CHEMICAL AND BIOLOGICAL INVESTIGATION OF THE STEM OF DICHAPETALUM CRASSIFOLIUM THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN CHEMISTRY BY HENRY AKWAFFO ONYAME [10246376] DEPARTMENT OF CHEMISTRY, UNIVERSITY OF GHANA. JUNE, 2015 University of Ghana http://ugspace.ug.edu.gh ii DEDICATION TO THE GLORY OF ALMIGHTY GOD AND ONYAME FAMILY University of Ghana http://ugspace.ug.edu.gh iii DECLARATION This is to certify that this thesis is the result of research undertaken by Henry Akwaffo Onyame toward the award of a Master of Philosophy Degree in Chemistry in the Department of Chemistry, University of Ghana. It has not been presented for a degree in this University or any other University. ………………………………….. Henry Akwaffo Onyame (Candidate) ……………………....... ……………………………. Dr. Dorcas Osei-Safo Dr. Mary Anti Chama (Supervisor) (Supervisor) University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGMENTS I am most grateful to God for bringing me this far. With profound gratitude I would to want to appreciate the invaluable contribution of my supervisors; Dr. Dorcas Osei-Safo and Dr. Mary Anti Chama for their helpful suggestions and constructive criticisms throughout this work. Special appreciation goes to Prof. Ivan Addae-Mensah whose involvement led to the successful completion of this work. I would want to thank Prof. Reiner Waibel and coworkers of the University of Erlangen for obtaining the NMR data of the isolates. I am grateful to my family, my parents and especially my immediate sister, Mrs. Elizabeth Kumashie and family for their financial assistance throughout this program. My sincere gratitude also goes to my pastor and wife, Rev. Samuel Akoto and Mrs. Eleanor Akoto for their prayers, encouragement, love and assistance throughout my life. I am very grateful to Mr. J. J. Harrison of the Chemistry Department for his advice and being a source of inspiration throughout my stay in this University. My heartfelt gratitude goes to Mr. Godwin A. Dziwornu also of the Chemistry Department without whose priceless assistance this work would not have been completed. I acknowledge the following individuals from Food and Drugs Authority; Mr. Ernest Afesey, Mr. Eric Kakari-Boateng, Mr. Nicholas Amoah Owusu and Mr. Isaac Adom for their assistance in obtaining IR spectra of my isolates. Special appreciation also goes to the following staff of Noguchi Memorial Institute for Medical Research; Mr. Joseph Otchere, Mrs. Yvonne Ashong, Mr. Joseph Quatey and especially to Mr. University of Ghana http://ugspace.ug.edu.gh v Edmund Dumashie for their assistance for the work done on the anti-schistosomiasis biological studies. I appreciate the assistance I received from the technical staff of the Chemistry Department, especially Mr. Bob Essien and also to the secretarial staff. I owe special appreciation to my colleagues; Claudine Fleischer, Fredrick Kumah, Roland Kofi Gyampoh, Shadrach Quanoo, Ehud Ashiawo and the rest of the staff of the Chemistry Department. I want to thank my wonderful brother, Mr. Gilbert Boafo and his family, Pastor Gabriel Dela, all the Adult Bible Class facilitators and the I. C. G. C. Wisdom Temple Choir for their support and love. To all others whose names I have not mentioned, I am grateful to you all and God Almighty bless you. University of Ghana http://ugspace.ug.edu.gh vi ABSTRACT In the present study, the stem of Dichapetalum crassifolium was investigated for its phytochemical constituents and their biological properties. The petroleum ether extract yielded friedelan-3-one, friedelan-3β-ol and a mixture of friedelan-3-one and friedelan-3β-ol. The ethyl acetate extract yielded pomolic acid, dichapetalin M and maslinic acid as well as the friedelins and a mixture of β-sitosterol and stigmasterol. No solid was isolated from the methanol extract. These compounds were identified and characterized using comparative thin-layer chromatography, comparative melting point, mixed melting point, IR, 1H and 13C NMR spectroscopy. In the light of recent investigations suggesting that the dichapetalins and some other triterpenoids have a broad spectrum of biological activities coupled with the urgent need of potential anti- schistosomal agents, the crude extracts as well as three isolates; friedelan-3-one, β- sitosterol/stigmasterol and dichapetalin M were screened for their potential egg hatch inhibition activity against Schistosoma mansoni and Schistosoma haematobium. The petroleum ether, ethyl acetate and methanol crude extracts gave IC50 values of 443.7 ± 0.04, 638.0 ± 0.08 and 893.7 ± 0.08 µg/ml respectively. Among the tested isolates, friedelan-3-one, β-sitosterol/stigmasterol and dichapetalin M gave IC50 values of 378.1 ± 0.23, 177.9 ± 0.10 and 191.0 ± 0.12 µg/ml respectively. The observed egg hatch inhibition activity of the most active isolates, the mixture of β- sitosterol/stigmasterol and dichapetalin M, were however found to be about 11 and 12-fold respectively less potent compared to that of the standard drug, praziquantel (IC50 = 15.47 ± 0.06 µg/ml), used in the study. University of Ghana http://ugspace.ug.edu.gh vii This study constitutes the first report of the chemical and biological investigation of this member of the family Dichapetalaceae. Among the compounds isolated, maslinic acid is reported for the first time from the plant family. University of Ghana http://ugspace.ug.edu.gh viii TABLE OF CONTENT Page DEDICATION ii DECLARATION iii ACKNOWLEDGMENTS iv ABSTRACT vi TABLE OF CONTENT viii LIST OF TABLES xii LIST OF SCHEMES xiv LIST OF FIGURES xv CHAPTER ONE Introduction 1.1 Description of Dichapetalum Crassifolium 1 1.2 The Dichapetalins as Potential Natural Anti-Cancer Agents 2 1.3 Schistosomiasis 5 1.4 Aim and Objectives 6 CHAPTER TWO Literature Review University of Ghana http://ugspace.ug.edu.gh ix 2.1 Ethnobotanical Uses of Some Selected Dichapetalum Species 7 2.2 Earlier Investigation into Some Toxic Dichapetalum Species and their Chemical Composition 9 2.3 The Dichapetalins and Their Biological Activities 12 2.3.1 Structural differences among the Dichapetalins 12 2.3.2 Biological Activities of the Dichapetalins 22 2.4 Dichapetalins from Other Sources and their Biological Activities 28 2.5 Other Compounds Isolated From Previously Investigated Dichapetalum Species 31 2.6 Schistosomiasis: A Major Neglected Parasitic Disease in Ghana 35 2.6.1 Control of Schistosomiasis 37 2.6.2 Drugs for Treating Schistosomiasis 37 2.6.3 Natural Products as Potential Source for the Control of Schistosomiasis 39 CHAPTER THREE Present Investigation 3.1 Summary 43 3.2 Investigation of the Extracts from the stem of D. crassifolium 44 3.2.1 Phytochemical Screening of the PE, EA and MeOH crude extracts 44 3.2.2 Investigation of the (PE) Extract 45 University of Ghana http://ugspace.ug.edu.gh x 3.2.2.1 Isolation and identification of friedelan-3-one, DCS-P1 46 3.2.2.2 Isolation and identification of friedelan-3β-ol, DCS-P2 49 3.2.2.3 Isolation of a mixture of friedelan-3-one and friedelan-3β-ol, DCS-P1P2 51 3.3 Investigation of the Ethyl acetate (EA) Extract 51 3.3.1 Purification and identification of friedelan-3-one, DCS-E1 54 3.3.2 Purification and identification of friedelan-3β-ol, DCS-E2 54 3.3.3 Purification and identification of a mixture of friedelan-3-one and friedelan-3β-ol, DCS-E1E2 and DCS-E3. 55 3.3.4 Purification and identification of a mixture of β-sitosterol and stigmasterol, DCS-E4 55 3.3.5 Purification of DCS-E5 and DCS-E6 (identified as pomolic acid) 58 3.3.6 Purification and identification of dichapetalin M, DCS-E7 66 3.3.7 Purification and identification of Maslinic Acid, DCS-E8 69 3.3.8 Purification and isolation of DCS-E9, DCS-E10 and DCS-E11 75 3.4 In Vitro Screening of the Crude Extracts, Friedelan-3-one, β-Sitosterol/stigmasterol and dichapetalin M For Anti-Schistosomal Activity 76 3.5 Conclusion 83 3.6 Recommendation 84 CHAPTER FOUR Experimental 4.1 Collection and Preparation of Plant Material 86 4.2 General Experimental Procedure 86 University of Ghana http://ugspace.ug.edu.gh xi 4.3 Chemicals and reagents 87 4.4 Solvent Extraction of the Plant Material 89 4.4.1 Extraction with Petroleum ether (40-60oC) 89 4.4.2 Extraction with Ethyl acetate 90 4.4.3 Methanol Extraction 90 4.5 Phytochemical Screening test procedure 90 4.6 Investigation of the Extracts 92 4.6.1 Petroleum ether (40-60oC) extract 92 4.6.2 Ethyl acetate (EA) extract 93 4.7 In Vitro Screening of Crude Extracts and some Solid Isolates for anti-schistosomal Activity 96 4.7.1 Schistosome Egg recovery and Concentration from infested urine sample by the modified Kotze et al Method 96 4.7.2 In vitro screening of test compounds against schistosome eggs using the 96-well plate Egg-Hatch Assay 97 APPENDIX I: IR data of isolates 99 APPENDIX II: 1H and 13C NMR data of isolates 112 APPENDIX III: Ethical Clearance 128 REFERENCES 129 University of Ghana http://ugspace.ug.edu.gh xii List of Tables Page Table 2.1 Ethnobotanical uses of selected Dichapetalum species 7 Table 2.2 Occurrence of the dichapetalins in investigated Dichapetalum species 13 Table 2.3 Cytotoxicity of dichapetalin A against some cancer cell lines 23 Table 2.4 Chemosensitivities of some dichapetalins on HCT116 and WM 266-4 cancer cell lines 25 Table 2.5 Potential anti-schistosomal drugs from plants (and natural product derived) sources 40 Table 3.1 Phytochemical screening test results on the crude extract of Dichapetalum crassifolium 44 Table 3.2 IR frequencies of DCS-P1 compared to that of Friedelan-3-one 47 Table 3.3 Comparison of IR data of DCS-P2 with that of Friedelan-3β-ol 50 Table 3.4 Infrared data of DCS-E4 compared to that of a reference sample 56 Table 3.5 IR data for DCS-E5 and pomolic acid, DCS-E6 58 Table 3.6 Comparison of 13C Chemical shifts of DCS-E6 (pomolic acid) with reference data 60 Table 3.7 Infrared data of DCS-E7 compared to that of dichapetalin M 66 Table 3.8 Comparison of 1H and 13C NMR data of DCS-E7 (dichapetalin M) with literature data 68 Table 3.9 1H and 13C chemical shifts of DCS-E8 (Maslinic acid) and reference data 73 University of Ghana http://ugspace.ug.edu.gh xiii Table 3.10A %EHI recorded for the duplicate test at different concentrations of extracts, pure compounds and standard, praziquantel. 78 Table 3.10B In vitro average %EHI values at different concentrations of extract, pure compounds and praziquantel. Data are Mean ± S. E. M 79 Table 3.10C In vitro half maximal inhibitory concentration (IC50) of test compounds 82 University of Ghana http://ugspace.ug.edu.gh xiv List of Schemes Page Scheme 1 The lethal synthetic pathway for the inhibition of Kreb’s Cycle 10 Scheme 2 Metabolic blocking of citric acid cycle by fluoroacetic acid 11 Scheme 3 Investigation of PE Extract 45 Scheme 4 Summary of work carried out on Ethyl Acetate extract 53 Scheme 5 Biosynthetic pathways of sterols required for normal plant development 57 University of Ghana http://ugspace.ug.edu.gh xv List of Figures Page Figure 1 Dichapetalins with methyl ester side chain 15 Figure 2 Dichapetalins with 5-membered lactone and allyl alcohol side chain 17 Figure 3 Dichapetalins with spiroketal side chain 19 Figure 4 Dichapetalins with lactol side chain 21 Figure 5 Structural differences between dichapetalins and acutissimatriterpenes 29 Figure 6 Dichapetalin-type triterpenoids isolated from Phyllanthus acutissima 30 Figure 7 Other compounds isolated from Dichapetalum species 33 Figure 8 Life cycle of Schistosoma species 36 Figure 9 Schistosomiasis drugs 38 Figure 10 Potential anti-schistosomal drugs 42 Figure 11a-c Expanded 1H NMR of DCS-E6 62-64 Figure 12 Expanded 1H NMR of DCS-E8 72 Figure 13 Graph of % Maximum Inhibition vrs Log [Crude Extract] 81 Figure 14 Graph of % Maximum Inhibition vrs Log [Test compounds] 81 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 Description of Dichapetalum Crassifolium Dichapetalum crassifolium Chodat (synonym: Dichapetalum crassifolium var. integrum Breteler) belongs to the family Dichapetalaceae1. Dichapetalaceae consists of angiosperms and comprises three genera; Dichapetalum, Stephanopodium and Tapura2. Dichapetalum Thouars is the major genus with 124 species out of which 86 are found in Africa, 19 in Asia and the rest in South America. Dichapetalum species are small tropical trees, shrubs or sometimes climbing plants, with leaves alternate, simple and individual, as well as small flowers and a nut or drupe3. The main centre of distribution of Dichapetalum in Africa is Central Africa, comprising Cameroun, Gabon, Equatorial Guinea, Republic of Congo, Democratic Republic of Congo, Central African Republic and the northern part of Angola4. D. crassifolium is distributed in West and Central Africa, Western Tanzania and Northern Zambia and thrives in rain forest at altitudes of 0-1700m1. In Ghana, D. crassifolium can be found in the Central, Western and Ashanti regions4. D. crassifolium is a large liana that can grow up to a height of 40m or more and could grow to a diameter of 5cm at the base. The lower parts of the stem may grow roots, the stem and branches have lenticels which are large and prominent. The leaves are lamina, usually grooved on both surfaces or hairless on either surface with elliptic to obovate shaped blades. The petioles may grow to 1-16mm long, the midrib of the leaves may be flat or rose above, more prominent beneath with 3-7 major lateral nerves on each side1,5. The plant has pedunculate inflorescence with up to 50 flowers. The flowers are often grouped on a short, leafless axillary shoot, and the developed flowers have 4-6mm long petals and stamens University of Ghana http://ugspace.ug.edu.gh 2 and 4-7mm long pistils. The peduncle is free from the petiole, 10-12mm long with small bracts and bracteoles which are less than 1mm long. The pedicel grows up to 5mm long with obtuse- truncate shaped calyx and erect sepals which group at the base. The sepals are 2.5-3mm in long. The petals are erect, entire or emarginated at the apex, narrowly oblong-obovate and 2.5-6mm long. Young fruits are seen on D. crassifolium in the middle of June and November. The grown fruits may be 1-3 lobed and 1-3 seeded. The 1-seeded fruits are ellipsoid to obovoid shaped, laterally compressed, 15-25mm long, 10-20mm broad, 10-18mm thick, obtuse at the apex and orange colored at maturity. The 2-3 seeded fruits develop deep cleft apically and laterally. The exocarp of the fruit is firm, 1-2mm thick with juicy mesocarp that is somewhat fibrous and adhered to the endocarp. The endocarp is bony, 1-2mm thick and strongly grooved outside but is hairy in the immature state. The seeds are subellipsoid, laterally compressed and 10mm long with brownish, smooth, glossy and strongly veined seedcoat1,5. To the best of my knowledge, no ethnobotanical use has been recorded for D. crassifolium and there has not yet been any chemical investigation of the plant. 1.2 The Dichapetalins as Potential Natural Anti-Cancer Agents. While earlier investigations into Dichapetalum species focused on the more toxic species, current investigations have and continue to concentrate on the entire chemical constituents of the less toxic ones including investigation of their biological activities. The presence of fluorinated compounds, mainly monofluoroacetate and fluorocarboxylic acids have been implicated in the toxicity of the Dichapetalum species6,7,8. University of Ghana http://ugspace.ug.edu.gh 3 Currently, there is increasing research into these Dichapetalum species due to the discovery of the novel phenylpyranotriterpenoid compounds named dichapetalins from the less toxic species9,10. The Dichapetalins constitute a small family of secondary metabolites of mixed biosynthesis in which a 2-phenylpyrano moiety is annulated to a dammarane-triterpene skeleton. The basic structure of a dichapetalin is biogenetically characterized by the addition of a C6-C2 unit, which probably might be derived from the shikimmic pathway to a 13, 30-cyclodammarane-type skeleton. The dichapetalin derivatives are distinguished by the nature of the side chain at C-179. In 1995, Achenbach10 and co-workers were the first to discover the first member of the group, dichapetalin A from the roots of D. madagascariense Poir and a year later they established its absolute configuration by X-ray crystallographic measurement11. Dichapetalin A was found to present strong anti-cancer activity against L1210 murine leukemia (EC90 = 1.7 × 10-10 M) but four fold less efficient towards KB carcinoma cells and murine bone marrow cells stimulated with GM- CSF (granulocyte-macrophage colony-stimulating factor) in vitro. In 1996, Achenbach and co- workers isolated seven other new dichapetalins, B - H but they exhibited poor or no cytotoxic activities. A recollection of the plant led to the isolation of dichapetalin M in 2008, which in a brine shrimp lethality test was found to be (LC50 = 0.011µg/ml) 28-fold more potent than dichapetalin A (LC50 = 0.31µg/ml). In the same test, dichapetalin C was active to some extent but dichapetalins D and F were inactive12,13. Fang14 et al in 1994 isolated 2 new dichapetalins, I and J in addition to A from the stem bark of D. gelonioides collected from the Philippines. A recollection of the plant in 2003 and subsequent chemical investigation led to the isolation of 2 other new members, dichapetalins K and L which were not isolated in the first collection. Dichapetalins K and L showed broad activity towards the University of Ghana http://ugspace.ug.edu.gh 4 human ovarian cancer cell line SW626 while dichapetalins A, I and J exhibited selective and cytotoxic activities (EC50 = 0.2 - 0.5 µg/ml) against the same cell lines. Tuchinda15 and co-workers have isolated five new dichapetalin-type triterpenoids from the aerial parts of Phyllanthus acutissima called acutissimatriterpenes A-E. The C-17 side chain and the presence or otherwise of a methylenedioxy unit in the phenylpyrano moiety distinguishes the acutissimatriterpenes from the dichapetalins. After testing the acutissimatriterpenes against a panel of six cancer cell lines namely, P-388 murine lymphocyte leukemia, KB human nasopharyngeal carcinoma, Col-2 human colon cancer, MCF-7 human breast cancer, Lu-1 human lung cancer and ASK rat glioma, acutissimatriterpenes A and B were found to be active active against murine lymphocyte leukemia giving EC50 = 0.4 and 0.5µg/ml respectively whereas acutissimatriterpene E showed significant activity against murine lymphocyte leukemia (EC50 = 0.005µg/ml) and moderate activity against human breast cancer (EC50 = 1.1µg/ml) and human lung cancer (EC50 = 3.1µg/ml). Acutissimatriterpenes C and D showed insignificant activities (EC50 > 5µg/ml) against all the cancer lines tested15. The structures of the dichapetalins and acutissimatriterpenes are showed on pages 15-21 and 29- 30 respectively. Towards the end of 2013, Long16 and co-workers isolated 6 new dichapetalins from D. mombuttense, D. leucosia, D. zenkeri, D. eickii and D. ruhlandii namely dichapetalins N–S, in addition to the previously isolated dichapetalins A, B, C, I, L and M and determined their structures based on spectroscopic methods. The cytotoxic and anti-proliferative profile of these dichapetalins against the human colorectal carcinoma HCT116 and human melanoma WM 266-4 cancer cell lines showed dichapetalin P to be more potent by 74-fold than dichapetalin A on WM 266-4 cells. University of Ghana http://ugspace.ug.edu.gh 5 Dichapetalin M so far has recorded the highest cytotoxic activity recording EC50 values of 0.007 and 0.05μg/ml on HCT1116 and WM 266-4 cell lines respectively.16 Chemical investigation of the root of D. filicaule led to the isolation of the most recent member of the group, dichapetalins X, in addition to dichapetalin A17. The anthelminthic activities of dichapetalin A and X were found to be 162.4 and 523.2 µg/ml respectively17. Jing18 and co-workers have isolated 14 new dichapetalins from the stem and leaves of D. gelonioides. From the different biological studies carried out by Jing et al, the dichapetalins showed antifeedant, nematicidal, antifungal, nitric oxide and anticholinestrase inhibitory activities, indicating the wide range of biological activities that the dichapetalins could exhibit. 1.3 Schistosomiasis Schistosomiasis, also known as bilharzia, bilharziosis or snail fever is a chronic parasitic disease caused by blood-dwelling fluke worms of the genus Schistosoma and is endemic in Africa, Asia and South America19. According to WHO, schistosomiasis is the second most prevalent tropical disease and it affects more than 200 million people worldwide20. In sub-Saharan Africa, the disease is said to cause the death of over 280, 000 people annually, and in Ghana, schistosomiasis is wide- spread in all the regions with overall prevalence estimated between 54.6-80%21,22. The disease therefore remains a considerable public health problem in these endemic regions. Currently, praziquantel is the only drug of choice for the control of schistosomiasis infection and resistance of some Schistosoma species to praziquantel has already been reported23. The classical strategy of alternating treatments to avoid the development of resistance is very crucial and so more recently there has been a growing interest in the scientific community to search for extracts and novel compounds from different plant sources with bioactivities to treat schistosomiasis University of Ghana http://ugspace.ug.edu.gh 6 infection.24,25,26. While many plant species from various families have been screened for their possible antischistosomal potencies, there has not been any report as regards members of the family Dichapetalaceae. 1.4 Aim and Objectives This current investigation was carried out as part of ongoing research into the less toxic species of the genus Dichapetalum aimed at isolating the phytochemicals present, with special focus on the dichapetalins and their biological properties. The species under study was D. crassifolium, whose phytoconstituents and their antischistosomal properties were investigated. The objectives were achieved as follows: I. Soxhlet extraction of the compounds present in the stem of D. crassifolium. II. Isolation and purification of the compounds by column chromatography and other separation and purification methods. III. Characterization of the pure isolates using spectroscopic methods. IV. Anti-schistosomiasis studies on both crude and some isolated compounds. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO LITERATURE REVIEW 2.1 Ethnobotanical Uses of Some Selected Dichapetalum Species In spite of rapid development in methods of organic synthesis in synthetic laboratories, medicinal plants continue to play significant roles in modern medicine due to their inherent distinct chemical and biological properties. In nature, plants are able to synthesize complex molecules from simple ones through highly specific reactions that they use for defense and communication. These complex compounds play significant roles not only as medicinal and pharmaceutical agents but as leads for synthetic optimization. Most species of Dichapetalum have been reported to have folkloric uses among which have been summarized in Table 2.1 below. TABLE 2.1: Ethnobotanical uses of selected Dichapetalum Species Dichapetalum Species Ethnobotanical Use D. cymosum26,27 Contains fluoroacetic acid which could be useful in anti-HIV infective therapy. D. braunii Engl.7 Grounded leaves for sore treatment D. barteri8 Rodenticide D. barbatum28 For treating wounds, possess antimicrobial activities. University of Ghana http://ugspace.ug.edu.gh 8 D. gelonioides18,29,30 Leaves for treating amenorrhea, used as coolant in treating mouth ulcers , raticide and rodenticide. D. rhodesium31 Tonic for infants, used as contraceptive and birth inducer. D. thunbergh32 Has an herbal and homeopathic property. D.macrocarpum, D. mossambicense and D. venenatum33 Used as arrow poisons D. madagascariense6 Treatment of jaundice, urethri tis, bacterial infections and viral hepatitis. Fruit pulp is edible, plant wood is used for domestic purposes. D. toxicarium34 Treating swellings, coughs, rheumatism, urethritis and chronic sores, used as rodenticide, sneeze powder for restoring consciousness. Fruit pulp is edible. D. pallidum35 Used for treating dysentery and diarrhea. Most of the Dichapetalum species have been reported to be toxic to livestock and humans including D. toxicarium, D. deflexum, D. tomentosum, D. braunii, D. cymosum, D. madagascariense, D. heudelotii, D. michelsonii, D. ruhlandii and D. stulmanii31, 36. The quest into their toxicity has received considerable attention from most researchers. University of Ghana http://ugspace.ug.edu.gh 9 2.2 Earlier Investigation into Some Toxic Dichapetalum Species and their Chemical Composition Fluorocarboxylic acids6,7 have been reported to be responsible for toxicity in some Dichapetalum species. Fluoroacetic acid [1] was identified as the main toxic compound in D. cymosum by Marais in 1944. His research further showed that fluoroacetic acid was in the lipid fraction27. Some other fluorocarboxylic acids responsible for the toxicity of the species of the genus Dichapetalum are ω- fluoro-oleic acid [8], ω-fluoro-palmitic acid [9], ω-fluoro-capric acid [10], ω-fluoro-myristic acid [11] and threo-18-fluoro-9,10-dihydroxystearic acid [12]7,37. Subsequent investigations showed that the African genus of the family Dichapetalaceae contains high levels of fluoroacetate which are in higher amounts in the roots and stem compared to the leaves8. O F OH Fluoroacetic acid [1] According to a South African report in 1996, D. cymosum38 was implicated in the mortality of eight percent of cattle. It has been reported that a sheep dies after feeding on one or two leaves of the plant, while three or four leaves would kill an ox. Marais reported the LD50 of the fluoroacetate to be 0.5mg/kg in the plant39. The primary point of action of fluoroacetate as proved by Peters is its interference with the metabolism of pyruvate in general and more specifically with aconitase. In the citric acid cycle, one step involves the conversion of acetyl CoA [2] and oxaloacetic acid [5] to citric acid by the enzyme citric synthase40. University of Ghana http://ugspace.ug.edu.gh 10 Scheme 1: The lethal synthetic pathway for the inhibition of Kreb Cycle CH 3 SCoA O Citrate synthase HO 2 C OH HO 2 C CO 2 H Aconitase HO 2 C HO 2 C CO 2 H H Energy HO 2 C CO 2 H O Acetyl CoA [2] Citric Acid [3] Cis-aconitic acid [4] Oxaloacetic acid [5] The citric acid [3] undergoes dehydration to produce aconitic acid [4] which undergo a series of metabolic reactions resulting in the release of energy from the cell. Oxaloacetic acid [5] is regenerated finally to enable the cycle to continue. Since all cells undergo the citric acid cycle, fluoroacetate is toxic to all cells by being activated to fluoroacetyl CoA [6] in the pathway responsible for generating metabolic energy41,42 (Scheme 2). The fluoroacetyl CoA [6] competes with acetyl CoA for the enzyme citrate synthase which is then converted to 2R, 3R-fluorocitric acid [7]40. The fluorocitric acid stereoisomer acts as a competitive inhibitor of the enzyme aconitase in the cycle, halting the regeneration of aconitic acid and in the process inhibits respiration and subsequently deprives the cell of energy. Kun and co-workers have suggested that fluorocitric acid binds irreversibly to the protein responsible for the transport of citrate into the mitochondria. Further accumulation of fluorocitrate results in calcium deficiency as a result of the formation of University of Ghana http://ugspace.ug.edu.gh 11 Scheme 2 Metabolic blocking of the citric acid cycle by fluoroacetic acid chelate with calcium ions in the serum of blood43. Though D. barteri and D. toxicarium also contain fluoroacetate, the main toxic carboxylic acid in D. toxicarium is ω-fluoro-oleic acid [8] since it constitutes the major component7. Furthermore, whereas fluoroacetate was found to be concentrated in the leaves, ω-fluoro-oleic is reported to be concentrated in the seeds of D. toxicarium7. Other fluorocarboxylic acids isolated from the seeds of D. toxicarium are ω-fluoro-palmitic acid [9], ω-fluoro-capric acid [10], ω-fluoro-myristic acid [11] and threo-18-fluoro-9,10-dihydroxystearic acid [12]7. F OH O - fluoro-oleic acid [8] F OH O F SCoA O COOH F HOOC H HO 2 C OH citrate synthase fluoroacetic acid fluoroacetyl-Coenzyme A [6] 2R, 3R- fluorocitric acid [7] Metabolic blocking of citric acid cycle (Kreb's cycle) aconitase [1] University of Ghana http://ugspace.ug.edu.gh 12 F OH O - fluoro-capric acid [10] F OH O - fluoro-myristic acid [11] F OH O OH OH H H threo-18-fluoro-9,10 dihydroxystearic acid [12] F OH O- fluoro-palmitic acid [9] 2.3 THE DICHAPETALINS AND THEIR BIOLOGICAL ACTIVITIES 2.3.1 Structural differences among the Dichapetalins The dichapetalins constitute a small family of secondary metabolites of mixed biosynthesis in which a 2-phenylpyrano moeity is annulated to ring A of a dammarane-triterpene skeleton [13]. The basic structure of dichapetalin [14] is biogenetically characterized by the addition of a C6-C2 unit, which probably might be derived from the shikimmic acid pathway to a 13, 30- cyclodammarane skeleton9. CH 3 CH 3 CH 3 CH 3 CH 3CH 3 CH 3 CH 3 Dammarane [13] O R H R' 18H 28 H OH Basic dichapetalin structure [14] { p h e n y lp ry a n o m o ie ty } 1' 6' 2' 7 17 11 12 B C D A A B C D University of Ghana http://ugspace.ug.edu.gh 13 A total of thirty-two (32) dichapetalin hybrid triterpenoids have been isolated from eight Dichapetalum species namely D. madagascariense, D. gelonioides, D. mombuttense, D. leucosia, D. zenkeri, D. ruhlandii, D. eickii and D. filicaule. Thirteen of these dichapetalins, A-M were isolated initially from two species namely D. madagascariense and D. gelonioides whiles the remaining nineteen were recently isolated from D. gelonioides, D. eickii, D. mombuttense, D. leucosia, D. zenkeri and D. filicaule. The table (Table 2.2) below shows the occurrence of the dichapetalins in the different Dichapetalum species investigated. TABLE 2.2: Occurrence of the dichapetalins in investigated Dichapetalum species. Dichapetalum speciesRef. Dichapetalin type isolated Plant Part D. madagascariense12,13 A [22], B [27], C [15], D [16], E [17], F [18], G [19a, b], H [19a, b], M [31]. Roots D. gelonioides14,18 A [22], I [23], J [24], K [25], L [26], [20], [21], [29], [30], [36], [37], [38], T [35], V [40], [41], [42], [43], [44] Stem bark/Leaves U [39] D. ruhlandii16 A [22]. Roots D. mombuttense16 A [22], L [26], N [28]. Roots D. leucosia16 C [15], I [26], P [34], S [33]. Roots D. zenkeri16 A [22], B [27], L [26], O [45], P [34], Q [46], R [47]. Roots University of Ghana http://ugspace.ug.edu.gh 14 D. eickii16 A [22], M [31]. Roots D. filicaule17 A [22], X [32]. Roots Significantly, dichapetalin A [22] has been isolated in all the Dichapetalum species investigated so far except D. leucosia. D. gelonioides13,18 has up to date produced the highest number of dichapetalins (total of 18), most of which are structural analogues of each other followed by D. madagascariense from which a total of nine dichapetalins have been isolated. It is also worth mentioning that majority of the dichapetalins have been isolated from the roots of the plants except for D. geloniodes14,18 where they were isolated from the stem bark and leaves of the plant. It is also reported that the stem bark of D. madagascariense indicated the presence of dichapetalin A [22] on TLC as part of a complex mixture while chemical investigation of both the root and stem of D. barteri44 did not show the presence of dichapetalins. The dichapetalins can be distinguished mainly by the nature of the side chain at C-17 which can be grouped into four namely methyl ester, lactone, spiro-ketal, and lactol side chains. I. Dichapetalins with Methyl ester side chain. Dichapetalins C [15], D [16], E [17], F [18], G [19a/19b], H [19a/19b], 7-dehydrodichapetalin G [20] and 7-dehydrodichapetalin E [21] have a methyl ester side chain. The methyl ester group consists of either; I. An open chain terminating in a primary alcohol for dichapetalins C [15] and F [18], or its stearic acid esterified analogue as applies to dichapetalin D [16], or University of Ghana http://ugspace.ug.edu.gh 15 II. The primary alcohol cyclizes with the oxo substituent at C-23 to give a 3-methyl furanyl moiety in dichapetalin E [17] or a cyclic methyl ketal in the case of dichapetalins G [19a/19b] and H [19a/19b] which are isomeric ketals with the 11,12-dihydro basic skeleton. Figure 1: Dichapetalins with methyl ester side chain. MeOOC O OH OH H CH 3 MeOOC CH 2 OCOC 17 H 35 CH 3 O OH H MeOOC CH 3 O OH OH H O MeOOC CH 3 H O MeOOC CH 3 H OMe OH O MeOOC CH 3 H OMe OH R 1 = OH, G or H(11,12-dihydro) [19a] R 1 = OH, G or H(11,12-dihydro) [19b] R 1 = O, 7-dehydro [20] O H CH 3 H R CH 3 CH 3 R 1 R R 1 = OH, Dichapetalin C [15] R 1 = OH, Dichapetalin D [16] R 1 = OH, Dichapetalin E [17] R 1 = O, 7-dehydro [21] R 1 = OH, Dichapetalin F [18]Dichapetalin basic structure [14] 1' 6' 17 11 12 7 University of Ghana http://ugspace.ug.edu.gh 16 As can be seen from Figure 1 above, the oxidation of the C7 hydroxyl group in dichapetalins E [17] and G [19b] results in 7-dehydrodichapetalin E [21] and 7-dehydrodichapetalin G [20] respectively. II. Dichapetalins with lactone side chain. Dichapetalins A [22], B [23], I [24], J [25], K [26], L [27], N [28], 21—dehydrodichapetalin Q [29] and 2’α-hydroxydichapetalin Q [30] possess a lactone side chain. Within the lactone side chain group, dichapetalins A [22], B [23], I [24], J [25], K [26], L [27] 21—dehydrodichapetalin Q [29] and 2’α-hydroxydichapetalin Q [30] have similar side chain comprising a 5-membered lactone with an allyl alcohol substituent. The differences in their structures are the presence of 11, 12- double bond, a 12-β-OH group, methoxy on the benzene ring of the phenylpyrano moiety and the cyclopropane moiety replaced by a tertiary methyl and a double bond. Dichapetalins J [25] and K [26] are methoxylated variants of dichapetalins I [24] and A [22] respectively which according to Fang14 et al are likely to be extraction artifacts due to the use of methanol as solvent. Dichapetalin B [27] is a hydroxylated analogue of dichapetalin A [22] at C22. 21—dehydrodichapetalin Q [29] and 2’α-hydroxydichapetalin Q [30] within the 5-membered lactone and allyl alcohol substituent group lack the cyclopropane moiety but instead have a tertiary methyl and double bond. Dichapetalin Q [46] and 21-dehydrodichapetalin Q [29] are similar but the presence of γ-lactone carbonyl group in the latter and lactol in the former distinguishes them. The presence of a hemiacetal group in 2’α-hydroxy-21-dehydrodichapetalin Q [30], which is the first to be reported in the Dichapetalum genus with a hydroxyl group at C2’, differentiates it from 21-dehydrodichapetalin Q [29]. University of Ghana http://ugspace.ug.edu.gh 17 O O CH 2 OH CH 3 R 2 CH 3 CH 3 H OH H H H CH 3 R 1 O Dichapetalin R 1 R 2 Other A [22] H H - B [23] H OH - I [24] H H 11, 12- dihydro 12-- OH J [25] OMe H 11, 12- dihydro 12-- OH K [26] OMe H - L [27] H H 11, 12- dihydro 28 19 18 11 12 17 7 22 26 24 1" 6' 1 O O CH 2 OH CH 3 CH 3 CH 3 H OH H H H CH 3 O H OH H R CH 3 R = H = 21-dehydrodichapetalin Q [29] R = OH = 2'- hydroxy-21-dehydrodichapetalin Q [30] Figure 2: Dichapetalins with 5-membered lactone and allyl alcohol side chain 2' 28 7 1819 21 26 University of Ghana http://ugspace.ug.edu.gh 18 Dichapetalin N [28] is the last member of the group with lactone side chain. It has a 5-membered lactone and α, β-unsaturated carbonyl on the side chain. It differs from dichapetalin A [22] by the presence of CHO group at C26 whereas dichapetalin A [22] has CH2OH group at this position. O O CH 3 CH 3 H OH H H CH 3 O CHO CH 3 Dichapetalin N [28] 26 III. Dichapetalins with Spiroketal side chain. Dichapetalins, M [31], P [34], X [32], have structural differences on C17 as already mentioned among the dichapetalins. The side chain of dichapetalin X [32] has a spiro-ketal ring with a β- hydroxybuturate substituent and a C22-OH group. Both dichapetalins M [31] and P [34] have the spiro-ketal ring in their side chain but the β-hydroxybuturate is replaced with a methyl ester. The other dichapetalins with the spiro-ketal moiety in their side chain are; dichapetalin S [33], dichapetalin T [35], dichapetalin U [39], dichapetalin V [40], 6α-hydroxydichapetalin V [41], 22- deoxy-4’’-methoxydichapetalin V [42], dichapetalin W [43] and 4’’-demethoxy-7-dichapetalin W [44]. University of Ghana http://ugspace.ug.edu.gh 19 O O O O CH 3 O O H H CH 3 CH 3 CH 3 H CH 3 OH O Dichapetalin P [34] O O O O OH O CH 3 H CH 3 H CH 3 OCOCH 3 OH H H CH 3 O O O O CH 3 O OH CH 3 CH 3 CH 3 CH 3 OH OH O Dichpetalin M [31] Dichapetalin X [32]  O O O O H H CH 3 CH 3 CH 3 H OH O CH 3 OH Dichapetalin S [33] O O O O CH 3 CH 3 CH 3 H CH 3 R 2 O R 3 R 1 H H H H R 1 = R 2 = H, R 3 = OAc = 22- deoxydichapetalin P [36] R 1 = H, R 2 = R 3 = OH = 25- De-O- acetyldichapetalin P [37] R 1 = R 2 = R 3 = OH = 25- De-O- acetyldichapetalin M [38] O O O O CH 3 CH 3 CH 3 H CH 3 O H H H H O OH O OH Dichapetalin T [35] Figure 3: Dichapetalins with a spiroketal side chain. University of Ghana http://ugspace.ug.edu.gh 20 O O O O CH 3 CH 3 CH 3 H CH 3 H H H H O OH O R 6 R 4 R 5 R 3 R 2 R 1 H H O H H OH Dichapetalin U [39] H H O OH H OH Dichapetalinn V [40] H OH O OH H OH 6hydroxydichapetalin V [41] OCH 3 H O H H OH 22- Deoxy-4''- methoxydichapetalin V [42] OCH 3 H O H OH H Dichapetalin W [43] H H OH H OH H 4''- Demethoxy-7-dihydrodichapetalin W [44] R 1 R 2 R 3 R 4 R 5 R 6 Dichapetalin Type 25 22 24 Figure 3: Dichapetalins with spiroketal side chain. Dichapetalins T [35] and U [39] are stereoisomers with structures similar to 22-deoxydichapetalin P [36] except at C25 where the acetoxy group in the latter is replaced by a β-hydroxy (4- hydroxyphenyl)- propanoyloxy group in the former. Dichapetalins T [35] and U [39] have structural difference only at C24 where the C24 methylene group in the spiro-ketal system occupies the α-position in the former but is in the β-position in the latter. In dichapetalin V [40], the methylene group at C22 in dichapetalin U [39] is replaced by a hydroxyl group. Dichapetalin V University of Ghana http://ugspace.ug.edu.gh 21 [40] is distinguished from 6α-hydroxydichapetalin V [41] by the presence of a hydroxyl group at the C6α position in the latter. Both 22-deoxy-4’’-methoxydichapetalin V [42] and dichapetalin