SECO NDARY M ETABO LITES FROM A LOCAL M EDICINAL PLANT - Gardenia erubescens. Stapf. & Hutch by ESTHER ADEBOLA ADELAKUN Submitted to thè faculty of Science in partial fulfilment of thè requirements for thè degree of DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN. March 1995. UNIVERSITY OF IBADAN LIBRARY 11 ABSTRACT. A detailed Chemical analysis of thè sterri of Gardenia erubescens Stapf. & Hutch. was carried out. The crushed stem was sequentialiy extracted with petroleum ether, ethyl acetate and methanol. Subsequently thè Petroleum ether, ethyl acetate and methanol extracts when subjected to phytochemical tests revealed ihe presence of steroids, flavonoids and saponins, while anthraquinones and alkaloids were not detected. The petroleum ether extract and thè n-butanol soluble fraction of thè methanol extract were further subjected to chromatographic analyses using a combination of column, TLC and preparative TLC. The TLC analysis of petroleum ether extract showed thè presence of steroids and flavonoids as thè major components. TLC analysis of thè n-butanol soluble fraction of methanol extract however revealed only thè presence of saponins. Purification of thè petroleum ether extract on column of silica gel afforded, after further purification by preparative TLC, five known compounds, one fraction containing isomerie mixture of two compounds and one new bisnortriterpenoid compound. From thè saponin fraction of thè n- butanol soluble fraction of methanol extract, five compounds were isolated and identified. Four of these compounds were known while one was a new_ oleanene-type compound. A total of fourteen compounds were isolated and identified from both thè petroleum ether extract and n-butanol soluble fraction of thè methanol UNIVERSITY OF IBADAN LIBRARY Ili extract of thè stems of Gardenia ernbescens. Characterization of thè known compounds was achieved through spectral correlation with authentic samples and/or derivatisation and comparison of melting points with thè known compounds. Full characterization of thè new compounds was accomplished by use of 2D nmr experiments - COSY and ^ H -^ c nmr correlation spectra, assisled by NOE difference spectroscopy. The known compounds isolated and identified from Gardenia ernbescens in this work were: •D-Mannitol •Stigmasterol •Stigmasterol-3p-D-glucopyranoside •5-Hydroxy-7,4'-dimethoxyflavanone •5-Hydroxy-7,4'-dimethoxyflavone •5-Hydroxy-7,3',4'-trimethoxyflavanone •3p-Acetoxyolean-12en-30-oic acid •Isomerie mixture of oleanolic and ursolic acids •3p,23-Dihydroxyolean-12-en-28-oic acid •2p,3(3-Dihydroxyolean-12-en-28-oic acid •2 p ,3 p ,23-Trihydroxyolean-12-en-2 8-oic acid and thè two novel compounds were: •Erubescenone and •3p ,23,24-Trihydroxyolean-12-en-28-oic acid. UNIVERSITY OF IBADAN LIBRARY IV ACKNOW LEDGEM ENTS I wish to start by expressing my thanks and sincere gratitude to my supervisor, Prof. J. I. Okogun who has been my mentor for more than fifteen years. I want to particularly thank him for his interest, eneouragement and understanding throughout thè course of this research work. I enjoyed a high level of intellectual freedom in my interactions with him and this was a source of motivation and incentive which kept me in high spirit particularly during thè difficult periods. He was like a father to me and as a daughter I have tried to live up to his expectations. This relationship greatly enhanced thè completion of this research work. I most sincerely thank him for his contributions and I will remain grateful to him. 1 most sincerely express my thanks to thè head of Chemistry department, University of Ibadan, Prof. D. A. Okorie, for thè eneouragement and thè facilities at our disposai in thè course of this work. My thanks also go to my lecturers in thè department of Chemistry, University of Ibadan, for their assistance, words of eneouragement and concem. I am particularly thankful to Prof. J. A. Faniran, Prof. E. K. Adesogan, Prof. A. A. Adesomoju, Prof. S. O. Ajayi, Dr. I. Iweibo, Prof. O. Ekundayo and Prof. O. Oderinde. They helped me in one form or another in thè course of this work and I am very grateful for their con tributions towards thè successful completion of this work. My thanks also go to thè entire technical staff of Chemistry department, University of Ibadan, for thè rapport and selfless assistance I enjoyed throughout. UNIVERSITY OF IBADAN LIBRARY V Mr. Tunde Abaire deserves to be mentioned and thanked. He single handedly pulverized thè plant materials used and also carried out thè first set of extraction. The assistance, guidance and concem of Messrs A. Adeyeye and O. Daramola, both of thè department of Chemistry, University of Ibadan, are also appreciated. Corning to thè University of Jos where I have been working for thè past twelve years, it is all thanks and appreciation to friends and colleagues for their good will and words of exhortation. In times of frustration and hopelessness, they gave me strength through their moral support. I am indeed very grateful for thè part you have played to see me through thè moments of anxiety and desperation. I am greatly touched by thè concern shown by my head of department, Assoc. Prof. K. I. Ekpenyong, who had been personally and ofFicially involved in seeing me through thè Ph.D. programme. I am also grateful to Assoc. Prof. M. M. Ekwemchi, who during his tenure as head of Chemistry department, was instrumentai to my nomination for thè World Bank Staff development programme. I also wish to thank thè former dean of Naturai Sciences, Prof. D. E. Ajakaiye, who was also greatly involved in securing overseas fellowship for me in order to facilitate speedy completion of my Ph.D. programme. I am sincerely grateful to my colleagues in thè department of Chemistry, University of Jos, for their co-operation and to thè entire technical and administrative staff for their unparalleled willingness to help at all times. My thanks also go to University of Jos authority for sponsoring my Ph D. programme and for nominating me for thè staff development programme under thè world bank loan scheme. UNIVERSITY OF IBADAN LIBRARY VI I am most grateful to Dr. Peter Ozo-Eson of thè department of Economics, University of Jos, for his invaluable assistance with thè computer. I also express my deep appreciation to Prof. S. A. Matlin and Dr O. W. Howarth, both of thè department of Chemistry, University of W arwick, Coventry, U.K., for their significant contributions with respect to structural elucidation of thè new compounds isolated in this work. I cannot but thank my parents - Pastor and Mrs. I. B. Owo, my brothers and sisters, my in-laws, for they all stood by me during thè years of struggle. I am grateful for your love and good wishes which have in no small way contributed to thè success of my work. My brother, Dr. J. O. Owojuyigbe, was my major benefactor for thè periods of my sojoum at Ibadan. I used his house as my hostel whenever I went to spend some weeks in thè University. I thank him greatly for all thè sacrifices he had to make just for my sake. I am indeed very grateful for thè love and care I enjoyed throughout thè period of my programme. I owe so much to my family. My children - Jumoke, Toyin, Sola and Femi were sources of inspiration to me. They always show great understanding whenever I had to be away for weeks. Even when I had to be away for nine months, all they did was to pray for me for thè successful completion of my research work. Their love and compassion gave me strength and determination to forge ahead in thè onerous task in my hands. I am very grateful to you all. My husband, Dr. Femi Adelakun, is thè greatest asset God gave me in this world. He was solidly behind me throughout. The love, care, encouragement and assuring support which I enjoyed ffom him gave me so much strength and hope which UNIVERSITY OF IBADAN LIBRARY VII sustained me till thè end of this work. I am sincerely grateful for your moral, emotional and financial support. Finally, I give all glory to God for hitherto He has helped me. The achievement so far has been largely due to providence. Each time one road closes, God opens another one. I will continue to praise Him and to testify to His goodness onto me all thè days of my life. I will always give thanks to thè Lord for He is great and His greatness is beyond human understanding. Esther Adebola Adelakun. March 1995. UNIVERSITY OF IBADAN LIBRARY Vili CERTEFICATION. I certify that this work was carried out by Mrs. E. A. Adelakun in thè department of Chemistry, University ofIbadan, Ibadan. Nigeria. SUPEF Prof. J. I. Okogun. BSc Special(London), Ph D. (Ibadan), D.I.C. (London), C. Chem., FRSC, FAAS, FCSN, FAS. Professor in thè department of Chemistry, University of Ibadan, Ibadan. Nigeria. March 1995. UNIVERSITY OF IBADAN LIBRARY IX DEDICATED ........ with love and thanks to my children - Adejumoke, Oluwatoyin, Olusola and Olufemi. UNIVERSITY OF IBADAN LIBRARY X TABLE OF CO NTENTS. Title. i Abstract. ii Acknowledgements. iv Certification. viii Illustrations X V List of tables. X X List of schemes. xxiii Glossary. X X V Chapter one. 1.1. Introduction. 1 1.2. Secondary metabolites and thè ecosystem. 7 1.2.1. Animai - Animai interactions. 7 1.2.2. Plant - Animai interactions. 12 1.2.3. Plant - Plant interactions. 18 1.2.4. Plant - Microbe interactions. 19 1.3. Early development in naturai products chemistry. 24 1.4. Phytochemical and pharmaceutical review of thè plant family Rubiaceae. 28 1.4.1. Naturai products from Rubiaceae plants other than Gardenia species. 28 UNIVERSITY OF IBADAN LIBRARY XI 1.4.2. Naturai products from rGadneipsecies. 42 1.5. The flavonoids. 60 1.5.1. Determination of thè structure of flavonoids. 62 1.5.2. UV/VIS spectroscopy. 63 1.5.3. Proton NMR spectroscopy. 66 1.5.4. NMR spectroscopy. 67 1.5.5. Mass spectrometry. 69 1.5.6. Biosynthesis of flavonoids. 71 1.5.7. Biological importance of flavonoids. 79 1.6. The triterpenoids. 80 1.6.1. Classification of triterpenes. 81 1.6.2. Tetracyclic triterpenes. 81 1.6.3. Pentacyclic triterpenes. 84 1.6.4. Degraded triterpenes. 85 1.6.5. Determination of structure of triterpenes. 86 1.6.6. Structure determination by degradative methods. 87 1.6.7. Ih nmr spectroscopy. 92 1.6.8. nmr spectroscopy. 95 1.6.9. Mass spectrometry. 99 1.6.10. Mass spectrum of oleanenes and ursenes. 100 1.6.11. Mass spectrum of A^4-taraxerenes. 106 1.6.12.28-Nor-Al 7(18).0leanenes. 108 1.6.13.Derivatives of friedelane. 109 UNIVERSITY OF IBADAN LIBRARY xii 1.6.14. Lupane derivatives. 109 1.6.15. Biosynthesis of triterpenoids. 112 1.6.16. Biosynthesis of tetracyclic triterpenoids. 115 1.6.17.B iosynthesis of pentacyclic triterpenoids. 118 1.6.18. Biosynthesis of phytosterols. 122 1.6.19. Biosynthesis of sterols in fungi. 127 1.6.20. Triterpenoids and sterols of invertebrates. 128 1.6.21. Pharmacological properties of triterpenoids. 131 1.7. Objectives of thè research work. 139 Chapter two Results and Discussion 142 2.1. Extraction and preliminary examination of extracts. 142 2.2. Isolation and purification of components from thè extracts of G. erubescens. 145 2.3. Derivatives of compounds isolated from G. erubescens. 150 2.4. NMR studies on thè triterpenoid components of G. erubescens. 152 2.5. Characterization of compounds from G. erubescens. 152 2.5.1. The structure of GS 1 152 2.5.2. Characterization of GSH 2 155 2.5.3. Determination of thè structure of GSH 26 159 2.5.4. Identification of GSH 41 161 2.5.5. Determination of thè structure of GSH 14 166 UNIVERSITY OF IBADAN LIBRARY Xlll 2.5.6. Strutture of GS 13 168 2.5.7. Determination of thè structure of GSH 24 170 2.5.8. Structure of GSH 32 179 2.5.9. Determination of thè structure of GSH 49 185 2.5.10. Elucidation of structure of GS A 5 190 2.5.11.Structure of GSA 8 196 2.5.12.Strutture of GSA 11 199 2.5.13. Strutture elucidation of GSA 16 204 Conclusion. 216 Chapter three 3.1. Experimental. 221 3.2. Preparation of detection reagents. 222 3.3. Extraction of plant material. 223 3.4. Treatment of extracts. 224 3.5. Phytochemical tests. 225 3.6. Analytical thin layer chromatography. 228 3.7. Column chromatographic analysis of petroleum ether extract. 230 3.8. Preparative thin layer chromatographic plates. 232 3.9. Chromatographic analyses of ethyl acetate and n-butanol soluble extracts. 234 3.10. Preparation of derivatives. 240 3.11. Determination of specific rotation for GSH 24, GSA 16 and GSA 16 triacetate. 242 UNIVERSITY OF IBADAN LIBRARY XIV 3.12. Spectral data on thè compounds isolated from G. erubesce2r4t5s. References. 255 UNIVERSITY OF IBADAN LIBRARY XV ILLUSTRATIONS. Figure. Title. Page. 1 IR spectrum of GS I 152b 2. Ih nmr spectrum of GS 1 152c 3. nmr spectrum of GS 1 153b 4. CIMS spectrum of GS 1 153c 5. IR spectrum of GS 1 hexaacetate 153d 6. 1H nmr of GS 1 hexaacetate 153e 7. 13C nmr of GS 1 hexaacetate 153f 8. EIMS of GS 1 hexaacetate 154b 9. IR spectrum of GSH 2 155b 10. 1H nmr spectrum of GSH 2 155c 11. ^H nmr ofG SH 2 155d 12. IH nmr of GSH 2 156b 13. ^H nmr of GSH 2 156c 14. Ih nmr ofG SH 2 156d 15. 13c nmr spectrum of GSH 2 157b 16. EIMS spectrum of GSH 2 158b 17. UV spectrum of GSH 2 159b 18. IH nmr spectrum of GSH 26 159c 19. 13c nmr spectrum of GSH 26 160b 20. UV spectrum of GSH 26 161b UNIVERSITY OF IBADAN LIBRARY XVI 21. EIMS of GSH 26 161c 22 nmr spectrum of GSH 41 161d 23. 13C nmr ofGSH41 162b 24. UV spectrum of GSH 41 163b 25. EIMS of GSH 41 163c 26. EIMS of GSH14 166b 27. IH nmr spectrum of GSH 14 166c 28. 13C nmr spectrum of GSH 14 166d 29. IR spectrum of GSH 14 166e 30. 1H nmr of GSH 14 acetate 167b 31. 13C nmr of GSH 14 acetate 167c 32. EIMS of GSH 14 acetate 167d 33. ÌH nmr of oxo-deriv. of GSH 14 168b 34. 13c nmr of oxo-deriv. of GSH 14 168c 35. EIMS of oxo-deriv. of GSH 14 168d 36. 1H nmr spectrum of GS 13 168e 37. 13C nmr spectrum of GS 13 169b 38. FABMS ofGS 13 169c 39. UV spectum of GSH 24 in CHCI3 170b 40. UV spectrum of GSH 24 in MeOH 170c 41. IR spectrum of GSH 24 170d 42. ^H nmr spectrum of GSH 24 170e 43. 13C nmr spectrum of GSH 24 171 b UNIVERSITY OF IBADAN LIBRARY XVII 44. DEPT spectrum of GSH 24 171 c 45. EIMS of GSH 24 172b 46. UV spectrum of GSH 24+NaOH in MeOH 173b 47. COSY spectrum of GSH 24 173c 48. IR spectrum of GSH 32 179b 49. 1H nmr spectrum of GSH 32 179c 50. DEPT spectrum of GSH 32 179d 51. CIMS of GSH 32 180b 52. IH nmr spectrum of GSH 49 185b 53. 13C nmr spectrum of GSH 49 185c 54. 13c nmr spectrum of GSH 49 185d 55. CIMS of GSH 49 185e 56. IH nmr spectrum of ursolic acid 186b 57. ^H nmr spectrum of oleanolic acid 186c 58. IR spectrum of GSA 5 191b 59. IH nmr spectrum of GSA 5 191 c 60. 13c nmr spectrum of GSA 5 191d 61. DEPT spectrum of GSA 5 191e 62. CIMS of GSA 5 191 f 63. IR spectrum of GSA 5 diacetate 192b 64. ̂H nmr of GSA 5 diacetate 194b 65. 13nmr of GSA 5 diacetate 194c 66. CIMS of GSA 5 diacetate 194d UNIVERSITY OF IBADAN LIBRARY xviii 67. 1H nmr spectrum of GSA 8 197b 68. nmr spectrum of GSA 8 197c 69. DEPT spectrum of GSA 8 197d 70. CIMS of GSA 8 197e 71. IR spectrum of GSA 11 199b 72. 1H nmr spectrum of GSA 11 199c 73. *3C nmr and DEPT of GSA 11 200b 74. CIMS of GSA 11 200c 75. IR spectrum of GSA 11 triacetate 202b 76. 1H nmr of GSA 11 triacetate 202c 77. 13C nmr of GSA 11 triacetate 202d 78. CIMS of GSA 11 triacetate 203b 79. IR spectrum of GSA 16 204b 80. 1H nmr spectrum of GSA 16 204c 81. 13C nmr spectrum of GSA 16 204d 82. DEPT spectrum of GSA 16 204e 83. CIMS of GSA 16 205b 84. IR spectrum of GSA 16 triacetate 206b 85. nmr of GSA 16 triacetate 206c 86. CIMS of GSA 16 triacetate 206d 87. IH nmr of GSA 16 methyl ester 206e 88. 13C nmr of GSA 16 methyl ester 206f 89. CIMS of GSA 16 methyl ester 206g UNIVERSITY OF IBADAN LIBRARY Ih nmr of GSA 16 triacetate methyl ester l^C nmr of GSA 16 triacetate methyl ester EIMS of GSA 16 triacetate methyl ester UNIVERSITY OF IBADAN LIBRARY XX LIST OF TABLES 0 Table Description Page 1. Value of higher plant medicinals in thè USA in 1973 3 2. Some Rubiaceous Anthraquinones. 31 3. Triterpenoids of Rubiaceae Plants. 38 4. Iridoid glycosides from Gardenia species. 45 5. Steriods and Triterpenoids from Gardenia species. 47 6. Flavonoid constituents of Gardenia species. 53 7. Classes of Flavonoids. 61 8. Major Absorption bands of Flavonoids. 63 9. Chemical shift data for C-2 and C-3 protons in flavones, isoflavones, flavanones and dihydroflavonols. 67 10. 1 Chemical shift data for C-4 signal in some flavonoid compounds. 68 11. 1 ̂ C Chemical shift characteristic of flavonoid functional groups. 68 12. Representative structures of tetracyclic triterpenes. 82 13. Basic C30 skeleton of thè different groups of pentacyclic triterpenes. 84 14. Examples of Quassinoids and Limonoids. 86 15. Chemical shift of highest C-methyl groups. 95 16. Absorptions of methyl esters. 96 UNIVERSITY OF IBADAN LIBRARY XXI 17. Absorptions of protons alpha to acetoxyl group. 97 i8. Absorptions of methyl groups of A^-oleanene. 98 19. 1 Chemical shift of common functional groups of triterpenoids. 98 20. Characteristic absorptions of olefinic carbon atoms of triterpenes. 99 21. Some biologically active triterpenes. 134 22. Structures of antitumour quassinoids. 137 23. Tumour inhibitory cucurbitacins. 138 24. Results of phytochemical tests on extracts of stems of G. erubescens. 143 25. 400 MHz nmr spectrum and NOES for erubescenone in CDCI3. 175 26 100 MHz nmr spectrum and ^ C - ^H correlations for erubescenone in CDCI3. 177 27. 400 MHz 1H nmr spectrum and NOES for 3p-acetoxyolean-l2-en-30-oic acid. 181 28 250 MHz Ih nmr spectra data for GSH 49, ursolic and oleanolic acids. 188 29. 100 MHz l^ c nmr spectra data for GSH 49, ursolic and oleanolic acids. 189 10. 400 MHz Ih nmr spectrum and NOES for 3(3,23- dihydroxyolean-12-en-28-oic acid. 195 UNIVERSITY OF IBADAN LIBRARY XXII 400 MHz nmr spectrum and NOES of 20,30,23- trihydroxyolean-12-en-28-oic acid. 400 MHz Ih spectrum and NOES for erubigenin in d5- pyridine. MHz spectra of erubigenin and its derivatives. Compounds isolated from stems of Gardenia erubescens Stapf. & Hutch. UNIVERSITY OF IBADAN LIBRARY xxm LIST OF SCHEM ES. Scheme. Description. Page I Major fragmentation pattern of flavonoids. 70 II Fragmentation pathways for flavones and flavanones. 72 III Biosynthesis of thè flavonoid basic skeleton. 73 IV Biosynthetic route for flavonols and cyanins. 75 V Biosynthesis of Isoflavones. 76 VI Biosynthesis of Rotenones. 77 VII Biosynthesis of Dalrubone. 78 Vili Fragmentation pattern of oleanenes and ursenes. 101 IX. Formation of ions e and g from oleanenes and ursenes. 104 X Formation of ion i from 15- ketoerythrodiol diacetate. 105 XI Fragmentation of methyl glycyrrhelate to give ion j. 105 XII Characteristic fragmentation of Al4-taraxerenes. 107 XIII Formation of ions 1 and 1' from A ^-ta rax e ren es . 108 XIV Fragmentation of A ^(^)-oleanenes to give ions p, o and o'. 110 XV Biosynthesis of squalene from all trans famesyl diphosphate. 114 XVI Biosynthetic route for thè synthesis of lanosterol. 116 XVII Biosynthetic route for dammaranes and euphanes. 118 UN VERSITY OF IBADAN LIBRARY XXIV XVIII Biosynthesis of lupanes, a- and (3-amyrms from protosterol carbonium ion II. 119 XIX Biosynthetic route for friedelin and glutinol. 120 XX Biosynthetic route for phytosterols. 123 XXI Alkylation of phytosterols at C-24. 126 XXII Biosynthesis of 24a- and -p alkyl sterols. 127 XXIII Biosynthesis of ergosterol. 129 XXIV Dealkylation of p-sitosterol into cholesterol by phytophagous insects. 130 XXV Biosynthesis of Ecdysone. 131 XXVI Fragment ions identified from mass spectrum of GSH 2. 158 XXVII Fragmentation pattern observed from thè mass spectrum of GSH 41. 164 XXVIII Plausible biogenetic route for erubescenone. 178 XXIX Fragment ions identified in thè mass spectrum of GSH 32. 184 XXX Fragmentation pattern in thè mass spectrum of GSA 5. 193 XXXI Fragment ions identified in thè mass spectrum of Erubigenin and its derivatives. 212 UNIVERSITY OF IBADAN LIBRARY XXV GLOSSARY General Abbreviations. TLC =.thin layer chromatography Prep. TLC = preparative thin layer chromatography UV = ultraviolet IR = infrared NMR = nuclear magnetic resonance DEPT = distortionless enhancement by polarization transfer NOE = nuclear overhauser effect COSY = correlated spectroscopy HETEROCOSY = heteronuclear correlated spectroscopy MS = mass spectroscopy EIMS = electron impact mass spectroscopy CIMS = Chemical ionization mass spectroscopy FABMS = fast atomic bombardment mass spectroscopy Rf = mobility relative to front nm = nanometers FT = fourier transform TMS = tetramethyl silane UNIVERSITY OF IBADAN LIBRARY XXVI Solvents and Reagents DMSO = dimethyl sulphoxide MeOH - methanol CHCI3 = chloroform EtOAc = ethyl acetate AC2O = acetic anhydride H2SO4 = sulphuric acid AgNC>3 = silver nitrate NaOH = sodium hydroxide HBr - hydrogen bromide AcOH = acetic acid LÌAIH4 = lithium aluminium hydride UNIVERSITY OF IBADAN LIBRARY CH APTER ONE INTRODUCTION During thè long history of mankind, man's views on thè cures of diseases have undergone radicai changes. The early medicines of man were obtained ffom naturai sources especially, thè vegetable kingdom. There is an abundance of plant species in thè world, and many are stili being discovered. The knowledge of thè use of these plants have accumulated over centuries of practice and were handed down by orai tradition from generation to generation. In thè course of civilization thè changes imposed by modem Science on man's sociocultural practices now seem to cause a change in thè disposition to such traditional values. However, some of thè practices and methods of thè use of these medicinal plants have survived to a greater extent in thè developing countries of thè world. The use of plants either singly or with other naturai products in thè treatment of diseases within an organized indigenous System constitutes part of what is known as traditional medicine and is defined "as thè sum total of all knowledge and practices whether explicable or not, used in diagnosis, prevention and elimination of physical, mental or social imbalance and relying exclusively on practical experience and observation handed down from generation to generation whether orally or in writing" 1. It naturally UNIVERSITY OF IBADAN LIBRARY 2 follows that thè people engaged in thè practice of traditional medicine are traditional medicai doctors. The enomous varieties of plants used in this sense are known as medicinal plants, and "a medicinal plant is defined as any plant which in one or more of its organs, contains substances that can be used for therapeutic purposes or which are precursors for thè synthesis of useful drugs"2. jealously Knowledge of thè uses of medicinal plants though / guarded by thè traditional medicai practitioners, has now become an area of great interest and exploitation in developing countries as Government in these countries now mobilize their people to be self-reliant in an attempt to save importation money through reduction in th è / J of foreign products. Although thè importance of naturally occuring drugs has long been recognised, it appears thè developed countries - Europe and North America, are increasingly shifting emphasis to synthetic drugs. However an analysis shown in table 1, as reported by Prof. Okogun 3 in his lecture on "Drag production efforts in Nigeria: Medicinal Chemistry Research and a missing Link", delivered to thè N.gerian Academy of Science, suggested a significant contribution from naturai products chemistry to drag production even in thè so called technologically advanced countries. But thè situation is different in developing countries as 60-85% of thè. population in every developing country rely on traditional medicine because it is cheaper and more accessible than modem medicine. Research in African traditional medicine had been sponsored by OAU since 1968. The UNIVERSITY OF IBADAN LIBRARY 3 OAU/STRC has continued to finance research in this area with regional co- ordinating centres scattered throughout Africa. The co-ordinating centre for West Africa is Ile-Ife^. The results of scientific investigations on medicinal plants continue to provide scientific proof of thè efFicacy of traditonal medicine. Table 1 Value of Higher Plant Medicinals in thè USA in 1973. = $ 3,000,000.00 Source of prescription Percentage 1973 1959 Higher Plant 25.2 25.2 Microbes 13.3 21.4 Animai 2.7 2.3 Total 41.2 48.9 A stage has therefore been reached whereby certain locai claims to specific medicinal activities of some of these plants cannot be regarded as superstition or simply ignored. It is important to continue to explore this valuable cultural legacy in Nigeria so as to pavé thè way for integration of traditional and modern medicine as it is now in China. In essence, it has UNIVERSITY OF IBADAN LIBRARY 4 become increasingly encouraging and necessary to subject many of these plants to scientific research so as to provide justification for their medicinal uses. This aspect of organic chemistry research is identified as naturai products chemistry. Naturai products chemistry in its different aspects is an ancient Science. For example, preparation of foodstuff, colouring matter, medicinals or stimulant are activities as old as mankind. When chemists took thè final jump in thè late 18th century from thè world of myths into modem Science, thè true properties of extracts obtained from nature became thè focus. They began to separate, purify and then analyse thè compounds produced in living cells. Separation methods were developed and without doubt naturai products chemistry has brought great stimuli to thè development of thè refined separation techniques available today, that is, column chromatography(CC), gas chromatography(GC), thin layer chromatography(TLC), high performance liquid chromatography(HPLC), paper chromatography(PC), electrophoresis, ion- exchange chromatography etc. These methods have made it possible to isolate compounds present in extremely small quantities. Structural elucidation was typically carried out by degradation to smaller fragments of known stmcture, but with computerised spectroscopy in its Service, naturai products chemistry has attained a highly refined status today. A large number of spectroscopic data correlating spectral properties with stmcture are available. These data give very valuable information about stmcture so much so that pure Chemical transformation can be reduced to a minimum or UNIVERSITY OF IBADAN LIBRARY 5 completely avoided. When thè amount of sample available is very limited and structure too complicated, X-ray crystallography is thè ultimate resource. While naturai products chemists are interested in secondary metabolites, thè biochemists are concemed mostly with products of primary metabolism. Primary metabolism refers essentially to thè photosynthetic and related processes producing thè carbohydrates, fats and proteins which are widely distributed. From thè compounds identified as components of primary metabolism, only a handful serve as source material for thè elaboration of thè thousands of known secondary metabolites. The most important is acetic acid, thè others are aromatic amino acids - tryptophan, phenylalanine and tyrosine, and thè aliphatic amino acids - omithine and lysine. When methionine is added as thè naturai methylating agent, thè list is virtually complete. Secondary metabolites are characteristic for thè particular biological group -family or genus or species. Apparently, thè synthetic process involved in thè production of secondary metabolites is related to thè mechanism of evolution of species. The specifìc pattern of constituents in species has been used for systematic determination. A characteristic feature of secondary metabolites is that their function is to a significant extent not always obvious. It is hard to believe that thè organism should allocate so large a proportion of metabolic resources for purposes void of sense. The production of secondary metabolites is found to be connected with several extemal factors such as growth, flowering, season, UNIVERSITY OF IBADAN LIBRARY 6 temperature, habitat etc. On this ground secondary metabolites are defìned as substances which play a prominent role in thè co-existence and co-evolution of species. The isolation of thè sex-attractant, bombycol, i from thè female silk moth was thè first evidence in support of thè idea that secondary metabolites produced in plants and animals have strong behaviour control on other species. This also signalled thè beginning of research on pheromones. The structures of several specific sex attractants are known today^. Examples are bombycol, I from Bombyx mori, methyl (E)-2,4,5-tetradecatrienoate, 2 from Acanthoscelides obtectus, exo-brevicomin, 3 from Dendroctonus brevicomio, (E,E)-3,7-dimethyl-2,6-decadien-l,10-diol, 4 from Danaus gilippus and cis- verbeno/l, 5 Vfrom ^Ips tVypogrWaphus. W ™ I, Bombycol. CO2CH3 2, M ethyl-(E )-2 ,4 ,5-tetradecatrienoate H 3, Exo-brevicomin. UNIVERSITY OF IBADAN LIBRARY 7 5, c'is*.\ferbenol. 1.2. SECONDARY METABOLITES AND THE ECOSYSTEM. Today thè role of naturai products with respect to co-existence and co-evolution of species has been an area of intense research activity. The established role of some naturai products in thè ecosystem are briefly discussed here in thè form of animal-animal, plant-animal, plant-plant and plant-microbe interactions. 