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Study of Phyto-Nutrients from Apricot and in Vivo Assessment of Their Bioactivity

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Study of Phyto-Nutrients from Apricot and in Vivo Assessment of Their Bioactivity STUDY OF PHYTO-NUTRIENTS FROM APRICOT AND IN VIVO ASSESSMENT OF THEIR BIOACTIVITY MUHAMMAD ASGHAR KHAN 13-arid-2141 Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2019 STUDY OF PHYTO-NUTRIENTS FROM APRICOT AND IN VIVO ASSESSMENT OF THEIR BIOACTIVITY by MUHAMMAD ASGHAR KHAN (13-arid-2141) A thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Biochemistry Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi Pakistan 2019 Dedicate this humble effort to my whole family whose prayers and affections are the source of strength and sign of success for my future & to my teachers especially my supervisor for guidance, support and encouragement CONTENTS Page List of Tables xiii List of Figures xiv List of Abbreviations xvii Acknowledgments xix Abstract 1 1 INTRODUCTION 3 2 REVIEW OF LITERATURE 15 2.1 APRICOT (Prunus armeniaca) 16 2.1.1 Taxonomy 17 2.2 ETHNOBOTANICAL USES 19 2.3 PHYTOCHEMISTRY OF APRICOT 20 2.4 MINERALS AND THEIR THERAPEUTIC 22 ROLE IN MEDICINAL PLANTS 2.5 REACTIVE OXYGEN SPECIES (ROS) 23 2.6 ANTIOXIDANTS 24 2.7 SYNTHETIC ANTIOXIDANTS 26 2.8 NATURAL ANTIOXIDANTS 29 2.9 PHENOLIC COMPOUNDS 30 2.10 FLAVONOIDS 30 2.11 ANTIOXIDANT IN-VITRO BIOLOGICAL ACTIVITY 33 2.12 APRICOT IN VIVO BIOLOGICAL ACTIVITY 36 2.13 ANTI MICROBIAL EFFECT OF MEDICINAL 36 PLANTS 2.14 ANTI-CANCER ACTIVITY OF MEDICINAL 39 PLANTS 2.15 CARBON TETRACHLORIDE INDUCED LIVER 40 DEMAGE Page 3 MATERIALS AND METHODS 42 3.1 FRUIT COLLECTION AND IDENTIFICATION 42 3.2 EXTRACTION AND PREPARATION OF FRUIT 42 EXTRACT 3.3 PROXIMATE ANALYSIS 42 3.3.1 Determination of Moisture Contents 43 3.3.2 Ash Content 43 3.3.3 Estimation of Crude Protein 43 3.3.3.1 Digestion 44 3.3.3.2 Distillation and Titration 44 3.3.4 Crude Fats Estimation 44 3.3.5 Crude Fiber Estimation 45 3.3.6 Determination of Carbohydrates 45 3.4 QUANTITATIVE DETERMINATION OF 46 PHYTOCHEMICALS 3.4.1 Determination of Alkaloid 46 3.4.2 Determination of Saponin 46 3.4.3 Total Phenolic Contents 47 3.4.4 Flavonoid Contents 47 3.4.5 Determination of Ascorbic Acid 48 3.4.6 Determination of Tannins 48 3.5 ASSAY OF β -CAROTENE FROM OIL 48 3.6 THIN LAYER CHROMATOGRAPHY (TLC) 49 3.7 ATOMIC ABSORPTION SPECTROMETRY 49 3.7.1 Wet Digestion of Sample 49 3.7.2 Metal Analysis 50 3.8 FLAVONOLS AND PHENOLIC ACIDS (HPLC) 50 3.8.1 Extraction/Hydrolysis 52 Page 3.8.2 HPLC System Analytical Conditions 52 3.9 FORIER TRANSFORM INFRARED 52 SPECTROSCOPY 3.10 GAS CHROMATOGRAPHY ANALYSIS 53 3.10.1 Extraction of Oil 53 3.10.2 Trans-esterification 53 3.10.3 GC Analysis 54 3.10.4 Quantification and Identification of Fatty Acids 54 3.11 ANTOXIDANT ACTIVITY 54 3.11.1 ABTs Method 54 3.11.2 DPPH Radical Scavenging Activity 55 3.11.3 Hydroxyl Radical Scavenging Activity 55 3.11.4 Hydrogen Peroxide Scavenging Activity 56 3.11.5 Ferric Ion Reducing Antioxidant Power (Frap Assay) 56 3.12 ANTIBACTERIAL ACTIVITY OF FRUIT 57 EXTRACT 3.12.1 Preparation of Inoculum 57 3.12.2 Agar Well Diffusion Method 57 3.13 ANTITUMOR POTATO DISC ASSAY 