PHARMACOGNOSTIC AND LACTOGENIC STUDIES OF THE LEAVES OF Stachytarpheta jamaicensis (L.) Vahl (VERBENACEAE) BY UDODEME OGECHUKWU HELEN PG/M.PHARM/13/64949 DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. FEBRUARY, 2015 PHARMACOGNOSTIC AND LACTOGENIC STUDIES OF THE LEAVES OF Stachytarpheta jamaicensis (L.) Vahl (VERBENACEAE) BY UDODEME OGECHUKWU HELEN PG/M.PHARM/13/64949 A PROJECT SUBMITTED TO THE FACULTY OF PHARMACEUTICAL SCIENCES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF PHARMACY (M. PHARM) SUPERVISORS: PROF. C.O. EZUGWU DR. (MRS.) U.E. ODOH DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA i TITLE PAGE Pharmacognostic and Lactogenic Studies of the leaves of Stachytarpheta jamaicensis (L.) Vahl(Verbenaceae). ii CERTIFICATION Udodeme Ogechukwu Helen, a Postgraduate Student with Registration Number PG/M.Pharm/13/64949 in the Department of Pharmacognosy and Environmental Medicines, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka has satisfactorily completed the requirement for the course work and research for the award of the Degree of Master of Pharmacy (M.Pharm) in Phytopharmacology. The work embodied in this report is original and has not been submitted in part or full for any other diploma or degree in this or any other University. ____________________ ____________________ Prof. C.O. Ezugwu Dr. (Mrs.) U.E. Odoh (Supervisor) (Supervisor) ___________________ ___________________ Prof. C.O. Ezugwu External Examiner (Head of Department) iii DEDICATION To God Almighty, my husband Aloysius and my children Aloysius Jnr, Michelle and Mary-Laveda with love and honour, I dedicate this work to them. iv ACKNOWLEDGMENTS It is my great pleasure to express my thanks to God for all He has done in my life and especially for giving me good health, strength and determination during these years of study. To him, I return all the Glory and honour for his work. During my research period, many individuals, laboratories and institutions have contributed to the successful completion of the study. My greatest respect goes to my first promoter,Prof. C.O. Ezugwu for his consistent input, professional and invaluable ideals into this project. I am highly indebted to you. With great pleasure and gratitude, I would like to thank, Dr. (Mrs.) U.E. Odoh for her intellectual stimulation, constant availability, unlimited support and devotion to my work. You are always available for consultations and your critical discussions have made this work a success. I greatly appreciate the vital role played by the University of Nigeria, Nsukka for granting me the admission and facilitating my program. Special acknowledgements also go to the Department of Pharmacognosy and Enviromental Medicines for allowing me to carry out the experiments in their laboratories and experimental sites. I would like to extend my thanks to all members of the International Centre for Ethnomedicine and Drug Development (Inter CEDD) and especially Mr. A.O. Ozioko for his constant assistance in determining the study plant collected during the field work. Reserved for special thanks are Mr. Nkem Austin Okorie of the Department of Pharmacology and Toxicology, Dr. Simeon Ikechukwu Egba (the medical laboratory technologists), Mr. Obioma Misheal Egeonu, Dr. Chinonso Ezeaso (of the Department of Pathology and Microbiology, Faculty of Veterinary Medicine) and my other friends whose assistance during the experimental aspects of this work is inestimable. My Profound gratitude goes to Dr. Parker Elijah Joshua of the v Department of Biochemistry, University of Nigeria, Nsukka for the quick touch he gave to the analysis of my work. I am also grateful to some other academic staff in the Faculty of Pharmaceutical Sciences who provided constructive suggestions that shaped the outcome of this work. Special thanks go to Prof. (Mrs.) S.I. Inya-Agha, Prof. J.M. Okonta, Prof. G.C. Onunkwo, Dr. C.E.C Ugwoke, Dr. T.C. Okoye, Dr. K.M. Tchimene, Pharm. M.A. Ezejiofor, Pharm. Rev. S.E. Ezea, for their inestimable academic support that led to the successful completion of this work. Special acknowledgement is due to Prof. P.N and Prof. (Mrs.) F.N. Okeke for their special interest, social guidance, unreserved support and encouragement. I honestly lack words good enough to describe all my appreciation. My appreciation also goes to Mr. Ebi for his invaluable input during the conceptualization of this study. I would like to extend my thanks to all members of the Department of Pharmacognosy and Environmental Medicines more especially, Mr. J.C. Ataogba for his great assistance. Also, I would like to appreciate all members in charge of Zoological Garden, University of Nigeria, Nsukka, for always being