Ganbold, Munkhchimeg (2010) Cytotoxicity of the Chinese herbal remedy 'Oldenlandia diffusa' and its anti-cancer effective constituents. (PhD thesis), Kingston University, .
Abstract
The main objective of the study was to investigate the bioactive components, their effect on HL60 leukaemic and Caco-2 colon carcinoma cancer cell lines and their bioavailability from a decoction of a Chinese Herbal Remedy OldenIandia diffusa, using a Caco-2 monolayer as a mimic for intestinal absorption. The HPLC separation method was set up by investigating parameters such as column type, mobile phase, isoeratic/gradient elution, flow rate, pH of buffer and detection wavelength. Using this method eleven fractions (F1-F11) were collected. Results from a cytotoxicity investigation using the CyQUANT NF, trypan blue and neutral red uptake assays showed that the decoction has a cytotoxic effect on HL60 (V=13.5±4.3%, n=3) and Caco-2 (V=50.0±1.4%, n=3) cancer cell lines. The most cytotoxic active fraction was F9 (V=62.2±7.3% (HL60) and V=32.l±7.9%, n=3 (Caco-Zj). DAPI staining and Western blotting (detection of cleaved-PARP) studies on the decoction and F9 fraction indicated that mode of cell death was apoptosis, which was mediated by a caspase cascade. Fraction F9 was separated into eight further fractions (compound- fraction-l to 8) by optimisation of the HPLC method. By using liquid-liquid extraction with ethyl acetate, eleven sub fractions (compound-fraction-l to 8 and ethyl acetate fractions (FEA-l to 3) were collected purely. All collected fractions were analysed by high resolution-MS and their MWs were determined as 268.07,238.07,242.24,280.38,256.24,282.25, 284.27, 456.36,550.17,328.22 and 330.24. In the most cytotoxic fraction F9, oleanolic acid and ursolic acid were isolated and identified in concentrations of 0.068 mg/g and 0.166 mg/g, respectively. FEA-l to 3 also showed cytotoxic effects on these cancer cells (V=53.3±2.2%, 55.4±4.4% and 50.6±11.3% (HL60) and V=63.4±13.5%, 45.8±5.9%, and 59.5±9.7%, respectively (Caco-2), Fraction FEA-l was identified as E-6-0-p-coumaroyl seandoside methyl ester (MW 550.17). Cytotoxicity assessment results showed that it has a growth inhibition effect on both cancer cell lines. Bioavailability after ingestion of the decoction was studied using 21-day grown Caco-2 monolayers. The post absorption sample (PAS) of the decoction and fraction F9 were shown to have a good permeability (P[sub]app=3.575xlO[sup]-6 cm/s). The PAS also has a cytotoxic effect on cancer cells (V=67.0±7.5%, n=3). When analysed by LC-MS, the most of the compounds that had previously been seen in the original fractions were again observed.
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