PHARMACOGNOSTIC AND LACTOGENIC STUDIES OF THE LEAVES OF jamaicensis (L.) Vahl ()

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).

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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)

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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 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.

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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 ------17 1.3.2.2 Herbal/Natural Galactogogues - - - - - 20 1.4 Factors that decreases lactation. - - - - - 22 1.4.1 Non-pharmacological factors ------22 ix

1.4.2 Pharmacological factors ------23 1.4.2.1 Synthetic Medications ------23 1.4.2.2 Herbs that may decrease milk supply - - - - 25 1.5 The Plant Stachytarpheta jamaicensis - - - - 25 1.5.1 Plant Taxanomy ------25 1.5.2 Description of the plant ------28 1.5.3 Geographical Distribution of the plant - - - - 29 1.5.4 Chemical Constituents of the plant. - - - - - 30 1.5.5 Ehnomedicinal Uses. ------31 1.6 Standardization of Herbal Medicines - - - - - 32 1.6.1 Pharmacognostic Standardization of herbal medicines - - 32 1.6.2 Macroscopical Standards ------33 1.6.3 Microscopical Standards ------34 1.6.3.1 Qualitative Microscopy ------35 1.6.3.2 Quantitative Microscopy ------35 1.6.4 Analytical Standards ------37 1.6.5 Structural Standards ------39 1.6.6 Physical Constants as standards - - - - - 39 1.7 Previous Pharmacological Studies - - - - - 40 1.8 Aims and Objectives of study ------42 CHAPTER TWO: MATERIALS AND METHODS - - - 43 2.1 Collection, Identification and Preparation of plant material - - 43 2.2 Drugs, Chemicals, Reagents and Equipment - - - - 43 2.3 Experimental animals ------44 2.4 Macroscopical Examination of the leaves - - - - 45 2.5 Microscopical Examination of the leaves - - - - 45 2.6 Determination of Analytical Standards - - - - - 47 x

2.7 Extraction ------50 2.8 Fractionation ------50 2.9 Phytochemical Analysis (procedure) - - - - - 51 2.10 Pharmacological Methods ------56

2.10.1 Acute Toxicity and lethality (LD50) test. - - - - 56 2.10.2 Effect of oral treatment of SJ crude methanol extract on serum prolactin (PRL) concentration ------57 2.10.3 Effect of oral treatment of SJ fractions on serum prolactin (PRL) concentration ------57 2.10.4 Effect of oral treatment of SJ crude methanol extract on mammary gland tissues ------58 2.10.5 Effect of oral treatment of SJ fractions on mammary gland tissues 58 2.11 Statistical Analysis ------59 CHAPTER THREE:RESULTS ------60 3.1 Macroscopical Examination of whole leaf - - - - 60 3.2 Microscopical Examination of the powered leaf. - - - 60 3.3 Analytical Standards of the leaf ------72 3.4 Percentage yield ------73 3.5 Phytochemical Tests ------74 3.6 Pharmacological Studies ------75 3.6.1 Toxicological Studies ------75 3.6.2 Effect of leaf Extract/Fractionson serum PRL Content - 76 3.6.3 Histological Studies of the mammary gland - - - - 84 CHAPTER FOUR: DISCUSSION AND CONCLUSION - - 91 4.1 Discussion ------91 4.2 Conclusion ------94 References Appendix

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LIST OF TABLES Table 1: Showing colostrum and milk production - - - - 10 Table 2: Result of quantitative microscopy - - - - - 71 Table 3: Result of analytical standards - - - - - 72 Table 4: Result of percentage (%) of extraction and fractionation processes 73 Table 5: Result of phytochemical analysis - - - - - 74

Table 6: Result of acute-toxicity (LD50) test of the crude methanol extract and its fractions ------75

Table 7: Result of the effect of methanol leaf extract of SJ on the Prolactin levels of treated rats. ------76

Table 8: Result of the effect of fractions of SJ on the Prolactin levels of treated rats. ------77

Table 9: Result of the effect of methanol leaf extract of SJ on the Prolactin levels of treated rats after priming with estradiol (E2). - - 78

Table 10: Result of the effect of fractions of SJ on the Prolactin levels of treated rats after priming with estradiol (E2). - - - 79

Table A1: Calculation of quantitative microscopy - - - - 104

Table A2: Acute toxicity test for (LD50) determination showing the number of cell deaths per dose. ------105

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LIST OF FIGURES Fig 1: Picture of anatomy of the lactating breast. - - - - 10

Fig 2: Picture of hormone levels of lactation. - - - - 11

Fig 3: Photograph of the plant showing leaves and flower clusters. - 29

Fig 4: Photograph of plant showing stems and paired leaves. - - 29

Fig. 5:Photograph of plant showing elongated flower clusters. - - 29

Fig. 6: Photograph of plant showing close-up of flowers. - - - 29

Fig. 7: Photomicrogragh of powdered leaf of SJ showing annular xylem vesselswith adjoining phloem parenchyma. - - - - 61

Fig. 8: Photomicrogragh of powdered leaf of SJ showing clustered palisade cells. ------61

Fig. 9: Photomicrogragh of powdered leaf of SJ showing epidermal cells with anticlinal sinuous wall. - - - - - 62

Fig. 10: Photomicrogragh of powdered leaf of SJ showing typical diacytic stomata. ------62

Fig. 11: Photomicrogragh of powdered leaf of SJ showing epidermal cells with cylindrical palisade cells. - - - - - 63

Fig. 12: Photomicrogragh of powdered leaf of SJ showing epidermal cells with robust multicellular uniseirate trichome. - - - 63

Fig. 13: Photomicrogragh of powdered leaf of SJ showing large phloem parenchyma cells. ------64

Fig. 14: Photomicrogragh of powdered leaf of SJ showing irregular shaped prism calcium oxalate. ------64

Fig. 15: Photomicrogragh of powdered leaf of SJ showing small bundle of fibres. ------65 xiii

Fig. 16: Photomicrogragh of powdered leaf of SJ showing spiral and annular xylem vessel. ------65

Fig. 17: Transverse section of the leaf showing the outlines of the Microscopical characters. Magnification x200. - - - 66

Fig. 18: Photomicrograph of TS of the lower epidermis of SJ showing anticlinal sinuous epidermal cell wall. - - - - 67

Fig. 19: Photomicrograph of TS of the lower epidermis of SJ showing multicellular uniseirate trichome and phloem parenchyma cells. 67

Fig. 20: Photomicrograph of the sectional view of TS of SJ showing bundle of phloem cells adjacent to xylem vessels. - - - 68

Fig. 21: Photomicrograph of the sectional view of TS of SJ showing bundle of scalariform-like xylem vessels. - - - - 68

Fig. 22: Photomicrograph of the sectional view of TS of SJ showing collenchymacells and annular xylem vessels. - - - 69

Fig. 23: Photomicrograph of the sectional view of TS of SJ showing stomata in the lamina of transverse section. - - - - 69

Fig. 24: Photomicrograph of the sectional view of TS of SJ showing typical phloem cells in the vascular system. - - - - 70

Fig. 25: Effect of methanol leaf extract of SJ on the serum Prolactin (PRL) levels of treated rats. ------80

Fig. 26: Effect of fractions of SJ on the serum PRL levels of treated rats. 81

Fig. 27: Effect of methanol leaf extract of SJ leaves on the serum PRL levels of treated rats after priming with Estrogen (E2). - - 82

Fig. 28: Effect of fractions of SJ leaves on the serum PRL level of treated rats after priming with Estrogen (E2). - - - - - 83

Fig. 29: Histological section of mammary gland from non-primed rat xiv

receiving distilled water, showing predominance of bare ducts (white arrow) and terminal end buds (TEBs) (black arrow) in a sea of adipose tissue (AT). ------84

Fig. 30: Histological section of mammary gland from an E2-primed rat receiving distilled water showing ductular proliferation (red arrow) with few budding of rudimentary alveoli. - - - - 84

Fig. 31: Histological section of mammary gland from a non-primed rat receiving 200 mg/kg BW of plant extract, showing ductular proliferation with moderate budding of rudimentary alveoli. - 85

Fig. 32: Histological section of mammary gland from E2-primed rat receiving 200 mg/kg BW of plant extract, showing tubuloalveolar differentiation containing proteinaceous materials in the lumen (mild). ------85

Fig. 33: Histological section of mammary gland from a non-primed rat receiving 400 mg/kg BW of plant extract, showing welldefined alveolar structure with lipid droplets within the alveoli. - - 85

Fig. 34: Histological section of mammary gland from E2-primed rat receiving 400 mg/kg BW of plant extract, showing tubulo-alveolar hyperplasia with protienaceous materials in the lumen of duct and alveoli (moderate). - - - - 86

Fig. 35: Histological section of mammary gland from a non-primed rat receiving 800 mg/kg BW of plant extract, showing welldefined alveolar structure with much lipid droplets in the alveoli. - 86

Fig. 36: Histological section of mammary gland from E2-primed rat receiving 800 mg/kg BW of plant extract, showing tubuloalveolar hyperplasia with proteinaceous materials in the lumen of duct and alveoli (high). - - - - - 86

Fig. 37: Histological section of mammary gland from non-primed rat receiving distilled water, showing predominance of bare ducts (white arrow) and terminal end buds (TEBs) (black arrow) in a sea of adipose tissue (AT). ------87

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Fig. 38: Histological section of mammary gland from an E2-primed rat receiving distilled water showing ductular proliferation with few budding of rudimentary alveoli. - - - - - 87

Fig. 39: Histological section of mammary gland from non-primed rats receiving ethyl acetate fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli. 87

Fig. 40: Histological section of mammary gland from non-primed rats receiving n- hexane fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli. 88

Fig. 41: Histological section of mammary gland from non-primed rats receiving n-butanol fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli .88

Fig. 42: Histological section of mammary gland from non-primed rats receiving water fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli. 88

Fig. 43: Histological section of mammary gland from E2-primed rats receivingethyl acetate fraction in a doseof 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts. - 89

Fig. 44: Histological section of mammary gland from E2-primed rats receivingn-hexane fraction in a dose of 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts. - 89

Fig. 45: Histological section of mammary gland from E2-primed rats receivingn-butanol fraction in a dose of 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts. - 90

Fig. 46: Histological section of mammary gland from E2-primed rats receivingwater fraction in a dose of 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts - 90

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ABBREVIATIONS PRL - Prolactin SJ - Stachytarpheta jamaicensis

E2 - Estrogen MER - Milk Ejection Reflex FIL - Feedback inhibitor of lactation EPG - Epidermal Growth Factor IGF-1 - Insulin-like Growth Factor 1 ACTH - Adreno-Corticotropic Hormone TSH - Thyroid Stimulating Hormone HPL - Human Placental Lactogen FSH - Follicle Stimulating Hormone LH - Luteinizing Hormone rBST - Recombinant Bovine Somatotropin TRH - Thyrotropin Releasing Hormone MEC - Mammary Epithelia Cells EPG - Epidermal Growth Factor BW - Body Weight AT - Adipose Tissue TEB - Terminal End Bud

DP - Ductular Proliferation

TAH - Tubulo-alveoli Hyperplasia

PM - Proteinaceous Materials

EDTA - Ethylene di-amine tetra-acetic acid 1

CHAPTER ONE GENERAL INTRODUCTION 1.1 LACTOGENESIS 1.1.1 Definitions Lactogenesis is the initiation of lactation (milk secretion), a process of functional differentiation which mammary tissues undergo when changing from non-lactating to a lactating state. This process is normally associated with the end of pregnancy and around the time of parturition (Wilde and Hurley, 1996). Lactogenesis may also be defined as the onset of milk secretion and includes all of the changes in the mammary epithelium necessary to go from the undifferentiated mammary gland in early pregnancy to full lactation sometimes after parturition (Neville et al., 2001). The literal meaning of the word is the production of milk by the mammary glands (Mifflin, 2004). Lactogenesis is the result of a series of cellular changes whereby mammary epithelia cells are converted from a non-secretory state to a secretory state. Therefore, lactogenesis is the process of milk production. It is initiated in the postpartum period by a fall in plasma progesterone, but prolactin (PRL) levels remain high. Lactation is the secretion of milk from the mammary glands of the breast (Guyton and Hall, 2006). There are four (4) stages of lactation (Riordan and Wambach, 2010) namely; 1. Mammogenesis (growth of the breast). 2. Lactogenesis (the functional change of the breast so they can secrete milk). 3. Galactopoiesis (maintaining the production of milk). 4. Involution (the termination of milk production). 2

Mammogenesis: The mature breast is mainly made up of adipose tissues; however the lactating breast has a greater proportion of glandular tissues. During pregnancy, the breast enlarges; the nipple pigment darkens; the skin becomes thinner and the veins in the breast become more prominent. In mammogenesis the ductal system grows and branches; the amount of connective tissues and supporting cell increases and fat is laid down in the breast. This is stimulated by the estrogen, growth hormone, PRL, insulin and the adrenal corticoids. Progesterone is involved in the last stages of mammogenesis after the ductal system has grown. It acts with the other hormones to develop the breast lobules and alveoli, and adapts the alveoli to have secretory properties (Husveth et al., 2011). Lactogenesis (induction of milk synthesis) is a process of differentiation whereby the mammary gland alveolar cells acquire the ability to secrete milk; it is conveniently defined by two-stage mechanism. The first stage of lactogenesis consists of partial enzymatic and cytological differentiation of the alveoli cells and coincides with limited milk secretion before parturition. The second stage begins with copious secretion of milk components shortly before parturition and extends throughout several days postpartum in most . The onset of copious milk secretion at parturition to meet the nutritional requirement of relatively well- developed neonates is a feature of lactation in all placental mammals (Husveth et al., 2011). Galactopoiesis (maintenance of lactation) requires an alveoli cell number, synthetic activity per cell and efficacy of the milk-ejection reflex (MER). After parturition, there is marked increase in milk yield. Galactopoiesis starts around 9 days after birth and finishes at the beginning of involution. It is the maintenance of milk secretion controlled by hormones. Breast size starts to diminish between 6 to 9 months after birth. The rate of milk formation normally decreases after 7-9 3 months; however milk production can continue for years if the child continues to suckle (Husveth et al., 2011). Involution: Involution is when the breast stops producing milk completely after weaning. It is the loss of secretory function of milk due to the accumulation of inhibiting peptides. It normally starts 40 days after the last breastfeed. The epithelia cells no longer require their secretory properties so they are removed by the process of apoptosis; a mode of physiological cell death (i.e. programmed cell death). In most species, the cessation of suckling or milking rapidly brings about mammary involution, which is characterized by a decrease in the number of mammary epithelia cells and also in the amount of secretory activity per cell and replaced by adipocytes (Husveth et al., 2011). 1.1.2 Stages of Lactogenesis There are basically two (2) stages of lactogenesis namely lactogenesis 1 and lactogenesis II (Riordan and Wambach, 2010). However, there is a third stage called lactogenesis III (also known as Galactopoiesis or simply lactation). (a) Lactogenesis 1: This is the first stage of lactogenesis which occurs during pregnancy, when the glands become sufficiently differentiated to secrete small quantities of specific milk components, such as casein and lactose. In humans, stage 1 occurs approximately midpregnancy and can be detected by the measurement of increased plasma concentrations of lactose and α- lactalbumin (Butte et al., 1992). After lactogenesis stage 1 has been achieved, the gland is sufficiently differentiated to secrete milk but the secretion is held in check by high circulating plasma concentrations of progesterone (Jensen, 1995) and possibly, in some species such as human, estrogen. The secretion product, often called colostrum which can be extracted from the breast of pregnant women, contains relatively high concentrations of sodium; chloride and protective substances, such as 4

immuglobulins and lactoferrin. Casein is not present and the lactose concentration is low at this time (Beerens, 1980). (b) Lactogenesis 11: This is the second stage of lactogenesis which is onset of copious milk secretion associated with parturition. In many species, such as cows, goats and rats, this stage begins before birth of the young, brought about by the sharp decrease in plasma progesterone that also initiates parturition. In humans, the progesterone level does not decrease postpartum but decreases approximately 10 fold during the first 4 days after birth accompanied by a programmed transformation of the mammary epithelium, which leads to transfer to the infant of 500 to 750 ml/day of milk by day 5 postpartum (Saarinen et al., 1977). This transformation requires a concerted change in several processes including changes in the permeability of the paracellular pathway between epithelia cells, changes in the secretion of protective substances such as immunoglobulins, lactoferrin and complex carbohydrates, and an increased rate of secretion of all milk components. Lactogenesis stage 11 can be monitored by changes in milk composition and volume in women and other species in which large milk samples can be obtained (Saarinen et al., 1977). The term colostrum and transitional milk traditionally used to describe the mammary secretion produced during the first 4 days and from days 4 to 10 postpartum respectively, do not define clear cut temporal changes in milk composition and are not useful distinctions. Lactogenesis stage 11 involves the formation of large amount of milk after parturition. It starts from day 3 postpartum to days 8. It is triggered by the reduction of progesterone. The breast becomes full and warm to produce large amount of milk. The initiation of stage 11 lactogenesis begins with the sudden withdrawal of pregnancy hormones at the delivery of the placenta. 5

