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Purification and identification of active compounds of Carpobrotus edulis against the reversal of resistance of human mdr1 gene transfected mouse lymphoma cells
In African traditional medicine, leaf juice of Carpobrotus edulis is used to treat diarrhoea, stomach cramps, eczema, herpes and allergies. A mixture of leaf juice, honey and olive oil in water is also used to treat Mycobacterium tuberculosis infections . The methanolic extract of C. edulis reverse resistance of mouse lymphoma cells that carry the human mdr1 gene to chemotherapeutic agents and increase the killing activity of Staphylococcus aureus infected macrophages [2,3]. In order to identify the compound(s) responsible for the above activity, the following purification protocol was preformed. The methanolic extract was extracted with hexane, ethyl acetate and chloroform. These extracts were tested for their ability to affect extrusion of rhodamine 123 from mouse lymphoma cells containing the human efflux pump gene mdr1, by flow cytometry (activity evaluation assay). The hexane extract, as the most active was subjected to purification procedures, including chromatography on polyamide, silica gel, and Sephadex LH-20 using different solvent systems. The content of the fractions and purity of the isolated compounds were checked by NP- or RP-TLC. Each fraction during the purification process was evaluated for activity. The isolated compounds were identified as triterpenes β-amyrin and oleanolic acid and monogalactosyldiacylglycerol, some of which have been previously described as having some anti-inflammatory properties or anti-mycobacterial and/or anti-microbial activity [4,5]. These compounds and purified fractions were evaluated for activity against human mdr1 gene transfected mouse lymphoma cells and found to be active to varying degrees. They should also be evaluated for ability to enhance the killing activity of human macrophages against intracellular bacteria, possibly by having direct inhibition of K+ and Ca2+ transport from the phagolysosome, thereby activating the hydrolytic machinery of the macrophage . Active compounds may prove to be useful lead compounds for inhibition of multidrug resistance of cancer cells and a possible benefit could be exploited in combination chemotherapy of chemotherapy resistant cancer. Further preclinical studies can, also, improve the design of anti-mycobacterial chemotherapy and efflux pump mediated mdr of important clinical bacterial pathogens.
Acknowledgements: Ana Martins and Marta Martins were supported by FCT grants SFRH/BD/19445/2004, SFRH/BD/14319/2003, respectively; Anikó Váradi for her precious technical contribution.
References: 1. National Biological Information Infrastructure, Global Invasive Species Database (GISD). www.invasivespecies.net/database, consulted in Mar 2008. 2. Martins, M, et al. (2005) Fitoterapia 96–99. 3. Ordway, D, et al. (2003) Phytother Res 17: 512–519. 4. Caldwell C, et al. (2000)J Nat Prod 63: 1611–1614. 5. Bruno, A, (2005) Eur J Pharm 524: 159–168. 6. Amaral L, et al. (2007) JAC 59: 1237–1246.