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Rubus Fruticosus and Verbascum Thapsus Medicinal Plants Collected from Dir (L) N.W.F.P

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Rubus Fruticosus and Verbascum Thapsus Medicinal Plants Collected from Dir (L) N.W.F.P Antioxidant and anti-inflammatory study of Rubus fruticosus and Verbascum thapsus medicinal plants collected from Dir (L) N.W.F.P. Pakistan Muhammad Riaz, B. Pharm. Department of Pharmacognosy Faculty of Pharmacy, University of Karachi Karachi-75270, Pakistan 2012 Antioxidant and anti-inflammatory study of Rubus fruticosus and Verbascum thapsus medicinal plants collected from Dir (L) N.W.F.P. Pakistan THESIS SUBMITTED FOR THE FULFILMENT OF THE DEGREE OF DOCTOR OF PHILLOSOPHY By Muhammad Riaz, B. Pharm. Supervised by Dr. Mansoor Ahmad, I.F. Meritorious Professor Department of Pharmacognosy Faculty of Pharmacy, University of Karachi Karachi-75270, Pakistan 2012 DIDICATED TO MY PARENTS i PUBLICATION FROM THESIS Riaz M , Ahmad M and Rahman N (2011). Antimicrobial screening of fruit, leaves, root and stem of Rubus fruticosus . J. Med. Plants Res ., 5(24): 5920-5924. ii CONTENTS i. Acknowledgements viii ii. Abstract ix iii. Khulasa xii 1. Introduction 01 I. Rubus fruticosus 03 II. Verbascum thapsus 07 2. Literature search i. Literature survey of Rubus fruticosus 13 ii. Therapeutic application of Rubus fruticosus 15 iii. Literature data for total phenols, anthocyanins and ascorbic acid 17 iv. Phytochemical literature survey of Rubus fruticosus 18 v. Structures of chemical constituents reported from R. fruticosus 23 vi. Literature survey of Verbascum thapsus 28 vii. Pharmacological literature survey of Verbascum thapsus 30 viii. Phytochemical literature survey of Verbascum thapsus 32 ix. Structures of chemical constituents reported from V. thapsus 38 3. Experimental 42 i. General/Materials 42 ii. Instruments 43 iii. Abbreviations 44 iv. Pharmacognostic evaluation/Standardization of drugs 45 v. Thin layer chromatography 48 vi. Pharmacological Analysis 48 vii. Anti-oxidant activity 48 viii. Anti-inflammatory activity 49 ix. Analgesic activity 50 x. Diuretic activity 51 xi. Gross behavioural activity 52 xii. Neuropharmacological Activity 54 iii xiii. Antimicrobial activity 56 xiv. Toxicity Studies 59 4. Results 60 5. Discussion 221 6. Conclusion 236 7. References 237 iv LIST OF TABLES ******************** Table 1a and 1b : An overview of chemical constituents identified by different chemical reagents 72 Table 2a and 2b : An overview of chemical constituents identified by different chemical reagents 74 Table 3 - 5: Fluorescence analysis of powder of Rubus fruticosus 76 Table 6 - 8: Fluorescence analysis of powder of Verbascum thapsus 77 Table 9: Fluorescence analysis of extracts of Rubus fruticosus 79 Table 10 : Fluorescence analysis of extracts of Verbascum thapsus 80 Table 11 : Rf values of Rubus fruticosus extracts by Thin layer chro- matography 81 Table 12 : Rf values of Rubus fruticosus extracts by Thin layer chro- matography 82 Table 13 and 14: Antioxidant activity of Rubus fruticosus and Verbascum thapsus extracts using DPPH assay 83 Table 15 and 16: Antioxidant activity of Rubus fruticosus and Verbascum thapsus extracts using ABTS and Nitric oxide assay 84 Table 17: Assessment of anti-inflammatory activity (Formalin induced inflammation) 85 Table 18: Assessment of anti-inflammatory activity (Carrageenan 86 induced inflammation) Table 19 and 20 : Effect of crude extract of on Hot plate Analgesiometer 87 in mice Table 21: Assessment of analgesic activity (Acetic acid 89 induced writhing) Table 22 and 23 : Effect of crude extract of on water bath (tail flick) in mice 90 Table 24: Diuretic activity of Rubus fruticosus and Verbascum thapsus (Various parts extracts) 92 Table 25-27: Behavioural response of Rubus fruticosus (Fruit) (100, 300, 500) in mice 93 Table 28- 30 : Behavioural response of Rubus fruticosus (Leaves) (100, 300, 500) in mice 96 Table 31- 33: Behavioural response of Rubus fruticosus (Root) (100, 300, 500) in mice 99 Table 34- 36: Behavioural response of Rubus fruticosus (Stem) (100, 300, 500) in mice 102 Table 37- 39: Behavioural response of Verbascum thapsus (Fruit) (100, 300, 500) in mice 105 Table 40- 42 : Behavioural response of Verbascum thapsus (Leaves) (100, 300, 500) in mice 108 Table 43- 45: Behavioural response of Verbascum thapsus (Root) (100, 300, 500) in mice 111 Table 46- 48: Behavioural response of Verbascum thapsus (Stem) (100, 300, 500) in mice 114 Table 49 and 50 : Assessment of neuropharmacological activity in v 30 minutes (open field and head dip activity) 117 Table 51 and 52 : Assessment of Exploratory activity (Rearing & cage cross) 119 Table 53 and 54: Assessment of Neuropharmacological activity (traction time) 121 Table 55 and 56: Assessment of Forced swimming activity 123 Table 57-64: Antibacterial activity of Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem extract) 125 Table 65: Antifungal activity of Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem extract) 129 Table 66-73: Insecticidal activity of standard drug, Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem) 129 Table 74-82: Anthelmintic