PHARMACOGNOSY OF SKIMMIA LAUREOLA (DC.) SIEBOLD. & ZUCC. EX WALP. AND ZANTHOXYLUM ARMATUM DC., FAMILY RUTACEAE Ph. D. THESIS BY BARKATULLAH DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR 2012 PHARMACOGNOSY OF SKIMMIA LAUREOLA (DC.) SIEBOLD. & ZUCC. EX WALP. AND ZANTHOXYLUM ARMATUM DC., FAMILY RUTACEAE BARKATULLAH A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BOTANY, UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN IN PARTIAL FULFILLMENT FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR 2012 DECLARATION The materials contained within this thesis are my original work and have not been previously submitted to this or any other university. BARKATULLAH CERTIFICATE OF APPROVAL This Dissertation, entitled “PHARMACOGNOSY OF SKIMMIA LAUREOLA (DC.) SIEBOLD. & ZUCC. EX WALP. AND ZANTHOXYLUM ARMATUM DC., FAMILY RUTACEAE.” submitted by Barkatullah is hereby approved and recommended as partial fulfillment for the award of Degree of Doctor of Philosophy in Botany. Professor Dr. Muhammad Ibrar __________________________ Research Supervisor, Department of Botany, University of Peshawar. Professor Dr. Muhammad Ibrar __________________________ Chairman, Department of Botany, University of Peshawar. External Examiner __________________________ DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR 2012 DEDICATION This Dissertation is dedicated to my father, late KHANIMULLAH, who not only raise and nurtured me but also taxed himself dearly over the years for my education and intellectual development. And To my mother, whose prayers are the source of motivation and strength during moments of despair and discouragement 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. After that my immense gratitude, heartiest thanks and deep regards to my research supervisor Prof. Dr. Muhammad Ibrar for his welcoming attitude, clear guidance, positive criticism and supportive approach which helped me to complete my research work with full expression of my capabilities. I greatly acknowledge the financial support provided by Higher Education Commission Islamabad for my studies. I am thankful to Prof. Dr. Farrukh Hussain, Prof Dr. F. M. Sarim, Prof. Dr. Addur Rashid, Mr. Zahir Muhammad, Mr. Ghulam Dastagir, Mrs. Tanvir Burni, Ms. Musarrat Jabeen, Mr. Rehman Ullah, Dr. Lal Badshah for their support and helping attitude. I am also grateful to my research fellows Mr. Zaman Sher, Mr. Ishfaq Hameed, Dr. Mohib Shah, Mr. Musharraf Khan, Ms. Tabassum Yaseen and Mrs. Shahida Naveed and also my lab fellows Ms. Maryam Ihsan, Ms. Ulfat samreen, Ms. Shambaleed and Ms. Sumera for pacing with me and maintaining a comfortable working atmosphere. 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. My deep regards are forwarded to Dr. Niaz Ali, Associate Professor, Khyber Medical University, Peshawar; Dr. Innayat ur Rehman, PCSIR Labs, Peshawar; Dr. Shaukat Hussain, KPK Agriculture University, Peshawar; Mr. Naveed Muhammad, Department of Pharmacy, University of Peshawar and Jan Ullah, Department of Chemistry, University of Peshawar, for providing professional guidance during my research work. I am also obliged to my friends Mr. Muhammad Pervez, Imtiaz Ahmed, Niaz Ali Ishtiaq Ahmed and Ghulam jelani for supporting me all the time. i Last but not the least I am extremely thankful to my Mother, my brothers Mr. Ikramullah, Ihsanullah, Inamullah, my sisters and especially my wife and kids for facing all the chores at home in my absence and for lending moral and social support which kept me going and doing well till completion of this thesis. This thesis is a product of various field visits, lab works, experimentations and analysis. Many people have contributed towards the completion of this work in one way or the other. Therefore I also acknowledge all those people from the core of my heart that have played a positive role for this achievement. BARKATULLAH ii PHARMACOGNOSY OF SKIMMIA LAUREOLA (DC.) SIEBOLD. & ZUCC. EX WALP. AND ZANTHOXYLUM ARMATUM DC., FAMILY RUTACEAE ABSTRACT Present study is about Skimmia laureola and Zanthoxylum armatum belonging to Family Rutaceae, comprises phytosociology, ethnobotany, pharmacognostic study, physicochemical and pharmacological activities of these plants. Phytosociological attributes of S. laureola were studied in six different localities of khyber Pukhtunkhwa, Pakistan showing that this plant grows gregariously in laomy and clay loamy soil at high altitude ranging from 2400- 3400 meters toward North facing slopes. S. laureola was found dominent in five out of six localities in association with a total of 44 plants with a density of 312 to 4437.5 hectare-1. Various other