Stage 11 occurs 2 to 3 days postpartum, paralleling the time when “the milk comes in”. This stage includes increase in blood flow, oxygen, glucose and citrate in the breast. The breast will begin to produce milk independent of infant suckling. Lactogenesis stages 1 and 11 are specifically controlled by hormones. (c) Lactogenesis 111:This stage was originally called galactopoiesis. But this can simply be called lactation. This is the production and maintenance of mature milk from day 9 postpartum until mom and baby decide to wean. The breast is not merely a passive container of milk. It is an organ of active production. When the infant suckles, a series of events takes place within the mother’s body. Once stage 11 lactogenesis has begun, continued milk production is governed by the infant. The more milk that is removed from the breast, the more milk will be produced. Milk production relies on the supply and demand principle. Also each breast works alone, if mom breastfeeds more from one breast, that breast will produce more milk than the other, which is why it is possible for a mom to breastfeed just from one breast (Nivelle et al., 2001). An intact hypothalamic pituitary axis regulating PRL and oxytocin levels is essential to the initiation and maintenance of lactation (Kent et al., 2003). This process of lactation requires milk synthesis and milk release into the alveoli and the lactiferous sinuses. When the milk is not removed, the increased pressure lessens capillary blood flow and inhibits the lactation process. Lack of suckling stimulation means lack of PRL release from the pituitary gland. Basal PRL levels that are enhanced by the spurts that results from suckling are necessary to maintain lactation in the first postpartum weeks. Without oxytocin, however, a pregnancy can be carried to term, but the woman will fail to lactate because she will fail to let-down. Lactogenesis 6 stage 3 is controlled by the endocrine system, but hormones do still play a role. 1.1.3 Physiology of the Breast Physiologically, the breast is an organ specialized for milk formation (lactation). Many additional changes are seen in the breast tissue during pregnancy and lactation due to the changes in hormones during those times (Hopkins, 2012). The physiology of the breast is directly dependent on parts of the body’s endocrine system. This system is essential in controlling the function of the human body.This is done by the production of hormones by these endocrine glands. The breast operates under multiple hormonal influences like estrogen, progesterone, thyroid hormones, growth hormones, cortical type hormones (Strand et al., 1995). These hormones are chemical messengers that circulate in the blood stream and acts on organs remote from their organ of primary secretion. With regard to breast function; At birth; the breast is just a little nipple bud with essentially no function. At puberty; the breast begins to enlarge under the influence of the hormones estrogen and progesterone. The hormonal flux associated with the menstrual cycle is dependent on estrogen and progesterone levels. During Pregnancy; the breast begins to do what we call proliferate or grow, the glands grow too. The nipple is stimulated when the baby sucks; muscular tissues surrounding the nipple cause it to become erect. When the nipple is stimulated, the brain’s pituitary gland secretes the hormone PRL (luteotrophic hormone) which triggers the breast milk gland cells to produce milk(lactogenesis). This does not occur until the baby and placenta are delivered. PRL is stimulated by estrogen and inhibited by progesterone. 7

Post Pregnancy; with the disappearance of the corpus luteum of pregnancy and the expulsion of the placenta, the levels of progesterone drop precipitously. Thus, the unopposed action of estrogen stimulates PRL production, which in turn stimulates the formation of milk. The feeding baby by its suckling action on the nipple expresses milk from the breast ducts, but it cannot get at the milk lying deep within the alveoli. Here again another hormone called oxytocin, comes into action. Stimulation of the nipples by suckling sends nerve impulses to the brain (hypothalamus). The brain in turn activates the pituitary to produce oxytocin which reaches the breast via the blood stream. 1.2. LACTATION: PHYSIOLOGY OFBREASTFEEDING: The breast undergoes dramatic changes in size, shape and function in association with puberty, pregnancy and lactation. These changes are critical to successful breastfeeding (Neville, 2001). Lactation describes the secretion of milk from the mammary glands and the period of time that a mother lactates to feed her young (Guyton and Hall, 2006). However, the process of lactation and the act of breastfeeding is quite complex, because a range of factors in the mother’s external and internal environment determines her breastfeeding efficacy. Her internal environment includes her physical and mental health, past experiences and intentions related to breastfeeding and body image, all of which impact her breastfeeding experience. Her external environment such as socioeconomic factor, her general physical environment and spousal, family and hospital staff support also influences breastfeeding success. And most importantly, the quality and quantity of maternal-infant interaction during the early postpartum period, sometimes described as the forth trimester, sets the stage for a successful breastfeeding experience (Neville et al., 2001). 8

1.2.1 How Breastfeeding Works The three major hormones in pregnancy are estrogen, progesterone and PRL. With the delivery of the placenta after the baby’s birth, estrogen and progesterone levels dramatically fall, whilst PRL levels remain high. There are PRL receptor sites within the acini cells and with a surge of the hormones on the sites; milk production begins (Hartmann et al., 1996). If the receptor sites are not primed early, some can begin to shut down which can affect future milk production. Colostrum is present in the breast from the later stages of pregnancy. The hormonal reflex associated with breastfeeding is as follows: • The baby suckles at the breast. • Nerve impulses are sent to the brain. • PRL is released into the blood stream via the pituitary gland. • PRL acts on the acini cells to make milk. As the milk producing hormone, PRL needs to be stimulated early and frequently to ensure long term milk production. At the same time oxytocin is working, • The baby suckles at the breast. • Never impulses are sent to the brain. • Oxytocin is released into the bloodstream via the pituitary gland. • Oxytocin works on the myoepithelial cells surrounding the acini cells to move milk through the breast. • The oxytocin effect can be temporarily inhibited by stress. • Basal levels are higher when the baby is near. The oxytocin reflex is also known as the “let down” reflex; mothers may experience a tingling or squeezing sensation in the breast when it occurs. Together 9

PRL and oxytocin can have an effect on a mother’s behaviour and feelings. They can trigger mothering behaviour and feeling of love and well being. For milk production to continue, milk needs to be removed effectively and frequently from the breast (Hartmann et al., 1996). Current research suggests that there are two (2) factors that control milk synthesis (Cox et al., 1996). (i) Milk contains a small whey protein called Feedback Inhibitor of Lactation (FIL) - the role of FIL appears to be to slow milk synthesis when the breast is full. This milk production slows when milk accumulates in the breast (and more FIL is present), and speeds up when the breast is emptier (and less FIL is present). (ii) The hormone PRL must be present for milk synthesis to occur. On the walls of the lactocytes (milk-producing cells of the alveoli) are PRL receptor sites that allow the PRL in the blood stream to move into the lactocytes and stimulate the synthesis of breast milk components. When the alveolus is full of milk, the walls expand/stretch and alter the shape of PRL receptors so that PRL cannot enter via those receptor sites- thus rate of milk synthesis decreases. As milk empties from the alveolus, increasing numbers of PRL receptors return to their normal shape and allow PRL to pass through thus rate of milk synthesis increases. The PRL receptor theory suggests that frequent milk removal in the early weeks will increase the number of receptor sites. More receptors sites means that more PRL can pass into the lactocytes and milk production capability would be increased (Hartmann et al., 1996). Three major factors are necessary to maintain the milk supply (MarieDavis, 2012); and they are (i) Intact neuro-hormonal pathways (ii) Suckling, breast stimulation and (iii)Milk removal. Factors I and II are controlled by neuro-endocrine system while III is controlled by autocrine system.

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1.2.2When The Breast Starts Making Milk. A woman’s breast starts getting ready to make milk when she becomes pregnant. Breast changes are caused by four main hormones. These hormones cause the ducts and glandular tissue (alveoli) to grow and increase in size (see the anatomy of breastfeeding in the image below).

Fig 1: Picture showing the anatomy of the breast

The breast starts to make the first milk colostrum, in the second trimester. Colostrum is thick and clear to yellow in colour. Once the baby and the placenta are delivered, the body starts to make more milk. Over the next few days, the amount of milk the breasts make will increase and the colour will change to appear more watery and white (Murphy, 2014) as seen in the table below. Table 1: Showing Colostrum and Milk Production

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1.2.3 Hormones Responsible For Lactation Lactation is influence by a complex hormonal milieu including reproductive hormones (estrogen, progesterone, placental lactogen, PRL, and oxytocin) and metabolic hormones (glucocorticoids, insulin, growth, and thyroid). The reproductive hormones act directly on the mammary gland, whereas the metabolic hormones act indirectly by altering endocrine response and nutrient flux to the mammary gland (Hovey et al., 2002). Ductal growth is primarily regulated by estrogen and growth hormone, and alveolar development requires progesterone, PRL, and possibly placental lactogen (Anderson et al., 1998). There are basically four (4) hormones that help the breasts make milk; estrogen, progesterone, PRL and oxytocin (Murphy, 2014). The body naturally knows how to adjust the level of these hormones to help the breast make milk, as seen in the drawing below.

Fig 2:Picture showing hormone levels of lactation

Estrogen and Progesterone These two (2) hormones prepare the breast to make milk and they are released by the placenta during pregnancy. They have two major roles. They increase the size and number of milk ducts in the breasts and also keep the body from making large 12 amounts of breast milk until after the baby is born. Once the baby is born and the placenta is delivered, these hormones decrease. This decrease signals the body that it is time to make milk. Progesterone This hormone influences the growth in size of alveoli and lobes. During pregnancy, the high levels of circulating progesterone inhibit the secretory process of the mammary gland before birth. Once the placenta is expelled after birth, progesterone levels declines rapidly and increasing PRL levels trigger the beginning of lactogenesis 11, which is the onset of copious milk secretion and production (Mohrbacher and Stock, 2003). Estrogen Estrogen stimulates the milk duct system to grow and differentiate. Like progesterone, high levels of estrogen also inhibit lactation. Estrogen levels also drop at delivery and remain low for the first several months of breastfeeding (Mohrbacher and Stock, 2003). Breastfeeding mothers should avoid estrogen based birth control methods, as a spike in estrogen level may reduce mother’s milk supply. Oxytocin This hormone is essential for milk removal from the mammary gland (Howie et al., 1981). Oxytocin contracts the smooth muscle of the uterus during and after birth and during orgasm(s). After birth, oxytocin contracts the smooth muscle layer of band-like cells surrounding the alveoli to release milk from the breasts. When the baby (or breast pump) begins to suck and draw the nipple into her mouth,afferent impulses from sensory stimulation of nerve terminals in the areola travel to the central nervous system (CNS) triggering the release of oxytocin from the posterior pituitary. In turn oxytocin is carried through the blood stream to the mammary gland where it interacts with specific receptors on the myoepithelial cells located 13 on milk-secreting cells (alveoli) and ducts, initiating contraction of the cells. This release causes milk to be squeezed out of the alveoli, into the ducts (milk canals) and out of the nipple, into the baby’s mouth (Uvnas-Moberg, 1996). This process is called letdown or milk ejection reflex (MER). Milk is continuously secreted into the alveoli of the breast however; to get the milk from the alveoli and into the duct, it needs to be ejected. Ejection is a neuronal and hormonal reflex involving oxytocin (Riordan and Wambach, 2010). PRL This hormone helps the breast to make milk. After the birth of the baby, its levels increase. Every time one breastfeeds or pumps, the body releases PRL. With each release, the body makes and stores more milk in the breast alveoli. If the level of this hormone gets too low, the milk supply will decrease. This is why it is important to breastfeed or pump right after delivery and then at regular time frames. High levels of PRL during pregnancy and breastfeeding increase insulin resistance, increase growth factor levels (IGF-1) and modify lipid metabolism in preparation for breastfeeding. During lactation, PRL is the main factor maintaining tight junctions of the ductal epithelium and regulating milk production through osmotic balance (Hartmann et al., 1996). Other hormones that influence lactation are; (a) Growth hormone: This is structurally very similar to prolactin and contributes to its galactopoietic function. (b) Adreno-cortico-tropic hormone(ACTH) and glucocorticoids. They have an important inducing function in several animal species. ACTH is thought to contribute to lactation as it is structurally similar to prolactin. Glucocorticoids play a complex regulating role in the maintenance of tight junctions. 14

