Investigation of skin permeation, ex vivo inhibition of venom-induced tissue destruction, and wound healing of African plants used against snakebites.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation causes 5000-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone every year. The inaccessibility of antiserum therapy is a vast problem, and only about 2.5% of the actual need for antiserum in Africa is covered. Numerous plants have shown in vitro inhibitory activity against one or more of the hydrolytic enzymes involved in snakebite-induced necrosis. However, a more thorough examination of the plant species in ex vivo and in vitro cell assay models is needed to test their ability to inhibit necrosis. MATERIALS AND METHODS: Extracts which had previously shown in vitro inhibitory activity against necrosis enzymes, were tested in an ex vivo air-liquid-interface model, and a wound healing scratch assay as well as for their ability to permeate the skin barrier and inhibit venom induced cell death. RESULTS: Of the 14 water extracts and 16 ethanol extracts tested at a concentration of 10 µg/mL, only the ethanol extracts of Tamarindus indica and Paullinia pinnata resulted in a small but significant increase in cell migration of around 10% compared to treatment with buffer after 24h treatment. The remaining extracts showed no effect, or they even delayed the cell migration compared to the treatment with buffer. After 48 h treatment, 10 of the tested extracts showed a decreased cell migration compared to no treatment. At a 100 µg/mL concentration all the extracts inhibited cell migration and five extracts killed some of the cells, while four extracts killed all the cells. Ten of the thirty extracts were tested in a Franz cell set-up but none of the extracts tested did permeate the skin barrier over a 48 h period, and will therefore be of very limited use topically in the initial treatment of snakebites in its present form. None of the extracts were able to directly interact with the enzyme to lower the cell toxicity of the venom. Two extracts, Dichrostachys cinerea and Grewia mollis, were tested in the ex vivo model, but none of them inhibited the tissue destruction caused by venom. CONCLUSION: On the basis of this study, topical treatment with plant extracts for snakebite-induced tissue necrosis cannot be recommended.

Hyaluronidase, phospholipase A2 and protease inhibitory activity of plants used in traditional treatment of snakebite-induced tissue necrosis in Mali, DR Congo and South Africa.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation, every year, causes estimated 5-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone. Antiserum is not easily accessible in these regions or doctors are simply not available, thus more than 80% of all patients seek traditional practitioners as first-choice. Therefore it is important to investigate whether the plants used in traditional medicine systems contain compounds against the necrosis-inducing enzymes of snake venom. MATERIALS AND METHODS: Extracts from traditionally used plants from DR Congo, Mali and South Africa were tested in hyaluronidase, phospholipase A2 and protease enzyme bioassays using Bitis arietans and Naja nigricollis as enzyme source. RESULTS: A total of 226 extracts from 94 different plant species from the three countries, Mali, Democratic Republic of Congo and South Africa were tested in phospholipase A2, proteases and hyaluronidase enzyme assays. Forty plant species showed more than 90% inhibition in one or more assay. Fabaceae, Anacardiaceae and Malvaceae were the families with the highest number of active species, and the active compounds were distributed in different plant parts depending on plant species. Polyphenols were removed in the search for specific enzyme inhibitors against hyaluronidase, phospholipase A2 or proteases from extracts with IC50 values below 100µg/ml. Water extracts of Pupalia lappacea, Combretum molle, Strychnos innocua and Grewia mollis and ethanol extract of Lannea acida and Bauhinia thonningii still showed IC50 values below 100µg/ml in either the hyaluronidase or protease bioassay after removal of polyphenols. CONCLUSION: As four of the active plants are widely distributed in the areas where the snake species Bitis arietans and Naja nigricollis occur a potential inhibitor of the necrotic enzymes is accessible for many people in sub-Saharan Africa.

Can phylogeny predict chemical diversity and potential medicinal activity of plants? A case study of Amaryllidaceae.

BACKGROUND: During evolution, plants and other organisms have developed a diversity of chemical defences, leading to the evolution of various groups of specialized metabolites selected for their endogenous biological function. A correlation between phylogeny and biosynthetic pathways could offer a predictive approach enabling more efficient selection of plants for the development of traditional medicine and lead discovery. However, this relationship has rarely been rigorously tested and the potential predictive power is consequently unknown. RESULTS: We produced a phylogenetic hypothesis for the medicinally important plant subfamily Amaryllidoideae (Amaryllidaceae) based on parsimony and Bayesian analysis of nuclear, plastid, and mitochondrial DNA sequences of over 100 species. We tested if alkaloid diversity and activity in bioassays related to the central nervous system are significantly correlated with phylogeny and found evidence for a significant phylogenetic signal in these traits, although the effect is not strong. CONCLUSIONS: Several genera are non-monophyletic emphasizing the importance of using phylogeny for interpretation of character distribution. Alkaloid diversity and in vitro inhibition of acetylcholinesterase (AChE) and binding to the serotonin reuptake transporter (SERT) are significantly correlated with phylogeny. This has implications for the use of phylogenies to interpret chemical evolution and biosynthetic pathways, to select candidate taxa for lead discovery, and to make recommendations for policies regarding traditional use and conservation priorities.

Cross-cultural comparison of medicinal floras used against snakebites.

ETHNOPHARMACOLOGICAL RELEVANCE: Envenomation causes an estimated 1.8-2.5 million incidences per year with a mortality level of 100-125,000 persons annually and more than 100,000 individuals suffer from severe complications, which may end in amputation of the attacked limb. The use of plants is a major part of the traditional practitioners' treatment of snakebites. MATERIALS AND METHODS: A database was created for plants used to treat snakebites worldwide. From this database, we selected five countries with a high number of entries and representing different cultures, geography and floristic zones: Brazil, Nicaragua, Nepal, China and South Africa. The datasets were analysed by regression and binominal analysis to see if any family or genus used against snakebites was overrepresented in the respective traditional medicinal systems relative to the abundance in the local flora. The families from the different geographical areas were compared to ascertain whether the same plant families are preferred by different peoples. RESULTS: Three 'hot' families (Apocynaceae, Lamiaceae and Rubiaceae) were recovered in at least two of the five compared countries in the regression analyses and one 'hot' family (Zingiberaceae) was recovered in two of the compared countries in the binomial analyses. Four out of five floras possess families identified as outliers in both regression and binomial analyses. Eight families were recovered by both the binomial and the regression analysis (40-62% of all highlighted families respectively). At the genus level, only Piper (Piperaceae) was recovered as a 'hot' genus in at least two floras. Seven genera were highlighted by both analyses (25-44% of the highlighted genera). CONCLUSIONS: Cross-cultural comparison of medicinal floras used against snakebites appears to be useful for highlighting candidate families and genera for further studies.

6-O-methylkrigeine, a new amaryllidaceae alkaloid from Nerine huttoniae Schönland.

A novel Amaryllidaceae alkaloid, 6-O-methylkrigeine, has been isolated from Nerine huttoniae and the configuration was elucidated by 1-D and 2-D (1)H-NMR, (13)C-NMR, HRMS and optical rotation.