0 at 230 nm. Mobile phase consisting of ethyl acetate:toluene (1:2 v/v) at a flow rate 1 mL/min. Pure phyllanthin and hypophyllanthin were separately weighed and dissolved in HPLC grade methanol to obtain the concentration 1 mg/mL. From these solutions, 400 μg/mL phyllanthin and 200 μg/mL of hypophyllanthin were prepared in the mobile phase. The extract was also weighed and dissolved in HPLC grade methanol to obtain the concentration 1 mg/mL and considered as sample. Aliquots of 0.25, 0.5, 1.0, 1.5, 2 and 2.5 mL volume of both phyllanthin and hypophyllanthin from the standard solutions were separately transferred to a series of 5 mL

volumetric #Modulators randurls[1|1|,|CHEM1|]# flasks and adjusted the volume to the mark with methanol in each flask to obtain 10–100 μg/mL and 5–50 μg/mL concentrations respectively. The sample solution was also diluted accordingly for the assay. Method was validation as follows3: (A) Linearity and limit of detection and quantification Six different concentrations of standard solutions were analyzed repeating three times (n = 3), mean value were employed at specified concentration

range. The linearity was evaluated using the least square method. Limit of detection (LOD) and limit of quantification (LOQ) were determined by the equation kSD/s, where k is a constant (3 for LOD and 10 for LOQ), SD is the standard deviation and s is the slope of the concentration/response graph. (B) Precision, robustness and accuracy The intra and inter-day precision were measured by assays of six replicate injections of the check details mixture of standard solutions at three concentration levels (10–5, 40–20 and 100–50 μg/mL). The intra-day assay with the interval of 4 h in 1 day while the inter-day assay precision, were performed over 6 days. Detection wavelength, proportion of the mobile phase, solvent brands, flow rate and column temperature were tested in the same day to evaluate robustness of the method. For each change the standard solution was injected

6 times. The accuracy of the extraction from method was determined by the method of standard addition. The standards of three different concentrations (80, 100 and 120%) were added into pre-analyzed samples and the amounts were estimated by measuring peak areas and by fitting these values to the straight-line equation of calibration curve. Acute toxicity study was done following the OECD guideline 423 with some modifications.2 The standardized MEPA was suspended in 1% CMC as vehicle. Following the 24 h of fasting, the animals were weighed and the suspension was administered orally at the doses of 300, 600, 2000 and 5000 mg/kg to test groups of rats, while the control group received CMC in the same volume using a ball-tipped stainless steel feeding needle.