(c) Thyroid Stimulating Hormone (TSH) is a very important galactopoietic hormone whose levels are naturally increased during pregnancy. (d) Human Placental Lactogen (HPL)- From the second month of pregnancy, the placenta releases large amounts of HPL. This hormone appears to be instrumental in breast, nipple, and areola growth before birth. (e) Follicle Stimulating Hormone (FSH) (f) Luteinizing Hormone (LH) (g) Insulin. The main role of this hormone appears to be in regulating nutrient fluctuation to the mammary gland by shunting nutrients away from traditional storage depositories, thereby making them more readily available for milk synthesis (Butte et al., 1999). (h) Thyroid hormone. This hormone is essential for effective milk production and in animals, appears to be necessary for mammary responsiveness to growth hormone and prolactin lactation (Hapon et al., 2003). 1.2.4 Milk Ejection Reflex This is the mechanisms by which milk is transported from the breast alveoli to the nipple. Suckling by the baby stimulates the Para ventricular nuclei and Supraoptic nerve in the hypothalamus, which signals to the posterior pituitary gland to produce oxytocin. Oxytocin stimulates contraction of the myoepithelial cells surrounding the alveoli, which already hold milk. This increased pressure causes milk to flow through the duct system and be released through the nipple. This response can be conditioned e.g. to the cry of the baby. Milk ejection is initiated in the mother’s breast by the act of suckling by the baby. The MER (also called let-down reflex) is not always consistent, especially at first. Once a woman is conditioned to nursing, let-down can be triggered by a variety of stimuli, including the sound of any baby. Even thinking about breastfeeding can stimulate this reflex, causing unwanted leakage, or both breasts 15 may give out milk when an infant is feeding from one breast. However, this and other problems often settle after two weeks of feed. Stress or anxiety can cause difficulties with breastfeeding. The release of the hormone oxytocin leads to the milk ejection or let down reflex. Oxytocin stimulates the muscles surrounding the breast to squeeze out the milk. Breastfeeding mothers describe the sensation differently. Some feel a slight tingling while others feel immense amounts of pressure or slight pain/discomfort, and still others do not feel anything different (Darly et al., 1993). A poor MER can be due to sore or cracked nipples, separation from the infant, a history of breast surgery, or tissue damage from prior breast trauma. 1.3 FACTORS THAT IMPROVES LACTATION A lot of factors improve lactation on mammals and these can be grouped into non-pharmacological and pharmacological factors. 1.3.1 Non-pharmacological factors (a)Breastfeeding self efficacy: This is defined as a mother’s belief that will be able to organize and carry out the actions necessary to breastfeed her infant. Higher levels of breastfeeding self-efficacy have been shown to predict longer and more exclusive breastfeeding in varied samples of women (McCarterSpaulding, 2012). Breastfeeding efficacy and social support have a theoretical relationship based on Bandura’s Social Cognitive Theory (Bandura, 1997). Using this theory as a framework, a person’s perception of self efficacy is influence by information received from various sources including; (1) Vicarious experience, such as role models. (2) Social or verbal persuasion, such as emotional support and encouragement. (b)Feeding Schedule: If the milk production is low, breastfeeding more often may be just the solution needed. Stimulating the breast through feeding can increase a mother’s milk production. Persistence is a key factor that affects milk production. 16

It may take a few weeks after the little one’s birth before the mother’s milk supply can meet the newborns needs. Studies have shown that fat levels in milk are higher when the time between feeding is shorter. (c)Nursing Time and Technique: As a baby breastfeeds, she may suckle at a lower pace because she is filling up or because she is falling asleep. Instead of nursing for a set amount of time, let the baby feed as long as she desires. Switching breasts during breastfeeding may also keep the newborn engage and eager to eat. Feeding from only one breast can cause the other breast to stop producing milk. (d)Diet: After giving birth, new mothers are often eager to hit the gym and start dieting to get back to pre-pregnancy condition. Exclusive breastfeeding can burn 200 to 250 calories a day and cutting your daily calorie intake too much can effect your milk production, according to KellyBonyata, a certified lactation consultant writing for Kellymom.com. A sudden drop in calories, rather than a gradual decrease can also impact milk production. (e)Professional Help: Learning the ropes of breastfeeding can sometimes leave new mothers frustrated and concerned. A doctor can refer you to a lactation consultant, a professional who can teach breastfeeding techniques and offer advice on increasing milk production. Improper breastfeeding techniques may affect the baby latch to mother’s breast, which could in turn affect how much milk the baby receives during feeding. It can also impact your milk production over the long term. (f)Increasing Milk Supply by Double-Nursing: After the baby has fed well and he seems finished, hold or carry him upright and awake for 10 to 20 minutes, allowing any trapped air bubbles to be burped up. This makes room for more milk. Then feed again on both breast before you let him go to sleep. Double nursing stimulates more MERs, thus increasing milk supply and its calorie content. 17

(g)Trust that Nature’s System Works: If one is nursing often enough and the baby is sucking effectively, one will make enough milk. It’s rare that a mother is unable to produce enough milk for her baby. And while it may seems that one’s life is stressful, mothers throughout history have breastfed their babies through war, famine and personal tragedies. The body nourished this baby through pregnancy so; there is no reason to think that one won’t succeed at breastfeeding. 1.3.2 Pharmacological Factors The pharmacological factors are also termed Galactogogues. These are medications or other substances believed to assist initiation, maintenance or augmentation of milk production. Milk production is a complex physiologic process involving physical and emotional factors and the interaction of multiple hormones, the most important of which is believed to be PRL. Galactogogues exert their pharmacologic effects through interactions with dopamine receptors, resulting in increased PRL levels and thereby augmenting milk supply. Through interaction with the hypothalamus and anterior pituitary, dopamine agonists inhibit while dopamine antagonists increase, PRL secretion and thereby milk production (endocrine control). Thereafter, PRL levels gradually decreases but milk supply is maintained or increased by local feedback mechanisms (autocrine control). Therefore, an increase in PRL levels is needed to increase, but not maintain, milk supply. More frequent and thorough emptying of the breasts typically results in increased milk production (Hartmann et al., 1996). Galactogogues are further subdivided into two categories; (A) Synthetic and (B) Herbal 1.3.2.1 Synthetic Galactogogues Among synthetic molecules used to increase lactation are (A) the dopamine antagonist, such as (i) antiemetics metoclopramide and domperidone (ii) antipsychotics sulpiride and chlorpromazine. (B) Hormone synthetic analogs such 18 as oxytocin, rBST, TRH, and medroxyprogesterone are also included in the synthetic galactogogues list. (A)Dopamine Antagonists These drugs block the dopamine 2 receptors (D2R) in the central nervous system which induces an increase of PRL synthesis in lactotrophic cells of the anterior pituitary. This high blood level of PRL increases milk protein synthesis rate and mammary epithelial cells (MEC) proliferation (Tabares et al., 2014.). i. Metoclopramide (Reglan) This is the most studied and most commonly used medication for inducing or augmenting lactation. This drug was originally commercialized in Europe as an antipsychotic and later in the US as a gastro kinetic agent that increases gastrointestinal motility. Its first reported use as a galactogogue was in 1975 (Guzman et al., 1979) and has been evaluated in many clinical trials (Anderson and Valides, 2007). In humans, 10 mg administered by oral route (PO) three times a day during 10 days increased milk production (Ingram et al., 2012). It is used in small animal veterinary medicine to treat cases of secondary hypogalactia or agalactia at doses of 0.1-0.2 mg/kg subcutaneously (SC) every 6-8 hours for 4-6 days (Kahn, 2007). ii. Domperidone (Motilium) Domperidone increases prolactin and udder milk supply. This is the only galactogogue proven effective through a small double-blind, placebo-controlled trial with fewer side effects. Women has enhanced lactation with 10 mg of domperidone orally three times daily (DaSilva and Knoppert, 2004). iii.Sulpiride (Egonyl) This is an antipsychotic (neuroleptic) medication that acts as a galactogogue by increasing prolactin-releasing hormone from the hypothalamus. A previous study reported an effective oral sulpiride dose of 50 mg every 8 hours for 4 weeks 19 in women with hypogalactia; in this investigation serum PRL concentrations increased during the first 2 weeks (Polatti, 1982). iv. Chlorpromazine Just like the sulpiride, it is an antipsychotic drug that acts as a galactogogue by increasing prolactin-releasing hormone from the hypothalamus. Chlorpromazine administered in doses of 15 mg/kg of body weight in rats during 5 days was effective in inducing lobuloalveolar growth and initiation of milk secretion initially primed with 10 µg estradiol daily for 10 days (Talwalker et al., 1960). (B) Hormone Synthetic analogs i. Oxytocin Oxytocin has been used in cattle with inadequate milk supply and is involved in causing milk let down (Riordan and Wambach, 2010). The major sites of expression of this peptide hormone are located in the magnocellular neuron region in the supraoptic and paraventricular hypothalamic nuclei (Gimpl and Fahrenholz, 2001). Oxytocin acts majorly by inducing the contraction of the myoepithelial cells via G protein receptor, and PLC is activated and induces the formation of diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3), by hydrolysis of membrane lipid phosphatidylinositol 4, 5-biphosphate (IP12). This IP3 induces intracellular Ca 2+ release, and this active Ca 2+- camodulin system triggers the activation of myosin light-chain kinase (MLCK) which initiates smooth muscle contraction in mammary myoepithelia cells (Gimpl and Fahrenholz, 2001). ii. Recombinant Bovine Somatotropin (rBST) The rBST approved in dairy cows is the 190 amino-acid variant with leucine at position 127, and it has an extra-methionine at the NH2 terminus (Etherton and Bauman, 1998). This hormone has direct effects on breast parenchyma and basal 20 metabolic rate. This promotes increase in milk synthesis, blood flow and viability of MEC, along with increase in insulin-like growth factor 1 (IGF-1) protein in liver and mammary tissues (Molento et al., 2002). iii. Thyrotropin Releasing Hormone (TRH) This peptide hormone is synthesized in the hypothalamus, stimulating the secretion of thyroid stimulating hormone (TSH) and PRL by the anterior pituitary (Tashjian, 1980). TRH is the principal physiological factor stimulating the fast release of PRL. The proposed mechanism of action of TRH is that the molecule binds to its receptor in lactotrophic cells of the pituitary gland and stimulates Ca2+/CaM release, which induces the PRL gene expression. Furthermore, the increase in intracellular CA2+/CaM stimulates the release of PRL stored in vesicles. This will increase PRL blood levels and promote more milk synthesis (Lachowicz et al., 1997). iv. Medroxyprogesterone This is a steroidal synthetic progesterone (a progestin). This drug causes hyperplasia of mammary secretory epithelium in macaques (Cline et al., 1998) and mice, with its activity being associated with epidermal growth factor (EGF) (Molinolo et al., 1998). 1.3.2.2 Herbal/Natural Galactogogues Some have been used in many cultures to stimulate milk production in women and in diary animals. Most of the galactogogues have not been scientifically evaluated but traditional use suggests their safety and some efficacy. The most commonly cited galactogogues of herbal origin are: Asparagus, Fenugreek, Brewer’s yeast, Blessed thistle, Milk thistle, Alfalfa. Others include: Anise, Astragalus Root, Boza, Burdock, Nettle, Fennel Seeds, Flax, Soapwort, Vervain, etc.

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Trigonella foenum graecum (Fenugreek) This is the most used commonly herbal galactogogue. It is a member of the leguminosae family that is cultivated in many parts of the world particularly in India, Mediterranean countries, North Africa, and Southern Europe. Reports indicate that seeds have mastogenic effect, stimulating growth of mammary gland. This plant is used around the world as galactogogue in women due to its phytoestrogens’ significant levels (McGuffin et al., 1997). Galega officinalis(Goat’s Rue): This is a traditional galactogogue, widely recommended in Europe, based on observations of increased milk supply when fed on cows in the 1990s (McGuffin et al., 1997). Silybum marianum(Milk thistle) has been used theoretically throughout Europe, but there are no randomized controlled trials to validate its use. The plant is still commonly known as St. Mary’s thistle in honor of the Virgin Mary. The American Herbal Products Association gives it a rating of 1, meaning that the herb may be safely consumed when used appropriately and does not contraindicate its use during lactation (McGuffin et al., 1997). Cnicus benedictus(Blessed thistle): Blessed thistle or milk thistle stimulates milk production, especially in combination with red raspberry leaves, milk thistle is also an antioxidant that is equal in potency to vitamins C and E. Besides, it can help to boost up liver function (McGuffin et al., 1997). Foeniculum vulgare(Fennel):Fennel seed stimulates milk flow. It increases udder milk production, but also helps to expel gas and aids in digestion and relieves Colic (McGuffinet al., 1997). Cumirium cyminum(Cumin seed):Cumin seed is a good digestive and galactogogue. Cumin seed can be used with jiggery (McGuffinet al., 1997). Borago officinalis(Borage):Borage is also an effective galactogogue and lactation herb, but it should not be used for longer than a week at a time as it contains an 22 alkaloid that may be harmful to the liver. It is a great herb for cooling fever and balancing the adrenals (McGuffin et al., 1997). Humulus lupulus(Hops):Hops have been used to increase milk production, but can cause depression when used over an extended period of time. The beer in some countries contains Hops that are helpful like galactogogues and lactation herbs (McGuffin et al., 1997). Garum bulbocastanum(Caraway):Black caraway aids milk production and helps in digestion disorders. It is helpful for postpartum as a galactogogue and lactation herbs (McGuffin et al., 1997). 1.4 FACTORS THAT DECREASES LACTATION There are a lot of factors that can negatively affect milk supply. These factors can be grouped into non-pharmacological and pharmacological. 1.4.1 Non-Pharmacological Factors (a)Delivery method: A study published in 2006 issue of the “Journal of Preventative Medicine and Public Health” found that women who delivered via cesarean section were less likely to breastfeed their babies than those who delivered vaginally. This could be attributed to a number of factors, such as the healing process or a baby with a low birth weight. (b)Fatigue: Motherhood can be exhausting. Lack of rest is a common cause of low milk supply. (c)Health: Illness or infection can decrease lactation. One needs to see a doctor to examine his/her health. If there is an infection, the doctor may need to prescribe an antibiotic. Other illnesses such as low thyroid function and anaemia can also cause a decline in the production of milk (Lawrence and Lawrence, 1999). (d) Caffeine: Soda, coffee, tea and chocolate are good in moderation, but large amounts of caffeine can dehydrate the body. This may decrease the amount of milk 23 one can produce. Too much caffeine can also affect the baby. Some of the caffeine is passed to the baby through breast milk. (e)Smoking: Smoking usually interferes with the release of oxytocin in the body, the hormone that stimulates the led-down reflex. (f)Alcohol: This is another substance that can inhibit the let-down reflex. It can also change the flavour of the milk, causing the baby to nurse less often and if the baby breastfeeds less often, much milk will not be produced. (g)Stress: Psychological stress can inhibit milk production. If you are concerned about privacy while breastfeeding, one might feel self-conscious or embarrassed which can interfere with let-down. Other causes of stress such as anxiety, pain, financial difficulty, and marital trouble can also contribute to a diminished milk supply. (h)Pregnancy: If one becomes pregnant while still breastfeeding, the hormones of pregnancy can cause a decrease in milk supply. (i)Diet: Diet and hydration have not been shown to cause a significant decrease in milk supply. Mothers all over the world can produce enough milk for their babies even when their diet is limited. However, a healthy diet and adequate hydration are important for your overall health (Lawrence and Lawrence, 1999). 1.4.2 Pharmacological Factors Below are lists of drugs that are contraindicated in lactation because they decrease milk production or cross the breast membrane and then affect the baby. They are grouped into two categories (Howard, 2001) namely; 1.4.2.1. Synthetic Medications (A) Medications that decrease milk production (1) Bromocriptine (2) Diuretics

