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-17 9. 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 CH3 SCoA O Citrate synthase HO2C OH HO2C CO2H Aconitase HO2C HO2C CO2H H Energy HO2C CO2H 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 HO2C 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. CH3 CH3 CH3 CH3 CH3CH3 CH3 CH3 Dammarane [13] O R H R' 18H 28 H OH Basic dichapetalin structure [14] {phen ylp ry an o m oiet y} 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 CH3 MeOOC CH2OCOC17H35 CH3O OH H MeOOC CH3O OH OH H O MeOOC CH3 H O MeOOC CH3 H OMe OH O MeOOC CH3 H OMe OH R1= OH, G or H(11,12-dihydro) [19a] R1 = OH, G or H(11,12-dihydro) [19b] R1 = O, 7-dehydro [20] O H CH3 H R CH3 CH3 R 1 R R1 = OH, Dichapetalin C [15] R1 = OH, Dichapetalin D [16] R1 = OH, Dichapetalin E [17] R1 = O, 7-dehydro [21] R1 = 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 CH2OH CH3 R 2 CH3 CH3 H OH H H HCH3 R 1 O Dichapetalin R1 R2 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 CH2OH CH3 CH3 CH3 H OH H H HCH3 O H OH H R CH3 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 CH3 CH3 H OH H HCH3 O CHO CH3 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 CH3 OO H H CH3CH3 CH3 H CH3 OH O Dichapetalin P [34] O O O O OH O CH3 H CH3 H CH3 OCOCH3 OHH H CH3 O O O O CH3 O OH CH3 CH3 CH3 CH3 OH OH O Dichpetalin M [31] Dichapetalin X [32]  O O O O H H CH3CH3 CH3 H OH O CH3 OH Dichapetalin S [33] O O O O CH3CH3 CH3 H CH3 R 2 O R 3 R 1 H H H H R1 = R2 = H, R3 = OAc = 22- deoxydichapetalin P [36] R1 = H, R2 = R3 = OH = 25- De-O- acetyldichapetalin P [37] R1 = R2 = R3 = OH = 25- De-O- acetyldichapetalin M [38] O O O O CH3CH3 CH3 H CH3 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 CH3CH3 CH3 H CH3 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] OCH3 H O H H OH 22- Deoxy-4''- methoxydichapetalin V [42] OCH3 H O H OH H Dichapetalin W [43] H H OH H OH H 4''- Demethoxy-7-dihydrodichapetalin W [44] R1 R2 R3 R4 R5 R6 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 CH3 H CH3 H H H CH3 OH CH3 H OH OH O O CH3 H CH3 H H CH3 OH H OH CH3 OH H CH3 O CH3 H CH3 H CH3 OH O OH OH H CH3 OH OHCH3 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 CH3 CH3 O H H H H OH O OCH3 CH3 O O OH CH3 O O O O OH O 28 H 19 H CH3 OCOCH3 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 OCH3 CH3 H O CH3 CH3 CH3 H OH H R 1 R 2 Acutissimatriterpene A: R1 = R2 = O-CH2-O [49] Acutissimatriterpene B: R1=R2 = H [50] O O O OCH3 CH3 R 1 R 2 O OH CH3 CH3 HCH3 Acutissimatriterpene C: R1 = R2 = O-CH2-O [51] Acutissimatriterpene D: R1 = R2 = 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. CH3 O CH2 CH3 H CH3 H COOH CH3 [53] O OH CH3 CH3 H CH3 H COOH CH3 [54] Figure 7: Other compounds isolated from some Dichapetalum species. University of Ghana http://ugspace.ug.edu.gh 33 CH3CH3 O CH3 CH3 H OH H CH3 CH3 CH3 Zeylanol [55] CH3CH3 O CH3 CH3 H OH H CH3 CH3 CH3 CH2 CH3 CH3 CH3 CH3H CH3 CH3 H H OH H COOH 28-hydroxyzeylanol [56] Betulinic acid [57] CH2 CH3 CH3 CH3 CH3 CH3 CH3H H H O COOH CH2 CH3 CH3 CH3 CH3H CH3 CH3 H H OH H COOH [58] Betulonic acid [59] O CH3 CH3 CH3 CH3 CH3 H CH3CH3 CH3 H H Friedelan-3-one [60] CH3 CH3 CH3 CH3 CH3 H CH3CH3 CH3 OH H H Friedelan-3-ol [61] CH3 CH3 CH3 CH3 CH3 CH3 H H CH3 H H O R CH3 O H OH OH O R = CHO = Canophyllal [62] R = CH2OH = Canophyllol [63] 2,3-dihydroxypropyloctadecanoate [64] Figure 7 continued. University of Ghana http://ugspace.ug.edu.gh 34 CH3 CH3 CH3 CH3 CH3 CH3 CH3 COOH OH OH Pomolic acid [65] CH3 CH3 CH3 CH3 HCH3 OH H CH3 CH3 CH3 CH3CH3 CH3 CH3 OH H H -sitosterol [67] Stigmasterol [66] O O CH3 CH3 CH2 CH3 O OH H H CH3 H OH OH CH3 H Pyracrenic acid [68] O O OOH OH OH OH OH CH2OH Sucrose [69] CH2OH CH2OH CH3 OH O Stearic acid [70] OH OH HH3CHN O OH CH3 O H NHCH3 N + CH3 CO2 - 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 CH3 N CH3 CH3 H Praziquantel [76] Oxamniquine [75] P Cl OH Cl Cl O OCH3 OCH3 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 OCH3 OHOH H3CO O O Curcumin [77] CH3 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 OO CH3 O O CH3CH3 CH3 H H Artesunate [81] O O O CH3 CH3 O O H CH3 CH3 H H Artemether [82] O O O CH2 H H O OH CH2 O CH2 CH2 O Vernodalin [83] N O O CH3 O CH3 O CH3 O H H Piplartine [86] OH O CH3 OH CH3OH OH O CH3 CH3 CH3 OH O OH O CH3 OH CH3O OH O CH3 CH3 CH3 OH O CH3 S S CH2 CH2 O Favaspidic acid [88] Desaspidin [89] Allicin [90] CH3 CH2OH CH3CH3CH3CH3 O N N CH3 OH O H Epiisopiloturine [84] CH3 CH3 CH2 CH3 CH3 H CH3 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  CHCl3/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 CH3 CH3 CH3 CH3 CH3 H CH3CH3 CH3 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 CH3 CH3 CH3 CH3 CH3 H CH3CH3 CH3 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 DCS-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 purpl