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Phytochemical, Nutritional and Biological Screening of Selected Plants of Hazara Farzana Phytochemical, Nutritional and Biological Screening of selected plants of Hazara by Farzana Gul Department of Biochemistry Faculty of Health Sciences Hazara University, Mansehra 2015 Phytochemical, Nutritional and Biological Screening of selected plants of Hazara Thesis submitted to The Department of Biochemistry Hazara University Mansehra In the partial fulfillment of the requirements for the degree of doctorate of Philosophy in Biochemistry Submittd by Farzana Gul Department of Biochemistry Faculty of Health Sciences Hazara University, Mansehra 2015 CERTIFICATE This thesis entitled, “Phytochemical, Nutritional and Biological screening of selected plants from Hazara” submitted by Ms Farzana Gul to the Department of Biochemistry, Faculty of Health Sciences, Hazara University, Mansehra, Pakistan, is accepted in its present form as satisfying the thesis requirement for the Degree of Doctor of Philosophy in Biochemistry. Supervisor: _____________________ Prof. Dr Mukhtiar Hassan Dean of Health Sciences External Examinar: ______________________ External Examinar: ______________________ Chairman: _____________________ Dated: _______________________ Dedicated to my family List of Contents Titles Page No. Acknowledgements……………………………..…………………..…i List of figures……………………………………..…………………...ii List of tables………………………………………..………………....iv Abstract …………………………………………….………………...vii Chapter 1 Introduction……………………………… ………..….…………..….1 1.1 Pimpenella stewartti .............................................................7 1.1.1 Biological classification/ Taxonomy..………………….8 1.1.2 Description………………………..…………………….8 1.1.3 Morphology……………………..……….……………..8 1.1.4 Distribution…………….…….…………………………9 1.1.5 Ethnobotinical uses…….…..……………………………9 1.1.6 Chemical composition…...……………………………..9 1.2 Malva neglecta…………….…..…………………………...10 1.2.1 Biological classification/ Taxonomy………………….11 1.2.2 Description…………………………………………….11 1.2.3 Distribution……………………………………………11 1.2.4 Morphology……………………………………………12 1.2.5 Edible uses……………………………….…………….12 1.2.6 Ethnobotanical uses……………………….…………...13 1.2.7 Other uses………………………………….…………..13 1.2.8 Chemical composition………………………………....14 1.3 Viola ordorata…………………………………………….15 1.3.1 Biological classification/ Taxonomy………………....16 1.3.2 Names in different languages………………………...16 1.3.3 Distribution…………………………………………...16 1.3.4 Description……………………………………………16 1.3.5 Part Used…………………………………………….17 1.3.6 Ethnobotanical uses………………………………….17 1.3.7 Chemical composition…………………………...….17 1.4 Phytochemicals………………………………………..….17 1.4.1 Primary metabolites…………………………………18 1.4.2 Secondary metabolites…………………………......18 1.4.2.1 Classes of secondary metabolites………….........19 1.5 Nutritional Studies………………………………………24 1.5.1 Moisture content……………………………………24 1.5.2 Ash content…………………………………………24 1.5.3 Crude Lipid………………………………………….25 1.5.4 Crude Fiber……………………………………….....26 1.5.5 Crude Protein………………………………………..26 1.5.6 Carbohydrates……………………………………….27 1.5.6.1 Functions of carbohydrates…………………….….27 1.5.6.1.1 Carbohydrates functions as primary source of energy………..……………………………..…....27 1.5.6.1.2 Carbohydrates functions as storage food………..27 1.5.6.1.3 Carbohydrate functions as framework in body.…28 1.5.6.1.4 Carbohydrate functions as anticoagulant....……..28 1.5.6.1.5 Carbohydrate functions as Antigen……………....28 1.5.6.1.6 Carbohydrate functions as Hormone………….….28 1.5.6.1.7 Carbohydrates provide raw material for industry..28 1.5.6.1.8 Miscellaneous Functions……………………….…29 1.6 Minerals Elements………………………………………...29 1.6.1 Principal Mineral Elements (Macro minerals)…….….29 1.6.2 Micro Minerals………………………………………..29 1.7 Biological Studies…………………………………….…..30 1.7.1 Antioxidants…………………………………….…...30 1.7.1.1 Synthetic Antioxidants……………………….……30 1.7.1.2 Natural Antioxidants………………………….…..31 17.1.3 Antioxidant activity determination assays………….31 1.7.1. 4 DPPH free radical scavenging assay………….….32 1.7.2 Antimicrobial Activity………………………….…..33 1.7.2.1 Extraction…………………………………..…..…34 1.7.2.2 Extraction solvents…………………………..……35 1.7.2.3 Extraction techniques…………………….....…….36 1.8 In vitro Antidiabetic study of plants……………………..37 1.9 Cytotoxic Activity of plants…………..……..…………..37 1.9.1 Toxicity testing against Brine shrimp…………..…..37 1.9.2 Phytotoxicity testing against lemna minor……..…..39 Chapter 2 Review of Literature……………………………………………………42 2.1 Medicinal plants………………………………………...42 2.2 Phytochemicals………………………………………….43 2.3 Nutritional Studies………………………………………46 2.4 Biological Studies……………………………………….48 2.5 Antimicrobial activities of plants………………………..50 2.5.1 Antibacterial activities of plants……………………50 2.5.2 Antifungal activities of plants………………………52 2.6 In vitro Antidiabetic study of plants…………………….53 2.7 Cytotoxic activity of plants………………………..