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(B) Chemotherapeutic Medications (1) Cyclophosphamide (2) Cyclosporine (3) Doxorubicin (4) Methotrexate (5) Gold salts (6) Propylthiouracil (7) Methimazole (C) Cardiovascular Medications (1) Avoid Atenolol and use other Beta Blockers only with caution. (2) Avoid Acebutolol (3) Amiodarone (D) Miscellaneous Medications (1) Dextroamphetamine (2) Lithium (3) Ergotamine (4) Metronidazole (5) Chloramphenicol (6) Potassium iodide (7) Phenindione (Anticoagulant) (E) Drugs of Abuse (1) Amphetamine (2) Cocaine (3) Heroin (4) Marijuana (5) Nicotine (6) Phencyclidine 25

1.4.2.2 Herbs That May Decrease Milk Supply Using large amounts of the following herbs and other natural remedies should be avoided while nursing because they have been known to decrease milk supply. These herbs are sometimes used by nursing mothers to treat oversupply or when weaning (kellyBonyata, 2011). • Juglans nigra (Black Walnut) • Stellaria media (Chick weed) • Geranium robertianum (Herb Robert) • Melissa officinalis (Lemon Balm) • Origanum vulgare (Oregano) • Petroselinum crispum (Parsley) • Mentha piperita/Menthol (Peppermint) • Vinca minor (Periwinkle Herb) • Salvia officinalis (Sage) • Rumex acctosa(Sorrel) • Mentha spicata (Spearmint) • Thymus serpyllum (Thyme) • Achillea millefolium (Yarrow) 1.5 THE PLANT Stachytarpheta jamaicensis 1.5.1Plant Plants are classified on the basis of their structure, function, development and evolutionary history. This system is credited to Carl Von Linn, a Swedish Naturalist of the Eighteenth Century. Following this system, Stachytarpheta jamaicensis is classified into: Kingdom : Plantae Phylum : Spermatophyta 26

Subphylum : Angiospermae Class : Dicotyledonae Order : Family : Verbenaceae Genus : Stachytarpheta Species : jamaicensis Geographical source : Eastern Nigeria (Udenu in Nsukka L.G.A) Common Names Blue porterweed; Blue snake weed; Brazilian tea; Jamaican porterweed; Jamaican vervain; Bastard vervain etc. Rat tail or Devil’s coach whip (English) Gajihan, Ngadi rengga (Indonesia) Verbena cimarrona (Spanish) Albaka (Philippines) Jia ma bian (Chinese) Vervaine (French) Kariyartharani (Hindi) Local Names Tsarkiyar kuusuu (Hausa) Agogo igun (Yoruba) Aran-umon (Efik) Characteristics Climate Tropical & subtropical regions Habitat Native bush lands, gardens Plant Habit Round Flower colour Blue-violet to magenta Odour characteristics 27

Morphological part used leaves Season of collection wet and dry season Time of collection morning Condition of collection before use fresh Loss on drying not more than 17.63% Storage condition cool dry place Packing material Amber coloured bottle. U.S.D.A zone 9b-11 (250F minimum) Plant type perennial Growth rate fast Light requirement full sun; light shade Flowering months all year but less from December through February Leaf persistence Evergreen Soil salt Tolerance High Salt spray Tolerance High Drought tolerance Medium Soil Requirements wide Nutritional Requirements Low Major pests none Typical Dimensions 1-3 feet tall by 3feet wide Propagation stem cutting, will self-seed Human Hazards None Wildlife butterfly attractant Uses ground cover, hanging basket, butterfly garden; background plant for shorter stature planting. 28

1.5.2Description of the Plant Stachytarpheta jamaicensis is an erect perennial herb, up to 1.2(2) m tall which is sometimes woody at the base, often dichotomously branched from the base and spreading. The young stems are obtusely quadrangular, and sparingly hairy. The leaves are arranged opposite, simple, obovate to oblong-elliptical, measure (2- ) 4-9 cm x (1- ) 2-5 cm, wedge-shaped to wing-like decurrent at base, with apex obtuse to slightly acute, with serrate-dentate margin, hairless above but sometimes sparingly hairy below. The leaves are subsessile to shortly-petiolate while the stipules are absent (Globinmed, 2012). The inflorescence is a spike, 15-50 cm long, solitary, cylindrical, stout and often flexuous. The rachis is up to 7 mm in diameter. The furrows of the half- immersed flowers are much narrower than the mature rachis. The peduncle is (0.5)1-2.5 (-3.5) cm long and hairless. The flowers are sessile, at first erect but later immersed in the thickened rachis. The sepal is compressed, completely embedded and about 5-7 mm long, while the petal is pale bluish, violet or purple, with a whitish spot at the throat, and saucer-shaped, with the tube about 1 cm long, slightly curved and 2-lipped. The upper lip is 2-lobed while the lower lip is 3- lobed. The lobes are subequal while the limb is about 8 mm wide. There are 2 fertile stamens and 2 staminodes. The ovary is superior, 2-locular with style endosperm (Globinmed, 2012). The fruit is a schizocarp, oblong-linear, measures 3-15 (-7) mm x 1.5-2 mm and is enclosed in the fruiting sepal. It splits at maturity into 2 hard mericarps where each mericarp is 1-seeded. The seed is linear and without endosperm (Globinmed, 2012).

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Fig 3: Photograph of the plant showing Fig 4: Photograph of plant showing leaves and flower clusters stems and paired leaves

Fig 5: Photograph of plant showing Fig 6: Photograph of plant showing close-up elongated flower clusters of flowers 1.5.3 Geographical Distribution of thePlant Stachytarpheta jamaicensis originates from the New World tropics and at present has a pantropical distribution (Globinmed, 2012). Blue porterweed is considered native to the South Florida Countries of Miami-Dade, Monroe, Collier and Lee. It grows on dunes, shell middens, pine rock lands but more commonly on disturbed sited. Its native range also includes the Caribbean, the Bahamas, Bermuda and Mexico, all throughout Central America to Brazil and Ecuador. It is now a pantropical weed present in east and West Africa, Madagascar, the Ryukyu Islands 30 of Japan, Taiwan, the Indian Subcontinent, Australia, Indonesia, Malaysia and on many pacific Islands (Gilman, 2011). 1.5.4 Chemical Constituents of plant Phytochemical studies have yielded flavonoids, triterpenes, monoterpenes, tannins, iridiods, phytosterols, aromatic acids, gamma amino butyric acid (GABA), dopamine and alkanes (Philippine Pharmacopoeia, 2004). -Phytochemicals isolated include epigenol-7-glucoronide, alpha-spinasterol, stachytarphine, uroslic acid, scutellarein and verbascoside (Philippine Pharmacopoeia, 2004). -A glucoside, stachytarphine has been isolated from the plant (Philippine Pharmacopoeia, 2004). -An iridiod glycoside, verbascoside or acetoside, has been isolated from the plant, shown to be a powerful antioxidant phytochemical (Philippine Pharmacopoeia, 2004). -A flavonoid, scuttelarain has been isolated, with cardioprotective, anti- inflammatory and antiviral actions (Philippine Pharmacopoeia, 2004). -Hopidulin, another flavonioids, is reported to be bronchodilator, antispasmodic and anti-asthmatic (Philippine Pharmacopoeia, 2004). -Study of leaves isolated a new lanostane triterpenoid 16β-(β-D-glycopyramosyl-3- 8, dihydroxylanstan-5, 22-diene-11-methroxy-1β-yl-6-0-(2, 3-dimethoxybenzoyl)- β-d-glycopyranoside (Okwu and Ohenhen, 2010). -Two novel steroidal glycosides were reported to have been isolated from the leaves of S. jamaicensis. The occurrence of steroidal glycoside in SJ may explain the use of the plant as a galactogogue (Okwu and Ohenhen, 2010).

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1.5.5 Ethnomedicinal Uses Stachytarpheta jamaicensis is used in ethnomedicine to treat numerous ailments. The root decoctions are abortive. Decoctions of leaves are vermifuge to children (Philippine Pharmacopoeia, 2004). In the Antilles, the expressed juice of fresh leaves is emetocathartic. Decoctions of leaves in enemas is used to expel intestinal worms; also used as purging vehicle for other vermifuges. Infusion of roots has been used for gonorrhea. Triturated fresh leaves have been used on ulcers. It is also used as maturative cataplasm for boils. Bruised leaves have been rubbed on sprains and bruises (Gilman, 2011). In Brazil, it is used for cough, fever, to expel worms and promote menstruation; as a diuretic and laxative. It is also use for rheumatism (Philippine Pharmacopoeia, 2004). In the West Indies, it is used to expel worms. The Creols use the leaf tea for dysentery. In North Nigeria, decoction is used for dysentery. It is also used as vermifuge. In Peru, it is used for diabetes. In Cuban herbal medicine, it is used as an abortive. In immigrant Haitian communities in Cuba, an infusion made from three whorls or tops of Stachytarpheta jamaicensis is used for children in the morning on an empty stomach as an anthelminitic (Philippine Pharmacopoeia, 2004). In traditional medicine, leaves and stem extract are used to prepare drugs for use as stomach tonic. It is also used for dyspepsia, allergies, asthma, fevers and liver problems (Philippine Pharmacopoeia, 2004). In phytomedicine, the leaves of Stachytarpheta jamaicensis are used for birth control, abortion, treatment of menstrual disorders and as a galactogogue (Okwu and Ohenhen, 2010). 32

Externally, it is used for ulcers, sores, cuts and wounds (Philippine Pharmacopoeia, 2004). 1.6 STANDARDIZATION OF HERBAL MEDICINES 1.6.1 Pharmacognostic Standardization of Herbal Medicines Standardization of drugs is the process of establishing or prescribing a set of peculiar identities, specific characteristics which are generally unique and of unshared qualities. Pharmacognostic Standardization of a drug is a process involving a series of laboratory experiments which reveal and assemble a set of inherent peculiar characteristics, such as, constant parameters, definite qualitative and quantitative value or specific and unique features on the bases of which similar herbal medicines, claimed to be the same, can be compared for the purpose of authenticity, efficacy, genuineness, purity, reproducibility and overall quality assurance. Various steps are involved in setting these Pharmacognostic Standards for the purpose of formulating a monograph of a crude drug. Uniformity of quality is promoted by the use of standards which are numerical qualities by which the quality of commodities may be assessed (Inya-Agha, 2006). Specific standards obtained through experimentation may be compiled into a monograph of selected medicinal plant. Monographs of selected medicinal plants can be assembled together to constitute a herbal pharmacopoeia. Herbal Pharmacopoeia carries an assurance of data for monitoring safety, efficacy, and reproducibility. The pharmacopoeia essentially provides parameters for any national drug regulatory requirements or dossier. Pharmacognostic Standardization of a herbal medicine is the provision of a standard official monograph to include the essential characteristics of the plant components. This is done for the purpose of correct identification and qualification such that any other sample of the plant at any other time can be compared with and related to the original sample when subjected to the recommended monograph. Such other samples can either be 33 accepted (if found to comply with the monograph) or rejected (if grossly below the standard). Besides the efficacy and safety, the provision of Pharmacognostical Standards is a pre-requisite for herbal formulation and clinical exploitation (Inya- Agha, 2006). 1.6.2Macroscopic Standards The Macroscopy of a crude drug includes its visual appearance to the naked eyes and its other sensory characteristics such as odour and taste sensations. Macroscopic analysis defines the establishment of morphological characteristics of plant products achieved by organoleptic evaluation. This involves recording features noted upon observing the specimen with or without the aid of a magnifying lens, as well as evaluation of the specimen using other sensory characters such as smell as well as touch. These tests provide size, colour and odour properties of the specimen being analyzed. Various crude vegetable drugs have been classified or grouped based on their characteristics. This identification and characterization processes can be carried out using various botanical examination techniques (Inya-Agha, 2006). These physical measurements may often provide the simplest and quickest indication for its identity, purity or quality when compared with the official monograph. Most herbal drugs fall under the following morphological classes: Barks (i.e. tissue in a wooden stem outside the cambium); Underground structures (i.e. roots, rhizomes, corms and bulbs); Whole herbs (all the above ground parts of the plant); leaves, flowers, fruits and seeds. The shape, size, texture or fracture -in the case of barks, roots or rhizomes; leaf arrangement, margins, venation, shape and surface description for leaves and for flowers; the presence or absence, and the type of calyx, corolla, androecium/gynoecium and inflorescence, type of ovary, placentation and seed description; they are all useful parameters for assessing the 34 identity, purity and to a certain extent, quality of the herbal material (Ewurum, 2009). 1.6.3 Microscopical Standards The microscopy of a crude drug provides relevant microscopical standards. Microscopical analysis gives the anatomical characteristics of the tissue obtained by transverse, radial and longitudinal sections. This helps to specify the taxonomic position of the crude drug in some cases (Inya-Agha, 2006). Microscopic analysis is certainly the most objective and reliable among the various pharmacognostic techniques of drug identification and standardization. Since it deals with tissue exposition and visualization under the microscope, essentially providing closer cellular discrimination in their intact natural arrangement as well as for qualitative measurement of the internal structures, no two plant species will posses exactly the same cellular patterns qualitatively and quantitatively in all respects (Ewurum, 2009). In fact, the use of microscopical and micrometric data (known as diagnostic characters) for example; types of cells- (parenchyma, sclerenchyma, collenchyma), size of the cells, trichomes, stomata pore, starch grains, aleuronic grains, fibres, pollen etc. is indispensable for the identification and quality control of herbal drugs especially in powdered forms. The basic layout tissues which is constant in crude drugs of stems, leaves, roots etc can be best ascertained by careful observation of the transverse, longitudinal (radial and tangential) and surface sections. The cells which are most useful for purposes of identification, standardization and quality include fibres, sclereids, trachieds, vessels, cork cells etc and also are least affected by drying process. The cell contents are detectable by using various micro chemical tests and with greatest importance are starch grains, calcium oxalate crystals, lignins, proteins, and fixed oils (Ewurum, 2009).