1.2.1. Animal-Animal Interactions. There have been reported cases where insects collect plant toxins for protection against predators. One such interaction is thè ability of Monarch butterflies (e.g. Damisp lexippus) to sequester, store and metabolize cardenolides of thè Asclepias spcei6 The cardenolide, uscharidin 6, which is a cardenolide of milkweed, has been shown to be metabolized by tissue UNIVERSITY OF IBADAN LIBRARY 8 homogenates of thè larvae of Monarch butterflies to thè more polar compounds - calotropin 7 and calactin, 8. The cardenolide content of these butterflies invoke a protective action through its emetic effect on predators. Similarly, thè adult butterflies of thè order- Danainae, sequester and store pyrrolizidine alkaloids from plant sources, using them both for defence and for manufacture of pheromones. The compound hydroxydanaidal, 9 has been identified as thè scent produced in thè coremata of Creatonotos gangis^, while 3-isopropyl, 3-t-butyl and 3-isobutyl-2-methoxypyrazine 10, 11 & 12 respectively have been identified in thè waming odour secretions of Danans plexippus and Zygaena ionicerae. The brown aphid - Aphis cytisorum also accumulates cytisine as a defence Chemical when fed on Petteria ramentacea'&. Other examples of sequestration of plant toxins include thè use of thè phenol, salicin 13, by thè Chrysomelid beetle - Chrysomela aenicollis. The larvae sequester it from thè leaves of Salix species, hydrolyse thè glucosidic linkage and oxidize thè aglycone to salicylaldehyde, which is thè principal toxin in their defence secretion. It is also known that some species of thè chrysomelidae have thè ability to sequester a metabolite of cucurbitacin D, 5 14 which is then used to protect themselves against predation by mantids . The marine world is not left out in this ecological chemistry, although it is less well developed. A few examples will be mentioned to illustrate thè complex ecological situation that existsbelow thè surface of thè seas and oceans. The dorid nudibranch, Dendrodoris limbata produces polygodial, 15 - UNIVERSITY OF IBADAN LIBRARY 9 a hot tasting sequiterpenoid - as an antifeedant against marine and fresh water fish. Other species like Glossodoris quadricolor sequester their defensive Chemicals from sponges they feed on. For example, thè ichthyotoxin of thè sponge, latrunculin B J_6, was identified in thè mucus secretion of thè mollusc, indicating that it is used as defensive a g e n t i Sponges may use their toxic secretion for their own purposes. Thus species of sighonodictyon burrow into living coral heads killing coral growth within a zone which extends for 1-2 cm. The toxin that is exuded is a guanidino- compound, T7. 6, Uscharidin. 7, Calotropin, 3' a-OH. ___________________________________ 8, Calactin, 3' [3-OH. UNIVERSITY OF IBADAN LIBRARY 10 10, R=CHMe2; i l , R=CMe3; 9, Hydroxydanaidal. 12, R=CH(Me)Et. OGhi CH2OH 13, Salicin. 14, Cucurbitacin D. CHO CHO 16, Latrunculin B. UNIVERSITY OF IBADAN LIBRARY 11 17, Guanidino-compound. ___________________________________ j_8, R=Ac; 19, R=H. 20, 5-en-3a-ol glucosyl). Likewise, fish may benefit from their own defensive secretions and avoid predation by larger animals, such as sharks^. The Pacific sole - pavonitms produces toxins 18-23, which are used to repel attack by sharkslO.On thè other hand, algae may be protected from fish by their secondary constituents^. It has been established that derivatives of phloroglucinol in thè brown algae Fucus vesiculosus and Ascophyllum nodosum effectively limit thè feeding of marine snail, Littorìna 12 UNIVERSITY OF IBADAN LIBRARY 12 1.2.2. Plant - Animai Interactions. The nature of ecological interactions between plants and animals bothers on protection against herbivores and insects by use of Chemicals as defence agents. The range of Chemicals used by plants and animals as toxins in response to predation or insect attack cuts across thè major classes of secondary metabolites. The toxins that occur in thè fruiting bodies of fungi are known to have protective role against herbivory. The known sesquiterpene dialdehyde, isovelleral 24, was obtained from Lentinellus ursinus^. Two other compounds, piperdial 25 and piperalol 26, were likewise found to be responsible for thè pungency of Lactarius piperatus, L. torminosus and Russula aqueletii. These three aldehydes bear an obvious structural resemblance to thè pungent principle, warburganol 27, which had been isolated from higher plants and shown to be antifeedant to Man and to insects 14. Sesquiterpene lactones, which occur mainly in members of thè family Compositae, are another group of sesquiterpenoids with defensive role against herbivory 15 The compounds deoxylactucin 28, lactucin 29 and lactupicrin 30, from Cichorium intybus have been implicated as defensive agents as they occur in high concentrations in thè most actively growing regions of thè pianti6. Some components of thè croton oil from Croton tiglium known to be skin irritants and co-carcinogens in animals were tested on thè larvae of thè mosquito, Culex pipiens. One of thè components, a diterpenoid 31_, caused 100% mortality in thè larvae at a concentration of O.óppml?. UNIVERSITY OF IBADAN LIBRARY 13 Furanocoumarins constitute another important class of plant toxins which are commonly found in plants of thè family Umbelliferae. These compounds are photomutagenic and photocytotoxic and it has been demonstrated that most of thè plants in this family are protected from herbivory by furanocoumarins 18. it was also observed that plants which contain angularly fused furanocoumarins 33, are more toxic than those with linear furanocoumarins 19, 32. A number of phenolic toxins have been identified. o- pentadecenyl- salicylic acid 34 and o-heptadecenylsalicylic acid 35, from thè exudate of Pelargonium hortorum were found to be moderately toxic to thè two-spotted spider mite, Tetranyclus 2ecturai 0 \ series of prenylated hydroquinones 36, have been detected in species of hPaceli nad are found to be responsible for thè adergerne effeets of these plants in Man^l. n 25, Piperdial, R=CHO 24, Isovelleral.______________________26, Piperalol, R=CH20H UNIVERSITY OF IBADAN LIBRARY 14 UNIVERSITY OF IBADAN LIBRARY 15 UNIVERSITY OF IBADAN LIBRARY 16 46, Nordihydroguaiaretic acid. The effect of tannins on insects and mammalian feeding continue to attract research interest. The popular concept that tannins are feeding deterrents to all kinds of herbivores and that tannins are deleterious to animals because they reduce thè digestibility of thè plant protein through complex formation needs to be modified. It has now been realized that tannins are no different from other classes of toxins in that herbivores can adapt to their presence in thè diet. Also thè deleterious dietary effects UNIVERSITY OF IBADAN LIBRARY 17 resulting from complex formation with plant protein may be modified by other chemicals22. '-he protective value of some alkaloids to plants is well documented. The most interesting group of alkaloids that have been studied recently are simple pyrrolidines, piperidines and indolizidines, which structurally resemble sugars. These compounds which are mainly found in legumes, have thè ability to inhibit animai glycosidases. Swainsonine 37, has recently been isolated from thè leaves of Astragalus 23 ancj js thè active principle of this plant which causes neurological disturbances in cattle and eventually death. Even thè well-known purine alkaloids, caffeine 38 and theobromine 39, now appear to have a defensive role in plants against attack by insects. The pattern of accumulation of caffeine and theobromine in plants of Coffea arabica is closely correlated with a proposed defence strategy, having very high concentrations of these compounds in young seedlings and during leaf development24. Phytoecdysones which are mimics of insect moulting hormones also provide a defence strategy by inhibiting thè process of moulting in thè larvae of insect invaders25 . The search for phytoecdysones in plants continues to yield new compounds. New reports include pinnatasterone 40, from Vitex pinnata(Vevbemceae)^, while abutasterone 4T, from Abuta velutina5 is also one of thè recent addition. In most cases, plants which contain ecdysones often contain other terpenoids that possess antifeedant or insecticidal properties. Within a given plant species, thè phytoecdysone UNIVERSITY OF IBADAN LIBRARY 18 defence may thus be only one component of a complex defensive strategy to repel insect invaders. Leaf-cutting ants constitute a serious menace to plants. These ants appear to be impervious to many secondary compounds which would in similar circumstances repel caterpillar or leaf miners. It is now obvious that plants provide immunity from attack by leaf-cutting ants by production of terpenoids which specifically stop invasion by ants27-32 The ant repellants range from simple monoterpenes such as p-ocimene^O through sesquiterpenoids such as caryophyllene epoxide^?, sesquiterpene lactones 42, 43 and diterpenoids^l, to triterpenoids such as jacquinonic acid 44 and azadirachtin 45. 1.2.3. Plant- Plant Interactions. Allelopathic effects seem to be an important factor which determines thè pattern of vegetation in naturai ecosystems. Allelopathic property is thè production of secondary metabolites by a plant which is used to control thè growth of other plants in its vicinity. A number of new classes of Chemicals have been identified as allelopathic agents in particular instances. The lignan, nordihydroguaiaretic acid 46, which occurs in thè leaves of Larrea tridentata has been implicated for thè well-marked allelopathic effect of this shrub on surrounding vegetation33. Another known allelopathic substance is juglone, which is thè toxic principle of walnut tree, Juglans regia. The well-known phenolic compounds such as p-coumaric and ferulic acids, have UNIVERSITY OF IBADAN LIBRARY 19 also been implicateci as allelopathic agents that are produced by thè bamboo Phyllostachys edulis,thè conifer Cryptomeria japonica and thè grass Setaria faberii34 1.2.4. Plant- Microbe Interactions. The interaction between plants and microbes usually takes thè form of production of toxins by thè microbes which renders thè host plant toxic with symptoms of disease condition or production of toxins by plants as Chemical defence agents against microbial infection. Depending on thè form of interaction, mycotoxins, phytotoxins or anti microbial agents are produced. Mycotoxins are secondary metabolites of fungi origin which contaminate certain plant tissues, e.g. groundnuts and cereals, as a result of infection. Mycotoxins pose a threat to animals eating such plant tissues because they tend to be highly poisonous. The best known group of mycotoxins are thè aflatoxins, which are formed by Aspergillus flavus. The aflatoxins are known to be phototoxic in their activity against animals while altemariol 47, which is a dibenzopyrone mycotoxin of Aiternaria species, has ateo been shown to be a photosensitizing a g e n t i Phytotoxins are ateo microbial metabolites but they are responsible for thè symptoms of disease in infected plants. Helminthosporoside, which has been shown as a mixture of three isomerie sesquiterpene glycosides 48-50, is thè toxin of thè sugar-cane pathogen, Helminthosporium 36 The fungal pathogen ( Aiternaria )acehino of thè water hyacinth, causes necrotic UNIVERSITY OF IBADAN LIBRARY 20 spots on its leaves by producing thè quinonoid toxin, alteichin 51_, while thè citrus disease fungus Aiternaria citri, wreaks its effects on lemon and lime trees by producing a complex of toxins^?. The most active being thè lactone, 52. UNIVERSITY OF IBADAN LIBRARY 21 UNIVERSITY OF IBADAN LIBRARY 22 60, Luteone, R1 = CH2CH=CMe2, R2= H; 6 i, Licoisoflavone A, R ' =H, R2 = CH2CH=CMe2. ( isoprenylated isoflavones ). Aiternaria solani also attacks thè potato plant through thè production of three related pyrone derivatives e.g. compound 53 and thè lactone, zinnolide^S 54. Several of thè phytotoxins that are produced by bacteria have been characterized. These are mostly amino-acid based. Perhaps typical of bacteria toxins is cronatine 55, which is a chlorosis-inducing toxin that is produced by Pseudomonas coronafaciens^. Anti microbial substances are however produced by some plants to prevent thè attack of microbes. The diterpenoids, sclareol, episclareol and ketopipimanool 56, which occur in Nicotiana glutinosa have proved to be effective antifungal agents^O Several flavanones that exhibit antifungal activity have also been reported. Pinocembrin 57, sakuranetin 58 and 6- isopentenyl-naringenin 59, have been shown to be very potent antifungal agents from p la n ts^ l-43 Some isoflavonoids have also been found to afiford significant antifungal protection. Examples are luteone 60 and licoisoflavone 61_, UNIVERSITY OF IBADAN LIBRARY 23 obtained from Lupin roots, which proved to be very potent by inhibiting thè growth of Cladosporium rhebuam44-46 The triterpenes have also been reported as antimicrobial agents. A well known example is thè presence of pentacyclic triterpene glycosides, avenacin 62a-d in thè roots of oats, which affords protection against thè fungus Ophiobolus graminis var. tritici. Avenacin has been identified as a mixture of four closely related glycosides^?. Microbial toxins of two tropical African plants deserve mention because of their further utilization by mammals. The leaves of Aspilia mossambicensis contain thè thiophene thiarubrineA 63, which is intensely antimicrobial. Its identification in thè plant explains why this species is widely used in native medicines to cure sores and other infections. Remarkably, it is also an anthelmintic and wild chimpanzees have leamt to swallow thè leaves whole, in order to rid themselves of nematodes and protozoan worms^S. The second plant is Maesa lanceolata, thè fruit of which has been shown to contain a particularly active antimicrobial agent, maesanin 64. The hot-water extract of thè fruit is used to prevent cholera4','. The knowledge acquired from various studies on secondary metabolites has been beneficiai to Man in view of thè exploitation of thè therapeutic properties of these compounds for a wide variety of application. UNIVERSITY OF IBADAN LIBRARY 24 1.3. EARLY DEVELOPMENT IN NATURAL PRODUCTS CHEMISTRY. Initially thè interest was simply in solving structural problems of naturai products and grouping them according to origin, pharmacological activity or structure but soon thè mass of information accumulated prompted chemists to see thè need for a more coherent view of biosynthesis. A few chemists took thè lead in thè painstaking search for thè hidden biosynthetic pathways and thè peep-hole into thè fascinating synthetic workshop of thè living celi was gradually opened. The recognition of biosynthetic principles is thè most significant development in naturai products chemistry. .CHO OCOR2 62 (a) Rl=OH, r 2= o-MeHNCgTLp (b) Rl=OH, R2=Ph. (c) Rl=H, R2= o-MeHNC6H4. (d) Rl=H, r 2= Ph. o s—s M e0\ J K . ACH2)9CH=CH(CH2)3Me 63, Thiarubrine A. O 64, Maesanin. UNIVERSITY OF IBADAN LIBRARY 25 Wallach and Ruzicka first recognized that thè terpenes had a common building block, thè isoprene unit and Winterstan and Trier suggested on good grounds that alkaloids were formed from a-amino acids. The biosynthetic routes of thè various classes of compounds have now been well mapped out and these routes have been subjected to various biomimetic studies with identification of precursors and intermediates. Biomimetic studies have also led to novel and elegant synthetic procedures. Although it has been thè tradition to confimi thè structure of a compound isolated from naturai source by synthesis, thè motivation nowadays for total synthesis is not so much thè question of confirmation but for thè challenge and new knowledge inherent in synthesising intriguing stmctures. The biochemical processes involved in thè manufacture of metabolites in thè living System are basically thè same as thè organic reactions used in organic chemistry. The stage for enzyme catalysed reaction is thè three- dimensional asymmetric surface of a protein. As a result of thè chiral environment thè products become enantiomeric. The link between primary and secondary metabolites is acetic acid. It occupies a centrai position as its thio ester - acetyl Co A, in thè metabolism of naturai products. One remarkable feature is that most metabolites originate from a very limited number of precursors. From acetic acid, mevalonic acid is derived, from which via 3,3-dimethylallylpyrophosphate and thè isomerie isopentenylpyrophosphate, thè isoprene unit and hence thè terpenoids are formed. From carbohydrates, shikimic acid is derived which is thè key to a UNIVERSITY OF IBADAN LIBRARY 26 wealth of aromatic compounds, while thè amino acids are precursors of thè large variety of nitrogen containing compounds. Several groups of metabolites have mixed pathways in which one principal pathway acts as a substrate for another metabolite from a different pathway. Thus flavonoids are derived from thè polyketide and acetate pathways. The indole alkaloids come from shikimate and acetate pathways. Naturai products were classified in thè past according to structure or biological origin as fatty acids, carbohydrates, terpenes, mould metabolites, etc. The biosynthetic scheme now groups thè compounds according to thè synthetic routes employed by thè celi as alkaloids, terpenoids, flavonoids, phenolics etc. There is of course overlap between thè two but this has not posed any serious problem. For many years, chemists were faced with thè task of tracing out thè pathways and thè mechanism of each step involved in thè biosynthesis of naturai products. In thè beginning accidental results contributed to thè elucidation of intermediate steps. Knoop postulated in 1904 that degradation of fatty acids occur via (3-oxidation. Collie also hypothesized in 1907 that thè reverse reaction, thè acetate condensation of Claisen type, is thè origin of naturally occurring phenolics. The breakthrough carne with thè work on mutants and with isotopically labelled compounds. The fundamental investigations were run with thè radioactive isotopes- ^H, ' and 32p But in recent years with thè advent of pulse Fourier-transform NMR UNIVERSITY OF IBADAN LIBRARY 27 spectroscopy, biosynthetic studies have witnessed a new explosive development. Radio tracing and mass spectrometry are thè most sensitive methods now. Labelled precursors are usually introduced into thè appropriate medium and thè product analysed. In order to locate exactly thè labelled atoms in a metabolite, it has to be degraded in an unambiguous way. The activity of each fragment isolated as CO2, acetic acid or other well defined small organic molecule is then measured. Controlled degradation of a big molecule is a very difficult and time consuming exercise. This method was skilfully demonstrated by Bloch et al in thè biosynthesis of cholesterol as being ultimately derived from acetate5®. Biosynthetic studies is a significant aspect of naturai products research. It has contributed in no small measure to thè increased synthetic ingenuity of chemists. UNIVERSITY OF IBADAN LIBRARY 28 1.4 PHYTOCHEMICAL AND PHARMACEUTICAL REVIEW OF THE PLANT FAMILY RUBIACEAE. 1.4.1. Naturai Products from Rubiaceae Plants otherthan species. Gardenia erubescens Stapf. & Hutch. belongs to thè family Rubiaceae. The plantein this family and specifically in thè genera Rubia, Galium and have long been known to contain substantial amounts of anthraquinones^l, with roots being especially rich sources of anthraquinones. These compounds are more often present as aglycones and sometimes in thè form of glycosides. As a result of their anthraquinone content many plants of thè Rubiaceae have been used for thè preparation of naturai dyes all over thè world. The best known for this purpose is madder, which is thè ground root of Rubia tinctorum L. from which alizarin is produced. Madder is used through thè complexing of alizarin with metal oxides to produce various coloured dyes^2. The synthesis and synthetic production of alizarin by Graebe and Liebermann in Germany killed thè earlier high revenue yielding French trade on naturally sourced alizarin^ The anthraquinones found in thè Rubiaceae do not have any laxative property54. Some of thè rubiaceous anthraquinones however exhibit very interesling biological in vitro activities such as antimicrobial55, hypotensive^b and antileukemic57-58 properties. In West and East Germany, extracts of thè roots of Rubia tinctorum L. have been used for treatment of kidney stones^4,59 UNIVERSITY OF IBADAN LIBRARY 29 The anthraquinones found in thè Rubiaceae constitute a homogeneous group of compounds, all being derivatives of thè tricyclic structure, 65. Table 2 shows few examples of anthraquinones 66-72 isolated from thè Rubiaceae. The compounds,oruwal, 66 and oruwalol, 67 were isolated by O 65. Adesogan et al^O-61 from Morinda lucida Benth. ,a species found in thè Western part of Nigeria. Another important genus of thè Rubiaceae is Cinchona species. These plants are cultivated for their bark which contains quinine, a potent antimalaria and quinidine, which is useful for cardiac arrhythmia in centrai South America. High yielding varieties of these trees have, for a long time, eamed useful foreign exchange for thè countries cultivating them. In malaria endemie countries of Africa, quinine was for a long time, thè drag of choice for treating this disease. Although synthetic antimalaria drugs like chloroquine have long replaced quinine in thè treatment of malaria because of its toxic side effeets such as impaired hearing on prolonged use, quinine is stili available as quinine hydrochloride UNIVERSITY OF IBADAN LIBRARY 30 solution or tablets and it is stili preferred particularly in thè treatment of chloroquine-resistant strains of malaria. The noreugenin-related alkaloids which is a class of chromone alkaloid have been reported in a genus of Rubiaceae family, that is Only two species in this genus were mentioned in thè review by Peter J. Houghton for their noreugenin-related alkaloid content. These are Schumanniophyton magnificum Harms. and S. problematicum. UNIVERSITY OF IBADAN LIBRARY 31 UNIVERSITY OF IBADAN LIBRARY 32 Table 2 Contd. UNIVERSITY OF IBADAN LIBRARY 33 Table 2 Contd. Ali thè chromone alkaloids reported from thè two plants are of noreugenin-pyridine or piperidine type. Three of these alkaloids were isolated ffom thè root bark of S. problem aticunfib . These are schumanniophytine 73, anicotinic acid noreugenin congener and two other unnamed noreugenin-piperidine-2-one derivatives. UNIVERSITY OF IBADAN LIBRARY 34 Subsequent phytochemical interest was shifted on S. magnificum Harms., a species that was first reported for its noreugenin alkaloid content by Okogun et. al^7. Their investigation on thè Chemical constituents was perhaps motivated by thè medicinal importance of thè plant in Nigeria where thè stem juice serves as an antidote for snakebite^. The two alkaloids first isolated by Okogun et. al. from S. magnificimi were schumannificine 74 and N-methylschumannificine 75. Houghton et. al69.5 later isolated ten other alkaloids, 76-84, in addition to thè first two mentioned above. Two of these, anhydroschumannificine 76 and N- methylanhydro-schumannificine 77, are analogues of 74 and 75. The structures of 74 and 75 were however revised on thè basis of NOE studies on thè N-methyl analogues of schumannificine. The structures 74-84 represent thè chromone alkaloids of Schumanniophyton magnificum Harms. 73. Schumanniophytine. UNIVERSITY OF IBADAN LIBRARY 35 R=CH3. Revised structure. T UNIVERSITY OF IBADAN LIBRARY 36 78, Rohitukine. CH3 HO O 80, N-Methylschumanniophytine. UNIVERSITY OF IBADAN LIBRARY 37 82, Schumaginine, R=H. 83, N-Methylschumaginine, R=CH3. 84, Schumanniofoside. Although Schumanniophyton is thè only genus of Rubiaceae in which chromone alkaloids have been isolated, it is possible that related genera that have shown thè presence of alkaloids in screening procedures might be new sources of thè noreugenin related alkaloids. There are a number of triterpenes that have been reported in Rubiaceae plants, some of which are used locally for medicinal purposes^O, A systematic investigation of thè Rubiaceae of Hong Kong revealed thè presence of thè common phytosterols, (3-sitosterol and stigmasterol, together with triterpene saponins and sapogenins^ 1 -74 From thè information available in thè literature, it seems thè quinovic acid derivatives are thè most common triterpenoids in Rubiaceae p lan ts^ l. UNIVERSITY OF IBADAN LIBRARY 38 Some of thè triterpenoids, 85-95. reported in Rubiaceous plants are represented in Table 3. Table 3 - Triterpenoids of Rubiaceae Plants. UNIVERSITY OF IBADAN LIBRARY 39 Table 3 Contd. UNIVERSITY OF IBADAN LIBRARY 40 Table 3 Contd. UNIVERSITY OF IBADAN LIBRARY 41 Table 3 Contd. UNIVERSITY OF IBADAN LIBRARY 42 Table 3 Contd. 1.4.2. Naturai Products from Gardenia Species. The plants of thè genus Gardenia appear not to be known to contain any anthraquinone for which thè family is well known, as there are no reports of isolation of anthraquinone from any Gardenia Species. There is also no report of thè presence of alkaloid in any of these species. Nevertheless, literature survey on Chemical constituents of Gardenia Species revealed these plants as rich sources of flavonoids, iridoids and triterpenes. A total of fifìteen species have so far been subjected to phytochemical analysis and research work on two of these - Gardenia jasminoides Ellis and G. lucida (Roxb) significanti dominated thè literature available on Gardenia plants. The breakdown of compounds isolated from Gardenia plants together with their sources are presented in tables 4-6 below. The tables give a vivid picture of thè various classes of naturai products in these plants. It would be difficult to use thè presence of any of thè three major classes of compounds as a taxonomic maker UNIVERSITY OF IBADAN LIBRARY 43 as thè tables visibly show that thè occurrence of these compounds do not follow any regular pattern in thè species. The only compound which has been reported in almost all thè species is D-mannitoli Given thè information available in thè tables, it is obvious that Gardenia plants may never approach thè significance of say thè Cinchona species with are respect to their therapeutic uses. The fruits of G. jasminoides Ellis very famous in Japan for its laxative property which has been attributed to thè iridoid constituents76-79 Qut 0f about eight iridoid glycosides 96-103. identified so far (table 4) in thè fruit of this plant, geniposide,_96 and genipin. 97 constitute thè major componentsi in addition to thè iridoid glycosides, thè leaf extract of G. jasminoides also contains a pseudoazulene iridoid, carbinal, 98 which has been identified as a potent anti-fungal compound80-81 This plant was also reported in China for its antifertility principles which was obtained from thè ethyl acetate extract of its flowers. Gardenoic acid, 104 (Table 5), thè active principle has been shown to be an early pregnancy terminating co m p o n en ti A significant proportion of thè constituents from these plants are triterpenoids, 104-119 (Table 5). Most of thè triterpenes reported have thè Oleanane type skeleton and this is indicative of thè preference for thè biosynthetic pathway leading to thè formation of Oleanane-type skeleton. As thè processes involved are enzyme controlled, it is expected that thè same type of enzymes should be present in thè species since their genetic makeup is similar, consequently simitar compounds are produced with same carbon skeleton which differ only in UNIVERSITY OF IBADAN LIBRARY 44 thè degree of substitution and thè extent of oxidative transformation of thè various substituents. The report on isolation of triterpenes of Gardenia plants has followed thè usuai style of research in triterpene chemistry, as it had been thè practice to simply identify thè triterpenes from their naturai sources as a matter of curiosity without any link to their therapeutic properties. However, thè emphasis is gradually shifting in thè direction of thè therapeutic importance of triterpenoids in recent times with thè discovery of anti-tumour and anti-leukemic properties within this group of compounds, particularly among thè quassinoids and limonoids. There is certainly a renewed interest in thè biological properties of triterpenes especially in Japan and China as more published data are now available on saponin constituents of medicinal plants^^-85 The flavonoid constituents of Gardenia plants also present a unique feature as most of thè flavonoids reported in these plants, 120-135 (table 6), are rare flavonoids with unusual A- and B-ring oxidation pattern. UNIVERSITY OF IBADAN LIBRARY 45 UNIVERSITY OF IBADAN LIBRARY 46 Table 4 Contd. c o 2h C 0 2Me HQ z » 0 h o h 2cx ^ \ j l u ^Glu 102. Gardoside^O. 103. Scandoside methyl esterno UNIVERSITY OF IBADAN LIBRARY 47 UNIVERSITY OF IBADAN LIBRARY 48 Table 5 Contd. UNIVERSITY OF IBADAN LIBRARY 49 Table 5 Contd. UNIVERSITY OF IBADAN LIBRARY 50 Table 5 Contd. UNIVERSITY OF IBADAN LIBRARY 51 Table 5 Contd. UNIVERSITY OF IBADAN LIBRARY 52 Table 5 Contd. UNIVERSITY OF IBADAN LIBRARY 53 Table 6- Flavonoid Constituents of Gardenia species. UNIVERSITY OF IBADAN LIBRARY 54 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 55 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 56 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 57 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 58 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 59 Table 6 Contd. UNIVERSITY OF IBADAN LIBRARY 60 1.5 THE FLAVONOIDS. The flavonoids are all structurally derived from thè parent substance, flavone, 136 which in tum has a structure based on thè chromone, 137 skeleton. O 136. Flavone. 137. Chromone. Most members of this class of naturai products are responsive for thè beautiful colours of flowers and fruits in nature. The flavones give yellow or orange colours and thè anthocyanins give red, violet or blue colours. The occurrence of this numerous class of oxygen heterocycles is restricted to higher plants and fems. Mosses contain a few flavonoid types but they are absent in algae, fungi and bacteria. The hydroxylation and methylation pattems appear to be genetically controlled, i.e. thè distribution of flavonoids is a useful tool for classification purposesl05. Biologically thè flavonoids play a major role in relation to insect pollinating and feeding on plants. The flavonoids are structurally characterized by having two hydroxylated aromatic rings A and B, 136 joined by a three carbon fragment giving a C 5 skeleton considered to be composed of two parts, Cg and C9 units. UNIVERSITY OF IBADAN LIBRARY 61 Some eleven major classes of flavonoids are recognised and within each group there are members at various oxidation levels. The major classes of flavonoids are presented in table 7. Table 7 - classes of flavonoids UNIVERSITY OF IBADAN LIBRARY 62 1.5.1. Determination of Structure of Flavonoids. Over thè years, UV spectroscopy has become a major technique for thè structure analysis of flavonoids. The main reason being that thè amount of structural information gained from a UV Spectrum is considerably enhanced by use of specific reagents which react with one or more functional groups on thè UNIVERSITY OF IBADAN LIBRARY 63 flavonoid nucleus. The second reason is that only a small amount of pure material is required. 1.5.2 UV/Visible Spectroscopy The UV spectra of most flavonoids consists of two major absorption maxima, one of which occurs in thè range 240-285nm (band II) and thè other in thè range 300- 400nm (band I). The band II absorption may be considered to be from thè A-ring benzoyl System and band I from thè B-ring cinnamoyl System. The position of absorption of bands I and II provide a guide to thè type of flavonoid. The major absorption bands for thè different classes of flavonoids is shown in table 8. The degree of hydroxylation of thè flavone skeleton determines to a large extent thè position of thè bands. Within thè same class, highly oxygenated members tend to absorb at longer wavelengths than those with fewer oxygen substituentslOó. Methylation or glycosylation of hydroxyl groups on thè flavonoid nucleus usually result in hypsochromic shifts, particularly of band I, while acetylation tend to nullify thè efifect of thè phenolic hydroxyl groups on thè spectra. Table 8 - Major Absorption Bands of Flavonoids. Band I (k,nm) Band II nm) Class of Flavonoid. 304-350 250-280 Flavones 352-385 250-280 Flavonols 300-340 245-270 Isoflavones UNIVERSITY OF IBADAN LIBRARY 64 Table 8 Contd. Band I (A., nm) Band II (À, nm) Class of Flavonoids. 300-340 270-295 Flavanones & Dihydro- flavonols 340-390 220-270 Chalcones 370-430 220-270 Aurones 465-550 270-280 Anthocyanidins & Antho-cyanins The position of hydroxyl groups and thè pattern of hydroxylation can be determined by thè use of specific reagents which induce structurally significant shifts in thè UV spectrum'07 The reagents commonly used are sodium methoxide (NaOMe), sodium acetate (AcONa), sodium acetate/boric acid (Na0Ac/H3B03), aluminium chloride (AICI3) and aluminium chloride/hydrochloric acid (AICI3/HCI). All hydroxyl groups on thè flavonoid nucleus are ionized to some extent by sodium methoxide. Hence for most hydroxylated flavonoids, shifts to longer wavelength are observed in both bands. The most significant and informative of thè shifts are viz. thè bathochromic shift of 40-65nm in band I without a decrease in intensity which indicates thè presence of a 4’-hydroxyl group in flavones and flavonols. Flavonols lacking 4'-hydroxylation also produce a 50-60nm red shift, but with a decrease in intensity. UNIVERSITY OF IBADAN LIBRARY 65 For flavanones, Isoflavones and dihydroflavonols, a consistent 35-40nm bathchromic shift of band II suggests thè presence of 5,7-dihydroxyl groups. Sodium acetate being a much weaker base than sodium methoxide tend to ionize only thè more acidic phenolic hydroxyl groups. Flavones and flavonols possessing a 7-hydroxyl group exhibit a 5-20nm bathochromic shift of band II vvhile thè presence of 7-hydroxyl group in isoflavones also produces a band II bathochromic shift of 6-20nm but 5,7-dihydroxy flavanones and dihydroflavonols give a shift of 60nm. A mixture of sodium acetate and borie acid is used for thè detection of o- dihydroxyl groups in flavonoids. Flavones and flavonols containing 0 -dihydroxyl groups in ring B give a 12-30nm bathochromic shift of band I while ring A ©- dihydroxyl groups give rise to lesser shifts. But thè isoflavones, flavanones and dihydroflavonols show a 10-I5nm bathochromic shift only in band II when o- dihydroxyl groups are present on ring A. Aluminium chloride chelates with functional groups such as thè 5-hydroxy- 4-keto, 3-hydroxy-4-keto and o-dihydroxyl groups. The reactions produce bathochromic shifts of one or both bands. The complex formed with 5-hydroxy-4- keto groups is stable in acid while thè complexes formed with o-dihydroxyl groups and thè 3-hydroxyl-4-keto groups decompose in acid medium. Hence treatment of a flavonoid solution with Aluminium chloride and then followed by addition of aqueous hydrochloric acid produces bathochromic shifts which may or may not disappear by addition of thè aqueous acid depending on thè type of groups involved in thè complex formation. UNIVERSITY OF IBADAN LIBRARY 66 The presence of a 5-hydroxyl group in a flavone gives a band I bathochromic shift of 35-55nm on addition of AICI3/HCI. Flavones with 3-or 3 and 5-hydroxyl groups however show a bathochromic shift in band I of 50-60nm. When a 5-hydroxyl group is present in isoflavones, a 10-14nm band II red shift is observed while flavanones and dihydroflavonols give a 20-26nm band II bathochromic shift on addition of AlCiyHCl reagent. 1.5.3 Proton NMR Spectroscopy Initially t.ve use of NMR for structural studies on flavonoids was confined to thè relatively non-polar flavonoids such as thè isoflavones and highly acetylated or methylated flavones which are soluble in solvents like CDCI3 and CCI4. However, thè introduction of DMSO-d^ and thè use of TMS-ether derivatives now make it possible for thè bulk of naturally occurring flavonoids to be studied by this method. A lot of information which has accumulated over thè period of studies on NMR spectroscopy of flavonoids have made it possible for NMR signals to be assigned tentatively to structural features. The distinguishing feature for such class of flavonoids seem to be endowed in thè signals originating from ring C. Hence flavones and isoflavones are readily differentiated by using thè position of C-2 and C-3 proton signals.Likewise, flavanones and dihydroflavonols are also ditTerentiated on thè basis of C-2 and C-3 proton signals (Table 9). The pattern of thè signals of thè aromatic rings A and B gives an indication of thè substitution pattern on these aromatic rings^^ UNIVERSITY OF IBADAN LIBRARY 67 The presence of hydroxyl protons are shown by signals in thè range 9.70- 12.40 ppm and thè disappearance of these signals on addition of D2O confirms their identity as hydroxyl proton resonance. Table 9 - Chemical shift data for C-2 and C-3 protons in flavones, isoflavones, flavanones and dihydroflavonols. Class of Compound H-2 (ppm) H-3 (ppm) Flavone 6.3 (s) Isoflavone 7 6-7.8 (s) Flavanone 5.0-5.5 (q) 2.8 (qq) Dihydroflavonol 4.8-5.0 (d) 4.1-4.3 (d) 1.5.4 «3C NMR Spectroscopy The application of 1 ̂ C-NMR spectroscopy in structural studies of flavonoids has assumed an interesting dimension, although it is essentially complementary to 1 H NMR. By spectral correlation, it is possible to determine thè structural features of an unknown flavonoid using a combination of 1H and 1 NMR data. Flavones, flavanones and dihydroflavonoids are readily distinguished by thè position of thè C4 s ig n a l^ The value of thè C4 signal is also indicative of thè substitution on C5 (Table 10). Generally thè carbon atoms of rings A and B fall into thè region for aromatic carbons with shifts corresponding to thè oxygenation pattern on thè rings. UNIVERSITY OF IBADAN LIBRARY 68 Table 10 - Chemical shift data for C4 signal in some flavonoid compounds. Compound. C4 (PPm) 5-Hydroxyflavone 181-182 5-Methoxyflavone or 5-Glycosides 176-177 5-Hydroxyflavanone/Dihydroflavonol 195-197 5-Methoxyflavanone or 5-Glycosides/ 189-192 Dihydroflavonol The Chemical shifts for thè different types of carbon present in flavonoids are presented in Table 11. Table 11 - Chemical shifts characteristic of flavonoid functional groups. Type of Carbon Chemical Shift (ppm) Carbonyl 200-170 Aromatic 140-125 (without oxygen substituent) 167-150 (C9,C7,C5) 110-90 (C6,C8,Ciò) Ethylenic 160-165 (with oxygen substituent) 100-110 (without oxygen substituent) Carbinolic 77-79 (C2 of flavanones) UNIVERSITY OF IBADAN LIBRARY 69 1.5.5 Mass Spectrometry The use of mass spectrometry has been a valuable tool in determining thè structures of flavonoids especially when small quantities of compounds are available. Electron impact (EI) mass spectrometry has been applied successfully to all classes of flavonoid aglycones and more recently to a number of different types of glycosides. Chemical ionization (CI) is less frequently utilized. It has only been applied to a few aglycones and gives few diagnostic fragments except for flavanones and dihydroflavonols. Fast atomic bombardment (FAB) on thè other hand is becoming popular as regards thè determination of thè structures of flavonoid glycosides. Most flavonoid aglycones give intense peaks for thè molecular ion [M+] which is often thè base peak. In addition to thè molecular ion, flavonoid aglycones also give major peaks for [M-H]+ and when methoxylated, [M - CH3]+ . With respect to flavonoid identification, thè most useful fragmentations have been found to be those which involve cleavage to give rings A and B fragments ̂ 6 The common fragmentions of flavonoids are labelled as pathway I and II as in scheme I. Pathway I corresponds to a retro-Diels-Alder cleavage and it produces two ions A ]+, and Bj + while pathway II gives a single charged species, B2+. UNIVERSITY OF IBADAN LIBRARY 70 Scheme I: Major fragmentation pattern of flavonoids Pathway I v \ c o Bi+- A l + - Pathway II B2+ The diagnostic fragmentation pattems for flavones and flavanones are illustrated in Scheme II. UNIVERSITY OF IBADAN LIBRARY 71 most Although thè base peak for flavonoids is thè molecular ion, [M+], peaks are usuaily prominent in thè spectra for [M -CO]+ and [A^ -CO]+ in addition to thè fragments A \ + and B]+. Substitution in thè A-ring can be detected by examining thè m/z value of thè A |+- fragment. Similarly, thè m/z value of B2+ fragment can be useful in determining thè substitution in ring B. 1.5.6 Biosynthesis of Flavonoids Studies on genetic aspect of colour and thè Chemical speculations on thè mode of formation of thè carbon skeleton of this class of compounds stimulated interest in thè biosynthesis of flavonoids. Tracer Studies were first applied to thè problem using intact plants or plant tissues. From thè results of thè studies, thè precursors were identified and this led to an understanding of some details of thè biosynthesis of flavonoids 105-106 The basic skeleton of thè flavonoids was found to arise from three malonyl CoA units, 148 and a cinnamoyl Co A, 149. The flavonoids are thus produced by folding of thè polyketide as shown in scheme III. UNIVERSITY OF IBADAN LIBRARY 72 +* ( Pathway I ) ^2 ( Pathway II ) f ^ Y ° À1+- ^ ^ ^ C = 0 +H [A i+ H ]+ 139. Flavanone Scheme D: Fragmentation pathways far flavones and flavanones UNIVERSITY OF IBADAN LIBRARY 73 Scheme III: Biosynthesis of thè flavonoid basic skeleton OCoA + 3 HOOCCH2COC0A 148 149 Polyketide 151. Flavanone 152. Flavone The ring A of most flavonoids has a phloroglucinol structure but it has been proved by labelling experiments that phloroglucinol is not on thè pathway nor is phloroglucinol cinnamate. Result of studies also showed that thè flavanone UNIVERSITY OF IBADAN LIBRARY 74 synthase complex is rather specifìc for p-OH-cinnamic acid which suggest that final hydroxylation of ring B takes place at thè C15 stage. O-methylation and glycosidation are also modifications taking place at thè final stages^^. The anthocyanidins are biosynthesized from flavanones via dihydroflavonols (scheme IV). The flavanones or chalcones are also precursors for thè isoflavones. The exact nature of this rearrangement is unknown but a plausible mechanism proposed by T orssel^5 involves formation of a diradicai which then undergoes radicai combination to a cyclopropanoid intermediate. The intermediate compound looses a proton to give thè isoflavones (scheme V). The rotenoids which are used as fish po isons and as insecticides, are structurally related to thè Isoflavones. The biosynthesis of rotenone has been studied in seedlings of Amorpha fruticosa. It was shown that rotenone is biosynthesized from formononetin, 163 by extra hydroxylation and methylation in ring B, 164 ,then hydrogen abstraction from thè methyl followed by radicai cyclization and isoprenylation gives rotenoic acid 165. Expoxidation of thè isoprenoid doublé bond of rotenoic acid, then cyclization via opening of thè epoxide and finally dehydration of thè tertiary alcohol gives rotenone 105 (scheme VI). UNIVERSITY OF IBADAN LIBRARY 75 157. A tiavonol Scheme IV: Biosynthetic route for flavonols and cyanins UNIVERSITY OF IBADAN LIBRARY 76 160, A diradicai 161. Cyclopropanoid intermediate HO O OH 162. Genistain (an isoflavone ) Scheme V. Biosynthesis o f Isoflavones UNIVERSITY OF IBADAN LIBRARY 77 Scheme VI. Biosynthesis of Rotenone Dalrubone is a rare flavonoid compound which has an unusual oxygenation pattern in that rings A and B appear to be reversed. The reversai has been explained as involving a 1,3-carbonyl transposition in thè chalcone in thè normal biosynthesis, effected by (3-oxidation, reduction and elimination of water (scheme VII), The proposed pathway was supported by results of feeding experiment^5 UNIVERSITY OF IBADAN LIBRARY 78 jCOCoA 148 149 HO HCN + ^ H<°VN V À y 1 HO O 168 H3CO\ ^ K ^ ° O 170 Scheme VII: Biosynthesis of Dalrubone The brief account of thè biosynthesis of flavonoids given above is inevitably selective. However an attempt has been made to mention thè important features of thè ftavonoid biosynthesis. UNIVERSITY OF IBADAN LIBRARY 79 1.5.7 Biological Importance of Flavonoids Flavonoids are phenolic and thè implication is that they can react with They speci fi c receptor groups primarily by hydrogen bonding*. have been identified to show a variety of physiological properties as a consequence of their ability to complex with enzymes and metal ions in thè biological System. Many unrelated types of secondary constituents have been associated with phytoalexin responses in plants, and flavonoids are not left out in this respect. Many phytoalexins have been identified to be flavonoids and most of them are pterocarpanslO^. Another property similar to phytoalexin activity is thè antibiotic effect of many llavonoid compounds. A good number of them have been examined for their antibacterial, antiviral and antihelmitic activities and they are found to be generally effective110. Flavonoids have also been implicated as anticancer agents. Several flavonoids are reported to be moderately effective against laboratory cultures of malignant celisi 05. Some flavonoids have also been reported to show oestrogenic activity. Isoflavones are thè only class of flavonoids so far recognised to possess this activity. The oestrogenic activity was attributed to thè stilbene-like structure of thè compounds which make them structurally similar to potent synthetic oestro gens like diethylstilboestrol. Flavonoids are also known to have beneficiai effects on vascular damage. This discovery was accidental. Recent research has revealed that flavonoids act on blood celi aggregation, a phenomenon that generally accompanies illness and UNIVERSITY OF IBADAN LIBRARY 80 injury^ 6 The action of flavonoids on celi aggregation is therefore consistent with thè beneficiai effects on capillaries and in disease condition since aggregation enhances symptoms of disease and induces pathology. 1.6 THE TERPENOIDS. The name terpene was initially given to thè group of compounds, mostly composed of thè fragrant principles of plants, recognised by Wallach to be built up of branched C5 units called isopentenyl or isoprene unit. Although isoprene unit was later perceived not to be thè functional unit used by nature, it nevertheless provided a useful device for rationalizing thè structures of many more complex compounds of higher molecular weight. The isoprene rule was subsequently formulated to ftccommodate a l l compounds found to be built up of C5 units. It States that terpenes are multiples of C5 units linked together head to tail. Based on thè number of C5 units, thè terpenes are classified as monoterpenes (Ciò) sesquiterpenes (C15), diterpenes (C20X Sesterterpenes (C25), triterpenes (C30) and tetraterpenes (C40). However, thè steroids and several other related compounds do not obey this rule apparently because degradation had occured, thè head to tail principle was violated or there had been rearrangements of thè originai skeleton. The terpenes house a wealth ofsignificant compounds. Most importantly, there are enormous number of terpenes which are physiologically very active compounds. The triterpenes which are built up from six isoprene units are formed by cyclization of squalene. Squalene is in tum formed from tail to tail condensation of UNIVERSITY OF IBADAN LIBRARY 81 two C15 units. Generally all triterpenes are recognised as being formed by cyclization of ali-trans squalene in various chair-boat conformational sequences. Structurally, thè triterpenes show limited variations in skeletal modifications when compared with thè lower terpenes like thè sesquiterpenes. A salient feature of nearly all triterpenes is thè equatorial hydroxyl group at C-3 as represented by thè basic tetracycic triterpene skeleton, 172. 28 HO 172 1.6.1. Classifìcation of Triterpenes. Triterpenes can be classified into two main groups, thè tetracyclic triterpenes that include thè sterols and thè pentacyclic triterpenes. 1.6.2. Tetracyclic Triterpenes. The group consists of thè sterols, lanostane, euphane and dammarane compounds. The various subgroups of thè tetracyclic triterpenes are illustrated in Table 12. UNIVERSITY OF IBADAN LIBRARY Table 12- Representative Structures of Tetracyclic Triterpenes. UNIVERSITY OF IBADAN LIBRARY 83 Table 12 Contd. UNIVERSITY OF IBADAN LIBRARY 84 1.6.3 Pentacyclic triterpenes. A number of groups can be distinguished within thè pentacyclic triterpenes. These include Oleanane, Ursane, hopane, lupane etc types. Table 13 shows thè characteristic C30 skeleton associated with each group. Table 13-. Basic C30 Skeleton of thè different groups of pentacyclic triterpenes. 183. Lupane 184. Hopane UNIVERSITY OF IBADAN LIBRARY 85 Table 13 Contd. 1.6.4 Degraded Triterpenes. The quassinoids and limonoids represent important groups of triterpenes in which extensive degradation had taken place. Consequently, they do not show any structural similarily with any of thè major groups already mentioned above. Hence it becomes imperative to treat them separately as unique groups of triterpenes. Few examples of these two groups of compounds, 189-192, are shown in table 14. UNIVERSITY OF IBADAN LIBRARY 86 Table 14-: Examples of Quassinoids and Limonoids. 1.6.5 Determination of structure of Triterpenes. Reports on studies of thè chemistry of triterpenes started to appear in thè early thirties. At that time thè laboratories were devoid of IR, NMR and Mass >pectrometers. Therefore thè characterization of compounds rested mainly on "leasuring m..p, b.p, 8, np> and using combustion analysis. UNIVERSITY OF IBADAN LIBRARY 87 Structure eludidation was largely accomplished by oxidative degradations and thè most important of all was thè pyrolysis in thè presence of sulphur or determine selenium. These reactions had been used to thè structures of cyclic sesquiterpenes and diterpenes. This experience together with employing thè 'Isoprene rule' as a connective guide-line in many cases led to establish thè correct constitution of thè triterpenes. Even thè relative configuration of thè substituents were determined without spectroscopy by correlation with known diterpenes 111. 1.6.6 Structure determination by degradative Methods. The structures of triterpenes were characterized mainly by degradative methods before thè advent of spectroscopy. Elucidation of structures of many pentacyclic triterpenes were carried out by dehydrogenation with selenium and identification of 1,8-dimethylpicene and 1,8- dimethyl-2-hydroxypicene as products of thè reaction. Compounds which gave these two products on dehydrogenation are taken to be structurally related to P- amyrin. The position of C=C and methyl groups in thè compounds were also determined through a series of Chemical transformations usually involving oxidative cleavage of thè C=C with subsequent pyrolysis which eventually produced fragments containing fewer rings. The products of these transformations were then correlated with known compounds 111. The structure of isomerie compounds generally presented challenging experience. However, through a sequence of Chemical transformations many of thè problems posed by such compounds were solved. For instance, thè structure UNIVERSITY OF IBADAN LIBRARY 88 elucidation of glycyrrhetic acid and thè identification of thè four soyasapogenols, A, C, B and D were particularly challenging problemsl 11. In recent times, due to thè availability of spectroscopic tools, structure elucidation by degradative methods has taken a new dimension. Only few Chemical transformations are now necessary because derivatives of unknown compounds are now frequently identified by spectral correlation with known compounds. The derivatives commonly employed in thè establishment of structures of triterpenes are acetate and methyl ester derivatives. While thè acetate derivatives help to confimi thè presence and number of hydroxyl groups, their spectral data together with thè melting point provide thè basis for comparison with thè spectra and melting point of authentic or known compounds 112. The methyl ester derivatives similarly confirm thè presence of carboxylic acid group and are also useful for comparison with available data on known compounds 113 Other derivatives such as oxo-compounds are often used in solving structural problems of unknown triterpenes 114. The location of carboxylic acid group on C 7 of an Oleanene or Ursene skeleton is easily established by thè formation of a y-lactonel 15, 193, from a A^Oleanene or Ursene compound, 194. UNIVERSITY OF IBADAN LIBRARY 89 C O O H Some other important transformations used in structural elucidation include conversion of unknown compounds into other known compounds for easy comparison. The reactions commonly used are simple organic reactions such as reduction, oxidation, dehydration, etc. For example bayogenin, 195 and castanogenin, J_96 were both reduced to thè same tetrahydroxyl compound, 197. This result provided a basis for comparison between thè two compounds 115. UNIVERSITY OF IBADAN LIBRARY 90 Another example is thè conversion of siaresinolic acid methyl ester, 198 into 19a -hydroxyerythrodiol, 111 by reduction with L1AIH4I16. The position of thè hydroxyl groups in thè compound 198 was thus identified by comparison with thè structure of 111. UNIVERSITY OF IBADAN LIBRARY 91 The nature of Chemical transformation used in structure elucidation tend to vary with thè structural problems presented by thè unknown compound. Sapogenins are commonly subjected to acid hydrolysis for full characterization of thè aglycone and thè sugar moieties. Where it is necessary to differenciate between a glycosidic and an ester linkage, both acid and alkaline hydrolyses are carried out^ 16-117 Chemical degradative method contributed significanti to structure elucidation of triterpenoids before spectroscopy became available. However, it was very diftìcult at that time, tos uchhasrtaactnecriezse compounds isolated in small quantities because large quantities of are usually needed to go through thè host of transformation sequences. Nevertheless, this method has contributed immensely to thè development of conformational chemistry, understanding of mechanisms and development of elegant synthetic methods. UNIVERSITY OF IBADAN LIBRARY 92 1.6.7 Ih Nuclear Magnetic Resonance Spectroscopy. The study of thè NMR spectra of triterpenoids was initiated with thè hope of providing a catalogue of spectra which would be useful in spectral correlations in thè structural elucidation of new compounds. Shamma et a l118 studied thè 1H NMR spectra of a series of pentacyclic triterpenes. They found distinct absorptions for methyl esters, acetoxyl groups and angular methyl groups. Certain other functional groups such as vinylic protons, protons a-to hydroxyl or acetoxyl and methylene protons were also found to be c lu e s important because they gave to certain structural features of thè triterpenes. A number of empirical rules emanated from thè results of their studies with respect to thè Chemical shift of common functional groups as a consequence of thè position of such groups and or its interaction with neighbouring groups. In thè case of C-methyl groups, it was noticed that thè Chemical shift of thè W2l S higest C-methyl group particularly indicative of thè position of thè carbomethoxyl function if present in thè molecule. The conclusion was that if a C- 28 carbomethoxyl function is present in a triterpene of thè ursane or oleanane series, thè highest C-methyl absorption peak appears upfìeld from 8 0.775. WSlS Alternatively, when C-28 position represented either by a hydroxymethylene, a methyl group or a lactone, thè highest C-methyl absorption peak appears downfield from 8 0.775. The lupanes were also found to conform to this rule (Table 15). The position of thè absorption of thè methoxyl moiety of a methyl ester is also partially indicative of thè relative position of thè carbomethoxyl group in thè triterpene molecule. The observation was that absorption of a C-28 methyl ester UNIVERSITY OF IBADAN LIBRARY 93 belonging to thè Oleanane or Ursane group is usualy upfield from 8 3.595 while thè carbomethoxyls located in other positions such as C-24 or C-30 absorb further downfield in thè region from 8 3.595-3.650 (Table 16). The reason for thè differences in thè positions of thè methyl ester absorptions is not known. It may be related to thè fact that thè C-28 carbomethoxyl function is extremely hindered or that this functional group is influenced by thè magnetic anisotropy of thè 12(13) doublé bond. Vinyl proton absorption of thè normal trisubstituted doublé bond, usually of 12|( 13) position in thè ursane and oleanane series was found to be in thè region between 84.93 and 85.50. If a terminal doublé bond is present, such as in lupane series, thè vinyl protons absorb at higher field around 4.30 to 5.07. Another useful and characterisitc absorption is thè vinylic methyl function, CH3-C=C. Normal methyl groups absorb from 80.63 to 81.50 but vinylic methyl peaks are found to appear between 81.63 and 81.8 and are usually sharp and well defined. Many triterpenes of thè lupane class have vinylic methyl groups and these are easily recognised. Thus betulin diacetate, melaleucic acid methyl ester and thurberogenin acetate exhibited vinylic methyl peaks at 81.67, 1.64 and 1.80 respectively. Acetoxyl protons are noted to give thè sharpest absorption of any function in thè triterpene serires, with majority of these protons absorbing between 81.92 and 81.97. There is however no clear differentiation between axial and equatorial acetoxyl functions in thè triterpenes as was thè case in cyclic polyol acetates^ ' 8 The acetoxyl groups of 1,2-glycols were observed to appear at higher fields (81.85- UNIVERSITY OF IBADAN LIBRARY 94 1.92) than thè aeetoxyl groups of analogous monoacetates. Thus vicinai glycols may be easily recognised by this method. The absorptions of protons a-to aeetoxyl goups usually appear between 8 3.65 to 5.60. This type of absorpstieocno fnadll airntyo two categories depending on whether thè aeetoxyl group is primary or *For secondary acetoxy groups, axial - protons are found between 84.00 to 4.75 while thè equatorial a-protons absorb at between 85.00 to 5.48. The-protons a- to primary aeetoxyl groups generally show absorptions at higher fields, beween 83.65-5.20. Protons -a- to vicinai aeetoxyl groups are found to appear at much lower field between 84.9-5.4, however, these values should be used with discretion as it is not easy to clearly difìferentiate between these protons if only thè one dimentional ^HNMR is considered (Table 17). Although thè methyl groups in triterpenes usually appear as sharp peaks at thè right end of thè spectrum, their absorptions are frequently found to overlap. The assignments to most of thè methyl groups are therefore not easy to make. From thè investigation of thè NMR spectra of some deuterated derivatives of thè - A* 2-Oleanene and A^-Ursene by Karliner and Djerassil 19, assignments were made for thè methyl groups in thè NMR spectrum of À^-Oleanene (table 18). The values shòwn in table 18 can only serve as a guide because thè positions of absorptions of angular methyl groups depend to a large extent on thè substitution pattern on thè carbon frame-work. UNIVERSITY OF IBADAN LIBRARY 95 Table 15 - Chemical Shift of Highest C-methyl groups. S/No. Triterpene Chemical Shift (ppm). 1. Arjunolic acid 0.683. methylester triacetate. 2. Echinocystic acid methyl 0JZ13. ester diacetate. 3 Oleanolic acid methyl 0.730 ester. 4. Ursolic acid methyl ester 0.735 acetate. 5. ct-Amyrin benzoate. 0.865 6. Erythrodiol diacetate. 0.875 7. Lupanol. 0.803 8. Betulin diacetate. 0.840 1.6.8 ]\MR Spectroscopy. 13C NMR has always played a complQnentary role to Ih NMR in structural elucidation of naturai products. In thè determination of structures of triterpenes it is significantly useful in providing a confirmatory evidence for thè presence of certain functional groups. The presence of thè functional groups listed in table 19 are readily recognised by mere inspection of NMR Spectrum of thè UNIVERSITY OF IBADAN LIBRARY 96 compounds. The table shows thè Chemical shifts of thè functional groups commonly encountered in triterpenoid compounds. Table 16 - Absorptions of Methyl Esters. S/No. Triterpene. Methoxyl Position of absorption. Carbomethoxyl function. 1. Ursolic acid 3.578 C-28 methyl ester. 2. Cochalic acid 3.570 C-28 methyl ester. 3. Echinocystic acid 3.595 C-28 methyl ester. 4. 11-Keto-a- 3.597 C-24 boswellic acid methyl ester. 5. Glycyrrhetic acid 3.645 C-30 methyl ester. UNIVERSITY OF IBADAN LIBRARY Table 17 - Absorptions of Protons Alpha to acetoxyl group. S/No. Triterpene. Absorptions of a- Type of Acetoxyl protons. group. 1. Erythrodiol 3.85 io diacetate. 2. Betulin diacetate. 4.05 io 3. Soyasapogenol B 4.22 io triacetate. 4. Chichipegenin 5.81 20 tetra-acetate. 5. Longispinogenin 5.62 20 triacetate. 6. Arjunolic acid 4.98 vicinal(2,3) methyl ester triacetate. 7. Asiatic acid 5.00 vicinal(2,3) methyl ester triacetate. 8. Al-Barrigenol 5.54 vicinal(l 5,16) penta-acetate. UNIVERSITY OF IBADAN LIBRARY 98 Table 18 - Absorption of Methyl groups of À^-oleanene. 5-Value 1.13 o.97 0.93 0.87 0.83 Methyl 27 25 24 23,29,30 26,28 grouP-__________________________________________________________ Table 19 - Chemical shifts of common functional groups of triterpenoids. Functional Group. Chemical Shift (ppm). C-OH(l°) 60-63 C-OH(2°) 71-74 C-OH(3°) 75- -COOH 180-182y 138-145\ H / 121-125 c\ — c h 2 105-110 The position of thè Chemical shift of olefinic carbons in triterpenoids usually gives a strong indication of whether it is an Oleanene, a ursene or a lupene UNIVERSITY OF IBADAN LIBRARY 99 compound as thè absorptions are characteristic of these classes of triterpenoids12{J~ 122 (Table 20). Table 20 - Characteristic Absorptions of Olefmic carbon atoms of Triterpenoids. Class of Position of Olefmic Carbon Compound. C l2 Cl3 C20 C29 Oleanene 122-126 140-145 - - Ursene 125-129 138-139.5 - - Lupene - - 150- 106-109 There has been a remarkable development in NMR spectroscopy in thè past decade. The use of 2D NMR spectra in form of COSY, DEPT, HET 2DJ, HETCOR etc. now provide additional powerfull tool for structure determination. 1.6.9 Mass Spectrometry. Mass spectrometry has been used in recent times. to an increasing extent for thè structure elucidation of triterpenoids. Before thè early 60s very little was known about thè mass spectra of this group of compounds apparently because of their low volatility. Budzikiewicz et a ll22 however took thè lead when they carried out investigation into thè mass spectra of some pentacyclic triterpenes. UNIVERSITY OF IBADAN LIBRARY 100 The possibilities and limitations of thè use of thè mass spectra as a diagnostic tool for unknown triterpenoids were also discussed. Since then, other reports 119 have been published on thè mass spectra of trierpenes and they seem to confimi thè observations made by Budzikiewicz et al. 1.6.10 Mass Spectrum of Oleanenes and Ursenes. The most characteristic fragmentation of all compounds of this class is thè retro-Diels-Alder cleavage of ring C when thè 12-13 doublé bond is present. The retro-Diels-Alder fragmentation produces species a, which is subject to further fragmentation and a neutral fragment b(Scheme Vili). Charge retention with thè fragment b can be observed to some extent but is usually of minor importance. Fragment a consists of rings D and E as shown by thè fact that substitution in rings A and B does not change thè mass, while alterations in rings D and E result in thè appropriate mass shifts. Thus thè unsubstituted parent hydrocarbon P-amyrin 199a yields fragment a of m/z 218. Methyl (3-boswellonate 19% gives a with same mass (m/z 218) as 199a while thè isomerie ether, methyl Oleanonate 199c with carbomethoxy group at C-17, exhibits species a at m/z 262 as does methyl 11- deoxyglycyrrhetate 199d. Erythrodiol diacetate 199e and thè isomerie diacetate 199f both give fragment a at m/z 276. This typical retro-Diels-Alder fragmentation leading to species a has become a useful diagnostic tool for thè presence of 12-13 doublé bond in thè oc- and P-amyrinsl 19. UNIVERSITY OF IBADAN LIBRARY Scheme Vili. Fragmentation pattern of Oleanenes and Ursenes. UNIVERSITY OF IBADAN LIBRARY 102 Ri R2 R3 R4 R5 199a. H2 Me Me Me H b 0 C02Me Me H Me c 0 Me CC>2Me Me H d (H)OHMe Me CC>2Me H e (H)OAc Me CH2OAc Me H f (H)OAc Me Me CH2OAc H Ion a further fragments to yield species c(Scheme Vili), Thus in methyl ' oleanoate 119c 59 mass units are lost to give species c while 15 mass units are lost in rings D and E of unsubstituted substances. The loss of methyl may not be exclusively from thè angular C-17 position but it has been documented that when thè C-17 substituent is a methyl group, its loss from species a is not very pronounced. However, when C-17 substituent is a carbomethoxy or lactone group thè intensity of species c equals or slightly exceeds that of a, while fragment c is several times more intense than a when thè angular substituent is CH2 OAc. Removai of these substituents from positions other that C-17 is also less pronounced. Consequently, loss of -COOMe group from a derived from 199d amounts to about 10% of thè intensity of a. Similarly for 199f. thè loss of CH2OAC from a does not exceed 25% of thè intensity of a, while thè abundance of species c from thè isomerie 199e is about ten times that of species a. Hence thè relative intensities of fragments a and c offer an important indication about thè attachment of a substituent at C-17. UNIVERSITY OF IBADAN LIBRARY 103 Species c decomposes to fragment f by loss of 70 mass unit. This cleavage is due to thè partial loss of ring E. Species c is always accompanied by a less intense ion d, 15 mass units lower. It is probably formed by a one-step process from species a (Scheme Vili). Species a is also accompanied by fragment e of relatively low abundance and containing 13 mass units less. It is probably formed by one hydrogen transfer and cleavage of an allylically activated bond (Scheme IX). Another important fragment, g contains rings A and B as determined by thè appropriate shifts upon substitution in these rings. In thè hydrocarbon p-amyrin, it is found at m/z 191 while in thè 3-ketone methyl oleanonate, it is shifted to m/z 205. A 3 - hydroxyl and 3-acetoxyl group causes shifts to m/z 207 and 249 respectively. The formation of g seem to involve transfer of one hydrogen atom (Scheme IX). Generally introduction of a keto group at C-6, C-16 or C-12 does not afifect thè fragmentation path of thè A^-unsaturated oleanenes and ursenes but only causes additional fragmentations. The existence of a carbonyl function dose to thè centers of principal fragmentation can change thè mode of cleavage as shown in thè mass spectra of 15-ketoerythrodiol diacetate 200 and methyl glycyrrhetate 201. 15- ketoerythrodiol diacetate does not show thè expected fragment at m/z 290 but its most abundant ion occurs at m/z 291. This ion i results from thè hydrogen transfer process similar to that observed in 15-keto steroids ' 24 (scheme X). UNIVERSITY OF IBADAN LIBRARY 104 Scheme IX. Formation of ions e and g from thè oleanenes and ursenes. In methyl glycyrrhetate thè expected fragment a (m/z 276) is present but in addition another fragment j at m/z 317 with higher intensity can be found. Its formation is also adduced to hydrogen transfer process (scheme XI). UNIVERSITY OF IBADAN LIBRARY 105 Scheme XI. Fragmentation of methyl glycyrrhetate to give ion j. UNIVERSITY OF IBADAN LIBRARY 106 1.6.11. Mass spectrum of A^-Taraxerenes. The mass spectra of Ibis group of pentacyclic triterpenes show a similar retro-Diels-Alder decomposition as observed with oleanenes and ursenes. The charged fragment k is also a diene but now comprises of rings A.B and C. Fragment k exhibits a mass which changes with alterations in rings A, B and C. Fragmeru k exhibits a mass of m/z 300 in taraxerone 202a. m/z 302 for taraxerol, 202b and m/z 344 for myricadiol diacetate, 202c due to thè alterations in thè C-3 substituent. lon k is accompanied by an ion k', 15 mass units lower, which is formed by thè loss of a methyl group, thè allylically activated one at C-8 (Scheme XII). UNIVERSITY OF IBADAN LIBRARY 107 202a. Rj= O, R2= Me b, Ri= (H)OH, R2= Me c, R]=(H)OAc, R2= CH2OAc Scheme XII. Characteristic R1 fragmentation o f Al4-Taraxerenes In addition to these two fragments, a very abundant fragment at m/z 204 for taraxerone and taraxerol is characteristic. The fragment 1, is derived from rings D and E as verified by thè spectrum of myricadiol which contains a rather small peak at m/z 262 but an abundant one at m/z 202 (I-CH3 COOH). The formation of 1 is proposed to involve thè fission of C-l 1-12 and C-8-14 bonds (Scheme XIII). UNIVERSITY OF IBADAN LIBRARY 108 Ioni Scheme XII1. Fomiation o f ions 1 and 1' from A14-Taraxerenes. Further fragmentation of 1 produces fragment I-CH3, or I-CH2 OAc depending on thè substituent on C-l 7 to give 1', m/z 189. 1.6.12 28-Nor-Àl708)-Oleanenes. The most abundant fragment ion is given by retro-Diels-Alder cleavage of ring D. 28-Nor-A^ 7(^)-oleanen-3-one and thè corresponding alcohol thus exhibit their most abundant fragment ion, 0 at m/z 163 (Scheme XIV). A strong peak at UNIVERSITY OF IBADAN LIBRARY 109 m/z 191 is also a common feature. This is assumed to involve migration of thè doublé bond to 13(18) position with subsequent decomposition to thè ion p (Scheme XIV). 1.6.13 Derivatives of Friedelane The friedelanes show very few characteristic fragmentations. A very prominent peak in thè spectrum of this class of compounds is due to species h at m/z 273 for thè friedelan-3-one derivative, 204. The Al 8-unsaturated analogs usually exhibit a very pronounced loss of methyl as a result of allylic activation of four quatemary methyl groups. 1.6.14. Lupane Derivatives. This series which contains a 5-membered ring E to which an isopropyl or isopropenyl group is attached show very pronounced loss of 43 mass unit (-C3H7) in certain members but becomes minimal in highly substituted derivatives. Saturated lupanes yield a species which corresponds to species g as thè most abundant fragment in addition to M-15 and M-43 peaks. In lupan-3-one, UNIVERSITY OF IBADAN LIBRARY 203a, R= O Ionp, m/z 191 b, R= (H)OH Iono' Scheme XIV. Fragmentation of Al7(18)„oieanenes to give ions p, o and o’ this fragment occurs at m/z 203. A fragment at m/z 191 is given structure m. A common characteristic feature is thè presence of very pronounced molecular ion. UNIVERSITY OF IBADAN LIBRARY I l i Two other fragments represented as n and q are frequently encountered in several related lupene compounds and their presence in a spectrum may offer valuable information and serve for identification purposes. In thè lupan-3-one spectrum, thè fragments coincide at m/z 218 but when C-3 carries a hydroxyl group, ion q occurs at m/z 220. UNIVERSITY OF IBADAN LIBRARY 112 The fragmentation of A^2_iUpenes do not show any resemblance to thè A^-oleanenes as they exhibit thè characteristic retro-Diels-Alder decomposition of ring C to a very small extent. It can be seen from thè detailed analysis of thè mass spectra of pentacyclic triterpenes that thè mass spectra of oleanenes and ursenes offer valuable information and that in many cases an unknown compound can be asigned to a certain structural type. In addition thè location of substituents can be narrowed down considerably!23 The mass spectra of lupane derivatives seem to be much less characteristic and only in simplest cases are a few fragments outstanding enough to offer useful information. Therefore only thè molecular weight and information about thè presence of certain functional groups can be derived in thè majority of members of thè lupane series. 1.6.15. Biosy nthesis of T riterpenoids. The formation of triterpenes has attracted interest over thè last couple of years. They are known to be biosynthesized from squalene which is derived from six isoprene units. The isoprene unit is in tum ultimately derived from mevalonic acid, which is formed from acetyl-coenzyme A via acetoacetyl CoA and 3(S)-3- hydroxyl-3-methylglutaryl-Co A (HMG-CoA)^ 25 Squalene is formed from two ali-trans famesyl diphosphate, 205 joind tail to tail. The 2,3-double bond of one famesyl diphosphate molecule is alkylated by another farnesyl diphosphate molecule with inversion of configuration and thè UNIVERSITY OF IBADAN LIBRARY 113 stereospecific elimination of Hx gives rise to thè cyclopropane moiety o f thè intermediate compound, presqualene, 206. In thè absence of NADPH thè intermediate compound accumulates but in thè presence of NADPH, thè cyclopropane rearranges by ring expansion and inversion of configuration at C-4, thè discrete cyclobutyl carbonium ion then collapses to thè linear ali-trans squalene 105,126-128, 207, (Scheme XV). The conversion of famesyl diphosphate into squalene is catalysed by squalene synthase. Parallel studies on thè different aspects of terpene and steroid chemistry gradually focussed thè interest around thè C30 hydrocarbon, squalene, as a conceivable progenitor of thè higher terpenoids. Squalene was first isolated from shark liver, sSlqauispp, but was latter found to be ubiquitously distributed. By folding this compound in certain models, thè basic triterpenoid skeleton can be constructed with thè angular methyls and side chain in correct positions. There exist also thè possibility that thè naturally occuring ali-trans squalene may isomerize at an olefmic centre at some stage of thè cyclization, but surprisingly there are few skeletal variations in thè triterpene series compared with thè sesquiterpene and thè diterpene series 129-130 UNIVERSITY OF IBADAN LIBRARY 114 i f Cyctobutyl carbonium ion 206'Presqualene diphosphate ♦ 207,Squalene Scheme XV. Biosynthesis of squalene from all trans famesyl diphosphate UNIVERSITY OF IBADAN LIBRARY 115 1.6.16. Biosynthesis of Tetracyclic Triterpenoids The first step in thè biosynthesis of sterols from squalene involves oxidation of squalene to fumish 3S-Squalene-2,3-epoxide in a reaction that requires molecular oxygen and NADPH. The reaction is catalysed by squalene epoxidase. Cyclization then follows giving thè tetracyclic sterol basic skeleton,J_72 which then undergoes rearrangements, oxidative and degradation processes leading to thè range of structurally varied groups of triterpenoids. Lansoterol, 208 is derived from 3S-Squalene-2,3-oxide in a chair boat-chair- boat conformation via thè protosterol carbonium ion I, 209 and a four step Wagner-Meerwein 1,2 shifts with elimination ofC-9 hydrogen (Scheme XVI). Considering thè required skeletal movements for cyclization it seems unlikely that thè whole process is fully concerted. The cyclization is more adequately viewed as involvng formation of a series of discrete carbonium ions, thè fate of which is controlled by thè enzyme surface. However, result of Chemical model experiements show that thè opening of thè epoxide ring receives considerable anchimeric assistance from adjacent doublé bonds*05,131. UNIVERSITY OF IBADAN LIBRARY 116 One further shift of C-9 Hp->Cg Hp and C-9 alkylation by C-19 leads to cycloartenol 210 containing a cyclopropane ring. It is generally accepted that cycloartenol is thè starting triterpene for thè biosynthesis of sterols in photosynthetic plants and algae while lanosterol performs thè equivalent role in animals and in thè few fungi that have been investigated 132-133 Further deep-seated rearrangement of protosterol carbonium ion I leads to thè cucurbitanes, 211 thè toxic principles of cucurbitaceous plants, a highly UNIVERSITY OF IBADAN LIBRARY 117 oxygenated group of tetracyclic triterpenes which have continued to attract interest because of their toxicity!34 Studies have shown that thè stereochemistry that is induced at C-20 in thè lanosterol formed is determined not by thè relative size of thè substituents at C-19 in thè analogues of squalene-2,3-epoxide but by thè stereochemical disposition of these substituents about thè À^-double bond^5 Two other tetracyclic triterpenoid groups are derived from thè protosterol carbonium ion II, 212. These are thè dammaranes and euphanes. The euphanes 213, differ from thè lanostanes in thè configuration around C-13,14 and 20, while thè dammaranes 214. retain thè protosterol carbonium ion II skeleton (Scheme XVII). UNIVERSITY OF IBADAN LIBRARY 118 1.6.17. Biosy nthesis of Pentacyclic T riterpenes. The pentacyclic triterpenes are mostly derived from protosterol carbonium ion II by expansion of thè ring D and cyclization with thè side chain. Lupeol 215, pseudo taraxasterol 216, P-amyrin 217 and their oxygenated derivatives have rearrangements within D and E rings only (Scheme XVIII). A large group of pentacyclic triterpenes are formed by backbone rearrangement UNIVERSITY OF IBADAN LIBRARY 119 217. P-Amyrin UNIVERSITY OF IBADAN LIBRARY 120 X X Scheme XIX. Biosynthetic route for Friedelin and 219, Friedelin Glutinol. involving multistep Wagner-Meerwein 1,2 shifts leading to friedelanes, glutinanes taraxeranes etc. (Scheme XIX). These rearrangements are thè result of a combination of factors, namely 1,3 diaxial steric interactions, conformational UNIVERSITY OF IBADAN LIBRARY 121 factors and stereoelectronic effects^3 Studies involving thè use of labelled precursors have provided strong evidence for thè biosynthesis of pentacyclic triterpenes from squalene-2,3-epoxide in higher plants. Ursolic acid22(7a 2a-hvdroxyursolic acid 226 band epi-maslinic acid 221 as their methyl esters, were examined by NMR spectroscopy following incroporation of ( 4 -^ C ) mevalonate or (1 ,2-^C 2 ) acetate into these acids in tissue culture of Isodon japon icus^^ .The labelling pattems observed confirmed thè biosynthetic route leading to thè formation of thè pentacyclic triterpenes. 220ii (Jrsolic acid, R=H. * * * 2a-Hydroxyursolic acid, R=OH The labelling pattems for carbons 4,23, and 24 in epi-maslinic acid is consistent with its formation from 3S-Squalene-2,3-oxide, with epimerization of thè 3p-hydroxyl function (at some stage) which must be formed initially. This evidence precludes thè idea that 3a-hydroxyl function is formed via cyclization of UNIVERSITY OF IBADAN LIBRARY 122 3R-Squalene-2,3-epoxidel33 Labelling experiments have also given a due as to thè particular stage in thè biosynthetic route when 12p proton is lost. The labelling pattern showed that 12J3 proton is lost from thè intermediate ion for Urs- 12-ene and Olean-12-enederivatives. 1.6.18. Biosynthesis of Phytosterols The biosynthetic transformation of squalene to thè À^-steroids such as p- sitosterol 100, stigmasterol 222, campesterol 223 and isofucosterol, 224, involve thè conversion of squalene into thè tetracyclic triterpenoid, cycloartenol, which is subsequently subjected to further degradative transformation to give thè sterols (Scheme XX). It has been suggested that thè known sequence of nuclear demethylation in higher plants reflects, in part, thè steric inaccessibility of thè 14 - methylcycloartenol. This steric crowding is relieved following thè isomerization of cycloeucalenol to obtusifoliol^ó (Scheme XX). Studies on thè course of biosynthesis of sterols in celi cultures of bramble (Rubus fruticosus)have provided powerful information which confirmed thè postulated sequence of biosynthetic route for À^-sterols. The major steroids of untreated cultures were thè A^-sterol, P-sitosterol, 70%, campesterol, 15% and ££X*OW*Ĥ l isofucosterol, 12% (Scheme XX). After four weeks of ° in thè presence of thè systemic fungicide, tridemorph, bramble cells no longer produced A^-steroids, owing to specific blocking of thè key enzyme that isomerizes cycloeucalenol, 225^ to furnish thè A^-sterol, obtusifoliol, 22 6 As a result cyclo-sterols such as 24- UNIVERSITY OF IBADAN LIBRARY 123 229 UNIVERSITY OF IBADAN LIBRARY 124 Scheme XX Contd. H 10 o (3-Sitosterol, R=Et 223, Campesterol, R=Me UNIVERSITY OF IBADAN LIBRARY 125 methvlene-cvcloartanol. 227, cycloeucalenol225aand pollinastanol derivatives, 22 5b constituted 80% of thè sterols that were p r e s e n t i . The naturai azasterol A25822B, 228. known to block nuclear reduction of steroidal 8,14-dienes in fungi also produced similar effect on bramble cells as a M 4_ sterols 229-230. accumulated^8 (Scheme XX). This observation provides powerful additional confirmation for thè postulated intermediacy of A*M4-sterols in thè biosynthesis of phytosterols 228, Azasterol(A25822 B). Alkylation at C-24 is a prominent feature of phytosterols. There are two extreme mechanistic descriptions (labelled a and b in Scheme XXI) for alkylation process but irrespective of thè route taken, it has been proved that 24- methylenesterol is an obligatory intermediate in thè formation of 24-ethyl- sterols139 (Scheme XXI). UNIVERSITY OF IBADAN LIBRARY 126 H* ( i ) S-adenosylmethionine(SAM). Scheme XXI_ Alkylation of ( ii ) SAM, Enz.-X phytosterol at C-24. Phytosterols also fall into two broad groups with respect to thè configuration at C-24. The 24ot-series, (3-sitosterol, stigmasterol, campesterol are suggested to be formed by isomerization of 24-methylenesterol to thè a24(25)_ isomer followed by stereospecific reduction to give thè 24a-alkyl sterol, route a (Scheme XXII). Two possibilities have been discussed for thè biosynthesis of 24 p-methyl sterols, namely reduction of a 24|3-methyl-A25-steroid (route b) or stereospecific reduction of a 24 -methyl-À23-steroidl40(route q . UNIVERSITY OF IBADAN LIBRARY 127 Scheme XXII. Biosynthesis o f 24-a and p-alkyl sterols. 1.6.19. Biosynthesis of sterols in Fungi. Demethylation of lanosterol is thè first major step in thè biosynthesis of ergosterol, 233 in yeast. Several commercial fungicides owe their antifungal activity, at least in part, to interference with thè biosynthesis of ergosterol by blocking thè 14a- UNIVERSITY OF IBADAN LIBRARY 128 demethylation of lanosterol or by blocking thè reduction of thè intermediate steroidal 8,14-diene^l. The removai of 14a-methyl is followed by oxidative demethylation of thè 4a-methyl group. Studies with a yeast mutant have confirmed that 4|3- methylsteroid cannot be demethylated^2 Transmethylation of thè 4p-methyl sterol 23d, involving S-adenosylmethionine gives fecosterol 23 Por episterol 23.6. Fecosterol is subsequently isomerized to episterol. The final steps in thè biosynthesis of ergosterol are thè reduction of ,,\24(28) double-bond and formation of A^ and A^2 doublé bonds (Scheme XXIII). 1.6.20. Triterpenoids and Steroids of Invertebrates. It is knovvn that many insects are incapable of biosynthesis of steroids de novo. In thè case of phytophagous insects, thè cholesterol that is required for celi membrane functions and for thè synthesis of hormones is acquired by dealkylation (at C-24) of dietary phytosterols. The degradative route is illustrated in Scheme XXIV for thè conversion of P-sitosterol into cholesterol, 238. The intermediates in Scheme XXIV - isofucosterol, 224 and fucosterol 239 could be formed (in principle) by hydroxylation of P-sitosterol at C-24 or C-28 followed by dehydration. However, observations from experiments 144 suggested that thè conversion of P-sitosterol into fucosterol and isofucosterol involves a dehydrogenation reaction rather than hydroxylation-dehydration sequence. The cholesterol is subsequently metabolised into thè ecdysteroids by a series of UNIVERSITY OF IBADAN LIBRARY 129 oxidative/reductive processes as illustrated by thè biosynthesis of 20- hydroxyecdysone, 240 in Scheme XXV. 208 235, Fecosterol 234, Zymosterol 233. ErgosteroL UNIVERSITY OF IBADAN LIBRARY 130 Marine invertebrates have made significant contribution to thè steroidal compounds. They have continued to provided novel and biosynthetically intriguing steroids!45 Scheme XXIV. Dealkylation of |3-sitosterol into cholesterol by phytophagous insects. Some of thè recent isolations include 4 ,2 7 -dinor-steroid' 46 ̂ 241. thè cyclopropanes!475 23H- isocalysterol 242 and 23, 24-dihydrocalysterol 243. mutasterol 244 and pulchrasterol'48 245. UNIVERSITY OF IBADAN LIBRARY 131 240 Scheme XXV. Biosynthesis o f Ecdysone. 1.6.21. Pharmacological properties of Triterpenoids. The medicinal importance of triterpenoids for thè most part is obscure as they do not show any impressive therapeutic properties like thè sesquiterpenes and thè diterpenes. However, there are a number of reports in thè literature on thè medicinal uses of some plants whose active principles have been identified as triterpenoids. UNIVERSITY OF IBADAN LIBRARY 132 245, Pulchrasterol. UNIVERSITY OF IBADAN LIBRARY 133 The triterpenoid saponins, dianosides A and B were isolated as thè principal analgesie constituents of Dianthus superbus L. Var. longicalycinus Williams 149 The flowers of Gardenia jasminoides Ellis which were used in Chinese folk medicine for birth control has also been reported to contain two cycloartane triterpenoids, gardenie and gardenoic acid B as thè active principles^ ̂ Makinde et al also reported that ursolic acid, isolated from Sp& dpthea campanulata. 15 0 b -c showed aruijnflamatory, antipyretic and anti-malaria properties. Table 21 presents some C-30 triterpenoids. 10^ 246-252. reported for their biological activities^ 50-154 In addition to thè afore mentioned C-30 triterpenoids, two other classes of triterpenoids are particularly very important classes with respect to their medicinal applications. They are thè quassinoids and thè cucurbitacins. The quassinoids have been implicated as potent antitumour agentsl^5-156 Some compounds in this class which are most active against thè P-388 lymphocytic leukemia are represented by compounds 253-258 (Table 22). The cucurbitacins have also been found to show antitumour properties. A number of new tumour inhibitory derivatives 259-261 have been described!57-158 (Table 23). Some derivatives are also reported to possess insect antifeedant activity. UNIVERSITY OF IBADAN LIBRARY 134 Table 21 - Some biologically active triterpenoids UNIVERSITY OF IBADAN LIBRARY 135 Table21 Contd. UNIVERSITY OF IBADAN LIBRARY 136 Table 21 Contd. As thè search for biologically active naturai products continues, an increasing number of new triterpenoids are being reported. It is particularly noteworthy that thè quassinoids have become thè focus of research interest in recent years. Although thè triterpenoids are not as popular as thè alkaloids or thè sesquiterpenoids in terms of biological activities, they have certainly offered a new direction and hope in thè search for biologically active naturai products for thè treatment of cancer in recent times. UNIVERSITY OF IBADAN LIBRARY Table 22 - Structures of some Antitumour Quassinoids. UNIVERSITY OF IBADAN LIBRARY 138 Table22 Contd. Table 23 - Some Tumour-inhibitory Cucurbitacins. 259, Datiscoside B. UNIVERSITY OF IBADAN LIBRARY 139 Table 23 Contd. 