Antituberculosis activity of flavonoids from Galenia africana L. var. africana

The recent increase in the incidence of tuberculosis (TB) with the emergence of multidrug-resistant (MDR) cases has lead to the search for new TB-drugs. Mycobacterium tuberculosis is a complex, resilient organism, and it is important to note that new drugs are required which can reduce TB’s six month treatment time and can be effective against drug-resistant strains of mycobacteria. Plants contain numerous biological active compounds, many of which have been shown to have antimicrobial activity. The search for biologically active extracts based on traditional use of plants is relevant due to the appearance of microbial resistance to many antibiotics and the occurrence of fatal opportunistic infections. Ethanol extracts of seven selected ethnobotanically South African medicinal plants (Artemisia afra, Dodonaea angustifolia, Drosera capensis, Galenia africana, Prunus africana, Syzygium cordatum and Ziziphus mucronata) were investigated for their antimycobacterial activity against two Mycobacterium species. The minimum inhibitory concentration (MIC) of ethanol extracts of A. afra, Dodonaea angustifolia, Drosera capensis and G. africana ranged from 0.781 to 6.25 mg/mL against a non-pathogenic strain of mycobacteria, ‘M. smegmatis’. G. africana showed the best activity, exhibiting an MIC of 0.781 mg/mL and a minimum bactericidal concentration (MBC) of 1.563 mg/mL against M. smegmatis. A drug sensitive strain of M. tuberculosis was found to be susceptible to the ethanol extracts of Dodonaea angustifolia and G. africana. (MICs 5.0 and 1.2 mg/mL respectively) when using the rapid radiometric-BACTEC method. The phytochemical analysis of G. africana led to the isolation and identification of three known compounds namely; (2S)-5,7,2'-trihydroxyflavanone, (E)-3,2',4'-trihydroxychalcone (not reported from natural sources) and (E)-2',4'-dihydroxychalcone. A novel chalcone ‘(E)-3,2',4'-trihydroxy-3'-methoxychalcone’ was also isolated from the ethanol extract of G. africana. Isolation of (2S)-5,7,2'-trihydroxyflavanone, (E)-3,2',4'-trihydroxychalcone and E)-3,2',4'-trihydroxy-3'-methoxychalcone was reported for the first time from this plant. The MIC of novel compound against M. tuberculosis was found to be 50.0 µg/mL whereas (2S)-5,7,2'-trihydroxyflavanone and (E)-3,2',4'-trihydroxychalcone exhibited an MIC of 100.0 µg/mL. During synergistic studies using (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone with the antituberculosis drug INH, it was found that the MICs of INH and the compounds were reduced sixteen and eight-fold respectively, resulting in a Fractional Inhibitory Concentration (FIC) of 0.1250 and 0.1875 respectively. The synergistic effect of two isolated compounds (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone) in in vitro studies also showed synergistic action, reducing their original MICs four-fold resulting in a FIC of 0.5. Since (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone from G. africana showed synergistic activity with INH, it is speculated that the compounds might have similar mechanism as that of INH. However, mechanistic studies on these compounds should be done in order to get an indication of the ‘flavonoids and chalcones’ interferences on mycolic acid synthesis, membrane synthesis and enzyme inhibition. Our investigation on the NADPH oxidase activity of (2S)-5,7,2'-trihydroxyflavanone with Mtr, found that this compound failed to exhibit any NADPH oxidase activity at 800 µM concentrations. Mtr is evidently not the target for the antimycobacterial activity of (2S)-5,7,2'-trihydroxyflavanone. Fifty percent inhibitory concentration of the ethanol extract of G. africana, and the two purified compounds, (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone were found to be 120.0; 110.3 and 80.2 µg/mL respectively against the U937 cells. The MIC of the ethanol extract of G. africana in U937 macrophages infected with M. tuberculosis was found to be 50.0 µg/mL indicating the extract’s intracellular antimycobacterial activity in real physiological conditions. The two purified compounds also showed good intracellular antimycobacterial activity. The MICs of (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone were found to be 100 and 50 µg/mL respectively. This study indicated that the intracellular activity of the ethanol extract is significant in macrophages. The activity might be due to M. tuberculosis being unable to avoid macrophage killing and its survival during phagocytosis, (including inhibition of phagosome-lysosome fusion, inhibition of the acidification of phagosomes, resistance to killing by reactive oxygen intermediates and modification of the lipid composition of the mycobacterial cell membrane, thereby altering its capacity to interact with immune or inflammatory cells). It can be concluded that the traditional use of G. africana for TB has been scientifically validated to some extent. Isolated compounds and the ethanol extract of the plant warrant further investigation for their potential as antimycobacterial agents. Since synergistic activity of purified compounds with existing antituberculosis drug INH, was significant, it will be worthwhile evaluating the efficacy of purified compounds in combination with TB-drugs in pre-clinical studies.Thesis (PhD)--University of Pretoria, 2009.Plant Scienceunrestricte

Evaluation of isolates and identified phenolics from Pelargonium Sidoides against Mycobacterium Tuberculosis, other bacteria and fungi

