A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20-40°C), (X2) flow rate (0.8-1.2 ml/min), (X3) acid concentration in aqueous phase (0-2%), (X4) organic solvent percentage at the beginning (40-50%), and (X5) at the end (50-60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1-4) and (X7) at the end (0-1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1 ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1-X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.
There is growing interest in the discovery of bioactive metabolites from endophytes as an alternative source of therapeutics. Identification of their therapeutic targets is essential in understanding the underlying mechanisms and enhancing the resultant therapeutic effects. As such, bioactive compounds produced by endophytic fungi from plants at the National Park, Pahang, Malaysia, were investigated. Five known compounds were identified using LC-UV-MS-NMR and they include trichodermol, 7-epi-brefeldin A, (3R,4S)-4-hydroxymellein, desmethyl-lasiodiplodin and cytochalasin D. The present study went on to investigate the potential anticancer effects of these compounds and the corresponding molecular mechanisms of the lead compound against human breast adenocarcinoma, MCF-7. For the preliminary screening, the cytotoxicity and apoptotic effects of these compounds against MCF-7 were examined. The compounds were also tested against noncarcinogenic hepatocytes (WRL68). The differential cytotoxicity was then determined using the MTT assay. Desmethyl-lasiodiplodin was found to suppress the growth of MCF-7, yielding an inhibitory concentration (IC50) that was seven-fold lower than that of the normal cells. The cytotoxic effect of desmethyl-lasiodiplodin was accompanied by apoptosis. Subsequent analysis demonstrated increased expression levels of caspase 3, c-myc and p53. Further, desmethyl-lasiodiplodin resulted in inhibition of monocyte chemotactic protein (MCP)-3, a cytokine involved in cell survival and metastasis. Hence, this study proposed that desmethyl-lasiodiplodin inhibited growth and survival of MCF-7 through the induction of apoptosis. This anticancer effect is mediated, in part, by upregulation of apoptotic genes and downregulation of MCP-3. As desmethyl-lasiodiplodin elicited minimal impact against normal hepatocytes, our findings also imply its potential use as a specific apoptotic agent in breast cancer treatment.