W [43] are methoxylated at C4’’ but there is hydroxyl group at C22 in 22-deoxy-4’’- methoxydichapetalin V [42] as opposed to the hydroxyl group at C24 in dichapetalin W [43]. Unlike dichapetalin W [43], 4’’-demethoxy-7-dichapetalin W [44] has no methoxy group and instead of the carbonyl group at C7 in dichapetalin W, it has a hydroxyl group in this position. IV. Dichapetalins with Lactol side chain. O O CH 3 H CH 3 H H H CH 3 OH CH 3 H OH OH O O CH 3 H CH 3 H H CH 3 OH H OH CH 3 OH H CH 3 O CH 3 H CH 3 H CH 3 OH O OH OH H CH 3 OH OH CH 3 Dichapetalin O [45] Dichapetalin Q [46] Dichapetalin R [47] Figure 4: Dichapetalins with lactol side chain. University of Ghana http://ugspace.ug.edu.gh 22 Dichapetalins O [45], Q [46] and R [47] have lactol in the side chain. Dichapetalins Q [46] and R [47] are first to be reported to contain a tertiary methyl and double bond instead of the cyclopropane ring of the dammaranes. Dichapetalin O [45] and A [22] structurally share a common backbone configuration but the presence of a hemiketal in dichapetalin O [45] as opposed to the lactone side chain in dichapetalin A [22] distinguishes them. 2.3.2 Biological Activities of the Dichapetalins Biological studies carried out on the dichapetalins including brine shrimp, anthelmintic, antifeedant, nematicidal, antifungal, nitric oxide and anticholinestrase inhibitory activities, anti- HIV and cytotoxicity studies have shown significant activities only with the dichapetalins with lactone or spiroketal side chain. Only dichapetalin C among the dichapetalins with methyl ester side chain has showed significant activity. In a brine shrimp lethality test, dichapetalin A [22] showed significant cytotoxic activity (LC50 = 0.31μg/ml) which is seven times higher than podophyllotoxin45, dichapetalin M [31] was found to be very active (LC50 = 0.011 μg/ml), twenty-eight times more potent than dichapetalin A [22]46. Dichapetalin A [22] has showed significant cytotoxic activities to different cancer cells in vitro with varying sensitivities in the respective cancer cell systems. It showed high selectivity against the human colorelectal carcinoma cell line HCT116 with EC50 greater than 10.1 μg/ml but broad selectivity against the human colorectal adenocarcinoma cell line Colo-25 with EC50 = 7.0 μg/ml. Against the human ovarian adenocarcinoma cell line, dichapetalin A [22] exhibited high sensitivity, SW626 (EC50 =0.2 μg/ml) and SKOV-3 (EC50 = 0.5μg/ml) but broad activity was observed for the human ovarian adenocarcinoma cell line OVcAR-3 (EC50 = 5.8 μg/ml). For the human Burkitt’s lymphoma leukemia cell line NAMALWA, dichapetalins A [22] was found to be University of Ghana http://ugspace.ug.edu.gh 23 highly selective (EC50 = 0.2 μg/ml) but broad sensitivity towards the human promyelocytic leukemia cell line HL-60 (EC50 = 6.4 μg/ml). Towards L1210 murine leukemia cell line, EC90 less than 0.0001μg/ml has been reported for dichapetalin A [22]. Broad to high selective anti-cancer activities have been exhibited by dichapetalin A [22] in the following cell lines: hormone- dependent cancer cell line LNCaP ( EC50 = 7.0 μg/ml), human prostate cancer cell lines NC1-H460 (EC50= 0.8μg/ml), DU 145 (EC50 = 7.6 μg/ml), human lung adenocarcinoma cell lines A549 (EC50 = 0.7 μg/ml), HOP-62 (EC50 = 0.5 μg/ml), Lu-1 (EC50 = 4.1μg/ml)14,16. The following cancer cell lines exhibited broad cytotoxic activities towards dichapetalin A [22]: the human melanoma WM266-4 (EC50 = 9.9μg/ml)13, human umbilical endothelial cells HUVEC (EC50 = 5.5 μg/ml), human neuroblastoma SKNSH (EC50 = 6.9 μg/ml), pancreatic adenocarcinoma cell lines BxPC 3 (EC50 = 1.8 μg/ml), renal adenocarcinoma TK10 (EC50 = 7 μg/ml), human breast adenocarcinoma cell line MCF-7 (EC50 = 2.1 μg/ml) and MDAMB-231 (7.6 μg/ml), human colon tumor cell lines Col-2 (EC50 = 6.1 μg/ml) and KM- 12 (EC50 = 5.1 μg/ml) and human breast ductal carcinoma T47 (EC50 = 1.3 μg/ml). However, the human breast cancer cell line was resistant towards dichapetalin A [22] recording EC50 greater than 20 μg/ml14,16. Long et al16 have profiled the cytotoxic activities of dichapetalin A against 16 cancer cell lines as shown in the Table 2.3 below. TABLE 2.3: Cytotoxicity of dichapetalin A [22] against some cancer cell lines16. Cell Line Tumor Type Dichapetalin A [EC50 (µg/mL)] HCT-116 Human colorectal carcinoma 0.1 NAMALWA Human Burkitt’s lymphoma 0.2 SKOV-3 Human Ovarian adenocarcinoma 0.5 University of Ghana http://ugspace.ug.edu.gh 24 HOP-62 Human lung cancer 0.5 A549 Human lung adenocarcinoma 0.7 NCI-H460 Human prostate carcinoma 0.8 T47D Human breast ductal carcinoma 1.3 BxPC3 Human pancreatic adenocarcinoma 1.8 KM-12 Human colon carcinoma 5.1 OVcAR-3 Human ovarian adenocarcinoma 5.8 HL-60 Human acute promyelocytic leukemia 6.4 Colo-205 Human colorectal adenocarcinoma 7.0 TK10 Human renal adenocarcinoma 7.0 MDAMB-231 Human breast adenocarcinoma 7.6 DU145 Human prostate carcinoma 7.6 WM 266-4 Human melanoma 9.9 They found out that the human colorectal carcinoma HCT116 (EC50 = 0.1µg/mL) recorded the highest sensitivity towards dichapetalin A [22] which was 68-fold greater than the most resistant to dichapetalin A [22] (human melona WM266-4 EC50 = 9.9µg/mL). They used these cancer cell lines as references to further profile the cytotoxic and anti-proliferative properties of dichapetalins B, C, I, L, M, O, P, Q, R and S. The results are summarized in table 2.4 below. All the tested dichapetalins indicated varying cytotoxic potencies on the two cancer lines investigated. More interestingly, dichapetalin P [34] was found to be four times more potent than dichapetalin A [22] on HCT116 cells and 74-fold more active on WM 266-4. Even though both dichapetalins A [22] and P [34] have lactone in their side chains, dichapetalin P [34] has a constrained and hence more stable lactone ring moiety provided by the spiro-ketal. This structural University of Ghana http://ugspace.ug.edu.gh 25 feature could be responsible for the remarkable activity of dichapetalin P [34]. The importance of the constrained lactone ring moiety side chain for activity is further illustrated by the fact that dichapetalin M [31], a 6α-hydroxy derivative of dichapetalin P [34] recorded the highest cytotoxic activities of EC50 = 0.007 and 0.05µg/mL on HCT116 and WM 266-4 cell lines respectively. Possibly the hydroxyl group in dichapetalin M [31] is required for activity due to its higher activity compared to that of dichapetalin P [34]. Dichapetalin B [27], initially isolated by Addae-Mensah and co-workers but never described in terms of biological activity was found nearly as active as dichapetalin P [34] on the two cell lines16. Dichapetalin N [28] was also very potent but dichapetalins O [45], Q [46] and R [47] which lack the lactone side chain showed decreased activity on the cell lines. Dichapetalin S [33] was found to be more active than dichapetalins L [26], O [45], Q [46] and R [47] on both cell lines probably due to the fact that dichapetalin S [33] shares some structural similarities to dichapetalins A [22] and P [34] which may be vital for activity. Table 2.4 below shows the chemosensitivities of some dichapetalins on HCT116 and WM 266-4 cancer lines as investigated by Long et al16. TABLE 2.4: Chemosensitivities of some dichapetalins on HCT116 and WM 266-4 cancer cell lines16. Dichapetalin type HCT116 [EC50 (µg/mL)] WM 266-4 [EC50 (µg/mL)] A [22] 0.1 9.9 B [27] 0.05 0.2 C [15] 0.3 2.1 I [23] 0.2 6.0 University of Ghana http://ugspace.ug.edu.gh 26 L [26] 0.4 2.0 M [31] 0.007 0.05 N [28] 0.05 0.9 O [45] 0.5 5.0 P [34] 0.04 0.2 Q [46] 1.5 15.9 R [47] 2.6 19.3 S [33] 0.3 0.8 In a separate study by Fang et al14 on the in vivo evaluation of dichapetalin A [22] (isolated from D. gelonioides collected in the Philippines) using the hollow fiber model at 1-6mg/kg doses, no significant growth inhibition was observed. Their observation further confirmed earlier investigations by Addae-Mensah and co-workers in 19969 as well as Achenbach et al in 199510. Thus though dichapetalin A [22] has showed interesting and significant cytotoxic activities in vitro, however results from in vivo studies are insignificant. In the anti-HIV activity test of dichapetalins A [22] and M [34] against HIV-1/IIIB in MT-4 cells using the Tetrazolium-base colorimetric assay, dichapetalins A [22] and M [34] elicited activities at concentrations toxic to the cells and therefore no significant and selective anti-HIV activity was observed46. In their recent report, Jing18 and co-workers tested the cytotoxic activities of dichapetalin U [39], V [40], W [43] and 7-dehydrodichapetalin E [21] against five cancer cell lines namely; HL-60 Human promyelocytic leukemia, SMMC-7721 Human hepatocellular cell line, A-549 Human alveolar basal-epithelial cell line, MCF-7 Human breast adenocarcinoma cell line and SW480 Human colon adenocarcinoma cell line. Dichapetalin V [40] recorded significant and broad cytotoxicity against all the tested cancer cell lines particularly for A-549 and MCF-7 (IC50 = 3 and University of Ghana http://ugspace.ug.edu.gh 27 3.5 μM respectively). Their experiment proved dichapetalin V [40] to be a better cytotoxic agent than cisplatin (IC50 = 8.3 and 16.1μM for A-549 and MCF-7 respectively) against A-549 and MCF- 7 cell lines. In the same experiment, dichapetalin U [39] showed selective activity towards A-549 (IC50 = 26.8 μM) and MCF-7 (IC50 = 8.2 μM) but dichapetalin W [43] did not show any activity against all the tested cancer lines. Moderate activities were observed for 7-dehydrodichapetalin E [21] but were broader compared to the recorded activities of dichapetalin U [39] against all the tested cell lines. Jing18 et al evaluated the feeding deterrent activities and nematicidal effects of the dichapetalins isolated from D. gelonioides. Dichapetalin A [22], 7-dehydrodichapetalin G [20] and 21- dehydrodichapetalin Q [29] exhibited antifeedant activities with EC50 = 3.1, 3.1 and 3.4μg/cm2 respectively against beet armyworm (Spoderata exugua). The observed activities were found to be comparable to that of commercial neem oil (1% azadirachtin) which gave EC50 = 2.7 μg/cm2 in the same experiment. The hydroxyl group at C7 (in dichapetalin A [22] and 21-dehydrodichapetalin Q [29]) and the tetrahydrofuran side chain of 7-dehydrodichapetalin G [20] are implicated in the increased anti-feedant activity since dichapetalins with p-methoxylated C-6’ phenyl ring in the same experiment recorded lower anti-feedant activities. In the nematicidal studies, all the compounds tested exhibited general nematicidal effects but were far less toxic to the nematodes compared with avermectin. Only 25-de-O-acetyldichapetalin P [36] and 4”-demethoxy-7- dihydrodichapetalin W [44] showed significant nematicidal activities causing 46.3 (± 3.6%) and 61.8 (± 5.1%) mortality respectively at 100μg/ml over 72hr period. Jing18 and co-workers evaluated the antifungal activities of dichapetalin A [22], 22- deoxydichapetalin P [36] and dichapetalin U [39] isolated from D. gelonioides against four pathogenic fungi namely; Colletotetrichum gloeosporioides, C. musae, Fusarium oxysporum f. sp. University of Ghana http://ugspace.ug.edu.gh 28 niveum and Rhizoctonia solani. Dichapetalin U [39] significantly inhibited the growth of all four fungal strains with the diameter of inhibition zone ranging from 12.4(±1.1) – 13.8(±1.0) mm. The antifungal activities observed for dichapetalin U [39] were comparable to that of nystatin against all the fungal strains. Dichapetalin A [22] and 22-deoxydichapetalin P [36] showed activities less potent than dichapetalin U [39] even though against C. musae, 22-deoxydichapetalin P [36] showed higher antifungal activity than dichapetalin A [22] and U [39]. Dichapetalin A [22] proved to be a potent inhibitor of microphage nitric oxide (NO) production with IC50 = 0.02μM compared to MG132 Proteasome inhibitor (IC50 = 0.1μM). However, compared to the proteasome inhibitor, dichapetalin A [22] is a weak acetylcholinestrase (AChE) inhibitor as dichapetalin A [22] measured AChE inhibition of 28.6 ±0.7 % while proteasome inhibitor recorded AChE inhibition of 59.9 ± 2.2 % in the same experiment20. In a separate study, dichapetalins A [22] and X [35] isolated from the roots of D. filicaule gave IC50 values of 162.4 and 523.2 µg/ml respectively in an anthelmintic activity test against clinical isolates of hookworm eggs17. 2.4 Dichapetalins from Other Sources and their Biological Activities Tuchinda15 et al in their search for anti-cancer agents from plants isolated five new phenylpyranotriterpenoids; Acutissimatriterpenes A [49] -D [52], and E [48] from the ethyl acetate extract of the aerial parts of Phyllanthus acutissima. Their work represents the first report of the isolation of dichapetalin-type triterpenoids with a spiro-ketal side chain from the genus Phyllanthus. University of Ghana http://ugspace.ug.edu.gh 29 O O CH 3 CH 3 O H H H H OH O OCH 3 CH 3 O O OH CH 3 O O O O OH O 28 H 19 H CH 3 OCOCH 3 OHH H 18 Acutissimatriterpene E [48] Dicahapetalin M [31] " 7 30 17 11 12 25 1" 1 19 11 12 7 30 17 25 6' 28 22 Figure 5: Structural difference between the dichapetalins and acutissimatriterpenes. The C-17 side chain and the presence or otherwise of a methylenedioxy unit in the phenylpyrano moiety distinguishes the acutissimatriterpenes from the dichapetalins as shown in Figure 5 above. As can be seen from the structures [48]-[52], acutissimatriterpenes A [49] and C [51] are methylenedioxy analogues of acutissimatriterpenes B [50] and D [52] respectively. Acutissimatriterpenes A [49] and B [50] are isometric ketals of acutissimatriterpenes C [51] and D [52] considering the side chain groups. Though acutissimatriterpenes A [49], B [50], and E [48] have similar tetrahydrofuran configuration, the only difference is the presence of hydroxylated alkane in E [35] at C22. Again the spiroketal side chains of dichapetalin M [31] and acutissimatriterpenes A [49], B [50], and E [48] are similar except at the C-25 where the acetoxy group in dichapetalin M is replaced by a methoxy group in A [49], B [50] and E [48]. University of Ghana http://ugspace.ug.edu.gh 30 O O O OCH 3 CH 3 H O CH 3 CH 3 CH 3 H OH H R 1 R 2 Acutissimatriterpene A: R 1 = R 2 = O-CH 2 -O [49] Acutissimatriterpene B: R 1 =R 2 = H [50] O O O OCH 3 CH 3 R 1 R 2 O OH CH 3 CH 3 HCH 3 Acutissimatriterpene C: R 1 = R 2 = O-CH 2 -O [51] Acutissimatriterpene D: R 1 = R 2 = H [52] Figure 6: Dichapetalin-type triterpenoids isolated from Phyllanthus acutissima. The isolated acutissimatriterpenes were tested against a panel of six cancer cell lines namely; P- 388 murine lymphocyte leukemia, KB human nasopharyngeal carcinoma, Col-2 human colon cancer, MCF-7 human breast cancer, Lu-1 human lung cancer and ASK rat glioma. The results according to Tuchinda et al indicated that acutissimatriterpene A [49] and acutissimatriterpene B [50] were active against murine lymphocyte leukemia giving EC50 = 0.4 and 0.5µg/ml respectively whereas acutissimatriterpene E [48] showed significant activities against murine lymphocyte University of Ghana http://ugspace.ug.edu.gh 31 leukemia (EC50 = 0.005µg/ml), human breast cancer (EC50 = 1.1µg/ml) and human lung cancer (EC50 = 3.1µg/ml). While the remaining cell lines did not show any significant activity, acutissimatriterpenes C [51] and D [52] showed insignificant activities (EC50 > 5µg/ml) against all the cancer lines tested. Results also from anti-HIV-1 activities employing cell-based cytotoxic and syncytium assays using MC99 virus and 1A2 cell line system showed various levels of activities for acutissimatriterpenes A-E (Selectivity index = >1.5- >8.1). For this particular study, it can be hypothetically stated that the presence of C22- OH and a non-substituted phenyl ring are crucial for activity. In an HIV-1 RT assay, acutissimatriterpenes A [49] and B [50] were moderately sensitive (> 50 to 70% inhibition at 200µg/ml) followed by acutissimatriterpenes D [52] and C [51] at 37% and 11% inhibition respectively, whiles acutissimatriterpene E [48] was the least active (-0.5% inhibition). Again the presence of an unsaturated C20-C22 and a non-substituted phenyl group may be important for the observed activities. These results as observed by Tuchinda and co-workers presents the only significant activities of the dichapetalins in anti-HIV studies. 2.5 Other Compounds Isolated From Previously Investigated Dichapetalum Species The stem bark of D. gelonioides14,18 afforded apart from dichapetalins A, I, J and K, two nordaboranditerpenoids; ent-16-nor-3-oxodolabra-1,4(18)-diene-2-ol-15-oic acid [53] and ent-16- nor-5α,13α(methyl)-2-oxodolabra-3-en-3-ol-15-oic acid [54], zeylanol [55], 28-hydroxyzeylanol [56] and betulinic acid [57]. Chemical investigation of the roots and stem of D. barteri44 even though did not show the presence of any dichapetalin, a new triterpene belonging to the friedo-oleanane family named 2-hydroxy-3- oxo-D:A-friedooleanan-29-oic-acid [58] was isolated in addition to betulinic acid [57], betulonic University of Ghana http://ugspace.ug.edu.gh 32 acid [59], friedelan-3-one [60], friedelan-3β-ol [61], canophyllal [62], canophyllol [63] and 7 homologous long chain esters of E-ferulic acids. The petroleum ether and acetone-chloroform extracts of the roots of D. filicaule17 also afforded friedelan-3-one [60] and friedelan-3β-ol [61], in addition to glycerol monostearate [64], pomolic acid [65] and a mixture of stigmasterol [66] and β-sitosterol [67] from the acetone-chloroform extract only. This presented the first report for the isolation of pomolic acid from the Dichapetalaceae17. Long16 et al isolated the abietic acid derivative pyracrenic acid [68] from the roots of D. mombuttense. The roots and stem back of D.madagascariense afforded friedelan-3-one [60] and friedelan-3β-ol [61] in addition to a mixture of stigmasterol [66] and β-sitosterol [67], zeylanol [55], sucrose [69], stearic acid [70] and potassium salts. In addition to fluoroacetate, phytosterols, tannins, organic acids, resins, two amino acids (namely N-methyl serine [71], N-methyl alanine [72]), and methyl pentoside trigonelline [73] have been isolated from D. cymosum47. CH 3 O CH 2 CH 3 H CH 3 H COOH CH 3 [53] O OH CH 3 CH 3 H CH 3 H COOH CH 3 [54] Figure 7: Other compounds isolated from some Dichapetalum species. University of Ghana http://ugspace.ug.edu.gh 33 CH 3 CH 3 O CH 3 CH 3 H OH H CH 3 CH 3 CH 3 Zeylanol [55] CH 3 CH 3 O CH 3 CH 3 H OH H CH 3 CH 3 CH 3 CH 2 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 H H OH H COOH 28-hydroxyzeylanol [56] Betulinic acid [57] CH 2 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H H H O COOH CH 2 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 H H OH H COOH [58] Betulonic acid [59] O CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 CH 3 H H Friedelan-3-one [60] CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 CH 3 OH H H Friedelan-3-ol [61] CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H H CH 3 H H O R CH 3 O H OH OH O R = CHO = Canophyllal [62] R = CH 2 OH = Canophyllol [63] 2,3-dihydroxypropyloctadecanoate [64] Figure 7 continued. University of Ghana http://ugspace.ug.edu.gh 34 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 COOH OH OH Pomolic acid [65] CH 3 CH 3 CH 3 CH 3 HCH 3 OH H CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH H H -sitosterol [67] Stigmasterol [66] O O CH 3 CH 3 CH 2 CH 3 O OH H H CH 3 H OH OH CH 3 H Pyracrenic acid [68] O O O OH OH OH OH OH CH 2 OH Sucrose [69] CH 2 OH CH 2 OH CH 3 OH O Stearic acid [70] OH OH H H 3 CHN O OH CH 3 O H NHCH 3 N + CH 3 CO 2 - N-methylserine [71] N-methylalanine [72] Trigonelline [73] Figure 7 continued. University of Ghana http://ugspace.ug.edu.gh 35 2.6 Schistosomiasis: A Major Neglected Parasitic Disease in Ghana. Infectious diseases are one of the leading causes of morbidity and mortality in the developing worlds. Majority of these diseases are caused by parasites that belong to the diseases of poverty or neglected tropical diseases. Among these parasites include the causative agents of trypanosomiasis, leishmaniasis, schistosomiasis, lymphatic filariasis and onchocerciasis24. Schistosomiasis is endemic to most tropical and sub-tropical regions of the globe as well as part of Asia and South America and according to the World Health Organization (WHO) report, it is the second most prevalent tropical disease after malaria20. More than 200 million people are infected with the disease world-wide, with higher prevalence occurring among children22. In the year 2000, a survey of the disease specific-mortality indicated that 70 million out of a total of 682 million people had experienced haematuria and 32 million, dysuria associated with schistosomiasis infection48. According to the report, 18 million out of those infected suffered bladder cancer and 10 million, hydronephrosis22. In Sub-Saharan Africa, it is estimated that 280,000 deaths are reported yearly due to schistosomiasis infection22. The disease therefore constitutes an important health problem in Africa22. In Ghana, the disease is wide-spread in all the regions and the prevalence of schistosomiasis infection is estimated between 54.8-60%, indicating that it is still a problem in some parts of the country23. Schistosomiasis is caused by blood-dwelling fluke worms of the genus Schistosoma, with S. mansoni (in Africa, Middle East, South America and the Caribbean), S. haematobium (in Africa and Middle East), and S. japonicum (in China, Indonesia, Philippines) as the main disease causing species19. The adult worms colonize the veins of either the portal vein system (in the case of S. mansoni and S. japonicum) or the urinary bladder plexus (S. haematobium) and can live for several years. Egg production is both responsible for both transmission of the parasite and aetiology of the University of Ghana http://ugspace.ug.edu.gh 36 disease. Schistosomal species are distinguished by differences in their morphology; both in parasite stage and their eggs, and by the intermediate snail host that supports the transmission of parasites19 (Figure 8). Figure 8: Life Cycle of Schistosoma species19 The infection of Schistosoma species occurs when the larvae of the parasites are liberated by the infected intermediate snail host, which once in contact with the definite human host, penetrates the skin. In the human host, the schistosomulae migrate to the liver via the bloodstream where they mature into adult male and female forms. After mating, the worms migrate to either the mesenteric intestinal veins or the venous plexus of the urinary system. The female worms then release the University of Ghana http://ugspace.ug.edu.gh 37 eggs which are able to pass the epithial of blood vessels and reach the intestinal lumen, the bladder or urethra lumen in order to be expelled by stools or urine19. 2.6.1 Control of Schistosomiasis Even though there is no consensus about the best strategy to control the disease, prevention and control strategies are based on improving the sanitation conditions and treating patients as well as controlling the intermediate vectors49. A combination of the approaches above is being considered for interrupting the life cycle of Schistosoma sp50,51. Current strategies have focused on the periodic treatment of people living at risk areas with anti-schistosomal drugs in order to reduce morbidity and transmission52. 2.6.2 Drugs for Treating Schistosomiasis Chemotherapy is the global strategy adopted in the fight against Schistosoma spp. infection. Current treatment of Schistosoma species infection relies on metrifonate [74], oxamniquine [75] and praziquantel [76]53. Metrifonate [74] or O,O- dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate was derived from an organophosphorus insecticide. It is recommended by WHO for treating S. haematobium infection since it has selective activity against the urogenital disease54. Metrifonate [74] acts as a reversible inhibitor of acetylcholinestrase, this inhibitory activity at low concentration causes a selective paralysis of the parasite’s muscles making it prone to being carried out by the bloodstream to either the liver (for S. mansoni) or to the lungs ( for S. haematobium). At higher concentrations, metrifonate [74] is toxic to humans, and at lower concentration S. mansoni unlike S. haematobium is able to go back to the mesenteric veins in the intestines to re-establish the infection53. University of Ghana http://ugspace.ug.edu.gh 38 N N O O H N + OH O - O N H CH 3 N CH 3 CH 3 H Praziquantel [76] Oxamniquine [75] P Cl OH Cl Cl O OCH 3 OCH 3 Metrifonate [74] Figure 9: Anti-Schistosomiasis drugs. Oxamniquine [75] or 1,2,3,4- tetrahydro-2-[isopropylamino]methyl(-)-7-nitro-6-nitro-quinoline methanol is reported to be a substrate of sulfotransferase that produces an ester which is able to react with nucleic acids and thereby interferes with replication and transcription processes in the Schistosoma spp.54. It causes an increased motility and tegument damage and it is more active against S. mansoni and hence recommended for treating S. mansoni infection. Due to its restricted use to S. mansoni and the high cost of production coupled to the emergence of resistant strains, oxamniquine [75] is not used in control campaigns even though it has less cytotoxic effects in humans compared to metrifonate [74]54. Praziquantel [76] or 2-(cyclohexanecarbonyl)-3, 6, 7, 11btetrahydro-1H-pyrazino[2,1-a]isoquinil- 4-one is the drug of choice for treating the three human pathogenic species of schistosoma-S. mansoni, S. japonicum and S. haematobium. It is a safe and low cost drug and has been used for over 2 decades for control strategies and patients treatment in countries like China, Brazil, Cambodia, Egypt, Morocco and Saudi Arabia55. The drug is suggested to induce membrane alterations, producing a Ca2+ entry in the muscle cells and paralysis in the contracted state56. The paralyzed parasites are then carried by the bloodstream. Due to its large scale usage for control programs over a long period, the resistance of S. mansoni to praziquantel has been reported in University of Ghana http://ugspace.ug.edu.gh 39 Egypt23, and also diminished efficacy as well as resistance of the other strains of schistosoma has been reported in the field and in the laboratory58,59,60. The classical strategy of alternating treatments to avoid the development of resistance is very crucial. Since vaccines, safe and affordable drugs are lacking to treat schistosomiasis especially in the tropics and sub-tropics, there has been a growing interest in the scientific community to search for extracts and new compounds from plant origin with anti-schistosomal properties61,62. 2.6.3 Natural Products as Potential Source for the Control of Schistosomiasis. Nature has always served as a unique source of structures of high phytochemical diversity, many of them showing interesting biological and medicinal purposes. It is estimated that over 70% of the African population still rely on traditional herbs for curative purpose. Plants have been used extensively in the treatment of various diseases including schistosomiasis especially in Africa and Asia61. The variety of the application of these herbal extracts indicates their ethno-pharmacological potential as sources of active compounds. Ndamba61 et al have documented a list of 47 plants from different plant families used in the treatment of urinary schistosomiasis. The extract of one of them, Pterocarpus angolensis (Leguminosae) was prepared as described by traditional healers in Zimbabwe. After treating some previously exposed mice with the extract from the stem bark of angolensis, the results were found to be similar to the efficacy of praziquantel62. Other researchers have also confirmed the anthelmintic potencies against schistosomula of S. mansoni of the extracts from the root and stem bark of Abrus precatorius (Fabaceae) and Elephantorrhiza goetzei (Mimosaceae)62. More recently, studies are focusing on the isolation, identification and validation of the active phytochemicals within the plant extracts. University of Ghana http://ugspace.ug.edu.gh 40 The most interesting antischistosomal compounds so far are derivatives of artemisinin; artemether [82] and artesunate [81]. Artemether has been found to be active against immature schistosomes and it has been suggested that artemether be used together with praziquantel for schistosomiasis treatment63. Table 2.5 below presents a brief review of natural products from plant and plant- derived sources with schistosomal properties. Table 2.5: Potential anti-schistosomal drugs from plants (and natural product-derived) sources. Compound/Source Relevant Data Curcumin [77] Curcuma longa64 Effective in vitro against S. mansoni adult worms; 100% mortality in both male and female worms at 50µg/ml, loss of motor activity, reduction in oviposition and separation of male and female worms. 2-hydroxychrysophanol [78] and Kwanzoquinone E [79] Hemerocallis fulva65 Effective in vitro against S. mansoni adult worms at 50 and 25µg/ml respectively. 35% and 80% mortality in male and female worms recorded for the former and latter respectively. Quercetin-3-O-β-D- rhamnoside [80] Schefflera vivosa66 Effective in vitro against S. mansoni worms at 100µg/ml, 25% mortality recorded in male and female worms, reduction in motor activity observed. Artesunate [81] and artemether [82] Artemisia annua63 Effective against immature schistosome in experimentally infested mice- 150-300mg/kg; 67-77% mortality in male and female. Reduction in the oviposition and motor activity, disruption in tegument. Vernodalin [83] Vernonia amygdalina67 Effective in vivo against S. mansoni in mice, inhibition of the oviposition and reduction in motor activity Epiisopiloturin [84] Pilocarpus mycrophyllus68 Effective in vitro against S. mansoni worms, 100% mortality observed at 300µg/ml in male and female worms, causes tegument disruption in the worms. University of Ghana http://ugspace.ug.edu.gh 41 Phytol [85] (Synthetic)69 Effective in vitro against S. mansoni worms; reduction in egg-laying observed at 25µg/ml. Piplartine [86] Piper tuberculatum70 Effective in vitro against S. mansoni worms; 100% mortality in male and female worms at 15.8µM, and 100% mortality in schistosomula. E/Z-Nerolidol [87] (Synthetic)71 Effective in vitro against S. mansoni worms; 100% mortality in male and female adult worms at 31.2 and 62.5µM, reduction in motor activity. Favaspidic acid [88] and Desaspidin [89] Dryopteris spp.72 Effective in vitro against S. mansoni worms; 100% mortality in male and female worms observed at 50 and 25µMrespectively. Reduction in motor activity and tegument disruption. Allicin [90] Alium sativum73 Effective against S. mansoni male worms in vitro; disruption of tegument observed at 10-20µg/ml. As indicated in Table 2.5, several in vitro studies have been conducted in search for new drugs or possible leads for the control of schistosomiasis. The extensive phytochemical investigations reveal the presence of potential anti-schistosomals from different plant classes with different functionalities like alkaloids, terpenes, amides and lactones among others. Even though little in vivo studies have been conducted compared to in vitro studies, the results from the in vitro studies are worth considering and should inspire the pathway to conducting in vivo studies to validate their antischistosomal properties. Also the search of new possible drugs must be intensified and optimized from other plant families like the Dichapetalaceae. Many phytochemicals from the Dichapetalum species investigated have revealed interesting pharmacological activities like anticancer, anthelmintic and antifeedant among others. Most of these compounds are terpenoids in nature and compared to the structures shown below, they may provide potential anti-parasitic agents or leads for the treatment of Schistosoma infections. The Dichapetalum species abound in the tropics and most of them have not been investigated. University of Ghana http://ugspace.ug.edu.gh 42 OCH 3 OHOH H 3 CO O O Curcumin [77] CH 3 OH OH O OOH 2-hydroxychrysophanol [78] OH O O OH OH OH Kwanzoquinone [79] O ORham O OH OH OH OH Quercetin-3-O-D-rhamnoside [80] O O O OH O O CH 3 O O CH 3 CH 3 CH 3 H H Artesunate [81] O O O CH 3 CH 3 O O H CH 3 CH 3 H H Artemether [82] O O O CH 2 H H O OH CH 2 O CH 2 CH 2 O Vernodalin [83] N O O CH 3 O CH 3 O CH 3 O H H Piplartine [86] OH O CH 3 OH CH 3 OH OH O CH 3 CH 3 CH 3 OH O OH O CH 3 OH CH 3 O OH O CH 3 CH 3 CH 3 OH O CH 3 S S CH 2 CH 2 O Favaspidic acid [88] Desaspidin [89] Allicin [90] CH 3 CH 2 OH CH 3 CH 3 CH 3 CH 3 O N N CH 3 OH O H Epiisopiloturine [84] CH 3 CH 3 CH 2 CH 3 CH 3 H CH 3 Nerolidol [87] Phytol [85] Figure 10: Potential anti-schistosomal drugs. University of Ghana http://ugspace.ug.edu.gh 43 CHAPTER THREE THE PRESENT INVESTIGATION 3.1 Summary The stem of D. crassifolium, collected from Kuntanase in the Ashanti Region of Ghana was chopped into small pieces and pulverized after air drying for one month. The pulverized plant material (5.0kg) was successively extracted with three different solvents: petroleum ether (PE), ethyl acetate (EA) and methanol (MeOH) using Soxhlet extraction. The process yielded 34g of PE, 55g of EA and 25g of MeOH crude extracts. Isolation and purification of the isolates from all extracts were achieved by column chromatography, thin layer chromatography (TLC) and recrystallization to yield 14 solids. Three (3) of these solids came from the PE extract, eleven (11) from the EA extract but none from the MeOH extract. The solids isolated from the PE extract were identified as friedelan-3-one, friedelan-3β-ol and a mixture of friedelan-3-one and friedelan-3β-ol by comparative TLC, co-melting point and by comparing their IR data with authentic samples and available literature data. These solids were also isolated both as pure compounds and as a mixture from the ethyl acetate extract. In addition to friedelan-3-one, friedelan-3β-ol and a mixture of the two, seven (7) other solids were isolated from the ethyl acetate extract. One of them (DCS-E4), was identified as the very common mixture of stigmasterol and β-sitosterol by co-TLC, co-melting point and IR data with reference samples and literature information. The solid DCS-E6 was identified as pomolic acid and another solid (DCS-E7) was identified as the previously isolated and identified University of Ghana http://ugspace.ug.edu.gh 44 phenylpyranotriterpenoid compound, dichapetalin M, also by co-TLC and melting point with authentic samples in addition to 1H and 13C NMR data. DCS-EM was obtained as a mixture of dichapetalin M and two other unknown solids based on TLC. Also, DCS-E8 was identified as maslinic acid after comparing its 1H and 13C NMR data with reference data. The identities of DCS- E5, DCS-E9 and DCS-E11 are unknown due to impurity and paucity of material. Investigation of the methanol extracts did not yield any solid. 3.2 INVESTIGATION OF EXTRACTS FROM THE STEM OF D. CRASSIFOLIUM 3.2.1 Phytochemical Screening of the PE, EA and MeOH Crude Extracts Phytochemical screening tests were carried out on the petroleum ether, ethyl-acetate and methanol crude extracts to determine the class of compounds present in them. The results of the phytochemical screening tests are summarized in Table 3.1 below. TABLE 3.1: Phytochemical Screening Test Results on the Crude Extracts of D. crassifolium. Class of compounds PE extract EA extract MeOH extract Alkaloids - - - Anthraquinones and anthracene - - - Flavonoids and leucoanthocyanins - - - Cardiac glycosides - - + Saponins - - - Tannins - + + Terpenoids + + + Legend: (+) = present and (-) = absent University of Ghana http://ugspace.ug.edu.gh 45 The results of the phytochemical screening test indicated that the chemical constituents of the stem of D. crassifolium are steroids in nature. 3.2.2 Investigation of the Petroleum Ether (PE) Extract The dried pulverized stem of the plant material (5.0kg) was defatted via Soxhlet extraction with petroleum ether (40-60oC) for 24 hours continuously. Concentration of the extract gave a total of 34.0g of a dark-greenish syrup-like crude matter coded as DCS-P. Phytochemical screening on DCS-P indicated the presence of terpenoids only. DCS-P was kept in the refrigerator until further investigation. Scheme 3 below summarizes how the phytochemicals present in the extract were isolated. Scheme 3: Investigation of PE Extract. DCS-P (23g) ML (13g) DCS-PA1 (9g) 21.5g, chromatographic separation of ML + DCS-PA1 100% PE EA  CHCl 3 /EtOH 1:1 DCS-PA2 (12.6g) DCS-PA3 (1.8g) Yellow oil 5.4g DCS-P1 DCS-P1P2 DCS-P2 180mg 40mg 80mg chromatographic separation PE/CHCl 3 25/75 Refrigeration University of Ghana http://ugspace.ug.edu.gh 46 Upon refrigeration, DCS-P was observed to have precipitate and supernatant portions. The supernatant (mother-liquor, ML) was filtered off and TLC in petroleum ether/ ethyl acetate (28:1), was run on both the solid portion DCS-PA1 and the mother liquor, ML. After staining with anisaldehyde spray reagent, the two portions revealed comparable spots (over 14) and hence were put together (21.5g), dissolved in chloroform and mixed with silica gel (6g) and air dried. This was later put on a glass column pre-loaded with silica gel (230g). The column was first eluted with 100% petroleum ether, then with petroleum ether and ethyl acetate mixtures until 100% ethyl acetate. The column was finally washed with chloroform/ethanol 1:1mixture. Fractions F021-F073, eluted with 10-30% of ethyl acetate in petroleum ether were combined based on their similar TLC profiles. This was coded DCS-PA2, which gave 4 spots on TLC (Petroleum ether/ethyl acetate: 14:0.5). The sub-fraction DCS-PA2 (12.6g), was again put on a smaller glass column pre-loaded with silica gel (80g) and eluted with petroleum ether/chloroform: 25/75 mixture. The column fractions yielded the solids DCS-P1 (120mg, 1 spot), DCS-P2 (70mg, 1 spot) and a mixture of these two solids coded DCS-P1P2 (20mg, 2spots) after recrystallization from ethanol. The 100% ethyl acetate fraction and the fraction obtained after washing the column (chloroform/ absolute ethanol 1:1) were also found to be the same on TLC and hence were combined (coded DCS-PA3) and safely kept in the refrigerator for future investigation. 