…….54 2.7.1 Toxicity testing against Brine Shrimp……………...54 2.7.2 Phytoxicity testing against Lemna minor…….…….55 Chapter 3 Materials and methods…………………………………………..…..….57 3.1 Plant Collection…………………………………..……...57 3.1.1 Drying of plant samples………………………....….57 3.1.2 Plant Extracts Preparation…………………...….….57 3.1.3 Hot water extraction………………………………..58 3.1.4 Solvent extraction………………………………….58 3.2 Methods…………………………………………….…...58 3.2.1 Preparation of solutions/reagents………………..…58 3.3 Qualitative Phytochemical analysis……..………………60 3.4 Nutritional analysis…………………………………..….62 3.5 Minerals analysis………………………………………..65 3.5.1 Determination of Minerals…………………….....65 3.5.2 Determination of phosphorous…………………....66 3.6 Biological studies……………………………………….66 3.6.1 Analysis of Phenolic compound.………..……….66 3.6.2 Analysis of Flavonoids………………………….67 3.6.3 Total Antioxidant activity………………………..68 3.6.4 DPPH Radical scavenging activity………………68 3.6.5 Microorganisms…………………………………..69 3.6.5.1 Screening of Antibacterial Activities……...........69 3.6.5.2 Antifungal assay…………………….…………..69 3.6.5.3 Statistical Analysis……………………..………..69 3.7 In vitro Antidiabetic Study of plants……………...…...70 3.8 Toxicity testing against brine shrimp………….……….70 3.9 Phytotoxic activity by using lemna minor plants……....71 3.9.1 Pytotoxicity test for crude extract………………...71 Chapter 4 Results and Discussion…………………………………………………73 4.1 Phytochemical analysis………………………………….73 4.2 Nutritional studies……………………………………….79 4.2.1 Mineral Analysis of selected plants by Atomic absorption spectrophotometery……………………87 4.3 Biological studies……………………………………….91 4.3.1 Analysis of Phenolic compounds………………….91 4.3.2 Determination of total flavonoid contents………..91 4.3.3 Free radical scavenging (DPPH) antioxidant activity……………………………………….93 4.3.4 Anti-microbial assays of selected plants…………96 4.3.4.1 Antibacterial assays of Pimpenella stewartti…....97 4.3.4.2 Antibacterial assays of Malva neglecta………...104 4.3.4.3 Antibacterial assays of Viola odorata………….110 4.3.5 Anti fungal Assays of selected plants……………117 4.3.5.1 Antifungal assays of Pimpenella stewariit.........117 4.3.5.2 Antifungal assays of Malva neglecta...................120 4.3.5.3 Antifungal assays of Viola odorata…………….123 4.4 In vitro Antidiabedic studies…………………….……124 4.5 Cytotoxic activity of plants…………………………….129 4.5.1 Brine shrimps lethality test………..……………...129 4.5.2 Phytotoxic activity by using lemna minor plants....131 ACKNOWLEDGEMENTS In the name of Allah most merciful and most beneficent Thanks to Almighty Allah (SWT) for giving me strength and courage to complete this task in time. I would like to thank my mentor, Professor Dr. Mukhtiar Hassan,Dean/ Chairman Department of Biochemistry Hazara University Mansehra, who shared with me a lot of his expertise and research insights and provided me excellent research environment to keep my concentration fixed on research obligation. I am thankful to all faculty members of department of biochemistry, especially Dr. Sajid Muhammad and Dr. Farooq for their co-operation. I am also thankful to Dr. Mudassar, Assistant Professor, Agriculture University Peshawar, for his guidance during experiments. I would like to show gratitude to Administrative staff of Biochemistry Department for their helpful attitudes. It gives me great pleasure in acknowledging the support and help of Dr. Ihsanullah, who continually and convincingly conveyed a spirit of adventure in regard to research. Without his guidance and persistent help this dissertation would not have been possible. I would like to thank all of my dear friends for all their encouragement and cooperation in compiling of this thesis. I would also like to say thanks to my family for their constant support throughout my whole life, for which I am sincerely grateful. I am grateful to my father for giving me the life, I ever dreamed. I can’t express my gratitude for my mother, whose unconditional love has been my greatest source of strength. I cannot end without saying how grateful I am to my husband, brothers and sisters especially Sehrish Khan, awho have given me a loving environment throughout my career. Farzana Gul i LIST OF FIGURES Figure No. Title Page No. 1.1 Pimpenella stewartii at flowering stage……………………….……7 1.2 Malva neglecta……………………………………………………..10 1.3 Viola odorata at flowering stage…………………………………..15 1.4 Artemia nauplii brine shrimp………………………………………38 1.5 Lemina minor (Duck weed)………………………………………..40 4.1 Mineral analysis of plants…………………………………………..90 4.2 Graphical representation of DPPH free radical scavenging activity of Malva neglecta, Pimpenella stewartii and Viola odorata………….…95 4.3 Comparative efficacy of extracts of Pimpenella stewartii against selected pathogenic bacterial strains (mm/5mgL-1)………………….100 4.4 Inhibition zones indicating antimicrobial activity of
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