58 3.14 CYTOTOXIC BRINE SHRIMP ASSAY 58 3.15 MTT BASED CYTOTOXICITY ASSAY 59 3.16 ANTI-DIABETIC ACTIVITY α-GLUCOSIDASE 59 INHIBITION 3.17 CCI4 TOXICITY STUDIES AND REMEDIAL 60 EFFECTS OF FRUIT EXTRACTS 3.17.1 Dissection of Animals 61 3.17.2 Tissues Histology 61 3.17.3 Microscopic Examination 62 Page 3.17.4 Biochemical Studies of Tissue Extract 62 3.18 CARDIO PROTECTIVE EFFECT RAT STUDY 63 DESIGN 3.18.1 Standard and Working Solutions 63 3.18.2 Sample Preparation 64 4 RESULTS AND DISCUSSION 65 4.1 BIOCHEMICAL ANALYSIS 65 4.2 PROXIMATE COMPOSITION 65 4.3 ESTIMATION OF PHYTOCONSTITUENTS 69 4.4 THIN LAYER CHROMATOGRAPHY OF 75 FRACTIONS 4.5 ATOMIC ABSORPTION SPECTROMETRY 76 4.6 FT-IR PROFILING 79 4.7 GC ANALYSIS 81 4.8 HPLC PHENOLIC PROFILE 85 4.9 QUANTITATIVE AMINO ACIDS PROFILE 92 4.10 ESTIMATION OF ANTIOXIDANT ACTIVITY 94 4.10.1 ABTS Radical Scavenging Activity 94 4.10.2 Ferric Ion Reducing Antioxidant Power (FRAP) 95 4.10.3 Hydrogen Peroxide (H202) Scavenging Activity 100 4.10.4 DPPH Radical Scavenging Activity 100 4.10.5 Hyaluronidase Inhibitory Activity 105 4.11 CYTOTOXICITY BRINE SHRIMP ASSAY 109 4.12 ANTITUMOR TEST OF APRICOT POTATO DISK 113 4.13 ANTIBACTERIAL ACTIVITY 116 4.14 ANTIPROLIFERATIVE ACTIVITY USING MTT 119 METHOD 4.15 BIOCHEMICAL CHANGES INDUCED BY 122 Page CARBON TETRAC HLORIDE 4.15.1 Effect of Fruit Extract on Liver Function Test 126 4.15.2 Effect of Extract On Antioxidant Enzyme And Total 127 Protein Level in Tissue Extract (Liver) 4.16 INTESTINAL ABSORPTION OF P-GP 132 SUBSTRATES SUMMARY 136 LITERATURE CITED 139 List of Tables Page 1 Atomic absorption spectrophotometer analysis 51 2 Proximate analysis percent of apricot samples 67 3 Estimation of phytochemicals 71 4 Phenolic contents, flavonoids and vitamin C per 100-gram of Fruit 73 5 RF values of standards under UV 365 nm on TLC 77 6 RF values of samples under UV 365 nm 78 7 Essential minerals composition mg/100g of apricot samples 80 8 Fatty acid profile of apricot samples identified by gas chromatography 86 9 Identification and quantification of polyphenols (HPLC) 89 10 Amino acids composition of apricot 93 11 ABTS Antioxidant activity of apricot 96 12 Scavenging potential of apricot (H2O2) 101 13 DPPH radical scavenging activity of apricot 106 14 Hyaluronidase inhibitory activity of fruit 110 15 Cytotoxicity brine shrimp assay of apricot 114 16 Antitumor potato disc analysis of apricot 117 17 Antibacterial Activity of apricot 120 18 Antiproliferative activity of apricot (%) viable cells after 03 Hours 123 19 Antiproliferative activity of apricot (%) viable cells after 06 Hours 124 20 Antiproliferative activity of apricot (%) viable cells after 24 Hours 125 21 Protective role of apricot samples extract on liver 128 22 Effect of apricot extract on antioxidant enzyme in lever cells 133 23 Talinolol in blood plasma (μg/ml) 134 List of Figures Page 1 Bioactive Phytochemicals in fruit having health benifits 18 2 Dangers of Reactive Oxygen Specie to human health 25 3 Free radicals and reactive oxygen 27 4 Ascorbic acid scavenging activity 28 5 Secondary Metabolites 31 6 Chemical structures of phenolic acids 32 7 Chemical structure of flavonoids 34 8 Apricot production statistics in major countries 66 9 Comparison of