3.2.2.1 Isolation and identification of friedelan-3-one, DCS-P1 DCS-P1 was obtained as white needle-like crystals after recrystallization from ethanol with melting point of 249-251oC. On TLC, DCS-P1 was observed as a single yellow spot with anisaldehyde spray reagent after heating. The yellow color lasted for a short time. The following Rfs were recorded in the following solvent systems: 100% CHCl3, Rf = 0.94; petroleum ether/EtOH University of Ghana http://ugspace.ug.edu.gh 47 (15:1), Rf = 0.79; petroleum ether/acetone (12:0.5), Rf = 0.82; petroleum ether/ ethyl acetate (14:0.5), Rf = 0.87 but the color faded quickly upon exposure to air. The spot also stained with iodine to give a dark grayish coloration which lasted temporarily. Comparative TLC and mixed melting point with a reference sample identified DCS-P1 as friedelan-3-one. The reference sample had a melting point of 250-252oC and showed the same Rf as DCS-P1 run in the solvent systems above. The IR data of the two compounds were comparable especially in the fingerprint region. The IR data of DCS-P1 and friedelan-3-one are presented in Table 3.2 below. TABLE 3.2: IR frequencies of DCS-P1 compared to that of friedelan-3-one. Frequency ( cm-1) of DCS-P1 Peak Intensity Frequency (cm-1) of friedelan-3-one17 Peak Intensity Interpretation 3003.19, 2926.86 2869.70 Strong 3003, 2971, 2926, 2869 Strong C-H stretch; alkanes 1715.54 Strong 1715 Strong C=O stretch; ketones 1462.80, 1389.39 Strong 1463, 1389 Strong C(CH3)2 bending vibration; alkanes O CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 CH 3 H H Friedelan-3-one 1 3 5 1 7 10 12 15 18 17 23 24 25 26 27 29 30 28 9 21 22 University of Ghana http://ugspace.ug.edu.gh 48 Friedelan-3-one, also called friedooleanan-3-one, DA-friedooleanan-3-one, friedelin, friedeline or 3-friedelanone has the IUPAC name (4R, 4aS, 6aS, 6aS, 6bR, 8aR, 12aR, 14aS, 14bs) – 4, 4a, 6a, 6b,8a, 11,11, 14a- octamethyl -2, 4, 5, 6, 6a, 7, 8, 9, 10, 12, 12a, 13, 14, 14b-tetradecahydro-1H- picen-3-one with a molecular weight of 426.73g/mol12. In the family Dichapetalaceae, friedelan-3-one has been isolated from D. barteri, D. madagascariense, D. gelonioides and D. filicaule. However, it has also been isolated from other different plant families among which are; Maytenus ilicifolia (Celastraceae)74, Ageratum conyzoides (Asteraceae)75, the stem bark of Hymenocardia acida (Hymenocardiaceae)76, Calophyllum cordata-oblongum (Calophyllaceae)77, root bark of Tripterygium hypoglaucum (Celastraceae)78 and from the plant Virola Calophylla ( Myristicaceae)79. Contrary to previous thought that terpenoids are biologically inactive, there is continuing evidence to confirm that terpenoids have broad spectrum of pharmacological activities coupled with low toxicity profile80. Apart from their use in most Asian countries for their anti-inflammatory, analgesic, antipyretic, hepatoprotective, cardiotonic, sedative and tonic effects80,81, several other activities including antioxidant, antimicrobial, antiviral, antiangiogenic, antiallergic, antipruritic, spasmolytic and cytotoxicity have been reported82, 83, 84. The anti-ulcerogenic activity of friedelan-3-one has been suggested by Queiroga74 et al after isolating it from Maytenus ilicifolia. Duraipandiyan85 and co-workers have suggested the potential antifungal effect of friedelan-3-one. Duraipandiyan as well as Kumari86 et al have investigated the anti-inflammatory activity of friedelan-3-one. Kumari and co-workers isolated friedelan-3-one in addition to surionol, daucosterol, ursolic acid, cycloeucaneol, nimbiol, sugiol and 3β- hydroxyglutin-5-ene from Cammiphora berryi and investigated their anti-inflammatory activity University of Ghana http://ugspace.ug.edu.gh 49 via an in vitro soybean lipoxygenase (SBL) assay. Among all the isolates, friedelan-3-one showed the highest significant activity with IC50 = 35.8µM. Erasmienne87 and co-workers also have confirmed the antiplasmodial activity of friedelan-3-one isolated from Endodesmia calophylloides. 3.2.2.2 Isolation and identification of friedelan-3β-ol, DCS-P2 DCS-P2 was obtained as a glassy-white, rod-like crystalline solid. It was soluble in chloroform and stained with iodine vapor to give a dark-brown coloration only upon longer exposure. The melting point was determined to be 272-274oC. It stained purple with anisaldehyde spray reagent on TLC upon heating. The purple color lasted for a longer time. The following Rf values were measured after running TLC on this solid in the following solvent systems: 100% CHCl3, Rf = 0.81; petroleum ether/EtOH (15:1), Rf = 0.46; petroleum ether/ acetone (12:0.5), Rf = 0.5, petroleum ether/ ethyl acetate (14:0.5), Rf = 0.61. After running comparative TLC and melting point on DCS-P2 and a reference sample, DCS-P2 was identified as friedelan-3β-ol based on its comparable Rf and melting point with the reference sample. The reference material had a melting point of 271-273oC. The IR data obtained for DCS-P2 was comparable to that of the reference sample. TABLE 3.3: Comparison of IR data of DCS-P2 with that of friedelan-3β-ol IR frequency (cm-1) of DCS-P2 Intensity of Peak IR Frequency (cm-1) of Friedelan-3β-ol17 Intensity of Peak Interpretation 3476.97 Strong 3479 Strong O-H stretch; alcohol 2932.17, 2870.58 Strong 2911, 2869 Strong C-H stretch; alkanes 1447.72, 1384.96 Strong 1451, 1385 Strong C(CH3)2 bend; alkanes University of Ghana http://ugspace.ug.edu.gh 50 OH CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 CH 3 H H Friedelan-3-ol 1 3 5 1 7 10 12 15 18 17 23 24 25 26 27 29 30 28 9 21 22 Friedelan-3β-ol, is synonymous with epi-Friedelinol, epifriedelanol, friedelinol and 3β-friedelinol has the IUPAC name (3S, 4R, 4aS, 6aS, 6bR, 8aR, 14aS)- 4, 14a, 6a, 6b, 8a, 11, 11, 14a- octamethyl- 1, 2, 3, 4, 5, 6, 6a, 7, 8, 9, 10,12, 12a 13, 14, 14b-hexadecahydropicen-3-ol. It has a molecular weight of 428.73g/mol12. From the plant family Dichapetalaceae, friedelan-3β-ol, like friedelan-3-one, has also been isolated from D. barteri, D. madagascariense, D. gelonioides and D. filicaule. In literature, friedelan-3β- ol has been isolated from other different plant families like Meytenus ilicifolia (celastraceae)74, Drynarai quercifolia (Polypodiaceae)87, Ulmus pumila L. (Ulmaceae)88, Ipomoea cairica (Convolvulaceae)89 and Myricaria paniculata (Myrtaceae)90. Apart from anti-ulcerogenic activity as suggested by Queiroga74 and co-workers for friedelan-3β- ol, Yang91 et al have suggested its possible use in controlling aging related diseases. 3.2.2.3 Isolation of a mixture of friedelan-3-one and friedelan-3β-ol, DCS-P1P2 The solid DCS-P1P2 was obtained as white needle-like crystals after recrystallization from ethanol. The melting point was determined to be 236-246oC. It showed 2 spots on TLC and stained yellow and purple with anisaldehyde spray reagent with the following Rfs when run in the University of Ghana http://ugspace.ug.edu.gh 51 following solvent systems: 100% CHCl3, Rf = 0.94, 0.81; petroleum ether/ EtOH (15:1), Rf = 0.79, 0.46; petroleum ether/acetone (12:0.5), Rf = 0.87, 0.51; petroleum ether/ ethyl acetate (14:0.5), Rf = 0.87, 0.61. DCS-P1P2 was identified as a mixture of fridelan-3-one (DCS-P1) and friedelan-3β- ol (DCS-P2) after running comparative TLC with DCS-P1, DCS-P2 and as authentic sample in the above solvent systems. Its lower melting point probably suggests the higher amount of friedelan-3-one than friedelan-3β-ol in the mixture as indicated by the sizes of the spots on TLC. The IR data for this mixture revealed prominent peaks at 3477, 1178 cm-1 for an OH stretch, a strong and sharp signal at 1710cm-1 for a carbonyl stretch and also the presence of a gem dimethyl group at 1449 and 1383 cm-1. 3.3 Investigation of the Ethyl Acetate (EA) Extract The defatted plant material was air dried and successively extracted with 10L of ethyl acetate for 24 hours. A total of 65g of crude extract was obtained after extracting 5.0kg of the plant material. The crude extract was dark-greenish in appearance and TLC in petroleum ether/ethyl acetate 10:3 indicated over 14 spots. This extract was coded DCS-EA and Scheme 4 below summarizes how work was carried out on the extract to obtain the isolates. DCS-EA was fractionated with wide increase in polarity into 13 fractions, DCS-EA1 - DCS-EA13. The similarity of some of the fractions on TLC resulted in the combination of DCS-EA1 and DCS- EA2 into DCS-EAA1, DCS-EA3 and DCS-EA4 into DCS-EAA2; DCS-EA5, DCS-EA6 and DCS-EA7 into DCS-EAA3; DCS-EA8, DCS-EA9 and DCS-EA10 into DCS-EAA4; DCS-EA11 and DCS-EA12 into DCS-EAA5 and finally elution with absolute ethanol gave fraction DCS- EAA6, giving a total of six fractions in all. University of Ghana http://ugspace.ug.edu.gh 52 The separate sub-fractions, DCS-EAA1 to DCS-EAA5 were each purified further by column chromatography. University of Ghana http://ugspace.ug.edu.gh 53 Scheme 4. Summary of Work Carried out on Ethyl Acetate extract. DCS-EA (40g) column chromatography PE/EA 10:1 PE/EA 10:3 PE/EA 10:6 PE/EA 1:1 PE/EA 1:6 100% EtOH DCS-EAA1 DCS-EAA2 DCS-EAA3 DCS-EAA4 DCS-EAA5 DCS-EAA6 Column; Recrystallization D S-E1 (18mg, 1 spot) DCS-E2 (15mg, 1 spot) DCS-E1E2 (15mg, 2 spots) F079-F0899 Column Recrystallization DCS-E3 (14mg, 2 spots) DCS-E4 (25mg, 1 spot) Column; Recrystallization DCS-E5 (15mg, 1 spot) DCS-E6 (45mg, 1 spot) Column; Recrystallization DCS-EM (40mg, 3 spots) DCS-E7 (20mg, 1 spot) DCS-E8 (32mg, 1 spot) DCS-E9 (10mg, 1 spot) DCS-E10 (5mg, 2 spots) Column; Recrystallization DCS-EAA6 supernatant F018-F025 F043-F048 Column DCS-E11 (242mg) PE/EA 1:3 supernatant University of Ghana http://ugspace.ug.edu.gh 54 3.3.1 Purification and identification of friedelan-3-one, DCS-E1 DCS-E1 was obtained as white needle-like crystals after recrystallization from ethanol. It was the first solid to precipitate from the sub-fraction DCS-EAA1 during the chromatographic separation. The melting point was determined to be 248-250oC and found to be soluble in chloroform. It appeared as a yellow spot (which faded quickly) on TLC after staining with anisaldehyde spray reagent in the following mobile phase compositions: 100% chloroform, Rf = 0.92; petroleum ether/ acetone (12:0.5), Rf = 0.80; petroleum ether/ethanol (15:1), Rf = 0.77; petroleum ether/ethyl acetate (14:0.5), Rf = 0.85. Based on the comparable Rf values, melting points and IR data of DCS-E1, DCS-P1 and with an authentic sample, DCS-E1 was identified as friedelan-3-one. The reference material had a melting point of 250-252oC. The IR data recorded significant peaks at 3003.19, 2926.86, 2869.70, 1715.54, 1462.80 and 1389.39 cm-1. 3.2.3.2 Purification and identification of friedelan-3β-ol, DCS-E2 The next solid from sub-fraction DCS-EAA1 during the chromatographic separation was DCS- E2, obtained as a glassy, white rod-like crystals with a melting point of 272-274oC. DCS-E1 gave a purple spot on TLC run in the following solvent systems when sprayed with anisaldehyde reagent: 100% chloroform, Rf = 0.79; petroleum ether/ethanol (15:1), Rf = 0.42; petroleum ether/acetone (12:0.5), Rf = 0.48; petroleum ether/ethyl acetate (14:0.5), Rf = 0.59. DCS-E2 does not fluoresce under UV light; it however stained with iodine over long exposure. Based on the comparable Rfs, melting points and IR data of DCS-E2, DCS-P2 and an authentic sample, DCS- E2 was identified as friedelan-3β-ol. The reference material had a melting point of 271-273oC. The IR data recorded significant peaks at 3476.97, 2932.17, 2870.58, 1447.72 and 1384.96 cm-1. University of Ghana http://ugspace.ug.edu.gh 55 3.3.3 Purification and identification of mixture of friedelan-3-one and friedelan-β-ol, DCS- E1E2 and DCS-E3 The solids DCS-E1E2 and DCS-E3 were similar in all respect to the already isolated solid DCS- P1P2. They were obtained as white crystals after recrystallization from ethanol and gave two spots; yellow and purple with comparable Rf values as DCS-P1P2. The IR data recorded for both were similar in addition to their comparable melting points. DCS-E1E2 and DCS-E3 were therefore identified as a mixture of fridelan-3-one and friedelan-3β-ol. 3.3.4 Purification and identification of a mixture of β-sitosterol and stigmasterol, DCS-E4 The next solid isolated from sub-fraction DCS-EAA2 was DCS-E4, obtained as white crystalline solid after recrystallization from petroleum ether/acetone mixtures and the melting point was determined to be 138-140oC. DCS-E4 gave a single orange spot with anisaldehyde spray reagent on TLC run in the following mobile phase compositions: 100% chloroform, Rf = 0.50; petroleum ether/ethanol (15:1), Rf = 0.20; petroleum ether/ ethyl acetate (10:4), Rf = 0.82; petroleum ether/ acetone (12:0.5), Rf = 0.21. The melting point recorded for the reference sample was 139-141oC. Based on comparable Rf values, melting point and IR data of DCS-E4 with a reference material, DCS-E4 was identified as a mixture of β-sitosterol [39] and stigmasterol [40]. The IR data of the reference sample and that of DCS-E4 is presented in Table 3.4 below. TABLE 3.4: Infrared values of DCS-E4 compared to that of β-sitosterol/stigmasterol mixture IR (KBr) vmax cm-1 DCS-E4 Peak Intensity IR (KBr) vmax cm-1 Ref.12,14 Peak intensity Interpretation 3434.43 Strong, broad 3434 Strong, broad O-H stretch, alcohol 2936.20, 2882.60 Strong, sharp 2933, 2886 Strong, sharp C-H stretch, alkane University of Ghana http://ugspace.ug.edu.gh 56 1639.76 Weak 1641.11 Weak C=C stretch, alkene 1463.99,1382.79, 1062.30 Medium 1464.23,1382.03, 1061 Medium C-H bending vibration, alkane CH 3 H CH 3 OH H CH 3 CH 3 CH 3 CH 3 3 4 5 6 7 8 1 2 9 10 19 11 12 13 14 17 15 16 20 21 22 23 28 24 25 27 29 26 CH 3 H CH 3 OH H CH 3 CH 3 CH 3 CH 3 3 4 5 6 7 8 1 2 9 10 19 11 12 13 14 17 15 16 20 21 22 23 28 24 25 27 29 26 -Sitosterol [39] Stigmasterol [40] The two compounds, β-sitosterol [39] and stigmasterol [40] structurally differ only at C22 – C23 due to unsaturation in the latter and as a result are often isolated together as a mixture. Also due to the electronic effects of the C=C carbons in stigmasterol [40], the chemical shifts of the protons and carbons in the environment of the unsaturation would be slightly higher (deshielded) than in β-sitosterol [39]. Steroids, being terpenoids are metabolites of isopentyl pyrophosphate oligomers and represent the largest group of phytochemicals. There are over 20,000 terpenoids in nature which occur in different plants including sea-weeds as well as wax-like coatings of various fruits and medicinal herbs like apples, cranberries, figs, olives, mistletoe and lavender among others92,93. Terpenoids are biosynthesized in plants by the cyclization of squalene, which is then converted to squalene 2, 3-oxide from which the precursor of phytosterols, cycloartenol [39a] is derived94. Cycloartenol [39a], a polycyclic sterol unique to plants, is alkylated at C-24 to produce a triene [39c] through a series of steps (Scheme 5), C-14 sterol reductase and a Ϫ8- Ϫ7 isomerase act on the University of Ghana http://ugspace.ug.edu.gh 57 triene to form 24-methylenelophenol, a branch point in the pathway. Parallel pathways lead to the membrane sterols sitosterol from which stigmasterol is derived95. Bulk sterols are integral parts of the membrane lipid bilayer, where, in conjunction with phospholipids, they regulate membrane permeability and fluidity. Sitosterol is believed to regulate membrane fluidity and permeability in plants membranes by restricting the mobility of fatty acyl chains in a similar manner to cholesterol in mammalian cells. It may be involved in how plants membranes adapt to changes in temperature95. Scheme 5: Biosynthetic Pathways of sterols required for Normal Plant Development. CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 2 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 2 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 OH CH 3 [39a] [39b] [39c] [39d] [39] [40] β-sitosterol, isolated from Cissus quadrangularis, according to Garinma96 et al has demonstrated anti-inflammatory and anti-hemorrhoidal activities. Chandler97 and co-workers have also confirmed the anti-hypercholesterolemic activity of β-sitosterol and stigmasterol. Stigmasterol has been reported to regulate the activity of the Na+/K+- ATPase in plants, similar to that of cholesterol in animal cells and again more specifically for cell differenciation and proliferation95. University of Ghana http://ugspace.ug.edu.gh 58 3.3.5 Purification of DCS-E5 and DCS-E6 (DCS-E6 identified as pomolic acid) DCS-E5 and DCS-E6 were the only solids isolated from sub-fraction DCS-EAA3. They were isolated as powdery white solids after recrystallization from a mixture of diethyl ether and chloroform (10/3) and the melting points were determined to be 264-266oC and 268-270oC respectively. DCS-E5 and DCS-E6 gave a purple spot with vanillin spray reagent on TLC run in petroleum ether/chloroform/acetone (7:3:5), Rf = 0.52. DCS-E5/DCS-E6 also gave a single violet spot with anisaldehyde spray reagent on TLC run in the following mobile phase compositions: 100% chloroform, Rf = 0.24; petroleum ether/acetone (10:3), Rf = 0.41; petroleum ether/diethyl ether (3:7), Rf = 0.58; petroleum ether/ethyl acetate (10:6), Rf = 0.87. The IR data recorded for both solids is presented in Table 3.5 below. Comparison of the 1H and 13C NMR data of DCS-E6 with those of a reference sample confirmed DCS-E6 as pomolic acid [65]. No other spectroscopic data was obtained for DCS-E5 due to paucity of material. Table 3.5: IR data for DCS-E5 and pomolic acid, DCS-E6 IR (KBr) vmax cm-1 DCS-E5 Peak intensity IR (KBr) vmax cm-1 DCS-E6 Peak intensity Interpretation 3436.94 Strong 3446.79 Strong O-H stretch 2927.99 Strong 2928.60 Strong C-H stretch, alkane 1691.94 1633.22 Strong Weak 1690.31 Strong C=O stretching vibration of carboxylic acid. 1461.71, 1385.91 Medium 1461.59, 1385.57 Medium C-H bending vibration, alkanes 1237.70,1157.91, 1029.73 Weak 1236.91,1157.03, 1029.73 Weak C-H bending vibrations University of Ghana http://ugspace.ug.edu.gh 59 O OH CH 3 CH 3 OH CH 3 CH 3 CH 3 OH CH 3 CH 3 28 30 24 23 25 26 6 27 15 29 3 2 18 20 21 22 16 7 Pomolic Acid [65] 12 13 9 4 10 14 19 8 17 The 13C-NMR data recorded for DCS-E6 revealed the presence of 30 carbons, with chemical shifts ranging from 15.1-180.6 ppm. The carbon signal at δC 180.6 was assigned to the carboxyl functional group at C-28 and this is further confirmed by the presence of broad, intense O-H stretching absorption in the recorded IR spectrum for DCS-E6. The downfield signals at δC 78.9 and 73.0 were assigned to C-3 and C-19 respectively. Even though these are sp3 carbons, the observed downfield shifts is as a result of their attachment to electronegative hydroxyl groups. The sp2-alkene type carbons, C-12 and C-13 were assigned chemical shifts of δC 129.1 and 138.0 respectively. Apart from C- 13 and C-19, the quaternary carbons were assigned chemical shifts of δC 41.1 (C-4), 38.6 (C-8), 37.5 (C-10), 47.1 (C-14) and 47.5 (C-17). Five of the tertiary methyl carbons were assigned the following chemical shifts; δC 23.6 (C-23), 16.5 (C-24), 15.4 (C-25), 16.0 (C-26) and 25.4 (C-27). The signals at δC 25.9 and 15.1 were assigned to C-29 and C-30 respectively. The 13C NMR assignment is summarized in Table 3.6 below. After assigning all the 1H and 13C-NMR signals of DCS-E6 and comparing with a reference data, DCS-E6 was clearly identified as the ursane-type triterpene