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Department of Botany University of Peshawar, Peshawar Pakistan PHARMACOGNOSTIC EVALUATION OF EHRETIA SERRATA ROXB. AND EHRETIA OBTUSIFOLIA HOCHT.EX.A.DC. FAMILY BORAGINACEAE BY ZILL-E-HUMA DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN 2013-14 AUTHOR’S DECLARATION I Zill-E-Huma hereby stated that my Ph.D. thesis titled “Pharmacognostic evaluation of Ehretia serrata Roxb. and Ehretia obtusifolia Hocht. Ex. A.DC. Family Boraginaceae” is my own work and has not been submitted to previously by me for taking degree from this University “University of Peshawar” or anywhere in the country/world. At any time if my statement is found to be incorrect even after my graduation the university has the right to withdraw my Ph.D. degree. ZILL-E-HUMA Date_______________ PLAGIARISM UNDERTAKING I solemnly declare that research work presented in the thesis titled “Pharmacognostic evaluation of Ehretia serrata Roxb. and Ehretia obtusifolia Hocht. Ex. A.DC. Family Boraginaceae” is solely my research work with non-significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and University of Peshawar towards plagiarism. Therefore I as an Author of the above titled thesis declare that no portion of my thesis has been plagiarised and any material used as reference is properly referenced/cited. I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the right to withdraw/revoke my PhD degree and that HEC and the University has the right to pub lish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis. Scholar/Author signature:_________________ ZILL-E-HUMA DEDICATION This Dissertation is dedicated to my PARENTS, who not only raise and nurtured me but also taxed himself dearly over the years for my education and intellectual development. and To my beloved brother Mr. ZIA MUHAMMAD, whose continuous guidance and help are the source of motivation and strength during moments of despair and discouragement. PHARMACOGNOSTIC EVALUATION OF EHRETIA SERRATA ROXB. AND EHRETIA OBTUSIFOLIA HOCHT.EX.A.DC. FAMILY BORAGINACEAE A Thesis Submitted to the University of Peshawar in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy In Botany By Zill-e-Huma Graduate study committee 1. Prof. Dr. Ghulam Dastagir Convener. 2. Prof. Dr. Siraj ud Din Member. 3. Dr. Zahir Muhammad Member 4. Dr. Tanvir Burni Member 5. Dr. Rasool Khan Member DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN 2014 PUBLICATION OPTION I hereby reserve the right of publication, including right to reproduce this thesis in any form for a period of 5 years from the date of submission. ACKNOWLEDGEMENT All praises are meant to Almighty Allah, The Creator, The Guider and the Sustainer, Who provided the courage, strength and vision to carry out this research work. It is a genuine pleasure to express my deep sense of thanks and gratitude to my mentor, philosopher and guide Prof. Dr. Muhammad Ibrar, Botany department, University of Peshawar. His dedication and keen interest above all his overwhelming attitude to help his students had been solely and mainly responsible for completing my work. His firmly advice, meticulous scrutiny, scholarly advice and scientific approach have helped me to very great extent to accomplish this task. I owe a deep sense of gratitude for my co-supervisor Asst. Prof. Dr. Barkatullah for his keen interest to me at every stage of my research work. His prompt inspiration, timely suggestions with kindness, enthusiasm and dynamism has enabled me to complete my thesis. I thank profusely to chairman of Botany department Dr. Ghulam Dastagir for his encouragement and support. I would like to thank to my respected teacher’s i.e Dr. Siraj-u-din, Dr. Zahir Muhammad, Mrs. Tanvir Burni, Mr. Rehman Ullah, Dr. Lal Badshah, Dr. Fazal Hadi and Dr. Sami Ullah for their support and helping attitude. I would like to thank to Dr. Memrez Khushal Gigyani, Research officer at VRI, Peshawar, for timely providing me all the necessary facilities and administrative support during my research work. I am also thankful to Dr. Farah khan PCSIR Peshawar, Dr. Sayyar Kazi and Mr. Iftikhar Department of Biotechnology, University of Agriculture, Peshawar for support and help. I am also grateful to my research fellows Mr. Ghulam Jilani, Mr. Shafqat Ali Khan, Dr. Maryam Ehsan, Mr. Inam Ullah, Mr.Najeeb Ur Rehman, Mubina Tariq, Sidra Haider, Umbreen Khattak, Ulfat Samreen, Naila Innayat, Palwasha Khan, Umbreen, Saima and Saiqa for pacing with me and maintaining a comfortable working atmosphere. i I would also like to acknowledge the contribution of office and para teaching staff of Department of Botany, University of Peshawar for helping me all the way throughout my study. It is privilege