Phytochemical and toxicological studies of some Botswanan plants used in traditional medicine
Traditional medicine is widely used in Botswana and in recent years a number of plant species have been submitted to the Botswana Police Forensic Science Laboratory by the Police as exhibits in cases of suspected poisoning by herbal medicine. The request would be for the forensic toxicologist to establish whether the plant material is toxic or not. In this study a selection of these plants are being investigated phytochemically and toxicologically. These include: Jatropha erythropoda Pax. (Euphorbiaceae), Cassia italica (E11.) Lam. Ex. (Leguminosae), Asclepiasfruticosa L. (Asclepiadaceae), Albizzia brevifolia Schinz (Leguminosae), Argemone mexicana L. (Papaveraceae) and Enicostemma axillare L. (Gentianaceae). A DNA-based test capable of identifying the species from powdered fragments of the plant material has also been developed in this project. Albizzia brevifolia, Enicostemma axillare and Jatropha erythropoda have not been investigated before; neither phytochemically nor toxicologically. Dichloromethane, methanol and water extracts of each of the plants were tested for cytotoxicity against a panel of four cell lines - three human and one murine cell line. While all extracts exhibited some degree of cytotoxicity, extracts from A. fruticosa were found to be the most toxic with LD50 values for the crude extracts of 1.3 -3.4. tg/ml. Phytochemical investigation of the extracts revealed the presence of a variety of secondary metabolites from the plants. A. brevifolia yielded terpenoids, phenolics, phenolic glycosides, a component of procyanidins, a lignan glycoside and sugars. E. axillare yielded terpenoids, a secoiridoid, and sugars, whereas A. mexicana yielded alkaloids. Investigation of C. italica and J. erythropoda revealed the presence of terpenoids, flavonoids, glycosides and sugars and that of A. fruticosa the presence of cardenolide glycosides. Among the compounds isolated and tested for toxicity, sanguinarine, an alkaloid from A. mexicana, was found to be the most toxic with an LD50 value of 0.22-1.4tg/ml1. The compound expresses toxicity by inhibiting Na/K ATPases and by intercalating with DNA bases and thus interfering with the replication process. Swertiamarin, the secoiridoid isolated from E. axillare, constituted about 10% of the dichloromethane extract of this plant, which showed significant toxicity. The plant also yielded swertiamarin as about 60% of the methanol extractive, which in contrast did not show any toxicity. Swertiamarin itself did not show toxicity at the levels tested, an indication that it is not responsible for the toxicity exhibited by the dichloromethane extract. However, secoirridoids such as swertiamarin might transform in vivo to toxic alkaloids. The phenolic compounds (and their glycosides), isolated from A. brevifolia, exhibited very weak or no toxicity, whereas the terpenoid, betulinic acid, showed some cytotoxicity. Another terpenoid, which was isolated from the plant, lupeol, is reported to be cytotoxic. The extracts of the plant showed significant toxicity, especially the methanol extract. The toxicity exhibited by betulinic acid could not account for the toxicity displayed by the extracts, particularly the methanol extract. This toxicity is perhaps due to other compound(s) that were not isolated or to synergistic activity. A DNA-based test has been developed for species identification using allele specific amplicons that show polymorphisms in the length of DNA sequences between two conserved primers. This is going to allow the species identification in cases where only small amounts of plant material are available, sometimes in mixture form. This will particularly be useful where there are no unique chemical markers to be used for identification. The test, is Polymerase Chain Reaction-based (PCR) and therefore very sensitive. Once the species is known more of it can be collected from the source or the wild to allow detailed toxicological and phytochemical work.