acid, pomolic acid [65]. University of Ghana http://ugspace.ug.edu.gh 60 Table 3.6: Comparison of 13C Chemical Shifts of DCS-E6 (Pomolic acid) with reference data. 13C No. DCS-E6 (δC in CD3OD/CDCl3) Reference data of Pomolic acid (δC in CD3OD/CDCl3)17 1 39.8 39.6 2 29.6 29.4 3 78.9 78.6 4 41.1 41.0 5 55.1 55.0 6 18.4 18.4 7 32.7 32.6 8 38.6 38.4 9 48.6 48.7 10 37.5 37.3 11 24.4 24.0 12 129.1 128.7 13 138.0 137.9 14 47.1 46.9 15 28.0 28.0 16 26.9 26.5 17 47.5 47.3 18 53.1 53.0 19 73.0 72.8 20 36.9 36.7 21 27.2 27.7 22 38.4 38.3 23 23.6 23.4 24 16.5 16.5 25 15.4 15.4 26 16.0 16.0 27 25.4 25.2 28 180.6 180.6 29 25.9 25.8 30 15.1 15.1 The 1H NMR (Figures 11a-c) recorded for DCS-E6 revealed several signals ranging from δH 1.2 - 2.2 for overlapping sp3-hybridized methylene and methine protons of pomolic acid. The signal at δH 5.35 (1H, t, J = 6Hz) was assigned to H-12 and the signal at δH 3.40 (1H, m) was assigned to H-3. For the methyl protons, H-29 was assigned δH 1.26 (3H, d, J = 7.2Hz), H-30 was assigned δH University of Ghana http://ugspace.ug.edu.gh 61 0.95 (3H, d, J = 12Hz) and H-26 was assigned δH 0.78 (3H, d J = 10.8Hz). These appeared as multiplets due to long range proton-proton coupling. The other methyl protons appeared as singlets and resonated at the following frequencies; δH 1.21 (H-24), 0.99 (H-25) and 0.92 (H-23). The prominent signal at δH 7.30 is due to residual CDCl3 in the solvent used in obtaining the spectrum. The chemical shift value of δH 2.49 (2H, ddd, J1 = 8.8, J2 = 9.6, J3 = 6.6Hz) was assigned to H-2, the downfield shift suggesting neighboring electronegative groups. The observed multiplicity at δH 2.49 could be explained by the fact that there was an initial coupling to the methylene protons at H-1 into a triplet and each signal of the triplet was further split into a doublet by the methine proton at H-3. The signal at δH 2.63 (1H, s) was assigned to H-18 and not H-9 primarily due to the electronegative neighboring environment (OH and COOH) of H-18 compared to H-9. The signal at δH 2.18 (1H, d, J = 7.8) was assigned to H-9. The signal at δH 3.21 (2H, dd, J1 = 15, J2 = 7.8) was assigned to H-11, the signal at δH 1.98(2H, d, J = 5Hz) was assigned to H-16 while the signal at δH 1.96 (2H, d, J = 4.6Hz) was assigned to H-22. University of Ghana http://ugspace.ug.edu.gh 62 Figure 11a : Expanded 1H NMR of DCS-E6. University of Ghana http://ugspace.ug.edu.gh 63 Figure 11b: Expanded 1H NMR of DCS-E6. University of Ghana http://ugspace.ug.edu.gh 64 Figure 11c: Expanded 1H NMR of DCS-E6. University of Ghana http://ugspace.ug.edu.gh 65 Literature is replete with several biological activity studies on pomolic acid (PA). For instance, PA isolated from the methanolic extract of Planchonella duclitan (Blanco) by Tzoug-Huei98 et al demonstrated cytotoxicity towards human colorectal carcinoma cell line HT29 and human breast carcinoma cell line MCF-7. They recorded IC50 (µM) values of 24.1 ± 4.2 and 32.4 ± 3.8 for PA towards HT29 and MCF-7 respectively, after treating the cells with different concentration of the test compounds over 72hr period. Also, Wu X-D99 et al have reported the moderate cytotoxic activity of PA towards HL 60, SMMC-7721, A-549, MCF-7 and SW480 cell lines using the MTT method. PA has also shown apoptotic and anti-proliferative properties against leukemia cell line HL-60, human gastric adenocarcinoma MK-1, human uterine carcinoma HeLa and murine melanoma B16F10 cells100,101. Kuang102 and co-workers have investigated the in vitro α-glucosidase inhibitory effect of PA among other triterpenoids isolated from the roots of Sanguisorba tenuifolia var. Alba. From their results PA demonstrated moderate α-glucosidase inhibitory effect with IC50 (mM ± SEM, mM) value of 1.84 ± 0.12. They therefore concluded that these ‘triterpenoids of the EtOAc-soluble fraction may be the potential anti-hypoglycemic agents in the plant as they have been shown to induce an anti-diabetic effect’. Among nine triterpene acids isolated from the ethanol extract of the leaves of Perilla frutescens, Banno103 et al investigated the anti-inflammatory effect of PA (in addition to anti-tumor promoting effects). After evaluating the anti-inflammatory effect of PA with respect to 12-O- tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1µg/ear) in mice, they observed an inhibition of inflammation value of ID50 = 0.12mg/ear. Also Estrada104 and co-workers have investigated the hypotensive and anti-platelets properties of pomolic acid on rats. University of Ghana http://ugspace.ug.edu.gh 66 To my best of knowledge, this is the second time pomolic acid is being reported to have been isolated from the Dichapetalaceae even though the compound has been isolated from different plants families over the past years. 3.3.6 Purification and identification of dichapetalin M, DCS-E7 The solid DCS-E7 was isolated as a white amorphous solid from sub-fraction DCS-EAA4. The melting point recorded for DCS-E7 is 282-284oC after recrystallization from a mixture of diethyl ether/chloroform (10/3). DCS-E7 stained violet on TLC when sprayed with either anisaldehyde or vanillin spray reagent in the following mobile phase compositions: petroleum ether/ethyl acetate (1:1), Rf = 0.74; petroleum ether/acetone/chloroform (7:5:5), Rf = 0.76; diethyl ether/acetone (7:3), Rf = 0.78; petroleum ether/acetone (10:8), Rf = 0.82. Comparable Rf values were obtained for DCS- E7 and a reference sample, dichapetalin M12 in the above solvent systems. The melting point of the reference sample was 280-282oC, comparable to that of DCS-E7. The IR data for both compounds (shown in Table 3.7) were also comparable especially in the fingerprint region except for the presence of a broad peak at 1618.38cm-1 of small intensity in the IR spectrum obtained for DCS-E7. The identity of DCS-E7 was further supported by comparing the 1H and 13C-NMR data with that of the reference sample. TABLE 3.7: Infrared data of DCS-E7 compared to that of a reference, dichapetalin M. IR (KBr) vmax cm-1 DCS-E7 Peak intensity IR (KBr) vmax cm-1 Dichapetalin M12 Peak intensity Interpretation 2976.95, 2943.43, 2882.64 Strong 2919, 2853 Strong C-H stretching vibration of alkanes 1770.96, 1750.12, 1699.79 Strong 1772, 1751, 1701 Strong C=O stretch of lactone, ester and α- hydroxy ketone 1368 Strong 1367 Strong C-H bend for alkanes University of Ghana http://ugspace.ug.edu.gh 67 1246 Strong 1244 Strong O-H bending vibration of alcohol 1098.27, 1039.35 Strong 1096.4, 1039.4 Strong C-O-C bending vibration of ether 694.96 Variable 691.1 Variable -C6H5 bending vibration The 13C-NMR (and 1H NMR, Table 3.8) spectrum for dichapetalin M showed a total of 38 signals from δC 14.4 to 213.7. The α-hydroxy ketone at C-7 (with a corresponding IR band at 1699.79cm- 1) resonated at a frequency of 213.7 and the signal at δC 174 was assigned to the carbonyl carbon, C-21, of the lactone ring. The following signals were recorded for the carbons of the benzene ring; δC 142.5 (C-1’’), 128.4 (2’’/6’’), 125.8 (3’’/5’’) and 127.4 (4’’). The alkenic carbons were assigned the following signals; 116.3 (C-2), 141.3 (C-3), 120.1 (C-11) and 131.6 (C-12). O O O O OH O CH 3 H CH 3 H CH 3 OCOCH 3 OHH H CH 3 Dichapetalin M [31] 25 23 21 11 12 17 30 19 18 28 7 1'' 6' 3'' 4'' 5'' 6'' 2'' 24 6 53 2 1 13 14 16 22 5' 2' 9 20 26 27 Seven sp3-hybridized carbons occurred downfield due to the deshielding effect of the oxygen atom attached to them. They are; δC 111.3 (C-23), 85.5 (C-25), 81.3 (C-6’), 78.5 (C-26), 72.1 (C-22) and 71.3 (C-6/2’). The spirocyclic ring carbon, C-23, has two deshielding oxygen atoms attached University of Ghana http://ugspace.ug.edu.gh 68 to it and the downfield chemical shift observed for C-25 is as a result of the electronegative acetoxy group attached to it. The downfield chemical shift observed for C-6’ is due to the attached benzene ring and the oxygen atom. The remaining twenty signals between δC 14.4-59.1 are for methyl, methylene and methane carbons. The following chemical shifts were assigned to the methyl carbons; δC 17.1 (C-18), 18 .2 (C-19), 22.0 (C-27), 26.7 (C-28) and 21.8 (AcCH3). All the remaining signals agreed well with literature data and the recorded melting point of 282-284oC for DCS-E7 further supports its identity as dichapetalin M. Table 3.8: Comparison of 1H and 13C NMR data of DCS-E7 (dichapetalin M) with literature data 13C No. DCS-E7 (CDCl3) δC/ppm δH/ ppm Dichapetalin M (CDCl3)12,13 δC/ ppm δH/ ppm 1 40.1 2.2a, 1.65a, d, 3.1Hz 40.0 2.19a, 1.64a 2 116.6 5.39, d, J= 4.8Hz 116.6 5.38, d, J = 7.0Hz 3 141.3 - 141.3 - 4 39.9 - 39.9 - 5 59.1 1.65a 59.1 1.64a 6 71.3 4.60, dd, J1= 10.4Hz, J2= 3.6Hz, 3.81(OH, d, J = 4.2Hz) 71.3 4.59,dd, J1= 13.0Hz, J2=4.5Hz, 3.79 (OH, d, J= 4.5) 7 213.7 - 213.7 - 8 47.1 - 47.0 - 9 52.1 - 52.1 1.97a 10 36.9 - 36.9 - 11 120.1 5.41 120.1 5.41, dd, J1= 10, J2= 2.5Hz 12 131.6 6.33, dd, J1= 8.3Hz, J2= 4.3Hz 131.6 6.33, dd, J1= 10, J2= 3Hz 13 31.6 - 31.6 - 14 36.5 - 36.5 - 15 26.7 1.99a, 2.2a 26.7 1.99a, 2.19a 16 23.6 1.78, m 23.7 1.32, 1.76, m 17 40.5 2.55, m 40.5 2.55, m 18 17.1 1.23, s 17.1 1.21, s 19 18.2 1.35, s 18.2 1.34, s 20 47.3 2.96, dd, J1= 8.3Hz, J2= 4.1Hz 47.3 2.96, dd, J1= 10, J2= 5.5Hz 21 174.0 - 174.0 - 22 72.1 4.18, d, 8.3Hz, 2.46 (OH, d, 8.3Hz) 72.1 4.18, dd, J1= 10Hz, J2 = 10Hz, 2.45 (OH, d, J = 10.0Hz) University of Ghana http://ugspace.ug.edu.gh 69 23 111.3 - 111.3 - 24 45.7 2.86, 2.52, d, J= 16.7Hz 45.7 2.86, 2.50, d, J = 15.0Hz 25 85.1 - 85.1 - 26 78.5 4.32, d, J= 8.3Hz, 3.98, d, J=8.3Hz 78.5 4.33, d, J = 10Hz, 4.09, d, J = 10Hz 27 22.0 1.70, s 22.0 1.70, s 28 26.7 1.54, s 26.6 1.53, s 30 14.4 1.28, d, J = 8.3Hz, 1.02, d, J=8.3Hz 14.4 1.28, d, J =6.0Hz, 1.02,d, J=6.0Hz 2’ 71.3 4.32, 3.92, d, 8.3Hz 71.3 4.32, 3.92, d, J = 10.0Hz 5’ 40.1 2.70, t, J= 11.4Hz, 2.25,dd, J1 = 11.4Hz, J2 = 2.1Hz 40.1 2.69,tbr, J = 13.5Hz, 2.25,dd, J1= 13.5, J2 = 2.5Hz 6’ 81.3 4.36a 81.3 4.34, dd, J1 = 11.5, J2 = 2.5Hz 1” 142.5 - 142.5 - 2’’/6’’ 128.4 7.41, d, J=3.6Hz 128.4 7.40, d, J = 7.5Hz 3’’/5’’ 125.8 7.35, t, J= 3.6Hz 125.8 7.35, t, J = 7.5Hz 4’’ 127.4 7.28, t, J= 3.6Hz 127.5 7.28, t, J = 7.5Hz AcCO 170.4 - 170.4 - AcCH3 21.8 2.06,s 21.8 2.08, s a Overlapping signals Dichapetalin M was first isolated in 2008 from the roots of D. Madagascariense13 and roots of D. eickii16 in 2013. In a brine shrimp bioassay its measured cytotoxicity of LC50 = 0.011µg/ml was approximately 200-fold more potent than podophyllotoxin45. Long16 et al have revealed dichapetalin M as the most potent cytotoxic member of the dichapetalins, recording cytotoxic activities of EC50 = 0.007µg/ml on HCT116 and EC50 = 0.05µg/ml on WM 266-4 cell lines respectively. 3.3.7 Purification and identification of Maslinic Acid, DCS-E8 The solid DCS-E8, white and powdery in appearance with a melting point of 256-258oC was isolated from sub-fraction DCS-EAA4 and was recrystallized from a mixture of chloroform/acetone (2:5). DCS-E8 stained purple with anisaldehyde spray reagent on TLC run in the following solvent systems: petroleum ether/ethyl acetate (1:1), Rf = 0.43; petroleum ether/acetone/chloroform (7:3:3), Rf = 0.54; diethyl ether/acetone (7:3), Rf = 0.78; petroleum University of Ghana http://ugspace.ug.edu.gh 70 ether/acetone (10:8), Rf = 0.78. The IR data recorded for DCS-E8 showed peaks at 3443.97 cm-1 (O-H stretching vibrations), 2941.14 cm-1 (C-H stretching vibration), 1692.67 cm-1(C=O stretching vibration, 1461.82 and 1385.04 cm-1 (C-H asymmetric bending vibration), 1270.26 cm- 1 (C-O-H bending vibration) and 1051.37cm-1 (C-O stretching vibration) . The 13C NMR spectrum obtained for DCS-E8 (Table 3.9) confirmed the presence of 30 carbons consisting of three sp2 hybridized-carbons (two of the alkene type and one C=O) and two sp3- hybridized carbons attached to hydroxyl groups. The other twenty-five carbons are six sp3- hybridized quaternary, seven methyl, nine methylene and three methine carbon signals. The signal at δC 180.8 was assigned to the carboxyl group at C-28 (Maslinic acid [91]) which is further supported by the strong C=O stretching vibration band (1692.67 cm-1) recorded in the IR spectrum of DCS-E8 for carboxylic acid. The signals at δC 122.2 and 144.1 were assigned to C-12 and C-13 respectively as these downfield shifts are expected for sp2-hybridized carbons of the alkene type, while the values at δC 68.8 and 83.7 were assigned to C-2 and C-3 respectively. Even though C-2 and C-3 are sp3-hybridized, the observed downfield shift is due to their attachment to the electronegative hydroxyl groups. The following chemical shifts were assigned to the methyl carbons; δC 28.7 (C-23), 16.6 (C-24), 16.8 (C-25), 17.0 (C-26), (C-27), 32.7 (C-29) and 23.6 (C-30). The signal at δC 46.5 was assigned to C-17 (downfield due to the electronegative COOH group) and the signal at δC 49.0 was assigned to C-9, which is downfield compared to the signal at δC 41.3 (C-18) supposedly due to the electronegative hydroxyl groups on the ring adjacent to C-9. From the 1H NMR spectrum recorded for DCS-E8 (in CDOD/CDCl3), the signal at δH 5.26 (1H) was assigned to H-12 on the sp2-hybridized carbon, C-12. The signal at δH 2.83(1H, dd, J1 = 6Hz, University of Ghana http://ugspace.ug.edu.gh 71 J2 = 13Hz) was assigned to H-18, the signal at δH 3.66 (1H, td) was assigned to H-2. The observed multiplicity is due to first coupling of the signal to the methylene protons at H-1, then each signal of the resulting triplet split further into a doublet by the methine proton at H-3. The downfield chemical shift of 3.66 (for a proton bonded to sp3-hybridized carbon) indicates the presence of electronegative bonded groups. The signal at δH 3.45 was assigned to H-3. The signal at δH 1.95 (2H, ddd, J1 = 11.6Hz, J2 = 9.6Hz, J3 = 9.6 Hz) was assigned to H-16 and the signal at δH 1.90 (1H, dd, J1 = 4.2, J2 = 7.8) was assigned to H-9 while the signal at δH 1.61 (2H, dd, J1= 11.3, J2 = 11.3 Hz) was assigned to H-11. Finally the following chemical shifts were assigned to the methyl protons; δH 1.14 (3H, s, H- 27), 1.02 (3H, s, H-23), 0.98 (3H, s, H-25), 0.93 (3H, s, H-30), 0.90 (3H, s, H-29), 0.81 (3H, s, H-26) and 0.78 (3H, s, H-24). O OH CH 3 CH 3 OH CH 3 CH 3 CH 3 OH CH 3 CH 3 28 24 23 25 26 6 27 15 3 2 18 21 22 16 7 Maslinic Acid [91] 20 29 30 12 13 9 The figure (Figure 12) below presents part of the expanded 1H NMR spectrum recorded for DCS-E8. University of Ghana http://ugspace.ug.edu.gh 72 Figure 12: Expanded 1H NMR of DCS-E8 University of Ghana http://ugspace.ug.edu.gh 73 Table 3.9: 1H and 13C Chemical Shifts of DCS-E8 (Maslinic acid) and reference data. 13C No. DCS-E8 ( in CD3OD/CDCl3) δC/ppm δH/ppm Literature data of Maslinic Acid ( in CD3OD)105 δC/ppm δH/ppm 1 47.7 48.2 - 2 68.8 3.66 (1H, ddd, J1= 10.3, J2= 8.5, J3= 6.5 Hz) 69.5 3.62 (1H, ddd, J1 = 11.3, J2 = 9.6, J3 = 4.5) 3 83.7 3.45(1H, d, 10.3Hz) 84.4 2.91(1H, d, J = 9.6) 4 39.3 - 40.5 - 5 55.4 - 56.7 - 6 18.5 -- 19.6 - 7 32.7 - 33.9 - 8 39.4 - 40.6 - 9 49.0 1.90 (1H, dd, J1=4.2, J2= 7.8Hz) 49.3 1.70 ( 1H, dd, J1= 14.0, J2 = 14.0Hz) 10 38.3 - 39.3 - 11 23.6 1.61 (2H, dd, J1 = 11.3, J2= 11.3Hz) 24.7 - 12 122.2 5.26 123.5 5.26 (1H, dd, J1 = 3.5, J2 = 3.5) 13 144.1 - 145.4 - 14 42.0 - 42.8 - 15 27.8 - 28.8 - 16 23.1 1.95(2H, ddd, J1=11.6, J2=9.6,J3= 9.6 Hz 24.0 2.02 (2H, ddd, J1 = 13.3, J2 = 13.3, J3 = 3.9) 17 46.5 - 47.7 - 18 41.3 2.83 (1H, dd, J1= 7.8, J2=8.3 42.8 2.86 (1H, dd, J1 = 14.1, J2 = 3.9) 19 46.3 - 47.3 - 20 30.8 - 31.7 - 21 34.0 - 35.0 - 22 33.2 - 33.9 - 23 28.7 1.02 (3H, s) 29.3 1.02 (3H, s) 24 16.6 0.78 (3H, s) 17.1 0.81 (3H, s) 25 16.8 0.98 (0.98) 17.5 1.01 (3H, s) 26 17.0 0.81 (3H, s) 17.8 0.82 (3H, s) 27 26.0 - 26.4 - 28 180.8 - 181.9 - 29 32.7 0.90 (3H, s) 33.6 0.91 (3H, s) 30 23.6 0.93 (3H, s) 24.1 0.95 (3H, s) University of Ghana http://ugspace.ug.edu.gh 74 Maslinic acid (also called crategolic acid) or (2α, 3β)-2, 3-dihydroxyolean-12-en-28-oic acid is widely distributed in natural plant sources. A number of researchers have assessed the biological activities of maslinic acid using different experimental modules and these have proved that maslinic acid exerts anti-tumor, anti-diabetic, anti-oxidant, cardioprotective, anti-HIV, neuroprotective, anti-parasitic and growth-stimulating effects106. Rufino-Palomares107 and co-workers have investigated the mechanism of maslinic acid with respect to its inhibitory effects on the growth of HT29 colon-cancer cells. Their result indicated that ‘treatment with maslinic acid resulted in a significant inhibition of cell proliferation in a dose- dependent manner and causes apoptotic death in colon-cancer cells. They found out that maslinic acid inhibits considerably the expression of Bcl-2 whilst increasing that of Bax; it also stimulates the release of mitochondrial cytochrome-c and activates caspase-9 and caspase-3’. According to them, ‘all these results points clearly to the activation of the mitochondrial apoptotic pathway in response to the treatment of HT29 colon-cancer cells with maslinic acid’. Lozano-Mena106 et al have recently comprehensively reviewed several researches relating to the biological importance of maslinic acid and ‘their results provide evidence of the potential of maslinic acid as a nutraceutical, not only for health promotion, but also as a therapeutic adjuvant in the treatment of several disorders’. Even though maslinic acid has been isolated from several other plants families, this current investigation reports for the first time, the isolation of this pentacyclic triterpene from the family of Dichapetalaceae. Even though the solid DCS-EM was isolated from this sub-fraction (DCS-EAA4), it was not pure and several TLCs run alongside the solids DCS-E6, DCS-E7 and DCS-E8 in the different solvent University of Ghana http://ugspace.ug.edu.gh 75 systems indicated that DCS-EM is a mixture of pomolic acid, dichapetalins M and maslinic acid. The melting point was determined to be 253-265oC. No attempt was made to purify it after a few trials proved futile. 3.3.8 Purification and isolation of DCS-E9, DCS-E10 and DCS-E11 Sub-fraction DCS-EAA5 was partitioned into four fractions; F018-F025, F028-F033, F036-F041 and F043-F048. The solid DCS-E9 was obtained from fraction F028-F033 after washing with diethyl ether. The solid was filtered off, dried and melting point measured to be 116-119oC. DCS- E9 stained pink with anisaldehyde on TLC run in petroleum ether/ethyl acetate (1:1), Rf = 0.16; petroleum ether/acetone/chloroform (7:5:5), Rf = 0.17; petroleum ether/acetone (10:8), Rf = 0.32; petroleum ether/ethanol (10:2), Rf = 0.45. The IR data obtained for DCS-E9 showed significant peaks at 3433.97cm-1 (strong, broad) for O-H stretching vibration of alcohol , 2918.33, 2849.26cm- 1 (strong, sharp) for C-H stretching vibration of alkanes , 1622.62cm-1 (strong) for C=O stretching vibration of ester and 1467.12 and 1384.51 cm-1(CH2, CH3 bending vibration of alkanes) and 1121.71cm-1 for CO-O bending vibrations. Initial 13C-NMR obtained for DCS-E9 predicted it to be a fatty acid, but due to the impure nature and paucity of the material, no further data was obtained to fully identify DCS-E9. The solid DCS-E10 (5mg, 4 spots) was obtained from the fraction F036-F041 via triturating with ethyl acetate. The solid stained with iodine but not with anisaldehyde spray reagent and it was whitish and powdery in appearance. The melting point was determined to be 124-137oC. No other data was obtained for this solid due to its impure nature and paucity of material. After eluting DCS-EAA6 with 50% ethanol solution, the resulting fractions were colloidal in nature making separation of solute impossible. Upon the addition of small amount of 0.1M HCl, University of Ghana http://ugspace.ug.edu.gh 76 the solute coagulated. The solute was filtered, dried to afford DCS-E11; brown in appearance and soluble only in chloroform/ethanol mixtures with a melting point of 175-178oC. On TLC in solvent systems with polarities close to 100% ethanol, it appeared as a streak and stained yellow to brown with anisaldehyde spray reagent. The IR data recorded revealed strong and broad peaks at 3390.53cm-1 for O-H stretch, 2920.13, 2850.01cm-1 for C-H stretch, 1653.30cm-1 for C=O stretch, and peak at 1515.18cm-1 for C=C aromatic. No further data was obtained on DCS-E11 due to its impure nature. 