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1.6.3.1 Qualitative Microscopy Qualitative Microscopy is concerned with the identification of the diagnostic characters present in the soaked, cleared “concised” crude drug materials or powdered crude drug with aid of the microscope. The purity of many unground drugs can be established or confirmed by an examination of calcium oxalate crystals, detailed structure of the trichomes and other features, for the detection of adulterants in powdered drugs, knowledge of microscopical structures are essential (Inya-Agha, 2006). 1.6.3.2 Quantitative Microscopy Quantitative microscopy gives a constant value for a given diagnostic character of particular species. When chemical and physical methods are inapplicable, as often happens with powdered drugs, in certain instances, one can determine the proportions of the substances present by means of the microscope using the lycopodium spore method. It involves accurate cellular micrometry of all the issues such as: palisade ratio, stomata number, stomata index, vein-islet number and veinlet termination number (Trease and Evans, 1996). i. Palisade Ratio: This is the average number of palisade cells beneath one epidermal cell, using four contiguous epidermal cells for the count. This ratio has been shown to be sufficiently constant to serve as a diagnostic character of species belonging to the same genus in certain instances (Trease and Evans, 1996). ii. Stomata Number:This is the average number of stomata per square millimeter of epidermis. It varies considerably with the age of the leaf. In recording results, the range as well as the average value should be recorded for each surface of the leaf and the ratio of values for the surface (Trease and Evans, 1996). 36 iii. Stomata Index:This is the percentage of the ultimate divisions of the epidermis of a leaf which has been converted into stomata. It is noted that while the stomata number varies considerably with the age of the leaf, stomata index remains constant irrespective of the age of the leaf. Thus if S = the number of stomata per unit area and E = the number of epidermal cells in the same unit area, then; Stomata Index; SI = S x 100 E + S 1 The figure so obtained is fairly constant for any species and can be used as a specific character (Trease and Evans, 1996). It is highly constant for given specie and may be determined on powdered samples. iv. Vein-islet Number: This is used to denote the minute area of photosynthetic tissues encircled by the ultimate division of the conducting strands. The number of vein-islets mm-2 is calculated from four contiguous square millimeters in the central part of the lamina, midway between the midrib and the margin. When determined on whole leaf, the area examined should be from the central part of the lamina, midway between the margin and midrib. It is independent of the size of the leaf and does not alter with the age of the plant (Trease and Evans, 1996). v. Veinlet Termination Number: This is the number of veinlet terminations per mm2 of the leaf surface. A veinlet termination is the ultimate free termination of a veinlet or branch of a veinlet. It also shares the same advantage as the vein-islet number (Trease and Evans, 1996).

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1.6.4 Analytical Standard Analytical Standards consist of the qualitative and quantitative analysis of the chief chemical constituents of crude drugs and also the determination of the various ash values as well as solvent extractive values in accordance with Pharmacognostical Methods (B.P., 2001). The percentage of fiber or stone cells in barks and roots, for example, Cascara, Rhubarb or Cinnamon could be characteristic. 1.6.4.1 Ash values a)Total ash value The ash content of a crude drug is the residue remaining after incineration of the crude drug. This represents the amount of inorganic salts adhering to or occurring naturally in the drug. At times, this may be extended to include inorganic matter added for the purpose of adulteration. Ash determination gives basis for the evaluation of the identity and purity of a crude drug. It also gives an idea of the crude drug’s extent of adulteration with inorganic matter (Trease and Evans, 1996). b)Water soluble ash values The water soluble ash is subjected to greater reduction than in the total ash. It is thus used in the detection of material exhausted by water and as such is an important indication for the presence of materials substituted for the genuine article (Trease and Evans, 1996). c)Acid-insoluble ash values Ash, which is insoluble in dilute hydrochloric acid, is referred to as acid- insoluble ash. Acid insoluble ash value is often preferred to total ash value. This preference is based on the fact that majority of crude drugs often contain calcium oxalates in large but variable amounts, and as such total ash values may vary within limits for genuine drug specimen. The total ash is therefore of no use in the detection of earthly matter adherent to such a drug specimen. Nevertheless, total 38 ash value is still of importance since its figures are useful in the exclusion of drugs which have been coated with lime, chalk or calcium sulphate to improve their appearance. However, since the calcium carbonate or oxide yielded by the incinerated oxalates is soluble in hydrochloric acid, variable constituents of the ash are thus removed by this means. This residue, known as acid insoluble ash, is then weighed, it is obvious that earthly matter is likely to occur with leaves which are densely pubescent, or are clothed with abundant trichomes, secreting resins or with root and rhizomes, or even earthly matter retained on them after heavy rain storms. Evidence of the presence of such excessive earthy matter can thus be obtained and established using this means (Trease and Evans, 1996). d)Sulphated ash value This is a process which entails the conversion of all oxides and carbonates to sulphates at higher temperatures. Sulphated ash produces a more constituent ash value (Trease and Evans, 1996). 1.6.4.2 Extractive yields Various methods have been employed in the identification and evaluation of crude vegetable drugs. It is pertinent to highlight the fact that, in most cases, the amount of drug soluble in a given solvent is an index of its polarity. Thus, on this basis, the determination of extractive yields finds its usefulness. The determination of ethanol-soluble extractive and water soluble extractive are used as a means of evaluating drugs whose constituents are not readily estimated by other means (Trease and Evans, 1996). 1.6.4.3 Moisture Content The most widely accepted method of moisture content determination of plant and food materials is the gravimetric methods, as modified by the Association of Official Analytical Chemist (AOAC) (1980). It is the most 39 important and widely used measurement in samples that absorb and retain water. Moisture content determination looks very simple in concept, but to practice the accurate determination is complicated by a number of facts which may vary considerably from one sample to another. Among the facts are the relative amounts of water available and the case with which the moisture can be removed. Air or vacuum oven drying at 70 0C– 80 0C are considered to be reliable methods provided that there is no chemical decomposition of the sample and water is the only volatile constituent removed. Sample should be dried to a constant weight (Trease and Evans, 1996). 1.6.5 Structural Standards These impose limits on the amounts of certain, named parts of the plant concerned which had been included carelessly or due to faulty collection as well as other foreign organic matter for example insects, fungi, hairs and contaminants brought about by pests and rodents, which constitute the materials deliberately added in partial or complete substitution of the genuine article with an intention to deceive. Appreciable amount of potent inorganic matter, animal excreta, insects or moulds may result in rejection of the drug. The amount allowable of any of these which can be fixed as a standard for any given drug must be determined in relation to the amount found experimentally for good commercial samples of such drugs (usually 2%). 1.6.6 Physical Constants as Standards The properties of crude drugs employed for the measurement of physical constants, include density, refractive index, iodine value, saponification value optical rotation or average mass of seeds and starch grains and after applicable to fixed oils, volatile oils, fats, waxes and resins.

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1.7 PREVIOUS PHARMACOLOGICAL STUDIES i. Antidiarrheal/Antimiorobial: The methanol extract of Stachytarpheta jamaicensis leaves showed significant antidiarrheal activity and moderate inhibitory activity against E.coli, Staph. epidermis and P.aeruginosa (Susidharan et al., 2007). Crude aqueous extract showed activity against B. subtilis, E. coli, C. albicans, S. aureus, P. aeruginosa, P. valgaris, and P. mirabilis. (Idu et al., 2007). Study showed more antimicrobial activity with the chloroform extract against gram positive organisms like S. aureus, E. faecalis, and B. subtilis. The chloroform and alcohol extracts showed antifungal activity against C. albicans and Saccharomyces cere viseae (Meena and Pitchai, 2011).

ii. Antioxidant/O2-Scavening Activity: Inhibitory effect of leaf extracts of Stachytarpheta jamaicensis (Verbenanceae) was done on the respiratory

burst of rat macrophages. Extract showed potent O2-scavening activity. Study suggests SJ may have potential pharmaceutical value for immunologic disease related to oxidative stress (Alvarez et al., 2004). iii. Anti-hypertensive/Bradycardic Effect: Some Cardiovascular Effects of the Aqueous Extract of the leaves of Stachytarpheta jamaicensisL. Vahl. The aqueous extracts of SJ leaves caused a dose-dependent drop in blood pressure and heart rate. The acute hypotensive effect could be partly caused by a negative chronotropic effect of direct effect on vascular smooth muscles (Almeida et al., 2001). iv. Antinociceptive/Anti-inflammatory: The study of the ethanol extract of SJ showed significant dose-dependent nociceptive activity in all nociceptive models tested. The extract showed significant anti-inflammatory activity in both acute and chronic models. The analgesic activity was assumed to be 41

modulated via peripheral and central mechanisms, partly involving the activation of the opioid receptor system (Sulaiman et al., 2009). v. Toxicity Study: A study on 20 Wister rats on the effect of powdered SJ leaves, using serum biochemistry and ultrasonography showed no toxicity, suggesting a wide therapeutic margin of safety (Idu et al., 2007). vi. Antimalaria: The ethanol extract of Stachytarpheta jamaicensis exhibited significant schizonticidal activity comparable to that of the standard drug, chloroquine. The antiplasmodial activity confirms its folkloric use in the treatment of malaria (Okonkon et al., 2008). vii. Anti-Dyslipidemia/Anti-Atherogenic: The effects of Stachytarpheta jamaicensis tea on plasma lipid profile and atherogenic indices were studied in rabbits. Treatment caused significant decrease in plasma total cholesterol, LDL, VLDL and triglycerides with also significant decrease in atherogenic indices. The result suggests the use of SJ tea in the management of primary and secondary dyslipidemia (Ikewuchi and Ikewuchi, 2009). viii. Cytotoxic: In the study on cytotoxic effect, leaf extract showed the highest inhibition on the growth of Hela cancer cells compared to the root and stem extracts (Putera and Shazura, 2010). ix. Hypoglycermic/Leaves: Study evaluated the hypoglycermic effectofan ethanolic extract of S. jamaicensis on blood glucose level of Streptozotocin induced diabetic rats. Result dose-dependent hypoglycermic activity almost equal to the standard drug metformin (Silambujanaki et al., 2009). x. Analgesic/leaves: Study evaluated the analgesic activity of various extract of dried leaves on acetic induced writing responses in Swiss albino mice. Result showed significant analgesic effect (Jagadish and Gopalkrishna, 2008). 42

xi. Wound Healing /leaves: Study evaluated the wound healing effect of a hydroalcoholic leaf extract of S. jamaicensis on streptozotocin induced diabetic rats. Results showed significant dose-dependent wound healing potential with a significant increase in percentage wound closure, tensile strength, hydroxyproline, Hexosamine, DNA and total protein content together with decrease in period of epithelization and blood sugar levels (Chitra et al., 2013). 1.8 AIM AND SCOPE OF STUDY This study is aimed at: i) Providing pharmacognostical standards for Stachytarpheta jamaicensis. ii) Ascertaining the scientific basis for the ethnomedicinal applications and uses of its leaves as a galactogogue. iii) Determining the safety of the plant and iv) Identifying the phytochemical constituents responsible for its effect.

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CHAPTER TWO MATERIALS AND METHODOLOGY

2.1 COLLECTION, IDENTIFICATION AND PREPARATION OF PLANT MATERIALS. The samples of Stachytarpheta jamaicensis(SJ) leaves were collected in Udenu Local Government in Enugu State of Nigeria in June, 2014. The plant was identifiedby Mr. A.O. Ozioko of the International Centre for Ethnomedicine and Drug Development (Inter CEDD), Nsukka, Enugu-State, Nigeria. SJ fresh leaves were air-dried in shade and pulverized and the leaf powder was used for extraction and pharmacognostic standardization. 2.2 DRUGS, CHEMICALS, REAGENTS AND EQUIPMENT

Accu-Bind Elisa Microwells (Monobind Inc. Lake forest, CA 92630, USA), Metoclopramide (NAFDAC Reg. No. 04-5946), Estradiol valerate injection USP (Cadila Healthcare Ltd, Ponda). All chemicals, solvents and reagents were of analytical grade. Chloral hydrate, mountant (Glycerine), 50 % Gelatin, Sodium hypochlorite, Hydrochloric acid, Phloroglucinol in hydrochloric acid and Iodine were used for microscopical analysis. Methanol (JHD), n-hexane (JHD), ethylacetate (JHD), n-butanol (JHD) and Water were used for extraction and fractionation.

Solution of crystalline CuSO4 in Sulphuric acid (Fehling’s solution I), solution of Rochelle salt and Potassium hydroxide (Fehling’s solution II), Potassium bismuth iodide solution (Dragendorff’s reagent), solution of iodine in potassium iodide (Wagner’s reagent), Potassium mercuric iodide solution (Mayer’s reagent), saturated solution of Picric acid (Hager’s reagent) Million’s reagent, 44

Naphthol solution in ethanol (Molisch’s reagent), -naphthol, Sulphuric acid

(H2SO4), Ammonium hydroxide (NH4OH), Chloroform, Sodium hydroxide (NaOH), carbon tetrachloride, Ferric chloride, Ethanol (30%, 90 %), Lead subacetate, Glacial acetic acid, Ethylacetate, Aluminum chloride and Olive oil were used for phytochemical test. Distilled water, 30 % Tween 80 and Arachis oil were used for pharmacological studies.

Sharp knife, mortar and pestle, sieve, funnel, gallons, cornical flasks, lypholizer (freeze dryer), beaker, nickel crucible, filter papers, aluminum foil, weighing balance, dessicator, stage micrometer, photomicroscope, micrometer disc, test tubes, measuring cylinder, bunsen burner, spatula, separating funnel, vacuum evaporator, electronic balance, stop clock, syringes and needle, photomicroscope, rotary evaporator, pipette, sledge microtome, centrifuge, EDTA bottles with and without EDTA inside, Dispensers, Microplate washers, Microplate reader with 450 or 620 nm filter, Absorbent paper, Microplate cover, Vacuum aspirator, Timer, Quality control materials and centrifuge were some of the equipment used for the experiment.

2.3 EXPERIMENTAL ANIMALS

Adult female Swiss albino rats (weighing 120-160 g) and mice (weighing 24-36 g) of either sex were obtained from Zoological Garden of the Department of Zoology, University of Nigeria, Nsukka. The albino rats were housed and mated with the male rats in a stainless steel metal cages with wood chips on the floor under standard laboratory condition with 12 h dark/light cycle so that they can become pregnant. They were fed with commercial feeds and tap water ad libitum. Following birth, the litters’ weights were recorded and culled to 7 litters per dam. 45

2.4MACROSCOPIC EXAMINATION OF THE LEAVES. The fresh leaves of SJ were visually examined. The macroscopic characters of the leaves which include type of margin, venation, base, apex, mid-rib, etc. were observed and noted. The organoleptic properties such as colour, odour and taste of the plant materials were also observed and noted. 2.5MICROSCOPIC EXAMINATION OF POWDERED LEAVES (a)Qualitative microscopy A sample of the leaf powder was placed on the slide, two drops of chloral hydrate solution was added to moisten the powder and also act as a clearing agent. The slide was passed across the flame of a bursen burner repeatedly until bubbles occurred. It was allowed to cool, the slide was covered with glycerin followed with cover slip and was viewed under the photomicroscope. The microscopic characters were observed and noted. (b) Quantitative Microscopy (i) Palisade ratio The powder of the leaf was cleared by boiling with chloral hydrate solution, mounted and examined with a 4 mm objective. A photomicroscope was employed so that the epidermal cells and the palisade cells lying below them were captured. First, a number of groups of each of four epidermal cells were traced and their outlines were inked in to make them more conspicuous. The palisade cells lying beneath each group were then focused and traced. The palisade cells in each group are those being included in the count, which are more than half covered by the epidermal cells; the value obtained divided by four gives the palisade of the group. The range of a number of groups from different particles was recorded (Trease and Evans, 1996). 46

(ii) Stomata number Fresh leaves were used and an approximate 50 % gelatin was liquefied on a water-bath and smeared on a hot slide. The fresh leaves were added, the slide inverted and cooled under a tap and after about 15-30 minutes, the specimen was stripped off. The imprint on the gelatin gave a clear outline of epidermal cells, stomata and fibres (Trease and Evans, 1996). (iii) Stomata index Pieces of leaf (not extreme margin or midrib) were suitably cleared and mounted; the lower surface was examined by means of a microscope with a 4 mm objective and an eye-piece containing a 5 mm square micrometer disc. Counts were made of the numbers of epidermal cells and of stomata (the two guard cells being considered as one unit) within the square grid, a cell being counted if at least half of its areas lie within the grid. Successive adjacent fields were examined until about 400 cells were counted and the stomata index value calculated from these figures. The stomata index was determined for both surfaces (Trease and Evans, 1996). (iv) Vein-islet number The leaves were cleared by boiling in chloral hydrate solution in a test tube and placed in a boiling water bath. A photomicroscope was set up and stage micrometer was used to divide the paper into squares of 1 mm2 using a 16 mm objective. The stage micrometer is then replaced by the cleared preparation and the veins were traced in four contiguous squares, in a rectangle of 1 mm x 4 mm. The vein-islet is numbered during the photomicroscope process (Trease and Evans, 1996).

