PHYTOCHEMICAL STUDIES ON CERTAIN SOUTH AFRICAN SPECIES OF THE RUTACEOUS GENERA AGATHOSMA WILLD. AND ZANTHOXYLUM L. A dissertation submitted to RHODES UNIVERSITY in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY by NATHAN FINKELSTEIN M.Sc., M.P.S. School of Pharmaceutlcal Sciences, Rhodes University, Grahamstown, Republic of South Africa. Decembe r, 1978. With esteem and gratitude this work is dedicated to my friend and former mentor, John M. CaZderwood. His interest afforded me the opportunity for academia advanaement, whiZe his inspiration evoked in me a Zove for naturaZ produat ahemistry. i i ACKNOWLEDGEHENTS This investigation was carried out in the Department of Chemistry, Rhodes University. The author wishes to express his sincere appreciation to Professor J. R. Nunn for making the necessary facilities available. Particular gratitude is also expres·sed to his supervisor, Professor D. E. A. Rivett, for his invaluable guidance, constant interest and inspiration throughout the study. The author is further indebted to: * Professor H. Parol Is and the lecturing staff of the Department of Chemistry for their helpful discussions and constructive suggestions; * Hr. A. W. Sonemann and the technical staff of the Department of Chemistry for their willing technica·l assistance; * Dr. A. Jacot Guillarmod and Hrs. E. Brink for the authentication of plant material, preparation of voucher specimens and advice on nomenclature; * Dr. I. C. Verdoorn and Hiss E. Retief of the Botanical Research Institute, Pretoria, for making available the facilities of the Institute's herbarium as well as useful botanical discussions; * Dr. P. G. Waterman of the Department of Pharmaceutical Chemistry, University of Strathclyde, Glasgow, Professor V. Deulofeu of the Universidad de Buenos Aires, Argentine, and Professor J. SlavIk of the Department of Hedical Chemistry, University J. E. Purkyn~, Czechoslovakia, for generous samples of authentic alkaloids and lignans; * Professor A. Cave and Dr. J. Vaquette of the Universite de Paris-Sud for their original spectra of· decarine; iii ,~ Mr. Gordon Young, Government Forester, Hogsback, for the collection and supply of plant material (Z. davyi(Verdoorn) Waterm.]; * Mr. M. J. Wells, Economic Botany Section, Botanical Research Institute, for arranging the collection and supply of plant material used in the Zantho~~um L. investigation; * Mrs. P. C. Estment for efficiently typing the final manuscript . Grateful appreciation for generous financial support is acknowledged to: * The Council for Sci"entific and Industrial Research; * The Foundation for Pharmaceutical Education; * Rhodes University; * Synthetico (Pty:) Ltd. iv CONTENTS Page Acknowledgements i i Contents iv List of tables, schemes and figures xi i Summary xi i j 1. INTRODUCTION 1.1 The plant family Rutaceae 2 1.2 The genus Agathosm2 Willd. 3 I • 2. I Phytochemical review 4 1.2.1.1 Volatile oil 4 1.2.1.2 Terpenoids 5 1.2.1.3 Sulphur compounds 7 1.2.1.4 Coumarins 8 1.2.1.5 Alkaloids 10 I .2.2 Chemotaxonomic aspects 10 1.3 The genus Zanthoxylum L. 12 1. 3. I Phytochemical review 18 1.3.1.1 Alkaloids 18 1.3.1.2 Lignans 22 1.3.1.3 Miscellaneous compounds 24 I . 3.2 Previous phytochemical studies on South African Zanthoxylum species 25 I .3.3 Medicinal properties 26 1. 3.4 Chemotaxonomy 28 2A. EXPERIMENTAL (AGATHOS~ WILLD. INVESTIGATION) 30 2A . I Preliminary extractions 30 v CONTENTS (continued) Page 2A. I . I Plant materials 30 2A.1.1.1 Agathosma puberuZa (Steud.) Fourc . 30 2A. 1. 1.2 Agathosma ovata (Thunb.) Pillans 30 2A. 1. 1.3 Agathosma apieuZata G. F. W. Mey 30 2A.I . I.4 Agathosma aZavisepaZa R. A. Dyer 31 2A. I .2 Drying and comminution 31 2A. 1. 3 Extraction 31 2A.2 Isolation and purification of crystalline material 32 2A.2.1 Small scale 32 2A.2.2 Large scale 32 2A.2.2 . 1 Fraction 8 33 2A . 2 . 2.2 Fractions 9 and 10 33 2A.2.3 "Alkal i-conversion" method 33 2A.3 Hydrogenation of puberul in 34 2A.4 Acetylation of "hydrogenated" puberulin 35 2A.5 Methylation of "hydrogenated" puberulin 36 2A.6 Ethylatlon of "hydrogenated" puberulin 36 2A.7 Prenylation of "hydrogenated" puberulin 37 2A . 