3.4 In Vitro Screening of the crude extracts, Friedelan-3-one, a mixture of β-Sitosterol and Stigmasterol, and dichapetalin M For Anti-Schistosomal Activity. Schistosomiasis is a neglected tropical parasitic disease caused by trematode parasites of the genus Schistosoma and affects several millions of people worldwide. Due to its widespread usage over the past four decades, there has been reported incidence of resistance of field and laboratory strains of Schistosoma against praziquantel, the only drug of choice for schistosomiasis treatment. Hence, the search for new and safe anti-schistosomal agents has intensified more recently. In the light of recent investigations suggesting that the dichapetalins have broad spectrum of biological activities18 coupled with the urgent need of potential anti-schistosomal agents, the crude extracts (petroleum ether, ethyl acetate and methanol extracts) as well as three isolates; friedelan-3-one, mixture of β-sitosterol and stigmasterol, and dichapetalin M from the stem of D. crassifolium were screened for their anti-schistosomal activities. The six samples with unknown anti-schistosomal activities were tested against field isolates of human Schistosoma eggs to ascertain their ovicidal properties. Thus, the in vitro anti-schistosomal activities of the test samples were carried out using the ovicidal method (Egg Hatch Assay, EHA) University of Ghana http://ugspace.ug.edu.gh 77 with praziquantel as the standard drug. Field isolates of human Schistosoma eggs were obtained after extracting them from urine samples collected from 120 participants at Tomefa, a rural community in the Ga West District around the Weija dam in the Greater-Accra Region of Ghana. Over 80% of the participants were school children aged between 8 and 14years. The modified Kotze108 et al technique was employed to identify, purify and extract the eggs from 4L of urine sample at the Noguchi Memorial Institute of Medical Research. The in vitro anti-schistosomal activity test was conducted in 96 well-plates. EHAs were set up by plating the purified egg suspension (50µl containing approximately 25 eggs) in a 96-well plate. Stock solutions were prepared (2mg/ml for each isolate and 5mg/ml for each crude extract) in DMSO for each test sample and by serial dilution (dilution factor = 0.5), different concentrations were prepared to the 7th well for each test sample. To each well already containing the egg solution, 50µl of test sample solution was added. The tests were conducted in duplicates. Water and praziquantel (2mg/ml stock solution) were used as negative and positive controls respectively. The plates were incubated at room temperature for 24 hrs after which eggs and unhatched eggs were counted using inverted light microscope and the percent egg hatch 100µl of formalin was added to each well to stop the hatching process. The number of hatched (larvae) inhibition calculated from the relation: % egg hatch inhibition (%EHI) = # unhatched unhatched + # larvae x 100 The %EHI was plotted against log of concentration. Extrapolation of the 50% EHI on the curve gave the half maximal inhibitory concentration (IC50) of the test samples. The tables below (Table 3.10 A and B) present the %EHI of the test samples at the different concentrations. University of Ghana http://ugspace.ug.edu.gh 78 Table 3.10A: %EHI recorded for the duplicate test at different concentrations of extracts, pure compounds and standard, praziquantel. % EHI Extracts Concentration µg/ml Petroleum Ether Ethyl Acetate Methanol 5000 83.3 87.5 92.8 100 100 100 2500 87.5 75 100 100 100 100 1250 66.7 83.3 78.6 87.5 66.7 66.7 625 66.7 75 83.3 66.7 53.8 47.6 312.5 23.0 25.0 33.3 54.5 14.3 33.3 156.25 40.0 36.4 44.4 50.0 16.7 0 78.125 14.3 14.3 50.0 41.7 16.7 25 Pure Compounds Concentration µg/ml Friedelan-3-one Stigmasterol/Sito sterol Dichapetalin M 2000 100 100 75 100 66.7 66.7 1000 80 80 72.7 72.7 75 66.7 500 66.7 50 83.3 66.7 50 50 250 50 66.7 66.7 75 54.5 47.6 125 16.7 0 44.0 0 38.5 0 62.5 0 25 25 25 14.3 0 31.25 0 14.3 0 11.1 0 16.7 Praziquantel Concentration µg/ml 2000 100 100 1000 100 100 500 100 100 250 100 83.3 125 83.3 83.3 62.25 90 77.8 31.125 76.9 75.0 University of Ghana http://ugspace.ug.edu.gh 79 Table 3.10B: In vitro average % EHI values at different concentrations of extracts, pure compounds and praziquantel. Data are mean ± SEM. Mean % EHI Extracts Concentration µg/ml Petroleum Ether Ethyl Acetate Methanol 5000 85.4 ± 2.1 96.4 ± 5.4 100 ± 0.0 2500 81.3 ± 6.3 100 ± 0.0 100 ± 0.0 1250 75.0 ± 5.8 83.1 ± 4.5 66.7 ±0.0 625 70.6 ± 3.9 75.0 ± 8.3 50.7 ± 2.9 312.5 24.0 ± 1.0 43.9 ± 10.6 23.8 ± 9.5 156.25 38.2 ± 1.7 47.2 ± 2.8 8.4 ± 4.0 78.125 14.3 ± 0.0 45.9 ± 6.2 20.9 ± 3.7 Pure Compounds Concentration µg/ml Friedelan-3-one Stigmasterol/Sitosterol Dichapetalin M 2000 100 ± 0.0 87.5 ± 12.5 66.7 ± 0.0 1000 80.0 ± 0.0 72.7 ± 0.0 70.9 ± 4.2 500 58.4 ± 8.4 75.0 ± 8.3 50.0 ± 0.0 250 58.4 ± 8.4 70.9 ± 4.2 51.1 ± 3.4 125 8.4 ± 4.2 22.2 ± 11.1 19.3 ± 9.7 62.5 12.5 ± 6.3 25.0 ± 0.0 7.2 ± 3.6 31.25 7.20 ± 3.6 5.6 ± 2.8 8.4 ± 4.2 Praziquantel Concentration µg/ml 2000 1000 100 ± 0.0 500 100 ± 0.0 250 91.7 ± 8.4 125.25 83.3 ± 0.0 62.5 83.9 ± 6.1 31.25 76.0 ± 1.0 University of Ghana http://ugspace.ug.edu.gh 80 Generally, the %EHI increased steadily with increasing concentration of test sample, thus the %EHI was concentration-dependent. At a concentration of 1250 µg/ml, all the crude extracts exhibited %EHI ˃ 50% with the ethyl acetate extract showing the highest %EHI of 83.3. The observed %EHI for the ethyl acetate and methanol extract were the same (100%) at a concentration of 2500µg/ml. The ethyl acetate extract demonstrated higher inhibition activity between the concentrations of 78.125-312.5µg/ml compared to the methanol extract. The petroleum ether extract showed %EHI of 85.4 at a concentration of 5000µg/ml and after two serial dilutions to a concentration of 1250µg/ml, it recorded %EHI of 75.0, higher than that of the methanol extract at the same concentration. Among the isolates, friedelan-3-one showed %EHI of 100% at a concentration of 2000µg/ml, comparable to that of the standard, praziquantel, at the same concentration. At this same concentration (2000µg/ml), the %EHI recorded for β-sitosterol/stigmasterol was 87.5, whiles dichapetalin M exhibited %EHI of 66.7. All the isolates showed %EHI ˃ 70% at a concentration of 1000µg/ml. The observed %EHI for friedelan-3-one at 1000 µg/ml was 80%, then after dilution to 500 µg/ml, the %EHI was 58%. Praziquantel exhibited the same %EHI of 100% at concentrations of 1000 and 500 µg/ml. At a concentration of 250µg/ml, the %EHI of β- sitosterol/stigmasterol was 70.9 whiles that of friedelan-3-one was 58.4, that of dichapetalin M was 51.1 and that observed for praziquantel was 91.7. Furthermore, between concentrations of 125-62.5µg/ml, the β-sitosterol/stigmasterol mixture was more potent than friedelan-3-one and dichapetalin M. The standard, praziquantel, was still significantly active at lower concentrations compared to the isolates and crude extracts. The calculated IC50 values for the test samples are presented in Table 3.10C below. University of Ghana http://ugspace.ug.edu.gh 81 Figure 13: Concentration-dependent effect of Petroleum ether, Ethyl acetate and Methanol extracts of the stem of D.crassifolium on the egg hatching of Schistosoma species (S. mansoni and S. haematobium) Each point is mean ± S.E.M (n=2). Figure 14: Concentration-dependent effect of isolates; friedelan-3-one, stigmasterol/sitosterol and dichapetalin M from the stem of D.crassifolium and standard, praziquantel, on the egg hatching of S. mansoni and S. haematobium species. Each point is mean ± S.E.M (n=2). University of Ghana http://ugspace.ug.edu.gh 82 Table 3.1C: In vitro half maximal inhibitory concentration (IC50) of the test samples Test Sample IC50 (µg/ml) ± S.E.M Crude Extracts 1. Petroleum Ether 443.70 ± 0.04 2. Ethyl Acetate 638.00 ± 0.08 3. Methanol 893.70 ± 0.08 Isolates 4. Friedelan-3-one 378.10 ± 0.23 5. β-sitosterol/stigmasterol 177.90 ± 0.10 6. Dichapetalin M 191.00 ± 0.12 Standard 7. Praziquantel 15.47 ± 0.06 Analysis from the GraphPad Prism graphs above (Figures 13 - 14) of the half maximal inhibitory concentration IC50 of the isolates gave the most effective inhibitory agent to be the mixture of β- sitosterol/stigmasterol, with an IC50 value of 177.9 ± 0.10 µg/ml followed by dichapetalin M with an IC50 value of 191 ± 0.12 µg/ml. Friedelan-3-one recorded an IC50 = 378.1 ± 0.23 µg/ml. The calculated IC50 for the standard praziquantel was 15.47 ± 0.06 µg/ml. Hence, the results show that praziquantel still remains a potent anti-schistosomal agent. The crude extracts; petroleum ether, ethyl acetate and methanol showed IC50 values of 443.7 ± 0.04, 638.0 ± 0.08 and 893.7 ± 0.08 µg/ml respectively. Thus, the petroleum ether crude extract exhibited the most effective in vitro ovicidal activity in the study among the crude extracts. Even though the ethyl acetate extract produced the compounds that exhibited high EHI activity - β-sitosterol/stigmasterol and dichapetalin M, it was not as active as the petroleum ether extract, University of Ghana http://ugspace.ug.edu.gh 83 which yielded only the less active friedelins. Possibly, the presence of polar constituents such as tannins in the ethyl acetate fraction as indicated in the phytochemical screening did not enhance its ovicidal activity. Also there may be poor synergistic effect among the constituents of the ethyl acetate extract, including the mixture of β-sitosterol and stigmasterol, and dichapetalin M. The methanol extract which contains mainly tannins showed the poorest activity suggesting that tannins may not influence the observed egg hatching activity. Previous investigations have confirmed the anti-schistosomal potencies of other terpenoids like phytol, where a reduction in egg-laying was observed at 25 µg/ml in S. mansoni worms69, also nerolidol71, where 100% mortality was observed in male and female adult worms at concentrations of 31.2 and 62.5µM respectively, and the effect of (+)-limonene epoxide on S. mansoni adult worms at a concentration of 25 µg/ml was similar to that of praziquantel, with reduction and mortality of all worms after 120hrs109. 3.5 CONCLUSION In the present study, the stem of D. crassifolium was investigated. The petroleum ether extract yielded friedelan-3-one, friedelan-3β-ol and a mixture of friedelan-3-one and friedelan-3β-ol. The ethyl acetate extract yielded pomolic acid, dichapetalin M and maslinic acid as well as the friedelins and a mixture of β-sitosterol and stigmasterol. No solid was isolated from the methanol extract. These compounds were identified and characterized using comparative thin-layer chromatography, comparative melting point, mixed melting point, IR, 1H and 13C NMR spectroscopy. The crude extracts as well as some of the purified isolates were screened for their potential Schistosoma species (S. mansoni and S. haematobium) egg hatch inhibition activities. The University of Ghana http://ugspace.ug.edu.gh 84 petroleum ether, ethyl acetate and methanol crude extracts gave IC50 values of 443.7 ± 0.04, 638.0 ± 0.08 and 893.7 ± 0.08 µg/ml respectively. Also among the tested isolates, friedelan-3-one, β- sitosterol/stigmasterol and dichapetalin M gave IC50 values of 378.1 ± 0.23, 177.9 ± 0.12 and 191.0 ± 0.12 µg/ml respectively. Hence among the crude extracts, the petroleum ether extract exhibited the most active egg hatch inhibition activity and also for the tested isolates, the mixture of β- sitosterol and stigmasterol was most active. The observed egg hatch inhibition activity of the mixture of β-sitosterol/stigmasterol and dichapetalin M were about 11 and 12 fold respectively less active compared to that of the standard, praziquantel (IC50 = 15.47 ± 0.06 µg/ml), used in the study. This study constitutes the first report of the chemical and biological investigation of the stem of Dichapetalum crassifolium. Maslinic acid is also reported for the first time from the Dichapetalaceae. 3.6 RECOMMENDATIONS Further spectroscopy is recommended to elucidate the structures of the unknown solids; DCS-E5 and DCS-E9. The use of modern chromatographic methods must be employed such as reversed- phase and HPLC instead of the conventional normal phase liquid chromatography to isolate more of the compounds present in the plant in future investigations. This is very important since as evidenced from the TLC analysis the isolated compounds are not the only compounds present in the plant. A comprehensive bioassay-guided isolation procedure should be adopted in order to extend the phytochemical and antimicrobial investigations and to ascertain the active phytochemicals present. Also, synergic effects among the crude extracts and isolated compounds should be investigated University of Ghana http://ugspace.ug.edu.gh 85 and in future investigations, in vivo studies should be included to confirm the in vitro anti- schistosomal results. University of Ghana http://ugspace.ug.edu.gh 86 CHAPTER FOUR EXPERIMENTAL 4.1 Collection and Preparation of Plant Material The plant material was collected from Kuntanase, a town located in the Ashanti Region of Ghana and identified as of D. crassifolium at the National Herbarium, Department of Botany (Specimen code: DCR001), University of Ghana. The stem was chopped into smaller pieces and air/shade dried for four weeks after which the dried material was milled. 4.2 General Experimental Procedure Soxhlet extraction was used as the extraction method of the pulverized stem of the plant. The organic solvents used were petroleum ether (40-60oC), ethyl acetate and methanol. Extraction was done for 24 hours continues for each solvent. The extracts, after each extraction cycle, were concentrated and dried under reduced pressure on Buchi Rotary Vacuum Evaporator and the recovered solvent used for the next extraction. The main purification procedure used for the isolation of the phytochemicals of each extract was solid-liquid column chromatography using silica gel 60 (Fluka; particle size 0.063-0.2mm) as the stationary phase material. Employing gradient elution technique, chromatographing was started off with petroleum ether (40-60oC) and the polarity of the mobile phase increased steadily until that mobile phase system which elutes the most polar constituents in the extract. Thin layer chromatography (TLC) using aluminium foil slides pre-coated with silica gel (0.2mm thick, Kiesegel 60 F254 Merck type) was employed to monitor the progress of the column University of Ghana http://ugspace.ug.edu.gh 87 chromatographic separation and also to determine the purity of eluates. Where necessary, recrystallization with appropriate solvents was used for further purification of isolates. Visualization of spots on TLC plates was by Ultraviolet (UV) light, iodine vapor, vanillin and anisaldehyde spray reagent. Melting points of pure isolates were determined with Stuart Scientific Melting Point Apparatus. Isolated known compounds were characterized by their appearance and retardation factor (Rf) on comparative TLC in different solvent systems with very different polarities and co-melting points. 1H and 13C Nuclear Magnetic resonance (NMR) data were obtained on 500MHz NMR spectrometer at the University of Erlangen, Germany. IR data was obtained on FT IR spectrometer at the Food and Drugs Authority, Ghana and visualization of spots under UV lamp was done using UVGL-58 Handheld UV lamp (254-365nm). 4.3 Chemicals and Reagents Wagner’s reagent 2.0g of potassium iodide and 1.27g of iodine were together dissolved in a little distilled water in a volumetric flask and the solution made up to 100cm3 with distilled water. The presence of alkaloids is indicated by the formation of large brown amorphous and flocculent precipitate after treating the hydrochloric acid solutions of the extracts with a few drops of reagent. Meyer’s reagent 1.36g of mercuric iodide is dissolved in distilled 60cm3 water and the resulting solution added to the one obtained by dissolving 5g of potassium iodide in 10cm3 distilled water and the resulting solution made up to 100cm3 with distilled water. The formation of a pale yellow precipitate when a few drops of the reagent are added to the test solution is indicative of the presence of alkaloids. University of Ghana http://ugspace.ug.edu.gh 88 Dragendorff’s reagent 8g of hydrated bismuth nitrate is dissolved in 20cm3 concentrated nitric acid and the resulting solution is slowly added to a solution of 27.2g of potassium iodide in 50cm3 of distilled water with stirring. Crystalline potassium nitrate which precipitates is filtered off and the filtrate made to 100cm3 with distilled water before being used as a reagent for testing for alkaloids in the test. The solution of the extract is made distinctly acidic with sulphuric acid and completely freed of any ethanol in which the alkaloid precipitates are soluble. Two or three drops of the reagent are used since some of the alkaloid precipitates are soluble in excess reagent. Formation of brown precipitate is indication of the presence of alkaloids. Anisaldehyde spray reagent 1cm3 of concentrated sulphuric acid is added to 50cm3 of glacial acetic acid. To the mixture is added 0.5cm3 anisaldehyde. The reagent is always prepared fresh and used as spray. The sprayed TLC plates are heated to 110oC for 10 minutes before visualization in visible light. Most organic compounds will stain as blue, violet, green, pink, yellow, red, cream and grey on a light pink background. Liebermann-Buchard reagent 5cm3 of redistilled acetic anhydride is cautiously mixed while cooling with 5cm3 of concentrated sulphuric acid. The resulting mixture is again slowly added, also while cooling on ice to 50cm3 of absolute ethanol. As a positive indication for the presence of terpenoids in an extract, this reagent when sprayed onto a TLC plate will produce pink to red spots after development followed by heating in an oven at a temperature of 110oC for 15 minutes and then examination under UV lamp. University of Ghana http://ugspace.ug.edu.gh 89 Iron (III) chloride solution 100cm3 of iron (III) chloride solution is prepared by mixing 10g of iron (III) chloride and 90cm3 of distilled water. Two to three drops of the solution are added to 3cm3 of a test solution. The presence of catechol-type tannins is indicated by the formation of a greenish-blue coloration. 