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(v) Veinlet termination number Pieces of the leaves were soaked in water and then were treated with sodium hypochlorite and 10 % hydrochloric acid. It was boiled with chloral hydrate solution for 5 minutes to clear. A camera was attached to the microscope and the leaf was mounted and examined. The veinlet was traced in four contiguous squares in a rectangle of 1 mm x 4 mm and each veinlet was numbered and noted (Trease and Evans, 1996). (vi) Transverse section of the leaf With the aid of the sledge microtome, a transverse thin section of the leaf was cleared in a solution of chloral hydrate, after which it was stained with 1 % phloroglucinol in hydrochloric acid, covered with glycerin and cover slip and viewed under the photomicroscope. In a second slide, the cleared thin section was stained with iodine, covered with glycerin and covers slip and was viewed under the microscope. The distribution of tissues and details of the structures of individual cells were photographed under the Photomicroscope. 2.6 DETERMINATION OF ANALYTICAL STANDARDS i. Determination of ash values a. Total ash values A tarred nickel crucible was placed in Mauffle furnace for about 15 mins, 35 0C, cooled in a desiccator for about one hour and the crucible was weighed

(W1). A 3.0 g of the powered material was placed into the nickel crucible and heated gently until all the moisture has been driven off and the plant material was completely charred (W2). The heat was slowly increased until the carbon has vaporized and the residue was free from carbon at 650 0C and the sample turns grey (white ash). The crucible was removed with crucible tong, cooled in a 48

dessicator, and reweighed (W3). The percentage ash content was determined by the relationship;

% Ash = Final weight of crucible (W3) – Initial weight of crucible (W1) x 100

Weight of sample and crucible (W2) – Initial weight of crucible (W1) 1 b. Water soluble ash value A nickel crucible was ignited to a constant weight at 450 0C, cooled and then weighed (W1). A 3.0 g of the material was placed and spread over the bottom of the crucible and then reweighed (W2). The plant material was incinerated to 450 0C by gradually increasing the heat until it was freed from carbon. The crucible was cooled in a desiccator and then reweighed. The contents of the crucible were transferred into a beaker; 5 ml of water was added into the beaker and then boiled for 5 mins. The mixture was filtered through an ashless filter paper, and both the residue and the filter paper were dried in an oven. The ashless filter paper containing the residue was compressed into the crucible and was subjected to heat until the ashless paper was eliminated. The crucible was reweighed (W3) and the differences were noted with formula.

% Water Insoluble Ash = Weight of sample and crucible (W2) – Initial weight of crucible (W1) x 100

Final weight of crucible (W3) 1 % water soluble Ash = % Total Ash - % Water Insoluble Ash c. Acid-insoluble ash value The ash obtained above was transferred into a beaker containing 25 ml of dilute hydrochloric acid and was boiled for 5 mins. The insoluble matter was collected in a sintered crucible and an ashless filter paper. The beaker and the crucible were washed repeatedly through the filter paper with hot water until it was free from acid. It was ignited to constant weight at about 500 0C.

49 d.Sulphated ash value A nickel crucible was ignited to a constant weight at 450 0C, cooled and weighed. A 3.0 g of the dried material was placed over the bottom of the crucible and then reweighed. The material was moistened with dilute sulphuric acid and then incinerated to 450 0C by gradually increasing the heat until it was free from carbon. The crucible was cooled in a desiccators and more dilute sulphuric acid was added. The heating was continued to about 800 0C with occasional cooling and reweighing until a constant weight was obtained. The % sulphated ash value was determined by difference of the two weights, thus;

% Sulphated Ash = Final weight of sample – Initial weight of sample x 100

Initial weight of sample 1 ii. Determination of extractive yields a. Alcohol soluble extractive value A 5.0 g of the material was weighed accurately and placed in a stoppered conical flask. A 100 ml of 90 % alcohol was added and the stopper of the conical flask was replaced firmly. The flask and its contents were shaken mechanically for about 6 hours and was allowed to macerate for another 18 hours and then filtered. The filtrate was collected and evaporated to dryness, and then the residue was dried to a constant weight at 105 0C. b. Water soluble extractive value A5.0 g of the material was weighed accurately and placed in a stoppered conical flask. A 100 ml of chloroform-water was added and the stopper of the conical flask was replaced firmly. The flask and its contents were shaken mechanically for 6 hours and were allowed to macerate for another 18 hours 50

and then filtered. The filtrate was collected and evaporated to dryness and then the residue was dried to a constant weight at 105 0C. iii. Determination of moisture content A preheated, tarred porcelain crucible was weighed and its weight with lid recorded (W1). A spatula full of the dried sample was introduced into the crucible and was reweighed, (W2). The sample was heated in an oven at the temperature of 65 0C for 12 hours, at intervals of 6, 3, 2, 1, hours until a constant weight, followed by cooling in a desiccator before reweighing. The constant weight, W3 was noted. The percentage moisture was calculated from the relationship.

% moisture = Weight of sample in crucible (W2) – Constant weight (W3) x 100

Weight of sample in crucible (W2) – Weight of crucible (W1) 1

Where W2 –W1 = weight of sample

W2-W3 = weight of moisture 2.7 EXTRACTION A 1.89 kg (1,890 g) of the ground leaves was macerated usingabsolute methanol in a stoppered container. The mixture was left for 72 hours with occasional shaking. It was then filtered througha plug of cotton wool or glass wool. The process was repeated exhaustively for complete extraction. The filtrate was then concentrated under reduced pressure using a rotary evaporator to obtain the crude extract. 2.8 FRACTIONATION This was conducted using a separating funnel. A miscibility studies was first done to ensure that the solvents were immiscible and to know which of the solvents floats on top. A 200 g of the methanol crude extract was properly re- dissolved in a given volume of water (polar solvent) and poured through the top with the stopcock at the bottom closed. An equal volume of n-hexane was added. 51

The funnel was then closed and shaken gently and the separating funnel was set aside to allow for the complete separation of the phases. The lid and the lower tap were then opened and the lower phase was released, by gravitation. When the lower layer (water mixture) has been removed, the stopcock was closed and the upper layer (n-hexane fraction) was poured out into another container. The lower layer (water mixture) was again re-introduced into the separating funnel with the stopcock at the lower end closed and the whole processes described above was repeated for ethyl acetate, n-butanol and water fractions respectively. The water fraction was also obtained at the end of the fractionation processes. The n-Hexane, ethyl acetate and n-butanol fractions (HF, EF and BF respectively) were concentrated under pressure while the water fraction (WF) was concentrated in an oven drier at a temperature of 20-30 0C. The fractions HF, EF, BF and WF were weighed and stored in the refrigerator for further experiment. 2.9PHYTOCHEMICAL ANALYSIS (PROCEDURE) Preliminary phytochemical analyses were carried out on the extract and fractions to detect the presence of phytoconstituents using standard methods (Harbourne 1973; and Evans, 1996) Test for Carhohydrates Molisch’s test A 0.1g of the powder was boiled with 2ml of distilled water and filtered. To the filtrate were added few drops of α-naphthol solution in ethanol (Molisch’s reagent). Concentrated sulphuric acid was then gently poured down the side of the test tube to form a lower layer. A purple interfacial ring indicates the presence of carbohydrates.

52

Test for Alkaloids A 20ml of 30 % sulphuric acid in 50 % ethanol was added to 2 g of the powder and heated on a boiling water bath for 10 minutes, cooled and filtered. 2 ml of the filtrate was tested with a few drops of Mayer’s reagent (potassium mercuric iodide solution), Dragendorff’s reagent (bismuth potassium iodide solution), Wagner’s reagent (iodine in potassium iodide solution), and (reagent) picric acid solution (1%). The remaining filtrate was placed in 100 ml separating funnel and made alkaline with dilute ammonia solution. The aqueous alkaline solution was separated and extracted with two 5 ml portions of dilute sulphuric acid. The extract was tested with a few drops of Mayer’s, Wagner’s, Dragendorff’s reagents. Alkaloids give milky precipitate with few drops of Mayer’s reagent; reddish brown precipitate with few drops of Wagner’s reagent; yellowish precipitate with few drops of (reagent) picric acid and brick red precipitate with few drops of Dragendorff’s reagent. Test for Reducing Sugars (Free) A 5 ml of a mixture of equal parts of Fehling’s solution I and II were added to 5 ml of aqueous extract and then heated on a water bath for 5 minutes. A brick red precipitate shows the presence of reducing sugar. Test for Glycosides (Combined reducing sugars) A 5 ml of dilute sulphuric acid was added to 0.1 g of the powder in a test tube, boiled for 15 minutes on a water bath, then cooled and neutralized with 20 % potassium hydroxide solution. 10 ml of a mixture of equal parts of Fehling solution I and II was added and boiled for 5 minutes. A more dense brick red precipitate indicates the presence of glycosides.

53

Test for Saponins A 20 ml of distilled water was added to 0.25 g of the powder and boiled on a hot water bath for 2 minutes. The mixture was filtered while hot and allowed to cool and filtrate was used for the following tests. (a) Frothing test A 5 ml of the filtrate was diluted with 15 ml of distilled water and shaken vigorously. A stable froth (foam) upon standing indicates the presence of saponins. (b) Emulsion test To the frothing solution was added 2 drops of olive oil and the contents shaken vigorously. The formation of emulsion indicates the presence of saponins. (c) Fehling’s test To 5ml of the filtrate was added 5ml of Fehling’s solution (equal parts of I and II) and the contents were heated on a water bath. A reddish precipitate which turn brick red on further heating with sulphuric acid indicate the presence of saponins. Test for Tannins A 1g of the powdered material was boiled with 20ml of water, filtered and used for the following tests.

(a) Ferric chloride test To 3ml of the filtrate were added few drops of ferric chloride. A greenish black precipitate indicates the presence of tannins. (b) Lead acetate test To a little of the filtrate was added lead acetate solution. A reddish colour indicates the presence of tannins. 54

Test for Flavonoids A 10ml of ethyl acetate was added to 0.2g of the powder and heated on a water bath for 3 minutes. The mixture was cooled, filtered and the filtrate was used for the following tests. (a) Ammonium hydroxide test A 4 ml of filtrate was shaken with 1 ml of dilute ammonia solution. The layers were allowed to separate. A yellow colour in the ammoniacal layer indicates the presence of flavonoids. (b) 1% Aluminum chloride solution test Another 4ml portion of the filtrate was shaken with 1ml of 1 % aluminum chloride solution. The layers were allowed to separate. A yellow colour in the aluminum chloride layer indicates the presence of flavonoids. Test for Resins (a) Precipitation test A 0.2 g of the powder was extracted with 15 ml of 96 % ethanol. The extract was then poured into 20 ml of distilled water in a beaker. A precipitate occurring indicates the presence of resins. (b) Colour test A 0.2 g of the powder was extracted with chloroform and the extract was concentrated to dryness. The residue was re-dissolved in 3 ml of acetone and another 3 ml concentrated hydrochloric acid was added. This mixture was heated on water bath for 30 minutes. A pink colour which changes to magenta red indicates the presence of resins. Test for Proteins A 0.5 g of the powder was extracted with 20 ml of distilled water and the filtrate was used for the following test.

55

(a)Million’s test To a little portion of the filtrate in a test tube was added two drops of Million’s reagent. A white precipitate indicates the presence of proteins. (b)Xanthoproteic reaction test A 5ml of the filtrate was heated with few drops of concentrated nitric acid. A yellow colour which changes to orange on addition of dilute sodium hydroxide indicates the presence of proteins. (c)Picric acid test To a little portion of the filtrate was added a few drop of picric acid. A yellow precipitate indicates the presence of proteins. (d)Biuret test A crystal of copper sulphate was added to 2 ml of the filtrate followed by 2 drops of potassium hydroxide solution. A purple or pink colour indicates presence of proteins. Test for fats and oil A 0.1 g of the powder was pressed between filter paper and the paper was observed. A control was also prepared by placing 2 drops of olive oil on filter paper. Translucency of the filter paper indicates the presence of fats and oil. Test for Steroids and Terpenoids A 9 ml of ethanol was added to 1 g of the powder and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. 5 ml of hot distilled water was added to the concentrated solution, the mixture was allowed to stand for I hour and the waxy matter was filtered off. The filtrate was extracted with 2.5 ml of chloroform using separating funnel. To 0.5 ml of the chloroform extract in a test tube was carefully added 1 ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface shows the presence of steroids. 56

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and heated with 3 ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the presence of terpenoids. Test for Acidic Compounds A 0.1 g of the powder was placed in a clear dry test tube and sufficient water added. This was warmed on a hot water bath and then cooled. The piece of water wetted litmus paper was dipped into the filtrate and the colour change on the litmus paper was observed. 2.10 PHARMACOLOGICAL METHODS

2.10.1 Acute-toxicity and lethality (LD50) test

The acute-toxicity and lethality (LD50) of the crude methanol extract and fractions was determined using the method described by Lorkes (1983) using the oral route. (i)Phase One:Determination of the toxic range The mice were divided into 3 groups of 3 animals each and treated with the crude methanol extract of SJ leaf at doses of 10, 100 and 1000 mgkg-1 body weight orally. They were observed for 24 hours for signs of toxicity. (ii) Phase two:Determination of lethality The doses used in this phase were determined by the number of deaths per dose recorded in phase one. Since no death occurred in all the doses of phase one, three different doses- 1600, 2900 and 5000 mgkg-1were administered orally to other groups of animals at one dose per animal. The treated animals were observed for number of deaths for 24 hours. The LD50of this test was determined by calculating the geometric mean of the least and most toxic doses i.e. the median lethal dose (LD50) was calculated using the second phase. All the phases and processes mentioned above were also repeated accordingly to determine the toxic range and lethality (LD50) of the fractions. 57

2.10.2Effect of Oral Treatment of SJ Crude Methanol Extract on Serum PRL Concentration Twenty-five (25) lactating dams weighing 180-250 g at the beginning of lactation and suckling seven pups were used for this experiment. They were divided into five groups of five lactating rats in each group (n = 5). Group I served as normal control and received distilled water (5 ml). Group II received the standard drug metoclopramide (5 mg/kg) (Alexandre et al, 2002) and Groups III – V received (200, 400 and 800 mg/kg body weight) of the extract respectively. All animals were treated daily and all groups received the extract and the drug for six days starting from day 4-9 of lactation (Vogel and Vogel, 1997). All administrations were done orally with a gavage syringe. On day 10 of the experiment, fasting morning blood samples were collected using a plain redtop capillary tube without anticoagulant. The blood was allowed to clot and the specimen was centrifuged. The serum obtained was stored at -20 0C until assay for PRL content using Prolactin kit. All the animals were sacrificed under chloroform anesthesia and then disposed properly. 2.10.3 Effect of Oral Treatment of SJ Fractions on Serum PRL Concentration Thirty (30) lactating dams weighing 140-250 g at the beginning of lactation and suckling seven pups were randomly selected and used for this experiment. The animals were divided into six groups of five lactating rats in each group (n = 5). Group I was treated orally with 5 ml of distilled water (normal control) while Group II received 5mg/kg metoclopramide (Alexandre et al., 2002) (standard drug). Groups III – VI received ethyl acetate (800 mg/kg), n-hexane (800 mg/kg), n-butanol (800 mg/kg) and water (800 mg/kg) BW of fractions respectively. All animals were treated daily and all groups received the fractions of SJ and the drug 58

(metoclopramide) for six days starting from day 4-9 of lactation (Vogel and Vogel, 1997). All administrations were done orally with a gavage syringe. On day 10 of the experiment, fasting morning blood samples were collected as previously described. All the animals were sacrificed under chloroform anesthesia and then disposed properly. 2.10.4 Effect of Oral Treatment of SJ Crude Methanol Extract on Mammary Gland Tissues Forty (40) virgin female rats aged 60-90 days were divided into 2 groups.

The first group first received a subcutaneous injection of estradiol (E2) in a dose of 10 µg/0.1 ml arachis oil twice daily for 2 days. Subsequently they were divided into 4 subgroups and received an oral administration of 5 ml distilled water, 200, 400 and 800 mg/kg BWof SJ extract twice daily for 5 days. The second group first received an oral administration of 5 ml of distilled water and was then divided into 4 subgroups receiving the same oral administration of the extract as the first group. On the 6th day, serum PRL was obtained for PRL content as previously described. All animals were anaesthetized with chloroform and the two inguinal mammary glands were removed or harvested. They were immediately fixed in 10 % formal- saline and embedded in paraffin after dehydration in a graded series of ethanol and xylene. Paraffin sections (5 µm) of the mammary glands were sliced and stained with Harris hematoxilin and eosin (Onuoha, 2010). Mammary gland structures were identified using a microscope coupled to an image analysis system. 2.10.5 Effect of Oral Treatment of SJ Fractions on Mammary Gland Tissues Fifty (50) virgin female rats aged 60 - 90 days were divided into 2 groups.

The first group first received a subcutaneous injection of estradiol (E2) in a dose of 10 µg/0.1 ml arachis oil twice daily for 2 days. Subsequently they were divided 59 into 5 subgroups and received an oral administration of 5 ml distilled water, ethyl acetate (800 mg/kg), n-hexane (800 mg/kg), n-butanol (800 mg/kg) or water (800 mg/kg) body weight of fractions twice daily for 5 days. The second group first received an oral administration of 5 ml of distilled water and was then divided into 5 subgroups receiving the same oral administration of the fractions as the first. On the 6thday, serum PRL was also obtained for PRL content and all animals were anaesthetized with chloroform. The two inguinal mammary glands were removed or harvested. The processes of fixation, dehydration, embedding, staining and identification of the mammary glands were done as previously described.

2.11 STATISTICAL ANALYSIS Data obtained were analyzed using one-way analysis of variance (ANOVA). Differences between means were considered significant at P < 0.05. Results were presented as mean ± standard error of mean(Mean ± SEM).

60

CHAPTER THREE RESULTS 3.1MACROSCOPIC EXAMINATION OF THE WHOLE LEAF The macroscopic examination of the whole leaf of Stachytarpheta jamaicensis showed the following; (i) Colour - Green and often have a slight bluish or grayish tinge. (ii) Margin - Serrate-dentate (iii) Apex - Bluntly acute/obtuse to slightly acute. (iv) Composition of lamina - Simple (v) Shape of lamina - Obovate (vi) Mid-rib - Raised at the lower surface but flat on the upper surface. (vii) Venation - Reticulate, Pinnate (viii) Base - Symmetrical (ix) Size - 2-12 cm long and 1-5 cm wide are borne on stalks (i.e. petioles) 5- 35 mm long. (x) Texture - Relatively thick and slightly fleshy. (xi) Surface - Hairless (Glabrous) or have a few hairs along the veins on the undersides (i.e. sparsely strigose). (xii) Odour - Characteristic. (xiii) Taste - Tasteless.

3.2MICROSCOPIC EXAMINATION (i)Microscopy of the Leaf Powder The microscopy of the powdered leaf of Stachytarpheta jamaicensis showed xylem vessels with phloem parenchyma cells & epidermal cells containing stomata, bundle of phloem parenchyma cells, epidermal cells with anticlinal sinuous wall, epidermal cells with diacytic type of stomata, palisade cells attached 61 to epidermal cells, unicellular & multicellular uniseirate trichomes, irregular shaped prism calcium oxalate, small bundle of fibres, and bundle of spiral & annular xylem vessels as shown in below.

Fig. 7: Photomicrograph of powdered leaf of SJ showing annular xylem vessel with adjoining phloem parenchyma

Fig. 8:Photomicrograph of powdered leaf of SJ showing clustered palisade cells 62

Fig. 9: Photomicrograph of powdered leaf of SJ showing epidermal cells with anticlinal sinuous wall

Fig. 10: Photomicrograph of powdered leaf of SJ showing typical diacytic stomata

63

Fig. 11: Photomicrograph of powdered leaf of SJ showing epidermal cells with cylindrical palisade cells

Fig. 12: Photomicrograph of powdered leaf of SJ showing epidermal cells with robust multicellular uniseirate trichome

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Fig. 13: Photomicrographof powdered leaf of SJ showing large phloem parenchymal cells

Fig. 14: Photomicrograph of powdered leaf of SJ showing irregular shaped prism calcium oxalate 65

Fig. 15: Photomicrographof powdered leaf of SJ showing small bundle of fibres

Fig. 16:Photomicrograph of powdered leaf of SJ showing spiral and annular xylem vessel 66

(ii) Transverse Section of the Leaf. The transverse section of the leaf of Stachytarpheta jamaicensis showed the outlines of the microscopical characters present in the powdered leaf with a magnification of x200 as shown below. However, the sectional view of each characters showed the upper and lower epidermis with anticlinal sinuous epidermal cell wall, multicellular uniseirate trichome hair attached to phloem cells, bundle of phloem cells adjacent to xylem vessel, bundle of scalariform-like xylem vessels, collenchyma cells with annular xylem vessels, stomata in the lumina of transverse section and typical phloem cells in the vascular system as shown in the figures below.

Upper epidermis Palisade cells

Spongy mesophyll Xylem vessels

Trichome

Collenchyma cells

Lower epidermis

Fig. 17: Transverse Section of the Leaf showing the outlines of the microscopical characters. Magnification x 200

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Fig. 18: Photomicrograph of TS of the lower epidermis of SJ showing anticlinal sinuous epidermal cell wall.

Fig. 19:Photomicrograph of TS of the lower epidermis of SJ showing multicellular uniseirate trichome and phloem parenchyma cells 68

Fig. 20: Photomicrograph of thesectional view of TS of SJ showingbundle of phloem cells adjacent to xylem vessel.

Fig. 21: Photomicrograph of thesectional view of TS of SJ showing bundle of scalariform-like xylem vessels.

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Fig. 22: Photomicrograph of thesectional view of TS of SJ showing collenchyma cells and annular xylem vessels

Fig. 23: Photomicrograph of thesectional view of TS of SJ showing stomata in the lamina of transverse section.

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Fig. 24:Photomicrograph of thesectional view of TS of SJshowing typical phloem cells in the vascular system

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(iii) Quantitative Microscopy of the Leaf The quantitative microscopy of the leaf of SJ values for palisade ratio, stomata number, stomata index, vein-islet number and veinlet termination number are as presented in Table 2. Table 2- Results of Quantitative Microscopy Parameters Values

Palisade ratio 4.42 ± 2.55

Stomata number:upper epidermis 105.67 ± 2.73 Lower epidermis 277.00 ± 17.08

Stomata index:upper surface 28.00 ± 2.31 Lower surface 21.00 ± 2.51

Vein-islet number 15.67± 0.66

Veinlet termination number 3.50 ± 0.00

Values shown are Mean ± SEM, n= 3

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3.3ANALYTICAL STANDARDS OF THE LEAF The analytical standard values of the leaf of SJ for the total ash, water soluble ash, sulphated ash, acid insoluble ash, alcohol soluble extractive value, water soluble extractive value and moisture content in percentage are as presented in Table 3. Table 3: Results of Analytical standards Parameters % composition

Total ash 11.85 ± 0.06

Water soluble ash 2.17 ± 0.00

Sulphated ash 8.80± 0.14

Acid insoluble ash 2.04 ± 0.02

Alcohol soluble extractive value 2.51 ± 0.15

Water soluble extractive value 4.85± 0.22

Moisture content 4.30 ± 0.02

Values of percentage composition shown are Mean ± SEM, n = 3

3.4 PERCENTAGE YIELD 73

The extraction process yielded the Crude Methanol Extract (CME) while the fractionation process yielded the n-hexane, ethyl acetate, n-butanol and water fractions with their respective percentage yield as shown in Table 4 below.

Table 4: Result of Percentage (%) yield of the extraction and fractionation processes

Sample Quantity (g) Yield (g) % Yield

Crude Extraction 1890.00 395.00 19.31

N-Hexane fraction 395.00 71.40 35.70

Ethyl acetate fraction 395.00 19.18 9.59

N-Butanol fraction 395.00 50.31 25.16

Water fraction 395.00 54.50 27.29

3.5PHYTOCHEMICAL ANALYSIS 74

The result of preliminary phytochemical analysis of the extract and fractions are as shown in Table 5 below. Table 5: Results of Phytochemical Analysis S/N Phytochemical Observations Constituents CME HF EF BF WF 1. Carbohydrates + - - + + 2. Reducing sugars + - - + +

3. Alkaloids + + + + + 4. Glycosides + - + + + 5. Saponins + - + + + 6. Tannins + - + + + 7. Flavonoids + - + + -

8. Resins + + + - - 9. Proteins + - + + + 10. Oils + + - - - 11. Steroids + + + + + 12. Terpenoids + + - - -

The key CME = Crude Methanol extract, HF = N-Hexane fraction EF = Ethyl acetate fraction, BF = N-Butanol fraction WF = Water fraction. - = not present += present 3.6PHARAMACOLOGICAL STUDIES 75

3.6.1Toxicological Studies (a)Acute Toxicity Test of the Crude Methanol Extract In the acute toxicity and lethality tests, result indicated no death in the two phases of the test as shown in Table 6. The LD50 was thus established to be > 5000 mg/kg. (b)Acute Toxicity Test of the Fractions The result of the acute toxicity and lethality test of n-hexane, ethyl acetate, n-butanol and water fractions indicated no deaths in the two phases of the test as shown in Tables 6. Their LD50’s were thus established to be > 5000 mg/kg.

Table 6: Representing the Result of Acute Toxicity (LD50) Test of the Crude Methanol extract and Fractions of SJ Leaves PHASE ONE DOSE (MG/KG) MORTALITY

10 0/3

100 0/3

1000 0/3

PHASE TWO

1600 0/1

2900 0/1

5000 0/1

3.6.2 Effect of Extract/Fractions on PRL Content 76

There was a significant (P<0.05) increase in serum PRL concentration of the lactating dams treated with the extract and fractions as compared to the controls.Serum PRL concentrations were also significantly (P<0.05) higher in the

E2- primed virgin rats treated with the extract and fractions, as compared to non- primed ones as shown in Tables 7 - 10 and Figs.25 - 28.

Table 7: Effect of methanol leaf extract of SJ on the Prolactin levels of treated lactating rats. Treatment Dose Prolactin levels Percentage increase (mg/kg) (ng/ml) in Prolactin levels (%)

Control 10.26± 0.18 Distilled water (5 ml) __

36.64± 0.58** Metoclopramide 5 72.00

Extracts 200 12.67± 0.51 19.02

14.66± 0.22* 400 30.01

16.13± 0.39* 800 36.40

Values are mean ± SEM, n = 5, *p < 0.05, **p<0.01

Table 8: Effect of the fractions of SJ on the Prolactin levels of treated rats 77

Treatment Dose (mg/kg) Prolactin levels Percentage increase (ng/ml) in Prolactin levels (%)

Control (distilled water 5ml) __ 10.05± 0.34 __

Metoclopramide 5 36.44± 0.42** 72.42

HF 800 25.66± 0.67* 61.00

EF 800 49.99 ± 3.12** 80.00

BF 800 30.89± 1.87** 68.00

WF 800 21.72± 0.20* 54.00

Values are mean ± SEM, n = 5, *p < 0.05, **p<0.01

Key HF = n-Hexane fraction EF = Ethyl acetate fraction BF = n-Butanol fraction WF = Water fraction Table 9: Effect of methanol leaf extract of SJ leaves on Prolactin level of treated rats after priming with Estrogen (E2) 78

Treatment Dose Prolactin Levels (ng/ml) Percentage increase in (mg/kg) Prolactin levels (%)

E2-primed Non-primed E2-primed Non-primed

Control(E2- primed) Distilled 11.00 ± 0.22 ______water

Control(Non- primed) Distilled 10.18 ± 0.18 ______water

Extract 200 11.75 ± 0.25 10.48 ± 0.35 6.40 2.90

400 13.78 ± 0.22 # * 11.08 ± 0.24 20.20 8.12

800 15.58 ± 0.20 # * 11.60 ± 0.35 29.40 12.24

Values are mean ± SEM, n = 5,# *p < 0.05

Table 10: Effect of fractions of SJ leaves on Prolactin level of treated rats after priming with Estrogen (E2) 79

Treatment Dose Prolactin Levels (ng/ml) Percentage increase in (mg/kg) Prolactin levels (%)

E2-primed Non-primed E2-primed Non-primed

Control(E2- primed) Distilled 10.79 ± 0.28 ______water

Control(Non- primed) Distilled 10.06 ± 0.24 ______water

HF 800 29.81 ± 0.28 # * 12.09 ± 0.09 64.00 17.00

EF 800 37.00 ± 0.28 # * 15.78 ± 0.22 71.00 30.10

BF 800 33.21 ± 0.34 # * 12.27 ± 0.52 68.00 18.01

WF 800 27.24 ± 1.42 # * 11.53 ± 0.36 60.40 13.00

Values are mean ± SEM, n = 5, # *p < 0.05

Key HF = n-Hexane fraction EF = Ethyl acetate fraction BF = n-Butanol fraction WF = Water fraction 80

Grp 1 = Control (distilled Water)

Grp 2 = Metoclo-treated (5mg/kg b.w.)