260, Datiscoside C. 1.7 OBJECTIVES OF THE RESEARCH WORK Gardenia erubescens Stapf. and Hutch. is a shrub found growing throughout thè savannah region in Nigeria. It is also found in Uganda and Sudan. The plant is used by thè natives in Northern Nigeria as aphrodis iacs and as a traditional remedy for a variety of other ailments particularly for thè treatment of gonorrhea, appetite abdominal disorders, loss of and insomnia. Its ffuits are edible and were reported to have been used to give strength on longer joumeys ̂ 59-160 UNIVERSITY OF IBADAN LIBRARY 140 Recently investigation into thè pharmacological effects of thè methanol and crude saponin extracts revealed that thè plant possesses interesting therapeutic potenti al as it showed sedative, analgesie, hypotensive and diuretic effect during in vivo tests carried out on rats, mice and cats by Hussain et a ll61 The medicinal application of G. erubescens has influenced interest in its Chemical constituents as hitherto there has been very little Chemical work carried out on this plant. Much have been reported on thè Chemical constituents of some Gardenia species. G. ansmjiodewas found to be rich in iridoid glycosides which were implicated as being responsible for thè laxative properties of its fruits while gardenoic acid was identified as thè early pregnancy terminating principle present in its flowers. Obviously, it is necessary to investigate thè Chemical constituents of Gardenia erubescens in order to identify thè organic metabolites present and thè result of such investigation could provide clues in rationalizing thè medicinal uses. It is along this perspective of providing scientific evidence for thè use of G. erubescens as a locai medicinal plant, that thè main objective of this research work is based. The trust of thè work is on isolation and characterization of secondary plant produets from G. erubescens with particular interest on thè saponin constituents as a means of providing scientific insight into its pharmacological properties. The work is also aimed at identification of novel structures as this constitutes a significai contribution to thè arsenal of naturai produets and also to scientific knowledge in generai. UNIVERSITY OF IBADAN LIBRARY 141 As this investigation was partly motivateci by thè absence of detailed documentation on thè Chemical constituents of G. erubescens, one of thè objectives of this work is also to achieve a detailed Chemical analysis of thè extracts of stems of G. erubescens Stapf. and Hutch. It was intended that hereafter thè isolated constituents would be individually subjected to bioassay experiments. UNIVERSITY OF IBADAN LIBRARY 142 CHAPTER TWO RESULTS AND DISCUSSION. 2.1 Extraction and Preliminary Examination of Extracts. The investigation into thè Chemical constituents of Gardenia erubescens was stimulated by thè pharmacological activities reported on thè crude extracts of thè roots and stembark of thè plant *61. The whole stem of G erubescens was used in this work. The plant material was collected in Jos, in September 1992 and identified by thè herbarium staff of thè School of Forestry Jos. A voucher specimen has been deposited in thè School of Forestry, Jos. The plant material was air-dried and pulverized before extraction was carried out. Each extraction was sequentially performed with petroleum ether for 24h in a doxhlet extractor and methanol for 24h or by soaking in 60% aqueous methanol for 72h after extraction with petroleum ether for 24h. A set of extraction was also carried out sequentially with petroleum ether for 24h, ethylacetate for 24h and finally with methanol for 24h. fr e e n is h-yellow gum. The yield of extracts ranged from 5-8g for every 2-3kg of plant material extracted. The methanol extract gave a dark brown gum on concentration and thè yield was on thè average 300g for 3kg of plant material. The ethylacetate extract gave a thick dark-green oil (~2.5g). The partitioning of thè methanol soluble UNIVERSITY OF IBADAN LIBRARY 143 fraction of methanol extract between water and n-butanol and then between water and ethylacetate gave n-butanol soluble and ethylacetate soluble extracts respectively. lOOg of thè methanol soluble fraction afforded 15g of n-butanol soluble fraction and about 1 g of ethylacetate soluble fraction. The various extracts, that is thè petroleum ether, ethylacetate, methanol, n- butanol soluble and ethylacetate soluble extracts were subjected to phytochemical tests for thè presence of flavonoids, steroids, alkaloids anthraquinones, saponins and tannins. The result of thè phytochemical screening is presented in Table 24. Table 24 - Result of Phytochemical Tests on Extracts of Stems of G. erubescens. Class of EXTRACTSi Compound. Petroleum Ethyl­ Methanol. n-Butanol EtOAc ether. acetate. soluble soluble. Flavonoid. + - - - - Steriod. ++ + + + - Alkaloid. - - - - - Anthra- - - quinone. Saponins. - ++ + - Tannins. - ++ - - - Negative, + Positive, ++ Srongly positive. UNIVERSITY OF IBADAN LIBRARY 144 Table 24 showed thè presence of flavonoids, steroids, saponins and tannins in thè extracts of thè stems of G. erubescens. The presence of flavonoid compounds only in thè petroleum ether extract suggested that relatively less polar flavonoid compounds are present and these are commonly thè methyl ether derivatives of thè hydroxyl group s on A and B rings of thè flavonoids. Alkaloids and anthraquinones wliich are very popular among thè members of Rubiaceae plants were also notably absent This is however in consonance with other Gardenia plants as there has been no report of thè presence of alkaloids and anthraquinones in any Gardenia plant. The result of thè test for steroids and saponins showed a substancial amount of these compounds to be present. The petroleum ether extract was strongly positive for thè presence of steroidal nucleus while thè methanol and n-butanol soluble extracts gave positive reaction for saponins. Tannins were present in thè polar extracts but much higher quantity was observed to be present in thè crude methanol extract while as expected, thè petroleum ether extract gave a negative response. The overall picture was that steroids and saponins constituted thè major components of Gardenia erubescens. In continuation of thè search for thè secondary plant products of G. erubescens, isolation, purification and characterization processes were performed on thè petroleum ether and methanol extracts. The petroleum ether extract was subjected to tic analysis on silica gel plates chromatoplate in various solvent systems and thè were sprayed with detection reagents for alkaloids, steroids and flavonoids. The results obtained by spraying thè chromatoplates with detection reagents complèment thè earlier observations UNIVERSITY OF IBADAN LIBRARY 145 from phytochemical tests carried out on dilute Solutions of thè crude extract. The presence of flavonoids and steroids were visibly corroborated. Flavonoid tic components appeared at Rf values - 0.72, 0.49 and 0.36 in EtOAc/Hexane, 1:3. The result of thè phytochemical analysis carried out on both crude methanol and n-butanol soluble extracts informed thè decision to limit thè tic analysis to n- butanol soluble fraction particularly as thè crude methanolp erxetrsaecnt ccoentained a lot of brown pigment. The sprayed chromatoplates confirmed thè ,__ of triterpenoids while other classes of compounds were not implicated. It was observed that 5-6 components located at Rf values 0.4-0.2 in thè solvent System, chloroform: methanol: water, 65:45:12, appeared as major triterpenoid components on thè chromatoplates. The results of thè tic analyses on thè petroleum and methanol extracts provided useful data which served as guide in thè isolation and purification of thè components of thè extracts of Gardenia erubescens. 2.2 Isolation and Purification of Components from thè extracts of G. erubescens. Column chromatrography and preparative tic served as a means of separation and purification of thè components from thè petroleum ether, ethylacetate and n-butanol soluble extracts of G. erubescens. Column chromatographic separation of thè petroleum ether extract was achieved on a column of silica gel G. (230-400 mesh) eluted with EtOAc/Hexane mixtures and further purification of thè isolated compounds where necessary was accomplished by preparative tic. Altogether, eight components were isolated and UNIVERSITY OF IBADAN LIBRARY 146 identified from thè petroleum ether extract. Six of thè components were obtained as single pure compounds while thè seventh fraction was composed of two isomerie compounds. The purity of each of thè compounds was confirmed by tic in one or two solvent systems. These compounds were labelled as GSH2, GSH 14, GSH 24, GSH 26, GSH 32 GSH 41 and GSH 49. The most abundant component was GSH 14 (~1 g), while GSH 49 (~500mg) was also obtained in high quantity relative to other components which were obtained in amounts less than lOOmg from 6- 1 Og of crude extract. GSH 2, GSH 26 and GSH 41 gave positive flavonoid reaction when thè chromatoplates were sprayed with 5% AlCtyMeOH reagent. Each of them appeared as dark absorbing spots in UV light before treatment and as bright yellow spots after treatment. GSH 14 gave a dark grey colour while GSH 24 gave brown colour and GSH 32, GSH 49, both gave reddish brown colour with acetic anhydride/sulphuric acid reagent. The compounds labelled as GSH 2, GSH 26 and GSH 41 were thus suspected to be flavonoid compounds and this had been confirmed by their spectral data. Also on thè basis of thè colour reaction with acetic anhydride/sulphuric acid reagent, GSH 14, GSH 24, GSH 32 and GSH 49 were identified as steroids and/or triterpenoid compounds. Their identities were unambiquously defined by their spectral data. The 1H and ' nmr spectra of GSH 49 suggested it as a mixture of two isomerie compounds. Column chromatographic analysis of ethylacetate extract performed with ethylacetate/hexane mixtures and then methanol/ethylacetate mixtures afforded one UNIVERSITY OF IBADAN LIBRARY 147 pure component which was eluted with 5% Methanol/ethylacetate Th.s co~?oun : which appeared as a single component on tic in 30% methanol/ethylacetate ( Rf value 0.28) and methanol-chloroform-water, 65:45:12 (Rf value 0.72) gave a dark brown colour with AC2O/H2SO4 reagent. The compound was later found to be identical to GS 13 (nmr spectra). From thè concentrated methanol extract, a white solid was obtained by tituration with methanol. The solid after washing thoroughly with methanol was recrystallized from methanol/water mixture and obtained as shining white crystals. This compound was labelled as GS 1 and was later identifìed by m.p. and spectral data as D-mannitol, m.p. 166-168°C. The mother liquor obtained from thè trituration with methanol of thè methanol extract was concentrated and partitioned between water and n-butanol in order to selectively extract thè saponins into thè n-butanol phase. The result of tic analysis of thè n-butanol soluble extract confirmed thè effective concentration of thè saponins in thè n-butanol phase as it showed up to six components which reacted positively with AC2O/H2SO4 and chlorosulphonic acid reagents. Chromatographic purification of thè n-butanol soluble extract on a column of silica gel using methanol/chloroform mixtures as eluting solvent produced a steroidal glucoside labelled as GS 13 which was eluted with 15% methanol/chloroform. Several attemtps were made to isolate thè remaining saponin components in thè extract. First, thè eluting mixture was changed to methanol- chloroform-water, 65:45:12 and then thè column was repeated using methanol- chloroform-water, 65:35:10. Secondly, thè n-butanol soluble extract was further UNIVERSITY OF IBADAN LIBRARY 148 treated to remove unwanted pigments (see experimental), thus obtaining a more concentrated saponin fraction. The column chromatographic processes were then repeated on thè treated n-butanol soluble extract but all efforts failed to yield any components. Although it is a common knowledge that highly polar components do not separate well on silica gel, there are reports in thè literature where saponin constituents of extracts were separated on a column of silica gel using methanol- chloroform-water (65:35:10)162-163 [t [s 0n thè basis of this information that preparative tic purification of thè n-butanol soluble extract was attempted since thè column chromatography failed. The preparative tic carried out in solvent systems, methanol-chloroform-water, 65:45:12 and 65:35:10, was also not successful. The implication of thè failure to achieve any level of separation of thè saponin components is that thè glycosides most likely contain oligosaccharides as thè sugar moiety, thus making thè saponins highly polar. The only documentation of thè presence and isolation of a saponin from a Gardenia plant was from thè work reported by Shukla et a l^ l. In their report, thè saponin isolated was -P-sitosterol-3- 0-J3-Dglucopyranosyl-(1—>4) - O-a-L-rhamnopyranoside from Gardenia lucida (Roxb.-), and it was obtained by solvent extraction. Generally, thè most popular and effective method of isolation of saponins from extracts involves thè use of polyamide as column materials with methanol/water mixtures as eluent. This method is frequently reported by thè Japanese working on saponins. Infact quite a substantial number of published work on saponins make use of chromatographic separation on a column of polyamide. UNIVERSITY OF IBADAN LIBRARY 149 seems Apparently this method to be thè only option left since thè two methods tried have failed. Unfortunately, thè polyamide material was not available and attempts to isolate thè saponins from thè n-butanol soluble extract was therefore abandone d . In thè light of thè problems enumerated above, thè saponin components were ' U ̂ “6 iU° n iso^ated and characterized as their aglycones. Mild acid hydrolysis (with 2% H2S0 4 /Me0 H) of n-butanol soluble extract produced thè aglycone mixture which showed three major triterpene spots (Rf values 0.52. 0.46, 0.40) on tic with 10% MeOH/CHCl3. Chromatographic purification of thè aglycone mixture on a column of silica gel eluting with MeOH/CHCl3 mixtures afforded four triterpene components which on further purification by preparative tic produced GSA 5, GSA 8, GSA 11 and GSA 16 as thè saponin aglycone present in thè n-butanol soluble extract of Gardenia erubescens. The total number of components thus isolated front thè methanol extract was six. These were GS 1, GS 13, GSA 5, GSA 8, GSA 11 and GSA 16. Apart from GS 1, thè rest gave positive reaction for steroid or triterpenes. UNIVERSITY OF IBADAN LIBRARY 150 2.3 Derivatives of Compounds Isolateti from G. erubescens. The identification of compounds from naturai sources is deemed not to be complete until thè functional groups present in such compounds are derivatized. The preparation of derivatives of a compound gives a strong evidence for thè presence of thè identified functional groups and this lends support for thè confirmation of thè structure of thè compound. The exercise requires that thè compound should be available in reasonable quantity, implying that for compounds available in very small quantities ( l - 10mg), it becomes difFicult to prepare derivatives. In thè pioneering years of thè development of isolation and characterization of naturai products, a number of compounds isolated in trace amounts were not fully identified. But with thè advent of FT nmr and thè application of COSY, NOE etc to structural problems, thè characterization of compounds available in few milligrams (up to lOmg) is effectively carried out now, in most cases, exclusively on thè basis of nmr studies and other spectral data (UV, IR and MS) available. Most of thè compounds isolated from both thè petroleum ether and n- butanol soluble extracts of Gardenia erubescens were in few milligrams (less than 50 mg). The most abundant being D-mannitol. The method of characterization of thè various compounds was therefore infuenced by thè quantity available for each compound. The compounds labelled as GSH 2, GSH 26, GSH 32, GSH 41 and GSA 8 were characterized by comparison of their spectral data and melting point with thè literature data. GSH 24 which was identified to be a new compound was subjected UNIVERSITY OF IBADAN LIBRARY 151 to exhaustive nmr studies to arrive at thè proposed structure. For GSH 49, identified as a mixture of ursolic and oleanolic acids, thè composition was established by direct comparison of spectroscopic data and Rf values with those of authentic samples. The other compounds, GSH 14, GS 1, GS 13, GSA 5, GSA 11 and GSA 16 were each subjected to one forni of Chemical reaction or another in addition to thè spectral data available. The acetates of GS 1, GSH 14, GSA 5 and GSA 11 were prepared by thè reaction with 1:1 mixture of pyridine/acetic anhydride. The acetate derivatives for each compound lend additional support for thè number of hydroxyl groups identified. For GS 13, characterization was achieved through acid hydrolysis to thè aglycone. The aglycone was subsequently identified by comparison of spectral data with that of GSH 14 and thè authentic sample and this eventually gave thè due to thè strutture of GS 13 which was found to be a 3(3-glucoside of GSH 14. GSA 16 was identified also as a new compound. It was subjected to detailed nmr studies together with preparation of derivatives. The acetate derivative gave a triacetate compound, indicating thè presence of three hydroxyl groups while thè reaction with diazomethane produced a methylester confirming thè presence of a carboxyl function. UNIVERSITY OF IBADAN LIBRARY 152 2.4 NMR Studies on thè Triterpenoid Components of Gardenia erubescens. The standard nmr spectra and 2D nmr spectra in particular were of tremendous assistance in elucidation of structures of GSH 32, GSA 5, GSA 8, GSA 11 and specifically thè structures of thè new compounds - GSH 24 and GSA 16. COSY and HETEROCOSY were thè 2D nmr spectra employed while DEPT and NOE difference spectra supplied complimentary datai 64-166 The spectral data obtained from these nmr studies were used, in combination with melting points and derivatization, to arrive at thè structures proposed for thè new compounds - GSH 24 and GSA 16. 2.5 Characterization of Compounds Isolated from Gardenia erubescens. 2.5.1 The Structure of GS 1 The IR spectrum of GS 1 showed thè presence of -OH group with an intense broad band at max 3300-3150cm 'l. The presence of thè hydroxyl group was further indicated by thè bands at 1262, 1147, 1092 and 1062 cu ri for C-O stretching vibrations (Fig 1). Information about thè number of protons present in thè compound was obtained from thè Ih nmr spectrum, a total of fourteen protons were shown to be present (Fig 2). Six protons appeared around 54.42-4.13 as 2H,d (54.42), 2H,t (64.35) and 2H,d (54.13). These are considered to be thè hydroxylic protons. The remaining eight protons appeared as a multiplet centered at 63.50. This Chemical shift is characteristic of carbinolic protons. The Ih nmr spectrum UNIVERSITY OF IBADAN LIBRARY CM-i UNIVERSITY OF IBADAN LIBRARY nmr spectrum of GS 1 UNIVERSITY OF IBADAN LIBRARY 153 clearly showed GS 1 to be a polyhydroxyl compound. The presence of hydroxyl groups was also visible from thè nmr spectrum showing peaks with Chemical shifts that are characteristic for both primary and secondary hydroxyl groups but at thè same time giving only three carbon signals (572, 70,64). This indicated a highly symmetrical molecule (Fig 3). Result of thè CIMS gave 183.0869 [M+l]+ which established thè formula C^H] 5O6 (requires 183.0864). The fragmentation proceeded with sequential loss of four molecules of water. The base peak being m/z 183 (Fig 4). Comparison of thè m.p 170°C with Lit. value (Lit. 166°-168°C)y-* gave strong indication that GS 1 was likely to be D-mannitol 262. a compound which is ubiquitously found among thè Gardenia plants. Further clarification about thè structure was achieved from thè hexaacetate derivative. The strong peak at Vmax 1743cm_l indicated thè presence of ester function and thè bands at 1267, 1224, 1121, 1089 cirri are C -0 stretching bands of thè ester function (Fig 5). Although there is a weak band at Vmax 3466 cm 'l, it was taken for thè presence of a minor contaminant as thè and nmr spectra (Fig 6 and 7) did not confimi thè presence of free hydroxyl group. There are peaks show ir^ree pajrs 0f aCetate groups (51.98,2.0,2.20) and thè Chemical shift ofthe carbinolic protons signifìcantly shifted downfìeld (Fig 6). The nmr spectrum also confìrmed thè presence of three pairs of acetate groups and also three pairs of carbinolic carbons, further indicating that thè molecule is highly symmetrical (Fig. 7). UNIVERSITY OF IBADAN LIBRARY 153b Fig. 3. nmr spectrum of GS 1 nw«*Wyj _L_ L 20 0 -2 0 UNIVERSITY OF IBADAN LIBRARY GS1 R.C.E.NH3 KW=182 ”” ” PT= 0° Cai lCfìl RAS)153c UNIVERSITY OF IBADAN LIBRARY 888 U 80Z- ì 8 8 9 i B30Z BBfrZ 00ZE 30017NIVERSITY OF IBADAN LIBRARY -09L'BL I •________ i____________ ,___________ j___________ i___________ !-------------- i-------------------i-----------------1----------------- 1--------------- J --------------- 1------------------- 1----------------- 1----------------- 1---------------- 1---------------1------------------ 1-----------------1----------------- 1----------------- 1— 5 . 0 4 . 0 3 . 0 2 . 0 1 . 0 PPM UNIVERSITY OF IBADAN LIBRARY Fig. 7. l^C nmr spectrum of GS 1 hexaacetate Fi-;:Il .001 AU PROG: ALIGCARB G A T E 2 4 - 1 0 - 9 3 Sf 6 2 . 9 9 6 Ci 1 2 5 0 0 . OOC SI 3 2 7 6 9 IO 3 2 7 6 8 sw 1 6 1 2 9 . 0 3 2 H2 'PT .984 P W 4.0 RO O.C AG 1 . 0 1 5 RG 2CC NS TE 297 C2 4 2 0 0 . 0 0 0 0 P 15H 00 LB 2 . 0 0 0 GB . 100 P P M / C M 5 . 3 7 5 5R 5 9 6 2 . 1 9 U 153fNIVERSITY OF IBADAN LIBRARY 154 The QMS also confirmed thè derivative to be a hexaacetate with m/z 435 [M+l ]+ peak. This corresponds to C ] T h e peaks at m/z (rei. int) 375 (100), 333(4), 289(3), 273(12) and 213(18) indicated progressive loss of acetic acid acetyl or unit from thè molecule (fig 8). The melting point of GS 1 hexaacetate was 117-119°C and that of D- mannitol hexacetate 263 (Lit. 121°c)93. The two values are comparable. Consequently, on thè basis of thè spectral data and melting point of GS 1 and its derivative, GS 1 hexaacetate, GS 1 was identified as D-mannitol. D- mannitol is a derivative of D-mannose in which thè aldehyde group of thè sugar has been reduced to a primary alcohol group. CH2OH <2H2OAc urlwn TrTi u THIPU* hT 7i AcU il Ac20/Pyr. rT iT u Uh irti ACU n IT IT THTU H OAc THT UAC c h 2o h CH2OAc 262 263 D-mannitol was reported for its low diuretic activity by Hussain et a ll61 UNIVERSITY OF IBADAN LIBRARY Fig. 8. EIMS of GS 1 hexaacetate •■93 13:07 DS90 GSD0002.6 RT= 01:36 +EI LRP i1-Au q -93 13:07 SA RMM 434 in TIC" 3934720 100:-;= 300464 ADELATUN FAB NBA RMM 434 375 10® 375 331r- 453-l T r~r y~r » t -j- i—i- r-4 -r-ì* i600 300 300 +740© UNIVERSITY OF IBADAN LIBRARY 155 2.5.2 Characterization of GSH 2 Vmax The IR spectrum of GSH 2 gave a weak band at/4331 cm 'l indicating thè presence of hydroxyl group while thè band at Vmax 3096 cm_l indicated thè presence of C=C-H stretch for aromatic System. This was further supported by thè bands at 1581 cm 'l and 1521 crrH which are C=C stretch bands for conjugated and/or aromatic System. With GSH 2 testing positive for flavonoid, thè band at 1629 crrr ' was suggested to be thè characteristic C=0 stretch band of flavonoids having a C-5 hydroxyl group. The bands at 1276, 1255, 1212, 1199, 1159, 1090, 1071 and 1029 cirH are characteristic C-0 stretching absorptions of aryl alkyl ethers. The absorptions naut c8l42e,u 8s29, 804, 746, and 733 cm 'l strongly suggested thè presence of aromatic (Fig 9). Both ^H and 1-̂ C nmr of GSH 2 showed peaks characteristic of flavonoid compounds. The presence of two aromatic rings with different substitution pattern was clearly evident in thè ^H nmr (Fig 10). Altogether thè * H nmr indicated thè presence of six aromatic protons, four being part of a para disubstituted benzene ring with Chemical shifts at 56.97 (2H, dd, J=9, 2 Hz) and 57.40 (2H, dd, J=9, 2 Hz). The splitting pattern (distorted doublet) is rem in iscent of AA'XX'System, 264 (Fig. 11). UNIVERSITY OF IBADAN LIBRARY 67.695 ----- - -----1--------- %T 61.731 Rise 49.402 43.237 37.073 ri-oH 30.908 24.743 18.579 I Fig. 9. IR spectrum of GSH 2 12.415 _______ _ ____ _ ________L _ J 1 ■ i 5250 4000 3200 2400 2000 1600 CK-i UNIVERSITY OF IBADAN LIBRARY [R\USER\FID1\665001,IR Date : 5.05.1993 Time: 17:28 Fig. 10. IHnm r spectrum ofGSH 2 U 155cNIVERSITY OF IBADAN LIBRARY F I U 1 \ 6 6 5 0 0 1 , ì k 5.05 » 19y 3 'rime: 17:34 Fig. 11. Ih nmr spectrum of GSH 2 0» * - 0 0 0 * 0 0 F 01 M 0 0 0 0 0 r- (M 'l 0 0 oo oo 0 0 0 F 0 0 0 01 r- 0 0 0 0 0 01 01 0> 01 01 01 01 K K 0- K K h 0 0 0 0 0 0 T _ ( p p m ) 7.4 7.3 7.2 7.1 7.0 6.9 UNIVERSITY OF IBADAN LIBRARY 156 The values ofJAX and JA'X' are thè sanie, approximately 7-10 Hz while thè values of JAX' and JA'X are also thè same but smaller, approximately 0-3Hz. The remaining two aromatic protons appeared at 66.02 (lH,d,J=3 Hz) and 66.05 (1 H,d,J= 3 Hz), suggesting a meta coupled aromatic protons (Fig 12). The peaks at 55.35 (IH, dd, J=3,10 Hz), 83.10 (IH, dd, J=1©,14 Hz) and 52.76 (IH, dd, J=3,14 Hz) suggested thè presence ofthree protons in thè C-ring of thè flavonoid (Fig 13). This implied thè absence of an olefinic bond between C-2 and C-3, that is, a flavanone structure was most likely. Inspection of thè splitting pattern of these three protons revealed an ABX System 265, in which thè equatorial and axial protons on C-3 form thè AB part. Typically JAB is about 12- 15 Hz, JBX (ax-ax) is about 5-10Hz and JAX (eq-ax) is about 2-3Hz. Using this as a guide thè proton at 55.35 was identified as Hx, thè proton at 83.10 as Hg and thè proton at 82.76 as H ^ The ^H nmr also revealed thè presence of two methoxyl groups at 83.79 (3H, S), and 83.82 (3H,S) and a highly cteshielded proton at 510.9 (IH, S) (Fig 14). UNIVERSITY OF IBADAN LIBRARY Fig. 12. 1H nm rspectrum ofG SH 2 N K) 0 © 0 * m in h iO 0 0 IO m m n 00 h m tM o q o o fO K) K) q C*S-0hO04ooo[)|af)tOnlO MKMDh Oh 40 0 0 N0COV 0 r-in C2 4 2 0 0 ,G00 CR 15^ 0?C T— H uB 2 . 0 0 0 GB . 10 J r / 4 0 . 0 0 rr SO 00 c i 230 O C 2 P J Q .0 0 9 - -:Z/0 11 25' .573 =»P(// M 4 . 000 SR 5 j q 3 . 13 UNIVERSITY OF IBADAN LIBRARY 158 MeO. V A .0 HC=CH- -OMe>c=o É3, m/z 134. HO A*1, m/z 166. Scheme XXVI. Fragment ions identified from mass spectrum o f GSH 2. The [M-CO]+ peak was also noticeable at m/z (rei. int.) 272 (2) but as it is obvious from thè relative intensity, it was not a prominent peak (Fig 16). UNIVERSITY OF IBADAN LIBRARY URR10HRT*ie* xl8 BQd-2 10-JUN-93 l i 46-B 08 2? 12-253J Ci* BpPi=8 1=8 . 7 y Hb=0 TIC=86234B8 flcnt fiRTLIH Sys RCE HlC GPE2 R.C.E.NH3 1̂ =380 PT= 8° CaUlCRL flR̂ 188. 301 98. 88. X 78. OinO i— H 60. 58. 48. 28. 10 . 135 8 .. x j u . X 1138 158 288 388 UNIVERSITY OF IBADAN LIBRARY 159 Additional evidence in support of thè flavanone structure was also obtained from thè UV spectrum. The dilute solution of GSH 2 in spectroscopic chloroform gave Xmax at 239.2nm and 290.5nm with a shoulder at around 335 nm (Fig 17). This is a characteristic absorption of flavanones. The absorption at 290.5nm (Band II) was due to ring A part of thè molecule while thè band at 335 nm (Band I) was due to thè dihydrocinnamoyl part of thè molecule. Band I is usually less prominent in flavanones and related compounds because of thè absence of conjugation between ring B and thè carbonyl group. Finally thè m.p. 112-113°C of GSH 2 compared very well with thè literature value (Lit. 115.5-115.9°C)167 for 5-hydroxyl 7,4-dimethoxyflavanone, 2.5.3 Determinatimi of Structure of GSH 26 The Ih nmr spectrum of GSH 26 looked simple with most of thè peaks showing between 86.2-7.9. The spectrum (Fig 18) also revealed thè presence of two methoxyl groups at 83.90 (3H,S) and 83.91 (3H,S). Inspection of thè peaks down field from 86.2 revealed a lot of similarities with thè ^H nmr spectrum of GSH 2, indicating that GSH 26 was also a flavonoid compound. The peaks at 86.38 (IH, d, J=2 Hz) and 86.50 (IH, d, J= 2 Hz) correspond to thè two aromatic protons on ring A of GSH 2, while protons at 87.02 (2H, dd, J- 9,2 Hz) and 87.87 (2H, dd, J= 9,2 Hz) also correspond to thè four protons on para disubstituted ring B of GSH 2. The ring C of GSH 26 is obviously dififerent from that of GSH 2 as thè absorptions due to thè protons in thè C-ring of GSH 2 were absent from thè spectrum of GSH 26. The peak at 86.60 (IH, S) suggested thè UNIVERSITY OF IBADAN LIBRARY U 159bNIVERSITY OF IBADAN LIBRARY Fig. 18. IH nmr spectrum of GSH 26 Ooinn ĉ£ [ J J i ' Ir + ? * * ' * * i I _ J ______ i______ i______ i--------- 1----------1--------- 1--------- I* i 8.0 6.0 5 . 0 4 . 0 3 . 0 PPM UNIVERSITY OF IBADAN LIBRARY 160 presence of a vinylic proton in C-ring. This implies that a doublé bond was present in ring C. As a consequence of this, GSH 26 was suspected to be thè flavone analogue of GSH 2. The 13C nmr spectrum (Fig 19) indicated thè presence of 17 carbon atoms just like GSH 2, however, thè Chemical shift for C-4, 8182.35, showed GSH 26 to be a 5- the hydroxyflavone (see table 10, p.66). The spectrum also showed . presence of two methoxyl groups (855.42 and 855.67) together with 15 other carbon atoms of thè flavnonoid skeleton. was The possible structure for GSH 26 therefore 267. 267 mention This structure has thè formula Ci 7 Hi 4 Ck. It is important tp that thè were down aromatic protons of both rings A and B . shifted _ field compared with those of was GSH 2. This -expected as there is extended conjugation of B -ring with thè a,p-unsaturated keto group in ring C. The UV. spectrum of GSH 26 gave À,max at 277,302, and 346 nm. This absorption is consistent with thè expected absorption bands of a flavone (see table UNIVERSITY OF IBADAN LIBRARY Fig. 19. nmr spectrum of GSH 26 SF 6 2 . 0 9 6 SY 5 2 . 0 01 1 2 5 0 0 . 0 0 0 SI 3 2 7 5 0 TD 3 2 7 5 0 SW 1 6 1 2 9 . 0 3 2 H Z / P T .904 PW 4.0 RD 1.000 AQ 1 . 0 1 6 RG 2 00 N S 2 3 0 6 0 TE 2 97 FW 2 0 2 0 0 02 4 2 0 0 . 0 0 0 DP 1 5H C PD o L B 2 . 0 0 0 GB . 100 ex 4 0 . 0 0 CY 0 . 0 FI 1 9 0 . 0 0 OP F2 5 0 . 0 1 0 P H Z / C M 2 2 0 . 1 2 0 P P M / C M 3 . 5 0 0 SR 5 9 6 2 . 