Anecdotal evidence of two South African Geranium species (Pelargonium reniforme and Pelargonium sidoides) from the United Kingdom with regard to plants being used against tuberculosis, which lacked scientific evidence’ prompted us to investigate these two plants for their antimicrobial properties. The German herbal remedy (‘Umckaloabo’) is prepared from these two plant species and is currently being sold for bronchitis. Acetone, chloroform and ethanol extracts were investigated against three bacteria (pathogens causing bronchitis), three fungi (fungal species associated with the upper and lower respiratory tract) and Mycobacterium tuberculosis. This is the first report on the extracts’ activity against Moraxella catarrhalis, and three fungi (Asperigilus niger, Rhizopus stolonifer and Fusarium oxysporum). Acetone and ethanol root extracts of P. sidoides and its combination with P. reniforme exhibited activity against bacteria at 5.0 mg/ml concentration. The fungi were significantly inhibited by the acetone and ethanol extracts of P. reniforme and the ethanol extract of P. sidoides at a concentration of 5.0 mg/ml. Antituberculosis activity was observed on acetone, chloroform and ethanol root extract of P. reniforme and chloroform extract of P. sidoides at 5.0 mg/ml concentration. The isolation and purification of compounds were attempted using two different approaches, of which the second approach resulted in isolation of four compounds and two flavonoids. One flavonoid (epigallocatechin) is isolated for the first time from P. sidoides. Laboratory investigations showed no activity of compounds isolated against M. tuberculosis. As Mycobacteria are intracellular pathogens, antimycobacterial activities may be due to either direct or indirect effects. Though the compounds in this study did not show antituberculosis activity, it can be speculated that the anecdotal evidence of TB-patients could be due to their immunostimulant activity.Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005.Plant Scienceunrestricte

Acute toxicity studies of the South African medicinal plant Galenia africana

Background: Medicinal plants are used by a large proportion of the global population as complementary and alternative medicines. However, little is known about their toxicity. G. africana has been used to treat wounds, coughs and skin diseases and is used in cosmetic formulations such as lotions and shampoos. Methods: The acute oral and dermal toxicity potential of G. africana was analyzed after a single administration of 300 and 2000 mg/kgbw for acute oral toxicity and 2000 mg/kgbw for acute dermal toxicity. Female Sprague- Dawley rats were used for the acute oral toxicity study whereas both male and female Sprague-Dawley rats were used for the acute dermal toxicity study. In the Episkin skin irritation test, the irritation potential of G. africana (concentrate) and G. africana (in-use dilution) extracts were assessed using the Episkin reconstituted human epidermis. In the dermal sensitization study, female CBA/Ca mice were treated with G. africana concentrations of 50, 100 and 200 mg/ml respectively. The vehicle of choice was dimethylformamide which acted as a control. Results: The results of the acute oral and dermal toxicity studies revealed that the median lethal dosage (LD50) for G. africana extract in Sprague-Dawley rats was considered to exceed 2000 mg/kgbw. In the irritation test, the G. africana (concentrate) and G. africana (in-use dilution) extracts were non-irritant on the Episkin reconstituted human epidermis. In the dermal sensitization study, the stimulation index (SI) values for the mice treated with the G. africana extract at concentrations of 50, 100 and 200 mg/ml/kgbw, when compared to the control group, were 1.3, 0.9 and 1.3 respectively. The open application of the extract at the various concentrations did not result in a SI of ≥ 3 in any group. Hence, it did not elicit a hypersensitivity response. Conclusion: These findings demonstrate that the acute toxicity profile for G. africana is acceptable and can subsequently be used for single use in the pharmaceutical and cosmetic industries

Bioprospecting the African Renaissance: The new value of muthi in South Africa

This article gives an overview of anthropological research on bioprospecting in general and of available literature related to bioprospecting particularly in South Africa. It points out how new insights on value regimes concerning plant-based medicines may be gained through further research and is meant to contribute to a critical discussion about the ethics of Access and Benefit Sharing (ABS). In South Africa, traditional healers, plant gatherers, petty traders, researchers and private investors are assembled around the issues of standardization and commercialization of knowledge about plants. This coincides with a nation-building project which promotes the revitalization of local knowledge within the so called African Renaissance. A social science analysis of the transformation of so called Traditional Medicine (TM) may shed light onto this renaissance by tracing social arenas in which different regimes of value are brought into conflict. When medicinal plants turn into assets in a national and global economy, they seem to be manipulated and transformed in relation to their capacity to promote health, their market value, and their potential to construct new ethics of development. In this context, the translation of socially and culturally situated local knowledge about muthi into global pharmaceuticals creates new forms of agency as well as new power differentials between the different actors involved

Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants

Seven ethnobotanically selected medicinal plants were screened for their antimycobacterial activity. The mininium inhibitory concentration (MIC) of four plants namely Artemisia afra, Dodonea angustifolia, Drosera capensis and Galenia africana ranged from 0.781 to 6.25 mg/mL against Mycobacterium smegmatis. G. africana showed the best activity exhibiting an MIC of 0.78 mg/mL and a minimum bactericidal concentration (MBC) of 1.56 mg/mL. The MICs of ethanol extracts of A angustifolia and G. africana against M. tuberculosis were found to be 5.0 and 1.2 mg/mL respectively. The mammalian cytotoxicity IC50 value of the most active antimycobacterial extract, from G. africana, was found to be 101.3 mu g/mL against monkey kidney Vero cells. Since the ethanol G. africana displayed the best antimycobacterial activity, it was subjected to fractionation which led to the isolation of a flavone, 5,7,2'-trihydroxyflavone. The MIC of this compound was found to be 0.031 mg/mL against M. smegmatis and 0.10 mg/mL against M. tuberculosis. This study gives some scientific basis to the 14 traditional use of these plants for TB-related symptoms. Copyright (C) 2008 John Wiley & Sons, Ltd