4.4 Solvent Extraction of the Plant Material 4.4.1 Extraction with Petroleum Ether (40-60oC) The pulverized stem of the plant (5.0kg), was extracted via Soxhlet extraction in batches. For each batch, 0.5g of plant material was extracted continuously for 24 hours using 3L of petroleum ether. After 2 successive extractions, the extract was filtered and the solvent was evaporated off on vacuum rotary evaporator to obtain the crude extract. A total of 34.0g of a dark-greenish syrup- like crude matter was obtained. The crude extract on TLC (Petroleum Ether/Ethyl acetate 10/1) indicated over 10 spots, some staining yellow, orange, pink, purple, blue and violet when sprayed with anisaldehyde reagent. Two spots were UV active, appearing as blue and bright green, these spots did not stain either with anisaldehyde or iodine. The anisaldehyde stain was used extensively throughout the petroleum ether crude extract investigation. 4.4.2 Extraction with Ethyl acetate After the petroleum ether extraction, the defatted plant material was air dried and the extraction repeated as described for the petroleum ether but this time ethyl acetate was the solvent used. The total weight of crude extract obtained after the Soxhlet extraction of 5.0kg of the plant material was 65.0g. TLC on the crude extract (Petroleum ether/ ethyl acetate 10/3) gave more than 10 spots, University of Ghana http://ugspace.ug.edu.gh 90 some staining yellow, pink, purple, blue and violet with anisaldehyde stain. Most of the spots observed for the petroleum extract were also observed for the ethyl acetate extract. 4.4.3 Methanol Extraction After the ethyl acetate extraction, the plant material was air dried and the extraction continued using methanol. A total of 2.5kg of plant material was extracted to give 23g of dark-reddish solid, very soluble in chloroform-ethanol mixture. TLC on the crude extract (Petroleum ether/ethyl acetate 10/6) gave several spots, some staining yellow, pink, purple and blue with anisaldehyde stain. Similar spots were also observed as ion the case of the ethyl acetate extract. 4.5 Phytochemical Screening Test Procedure The petroleum ether extract, ethyl acetate extract and methanol extract were screened separately for alkaloids, anthraquinones and anthracene derivatives, flavonoids and leuco-anthocyanins, cardiac glycosides, terpenoids, tannins, and saponins as follows: Test for alkaloids About 0.1g of each of the crude extracts in separate test tubes was added to 5cm3 of 2M HCl solution. This was stirred, warmed and filtered. The filtrate from each extract was then divided into three test tubes. To one portion of each test solution was added Dragendorff’s reagent; to another portion, Meyer’s reagent and to the remaining portion, Wagner’s reagent. The absence of a yellowish or reddish brown precipitate in the test tubes indicated the absence of alkaloids. Test for flavonoids and leucoanthocyanins. About 0.1g solid of each of the crude dried extract was added to 15cm3 of 80% ethanol. The resulting solution was filtered and the filtrate divided into two separate test tubes. To one portion University of Ghana http://ugspace.ug.edu.gh 91 was added magnesium turnings followed by 0.5cm3 concentrated hydrochloric acid and observed color changes within 10 minutes. 5cm3 of concentrated hydrochloric acid was added to the other portion and warmed for 5 minutes. The presence of light pink color in both extracts indicated the presence of flavonoids. Test for tannins About 0.5g of each extract was dissolved in 10cm3 of aqueous methanol and the solution divided into two portions. To one portion, was added freshly prepared iron (III) chloride solution and to the other portion nothing was added to serve as blank. The formation of a dark greenish-blue coloration on addition of iron (III) chloride indicates the presence of tannins. Test for saponins The presence of saponins in the extracts was indicated by the fact that there was foaming when some amounts of the extracts were each shaken with water. The foaming disappeared after the test tube had been set aside for about an hour. Salkowski’s test for cardiac glycosides About 0.5g of each extract was dissolved in 2cm3 of chloroform in a test tube. Concentrated sulphuric acid was carefully added down the side of the test tube to form a lower layer. Formation of a reddish brown color at interface indicates the presence of the glycone portion of cardiac glycosides. University of Ghana http://ugspace.ug.edu.gh 92 Test for anthraquinones and anthracene About 0.5g of each extract was dissolved in 30cm3 of distilled water and filtered. The filtrate was shaken up with 10cm3 benzene in a separating funnel and allowed to stand. The benzene layer was transferred into a test tube and shaken with 5cm3 of 2M ammonia solution. The absence of red color in the ammonia layer was indicative of the absence anthraquinones in the extract. 4.6 Investigation of the Extracts 4.6.1 Petroleum ether (40-60oC) Extract A total of 34g of crude extract was obtained after the Soxhlet extraction and concentration on the vacuum rotary evaporator. This was coded DCS-P and kept in the refrigerator. After few days, DCS-P was observed to have solid precipitate and liquid supernatant portions. The supernatant (mother-liquor, ML) was filtered off and TLC in Petroleum ether/ ethyl acetate, 14:0.5 was run on both the solid portion DCS-PA1 and the mother liquor, ML. After staining with anisaldehyde, the two portions revealed comparable spots and hence were put together (20g), dissolved in chloroform and mixed with silica gel (6g) and air dried. This was later put on a glass column pre- loaded with silica gel (230g). The column was first eluted with petroleum ether 100%, then with petroleum ether and ethyl acetate mixtures until 100% ethyl acetate. The column was finally washed with chloroform/ethanol 1:1mixture. The eluates were collected in test tubes, 10 ml each. TLC was run on the eluates to monitor the progress of the chromatographing and to combine comparable eluates. At 10% of ethyl acetate in petroleum ether, the fractions F021-F073 were combined based on their similar TLC profiles. This was coded DCS-PA2, which gave 4 spots on TLC (Petroleum ether/ethyl acetate: 14/0.5). The sub-fraction DCS-PA2 (5.6g), was again put on a smaller glass University of Ghana http://ugspace.ug.edu.gh 93 column pre-loaded with silica gel (80g) and eluted with petroleum ether/chloroform: 25/75 mixture. The eluates were collected in smaller test tubes in volumes of 5ml. The column fractions yielded the solids DCS-P1 (60mg, 1 spot), DCS-P2 (40mg, 1 spot) and a mixture of these two solids coded DCS-P1P2 (20mg, 2spots) after recrystallization from ethanol . The 100% ethyl acetate fraction and the fraction obtained after washing the column (chloroform/ absolute ethanol 1:1) were also found to be the same and hence were combined (coded DCS-PA3) and safely kept in the refrigerator. All other isolates were oily in nature. 4.6.2 Ethyl Acetate (EA) extract The ethyl acetate extract (40g) was loaded onto a chromatographic glass column pre-loaded with silica gel (470g) and eluted quickly with wide increase in polarity, starting from petroleum ether/ethyl acetate mixtures of 10:1, 10:3, 10:6, 1:1, 1:3, followed by 100% ethyl acetate and finally with absolute ethanol. In all 13 fractions were obtained initially, DCS-EA1 - DCS-EA13, but the similarity of some of the fractions on TLC resulted in the combination of DCS-EA1 and DCS-EA2 into DCS-EAA1, DCS-EA3 and DCS-EA4 into DCS-EAA2; DCS-EA5, DCS-EA6 and DCS-EA7 into DCS-EAA3; DCS-EA8, DCS-EA9 and DCS-EA10 into DCS-EAA4; DCS-EA11 and DCS-EA12 into DCS- EAA5, thus six fractions in all. The fraction DCS-EAA1 (4.8g) was loaded onto a column pre-loaded with silica gel (75g) and eluted with petroleum ether/chloroform 25/75 throughout. The column fractions yielded the solids DCS-E1 (18mg, 1 spot), DCS-E2 (15mg, 1 spot) and a mixture DCS-E1E2 (15mg, 2 spots) after recrystallization from absolute ethanol. The solids DCS-E1and DCS-E2 spotted yellow and purple respectively with anisaldehyde stain on TLC run in petroleum ether/ethyl acetate (14:0.5). The University of Ghana http://ugspace.ug.edu.gh 94 solid DCS-E1E2 spotted yellow and purple in the same solvent system on TLC with the same Rf values as the solids DCS-E1 and DCS-E2. The last fraction from the column, F071-F089, was safely kept in the refrigerator for future investigation. The fraction DCS-EAA2 (3.6g) was loaded on a glass column pre-packed with silica gel (75g) and eluted with petroleum ether/ ethyl acetate (10:1). The eluates were collected in volumes of 5ml and combined based on the TLC profiles. The column fractions (F010-F015) yielded the solid DCS-E3 (14mg, 2 spots) which indicated similar TLC profiles as DCS-P1P2 and DCS-E1E2 after running them in different solvent systems. The column fractions (F018-F024) yielded the solid DCS-E4 (25mg, 1 spot), this solid indicated a spot which stained purple with anisaldehyde spray on TLC in the solvent system; petroleum ether/ethyl acetate (10:4). The fraction DCS-EAA3 (4.2g) was loaded on a glass column pre-packed with silica gel (75g) and slowly eluted with petroleum ether/ethyl acetate 10:2 then finally with 30% ethyl acetate in petroleum ether. The column fraction yielded the solid DCS-E5 (45mg, 1 spot) which stained blue with anisaldehyde spray on TLC run in petroleum ether/ethyl acetate (10:6). The fraction DCS-EAA4 (7.4g) was loaded on a glass column pre-packed with silica gel (120g) and slowly eluted starting with petroleum ether/ethyl acetate 10:3 and finally with 40% ethyl acetate in petroleum ether. The solid DCS-E6 (55mg, 3 spots) precipitated from fractions F025- F040, were filtered and recrystallized from a mixture of diethyl ether/chloroform (10:3). From fractions F054-F071, the solid DCS-E7 (25mg, 1 spot) precipitated and was recrystallized from a mixture of chloroform/diethyl ether (2:5). DCS-E7 stained violet with anisaldehyde spray on TLC run in petroleum ether/ ethyl acetate (10:6). The fractions F075-F091 after concentrating on the vacuum rotary evaporator and recrystallization from 2/5 mixture of chloroform/diethyl ether University of Ghana http://ugspace.ug.edu.gh 95 afforded the solid DCS-E8 (32mg, 1 spot). DCS-E8 stained purple with anisaldehyde spray on TLC run in petroleum ether/ethyl acetate (10:6). The last fraction, F093-F114 was combined with sub-fraction DCS-EAA5. DCS-EAA5 (3.8g) was loaded on a glass column pre-packed with silica gel (80g) and eluted slowly with chloroform/ absolute ethanol 95/5 throughout. Based on TLC, four fractions; F018-F025, F028-F033, F036-F041 and F043-F048 were obtained. The solid DCS-E9 (10mg, 1 spot) was obtained from fraction F028-F033 after washing with diethyl ether three times. The solid was filtered off and dried. DCS-E9 stained pink with anisaldehyde on TLC run in petroleum ether/ethyl acetate (1:1) and petroleum ether/acetone/chloroform (7/5/5). When sprayed with anisaldehyde, a grey spot was visualized but was observed as pink when heated. No solid precipitated from fraction F043-F048, there appeared to be solid in fraction F018-F025 but all effort to get the solid even in the smallest amount proved futile. TLCs were run on this fraction in different solvent systems to establish the veracity of the presence of a solid in this fraction. The solid DCS-E10 (5mg, 2 spots) was obtained from the fraction F036-F041via triturating with ethyl acetate since it was very soluble in acetone. The solid stained with iodine but not anisaldehyde and it was dirty white and powdery in appearance. After eluting DCS-EAA6 with 50% ethanol solution, the resulting fractions were colloidal in nature making separation of solute impossible. Upon the addition of small amount of 0.1M HCl, the solute coagulated. The solute was filtered, dried to afford DCS-E11; brown in appearance and soluble only in chloroform/ethanol mixtures. On TLC, it appeared as a streak and stained yellow to brown with anisaldehyde spray reagent. University of Ghana http://ugspace.ug.edu.gh 96 4.7 In vitro screening of crude extracts and some isolates for anti-schistosomal activity 4.7.1 Schistosome Egg recovery and Concentration from infested urine samples by the modified Kotze et al108 Method. Materials and Reagents 1. Urine 2. 50ml Falcon tubes 3. 50ml of 0.9% Saline 4. 40ml of 0.015% Brij-35 5. 2 × 250ml beakers 6. 180, 150 and 80µm sieves ( Nonaka Rikaki Co. Ltd, Japan) 7. Centrifuge 8. Aspirator Field isolates of human Schistosoma eggs were obtained after extracting them from urine samples collected from 120 participants at Tomefa, a rural community in the Ga West District around the Weija dam in the Greater-Accra Region of Ghana. Ethical clearance for the anti-schistosomal test was obtained at the Noguchi Memorial Institute of Medical Research (Appendix III, pg. 128). Over 80% of the participants were school children aged between 8 and 14years. Urine samples that were positive for schistosome eggs were pooled into 50ml falcon tubes and centrifuged at 500g for 5 minutes. The supernatant was discarded and the deposits suspended in 50ml of 0.9% saline and centrifugation repeated. The sediment was suspended in 40 ml of 0.015% Brij-35 and shaken vigorously. It was centrifuged again at 500g for 5 minutes. The supernatant was discarded and the sediment re-suspended in saline into a 200ml beaker. More saline was added to the 150ml mark. University of Ghana http://ugspace.ug.edu.gh 97 The suspension was filtered through a stack of three sieves: 180 µm, 150 µm and 80 µm, respectively (Nonaka Rikaki Co. Ltd, Japan). The stack of sieves was thoroughly flushed with a jet of saline to wash the eggs. To prevent air lock, the 150 m and 80 m sieves were occasionally separated. At the end of washing, the 80 µm was removed and inclined at approximately 45o to the horizontal and a jet of saline applied to it to wash off the eggs into a beaker. The suspension was centrifuged at 200g for 10 minutes and left for 30 minutes after which the supernatant was aspirated down to 5ml and egg count was estimated in 20l of solution. 4.7.2 In vitro screening of test compounds against schistosome eggs using the 96-well plate- Egg Hatch Assay. From the 5ml concentrated schistosome egg suspension, three 30µml the suspension was pipetted unto a slide and observed under light microscope. The number of eggs per 30µml was noted and hence the average number of eggs per 30µml determined. From this result the approximate number of eggs in the 5ml concentrated egg solution was calculated. The test compounds; DCS-P1, DCS-E4, and DCS- E7 (2mg for each) and then 5mg each for the crude extracts (petroleum ether, ethyl acetate and methanol) were dissolved in 1ml of 5% DMSO to give 2mg/ml (for each test isolate) and 5mg/ml (for each crude extract) stock solutions. 100µl of the stock solution of each test compound was pipetted in duplicate into the first wells. By using serial dilution, 50µl dilute solutions of the test compounds were prepared in decreasing order of concentration to the seventh well. Then 50μl of egg solution was added to each well and the presence of eggs ascertained under the inverted microscope. Water and praziquantel (2mg/ml stock solution) were used as negative and positive controls respectively. University of Ghana http://ugspace.ug.edu.gh 98 Egg Hatch Assays was incubated for 24 hours at ambient temperature and the process of egg hatch was stopped by the addition of 100ml of formalin to each well. The number (#) of hatched eggs and unhatched eggs (larvae) was counted using inverted light microscopy. The percent hatch inhibition (% EHI) was calculated as: % egg hatch inhibition (%EHI) = # unhatched unhatched + # larvae x 100 The %EHI was plotted against log concentration. Extrapolation of the 50% EHI on the curve gave the half maximal inhibitory concentration (IC50) of the test sample. University of Ghana http://ugspace.ug.edu.gh 99 APPENDIX I: IR Data of Isolates Page IR spectrum of friedelan-3one, DCS-P1 100 IR spectrum of friedelan-3β-ol, DCS-P2 101 IR spectrum of friedelan-3-one, DCS-E1 102 IR spectrum of friedelan-3β-ol, DCS-E2 103 IR spectrum of mixture of friedelan-3-one and friedelan-3β-ol, CPC-S1 104 IR spectrum of mixture of stigmasterol and β-sitosterol, DCS-E4 105 IR spectrum of DCS-E5 106 IR spectrum of DCS-E6 107 IR spectrum of DCS-E7 108 IR spectrum of DCS-E8 109 IR spectrum of DCS-E9 110 IR spectrum of DCS-E11 111 University of Ghana http://ugspace.ug.edu.gh 100 IR s p ec tr u m o f fr ie d el an -3 o n e, D C S -P 1 University of Ghana http://ugspace.ug.edu.gh 101 IR s p ec tr u m o f fr ie d el an -3 β -o l, D C S -P 2 University of Ghana http://ugspace.ug.edu.gh 102 IR s p ec tr u m o f fr ie d el an -3 -o n e, D C S -E 1 University of Ghana http://ugspace.ug.edu.gh 103 IR s p ec tr u m o f fr ie d el an -3 β -o l, D C S -E 2 University of Ghana http://ugspace.ug.edu.gh 104 IR s p ec tr u m o f m ix tu re o f fr ie d el an -3 -o n e an d fr ie d el an -3 β -o l, C P C -S 1 University of Ghana http://ugspace.ug.edu.gh 105 IR s p ec tr u m o f m ix tu re o f st ig m as te ro l an d β -s it o st er o l, D C S -E 4 University of Ghana http://ugspace.ug.edu.gh 106 IR s p ec tr u m o f D C S -E 5 University of Ghana http://ugspace.ug.edu.gh 107 IR s p ec tr u m o f D C S -E 6 , p o m o li c ac id University of Ghana http://ugspace.ug.edu.gh 108 IR s p ec tr u m o f D C S -E 7 , d ic h ap et al in M . University of Ghana http://ugspace.ug.edu.gh 109 IR s p ec tr u m o f D C S -E 8 , M as li n ic a ci d University of Ghana http://ugspace.ug.edu.gh 110 IR s p ec tr u m o f D C S -E 9 University of Ghana http://ugspace.ug.edu.gh 111 IR s p ec tr u m o f D C S -E 1 1 University of Ghana http://ugspace.ug.edu.gh 112 APPENDIX II: 1H and 13C NMR data of Isolates Page 13C NMR data of DCS-E6, pomolic acid 113 1H NMR data of DCS-E6, pomolic acid 114 1H NMR data of DCS-E7, dichapetalin M 115 13C NMR data of DCS-E7, dichapetalin M 116 Expanded 1H NMR data of DCS-E7, dichapetalin M 117-120 13C NMR data of DCS-E8, maslinic acid 121 1H NMR data of DCS-E8, maslinic acid 122 Expanded 1H NMR data of DCS-E8, maslinic acid 123-127 University of Ghana http://ugspace.ug.edu.gh 113 University of Ghana http://ugspace.ug.edu.gh 114 University of Ghana http://ugspace.ug.edu.gh 115 University of Ghana http://ugspace.ug.edu.gh 116 University of Ghana http://ugspace.ug.edu.gh 117 University of Ghana http://ugspace.ug.edu.gh 118 University of Ghana http://ugspace.ug.edu.gh 119 University of Ghana http://ugspace.ug.edu.gh 120 University of Ghana http://ugspace.ug.edu.gh 121 University of Ghana http://ugspace.ug.edu.gh 122 University of Ghana http://ugspace.ug.edu.gh 123 University of Ghana http://ugspace.ug.edu.gh 124 University of Ghana http://ugspace.ug.edu.gh 125 University of Ghana http://ugspace.ug.edu.gh 126 University of Ghana http://ugspace.ug.edu.gh 127 University of Ghana http://ugspace.ug.edu.gh 128 Appendix III: Ethical Clearance University of Ghana http://ugspace.ug.edu.gh 129 References 1. 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