activity of standard drug, Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem) 134 Table 83-91: Brine shrimp lethality test of Rubus fruticosus , Verbascum thapsus and standard 143 vi LIST OF GRAPHS ********************* Graph 1 : Anti-oxidant activity determination of Rubus fruticosus, Verbascum thapsu s 148 Graph 2: Anti-inflammatory activity (Formalin induced inflam- 152 mation) of Rubus fruticosus and Verbascum thapsu s Graph 3: Anti-inflammatory activity (Carrageenan induced inflam- 156 mation) of Rubus fruticosus and Verbascum thapsus Graph 4: The effect of hot plate activity of Rubus fruticosus and Verbascum thapsu s 160 Graph 5: Analgesic activity of Rubus fruticosus and Verbascum thapsu s by acetic acid induced writhing test 164 Graph 6: Analgesic activity of Rubus fruticosus and Verbascum 168 thapsu s through tail flick water bath Graph 7: Open field and head dip activity of Rubus fruticosus 172 and Verbascum thapsus Graph 8: Cage cross and rearing activity of Rubus fruticosus 176 and Verbascum thapsus Graph 9: Effect of Rubus fruticosus and Verbascum thapsu s 180 on mobility time Graph 10: Antibacterial activity of Rubus fruticosus and 184 Verbascum thapsus Graph 11: Brine shrimp lethality test of Rubus fruticosus, 216 Verbascum thapsus and standard LIST OF FIGURES ********************** Figure 1: TLC finger prints of Rubus fruticosus, Verbascum thapsus 146 vii ACKNOWLEDGEMENTS I would like to express my eternal gratitude to my thesis supervisor Prof. Dr. Mansoor Ahmad for his constant and strong knowledge, inspiration and indefinite kindness. Without his continuous inspiration this work would not have been accomplished at this moment. He shall always remain an unstinting inspiration for me, for the rest of my life. His valuable suggestions as final words during the research work are greatly acknowledged. His kind thoughts and wishes have been tremendous comforts to my whole research work. I am also thankful to the respected Chairman, Department of Pharmacognosy, Prof. Dr. Iqbal Azhar and the respected Dean Faculty of Pharmacy, Prof. Dr. Ghazala Hafeez Rizwani . I offer my heartiest gratitude to all teaching faculty for their kindness, help and support, which made my stay in department of Pharmacy, University of Karachi, and a momentous period of my life. I am also grateful to Higher Education Commission Islamabad, Pakistan for financial support. I also offer my sincere gratitude to Dr. Mehjabeen and Dr. Noor Jahan , who being my seniors helped me in conducting pharmacological part of my research work. I will never forget inspiration and kind attitude Mr. Zahid Khan to me in lab. I heartily acknowledge their kindness and support. I would also thankful to the lab staff of the Department of Pharmacognosy, especially Mr. Noman-ul-haq and Tauseef Ahmed for their help during work. My sincere thanks are due to my colleagues and friends, Mr. Najam Ur Rehman and Mr. Rizwan Ahmad for providing me enormous support during the whole time. I owe special thanks to them for completing this assignment. Last, but not the least my Parents and Family that with their continuous support outlined my academic career. Their prayers and love made many of my academic dreams turning into glaring realities. Without their love and support this work would have been a distant point in my life. Muhammad Riaz viii ABSTRACT The aim of this study is not only to preserve the traditional information but also to update and expand this knowledge of medicinal plants according to modern parameters. In this regard two species Rubus fruticosus (Rosaceae) and Verbascum thapsus (Scrophulariaceae) were collected from Northern area Dir (KPK formally called NWFP) of Pakistan which is very rich in medicinal plants, not only to evaluate but to validate their traditional uses according to advanced screening techniques. Methanolic extract of each part of both species was investigated and evaluated for anti-inflammatory, antioxidant potentials, pharmacognostic, neuropharmacological, gross behavioral studies, toxicity studies and diuretic action. Pharmacognostic studies The presence of carbohydrates, triterpenes, tannins, flavonoids and sterols were detected in R. fruticosus and V. thapsus during chemical screening. Thin layer chromatographic plates were also developed for the extract of each part of the plant. The Rf value of each spot was calculated during experiment, for R. fruticosus fruit (0.38,0.42,0.59,0.66,0.73,0.81) for leaves (0.33,0.62,0.64,0.72,0.75) for root (0.38, 0.43) and for stem extract (0.36, 0.43) in Ethyl acetate, methanol and water system. The Rf values in the same solvent system were calculated for V. thapsus fruit (0.26,0.32, 0.46,0.59,0.78) for leaves (0.14,0.22,0.32) for root (0.36,0.46,0.48,0.52, 0.59,0.78) and for stem extract (0.17, 0.39,0.4,0.52, 0.59,0.69,0.78). Antioxidant activity Antioxidant studies using DPPH, ABTS and Nitric oxide free radical spectrophotometeric methods showed the antioxidant capacity order on % free radical scavenging basis for R. fruticosus various parts extracts as fruit > leaves > root > stem and for V. thapsus root > fruit > leaves > stem.
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