to thank to my sisters, Miss Haleema Begum, Sadia Noreen, Mehreen Zaman and my Brother in Laws Zia Muhammad and Gohar Ali for their kind help and encouragement throughout my study period. I want to show my deepest love to my mother for playing the role of mother, guider, supporter and mentor in fulfilling of my dreams. ZILL-E-HUMA ii ABSTRACT The current investigation is carried out to detect the pharmacognostic, physicochemical and pharmacological evaluation of leaf, stem bark and root of Ehretia serrata Roxb. and Ehretia obtusifolia Hochst. Ex A. DC which belongs to the family Boraginaceae. The morphological study showed that E. serrata is a small size deciduous tree, less than 10 m tall, locally named as puran and punna in Urdu. Its Leaf is simple with oblong shape (5-16 cm long and 2.8-6.5 cm wide), serrulated margins and glabrous surface. E. serrata contains drupe globose type fruit with yellowish green-light color when unripe and black when ripe. Stem bark is irregular with up to 2.5 cm wide, dark brown outer color and whitish golden inner surface color. E. obtusifolia is a deciduous small shrub or small tree up to 6 m tall; straggling or with several branches from the base. The Leaf is oblong-elliptic or oboviate with obtuse apex, entire margins and reticulate venation. Microscopic study of E. serrata showed the presence of conical trichomes and anomocytic type of stomata on both surfaces where as anisocytic type of stomata was observed only on the lower epidermis with stomatal index 36.7μ. Upper surface showed 30.4 μ stomatal index. Other parameter includes palisade ratio (6.4+0.894 per mm2), vein Islet number (19.4+1.79 per mm2), stomata number (21.8+1.78 per mm2) and vein termination number (10.6+1.987 per mm2). A Similar microscopic anatomical study was carried out of leaf of E. obtusifolia and we observed two types of trichomes i.e simple conical trichomes and capitate glandular trichome on both surfaces. Stomatal cluster was observed only on the lower epidermis with three types of stomata i.e anomocytic, anisocytic and hemiparacytic. Other observed parameters were palisade ratio (6.2 + 0.81 per mm2), vein islets number (18.6 + 1.14 per mm2), vein termination number (8.2 + 0.828 mm2), stomatal number (21.8 + 1.78 per mm2) and stomatal index (35.7 + 6.12 μ). Stem bark anatomy of both experimented plants showed clear layers of cork, cork cambium, phloem elements, medullary rays and tracheids, etc. Fruit anatomy of both plants showed a clear distinction between three layers of pericarps and seeds. Powder drug microscopy of leaf, stem bark and fruits of E. serrata and E. obtusifolia was carried out and observed stomata, epidermal cells, fibers, tracheids and vessels, etc. iii Results of qualitative preliminary phytochemical screenings of examining parts of E. serrata and E. obtusifolia are given, presenting the presence of phenol compounds, starch, protein, reducing sugar, steroids, terpenoids, fats, aldehydes, anthraquinones, glycosides, amino acids, saponins, alkaloids, tannins, phlobatannin. Similarly, results of a fluorescence study of leaf, stem bark and fruit of E. serrata and E. obtusifolia are also given. Results of elemental analysis showed the presence of some essential elements in all examined parts of these two plants. The most abundant elements found in all three parts of E. serrata and E. obtusifolia were Mg, Na, and Fe and which may contribute to analgesic and anti-inflammatory effects of these plants. Proximate analysis of both plants showed carbohydrate, proteins, fibers, fats and moisture in fairly large amounts. The methanolic extracts of E. serrata and E. obtusifolia leaf, stem bark and fruit were non-toxic against Swiss albino mice. Methanolic extract of leaf of E. serrata showed the highest (P<0.01) cytotoxic activity (83.3%) with the LD50 values 15.29 µg/mL at dose level 1000 µg/mL and in E. obtusifolia the fruit methanolic extract showed highest (P<0.01) (80.0%) cytotoxic activity at 1000 µg/mL with an LD50 value of 16.07 µg/mL. All experimented parts of both plants showed non-significant (P>0.05) Phytotoxic activity at all dose levels of 10, 100 and 1000 µg/mL. The anti-oxidant effect of all parts (leaf, stem bark and fruit) of both plants was dose dependent. High (94.1%) DPPH inhibition was shown by stem bark extract of E. serrata at dose level 1000 µg/mL after 60 min and fruit extract of E. obtusifolia (85.01%) at level 1000 µg/mL after 30 minutes. Based on IC50 (µg/mL) stem bark of E. obtusifolia and leaf of E. serrata showed significant values as compared to ascorbate. Both plant methanolic extract was not that much more effective against bacterial strains inhibition.
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