helpful and supportive. To my fellow colleague, Miss Edidiong Udofot Esua, I say well done for making the program a worthwhile experience. My heartfelt thanks also go to my lovely parents, Chief J.O and Mrs. F.N Okoye-Oti, for they have been a great source of inspiration. Thank you for your love, prayers and support. Finally, I am greatly indebted to my dear husband Aloysius and my children Aloysius Jnr, Michelle and Mary-Laveda for their support, encouragement, patience and understanding during these periods of this study. I remain forever grateful to you all, and, may God richly reward you. vi You are all wonderful people. Udodeme, Ogechukwu Helen. University of Nigeria, Nsukka, 2015. vii ABSTRACT Based on the ethnomedicinal use and belief that Stachytarpheta jamaicensis (SJ) can increase milk supply in lactating women, experimental studies were carried out to determine the effect of crude methanol extract (CME) and fractions of SJ leaves on serum PRL levels. The pharmacognostic standards, preliminary phytochemical analysis and acute toxicity study were also carried out. The fresh leaves were collected, cleaned, air-dried and pulverized. A photomicroscope was used in the qualitative microscopic recognition of the characteristic cell types and cell contents present in the powdered leaf. The quantitative microscopy of the leaf of SJ showed the values of palisade ratio, stomata number, stomata index, vein-islet number and vein termination number to be 4.42 ± 2.53, (105.67 ± 2.73, 277 ± 17.08), (28.00 ± 2.31, 21.00 ± 2.51), 15.67 ± 0.66 and 3.50 ± 0.00 respectively. For the analytical standards; 11.85 ±0.06, 2.17 ± 0.00, 8.80 ± 0.14, 2.04 ± 0.02, 2.51 ± 0.15, 4.85 ±0.22 and 4.30 ± 0.02 were obtained for total ash, water soluble ash, sulphated ash, acid insoluble ash, alcohol soluble extractive value, water soluble extractive value W and moisture content respectively. The extraction process yielded 19.31 % /W of W W the CME while the fractionation process yielded 36.70 % /W, 10.00 % /W, 26.15 W W % /W and 27.80 % /W of n-hexane fraction (HF), ethyl acetate fraction (EF), n- butanol fraction (BF) and water fraction (WF) respectively. The qualitative phytochemical analysis of the CME and fractions showed the presence of carbohydrates, reducing sugars, alkaloids, glycosides, saponins, tannins, flavonoids, resins, proteins, steroids and terpenoids. The acute toxicity (LD50) studies of CME, HF, EF, BF and WF were > 5000 mg/kg. The lactogenic activity studies of SJ leaves were carried out by checking the effect of their oral treatment on serum prolactin (PRL) secretion and release in rats.The CME of SJ produced an appreciable increase in serum PRL level when compared to the control in a dose- dependent manner. However, the ethyl acetate fraction exhibited the highest when compared to the control with potency greatly higher than the standard drug metoclopramide. The order of activity of the fractions is EF (800 mg/kg) > BF (800 mg/kg) > HF (800 mg/kg) > WF (800 mg/kg). The E2-primed groups showed high serum PRL concentration especially the groups receiving 800 mg/kg of the extract and ethyl acetate fraction respectively. The histological findings of the rat mammary gland tissues were shown to stimulate mammary gland development and differentiation of the lobulo-alveolar system from the lobular buds with milk secretion (proteinaceous materials) within the lumen of the alveoli and ducts. The largest tubulo-alveolar hyperplasia with were observed in the E2-primed groups receiving 800 mg/kg of extract and ethyl acetate fraction respectively. Stachytarpheta jamaicensis thus possesses galactogenic property which supports its traditional usage in lactating women with a wide therapeutic index. viii TABLE OF CONTENTS Title Page - - - - - - - - - - i Certification - - - - - - - - - ii Dedication - - - - - - - - - - iii Acknowledgments - - - - - - - - iv Abstract - - - - - - - - - - vii Table of Contents - - - - - - - - - viii List of Tables - - - - - - - - - xi List of Figures - - - - - - - - - xii Abbreviations - - - - - - - - - xvi CHAPTER ONE:GENERAL INTRODUCTION - - - 1 1.1 Lactogenesis - - - - - - - - 1 1.1.1 Definitions - - - - - - - - 1 1.1.2 Stages of Lactogenesis - - - - - - - 3 1.1.3 Physiology of the breast - - - - - - 6 1.2 Lactation: Physiology of breastfeeding - - - - 7 1.2.1 How Breastfeeding works. - - - - - - 8 1.2.2 When the breast starts making milk - - - - - 10 1.2.3 Hormones responsible for Lactogenesis - - - - 11 1.2.4 Milk Ejection Reflex - - - - - - - 14 1.3 Factors that improves lactation - - - - - - 15 1.3.1 Non-Pharmacological factors - - - - - - 15 1.3.2 Pharmacological factors - - - - - - 17 1.3.2.1 Synthetic Galactogogues