.1 Qualitative phytochemical analysis of Acalypha ciliate leaves 33 2.4.2 Test for carbohydrates 33 2.4.3 Test for reducing sugars 33 2.4.4 Test for tannins 34 2.4.5 Test for steroids 34 2.4.6 Test for Saponins 34 2.4.7 Test for flavonoids 34 2.4.8 Test for alkaloids 34 2.4.9 Test for glycosides 35 2.4.10 Test for cyanogenic glycosides 35 2.5.1 Quantitative phytochemical analysis 35 2.5.2 Determination of hydrogen cyanide 35 2.5.3 Determination of alkaloids 35 2.5.4 Determination of flavonoids 35 2.5.5. Determination of steroids 36 2.5.6 Determination of tannins 36 2.5.7 Determination of reducing sugar 36 2.5.8 Determination of glycosides 36 2.5.9 Determination of soluble carbohydrates 37 2.5.10 Determination of saponins 37 2.6.1 Determination of median lethal dose (LD50) 37 2.7.1 Experimental design 37 9 2.7.2 Test for glucose level 38 2.8.1 Liver function tests 38 2.8.2 Assay of alanine aminotransferase (ALT) 38 2.8.3 Assay of aspartate aminotransferase (AST) 39 2.8.4 Assay of alkaline phosphatase (ALP) 39 2.9.1 Determination of cholesterol 40 2.9.2 Determination of low density lipoprotein (LDL) cholesterol 40 2.9.3 Determination of high density lipoprotein (HDL) cholesterol 40 2.9.4 Determination of triacylglycerol (TAG) 41 2.10.1 Determination of serum urea 41 2.11.1 Statistical analysis 42 CHAPTER THREE: RESULTS 3.1 Qualitative phytochemical constituents of Acalypha ciliata leaves 43 3.2 Quantitative Phytochemical constituents Acalypha ciliata leaves 45 3.3 Colony forming unit of bacteria (CFU) 47 3.4 Sensitivity test 49 3.5 Duration of antibiotic activity of aqueous and methanol extracts of Acalypha ciliata leaves 51 3.6 Minimum inhibitory concentration of aqueous and methanol extracts of Acalypha ciliata leaves 53 3.7 Minimum bactericidal concentration of aqueous and methanol extracts of Acalypha ciliata leaves 55 3.8 Median lethal dose (LD 50 ) 57 3.9 Effects of methanol extract of Acalypha ciliata leaves on glucose concentration of alloxan-induced diabetic rats.