8 Acid fission of ether linkage of puberulin 37 2B. EXPERIMENTAL (ZANTHOXYLUM L. INVESTIGATION) 38 2B. I Preliminary extractions 39 2B . 1. I Plant materials 39 2B.1.1.1 Zanthoxytum davyi (Verdoorn) Wate.rm. 39 2B.1.1.2 ZanthoxyZum thorncroftii (Verdoorn) \~aterm. 39 2B . 1.1.3 ZanthoxyZum humiZe (E. A. Bruce) Waterm. 40 2B. I .2 Drying and comminution 40 vi CONTENTS (continued) Page 2B.1.3 Extraction 40 2B.2 Isolation and characterization of compounds 41 2B.2.1 Z. davyi concentrated extracts 41 2B.2.1.1 n-Hexane extract of stem bark (ZO/SB1) 41 2B.2.1.1.1 Acetylation of lupeo1 42 2B.2.1.1.2 Benzoylation of lupeol 42 2B.2.1.1.3 Saponification of lupeol acetate 43 2B.2.1.2 Chloroform extract of stem bark (ZO/SB2) 43 2B.2.1.2.1 Yellowish precipitate (ypl) 43 2B.2.1.2.2 Chloroform fraction and washings (CFMI) 44 2B.2.1.2.3 Liquid-liquid extraction of combined acid filtrate (AF1) 44 2B.2.1.2.4 Conversion of crystals NF/3 to iodide salt 45 2B.2.1.3 Methanol extract of stem bark (ZO/SB3) 46 2B.2.1.3. I Horizontal cellulose chromatography of alkaloidal residue (R j ) 46 2B.2.1.3.2 Fractions - 8 47 2B.2.1.3.3 Fractions 9 - 17 47 2B.2.1.3.4 Fractions 18 - 32 47 2B.2.1.3.5 Fractions 33 and 34 47 2B.2.1.3.6 Fractions 35 - 51 48 2B.2.1.4 n-Hexane extract of root bark (ZO/RB1) 48 2B.2.1.4.1 Fraction 10 49 2B.2.1.4.2 Fractions 9 and 11 (bulked) 49 2B.2.1.4.3 . Fractions 12 and 13 (bulked) 50 2B.2.1.4.4 Acetylation of B-sitostero1 (NF/6) 50 2B.2.1.4.5 Fractions 14 - 16 (bulked) 51 2B.2.1.4.6 Fractions 17 - 36 51 vi j CONTENTS (continued) Page 2B.2.1.5 Chloroform extract of root bark (ZO/RB2) 51 2B.2.1.5. 1 Yellow deposit Y02 51 2B.2.1 . 5.2 Residue R2 51 2B.2 . 1. 5.3 Conversion of R2 to iodide salt 52 2B.2 . 1. 6 Methanol extract of root bark (ZO/RB3) 52 2B . 2. 2 Z. thorncroftii concentrated extracts 52 2B.2.2.1 Light petroleum (b.p.400-600) extract of root bark (ZT/RBJ) 52 2B . 2.2. 1.1 Orange precipitate (OPl) 53 2B.2.2.1 . 2 Combined acid filtrate (AF2) 53 2B . 2. 2. 1.3 Fractions P2 and P3 54 2B . 2.2.2 Chloroform extract of root bark (ZT/RB2) 54 2B.2.2. 2.1 Yellow precipitate (YP2) 54 2B . 2.2.2.2 Combined acid filtrate (AFJ) 55 2B . 2. 2. 2. 3 Column chromatography of remaining ZT/RB2 55 2B.2.2.2.4 Fractions 23 - 26 56 2B.2.2.2.5 Conversion of R3 to the hydrochloride 57 2B . 2.2.2.6 Acetylation of R3 58 2B.2 . 2.2.7 Methylation of R3 59 2B.2.2.2.8 Fraction 31 60 2B.2.2.2.9 Fraction 33 60 2B.2.2.2.10 Yellow insoluble solid (YP3) 60 2B.2 . 2.3 Methanol extract of root bark (ZT/RB3) 61 2B.2.2 . 3. I Low .pressure liquid chromatography of ZT/RB3 62 2B.2 . 2.3. 2 Fractions 57 - 66 63 2B.2.2.3. 3 Fractions 84 - 86 63 2B . 2.3 Z. humiZe concentrated extracts 64 2B.2 . 3. 1 Light petroleum (b . p. 40°- 60°) extract of root bark (ZH/RB1) 64 vii i CONTENTS (continued) Page 2B.2.3.1.1 Orange precipitate (OP2) 64 2B.2.3.1.2 Fractions 1 - 3 65 2B.2.3.1.3 Fractions 12 - 15 65 2B.2.3.1.4 Fractions 16 - 17 65 2B.2.3.1.5 Isolation of chelerythrlne chloride from OP2 66 2B.2.3.1.6 Combined acid filtrate (AF4) 67 2B.2.3.1.7 Low pressure liquid chromatography of E2 68 2B.2.3.1.8 Fractions 17 - 23 68 2B . 2 . 3. I • 9 Frae t 1on s 30 - 38 68 2B.2.3.I.l0 Fractions 50 - 59 69 28.2.3.2 Chloroform extract of root bark (ZH/RB2) 69 2B.2.3.2. I Fractions 19 - 23 (bulked) 70 2B.2.3.3 Methanol extract of root bark (ZH/RB3) 71 28.2.3.3.1 Conversion of candlclne chloride to Iodide salt 73 28.2.3.3.2 Column chromatography of R4 73 2B.2.3.3.3 Fraction 4 74 3A· DISCUSSION (AGATHOS~ WILLD. INVESTIGATION) 75 3A. I Coumarlns 75 3A.2 Extraction and purification of puberulln 76 3A.3 Locality of puberulln 76 3A.4 Structure elucidation of puberulln 77 3A.S Coumarlns with I.soprenold substltuents 84 38. DISCUSSION (ZANTHOXYLUM I.. INVESTIGATION) 88 3B.1 Preliminary extractions and separations 88 ix CONTENTS (continued) Page 3B. 1 . 1 Materials 88 3B.l . 2 Extraction 88 3B.2 Screening of concentrated extracts 89 3B.2.1 Alumina chromatograms 90 3B . 2.2 Silica Gel G chromatograms 90 3B.2. 3 Cellulose chromatograms 90 3B . 2.4 Detection of compounds 91 3B.3 Compounds isolated from Z. davyi extracts 92 3B.3.1 Stem bark extracts 92 3B . 3.1 .1 Concentrated n-hexane extract (lD/SB1) 92 Lupeol 93 3B .