Antiglycation and Antioxidant Potential of Selected Plant Materials By Asia Atta M.Phil. (UAF) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015 DECLARATION I hereby declare that the contents of the thesis, “Antiglycation and antioxidant potential of selected plant materials” are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/equations/formulae/protocols etc). I further declare that this work has not been submitted for award of any diploma/degree. The University may take action if the information provided is found inaccurate at any stage. Asia Atta 2002-ag-743 The Controller of Examinations, University of Agriculture, Faisalabad. “We, the Supervisory Committee, certify that the contents and form of the thesis submitted by Ms. Asia Atta, Regd. No. 2002-ag-743, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of Ph.D. degree”. Supervisory Committee 1. Chairman __________________________ Prof. Dr. Munir Ahmad Sheikh 2. Member __________________________ Dr. Muhammad Shahid 3. Member __________________________ Prof. Dr. Tanweer Khaliq DEDICATION To My loving Parents, Who mean the world to me I dedicate this thesis to the best gift given to me from almighty Allah, My Parents and my entire family Who always provide me compassion throughout my life and my success is really the fruit of their prayers ACKNOWLEDGMENT In the name of Allah, the merciful, the beneficent I have the pearls of my eyes to admire countless blessings of Allah Almighty because the words are bound, knowledge is limited and time of life is too short to express his dignity. Then the trembling lips and wet eyes praise the greatest man of Universe, the last messenger of Allah, HAZRAT MUHAMMAD (PBUH), whom Allah has sent as mercy and the illuminating torch for the world. I deem it my utmost pleasure to avail an opportunity to express my gratitude to my honorable supervisor, Dr. Munir Ahmad Sheikh, Department of Biochemistry , University of Agriculture, Faisalabad for his kind behavior, moral support and constructive criticism during my study period. I am passionately thankful to Dr. Muhammad Shahid, (Associate professor, Department of Biochemistry) for his sincere and encouraging attitude, guidance and moral support throughout my PhD studies. I am extremely obliged to Prof. Dr. Tanweer khaliq (Chairman, Department of physiology and pharmacology, UAF) for his special attention with regard to my research work and exam. I would like to express my gratitude and profound admiration to my International Research Support Initiative Program (IRSIP) supervisor Dr. Hang Xiao (Associate profess) and his team for their immense support toward my research work in the Department of Food Science, University of Massachusetts, Amherst (MA), USA. I also ought to financial support (Indigenous scholarship and IRSIP) provided by Higher Education Commission (HEC), Islamabad, Government of Pakistan without this financial support it could not possible for me to complete my doctoral studies. At the same time I express my special thanks to my research groups of Medicinal Biochemistry Lab, Department of Biochemistry, UAF and Department of Food Science, University of Massachusetts, Amherst (MA), USA for their unreserved love, benevolent help and encouragement during the whole studies and research. I need to thank all my near and dear friends and fellows, Dr. Sumaira Sharif, Dr. Mazhar Abbas, Dr. Ghulam Mustafa, Dr. Sammar Abbas, Dr. Ali Raza, Dr. Abid Ali, Dr. Nazia kanwal, Dr. Sumia Akram, Dr. Mushtaq, Salman bhai, Ali bhai and Sir Shamshad, Runa khan, Saira and Andleeb for their love providing amenities and sincere friendship. I pay my special thanks to Nadeem bhai for his unreserved affection, care and sincere prayers for my success. May ALLAH bless them with success and happiness, Ameen! Many thanks are due to my nephews M. Zaighum, Zaryab, Aalyan, M. faateh and to my nieces Meerab, Wardah and Irha for their innocient concern which give me heartiest pleasure. I express special appreciation to Uncle Iqbal Hussain Qureshi, for his unreserved affection guidance, encouragement and everlasting prayers. I owe immense feelings of love and thanks to my affectionate parents as their prayers are always behind my each and every success and for my entire family for being there in times of need, their great patience, continuous encouragements and untiring efforts while I did this work. A special thanks to my siblings for bearing me throughout my degree. All of my prayers, good habits and sacred emotions are for them. May Allah bless them all here and hereafter (Amin). Asia Atta TABLE OF CONTENTS Sr. No. TITLE Page No. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 8 2.1 Concepts of functional and nutraceutical foods 8 2.2 Oxidative stress and safety concerns 10 2.3 Phytoconstituents as antioxidant 11 2.3.1 Tocopherols composition 12 2.3.2 Phenolic content 12 2.3.3 Flavonoids 13 2.3.4 Carotenoids 14 2.3.5 Alkaloids 14 2.3.6 Saponins 15 2.4 Antioxidant capacities of plant materials 15 2.4.1 Antioxidant activities of vegetables 15 2.4.2 Antioxidant activities of dry fruits 16 2.5 Anticancer potential of selected plant materials 17 2.5.1 Anticancer potential of vegetables 17 2.5.2 Anticancer potential of dry fruits 19 Glycation and formation of advanced glycation end 2.6 19 products 2.6.1 Advanced glycation end products 20 2.6.2 Intracellular AGEs and their harmful effects 21 2.6.3 Mechanisms of actions of AGEs 22 i 2.6.3.1 Cross linking extracellular matrix proteins 22 2.6.3.2 Crosslinking intracellular proteins 22 2.6.3.3 RAGE dependent effects of AGEs 22 Protein modification to form Amadori in diabetes & its 2.6.4 24 complications 2.7 Carbohydrate hydrolyzing enzymes inhibition 25 Biochemistry of carbohydrate hydrolyzing enzymes and their 2.7.1 26 inhibitors 2.7.2 Inhibitors of carbohydrate hydrolyzing Enzymes from plants 27 Phytoconstituents with carbohydrate hydrolyzing enzymes 2.7.3 27 inhibitory activity 3 MATERIAL AND METHODS 29 3.1 List of chemicals 29 3.2 Instruments used 29 3.3 General procedure 30 3.3.1 Collection and identification of plant materials 30 3.3.2 Grinding and sample size/extraction 31 3.3.2.1 Extraction of free phenolic compounds 31 3.3.2.2 Extraction of bound phenolic compounds 32 3.3.3 Proximate analysis of plants materials 32 3.3.3.1 Estimation of crude protein 32 3.3.3.2 Estimation of crude fats 33 3.3.3.3 Estimation of crude fibers 33 3.3.3.4 Determination of ash contents 34 3.3.3.5 Determination of total carbohydrates 34 3.3.3.6 Total energy 34 3.3.4 Mineral analysis 34 ii 3.3.5 Phytochemistry of plants 35 3.3.5.1 Qualitative analysis 35 3.3.5.2 Quantitative analysis 35 3.3.6 Antioxidant activity of plant extracts 36 3.3.6.1 Total phenolic contents (TPC) of selected plant extracts 36 3.3.6.2 Total flavonoids content (TFC) of selected plant extracts 37 3.3.6.3 DPPH radical scavenging assay of selected plant extracts 37 3.3.6.4 Determination of reducing power of selected plant extracts 37 3.3.7 Brine shrimp lethality assay 37 3.3.7.1 Lethality concentration determination 38 3.3.8 Thrombolytic assay 38 Measurement of inhibition of cell proliferation by plants 3.3.9 39 extracts 3.3.9.1 Cell lines and culture 39 3.3.9.2 In vitro anti-proliferative assay 39 3.3.10 Antiglycation activity 40 3.3.10.1 Non-enzymatic protein glycation 40 Spectrophotometric analyses of nitro-blue tetrazolium (NBT) 3.3.10.2 40 reductive assay 3.3.10.3 Spectrophotometric analyses of Girard-T assay 40 3.3.10.4 AGEs analysis 40 3.3.11 Enzyme assay 41 3.3.11.1 α-Glucosidase assay 41 3.3.11.2 α-Amylase inhibition assay 41 3.3.12 Statistical analysis 42 4 RESULTS AND DISCUSSION 43 Extraction and % age yield of extracts from selected 4.1 43 plant materials iii 4.2 Proximate analysis of selected plant materials 45 4.2.1 Proximate analysis of selected vegetables 46 4.2.2 Proximate analysis of selected dry fruits 47 4.3 Mineral Analysis of the selected plant materials 48 4.3.1 Mineral analysis of selected vegetables 49 4.3.2 Mineral analysis of selected dry fruits 50 4.4 Preliminary phytochemical analysis 53 4.5 Quantitative analysis of phytochemicals 54 4.5.1 Quantitative analysis of phytochemicals of vegetables 54 4.5.2 Quantitative analysis of phytochemicals of selected dry fruits 55 4.6 Antioxidant potential of plant materials 57 4.6.1 Antioxidant concentration of selected plant materials 57 4.6.1.1 Total phenolic contents 57 4.6.1.2 Total flavonoid content (TFC) 59 4.6.2 Antioxidant activities of plant materials 60 4.6.2.1 Reducing power assay 60 4.6.2.2 DPPH radical scavenging activity 61 Toxicological screening of plant materials against brine 4.7 64 shrimp nauplii 4.7.1 Brine shrimp lethality bioassay of vegetables 64 4.7.2 Brine shrimp lethality bioassay of dry fruits 65 4.8 Thrombolytic activity of plant materials 66 Thrombolytic activity of methanolic extracts of selected 4.8.1 67 vegetables Thrombolytic activity of methanolic extracts of selected dry 4.8.2 67 fruits 4.9 Antiproliferative activity of selected plant materials 68 4.9.1 Antiproliferative activity of selected vegetables 69 4.9.2 Antiproliferative activity of selected dry fruits 70 4.10 Antiglycation activities of selected plant materials 72 iv 4.10.1 Effect of selected plant materials on the reduction on NBT 74 4.10.1.1