ecological, ethnobotanical and commercial aspects of the plants are also worked out. Similar studies of Z. armatum showed that the plants grows in association with 51 species on the North and North West slopes in the foothills of khyber Pukhtunkhwa at an elevation of 850- 1600 meters with a density ranging from 560 to 1020 hectare-1. Pharmacognostic study included leaf and bark of S. laureola and leaf, bark and fruit of Z. armatum. Leaf of S. laureola is punctate with glabrous surfaces. Transverse section of the leaf through the midrib region showed usual bifacial structure with prominent oil cavities in the midrib regions. Other leaf features such as palisade ratio (7.8±0.21), vein islets number (15.4±0.63 per mm2), vein termination number (19.1±0.43 per mm2), stomatal number (196.1±3.07 per mm2) and stomatal index (12.96±0.14) were worked out. Eight different stomatal types were detected, in which actinostephanocytic was the most frequently occurring one . Stomatal cluster was also observed. Morphology and anatomy of Z. armatum was also carried out. Leaf of this plant lacks any type of trichome, where as in the midrib region, prominent oil cavities were observed. Palisade ratio (8.2±0.32), vein islets number (16.8±0.64 per mm2), vein termination number (11.3±0.47 per mm2), stomatal number (122.1±4.32 per mm2) and stomatal index (12.32± iii 0.26) were cworked out. Anatomy of Z. armatum fruit showed two portion i.e. fruit wall and seed, the later being non endospermic and contained small elongated embryo. Powder drug microscopy of the parts was carried out. Ash analysis for both plants were carried out. Results of qualitative and quantitative preliminary phytochemical screenings of selected parts of S. laureola and Z. armatum are given, showing the presence of carbohydrates, proteins, alkaloids, phytosterols, triterpenoids, phenols, flavonoids, tannins, anthocyanins, saponins, glycosides, fixed and volatile oils. Similarly results of extractive values and fluorescence analysis are also given. Elemental analysis showed presence of Zn, Mn and Cr in fairly good amounts and may contribute to hypoglycemic effect of these plants. Correlations among various elements were also determined. Proximate analysis of both plants showed carbohydrate, proteins, fibers, fats and moisture contents in fairly large amounts. Results of essential oils obtained from the leaves of S. laureola (SVO) and Z. armatum (ZVO) and fixed oils (ZHO and ZEO) from the fruit of Z. armatum, extracted with different solvents were evaluated for physicochemical characteristics including color, odor, % yield, density, optical activity, refractive index, specific gravity, carbon residue, absolute viscosity, kinematic viscosity, total acid number, iodine number and saponification value are presented. A total 31 different components in SVO and 34 in ZVO, 14 in ZHO and 14 in ZEO were identified through GC-MC analysis and their percent concentration is given. Toxicological studies showed that both plants are safe for human use. Antipyretic effect was found dose dependant. SLE showed maximum antipyretic action of 72.31%, ZLE showed 85.42 percent pyrexia inhibition of whereas ZFE showed a maximum antipyretic action of 83.84%. Hypoglycemic effect of SLE was found to be dose dependent and like the standard allopathic drug and it induced reduction of blood glucose level after 2 hour of dose administration. All the doses showed significant reduction (p < 0.05) in glucose level at 6th hour post administration, but 300 mg/Kg body weight dose showed maximum reduction of blood glucose level at 6 hour (80.54±0.04). iv Antispasmodic effects of ethanolic and n-hexane extracts of leaf (SLE, SLH), bark (SBE, SBH) and leaf essential oil (SVO) of S. laureola and ethanolic and n-hexane extracts of leaf (ZLE, ZLH), bark (ZBE, ZBH), fruit (ZBE, ZBH) and leaf essential oil of Z. armatum on the isolated rabbit jejunum for both spontaneous and KCl induced contractions showed that SVO and ZVO were the most efficient one, causing 100 % relaxation of the smooth muscles at very low concentrations, thus providing a scientific proof for its ethnopharmacological use as an antispasmodic drug. The two plants were also evaluated for cytotoxic, phytotoxic, antibacterial and anti fungal activities. SLE, SLH and SVO of S. laurola and ZBE, ZBH and ZVO of Z. armatum showed outstanding cytotoxic results with LD50 values of 5.34, 7.44, 11.01, 16.79 17.06 and 15.90 µg/ml. respectively. Lemna minor phytotoxicity asssay of SBE showed 100% inhibition at 1000 μg/ml followed by SBH, SLE and SLH with FI50 of 25, 1.38, 4.54 and 8.67 μg/ml respectively. ZBE, ZFE and ZLH also showed excellent