1 9 i 120 TiO 100 90 80 70 60 PPM UNIVERSITY OF IBADAN LIBRARY 161 8). The 277nm absorption (band II) is due to thè ring A portion of thè molecule while 302-346nm absorption (band I) is due to thè cinnamoyl part (Fig 20). The band I of GSH 26 is more intense than band II as a result of thè conjugation which exists between rings B and C while thè opposite was thè case with GSH 2. CIMS confirmed thè formula H14 O5 giving m/z 299.0919 [M+l]+ (C 17 H15 O5 requires 299.0915). The peak at m/z 269 represented [M-l ]-CO fragment. The expected peaks from thè major fragmentation pathway for flavonoids were not prominent (Fig 21) but thè peak at m/z 166 (fragment A j+) seem to confimi that GSH 2 and GSH 26 carry thè same substituents on ring A (Scheme XXVI). The spectral data on GSH 26 supported thè structure as a flavone analogue of GSH 2. GSH 26 is consequently identified as 5-hydroxy 7,4'-dimethoxyflavone, 267. The melting point of GSH 26 was not determined due to thè trace amount isolated. 2.5.4 Identification of GSH 41. Inspection of thè ^H nmr spectmm of GSH 41 revealed marked similarity with that of GSH 2 but with a little difference. The spectmm showed thè presence of three methoxyl groups instead of two present in GSH 2. This difference was also reflected in thè splittings of ring B protons (Fig 22), implying that thè additional methoxyl group was probably on ring B. GSH 41 is undoubtedly a flavanone. The peaks at 62.83 (IH, dd, J= 3,14), 83.13 (IH, dd, J-1Q J3) and UNIVERSITY OF IBADAN LIBRARY +■ \ ■ I UNIVERSITY OF IBADAN LIBRARY m 183 M*3 8̂ ~id 8B2-HH EHH'TD'W 320 ÌM-$k Hiuua-M 08£t0ES*3Il 8=*H *'•2*5=1 9=Hrfg *•13 reS2-2I -0fi=9*58̂ { £S-6 requires 329.1020). In addition to this, thè mass spectrum also showed peaks characteristic of thè fragmentation pattern of flavonoids (Fig 25). The peaks at m/z (rei. int.) 330 (20), 298 (2), 193 (10), 164 (60) 151 (100) and 138 (15) could be obtained from thè fragmentations represented in Scheme XXVII. As shown in Scheme XXVII, thè fragmentation pattern of GSH 41 was slightly difFerent from that of GSH 2. The fragment B2+ appeared as thè base peak and B3+ fragment also gave very intense peak. The presence of thè fragments B3+ and A2+ provided a strong evidence for thè similarity in GSH 2 and GSH 41. The fragment A j+ which was visible in thè EIMS of GSH 2 was not observed in thè EIMS of GSH 41, however, fragment m/z 138 could in principle UNIVERSITY OF IBADAN LIBRARY Fig. 24. UV spectrum of GSH 41 + UNIVERSITY OF IBADAN LIBRARY sror at -** re I UNIVERSITY OF IBADAN LIBRARY tr*t 164 OMe À \, nVz 166 -c2h2 f -CO irtz 138. w irte 138. Scheme X X V II. Fragmentation pattern observed from thè mass spectrum o f GSH 41. UNIVERSITY OF IBADAN LIBRARY 165 be derived from A j+ by less of CO, but it is also possible for B3+ to give rise to thè same fragment by loss of C2H2. The evidence from thè spectral analysis unequivocally proved that GSH 41 is a 5-hydroxy-7, 3', 4' - trimethoxyflavanone, 269. The compounds 266, 267. and 269 are being reported in Gardenia plant for thè first time, although they are not new compounds. The compound 266, is a 7,4' -dimethyl ether of naringenin and compound 269, is a 7, 3', 4' - trimethyl ether of eriodictyol. The most common flavonoid reported in many Gardenia plants is Garden in, 120 a highly methoxylated flavone. This tendency to produce methoxylated flavonoid compound was also displayed in thè compounds from Gardenia erubescens as dimethoxyl and trimethoxyl flavonoid compounds were thè only flavonoid compounds isolated. The presence of flavanones in Gardenia erubescens is significant hearing in mind thè pharmacological properties of flavanones as they commonly possess antimicrobial activity. The presence of these flavanone compounds may be responsible for thè smooth appearance of thè stem of this plant probably because thè flavanones offer protection from thè attack of pathogenic agents. It is possible that thè stem bark of this plant could serve as a remedy in thè treatment of some skin diseases although ethnomedical reports did not mention any application in this regard. UNIVERSITY OF IBADAN LIBRARY 166 The report on thè treament of gonorrhoea could not possibly be attributed to thè flavanone component because aqueous extracts were reportedly used for such treatment. 2.5.5 Determination of Structure of GSH 14. The mass spectrum of GSH 14 (fig 26) established thè formula C29H48O with m/z 412.3705 [M+] (C29H48O requires 412.3693). The compound gave a positive Liebermann-Burchard reaction, suggesting a steroid structure. The nmr spectrum (Fig 27) gave further support for thè steroidal structure as majority of thè peaks were found upfield. The peaks at 55.15 (2Hm) and 55.36 (lH,d, J=5) revealed thè presence of olefmic protons while thè signal at 53.57 (IH, m) indicated thè presence of carbinolic proton. The 2H multiplet at 55.15 suggested a symmetrically substituted doublé bond. The nmr spectrum (Fig 28) revealed thè presence of a secondary hydroxyl carbon ( 5C, 71.7) and four olefmic carbon atoms ( 5C, 121.6, 129.2, 130.2, and 140.6) The four olefmic carbon atoms confirmed thè presence of two doublé bonds in thè compou nd. The number of carbon signals in thè l^C nmr spectrum added up to only twenty-six as it is common for some peaks to overlap. The indication for thè presence of hydroxyl group was also visible in thè IR spectrum (Fig 29) with bands at Vmax 3426 cm‘l (-OH Str), 1063 cm"l (C-0 Str). It was also obvious from thè IR spectrum that thè bulk of thè compound is majorly hydrocarbon in composition. The presence of doublé bond was also indicated by UNIVERSITY OF IBADAN LIBRARY O v» ! I! UNIVERSITY OF IBADAN LIBRARY PPM UNIVERSITY OF IBADAN LIBRARY Fig. 28. nmr spectrum of GSH 14 .1 J i 130 120 110 100 90 80 70 60 50 40 30 20 PPM UNIVERSITY OF IBADAN LIBRARY P991 UNIVERSITY OF IBADAN LIBRARY 167 thè weak bands at 1690 and 1660 cm*' together with thè band at 970 cm‘ 1 which is characteristic of trans substituted 22,23 doublé bond of a steroid structure. The compound had a m.p. 166-169°C (Lit. 170°C)168 On thè basis of thè spectral data and melting point, GSH 14 was suspected to be stigmasterol, 270. H It formed a monoacetate which had a melting point 140-142°C (Lit 144- 144°q 168. The IH nmr spectrum of thè acetate (Fig 30) showed thè acetate peak at 62.05 (3H,S) with thè down field shift of thè secondary carbinolic proton at 6 4.63 (lH,m). The remaining part of thè spectrum appeared thè same as in thè sterol. The carbonyl carbon of thè acetate group appeared at 5C, 170.46 in thè nmr spectrum (Fig 31). The mass spectrum showed thè molecular ion [M+] at m/z 454 which corresponded to C31H51O2. The M-60 peak at m/z 394 (100) is characteristic of acetate derivatives (Fig 32). Oxidation of thè compound with pyridinium chloro-chromate produced a mixture of two isomerie OXO-compounds. The mixture was not further purifled, UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY Fig. 31. nmr spectrum of GSH 14 acetate UÀ it 2t>-i a-y.i SF 6 2 . 8 9 6 01 1 2 5 0 0 . 0 0 0 SI 3 2 7 6 8 TD 3 2 7 6 3 SW 1 6 1 2 9 . 0 3 2 H 2 / P T .934 PW 4.0 RD 0.0 AQ 1.016 R G 200 NS 2 1 2 4 3 TE 297 o 02 4 2 0 0 . 0 0 0 DP 15H CPD r-H L3 2 . 0 0 0 GB . 100 P P M / C M 4. 2 5 0 SR 5 9 6 3 . 1 8 t J L i _jjJì , Uu jiiiliLu-̂ 100 90 P P M UNIVERSITY OF IBADAN LIBRARY Fig. 32. EIMS of GSH 14 acetate I UNIVERSITY OF IBADAN LIBRARYPZ.91 168 however, some useful deductions were made on thè basis of thè spectra of thè oxo- compound. The nmr spectrum (Fig 33) revealed thè disappearance of thè carbinolic proton implying that thè secondary hydroxyl group had been converted to thè carbonyl group. The nmr spectrum also revealed thè presence of keto group (8C, 202) but no carbinolic carbon was visible (Fig 34). The molecular ion [M+] at m/z 410 which correspondes to C29H46O (Fig 35) also suggested thè conversion of a secondary hydroxyl group to a keto group. The evidence from thè spectra of both thè acetate and thè oxo-compound supported a monohydroxyl steroid compound. Full identifìcation of GSH 14 was therefore carried out by comparison of melting point, spectral data and Rf value with stigmasterol, thè authentic sample. The melting point of mixed sample (GSH 14 + authentic sample) was 168°C (Lit. 170°C). TLC analysis of thè mixed sample (using EtOAc/Hexane, 1:3 and 1:2) gave one component with Rf values 0.42 and 0.61 respectively. Comparison of thè and nmr spectra of GSH 14 and thè authentic sample revealed vividly that GSH 14 is thè same compound as stigmasterol. On thè basis of available data, GSH 14 was identified as stigmasterol, 270. 2.5.6 Structure of GS 13 GS 13 was identified as a glycoside from thè information obtained from analysis of thè 1H and * nmr spectra of thè compound. The presence of carbinolic protons in thè region 84.0-4.63 was evident of a polyhydroxylic compound (Fig 36). The peak at 85.36 idicated thè presence of a doublé bond. The UNIVERSITY OF IBADAN LIBRARY Fig. 33. nmr spectrum of GSH 14 oxo-deriv. U 168bNIVERSITY OF IBADAN LIBRARY Fig. 34. l^ c nmr spectrum of GSH 14 oxo-deriv. 01 1 2 5 0 0 . 0 0 0 SI 3 2 7 6 8 TD 3 2 7 6 8 sw 1 6 1 2 9 . 0 3 2 HZ/'PT .984 PW 4.0 RQ C.C AG 1 . 0 1 6 RG 2 00 NS 4 5 COO TE 2 9 7 02 4 2 0 0 . 0 0 0 DP 1 5 H e p a L0 2 . 0 0 0 GB . 100 P P M / C M 5 . 3 7 5 SR 5 9 5 2 . 1 9 UNIVERSITY OF IBADAN LIBRARY Fig. 35. EIMS ofGSH 14 oxo-deriv. UNIVERSITY OF IBADAN LIBRARY P891 Fig. 36. nmr spectrum of GS 13' U 168eNIVERSITY OF IBADAN LIBRARY 169 anomeric proton of thè sugar molecule could not be locateci on thè 1H nmr spectrum because of thè slight wetness of thè pyridine-d5, giving a broad water peak about thè same region for thè aromeric proton. The remaining peaks which appeared upfield suggested a steroidal nucleus. The nmr spectrum gave more information about thè structure of GS 13 as it showed peaks for six sugar carbons with one additonal carbinolic carbon and four olefinic carbon signals (Fig 37). The spectrum therefore revealed thè sugar moiety as a hexose and that thè steroidal nuleus contained two doublé bonds. The Chemical shifì of thè sugar carbons, 5C, 102.6,7§«5£*7S .l6*75*4-0 , 7 1 . 7 5 and 6 2 .8 8 are characteristic a b so rp t io n s o f g lu c o s e c a r b o n a t o m s ^ ^ " * The position of attachment of thè sugar molecule was on C-3 and this resulted in a downfield shift of thè C-3 carbon, 8C, 78.67. Information about thè mass of thè compound could not be obtained with EIMS or CIMS. The FABMS likewise did not give a molecular ion peak (Fig 38). A fragment at m/z 411 was however identifìed as a [M-glc] peak, suggesting thè aglycone to be a steroid with molecular mass 412. GS 13 was finally subjected to acid hydrolysis in aqueous methanol. 1H and 13C nmr spectra of thè aglycone obtained was found to be identical to thè spectra of GSH 14. The aglycone also gave a positive Liebermann-Burchard reaction, confirming thè presence of a steroidal nucleus. Considering thè available spectral data on GS 13 and its aglycone, GS 13 was identifìed as stigmasterol 3p-glucopyranoside, 271. UNIVERSITY OF IBADAN LIBRARY Fig. 37. 13C nmr spectrum of GS 13 . 22 7 . 200 . 16 vQo\ UNIVERSITY OF IBADAN LIBRARY 9E 71 41 21 01 8 6 4 2 9 .7 .5 .3 . 1 . é c .( UNIVERSITY OF IBADAN LIBRARY 170 H H 2.5.7 Determination of Structure of GSH 24 GSH 24 was isolateci from thè petroleum ether extract of Gardenia ernbescens and it crystallised out as pale yelow crystals ffom ethylacetate/hexane mixture. It has a m.p. 203-205°C and +61 (C, 2.0, CHCI3). The compound also showed a strong absorption in thè VU region. A dilute solution of GSH 24 in CHCI3 gave A,max 282 nm (fig 39) while >*max 280 was obtained with methanol as solvent (fig 40). The UV spectrum gave indication of thè presence of a conjugated chromophore in thè compound. Its IR band, Vmax 3583-3337 cm 'l showed thè presence of hydroxyl group and thè band at 1662 and 1636 cm_l indicated thè presence of isolated and conjugated doublé bonds (fig 41). The absorptions in thè region 1174-1072 cm 'l due to C-0 stretch provided additional evidence for thè presence of hydroxyl group. The 1H nmr spectrum (fig 42) revealed thè presence of five methyl groups at 80.83 (3H,S), 50.93 (3H,S), 61.33 (3H,S), 81.39 (3H,S) and 51.87 (3H,d, J=1.9). There were also five peaks appearing downfield from 84.6, each corresponding to one UNIVERSITY OF IBADAN LIBRARY CM Fig. 39. UV spectrum of GSH 24 in CHCI3 t j j + 4- C< LTI _ UNIVERSITY OF IBADAN LIBRARY qoz.i Fig.. 40 UV Spectrum of GSH 24 in MeOHU 170cNIVERSITY OF IBADAN LIBRARY 345 G20 835 1 70 445 720 935 270 Fig. 41. IR spectrum of GSH 24 545 820 5 4800 3208 2400 2800 1680 1200 800 Ak Ch-l UN 170dIVERSITY OF IBADAN LIBRARY PPM UN 170eIVERSITY OF IBADAN LIBRARY 171 proton absorption - 55.99 (IH, bs), 85.67 (lH,bt), 85.40 (lH,bt), 54.86 (lH,bs) and 84.74 (lH,bs). The peaks at 54.86 and 84.74 suggested thè presence of terminal olefmic bond while thè peaks at 85.67 and 85.40 indicated thè presence of two other doublé bonds not coupled to each other. The presence of a,(3-unsaturated keto group was evident from thè nmr spectrum (fig 43) with thè peak at SC, 193.7. A total of eight olefinic carbons were also revealed in thè spectrum, of which five were found to be quaternary, two tertiary and one primary from thè DEPT spectrum (fig 44). A carbinolic carbon (8C, 75.0) found to be quaternary (DEPT spectrum) could also be seen in thè nmr spectrum, thus suggesting thè presence of a tertiary hydroxyl group in thè molecule. Altogether, twenty-eight carbon signals were present in thè l^ c nmr spectrum. The Àmax from thè UV spectrum suggested that an a,p~unsaturated keto group alone cannot account for thè strong absorption. For a six membered ring a,|3-unsaturated ketone of thè type 272, thè calculated /.max is 239 nm. The IH broad singlet at 5.99 however offered thè possibility of a diosphenol function, 273. in thè molecule. HO 272 273 UNIVERSITY OF IBADAN LIBRARY c. / 20200 4200 .000 15H CPD 2.000 . 100 400..00 0 * 200 .002P Fig. 43. l^C nmr spectrum of GSH 24 1 0 .004P /CM 29 8 .75 2 M/CM 4 .75 0 5963 .18 X _ _ L _ 190 180 170 160 150 140 130 120 110 1 0 0 90 80 70 60 50 PPM U UM rO M »-< X C D LD OfXjaL UNIVERSITY OF IBADAN LIBRARY Fig. 44. DEPT spectrum of GSH 24 o « . U m ìjt&frfiV' 33.3 j? 70.3 Mj UNIVERSITY OF IBADAN LIBRARY 172 The calculated A-max for thè enol chromophore is 274nm, this obviously is comparable to thè observed Àmax, 282 nm. Its high resolution EIMS established thè formula C28H38O3 witn m/z 422.2930 [M+] (C2 8 H3 8 O3 requires 422.2900). The presence of a second hydroxyl group is thereby implicated. The doublé bond equivalent found, based on thè formula, C28H38O3 was ten DBE. Four olefmic bonds and one keto group DBE have so tar been identifìed, leaving five - to be accounted for. The only option left is for thè compound to have a pentacyclic structure. The presence of a 1,1- disubstituted doublé bond together with a pentacyclic structure suggested thè compound to be a lupene. Another evidence for thè lupene skeleton was thè presence of an intense molecular ion in thè EIMS (fig 45) and this is a characteristic feature of thè lupene series^3 The loss of water from thè molecular ion, m/z 404 [M+-1 8], is a further prove for thè presence of hydroxyl group. It is however not possible to draw far reaching conclusions from thè mass spectrum as thè fragmentations did not produce useful diagnostic ions probably due to thè high degree of unsaturation in thè molecule, although lupenes are known to give mass spectra which offer very little diagnostic information except for thè mass of thè molecular i o n 123 Evidence for thè presence of thè enol fiinction was sought from UV studies on thè compound. Comparison of thè UV spectrum of methanol solution of GSH 24 and that obtained after addition of a small drop of dilute solution of sodium hydroxide produced no due. A shift of thè À,max to longer wavelength was UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY 173 expected on addition of sodium hydroxide solution but no shift was observed. although there was an increase in intensity of absorption (fig 46). However, this observation can not be used to suggest thè absence of thè diosphenol function as thè compound was only sparingy soluble in methanol. The result obtained by treatment of thè chromatoplate of thè compound with ferric chloride did not also give any useful due because a greyish black colour was observed instead of thè brown colour usually obtained with cucurbitacins with diosphenol function 171. The structure of GSH 24 was finally established by nmr studies. The assignment of thè 1H nmr spectrum was supported by 2D COSY (fig 47) and by use of NOE difference spectroscopy (table 25). The l^C nmr assignments were supported by CPD, 1-bond and long range l^C -lff correlation spectra (table 26). GSH 24 was identified as a bisnortriterpene lupenol. This we have named erubescenone, 274. UNIVERSITY OF IBADAN LIBRARY Fig. 46 UV Spectrum of GSH 24 Wavelength (nm) U 173bNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY 174 From available literature report, lupenol and betulinic acid had been reported in thè Rubiaceae plants of Hong Kong70,73 but no lupene compound had been reported in Gardenia plants. This work reports thè presence of a lupene compound in Gardenia plant for thè First time. There is also no report of any compound with same structure as erubescenone in thè literature from any source, hence this work reports erubescenone as a novel compound. Erubescenone has two carbon atoms less than thè normal lupenes and added to this is thè unusually high number of doublé bonds, both of which present a unique feature. A possible biogenetic pathway for erubesceone is outlined in Scheme XXVIII. The biogenetic formation of erubescenone could possibly involve oxidative degradation of betulinic acid derivative through loss of C-17 carboxylic acid group as formic acid. UNIVERSITY OF IBADAN LIBRARY 175 Table 25 - 400MHz ' H NMR spectrum and noes for erubescenone in CDCI3. Position. 5/MuItiplicity. J( Hz ). Couplings to NOEs to la 2.12 d 16.5 IP, 11,24 ip , 5a, 9a, 11 ip 2.65 d 16.5 l a la , 24 3-OH 5.99 bs 5a, 6a, 6p, 23 5a 2.40 m 6a, 6|3, 11 6a 1.88 m 5a, 6(3, 7a, 7 P 6P 1.47 ddd 2.9,3.3 5a, 6a, 7a 7a 1.65 dd 3.7,13.2 6a, 6p, 7p 6a, 7p, 26 7p 1.54 dm 3.3,13.2 6a, 7a 9a 1.97 s l la /p , 25 la , 5a, 12, 24 25,26 I l a 2.01 m la , 5a, 9a H P 2.05 12 5.40 bt 18p 27Z 1 l,18p, 28 15a 1.71 dm 17 15P, 16, 18p 22p 15p 2.36 m 15a, 16, 18p, 26 16 5.67 bt 15a, 15P, 18p 15a, 15p, 22a 22P UNIVERSITY OF IBADAN LIBRARY 176 Table 25 Contd. Position. 5/Multiplicity. J(Hz). Couplings to NOES to 18(3 2.63 bs 12 15a, 15P, 12, 22p, 28 16, 22 p 19p 1.65 bs 21 p, 22a, 22p 27E, 27Z -OH 21a 2.50 m 6.13 21P, 22a, 22p 27E, 27Z 21 p 2.31 m 21a,22a, 22p 27E, 27Z 22a 2.33 m 21a,21p ,22p 22 p 2.01 m 15a, 16, 18p 21a, 21 p, 22a 23 1.87 d 1.9 5a 24 0.93 m la IP, 6 p ,9 a , 25 25 0.83 s 9a 6p, 7p ,9a , 11 15p, 18p,24 26 1.33 s 15p 7a, 9a, 15a 27Z 4.74 bs 21a, 21 p, 27E 21 p, 27E 27E 4.86 bs 21a, 21 p. 27E 28, 27Z 28 1.39 s 12, 18P,27E UNIVERSITY OF IBADAN LIBRARY 177 Table 26- 100MHz NMR spectrum and ^C -^H correlations for erubescenone in CDCI3. c 6 type coupling to number 1-bond long range 1 51.2 c h 2 la , 2(3 24 2 193.7 00 la ,2p 3 143.4 c= 23,ip,3-OH 4 130.6 c= 23,5,3-OH 5 48.6 CH 5 6a,6p,23,24 6 20.7 c h 2 6a,6p la ,5 7 32.2 c h 2 7a,7p 25 8 39.2 c 7a,7p,9,25,26 9 43.6 CH 9 7P,11,24,25 10 41.4 C la,ip,5,9,24 11 23.6 c h 2 1 la , l lp 12 12 125.7 HC= 12 18 13 137.2 C= 11,18,26 14 40.8 c 12,25,26 15 32.6 c h 2 15a,15p 26 16 122.5 HC= 16 15a,15p 17 134.3 C= 18,22p 18 56.0 CH 18 12,16,28 19 75.0 C-OH 18,27E,27Z, 28 20 153.7 C= 21a,2ip,28 21 33.6 c h 2 21a,21p 27E,27Z 22 35.9 c h 2 22a,22p 16,21a 23 13.2 c h 3 23 24 13.9 c h 3 24 la ,5 25 17.2 c h 3 25 7a,l l a 26 25.1 c h 3 26 27 106.7 =c h 2 27E,27Z 28 23.7 _CH3_________ 28 UNIVERSITY OF IBADAN LIBRARY 178 One of thè C-4 methyl groups is subsequently lost. The enol function in ring A is probably formed en route to C-4 demethylation via thè a-diketone. 274 UNIVERSITY OF IBADAN LIBRARY 179 Erubescenone has ring A which carries a diosphenol function and this is a characteristic feature of some cucurbitacins. It is therefore logicai to regard this compound as a potential cytotoxic agent. 2.5.8 Structure of GSH 32. GSH 32 was obtained as clusters from ethyl acetate and had a m.p. 246- 248°C (Lit. m.p. 242°C )172. its ir showed thè presence of an ester group, Vmax 1732 cm-l. The bands at Vmax 3300-3150 crrr ' (OH str) and 1694 c n r ' both indicated thè presence of carboxyl group (Fig. 48) while bands at 1253 cm 'l, and 1029 cirri gave further indication of thè presence of both carboxyl and ester groups. 'Hnmr spectrum also indicated thè presence of thè acetate group, 82.03 (3H,S), seven methyl groups, 80.84 (3H,S), 0.85 (3H,S), 0.89 (3H,S), 0.91 (3H,S), 0.93 (3H,S), 1.11 (3H,S); a carbinolic methine proton, 84.48 (lH,t, J=8Hz) and an olefinic proton, 85.26 (lH,t, J=3.5) (Fig 49). The presence of seven angular methyl groups suggested a pentacyclic triterpenoid skeleton, while thè down field appearance of thè carbionolic proton indicated thè acetate group to be on C-3 A total of 32 carbon signals appeared on thè nmr spectrum. Nine of these were quatemary as shown by DEPT spectrum (Fig 50), ten were methylene groups, five were methine groups and thè rest eight were methyl groups. The signals at 8C, 170.95 and 183.45 confìrmed thè presence of acetate and carboxyl groups. The signals at 8C, 122.43 and 143.48 were absorptions of thè olefinic UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY Fig. 49. 1H nmr spectrum of GSH 32 < . I 1 . UNIVERSITY OF IBADAN LIBRARY T3 Fig. 50. DEPT spectrum of GSH 32 UNIVERSITY OF IBADAN LIBRARY 180 carbon atoms and this gave a strong indication of an oleanen-type skeleton for thè compound. CIMS (Fig 51) gave m/z 499 [M+l]+ . Accurate mass obtained for [M+l] - 60 peak (m/z 439) was 439.3590 which corresponded to C30 H47 O2 (requires 439.3564). The fragmentation pattern revealed in thè CIMS spectrum seem to suggest an olean-12-ene skeleton because thè fragments at m/z 248, 235,205 and 191 correspond to fragments a,e c and d respectively and these are diagnostic fragments produced by retro-Diels Alder cleavage of ring C of A^-oleanene or A^-ursene skeleton (Scheme Vili). The presence of fragment at m/z 248 also provided a strong evidence for thè location of thè carboxyl group on D/E rings Given thè information available from thè spectral data, thè structure, 275 could be suggested for GSH 32. The exact location of thè carboxyl group was determined from NOE and lH- 1 correlation spectra (table 27) and C-30 was identified as thè position occupied UNIVERSITY OF IBADAN LIBRARY IVU- 438 423 I 452 508 URR14RRTÌ76* xie Bgd=2 17-JUN-93 13 = 27+882:04 12-253J Cl* BpR=8 1=2.5m Hx=8 T1C=6455800 ficnt WRTL1N Sys=RCE G32 R.C.E.NH3 RW=? PT= 0° CaUlCfil ♦xio*o «5 439 393 423 1 499 516 379 303 J L i l 483 ,A .Jkri iS r 488 500 UNIVERSITY OF IBADAN LIBRARY 181 by thè carboxyl group. The full structure of GSH 32 is therefore 3p-acetoxyolean- 12-en 30-oic acid, 276. Table 27 - 400 MHz IH NMR spectrum and NOES for 3p-acetoxyolean-12-en-30- oic acid Position 5/multiplicity J(Hz) NOES to la 1.04 m ip 1.55 d 12 2a 1.95 d 12 2P 2.0 m 3a 4.48 t 11 2a/p ,ip ,5a 5a 0.84 d 6a 1.52 d 6P 1.48 m l a 1.49 t 11 7P 1.32 d 11 9a 1.53 m lla /p 1.90 m 12 5.26 t 3.5 18p ,lla /p 15a 1.06 d 12 15p 1.70 t 12 16a 1.95 t 12 UNIVERSITY OF IBADAN LIBRARY 182 Table 27 Contd. Position. 5/Multiplicity. J(Hz). NOES to 160 1.62 m 180 2.80 dd • 4,10 12,11,19 19oc 1.14 t 12 190 1.68 d 12 21a 1.16 t 12 210 1.40 d 12 22a 1.80 d 12 220 1.60 t 12 23 0.85 s 3a,6a,7a,70 24 0.84 s 60,25,CH3COOH 25 0.93 s 10,60,1 la /0 ,24,26 26 0.73 s 70,1 la /0 ,150,25 27 1.11 s 7a,12,16a 28 0.89 s 220,210 I 29 0.91 180,19a,21a,22a CH^COO 2.03 s UNIVERSITY OF IBADAN LIBRARY 183 The spectral data and melting point of GSH 32 compared fairly well with thè literature data on 3p-acetoxyolean-12 en-30-oic acid. The fragmentation pattern observed in thè CIMS spectrum of GSH 32 is illustrated in Scheme XXIX. COOH CHjCOO The presence of 3p-acetoxyolean-12-en-30-oic acid is being reported for thè first time in a Gardenia plant. As thè Gardenia plants have been shown to be rich sources of oleanene-type triterpenoiods, thè presence of this compound is an indication of thè possibilities for a variety of secondary modifications on thè oleanene skeleton. UNIVERSITY OF IBADAN LIBRARY 184 Scheme XXIX. Fragment ions identified in thè mass spectrum o f GSH 32. UNIVERSITY OF IBADAN LIBRARY 185 2.5.9. Determinatici! of structure of GSH 49 GSH 49 gave a positive Liebermann-Burchard reaction for triterpene. The 1H nmr spectrum showed peaks for vinylic protons, 85.15 (2H, dt), carbinolic protons, 8311 (2H m) and an unusually high number of peaks for thè methyl groups (fig 52). The nmr showed peaks which appeared in pairs (Fig 53). Two peaks appeared for thè carboxyl carbon at 8C, 180.19 and 179.90, there were also s i g n a l s two pairs of for thè olefinic carbons at 8C 144.85, 139.30 and 122 .5 7 124.29. Similarly many of thè signals for thè CH, CH2 and CH3 carbon atoms appeared in pairs (Fig 54). Analysis of thè 1H and 1 nmr spectra gave an impression of a triterpenoid with two carboxyl groups and two olefinic bonds. But considering thè total number of carbon atoms obtained from thè nmr spectrum, it is obvious that this fraction can not be a pure compound. The appearance of signals in pairs suggested a mixture of two isomerie compounds. The CIMS of this fraction however did not show two molecular ions. Only one molecular ion, m/z 457 [M+l]+ peak was produced (Fig 55). Accurate mass measurement gave m/z 457.3680 for [M+l]+ peak. This corresponds to C30 H49 O3 (requires 457.3669). The fragments m/z 248,235,205, and 191 are characteristic of A^-oleanene or A^-ursene skeleton and suggested that this fraction contained oleanolic acid and one other triterpene compound. The peaks at m/z 439 [M-18], 411 [M-46] and 393 [M-60] supported thè presence of hydroxyl and carboxyl groups. UNIVERSITY OF IBADAN LIBRARY Fig.52. 1H nmr spectrum of GSH 49 UNIVERSITY OF IBADAN LIBRARY Fig. 53. 13C nmr spectrum of GSH 49 UNIVERSITY OF IBADAN LIBRARY Fig. 54. 13C nmr spectrum of GSH 49 sw 1 6 1 2 9 . 0 3 2 H Z / P T .984 PW 4.0 RD 1 . 0 0 0 AG 1 . 0 1 6 RG 2 00 NS 2 3 4 4 5 TE 2 9 7 FW 20 2 0 0 02 4 2 0 0 . 0 0 0 DP 15H C P D Tm3 LB 2 . 0 0 0 GB . 100 00 ex 4 0 . 0 0 CY 0 . 0 FI 6 0 . 0 0 0 P F2 1 0 . 0 0 8 P H Z / C M 7 8 . 6 0 7 P P M / C M 1 . 2 5 0 SR 5 9 1 9 . 5 9 iwV/V̂1 V %M/W n/"W *,v "*— tir UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY ■v : - v >- S* *&>’- ■ ■. ■* * -S r . ri* ■ Ss • • s - .. -s#v - V T •«• IV .’f 'Tì • . ■ s - ù.: i 1** V .% V ?X ^ V k ì 1 ■. i .. ±. Si; '4 > : S- *.r.~ ; V-, «V-* ‘W < \ z i ? ? # & _ r- ■>. *■ v , - , ',■ •14' ' • * / . ’■ , « '-' ’̂ r J " . ' *'*%%*••*V -1 * i b : & 4 ' f » . • > .x V ;v. r r « ' V X . v - , . 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' ■ v - v . ; : .• . ‘C i* : , ̂ -•■ ; * • ì a , -*•.-.• - V 'p ^ j^ v v - y fr : '* • *' :?r^ . •■’... . SA - - . ,:> " - ^ ' ^ 7 '^7 ‘ À A . . \ , i 4 À V ® f - . . i s r ; -A i 'i ìl . - V’.' V ‘.J . « -.v^vV: 4J*‘ A ^ . v £ ‘-k- .'jrf; 7 * . --v;"- J*«5*®'.74"' ■ & - acid • f e f - 5 ^ j ì « . , ' r ^ i i t e d M 4. :•*-* T i ì ^ B v s i a H f a r ’ 1 ■'*; * C 7)^,1 ̂ '- ■' • * i 4 ’ \ : 4 . *1 *U- '=*. 3 - • -v \ ? f e : ' ; Ì ^ v ^ = . v ' i w v - ‘ -.'v *a ì ? U : •>> J «A aW '4 •T / , " - - 7 7 U s . • *• >. . '. “• •• -»-Tr . :4 .-' ‘ i. ' .-. z ' 7 . 1" vJ^' - ?X i;4 - ^ v 4 ^ T A 7 ĉ V f - -r-*: : ■“ :‘ ’-'• A f i W 'A ì & é \ N, nmr ìe "■ «’ ■ A- • v Wv ‘ i ■ V,-v ■ * t ' '-.*4. ** • ■ »^J -m • ‘ *■ - ... -V ' . .-■* :v*. . ^ 1 5 : - 'A : X - ; A *■* Ì - * '"*”’ i ^ .H •’ t-"*;. ' .'* "X'.^L;. _ >%,-X • ' HÉ-« — -jì.-i. - * i - . v '? ' ' ■•’-j -t, :>* • •* • ; •- ̂ -"StiC5. X S i;- - > • ursolic •> V v -■rtà-f. 'ibriTJ .»> • » ■ > J :< ..*■ -i.1- .■. . ';• ;-• À ^ ' ^ ■* • ’ ■ ■;r n \ s m.y * j i .•* ,•; ;< .■ .;■ • ; •;. ;•-■ i y ■**-?■ 7 -> -X V y . A’v : - y » / ; -- xy \ ' . 4 V:• v *;' ' V ? m ' ‘. — .. . ; ^ ' s v ^ r i i ì i K f v i ■ .rii. »T ■ « ^ . . •• - • : i . •• i . r . •, . ■ «* . . - ■ . : ._■• _.. ,r_ —..- .• • » . .■ » , A ’- X A ' • 4 ' - ; y ' . ' • ; . m a 7 : ^ " 4 - . ; ■ * : . ' • r X S A A T v - • - • ' - • - UNIVERSITY OF IBADAN LIBRARY _L___i— I , , , . 1 ___ .___ i----- 1----- !----- 1----- »----- »----- 1---- 1— 5*0 4 . 0 3 . 0 2 . 0 1 . 0 PPM U 186bNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY 187 peaks associated with each isomer. This was easily assessed from thè 1JC nmr spectrum. The relative intensity of thè carboxyl carbon signals ( 5C, 180.19 and 179.90) were equal. Likewise most of thè pair of signals appearing upfìeld showed equal relative intensities. The obvious conclusion is that GSH 49 contained equal amounts of ursolic 277 and oleanolic 278 acids in thè mixture. This is thè first report of thè presence of an ursene compound in Gardenia plants. All thè triterpene compounds of Gardenia plants reported in thè literature occurrence belong to thè Oleanene series. This is however not a strange because quinovic acid, which had been reported as a common triterpenoid of Rubiaceae plants is of thè ursene series. UNIVERSITY OF IBADAN LIBRARY 188 Table 28- 250 MHz Ih NMR Spectral data for GSH 49, Ursolic and Oleanolic acids. Position GSH 49 Ursolic acid Oleanolic acid 3a 3.11( IH, m ) 2.91(lH,t,J=8Hz) 3.10(lH,t,J=7- 10Hz) 12 5.15 (H,bs) 4.94(lH,t,J=3Hz) 5.14(lH,t,J=3Hz) 18 2.96(lH,dd,J=4, 2.08(lH,d,J=13 2.95(lH,dd,J=4, 10Hz) Hz) 10Hz) 2.29(1 H,d,J=ll Hz) Mej 0.93 0.69(6H,d,J=4Hz) 0.93(3H,s) Me2 0.90 0.50(6H,s) 0.89(3H,s) Me3 0.88 0.47(6H,s) 0.67(6H,s) Me4 0.70 0.34(3H,s) 0.66(3H,s) Mes 0.68 0.60(3H,s) Me6 0.67 0.54(3H,s) Me7 0.64 Mes 0.61 Me9 0.60 Meio 0.54 UNIVERSITY OF IBADAN LIBRARY 189 Therefore, on biogenetic grounds, thè presence of ursolic acid in G.erubescens is a possibility, as plants of thè same family naturally possess inherent potential to produce similar naturai products, although a number of factors, such as geographical location and genetic variation may influence thè production of certain naturai products in preference to some other compounds. Table 29- 100 MHz 13C NMR data for GSH 49, Ursolic and Oleanolic acids. Carbon UGSH 49 Ursolic acid °GSH 49 Oleanolic acid 1. 39.09 38.4 38.95 38.25 2. 24.93 24.44 26.17 25.50 3. 78.14 77.43 78.14 77.38 4. 39.50 38.80 39.39 38.71 5. 55.83 55.13 55.83 55.13 6. 17.52 18.10 18.80 18.12 7. 33.59 32.90 34.24 33.54 8. 39.50 38.80 39.77 39.07 9. 48.06 47.35 48.06 47.44 10. 37.30 36.60 37.40 36.69 11. 23.64 22.95 23.92 23.10 12. 124.29 124.97 122.57 1 2 1 . 8 8 13. 139.27 138.58 144.85 144.14 14. 42.52 41.81 42.19 41.48 15. 28.80 28.15 28.70 28.11 UNIVERSITY OF IBADAN LIBRARY 189b T.ìhle 29 Cont’d GSH 49 Chemical Shifts of Carbons for thè Oleanolic acid component of thè mixture. UGSH 49 Chemical shifts of Carbons for thè Ursolic acid component of thè mixture. The 13C NMR data for GSH 49 and thè pure compounds - oleanolic and ursolic acids, as presented in table 29 unequivocally shows that thè 13C NMR spectrum of GSH 49 contained signals for thè carbons of both oleanolic and ursolic acids. The data shown in table 29 compares fairly well with a similar table for thè 13C NMR data of thè methyl ester of a mixture containing oleanolic and ursolic acids and those of thè methyl esters of pure oleanolic and ursolic acids as reported by Ngonela, S.A. in his diesis. 173*176 The obvious differences were thè upfield shift of thè C-28 carbon signal and thè presence of a carbon signal for thè methoxyl group in thè methyl ester derivatives. 173 However, table 29 shows consistent downfield shifts in thè 5-value of thè carbon signals for thè mixture as compared with thè pure components. This trend was not observed in thè table for thè methyl ester derivatives, as thè 5 -values for thè carbon signals of thè mixture and thè pure compounds were almost thè same.173 UNIVERSITY OF IBADAN LIBRARY 1 8 9 c The shifts as they appear in table 29 could be attributed to thè possibility of intermolecular H-bonding between thè Oleanolic acid molecules and ursolic acid molecules in thè mixture. This type of interaction is not likely to be strong in thè methyl ester derivatives as they do not contain carboxyl groups. Hence, there were no shifts observed in thè 5-values for thè carbon atoms of thè mixture of methyl ester derivatives of oleanolic and ursolic acids when compared with thè 5-values of thè carbon atoms of thè pure methyl oleanoate and methyl ursolate. UNIVERSITY OF IBADAN LIBRARY 190 Table 29 contd. Table UGSH 49 Ursolic acid °GSH 49 Oleanolic acid 16. 17.52 18.10 18.80 18.12 17. 46.69 47.35 48.13 48.98 18. 53.56 52.86 42.03 41.32 19. 39.98 39.28 46.50 45.79 2 0 . 39.39 38.71 30.00 30.29 2 1 . 31.08 30.40 33.27 32.51 2 2 . 37.46 36.77 30.00 30.30 23. 28.34 28.00 28.12 27.63 24. 16.57 15.92 16.57 15.90 25. 15.68 15.01 15.55 14.90 26. 17.52 18.10 17.47 16.75 27. 23.76 22.95 23.83 23.10 28. 179.90 179.23 180.19 179.51 29. 17.07 16.87 30.97 30.30 30 21.41 20.77 18.80 18.12 2.5.10 Elucidation of Structure of GSA 5 The first indication that GSA 5 is a triterpenoid was from thè positive Liebermann-Burchard reaction. The absorptions at Vmax 3450 and 3330 cm ' 1 in thè IR Spectrum suggested thè presence of hydroxyl group. The Strong peak at UNIVERSITY OF IBADAN LIBRARY 191 Vmax 1698 cur* also suggested thè presence of carboxyl group and thè bands at 1304-1016 cm-1 supported thè presence ofhydroxyl and carboxyl groups (Fig 58) The Ih nmr spectrum showed six methyl groups as singlets (Fig 59) Three carbinolic protons were also visible at 83.57 (IH, d, J=10.4Hz) and 83.86 (2H,m). The peak at 85.15 (IH, t,J=3Hz) represent an olefmic proton. The nmr spectrum gave signals for thirty carbon atoms (Fig 60) and as shown by DEPT spectrum (Fig 61), they consist of eight quatemary, five methine, eleven methylene and six methyl carbon atoms. The two carbi nolic carbons were also revealed as one primary and thè other secondary. The signals at 8C, 121.06 and 143.33 supported thè presence of olefmic bond while thè signal at 8C, 178.72 also supported thè presence of carboxyl group. From thè Chemical shifìs of thè olefmic carbons 8121.06 and 8143.33 (see table 20, p.97) and taking into consideration thè fact that thirty carbon atoms were present, GSA 5 appeared to be a dihydroxyl triterpenoid with oleanene skeleton. The 1H nmr signal at 83.57 (lH,d, J=10.4Hz) could be attributed to one of thè methylene protons of thè primary hydroxyl group. The signal for thè second proton seem to overlap with thè carbinolic methine proton at 83.86 (2 H,m). An insight into thè probable location of thè carboxyl and primary hydroxyl groups was obtained from analyis of thè mass spectrum. Further evidence for thè presence ofhydroxyl groups was provided by thè loss of mass units 17 and 18 consecutively from thè molecular ion - m/z 455 [M-17] and 437 [455-H20] and thè loss of CO group (Fig 62). The accurate mass measurement for M-17, m/z 455.3532 corresponds to C30 H47 O3 (requires 455.3513). The EIMS also showed UNIVERSITY OF IBADAN LIBRARY 68.208 59 .682 51 .156 42.630 34 .104 r 25.578 17.052 8 .526 0 . 0 0 0 5250 4000 3200 2400 2000 1600 1200 800 490 CM UNIVERSITY OF IBADAN LIBRARY PPM U 191cNIVERSITY OF IBADAN LIBRARY fò 180 PPM f- UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY m ii Bài Jv iti oc: lii 1 ‘16 se tu :>sgai 8t m He 06 SS1* 8̂ 85 ìiìr 'vO U ►-+> '88 '88 '881 19310*3 ,0»19 t m e iirn T B 5 usa 339 sfis HI1iay=M 88338^=311 8=UH * £ ' 1 0 9-!i 9̂ ♦n resaci iNaoosoi Eg'cOS-st r>pt)g aiH +3attiui46jaaH 88t> 886 803 801 ~>— JU 8 ai 83 86 8t> 85 U ÙC UNIVERSITY OF IBADAN LIBRARY 192 peaks at m/z 248, 203, 189 and 133. These are diagnostic fragments which confirmed an olean-12-ene skeleton. Information about thè position of substituents on thè triterpene skeleton was also obtained by considering these fragments whose masses changed based on thè position of substituents. As illustrated in Scheme Vili, p. 101, thè type of substituent on rings D/E determines thè mass of fragment ion a . Given thè mass m/rzi 2n4g8p awsa ison a in this compound, it then follows that thè only group present on D/E thè carboxyl group. Therefore, by inference, thè primary hydroxyl group can be on C-23, C-24, C-25, or C-26, most commonly thè primary hydroxyl groups are located on C-23 or C-24. The position of thè primary hydroxyl group was confirmed to be on C-23 by NOE and ^H-l^C correlation spectra (table 30). The analysis made so far led to thè structure 279 for GSA 5. Interpretation of thè mass spectrum based on this structure is shown in Scheme XXX. The structure of GSA 5 was confirmed by thè preparation of thè diacetate. The IR spectrum of thè acetate (Fig 63) showed a strong band at Vmax 1739 cm- l for C=0 UNIVERSITY OF IBADAN LIBRARY CM-1 UNIVERSITY OF IBADAN LIBRARY 193 stretch of acetate. Its ' H nmr revealed two acetate signals at 81.96 and 82.01 suggesting thè derivative to be a diacetate. The three carbinolic protons are — 1+ ▼ m/z409 COOH Ion c, m/z 205 Scheme XXX. Fragmentation pattern in thè mass spectrum of GSA 5. UNIVERSITY OF IBADAN LIBRARY 194 now clearly identifìed and appeared more downfield - 53.63 (lH,d, J=11.6Hz), 53.80 (lH,d, J= 11.6Hz) and 54.73 (lH,m) fig 64. The 1 nmr spectrum also showed two signals for C=0 of acetate ( 5C 170.61 and 170.91) together with thè carboxyl carbon (fig 65). Its CIMS gave ions at m/z 574 [M+NH4]+, 510 [M-64] 451 [510-AcO], 248, 203, 189 and 133. (Fig 66). The presence of thè ion a with m/z 248, also implied thè absence of thè hydroxyl groups on rings D/E. Final proof for thè structure of GSA 5 was obtained from NOE and ' H - ^ c corrleation spectra (table 30). UNIVERSITY OF IBADAN LIBRARY i 4 . 0 3 . 0 2.0 1.0 PPM U 194bNIVERSITY OF IBADAN LIBRARY HZ/PT ÌPB-; PW 4.0 RD 0.0 AG 1.015 R3 2 5'. 235: TE 2.- 02 4200.CCO DP 15H CPD LB 2.eoe Fig. 65. nmr spectrum of GSA 5 diacetate GB I i00 PPM/CM 4.500 SH 5962.19 o _ J __________________ !__________________ I__________________ !_________________ !__________________!_______________ I__________________I_____________________ 1_______________ I__________________!_____________________1________________ L_ 180 170 160 150 140 130 120 110 100 90 80 70 60 ___________________________________________________ _______________________________BPM_______________ ____ ____________ _______ UNIVERSITY OF IBADAN LIBRARY Fig. 66. CIMS of GSA 5 diacetate UNIVERSITY OF IBADAN LIBRARY 195 Table 30- 400 MHz iHNM R spectrum andNOES for 3p-,23-dihydroxyolean-12- en-28-oic acid. Position 6/Multiplicity J (H z) NOES to la 1.05 m ip 1.62 d 12 2a 1.98 d 12 2(3 2.00 m 3a 3.86 m 5a 1.58 d 12 6a 1.79 d 12 6(3 1.48 dd 12,12 24.25,26,6a,7a l a 1.69 m 7P 1.32 d 11 9a 1.80 m lla /p 2.00 m 12 5.15 t 3 lla/p ,18p,26 15a 1.19 d 12 15p 2.10 t 12 26,15a,16p 16a 2.10 m 16p 1.92 m 18p 3.00 dd 4,10 12,19p,22p,30 19a 1.82 t 12 UNIVERSITY OF IBADAN LIBRARY 196 Table 30 Contd. Position. 6/Multiplicity. J(Hz). NOES to 190 1.30 d 12 21a 1.45 d 12 29,19cc 210 1.21 d 12 22a 1.90 t 12 220 2.08 d 12 23a 3.86 d 10.4 23b 3.57 d 10.4 23a,5a,6cc,3a 24 0.71 s 23a,23b,3a 25 0.64 s 10,20,1 loc/0 26 0.70 s 23a &b 27 0.88 s 12,16a,9a,7a, 180 29 0.61 s 19a,21a 30 0.69 s 180,220 From thè analysis of thè spectral data available on GSA 5 and its diacetate 280. it was identified as 30, 23-dihydroxyolean 12-en-28-oic acid, 279. 2.5.11.Structure of GSA 8 This compound al so gave a positive Liebermann-Burchard reaction for triterpenoid. UNIVERSITY OF IBADAN LIBRARY 197 Its 1H mnr spectrum revealed thè presence of seven methyl groups which appeared as singlets (Fig 67). There were also two carbtnolic protons at 83.12 (lH,d, J=4Hz) and 84.06 (IH, dt, J=3 Hz) together with an olefinic proton at 85.18 (1 Ht, J=3 Hz). The 1 3C nmr spectrum confirmed thè presence of two carbinolic carbons at 8C, 70.76 and 70.68, indicating thè presence of two hydroxyl groups (Fig 68). DEPT spectrum showed thè two carbinolic carbons to be methine carbon atoms (Fig 69), suggesting that thè two hydroxyl groups were secondary. The presence of carboxyl and olefinic groups were also supported by signals at 8C 179.54, 8122.03, and 8144.19. The CIMS showed evidence for thè presence of hydroxyl and carboxyl groups with fragment ions m/z 455 [M-17], 437 [455-H20] and 409 [437-CO] with thè molecular ion peak at m/z 472 (Fig 70). The fragment ions m/z 248,203, 189 and 133 supported olean-12-ene skeleton and at same time gave an indication of thè presence of only thè carboxyl group on rings D/E. The molecular mass m/z 472 corresponds to thè formula C30H48O4. The multi pi icity of thè carbinolic protons suggested a vicinai diol. If there are no hydroxyl groups on rings D/E, then thè other alternative location would be on ring A since C-3 always carry a hydroxyl group. The location of th e h y d r o x y l groups on ring A is illustrated with structure 281. The two hydroxyl groups being located on C-2 and C-3. UNIVERSITY OF IBADAN LIBRARY Fig. 67. nmr spectrum of GSA 8 U 197bNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY Fig. 69. DEPT spectrum of GSA 8 *— y—»— —J 73 53 5 3 UNIVERSITY OF IBADAN LIBRARY PZ.6I Fig. 70. CIMS of GSA 8 3 9 5 4 8 9 4 ? ? 3 0 0 5 0 8 I M 1 f t T f l 4 8 * s U 8 0ùd~~38 2 6 ~ D C Ì -9 3 14 5 9 -8 03 41 1 3 -2 5 3 J C i* epR =e 1 * 1 .4 m Hw=8 T IC = 5 8 2 4 5 8 8 f ic n t R f l T U H S y s : flCE GSfìo B .C . E . N H 3 K H -4 7 8 P T = 0° C à L U C f t l 4 3 ? 4 55 4 8 9 4?3 , % 11 , 1. «►-*- ~j— ***- 3 8 0 400 UNIVERSITY OF IBADAN LIBRARY 198 The multiplicity of Ha is 1 H,d (83.12 J=4Hz) being split by Hg. But Hg has a multiplicity resulting from coupling with Ha and thè two adjacent methylene proton s on C1 hence it was split into doublé triplet (84.06 J=3Hz). The coupling Constant in thè C-3 hydrogen signal was used to determine thè configuration of thè hydroxyl groups on C-2 and C-3 according to thè deductions of Cheung et a l ^ 1.It was reported that splitting in thè C-3 hydrogen signal has J=2- 3 Hz in 2a,3a-dihydroxy compounds and J=4-5 Hz in 2p,3p-dihyroxy compounds. The J value for C-3 proton signal of GSA 8 was 4Hz and accordingly suggested thè configuration at C-2 and C-3 to be 2p,3p-dihydroxyl. This compound was found to share thè same feature with GSA 11 with respect to substitution on C-2 and C-3. The configuration of thè hydroxyl groups on ring A of GSA 11 was determined from NOE and ^ H -^ c correlation spectra as 2p,3P, 23-trihydroxyl, hence by extension thè 2p,3(3-confìguration of thè hydroxy groups on ring A of GSA 8 was confirmed. The structure of GSA 8 based on thè spectral data obtained was proposed to be 2p,3P-dihydroxyolean-12-en-28-oic acid, 282. UNIVERSITY OF IBADAN LIBRARY 199 HO HO This structure could not be confirmed further by derivatization due to thè probable small quantity isolated. However, it is most that thè structure ' correct because GSA 8 shares similar features with GSA 5 and GSA 11, which have b een identified as olean-12-ene-28-oic acid derivatives. 2.5.12 Structure of GSA 11 GSA 11 was thè third triterpene aglycone isolated from thè stems of G. erubescens. It also gave a positive Liebermann-Burchard reaction, The IR spectrum (Fig 71) showed bands for hydroxyl groups at Vmax 3500, 3465 cm_1 and thè carboxyl group at Vmax 1677 c m 'l . nmr spectrum revealed signals for six angular methyl groups and four carbinolic protons (Fig 72). The proton signals at 63.38 (IH, d, J=11 Hz) and 83.83 (IH, d, J=11Hz) appeared to be that of geminai protons (large couplirig Constant). The other two carbinolic protons at 83.93 (lH,d, J=4Hz) and 84.18 (1H, dt, J=3Hz) were similarly coupled to each other and thè J UNIVERSITY OF IBADAN LIBRARY CM-1 UNIVERSITY OF IBADAN LIBRARY Fig. 72. IH nmr spectrum of GSA 11 j PPM UNIVERSITY OF IBADAN LIBRARY 200 value suggested they were vicinai protons. The singal at 85.17 (IH, t, J=3 Hz) represented an olefmic proton. The 1 nmr and DEPT spectral data indicated that GSA 11 possessed six methyls, ten methylenes, six methines and eight quatemary carbons (Fig 73). It also revealed thè presence of three carbinolic carbons, two of which were secondary and one primary. The signal at 8C, 180.19 supported thè presence of carboxyl group. The molecular ion from CIMS had m/z 489.3580 [M+l]+. This corresponds to C30H49O5 (requires 489.3567). The mass spectrum also showed loss of water molecules from three consecutive fragment ions m/z 471 [M-17], 453 [471-HgO], 435 [453-H20] and also a loss of CO from fragment ion m/z 4 f 5 (Fig 74). The presence of thè characteristic fragment ions of thè olean-12-ene skeleton gave an indication that GSA 11 is an oleanene compound and this was already implicated from thè Chemical shifts of thè olefmic carbon atoms. One additional powerful information obtained from thè CIMS was thè presence of fragment ion m/z 248. Its presence suggested that only thè carboxyl group is present on D/E rings. The location of thè three hydroxyl groups was resolved to be on ring A from NOE, COSY and ^H -^C correlation spectra (table 31). The structure that is consistent with available data on GSA 11 is 2p,3[3,23-trihydroxyolean-12-en- 28-oic acid, 283. UNIVERSITY OF IBADAN LIBRARY Fig. 73. 13C nmr and DEPT spectrum of GSA 11 _1------------------------ 1-------------------------- i-------------------------- 1_________________ I__________________L n 73 G3 53 43 33 U 200bNIVERSITY OF IBADAN LIBRARY — o co xx un co **u co co — ' >30 «33 CO *30 C D *3D *30 *30 >30 CT> r o TSZ r<0o*7~33S» 3370 r o *30 OD 3Ó — » 3m0 •awfc,V—*• CO «—« ro » ro0-> m CO 33T 'sJ 33 3» CO C X 1.1—1 ro ~T~ 35! "»♦ 33 fi >> cs> ro •tv U3 CO e x CO » ro ■..4 co w-* CO *> rv> ru rrov co f Cro3Cr/oJr ■ ■> co » cr? ro . I ru C«D- sr—li?_ L - o - > COSD ro J . ---------- ------------------------- P *CO 5 re* -T3 33•C ro -«- * 3—04 *C:_O *C OOJ30- co r<——» r&. 2F5W3TJL Ci* ostile lr»l=? FT= I8N® $CaqLS :JfCìfnll HfìR UNIVERSITY OF IBADAN LIBRARY 206 The IR spectrum indicateci thè presence of ester group with a strong band at Vmax 1747cm-l . A weak band at Vmax 330-3200 c n r ' for O-H stretch for thè carboxyl group and its C=0 band at Vmax 1697 cm-l were also present (Fig 84). Its 1H nmr showed signals for three acetate methyls at 61.95, 1.98 and 2.03. The carbinolic proton signals showed significant shifts, with H-3 proton moved downfield (Fig 85). The formation of thè triacetate derivative confirmed thè presence of three hydroxyl groups in GSA 16. The CIMS of thè triacetate gave thè [M+NH4]+ ion at m/z 632.416, which corresponds to C36H5gOgN (requires 632.415) and [M+l]+ ion at m/z 615.390, which corresponds to C36H55O8 (requires 615. 388). The presence of thè diagnostic ions m/z 248, 204 and 192 gave further support for thè absence of any of thè three hydroxyl groups on ring D or E (fig 86). Given thè molecular formula of thè triacetate as C36H54O8, it follows that thè molecular formula of GSA 16 would be C3()H4g05. The methyl ester derivative of GSA 16 confirmed thè presence of carboxyl group in thè compound. The 1H nmr spectrum showed one methoxyl group (Fig 87) at 63.37 (3H,S) while nmr spectrum confirmed thè formation of a methyl ester with thè carboxyl carbon signal shifted upfield, 6C, 178.0 (fig 88). The CIMS also confirmed thè formation of a methyl ester with thè [M+l ]+ ion peak at m/z 503 (fig. 89). The peaks at m/z 262, 203 are fragment ions a and c respectively. The shift of thè mass of ion a from m/z 248 in thè free acid to m/z 262 in thè methyl ester confirmed thè presence of thè carboxyl group on rings D/E. UNIVERSITY OF IBADAN LIBRARY 68.208 59.582 51.156 42.630 34.104 X) < > < C O O H \ f i r^COOH HO ^ ^ ch2oh hoh2c ^ ^ ch2oh 2 p ,3 (3,23-T rihydroxyolean-12-en-2 8- oic acid.(Bayogenin). (GSA 11) 3 p ,23,24-T rihydroxyolean-12-en-2 8- oic acid (Erubigenin). (GSA 16) UNIVERSITY OF IBADAN LIBRARY 221 CHAPTER THREE 3.1 EXPERIMENTAL. The 250MHz, 400MHz NMR and 100 MHz 13C NMR spectra were recorded as dilute Solutions in d5-pyridine, DMSO-d^ or CDCI3 on a Bruker ACP400 and 250 instrument with tetramethylsilane as internai reference. IR Spectra (Nujol mulls) were recorded on a Perkin Elmer 1720X FTIR. Optical activity was measured on an AA-1000 polarimeter. UV spectra were recorded on Unicam 8700 series UV/VIS spectrometer and melting points were taken on Gallenkamp apparatus and were uncorrected. EI, CI and FAB mass spectrometer spectra were run on a Kratos MS-902 doublé focusing instrument at 70 eV. Precoated silica gel Kieselgel 60 F254 plates were used for analytical TLC, silica gel a for thinlayer chromatography was used for preparative TLC and silica gel G. (230-400 mesh) was used for column chromatography. All solvents used in this work were redistilled. Petroleum ether used refers to thè 60-80°C boiling range. The Chemicals, sodium hydroxide, Aluminium chloride, magnesium sulphate, bismuth nitrate and ferric chloride were obtained fronti suppliers. Detection reagents used for phytochemical test for alkaloid, was Dragendorffs reagent, chlorosulphoric acid reagent and Acetic anhydride/H2S04 reagent for steroids. Flavonoid tests were carried out with NH3 vapour, aluminium chloride reagent and magnesium/concentrated hydrochloric acid reagent. Ferric UNIVERSITY OF IBADAN LIBRA Y 223 3.2.4. Aluminium Chloride Reagent 5% solution of aluminium chloride in methanol was prepared by dissolving 5g of aluminium chloride in 10 mi of distilled water and thè solution made up to lOOml with methanol. 3.2.5. Sulphuric Acid Reagent Equal volume of methanol and concentrated sulphuric acid were mixed together to give 50% methanolic sulphuric acid. 3.2.6 Liebermann-Burchard Reagent (adapted for TLC) 2 mi concentrated sulphuric acid, 40ml acetic anhydride and 100 mi chloroform were mixed together to make thè spray reagent. 3.3 EXTRACTION OF PLANT MATERIAL The stems of Gardenia erubescens were airdried and then pulverized. 3kg of thè pulverised stems was extracted in aSoxhlet extractor with petroleum ether for 24hr. This was followed by extraction with methanol for 24hr. Subsequent extractions were carried out with some modifications. For one batch of thè extraction, 2kg of thè pulverized stems was extracted with petroleum ether for 24hrs, then ethylacetate for 24hr and finally methanol for 24hr. Another extraction was carried out with 4kg of thè pulverised stem. The plant material was First extracted with petroleum ether for 24hr after which thè UNIVERSITY OF IBADAN LIBRARY 224 defatted plant material was soaked in 60% aqueous methanol for 72hr. The aqueous methanol extract was filtered before further treatment. 3.4 TREATMENT OF EXTRACTS 3.4.1 Petroleum ether Extract I h ree sets of extractions were carried out and they were concentrated in vacuo as at when extration was carried out. The first petroleum ether extract was concentrated on a water-bath to a dark greenish-yellow oil which became a dark greenish-yellow gum on cooling. Before thè extracts were subjected to analysis, they were taken up into chloroform and thè insoluble component filtered off. The filtrate was rotary evaporated and these were used as thè petroleum ether extracts. The yield of thè extracts was about3-5g. 3.4.2 Ethyl acetate Extract. Concentration of thè ethyl acetate extract gave a thick dark green oil (syrup) (2.5g). This extract was obtained once as it proved to be uninteresting 3.4.3 Methanol Extract. The first extraction carried out with methanol yielded on concentration on a water-bath a thick dark brown gum (300g). UNIVERSITY OF IBADAN LIBRARY 225 The extracts that were obtained from thè subsequent extractions carried out with 60% aqueous methanol were concentrated in vacuo to a reddish brown solid weighing about 200g and 250g respectively. Tituration of thè concentrated methanol extracts with methanol precipitated a white crystalline solid which was filtered off and washed several times with methanol and then recrystallized from MeOH/E^O mixture. The methanol mother liquor of thè extract was again concentrated in vacuo. 1 OOg of thè extract was dissolved in 250ml of distilled water and thè mixture was extracted with 3x lOOml n-butanol. The n-butanol soluble fractions were combined and concentrated in vacuo to a yellowish brown solid (~15g). This was used as n-butanol soluble fraction of methanol extract in subsequent analysis. The aqueous phase was again also extracted with 50ml portions of ethylacetate, four times and thè fractions combined and concentrated in vacuo to a brown gum (~1 g). The aqueous phase was fmally discarded as it contained water soluble substances which are difficult to isolate. 3.5 PHYTOCHEMICAL T E S T S ^ l 'S . The petroleum ether, ethyl acetate, n-butanol soluble and ethyl acetate soluble extracts were separately tested for thè presence of flavonoids, steroids, alkaloids, anthraquinones and saponins. UNIVERSITY OF IBADAN LIBRARY 226 3.5.1 Phytochemicl analysis of Petroleum ether Extract. i. Flavonoid test A dilute solution of thè extract in a mixture of chloroform/methanol was made. To this solution magnesium tumings (few pieces) were added followed by addition of concentrated hydrochloric acid. A dark red colour was observed. ii. Steroid test Liebermann-Burchard test was carried out. A small quantity of extract was dissolved in 2ml acetic anhydride in a test-tube. This was cooled in an ice-bath before sulphuric acid was carefully added. There was a colour change from violet to green in thè upper layer. iii. Alkaloidtest A dilute solution of thè extract was made with chloroform/methanol mixture. Few drops of Dragendorffs reagent was added. There was no turbidity or precipitation observed. iv. Anthraquinone test The Bomtrager's test was used. About 0.2g of thè extract was dissolved in lOml of benzene. The solution was filtered and 5ml dilute ammonia solution was added. The mixture was transfered into a 50ml separating funnel and shaken. The ammoniacal phase remained yellowish green. UNIVERSITY OF IBADAN LIBRARY 227 3.5.2 Phytochemical analysis of Ethyl acetate extract. i. Steroid test Liebermann-Burchard test was carried on a dilute solution of thè extract in acetic anhydride. The upper layer of thè reaction mixture tumed green. ii. Othertests The test for alkaloid, flavonoid, and anthraquinones were also carried out. The observations were negative for these compounds. 3.5.3 Phytochemical analyses of methanol extract, n-Butanol soluble and ethyl acetate soluble extracts. i. Flavonoid test. Dilute solution of each extract was prepared in a test-tube and thè Shinoda test carried out. For thè methanol extract, colour of thè solution remained brownish. The n- butanol soluble extract also did not give any colour change while there was a colour change ffom pale brown solution to yellowish brown in case of thè ethylacetate soluble extract. ii. Steroid test Liebermann-Burchard test was carried out on each of thè extracts. The methanol extract gave a pale pinkish colouration at thè interface which latter tumed reddish, while a violet colour was observed for thè n-butanol soluble extract which also tumed reddish with time. There was no visible reaction with thè ethylacetate soluble extract. UNIVERSITY OF IBADAN LIBRARY 228 iii. Alkaloid test. Dilute Solutions of each of thè extracts were tested for alkaloid. The results were negative. iv. Tannins test A few drops of 1% solution of ferric chloride was added to Imi methanol solution of each extract in a test tube. A dark green colouration was observed for thè methanol extract. The n-butanol soluble did not show any visible reaction but a green colouration was observed for thè ethyl acetate soluble extract. v. Saponin test Frothing test was carried out on thè crude methanol extract and thè n- butanol soluble extract. A small quantity of each extract was disolved in 2ml distilled water in a test-tube and shaken. Both extracts produced frothing which persisted on warming in a hot water-bath. 3.6 ANALYTICAL THIN LAYER CHROMATOGRAPHY (TLC). 3.6.1 TLC analysis of Petroleum ether extract. A moderately concentrated solution of thè petroleum ether extract in chloroform was applied on precoated silica gel plates (cut to size 2.5 by 10 cm) with thè aid of capillary tube and developed in thè following solvent mixtures. (a) CHCl3:Hexane, 1:3; (b) di isopropyl ether; (c) diisopropyl ether: acetone, 75:30; (d) ethylacetate:Hexane, 1:3 and (e) ethylacetate:hexane, 1:1. UNIVERSITY OF IBADAN LIBRARY 229 The chromatoplates from each solvent System were separately viewed under thè UV, in iodine tank and also sprayed with detection reagents for alkaloids, steroids and flavonoids (fresh plates were prepared for thè different analyses). In each case one or two spots were visible as flavonoid spots which appeared as dark spots in UV but changed to bright yellow spots after treatment while a number of spots appeared as steroids but there was no visible alkaloid spot. For all thè solvent systems used, thè extract showed several spots on thè b u t chromatoplate thè ethylacetate: Hexane (1:3) mixture gave thè best resolution showing not less than ten overlapping spots with iodine vapour. 3.6.2 TLC analysis of n-butanol soluble extract. The composition of n-butanol soluble fraction of methanol extract was examined by subjecting thè extract to TLC analysis in a number of solvents. A reasonably concentrated solution of thè extract in methanol was spotted on thè TLC plates and developed in thè following solvent systems: a) Chloroform:methanol,l :3,1:1, and 65:45 b) Chloroform-methanol-water 65:45:12 and 65:35:10 c) Dichloromethane-methanol-water 40:10:1 d) Ethyl acetate-pyridine-water 10:4:3 e) Ethyl acetate-pridine-water methanol 16:4:2:1 f) n-Butanol-acetic acid-water 3:1:1 and 4:1:5 The spots on thè chromatoplates from thè different solvent systems were detected by: UNIVERSITY OF IBADAN LIBRARY 230 i) Spray reagents for alkaloids, steroids and flavonoids. ii) Visualising in UV and iii) lodine vapour. The alkaloid and flavonoid spray reagents gave negative results, while many of thè spots reacted positively with chlorosulphonic acid reagent giving grey to brown colours. Most of thè steroid spots observed were concentrated at around Rf values 0.4 and below. On each chromatoplate several spots were visible with many overlapping. Most of thè solvent Systems dragged thè components in a tailing fashion but thè chromatoplates developed in chloroform-methanol-water 65:45:12 and 65:35:10 showed thè best resolution. 3.7 COLUMN CHROMATOGRAPHIC ANALYSIS OF PETROLEUM ETHER EXTRACT. lOg of thè petroleum ether extract was purifed on a column of silica gel (400g) packed in hexane. The colum was developed in hexane. Elution continued with 2% EtOAc/Hexane mixture. 