446 CHAPTER 8 CONCLUSIONS The Menispermaceae forms a well-defined family within the order Ranunculales and consists of five tribes. The South African Menispermaceae consists of seven genera and 13 species. The seven genera occur in four of the five tribes, namely: Pachygoneae - Albertisia; Anomospermeae - Tiliacora; Tinosporeae - Tinospora and Menispermeae - Antizoma, Cissampelos, Cocculus and Stephania. The genus Antizoma with its two species A. angustifolia and A. miersiana and Cissampelos capensis are endemic to southern Africa. The other non-endemic species are Albertisia delagoensis, Cissampelos hirta, C. mucronata, C. torulosa, Cocculus hirsutus, Stephania abyssinica, Tiliacora funifera, Tinospora caffra, T. fragosa and T. tenera. During this study all 13 species occurring in southern African were observed in their natural known habitats and also in new localities. Research done at most of the important herbaria in South Africa supported species identification and recording of ethnobotanical and locality information. This research also contributed to the creation of new distribution maps of the 13 species in South Africa. A key based on leaf morphology was developed for the 13 southern Africa species. Some rare characteristics which occur in the South African species include the dorsal spur at the insertion of the petiole in Antizoma angustifolia, zygomorphy in Antizoma and Cissampelos and the many stamens present in Albertisia delagoensis flowers. The number of carpels in this family ranges between three and twelve, but only one carpel occurs in the South African species of the genera Antizoma, Cissampelos and Stephania. Most members of the family are climbers, but Antizoma angustifolia, Antizoma miersiana and Cissampelos capensis have adapted to dry habitats and contain xeromorphic characteristics. Based on the morphology, growth habit and alkaloid patterns of the two Antizoma species it is proposed that they be included in the genus Cissampelos. The ethnobotanical survey on the Menispermaceae family confirmed its importance for medicinal uses. World-wide species from 41 genera were recorded to be used for various ailments. In southern Africa and Africa, Cissampelos species were by far the most frequently used medicinal plants. In Africa it was followed by Stephania, Tinospora and Triclisia. In the rest of the world Menispermaceae is used to the same extend as in Africa, except that Tinospora 447 is the most used genus, followed by Cissampelos. Many similarities exist between the types of ailments treated with the genus Cissampelos in southern Africa, Africa and the rest of the world. The most important uses are for stomach problems, menstrual problems, pregnancy related problems, as a diuretic, for wounds and ulcers. Tinospora is mostly used as an anthelmintic, for arthritis and rheumatism, diabetes, fever, malaria, wounds, ulcers and as a tonic. Ethnobotanical research undertaken on the 13 South African species in the north-eastern parts of KwaZulu-Natal and the eastern parts of the Karoo, recorded many new uses and confirmed some uses previously documented. It was also confirmed that the use of plants as medicines are still commonly practised in the rural areas of South Africa and that there is still much scope for fieldwork to record traditional uses in these areas. Cissampelos capensis is medicinally the most used species in South Africa, followed by Cissampelos mucronata. Stomach problems are the most recorded ailments treated with Albertisia delagoensis, both Antizoma species and all four Cissampelos species. Cissampelos capensis and Stephania abyssinica are very important plants for “blood purification”. No medicinal uses were recorded for Cocculus hirsutus in South Africa, but it is known for its magical properties, similar to Cissampelos capensis, Cissampelos torulosa, Stephania abyssinica and Tinospora fragosa. Many new medicinal uses were recorded for Albertisia delagoensis, which was previously practically unknown. The three Tinospora species are not very important medicinal plants in South Africa. The family Menispermaceae is well known for its curare-like properties, but in South Africa Tinospora caffra is the only species recorded for this use. Cissampelos capensis is preferred by the Khoisan and Cissampelos mucronata by the Zulu. The differences in the medicinal uses of these Cissampelos species could be linked to the difference in their geographic distribution. The family Menispermaceae contains 22 different alkaloid types of which the bisbenzylisoquinoline, aporphines and protoberberines are considered good chemical markers for this family. During this study the major alkaloids in seven species of the genera Albertisia, Antizoma and Cissampelos were isolated and identified by means