Micropropagation and pharmacological evaluation of Boophone disticha.
Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa.