A BIOSYSTEMATIC STUDY OF THE GENUS SUTHERLANDIA Br. R. (FABACEAE, GALEGEAE) by DINEO MOSHE DISSERTATION presented in fulfilment of the requirements for the degree of MAGISTER SCIENTIAE in BOTANY at the FACULTY OF NATURAL SCIENCES of the RAND AFRIKAANS UNIVERSITY SUPERVISOR: PROF B-E VAN WYK CO-SUPERVISOR: MRS M VAN DER BANK DECEMBER 1998 OPSOMMING 'n Biosistematiese studie van die genus Sutherlandia (L.) R. Br., 'n relatief onbekende genus met verwarrende geografiese vorme, word aangebied. Die spesies van Sutherlandia is almal endemies aan Suidelike Afrika. Die spesies is naverwant en probleme rondom hul taksonomie word bespreek. Enkele morfologiese kenmerke wat nutting is om spesies te onderskei, word geIllustreer en in detail bespreek. Morfologiese inligting word gebruik om infrageneriese verwantskappe te ondersoek in 'n fenetiese ontleding van 51 geografies-geIsoleerde bevolkings. Sutherlandia het tradisionele gebruike, hoofsaaklik as behandeling teen interne kankers en as 'n algemene tonikum. 'n Ondersoek van chemiese verbindings is gedoen en die resultate word geIllustreer en in tabelle aangebied. Die aard van hierdie studie het nie gedetailleerde mediese ondersoeke toegelaat nie, maar die medisinale waarde van Sutherlandia en die waargenome chemiese verbindings word toegelig. Daar word voorgestel dat die anti-kanker aktiwiteit hoofsaaklik toegeskryf kan word aan die hob vlakke van kanavanien, 'n nie-proteIen aminosuur, in die blare van die plant. Kanavanien, 'n analoog van arginien, is bekend vir sy aktiwiteit teen gewasvorming. Die waarde van die plant as 'n bitter tonikum hou waarskynlik verband met die teenwoordigheid van triterpendiede, sommige waarvan waarskynlik ook ander voordelige uitwerkings het. Ensiem-elektroforese is gedoen om genetiese verwantskappe tussen die talryke streeksvorme van Sutherlandia te ondersoek. 'n Studie van 19 bevolkings het aangetoon dat hulle almal naverwant is en dat 'n meer konserwatiewe behandeling van die taksons nodig is. Die aantal taksons word dus verminder. 'n Volledige taksonomiese hersiening van die genus word aangebied. Die aantal spesies word verminder van ses na twee, naamlik S. frutescens en S. tomentosa. Eersgenoemde word in drie subspesies verdeel, naamlik subsp. frutescens, subsp. microphylla en subsp. speciosa. Sommige streeksvorme word beskryf en gelllustreer, maar hulle word nie as formele taksons erken nie. 'n Sleutel tot die spesies, subspesies en streeksvorme word voorsien, en die nomenklatuur, tipifisering, beskrywing en geografiese verspreiding van elke takson word verskaf. Die multi-dissiplinere benadering van hierdie studie het gelei tot 'n beter begrip van die morfologiese, chemiese en genetiese variasie in hierdie onbekende, maar potensieel waardevolle medisinale en sierplant. II A BIOSYSTEMATIC STUDY OF THE GENUS SUTHERLANDIA R. Br. (FABACEAE, GALEGEAE) SUMMARY A biosystematic study of the genus Sutherlandia (L.) R. Br., a poorly studied genus with confusing geographical variants, is presented. The species of Sutherlandia are all endemic to southern Africa. The species are very closely related and problems regarding their taxonomy are discussed. A few morphological characters that are useful in distinguishing amongst species are illustrated and discussed in detail. Morphological data are used to investigate infrageneric relationships in a phenetic analysis of 51 geographically separated populations. Sutherlandia has traditional medicinal uses, mainly as an anti-cancer treatment for internal cancers and as a general tonic. A survey of chemical compounds was done and the results are illustrated and presented in tables. The nature of this study did not allow detailed medical investigations, but the medicinal value of Sutherlandia and the compounds detected are highlighted. It is suggested that the anti-cancer activity can mainly be ascribed to the high levels of canavanine, a non-protein amino acid, in the leaves of the plant. Canavanine, an arginine analogue, is known for its antitumourigenic properties. The value of the plant as a bitter tonic is probably related to the presence of several triterpenoids, some of which may well also have other beneficial effects. Enzyme electrophoresis was done to explore genetic relationships amongst the numerous regional forms of Sutherlandia. A study of 19 populations showed that they are all closely related and that a more conservative treatment of the taxa is called for. As a result the number of taxa is reduced. A complete taxonomic revision of the genus is presented. The number of species is reduced from six to two, namely S. frutescens and S. tomentosa. The former is divided into three subspecies, namely subsp. frutescens, subsp. microphylla and subsp. speciosa. Some regional forms are described and illustrated, but these are not formally recognised as taxa. A key to the species, subspecies and regional forms is provided, and the. nomenclature, typification, description and geographical distribution for each of the taxa are given. III The multidisciplinary approach of this study provided a better understanding of the morphological, chemical and genetic variation in this relatively poorly known but potentially valuable ornamental and medicinal plant. IV TABLE OF CONTENTS OPSOMMING SUMMARY III INTRODUCTION. 