5% EtOAc/Hexane mixture eluted a white grease and further elution gave thè carotenoid fraction as orange-red oil(~3g). Elution with 8% ethyl acetate/hexane gave a white solid. This fraction, GSH 2 (~50mg) was lightly coloured by thè carotenes but thè orange colouration was successfully removed by washing thè solid with hexane and TLC in 5%, 8%, 10% EtOAc/Hexane showed only one spot. When thè solvent mixture was changed to 10% ethyl acetate UNIVERSITY OF IBADAN LIBRARY 231 hexane, fractions 9-20 collected contained one major steroid spot (TLC) with other minor contaminants which were mostly thè carotenes. These fractions were then combined as GSH 14 and subjected to further purification on preparative TLC. 15% ethyl acetate-hexane gave fractions 22-38 which contained three components. Fractions 22-25 contained two UV active spots (TLC) with thè spot having higher Rf value (Rf 0.63) as thè major component, GSH 24. The minor contaminant '/t■e’ :v.‘ which gave a positive flavonoid test (TLC) was removed from GSH 24 by preparative TLC. Fractions 26 and 27 were pure (TLC) Rf value 0.50 in 10% EtOAc/Hexane and found to be similar to thè contaminant of GSH 24. The TLC showed one spot which tested positive for flavonoid. This component was labelled GSH 26. The quantity obtained was in trace amount. Fractions 31-38 also happened to be contaminated with GSH 26. Therefore further purification of this fraction, GSH 32 , which has steroid (TLC, Rf value 0.42) as thè major component was carried out by preparative TLC. The chlorophylls carne down with 20% ethyl acetate/hexane and with thè chlorophylls also carne a component which was UV active and also gave positive flavonoid test (TLC). Fractions 40-43 contained this compound as thè only component but with heavy colouration with chlorophyll pigment. The solution of this fraction in chloroform was treated with activated charcoal to give a pale green solid - GSH 41 (~25mg). The column was continued with 25% ethylacetate/hexane and thè fractions collected, 48-60, showed one major steroid spot (TLC) together with chlorophyll pigment. These fractions were combined and rechromatographed on a s h o r t column. UNIVERSITY OF IBADAN LIBRARY 232 of silica gel eluted with 20% and 25% ethyl acetate/hexane mixtures. The pure fractions collected were stili slightly coloured with chlorophyll. This component was labelled as GSH 49. GSH 49 was later found (^H and l^C NMR) to be a mixture of two isomerie compounds and an attempt was made to separate thè components. 30%-35% ethyl acetate/Hexane eluted an orange oily material. The column was terminated with 50% ethylacetate-hexane as fractions collected were uninteresting. 3.8 PREPARATIVE THIN LAYER CHROMATOGRAPHIC PLATES. The 20 by 20 cm standard plates were used to prepare thè preparative plates. A slurry of silica gel G for thinlayer chromatography was prepared by mixing silica gel (250g) with 450 mi distilled water was used for coating up to seven standard plates with 1 mm thickness. The prepared plates were activated ovemight at 110 C and allowed to cool before use. All thè preparative TLC carried our were with freshly prepared plates. 3.8.1. Prep. TLC for GSH 14 About lg of GSH 14 was purified by Pred. TLC. Each piate was loaded with about 200mg of thè substance as it contained only coloured contaminant. The plates were developed in 5% ethylacetate/hexane mixture. The orange colour moved with thè solvent front and was easily removed from thè piate. The steroid bands were scrapped and extracted with chloroform. The chloroform solution was evaporated to dryness in vacuo and thè compound recrystallized from UNIVERSITY OF IBADAN LIBRARY 233 hexane/ethylacetate mixture as white needles (~0.6g). Purity was checked by TLC in 5% and 10% ethylacetate/hexane mixture. 3.8.2. Prep. TLC for GSH 24 Less than 40 mg of this substance was available. The whole quantity was dissolved in Imi chloroform and loaded onto two preparative plates. The plates d e v e lo p e d were with 10% ethylacetate/hexane mixture. The major UV active band ( Rf value = 0.61) was scrapped and extracted with chloroform. The chloroform extract was concentrated to a small volume, then transfered into a small sample vai where thè solvent was completely removed in vacuo. TLC in 10% ethyl acetate/hexane showed one spot. The chromatoplate was also sprayed with ferric chloride reagent and heated in thè oven for about five minutes. A greyish black colour developed. Weight of GSH 24 recovered was ~25mg. 3.8.3. Prep TLC for GSH 32 The amount of substance available was less than 50mg. This was dissolved in 1 mi chloroform and loaded on two preparative plates. The plates were developed in 10% ethyl acetate/hexane mixture. The UV active bands (minor contaminanti were removed and steroid bands (Rf value = 0.42) were scrapped and extracted with chloroform. Complete evaporation of solvent gave GSH 32 as white solid ( 35mg). Purity was confirmed by TLC in 10% ethyl acetate/hexane mixture. UNIVERSITY OF IBADAN LIBRARY 234 3.8.4. Prep TLC for GSH 49 i) By normal preparative plates. Four preparative plates were each loaded with about 50mg of GSH 49 dissolved in chloroform/methanol (1:1). The plates were allowed to run continuously in 10% MeOH/CHCl3 mixture for 3hrs. The reference piate was treated with 50% methanolic sulphuric acid reagent. It was observed that there was no resolution. ii) By silver nitrate-impregnated preparative plates The silica gel slurry was made by adding AgNC>3 (10% w/w) to thè silica gel powder. This was used to prepare thè silver nitrate impregnated preparative plates. The purification was repeated as for thè normal preparative plates. This mixture could not be resolved. 3.9. CHROMATOGRAPHIC ANALYSES OF ETHYL ACETATE AND N-BUTANOL SOLUBLE EXTRACTS. 3.9.1. Column chromatographic analysis of Ethyl acetate Extract. 2g of ethylacetate extract was chromatographed on a column of silica gel (150g) packed in Hexane. The column was eluted with ethylacetate/hexane mixtures 10%, 15%, 20%, 25%, 35%, 50%, 70% and 100% ethylacetate. The fractions collected contained greenish oily material. UNIVERSITY OF IBADAN LIBRARY 235 Elution of thè column was continued with 5% MeOH/EtOAc, 10% MeOH/EtOAc and was terminated with 15% MeOH/EtOAc. 5% MeOH/EtOAc eluted fractions containing one major component. The fractions were combined to give a pale brown solid. This was titurated with acetone to give a pale white solid (~10mg). It gave only one spot on TLC (methanol: chroform: water (65:45:12) and 30% MeOH/CHCl3). This compound was later found to be identical to GS 13 (NMR). 3.9.2. Column chromatographic analysis of n-butanol soluble extract. T he n-butanol soluble fraction (6g) of methanol extract was subjected to chromatographic analysis on a column packed with silica gel (300g) (230-400 mesh) in chloroform and thè column run with mixtures of CHC^/MeOH in increasing order of polarity. Elution with 2% MeOH/CHCl3 brought down chlorophyll pigment while 4% MeOH/CHCl3 eluted some yellowish oily substance. A brownish solid substance carne down with 15% MeOH/CHCl3. TLC analysis in 30% MeOH/CHCl3 showed it as one component. The colouration was removed by tituration with ethylacetate. This was labelled as GS 13 (quantity was very small). The column elution continued with 20%, 25% 30%, 35%, 40%, 45%, 50% and 75 MeOH/CHCl3 mixmtiurxets,u bruets thè fractions eluted contained brownish gurmsy material which contained of substance^TLC). The fractions eluted with 50% MeOH/CHCl3 showed three major components on TLC when sprayed with UNIVERSITY OF IBADAN LIBRARY 236 chlorosulphonic acid reagent and thè NMR of this impure fraction indicated thè presence oftriterpenoid components. The column was thus not successful. The column was also rep eated using mixtures of solvents: (i) chloroform- methanol-water, 65:45:12 and (ii) chloroform-methanol-water, 65:35:10. These two solvent mixtures were used to run. two separate columns. Both columns were not successful. 3.9.3. Treatment of n-butanol soluble Extract. The n-butanol soluble extract (lOg) was further subjected to treatment in order to rid thè saponins of pigments and less important components. This was achieved by precipitation of thè saponins from thè methanol solution by using chloroform. The precipitate (saponin content) was collected by filtration and sucked dried to a dark brown solid (6g). TLC analysis of thè treated n-butanol soluble fraction showed thè presence of 4-6 saponin components. 3.9.4. Column chromatographic analysis of thè treated n-butanol soluble extract. The treated n-Butanol soluble fraction was subjectedto column chromatography. 3g of thè extract was chromatographed on a column of silica gel (250g) packed in chloroform. Elution was carried out with chloroform/methanol mixtures. The fractions collected were brownish gumy substances containing mixtures of components. UNIVERSITY OF IBADAN LIBRARY 237 The column was repeated with chloroform-methanol-water, 65: 35: 10 mixti; e :ut thè result was not satisfactory. 3.9.5 Preparative TLC for treated n-BuOH Soluble Extract. treated n thè n-butanol was dissolved in lOml methanol. Each preparative lo a d e d . s with about 50mg of extract. The plates were developed in : .n-methanol-water, 65: 45: 12 (best mixture, see TLC analysis of methanol . A reference piate which was sprayed with chlorosulphonic acid reagent as used as a maker for each set of plates run. Two bands were only visible from : e preparative plates. The two bands (Rf values 0.36 and 0.44) were removed into separate flasks and were extracted with MeOH. The methanol extracts on concentration did not contain any saponin (TLC and NMR). Another set of preparative plates were prepared and used in thè solvent System chloroform-methanol-water, 65: 35: 10 containing 20% ammonium hydroxide. The bands also overlapped. When they were removed, extracted with MeOH and thè solution concentrated, there was no trace of any saponins. Apparently thè saponins were not being extracted from thè silica gel. 3.9.6 Acid Hydrolysis of thè treated n-butanol soluble fraction. lOg of thè extract was dissolved in lOOml methanol and lOOml 4% methanolic sulphuric acid was added to give 2% sulphuric acid in thè methanol solution. The mixture was refluxed on a steam bath for 6hr. After cooling, thè reaction mixture was poured into 500ml distilled water and thè aqueous mixture UNIVERSITY OF IBADAN LIBRARY 238 extracted with ethylacetate. The ethylacetate extract was washed with water, dried with MgS0 4 and then concentrated in vacuo. About 3.8g of aglycone was recovered. The process was repeated for four sets of methanol extracts obtained in thè course of this work. The various weights of aglycone obtained were 3.8g, 3.6g, 2.1g and 5.4g. 3.9.7 Acetylation of n-butanol soluble Aglycone. 2 g of thè crude aglycone was acetylated with 60ml pyridine/acetic anhydride (1:1) mixture for four days. The reaction was stopped by addition of 30ml methanol to destroy excess anhydride. The reaction mixture was concentrated, diluted with water and then extracted with chloroform. The chloroform extract was washed with water, dried with MgSC>4 and thè solvent removed in vacuo giving 1 5g of crude aglycone acetate. 3.9.8. Column chromatographic analysis of n-butanol soluble aglycones. 5g of crude aglycone mixture was chromatographed on silica gel (300g) packed in chloroform. Column elution was carried out with chloroform/methanol mixtures. For 2% methanol/chloroform thè fractions collected contained brown pigment. Fractions 5-10 were collected from elution with 4% methanol/chloroform. Fractions, 5,6 and 7 were combined on thè basis of TLC (in 2 % and 5% MeOH/CFICl3). These fractions contained one major component with minor contaminants (pigment). Fractions 8,9,10 however showed only one pure component with similar Rf value (0.32) to thè component in 5,6 and 7. Combined UNIVERSITY OF IBADAN LIBRARY 239 fractions, 8,9,10 gave GSA 8 ( ~15mg). The combined fractions 5,6 and 7 w ere later purified by preparative TLC to give GSA 5. Further elution with 6% methanol/chloroform gave fractions 11-25. Fractions 11, 12, 13 and 14 showed one single component (TLC) but was contaminated by brown pigment. Recrystallization in 50% methanol/chloroform gave white salt-like crystals labelled as GSA 11 ( ~20mg). Fractions 16, 17 and 18 contained another brownish solid which has similar Rf value (0.25 in 10% MeOH/CHCl3 ) with thè component of Fractions 11, 12 13 and 14. The combined w ere fractions 16, 17 and 18 . further purified by preparative TLC as GSA 16. 3.9.9 Preparative TLC for GSA 5 and GSA 16 The combined fractions for GSA 5 and GSA 16 were concentrated and redissolved in Imi 50% methanol/chloroform. Each compound was loaded separately on two preparative plates and thè plates were developed in 30% methanol/chloroform mixture. The pigment contaminants did not move from thè origin. The area above thè pigment band was scrapped and extracted with 50% methanol/chloroform mixture giving GSA 5 ( ~50mg) as white crystals and GSA 16 ( ~40mg) as white powder. 3.9.10.CoIumn Chromatographic analysis of Acetylated Crude Aglycones. About 1,4g of crude aglycone acetate was chromatographed on silica gel (150g) column. The column was developed in hexane and elution continued with ethylacetate/hexane mixtures. Elution with 5%, 10%, 15%, 20%, 25% up to 75% UNIVERSITY OF IBADAN LIBRARY 240 ethylacetate/hexane gave fractions containing mixtures of components (TLC). The column was discontinued. 3.10. Preparation of Derivatives. i) GS 1 hexaacetate lg of GS 1 was dissolved in 50ml pyridine/acetic anhydride (1:1) mixture. The reaction mixture was kept for three days. Excess acetic anhydride was destroyed by addition of 20ml methanol. The solvent was removed and residue diluted with water. The aqueous mixture was then extracted with chloroform. The chloroform extract was washed with distilled water, dried with magnesium, sulphate and thè solvent removed completely. The product was redissolved in chloroform and left to crystallize out, giving white heavy crystals of GS 1 hexaacetate (1 2g). ii) GSH14acetate 0.5g of GSH 14 was dissolved in 1:1 mixture of pyridine/Ac20 (30ml). The reaction mixture was left ovemight and thè usuai work-up carried out. The acetate (0.3g) was redissolved in chloroform and left to crystallize out. iii) GSH 14 Oxo-compound 50mg of GSH 14 was added to 50mg of pyridinium chlorochromate in lOml dried dichloromethane. The reaction was stirred at room temperature for 2hr and thè reaction was minotored by TLC (in ethylacetate/hexane, 1:3). The reaction UNIVERSITY OF IBADAN LIBRARY 241 mixture was fìltered when reaction had been completed (TLC) and thè filtrate diluted with water. The aqueous mixture was extracted with diethyl ether. iv) GSA 5 diacetate 25mg of GSA 5 was dissolved in 20ml 1:1 mixture of pyridine/AC20 mixture. The reaction was left for two days and thè usuai work-up carried out. The crude acetate ( 15mg) was redissolved in chloroform and left to crystallize out. v) GSA 11 triacetate 20mg of GSA 11 was dissolved in 20ml 1:1 mixture of pyridine/AC20. The same procedure as for GSA 5 diacetate was used. The crude GSA 11 triacetate (lOmg) was redissolved in CHCI3 and left to crystallize out. vi) GSA 16 triacetate 20mg of GSA 16 was acetylated according to thè procedure for GSA 5 and GSA 11. The crude GSH 16 triacetate was further purified by preparative TLC. The preparative piate was run in ethyl acetate/hexane, 1:3 mixture. The band was extracted with chloroform and thè solvent removed in vacuo. The product (12 mg) was then redissolved in chloroform and left to crystallize out. UNIVERSITY OF IBADAN LIBRARY 242 vii) OSA 16 methvl ester The methvl ester was preparaci by reacting thè acid with diazomethane. The diazomethane was generated from thè diazogen, N-methyl-nitroso-p-toluene suipr.onamide by thè method described by Paolo Lombardi1 s0. The diazomethane was carried into thè reaction flask containing lOmg of GSA 16, dissolved in 5ml methanol, by a streani of nitrogen gas. The reaction was monitored by TLC (10% MeOH/CHCl3 and EtOAc/Hexane 1:3) and stopped when all thè acid molecules had reacted. The solvent was then removed and GSA 16 methyl ester was recoveed as white solid. vii) GSA 16 triacetate methyl ester The GSA 16 methyl ester was acetylated by thè usuai method. The GSA 16 triacetate methyl ester (3-5mg) was obtained as a pale yellow gum. Further purifìcation was hindered by thè small quantity available. 3.11. Determination of Specific rotation for GSH 24, GSA 16 and GSA 16 triacetate. The weighed samples GSH 24 (20mg), GSA 16 (30mg) and GSA 16 triacetate (20mg) were dissolved in Imi spectroscopic chloroform or methanol as appropriate. The observed rotation was measured with thè AA-1000 polarimeter. The specific rotation was then calculated from thè observed rotation using thè expression, UNIVERSITY OF IBADAN LIBRARY 243 [oc]d = ctp 2 xC C= Conc. in g/ml, a D is observed rotation length of tube = 2 decimeter GSH 24 a 20p = + 2.4401 C = 0.02g /mi (CHCI3 ) [a]20D = 2.4401 2x0.02 [ct]20p = + 61 GSA 16 a 22p = + 4.5604 C = 0.03g/ml (MeOH) [q]22p = 4 5604 2x0.03 [a]22p = + 76 UNIVERSITY OF IBADAN LIBRARY GSA 16 triacetate a 22D = + 3.6022 C = 0 .0 2 g/ml (CHCI3 ) - 3.6022 2 x 0.02 [a]22o = + 90 UNIVERSITY OF IBADAN LIBRARY 245 3.12. SPECTRAL DATA ON THE COMPOUNDS ISOLATED FROM erubescens. 3.12.1 GS 1 D-Mannitol (GS 1) white crystals, m.p. 170°C (Lit. 166-168°C)93. IR Vmax i Nujol). 3300-3100, 2927, 2825, 1377, 1351, 1262, 1147, 1092, 1026, 933, 891 and 171 cm_l IH nmr (DMSO-d6) 64.42 (2H,d,J=6.0Hz) 4.35 (2H,t,J=6.0Hz) 4.13 (2H,d,J=8.9Hz) 3.50 (8H, m) 13c nmr (DMSO-d6>: 672, 70, 64 EIMS M/Z (rei. int.) 183.0869 (100) [M+l]+ (C6Hi506requires 183.0864), 165(10), 147(5), 111(2), 99(2) Mannitol hexa acetate. White crystals, mp. 117-119°C (Lit. 1210C)yA IR Vmax (Nujol) 2926, 2854, 1742, 1456, 1373, 1225, 1089, 1071, 1059, 1034, 992, 907, 863, 722 and 620 cm“l. IH nmr (CDC13) 61.98 (6H,S) 2.0 (6H,S) 2.2 (6H,S) 4.03 (2H,dd, J=5,7Hz) 4.18 (2H,dd, J=3,10Hz), 5.03 (2H,m) 5.37 (2H, dd, J=l,9Hz) 13C nmr (CDCI3) 20 (CO-CH3), 62, 67, 68, 169, 170, 171. EIMS m/z (rei. int.) [M+l]+ 435(10), 375 (100), 333 (5), 289(3), 273(12), 213(8), 153(55), 136(48), 77(55). UNIVERSITY OF IBADAN LIBRARY 246 3.12.2 GSH 2 m.p. 111-113°C (lit. 115.5-115.90C)lb / . IR Vmax (nujol) 4331, 3096, 2925, 2854, 1629, 1581, 1521, 1490, 1436, 1374, 1255, 1184, 1159, 1090, 961,804, 733crrrl U V Xmax (CHCI3) 239.2nm and 290.5nm IH nmr (CDCI3) 52.76(1H,dd,J=3Hz), 3.10(lH,dd,J=14,4Hz), 3.79(3H,S), 3.82(3H,S), 5.35(1 H,dd,J=3-6,10Hz), 6.97(2H, dd, J=9,2Hz), 7.40(2H,dd,J=9,2Hz), 10.90(1H,S); 6.02(1 H,d,J=3Hz), 6.05(lH,d,J=3Hz). 13C nmr. 5C2, 78.8; C3, 43.1; C4, 195.9; C5, 164.0; C6, 94.9; C7, 167.8; C8, 94.1; C9, 162.8; CIO, 103.0; C l, 130.3; C2’& C6’, 127.6; C 3 '& C5', 114.1. EIMS/mz (rei. int) [M+] 300 (45) (Acc. mass 300.0998), 283(5) 272(2), 257(3), 193(20), 166(20), 134(100), 121(60), 91(45). 3.12.3 GSH 14 m.p. 166-168°C (Lit. 170<>C)lf>« IR Vmax (Nujol) 3426, 2954, 2925, 2854, 1690, 1660, 1462, 1378, 1063, 970 cm-l IH nmr (CDCI3) 63.57(lH,m), 5.15(2H,m), 5.36(lH,d,J=5Hz), 0.69(3H,S), 0.71(3H,S) 13 Cnmr (CDC13). 611.9, 12.1, 19.3, 19.7, 20.9, 21.1, 24.3, 25.3, 29.1, 31.6, 31.8, 37.2, 38.7, 39.7, 40.4, 42.1, 42.2, 50.0, 51.1, 56.7, 56.8, 71.7, 121.6, 129.2, 130.2, 140.6. EIMS. m/z (rei. int) [M+] 412 (10) (acc. mass 412.3705), 408(5), 351(5), 255(20), 213(15), 159(30) 105(40), 81(50) 55(100). UNIVERSITY OF IBADAN LIBRARY 247 GSH 14 acetate m.p. 140-142°C (Lit= 144-144.6°C)168. IH nmr (CDCI3) 50.68 (3H,S) 0.71 (3H,S) 2.05 (3H,S), 4.63 (lH,m) 5.15 (2H,m) 5.40(lH,d,J=5Hz) 13c nmr (CDCI3) 511.74, 1187, 11.92, 12.15, 18.87, 19.20, 20.90, 20.99,21,35, 27.64, 31.73, 31.76, 36.47, 36.86, 37.99, 40.40, 49.89, 55.79, 73.87, 122.53, 138.21, 139.52, 170.46 EIMS m/z (rei. int.): 454(1) [M+], 394(100), 255(70), 159(40), 147(62), 105(55), 81(84). GSH 14 - Qxo-compound IH nmr (CDCI3) 55.15 (2H,m) 5.35 (lH,d,J=5Hz) 5.73 (1H,S), 6.18 (1H,S). 13C nmr (CDCI3): 5122.81, 129.23, 138.13, 171.76, 199.72 and others. EIMS. m/z (rei. int.): 410(30) [M+], 27(40), 229(30), 124(40), 81(60), 55(100). 3.12.4 GSH 26 UV Xmax (MeOH): 277.6, 303.0, 346.1 nm *H nmr (CDCI3). 53.90(3H,S) 3.91(3H,S), 6.38(lH,d,J=2Hz), 6.50 (lH,d,J=2Hz), 6.60 (1H,S) 7.02(2H,dd,J=9,2Hz), 7.87(2H,dd,J=9,2 Hz) 13C nmr (CDCI3) 555.42 (OCH3), 55.67 (OCH3), 92.52(C6), 97.92(C8), 104.27(C3,C10), 114.40, C3', C5'), 123.50(C1' ), 127.94(C2', C6'), 157.61 (C9), 162, 162.11 (C5), 162.49 (C2), 163.93 (C41 ), 165.33 (C7), 182.35 (C4). UNIVERSITY OF IBADAN LIBRARY 248 EIMS. m/z (rei. int.) [M+] 298(60) (acc. mass 299.0910), 269(20), 255(10), 241(2), 213(5), 166(5), 129(20), 97(30), 83(45), 69(90), 55(100). 3.12.5 GSH 24 M.P 203-205°C., [ a ]^ud + 61 (C,2.0, CHCI3). IR Vmax (Nujol): 3583, 3565, 3337, 1662, 1636, 1457, 1377, 1244, 1174, 1162, 1072, 930, 900, 836 and 774 cnr 1. UV Àmax (CHCI3) 282 nm, /.max (EtOH) 280nm 1H nmr (CDCI3): 50.83(3H, S), 0.93(3H, S), 1.33(3H, S), 1.39(3H, S), 1.87(3H,d,J=1.9Hz), 4.74(lH,bs), 4.86(lH,bs), 5.40(lH,bt), 5.67(lH,bt), 5.99(lH,bs). 13c nmr (CDCI3): 513.2, 13.9, 17.2, 20.7, 23.6, 23.7, 25.1, 32.2, 32.6, 33.6, 35.9, 39.2, 40.8,41.4, 43.6, 48.6,51.2, 56.0, 75.0, 106.7, 122.5, 125.7, 130.6, 134.3, 137.2, 143.4, 153.7, 193.7. EIMS m/z (rei. int.): 422.2930 [M+l (C28H38O3 requires 422.2900) (60), 404(12), 379(4), 339(20), 219(18), 201(20), 183(28), 151(50), 132(50), 119(100), 105(70), 91(80), 55(58). 3.12.6 GSH 32 Clusters from ethyl acetate, m.p. 246-248°C (Lit. 242°C)i 69 IR Vmax (Nujol). 2924, 2854, 1732, 1696, 1463, 1377, 1253, 1029, 722 cm-1 IH nmr (CDCI3) 80.73(3H,S), 0.84(3H,S), 0.85(3H,S), 0.89(3H,S), 0.91(3H,S), 0.93(3H,S), 1.11(3H,S), 2.03(3H,S), 2.80(lH,dd,J=3,10Hz) 4.48(1 H,t,J=8Hz) 5.26(1 H,t,J=3.5Hz). UNIVERSITY OF IBADAN LIBRARY 249 13c nmr (CDCI3) 815.27, 16.54, 17.04, 18.05, 21.21, 22.76, 23.27, 23.40, 23.46, 25.78, 27.54, 27.92, 30.55, 32.31, 32.40, 32.94, 33.65, 36.86, 37.57, 37.93, 39.14, 40.81, 41.43, 45.70, 46.41, 47.42, 55.16, 80.80, 122.43, 143.48, 170.95, 183.45. CIMS m/z m/z (rei. int.) 499 [M+l]+, 439(100) ([M+l]+ - 60) acc. mass 439.3590(C3oH4702 requires 439.3564), 393(10), 248(12), 205(12), 191(45). 3.12.7 GSH 41 m.p. - 122 - 123°C. U.V. Àmax (CHCI3): 239.5, 289.lnm. IH nmr (CDCI3): 82.83 (lH,dd., J=3,14Hz), 3.13(1H, dd, J=13,4Hz), 3.82(3H,S), 3.92(3H,S), 3.94(3H,S), 5.36(lH,dd,J=3,10Hz), 6.93(lH,dd, J=9), 7.01(2H,m), 6.07 (lH,d,J=2Hz), 6.08(lH,d,J=2Hz). 13C nmr (CDCI3): 843.23(C3), 55.58 (OCH3), 55.89 (2x OCH3) 79.12 (C2), 94.18 (C8), 95.03(C6), 103.03 (CIO), 109.33 (C2), 111.10 (C5'), 118.75 (C6'), 130.71 (CI'), 149.23 (C3'), 149.45(C4'), 162.71(C9), 164.06 (C5), 167.89 (C7), 195.81 (C4). EIMS m/z (rei. int.) [M+] 330 (20), 329.1025 [M-l]+(C igH 1706 requires 329.1020), 298(2), 193(10), 164(60), 151(100), 138(15), 103(15), 91(25), 77(25). 3.12.8 GSH 49 Ih (nmr) (d5-pyridine). See table 28, p. 188. 13C (nmr) (d5-pyridine). See table 29, p.189. CIMS m/z (rei. int.) 457.3680 (15) [M+]+ (C30H49O3 requires 457.3669), 439 (95), 411 (50), 393 (5), 249(40), 205(30), 191(100). UNIVERSITY OF IBADAN LIBRARY 250 3.12.9 GS 13 Amorphous powder, m.p. 315-318°C. IH nmr (pyridine-ds): 50.67 (3H,S), 0.69(3H,S), 0.89 (6H,d,J=lHz), 0.95 (6H,S), 4.0-4.63 (sugar protons), 5.36 (IH, m). 13C nmr (pyridine-d5) sugar carbons - 8102.62 (CI'), 75.40 (C2% 78.16 (C3'), 71.75 (C4'), 78.56 (C5’), 62.88 (C6'). Aglycone carbons - 512.03, 12.20, 12.59, 19.27, 19.48, 20.04, 21.34, 21.53, 23.45, 24.57, 25.77, 26.42, 29.51, 30.31, 32.11, 32.23, 37.53, 39.39, 40.00, 46.09, 50.39, 56.29, 56.88, 78.67, 121.99, 129.51, 138.89, 140.96. FABMS m/z: 411 [M-glc], 383, 355, 303, 248, 217. 3.12.10 GSA 5 Needles from methanol/chloroform mixture, m.p. 295-297°C. IR Vmax 3450, 3330, 2926, 2854, 1698, 1462, 1377, 1304, 1268, 1208, 1190, 1076, 1038, 1016, 973, 722 cirri. !h nmr (pyridine-d5): 50.61(3H,S), 0.64(3H,S), 0.69(3H,S) 0.70(3H,S), 0.71 (3H,S), 0.88(3H,S), 2.96(lH,dd,J=4,10Hz) 3.57 (lH,d,J-10.4Hz), 3.86(2H,m), 5.15(lH,t,J=3Hz). l3C nmr (pyridine-d5): 811.61, 14.45, 15.97, 17.07, 22.18, 22.24, 22.33, 24.64, 26.16, 26.82, 29.43, 31.45, 31.69, 31.72, 32.69, 35.71, 37.26, 38.24, 40.48, 40,66, 41.37, 44.93, 45,13, 46.63, 47.08, 66.37, 71.87, 121.06, 143.33, 178.72. CIMS m/z (rei. int ): 472(2) [M+], 455(40), 437(70) and EIMS m/z (rei. int.) 248(20), 203(100), 189(15), 133(30). UNIVERSITY OF IBADAN LIBRARY 251 GSA 5 Diacetate Melting point. 184°C IR Vmax (Nujol): 2925, 2854, 1739, 1696, 1463, 1376, 1376, 1245, 1034, 920, 735 c n r1. IH nmr (CDC13): 60.68(3H,S), 0.76(3H,S), 0.84(3H,S), 0.86(3H,S), 0.90(3H,S), 1.05(3H,S), 1,96(3H,S), 2.01(3H,S), 2.75(lH,dd,J=4,10Hz) 3.63(lH,d,J=11.6Hz), 3.80(lH,d,J=l 1.6Hz), 4.73(lH,m) 5.21(lH,t,J=3Hz). (CDCI3): 512.96, 15.70, 16.96, 17.77,20.84,21.13,22.82,23.25,23.45, 25.67, 27.47, 30.54, 32.12, 32.29, 32.94, 33.64, 36.68, 37.55, 39.12, 40.38, 40.84, 41.38, 45.65, 46.38, 47.55, 47.69, 65.29, 74.38, 122.33, 143.47, 170.61, 170.91, 183.37. CIMS m/z (rei. int.): 574(20) [M+NH4]+, 510(18), 497(60), 451(22), 437(60), 248(100), 203(60), 189(45), 133(20). 3.12.11 GSA 8 Needles from methanol/chloroform mixture, m.p. 273-275°C IH nmr (pyridine-d5): 50.56(3H,S), 0.64(3H,S), 0.75(3H,S), 0.90(3H,S), 0.94(3H,S), 1.02(3H,S), 1.25(3H,S). 2.97 (lH,dd, J=4,9Hz), 3:12(lH,d,J=4Hz), 4.06(1 Hd,t, J=3Hz), 5.18(1 H,t,J=3Hz). 13C nmr (pyridine-d5): 515.94, 16.81, 17.50, 17.96, 23.08, 23.29, 25.57, 27.58, 29.59, 30.29, 32.60, 33.55, 36.70, 38.14, 39.23, 41.36, 41.66, 44.26, 45.80, 46.02, 47.85, 55.30, 70.76, 77.68, 122.03, 144.19, 179.54. CIMS m/z (rei. int.): 472(5) [M+] 455(50), 437(75), 409(10), UNIVERSITY OF IBADAN LIBRARY 252 EIMS, m/z (rei. int ): 248(100), 203(90), 189(20), 133(40). 3.12.12 GSA11 White crystals from methanol/chloroform mixture, m.p. 320-325°C (lit. 328-330°C)115. IR Vmax 3465, 3280, 2925, 3280, 2925, 2854, 1677, 1462, 1377, 1046, 722 cm 'l IH nmr (pyridine-d5): 80.56(3H,S), 0.64(3H,S), 0.75(3H,S), 0.90(3H,S), 1.02(3H,S), 1.25(3H,S), 2.0(lH,dd,J=2,l 1.4Hz), 2.96(lH,dd,J=4,9Hz), 3.98(lH,d,J=11.0Hz), 3.83(lH,d,J=llHz), 3.93(lH,d,J=4Hz), 4.18(lH,dt,J=3.3Jz), 5.17(lH,t,J=3Hz). 13C nmr (pyridine-ds)): 814.56, 17.25, 17.50, 18.28, 23.67, 23.73, 23.98, 26.21, 28.24, 30.92, 32.99, 33.20, 34.18, 37.22. 39.86, 41.99, 42.31, 42.42, 44.88, 46.40, 46.64, 48.13, 48.55, 67.62, 71.60, 72.99, 122.72, 144,84, 180.19. CIMS m/z (rei. int.): 489(10) [M]+ (acc. mass 489.3580), 471(100), 453(85), 435(30), 407(20), 248(40), 204(40), 191(35), 173(20). GSA 11 triacetate Melting point, 248-251«C (Lit 257-2590Q115. IR Vmax. 2926, 2854, 1747, 1696, 1463, 1376, 1240, 1159, 1041, 920, 821, 734 cm-l IH nmr (CDCI3): 80.70(3H,S), 0.84(3H,S), 0.86(3H,S), 0.96(3H,S), 1.04(3H,S), 1.16(3H,S), 1.94(3H,S), 1.99(3H,S), 2.01(3H,S), 2.78(lH,dd,J=4,9Hz), UNIVERSITY OF IBADAN LIBRARY 253 3.61(lH,d,J=llHz), 3.80 (lH,d,J=l 1Hz), 4.86(lH,d,J=4Hz), 5.21(lH,t,J=3Hz), 5.34(1 H,bd,J=3Hz) 13Cnmr(CDCl3): 813.67, 16.38, 17.13, 17.53,20.71,20.83,21.19,22.64,23.31, 23.79, 27.37, 30.56, 32.08, 32.31, 32.95, 33.64, 36.57, 39,23, 39.95, 40.80, 41.41, 41.52, 45.59, 46.38, 47.54, 48.07, 65.42, 69.52, 71.86, 122.20, 143.62, 170.03, 170.28, 170.79, 183.69. CIMS m/z (rei. int.): 632(100), [M+NH4]+, 586(10), 555(20), 513(10), 435(10), 248(50), 204(25), 81(10). 3.12.13 GSA 16 Erubigenin7 White powder, m.p. 300-302OC, = +76(C,3.0, MeOH). IR Vmax (Nujol): 3375, 3300, 2924, 2854, 1696, 1462, 1378, 1238, 1021 cm-1 IH nmr (d5-pyridine) see table 32, p.207. nmr (d5-pyridine) see table 33, p.210. CIMS m/z (rei. int.): 489(20) [M+l]+, 471(18), 453(40) 435(20), 407(10), 338(10), 248(24), 203(30), 191(30), 177(45), 133(100), 73(45), 58(35). Triacetate: m.p. 206-208<>C, [a]22D = + 90 (C, 2.0, CHCI3). IR Vmax (Nujol): 3300-3200, 2924, 2854, 1742, 1697, 1462, 1377, 1234, 1046, 909, 803, 736 cm‘l ÌH nmr (CHCl3-d): 80.68(3H,S), 0.84(3H,S), 0.86(3H,S), 0.90(3H,S), 1.05(3H,S), 3.96( 1 H,d,J= 11.8Hz), 4.15(2H,d,J=13.7Hz), 4.35(lH,d,J=12Hz), 5.22(lH,bt, J=3- 5Hz). nmr (CHCl3-d): see table 33, p.210. UNIVERSITY OF IBADAN LIBRARY 254 CIMS (NH3) m/z: 632.416 [M+NH4I+ (C36H5808N requires 615.338), 569, 555, 509, 435, 248, 204, 192, 163, 119, 85, 58 (100). Methyl ester !h nmr (ds-pryridine): 60.52 (3H,S), 0.55 (3H,S), 0.58(3H,S), 0.70(3H,S), 0.83(3H,S), 3.37(3H,S), 3.68(1,H,m), 3.92(lH,d,J=10.9Hz), 4.05(lH,m), 4.32(lH,d,J=l 1Hz), 5.06 (lH,bt, J=34Hz), 3(0H) at 65.17, 5.97 and 6.39. 13C nmr (d5-pyridine): see table 33, p.210. CIMS m/z: 503 [M+l]+, 453, 429, 393, 355, 295, 281, 262, 221, 203, 147, 84(100), 73, 56. Methyl ester triacetate IH nmr (CHCl3-d): 50.65 (3H,S), 0.83(3H,S), 0.86(3H,S), 0.91(3H,S), 1.04(3H,S), 1.94(3H,S), 1.99(3H,S), 2.02(3H,S), 3.55(3H,S), 2.95(lH,d,J= 11.8Hz), 4.15(2H,d,J=10.3Hz), 4.36(lH,d,J=12Hz), 5.22(lH,bt, J=3Hz). 13c nmr (CHCl3-d) - see table 33, p.210. EIMS m/z 628.397 [M+] (C37H5608 requires 628.396), 568, 503, 429, 355, 262, 221, 203, 189, 105, 83(100), 47, 43. UNIVERSITY OF IBADAN LIBRARY 255 REFERENCES 1. Sofowora, A , 1984. Medicinal Plants and Traditional Medicine in Africa. Spectrum books Ltd. Ibadan and John Willey and sons. Pg 2. 2. Sofowora, A , 1984. Medicinal Plants and Traditional Medicine in Africa. Spectrum books Ltd. Ibadan and John Willey and sons Pg 6 3. Okogun, J. 1,1985 . "Drag Production efforts in Nigeria: Medicinal Chemistry Research and a missing link" . Lecture delivered to thè Nigerian Academy of Science, 13th Aprii. 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