various proximate contents 67 10 Comparison of various phytochemicals contents 71 11 Tannic acid calibration curve 72 12 Standard concentration-response curve of β- carotene 72 13 Comparison of TPC, flavonoids and saponins in fruit 73 14 Standard concentration- response curve gallic acid 74 15 Standard concentration-response curve of quercetin 74 16 Comparison of elemental analysis of fruit 80 17 FTIR interferogram of GAFS 82 18 Functional groups spectrum of GAFS 82 19 FTIR interferogram of QAFS 83 20 Functional groups spectrum of QAFS 83 21 FTIR interferogram of AAFS 84 22 Functional groups spectrum of AAFS 84 23 Comparison of methylated fatty acids 86 24 Fatty acid profile of apricot identified by gas chromatography 87 25 HPLC chromatogram of standard polyphenols 89 26 HPLC chromatogram of GAFS polyphenols 90 27 HPLC chromatogram of QAFS polyphenols 90 Page 28 HPLC chromatogram of AAFS polyphenols 91 29 ABTS Concentration-response curve of BHT 96 30 ABTS Concentration-response curve of GAFS 97 31 ABTS Concentration-response curve of QAFS 97 32 ABTS Concentration-response curve of AAFS 98 33 Comparison of H2O2 scavenging activity 102 34 H2O2 scavenging concentration-response curve of ascorbic acid 103 35 H2O2 scavenging concentration-response curve of GAFS 103 36 H2O2 scavenging concentration-response curve of QAFS 104 37 H2O2 scavenging concentration-response curve of AAFS 104 38 DPPH concentration-response curve of QAFS 107 39 DPPH concentration-response curve of AAFS 107 40 DPPH concentration-response curve of GAFS 108 41 DPPH concentration-response curve of Gallic acid 108 42 Hyaluronidase inhibitory activity concentration-response curve of 111 GAFS 43 Hyaluronidase inhibitory activity concentration-response curve of 111 QAFS 44 Hyaluronidase inhibitory activity concentration-response curve of 112 AAFS 45 Hyaluronidase inhibitory activity concentration-response curve of 112 catechin as standard 46 Cytotoxicity brine shrimp assay concentration-response curve of 114 GAFS 47 Cytotoxicity brine shrimp assay concentration-response curve of 115 QAFS 48 Cytotoxicity brine shrimp assay concentration-response curve of 115 AAFS Page 49 Antitumor test concentration-response curve of GAFS 117 50 Antitumor test concentration-response curve of QAFS 118 51 Antitumor test concentration-response curve of AAFS 118 52 Comparison of anti-bacterial activity of apricot samples 121 53 Percent viability of cells after 03 hours 123 54 Percent viability of cells after 06 hours 124 55 Percent viability of cells after 24 hours 125 56 Histopathology of liver 130 57 Talinolol concentration time curve of apricot 134 List of Abbreviations AA Ascorbic acid AAE Ascorbic acid equivalent AAS. Atomic absorption spectrometer AEAC Ascorbic acid equivalent antioxidant activity AOAC Association of Official Analytical Communities AOCS Association of American Oil Chemists BW Body weight CA Cluster analysis Carb Carbohydrates CF Crude fats CFb Crude fibers CP Crude proteins CRM Certified reference material DM Dry matter DPPH 2, 2-diphenyl-1-picrylhydrazyl EDTA Ethylene diamine tetra acetic acid EV Energy value FAO Food and Agriculture Organization of the United Nations Fib.
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