Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC

Ganbold, Munkhchimeg, Barker, James, Ma, Ren, Jones, Lucy and Carew, Mark (2010) Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC. Journal of Ethnopharmacology, 131(2), pp. 396-403. ISSN (print) 0378-8741


AIM OF THE STUDY: Oldenlandia diffusa is a traditional Chinese herbal remedy with known cytotoxic activity in vitro and in vivo. The aim of the study was to identify the most cytotoxic constituents of a water extract (a decoction is traditionally used as a treatment) by HPLC and activity-guided fractionation. The bioavailability of the decoction and certain fractions, and the mode of cell death induced by these mixtures, were also investigated. MATERIAL AND METHODS: A decoction of O.diffusa was prepared and separated by HPLC into eleven fractions (F1-11) for testing on the growth of HL60 leukaemia cells; two of the most active fractions were also tested on Caco-2 colon cancer cells. Cell viability was measured by trypan blue exclusion, DNA content (Cyquant NF assay) and neutral red uptake. Evidence of apoptosis was gained from cells stained with the nuclear dye DAPI, and detection of cleaved poly (ADP-ribose) polymerase (PARP) by Western blot. RESULTS: Fraction 9 was found to be the most active fraction, and, along with the decoction, induced apoptosis. Cells stained with DAPI showed a decrease in cell size and nuclear fragmentation characteristic of apoptosis. Detection of cleaved PARP further confirmed induction of apoptosis by O.diffusa decoction and fraction 9. The bioavailability of O.diffusa was investigated by production of post-absorption samples using Caco-2 intestinal epithelial monolayers. Addition of post-absorption samples (taken from the basolateral side after 3 hr incubation with the decoction on the apical side) inhibited the growth of HL60 cells, and suggested a degree of bioavailability. The constituents in fraction 9 were further separated by HPLC and eight major compounds were identified by LC-MS: two of these were ursolic acid (UA) and its enantiomer oleanolic acid (OA). Concentrations of UA and OA in the decoction were then calculated by reference to the area of the peaks of UA and OA found in F9. The post-absorption sample of F9 contained six of the eight constituents in the original pre-absorption fraction 9. CONCLUSIONS: Taken together, the results suggest that certain constituents, possibly including ursolic/oleanolic acid, may be bioavailable and at sufficient concentration to induce apoptosis in cancer cells in vitro through a mechanism including the cleavage of PARP.

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