of NMR and HPLC. The HPLC retention times for seventeen isolated and identified alkaloids were used as standards for the population study. These seven species were intensely studied because very little was known about the alkaloids that occur in them. The alkaloids in Cocculus hirsutus, Stephania abyssinica and Tiliacora funifera are well documented and were therefore not isolated and identified. Tinospora fragosa's complexity caused impure alkaloid isolation, which has prevented NMR identification of any alkaloids. Alkaloids in Tinospora caffra and Tinospora tenera could not be 448 identified during this study, because of the lack of sufficient plant material and the small yields obtained. The seventeen alkaloids isolated during this study were: glaziovine, crotsparine, pronuciferine (proaporphine); bulbocapnine, dicentrine, lauroscholtzine (aporphine); reticuline (benzyltetrahydroisoquinoline); cissacapine, 12-O-methylcurine, cocsoline, cocsuline, cycleanine, cycleaneonine, insularine, insulanoline, O-methylcocsoline (bisbenzyltetrahydroiso- quinoline alkloids) and salutaridine (morphinane). None of them were novel alkaloids. Most of the alkaloids in the 13 South African Menispermaceae species were very complex and difficult to isolate. The HPLC method used for identification of the alkaloids in the crude alkaloid extracts gave good results, provided that the pH used in the solvent buffer stays constant. Large differences in alkaloid yields were found between the leaves, stems and rhizomes. Different alkaloid types were also present in the leaves and rhizomes of the same species. Qualitative and quantitative differences were found in alkaloid content in plants from the same population. This might be the result of the different developmental stages of plants in the same population. Seasonal changes seem to influence the quantitative presence of alkaloids, whereas their qualitative variation can possibly be explained by particular biochemical pathways in alkaloid synthesis being switched on or off as a result of environmental influences. Cissampelos, Cocculus, Stephania and Tinospora can be distinguished from each other on the basis of their unique alkaloids. Albertisia, Antizoma and Tiliacora have no unique alkaloid types. All thirteen species studied have unique alkaloids that distinguish them from the other species in their respective genera. This study mostly focussed on the genera Antizoma and Cissampelos and therefore a detailed comparative study was done on their alkaloids. It was found that the number of similarities equals the number of differences between the six species, with crotsparine as a major alkaloid unique in the two Antizoma species and dicentrine occurring as a unique major alkaloid in the four Cissampelos species. Bulbocapnine and cycleanine, the two main alkaloids in Antizoma miersiana were practically absent (only trace amounts) in Antizoma angustifolia and pronuciferine, a main alkaloid in the last species, was absent in Antizoma miersiana. Clear differences were found between Cissampelos capensis coastal and inland forms, with bulbocapnine and salutaridine present as main alkaloids in the coastal form and absent in the inland form. 12-O-Methylcurine occurred as the main alkaloid unique in Cissampelos capensis (both forms) and pronuciferine occurred as the main alkaloid in Cissampelos hirta. Unique unidentified alkaloids were found in Cissampelos 449 mucronata leaves and stems (X7) and in Cissampelos torulosa leaves (X2). Where alkaloids are used in chemotaxonomy it is important to mention the plant part used, the stages of development, the season of the year, the geographical distribution and genetic variation during sampling. One of the aims for this study was to investigate the generic status of Antizoma. This was done by cladistic analysis of morphological and alkaloid data for the two Antizoma and four Cissampelos species. The cladistic analysis of the morphological data strongly indicated that Antizoma is nested within the genus Cissampelos, being sister to C. capensis. This confirmed that Antizoma might be a drought-adapted clade within Cissampelos. Cladistic and phenetic analyses of the alkaloid data partly support the current generic status of Antizoma and partly support the morphological outcome as to the generic status of Antizoma. There is enough evidence to consider the transfer of A. angustifolia and A. miersiana back into the genus Cissampelos. The alkaloid data should therefore not be used singly for taxonomic purposes, but should be considered together with other characteristics of plants. Thus, the correct names for the two Antizoma species in Cissampelos will be C. angustifolius Burch. (1822) and C. miersiana (Harv.) Th. Dur & Schinz. (1898). Most of the medicinal uses