1 MATERIALS AND METHODS 2 2.1 Morphological characters. 2 2.2 Chemical compounds 4 2.3 Enzyme electrophoresis 10 2.4 Phenetic analysis 14 MORPHOLOGICAL CHARACTERS 15 3.1 Habit 15 3.2 Leaves 17 3.3 Inflorescence and flowers 20 3.4 Fruits and seeds 26 ENZYME ELECTROPHORESIS 31 CHEMICAL CHARACTERS 39 5.1 Alkaloids 40 5.2 Monoterpenoids 40 5.3 Flavonoids 41 5.4 Triterpenoids 43 5.5 Amino Acids 48 5.6 Pinitol 53 MEDICINAL VALUE OF SUTHERLANDIA 66 6.1 Historical background 66 6.2 Importance of triterpenoids 67 6.3 Importance of amino acids 68 Canavanine 68 Arginine and the nitric oxide pathway 69 y-Aminobutyric acid (GABA) 70 6.4 Pinitol 70 PHENETIC ANALYSIS 72 8. GENERAL CONCLUSIONS 82 9.TAXONOMY 84 9.1 Historical overview 84 9.2 The genus Sutherlandia 85 9.3 Key to the species and subspecies of Sutherlandia 86 9.4 The species and subspecies of Sutherlandia 87 ACKNOWLEDGEMENTS 105 10. REFERENCES 106 LJ CHAPTER 1 INTRODUCTION The genus Sutherlandia R. Br., commonly known as cancer bush, comprises six species, all of which are endemic to southern Africa (Phillips & Dyer, 1934). The taxonomically significant characters to distinguish the species are the habit, the shape of the pods and the shape and pubescence of the leaflets. Sutherlandia species have both medicinal and horticultural uses. Leaf infusions of S. frutescens (L.) R. Br. are used to treat stomach, intestinal and uterine ailments. It is also used as a cough remedy, as a tonic (Smith, 1895; Van Wyk et al., 1997), to relieve eye ailments and chicken pox (Watt & Breyer-Brandwijk, 1962) and most interestingly, to treat internal cancers (Smith, 1895; Dykman, 1908; Van Wyk et al., 1997). The vernacular names refer to the use as a cancer cure, and there are several anecdotes to support this claim, but so far no published scientific evidence (Gabrielse, 1996). Canavanine, a non-protein amino acid with known antitumourigenic properties, has been extracted from seeds of S. frutescens (Bell, 1958) and further investigations seem worthwhile. The species were grown as ornamentals in England as early as 1683 (Curtis, 1792). To this day, Sutherlandia plants are popular in gardens in many parts of the world (Bailey, 1976; Mabberley, 1987; Griffiths, 1992). Phillips & Dyer (1934) highlighted the problems associated with the taxonomy of this genus. It was still unclear whether there are six species, two, or perhaps only one. The taxonomy of Sutherlandia is confused because the taxa often grade into each other, but at most localities individual species are quite easy to recognise (Phillips & Dyer, 1934). They can only be distinguished by a combination of characters and even then this is not always conclusive (Schrire & Andrews, 1992). The existence of morphologically integrated populations also led Phillips & Dyer (1934) to identify the need for a genetic study of the genus Sutherlandia. The aim of this study was to revise the taxonomy of all the confusing geographical variants of this relatively poorly known genus, by studying morphological and genetic variation at the population level. A further aim was to study chemical compounds of the genus, not only to find possible chemotaxonomic characters, but also to explore a possible rationale behind the traditional medicinal uses of Sutherlandia. 1 CHAPTER 2 MATERIALS AND METHODS 2.1 Morphological characters Herbarium material The large collections of specimens in the National Herbarium, Pretoria (PRE) and the Rand Afrikaans University Herbarium (JRAU) were used. Apart from morphological data, the collections provided locality information for plotting distribution maps of the species and infraspecific taxa. Fieldwork A field trip was undertaken to the Northern, Western and Eastern Cape and Free State Provinces to study plants in situ and to collect fresh material for analysis. Fresh flower, fruit, stem and leaf material was fixed in FAA (Sass, 1958). Voucher specimens were collected at each population. Photographs were taken to illustrate habitat, habit and gross morphology. Morphology The herbarium specimens from different localities/populations were categorized into operational taxonomic units (OTU's). At least six specimens from each locality/population were selected where possible to represent