Grp 3 = extract-treated (200 mg/kg b.w.)

Grp 4 = extract-treated (400 mg/kg b.w)

Grp 5 = extract-treated (800 mg/kg b.w.)

Values are mean  SEM, n = 5, *p < 0.05, **p<0.01

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Grp 1 = Control (distilled Water)

Grp 2 = Metoclo-treated (5mg/kg b.w.)

Grp 3 = HF-treated (800 mg/kg b.w.)

Grp 4 = EF-treated (800 mg/kg b.w

Grp 5 = BF-treated (800 mg/kg b.w.)

Grp 6 = WF-treated (800 mg/kg b.w.)

Values are mean  SEM, n = 5, *p < 0.05, **p<0.01

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# *

# *

Fig. 27: Serum PRL content before and after priming with estradiol (E2). Values are the mean ± SEM. *Statistically significant compared to the control, # Statistically significant compared to the E2-primed control group (ANOVA, at least P<0.05). Non-primed: not previously treated with E2. Primed: previously treated with E2. Group 1: control group receiving 5ml distilled water (orally, 5days), Groups 2, 3, and 4: treated groups receiving 200, 400 and 800 mg of plant extract/kg BW (orally, 5days) respectively.

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# * # *

#*

# *

Fig. 28:Serum PRL levels before and after priming with estradiol (E2) Values are the mean ± SEM.*Statistically significant compared to the control, # Statistically significant compared to the E2-primed control group (ANOVA, at least P<0.05).Non-primed: not previously treated with estradiol. Primed: previously treated with estradiol. Group 1: control group receiving 5ml distilled water (orally, 5days), Groups 2, 3, 4 and 5: treated groups receiving ethyl acetate (800 mg/kg), n-hexane (800 mg/kg), n-butanol (800 mg/kg) and water (800mg/kg) BW of fractions (orally, 5days)respectively.

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3.6.3 Histology of Mammary Tissue

The histological sections showed evidence of secretions into ducts of the E2- primed groups receiving the extract which was in a dose-dependent manner when compared to the non-primed rats as shown in Figs. 29 – 46.

AT

AT

Fig. 29: Histological section of mammary gland from non-primed rat receiving distilled water, showing predominance of bare ducts (white arrow) and terminal end buds (TEB) (black arrow) in a sea of adipose tissue (AT).

AT

Fig. 30: Histological section of mammary gland from an E2-primed rat receiving distilled water showing ductular proliferation (red arrow) with few budding of rudimentary alveoli.

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Fig. 31:Histological section of mammary gland from a non-primed rat receiving 200 mg/kg BW of . plant extract, showing ductular proliferation with moderate budding of rudimentary alveoli.

Fig. 32: Histological section of mammary gland from E2-primed rat receiving 200 mg/ kg BW of plant extract, showing tubuloalveolar differentiation containing proteinaceous materials in the lumen (mild).

Fig. 33:Histological section of mammary gland from a non-primed rat receiving 400 mg/kg BW of plant extract, showing well defined alveolar structure with lipid droplets within the alveoli. 86

Fig. 34:Histological section of mammary gland from E2-primed rat receiving 400 mg/kg BW of plant extract, showing tubuloalveolar hyperplasia with protienaceous materials in the lumen of duct and alveoli (moderate).

Fig. 35:Histological section of mammary gland from a non-primed rat receiving 800 mg/kg BW of plant extract, showing welldefined alveolar structure with much lipid droplets in the alveoli.

Fig. 36: Histological section of mammary gland from E2-primed rat receiving 800 mg/kg BW of plant extract, showing tubuloalveolar hyperplasia with protienaceous materials in the lumen of duct and alveoli (high). 87

AT

Fig. 37:Histological section of mammary gland from non-primed rat receiving distilled water, showing predominance of bare ducts (white arrow) and TEB (black arrow) in a sea of adipose tissue (AT).

Fig. 38: Mammary gland from an E2-primed rat receiving distilled water showing ductular proliferation with few budding of rudimentary alveoli

Fig. 39: Histological sections of mammary gland from non-primed rats receiving ethyl acetate fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli. 88

Fig. 40: Histological sections of mammary gland from non-primed rats receiving n-hexane fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli.

Fig. 41: Histological sections of mammary gland from non-primed rats receiving n-butanol fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli. .

Fig. 42: Histological sections of mammary gland from non-primed rats receiving water fraction in a dose of 800 mg/kg BW, showing ductular proliferation with moderate budding of rudimentary alveoli.

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Fig. 43: Histological sections of mammary gland from E2-primed ratsreceivingethyl acetate fraction in a dose of 800 mg/kg BW showing alveolar hyperplasia with dilated alveolar lumens and ducts. The alveoli and ducts contain proteinaceous secretory materials.

Fig. 44: Histological sections of mammary gland from E2-primed ratsreceivingn-hexane fraction in a dose of 800 mg/kg BW showing alveolar hyperplasia with dilated alveolar lumens and ducts. The alveoli and ducts contain proteinaceous secretory materials.

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Fig. 45: Histological sections of mammary gland from E2-primed ratsreceivingn-butanol fraction in a dose of 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts. The alveoli and ducts contain proteinaceous secretory materials.

Fig. 46: Histological sections of mammary gland from E -primed ratsreceivingwater fraction in a 2 dose of 800 mg/kg BW, showing alveolar hyperplasia with dilated alveolar lumens and ducts. The alveoli and ducts contain proteinaceous secretory materials.

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CHAPTER FOUR DISCUSSION AND CONCLUSION

4.1 DISCUSSION Milk produced by the mammary gland of humans and other mammals is an essential commodity which is the optimal source of nutrition for the newborn infant. The first milk (colostrum) contains relatively high concentrations of sodium chloride and protective substances such as immunoglobulins and lactoferrin while the copious milk secreted contains proteins, lipids, carbohydrates, micronutrients and trace elements required for growth, development and immune protection (Beerens, 1980). The mammary glands can only secrete milk when there is a delicate balance between the reproductive and metabolic hormones however; this must be controlled by FIL and the PRL receptor sites (Cox et al., 1996). The dramatic change in size, shape and function in association with puberty, pregnancy and lactation which the breast undergoes are critical to successful breastfeeding (Neville, 2001). Pharmacognostical standardization of Stachytarpheta jamaicensis showed that it has a set of peculiar identities, specific characteristics which are generally unique and of unshared qualities. With the macroscopical standards, the macroscopic features of the leaves such as margin, venation, base, apex, midrib etc. were seen to be serrate-dentate, reticulate/pinnate, symmetrical, bluntly acute and raised at the lower surface but blunt at the upper surface respectively. The organoleptic properties such as colour, odour and taste of the plant material were also seen to be green, characteristic and tasteless respectively. From the quantitative microscopy, there was presence of numerous stomata on the lower epidermis of SJ while the upper epidermis had few scattered stomata cells. This characteristic feature was used in the determination of stomata number, stomata 92 index, vein-islet number, vein-islet termination number and palisade ratio. It was observed that palisade cells were seen only in the upper epidermis indicating that SJ is a terrestrial plant. The stomata index of the lower epidermis was less than that of the upper epidermis (values 28.00± 2.31 and 21.00 ± 2.51 respectively). This is because; the epidermal cells of the lower epidermis were not increasing measurably with the stomata cells. The analytical standards of SJ leaves were within pharmacopoeia standards and it could be used as a reference guide for the identification and assessment of their quality and purity. The constituent of SJ leaves were very soluble in both alcohol (2.51± 0.15) and water (4.85± 0.22)with the highest solubility being in alcohol. This shows that the drug would be better extracted using water than alcohol. The value of the total ash was low (11.85 ± 0.06) indicating that little amount of inorganic matter adhered to the crude drug. The water soluble, acid insoluble and sulphated ash values (2.17 ± 0.00, 2.04 ± 0.02, 8.80 ± 0.14 respectively) were within the limits in the standard crude drug monograph and this gives evidence of the presence of minute earthy matters. The moisture content value (4.30 ± 0.02) shows possible hydrolysis and degradation of the active components when exposed to air. These values are useful parameters for inclusion in the monograph of the plant. Phytochemical analysis of the extract and fractions revealed the presence of phytoconstituents such as alkaloids, carbohydrates, reducing sugars, flavonoids, steroids, saponins, terpenoids, tannins, glycosides, resins, proteins, oils and acidic compounds. The presence of these phytochemical constituents has been reported in S. jamaicensis (Idu et al., 2007). The pharmacological activities of most plant extracts can be traced to these bioactive consitutents. Steroids, glycosides and triterpenoids have been implicated in lactogenic activities of plant extracts (Okwu and Ohenhen, 2010) and their presence in the extract and fractions only supported their observed pharmacological activities. 93

The acute toxicity (LD50) of SJ leaves in albino rats was found to be > 5000 mgkg-1. The plant crude methanol extract and fractions were characterized by a very low degree of toxicity. This implies that the leaves of S. jamaicensisare non- toxic and relatively safe. The lactogenic studies shows that; the methanol leaf extract and fractions of Stachytarpheta jamaicensis produced an appreciable increase in serum PRL level when compared to the control in a dose-dependent manner and as such exhibited an activity by increasing the serum PRL level in lactating rats as seen in (Fig. 25). The lactogenic activity displayed by the highest dose of methanol leaf extract (800 mgkg-1) when compared with that of metoclopramide (commercial hyperprolactinemia-inducing agent) showed a very wide significant statistical difference in serum PRL level. Interestingly, the ethyl acetate fraction amongst the other fractions of Stachytarpheta jamaicensis leaf produced a high significant increase in serum PRL level when compared to the control with potency greatly higher than the standard drug metoclopramide. The ethyl acetate fraction of Stachytarpheta jamaicensis leaves exhibited a lactogenic activity by increasing the serum PRL in lactating rats and as such, this may be responsible for the lactogenic activity displayed by the methanol leaf extract of Stachytarpheta jamaicensis;the mechanism of action is still unknown. The extract and fractions showed a progressive but significant increase in lactogenic activity. The order of activity of the fractions at 800 mgkg-1 are in the order of Ethyl acetate > n-Butanol > n- Hexane > water fractions respectively as seen in (Fig. 26).

With respect to the E2-primed animals, the highest serum PRL concentrations were observed in the groups receiving 800 mgkg-1 of extract (fig. 27) and the groups receiving 800 mgkg-1 of ethyl acetate fraction (fig. 28). Histologically, the extract and fractions of SJ were able to stimulate mammary gland development and differentiation of the lobulo-alveolar system 94 from the lobular buds with milk secretion within the lumen. The mammary glands of non-primed rats receiving distilled water (Figs. 29 & 37) showed a predominance of bare duct system and terminal end buds (TEBs) suspended in a sea of adipose tissue (AT). Although the mammary gland of the E2-primed animals receiving distilled water consisted of ductular proliferation (i.e. ducts branching into ductules) (Figs. 30 & 38), no alveolar development was observed.

In all E2-primed animals receiving the extract and fractions, ductule branching with alveolar hyperplasia was observed as well as proteinaceous secretory materials in the alveoli. There was evidence of secretion into the ducts of the E2-primed groups receiving the extract and it showed a dose-dependent result.

Also, the E2- primed groups receiving the fractions also showed evidence of secretions into the ducts. The largest tubulo-alveolar hyperplasia with proteinaceous materials secreted in the lumen of alveoli and ducts were observed in the E2-primed group -1 receiving 800mgkg of extract (Fig. 36) and the E2-primed group receiving 800mgkg-1 of ethyl acetate fraction (Fig. 43). Further investigations have to be made to provide understanding of the mechanism of action of the extract on mammary gland development. 4.2 CONCLUSION In conclusion, it can be stated that; the leaves of SJ have been identified, evaluated as well as standardized and may possibly qualify to be included in the official crude drug monographs. The crude methanol extract and fractions of SJ effectively increased serum PRL levels and stimulated PRL synthesis and release in the rats; with the ethyl acetate fraction showing the highest and most potent activity. The lactogenic activity establishes a rationale for the ethnomedicinal applications and uses of SJ leaves as a galactogogue in lactating women. The lactogenic properties of the plant reside in the phytoconstituents therefore; further 95 studies are required to isolate and purify the particular bioactive constituent responsible for the observed lactogenic effect and mechanism of action on mammary gland development. The acute toxicity studies characterized by SJ extract and fractions show that it has a wide safety index which makes it safe for human consumption. Also, a subchronic toxicity studies needs to be carried out on the extract and fractions to assess the liver enzymes and kidney metabolites for liver and renal function test. It is worthy of note to specify here that this is the first work being carried out on Stachytarpheta jamaicensis leaves in terms of lactogenic activity of the plant and as such no previous studies on the plant extract and fractions has been tested as a galactogogue in literature.

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APPENDIX TABLEA1: Calculations of quantitative microscopy Quantitative Standards Morphological Parts Parameters Leaf Palisade ratio Group 1 Group 2 Group 3 Number of palisade square millimeter 21/4 16/4 16/4 Average number 13.25/3 = 4.42 Stomata number Group 1 Group 2 Group 3 Number of stomata per square millimeter 104 111 102 Average number (upper surface) 317/3 = 105.67

Number of stomata per square millimeter 253 310 268 Average number (lower surface) 831/3 = 277.00 Stomata index Group 1 Group 2 Group 3 Number of epidermis (upper epidermis) 43 53 54 Number of stomata (upper epidermis) 21 17 21 Stomata index = s 21 17 21 x 100 E + s 43+21 53+17 54+21 Average number 84/3 = 28 % Number of epidermis (lower epidermis) 26 58 96 Number of stomata (lower epidermis) 9 13 22 Stomata index = s 9 13 22 x 100 E + s 26+9 58+13 96+22 Average number 63/3 = 21 % Vein-islet number Group 1 Group 2 Group 3 Number of vein –islet per square millimeter 17 15 15 Average number 41/3 = 15.67 Vein termination number Group 1 Group 2 Group 3 14/4 14/4 14/4 Average number 10.5/3 = 3.5 105

Table A2: Acute toxicity test for LD50 determination showing the number of cell deaths per dose group. PHASE ONE DOSE (MG/KG) MORTALITY

10 0/3 100 0/3 1000 0/3 PHASE TWO

1600 0/1 2900 0/1 5000 0/1

Where fraction = Number of dead animals Total number of animals used

LD50 = > 5000 mg/kg