Quantitative Thin Layer Chromatography Analysis, Antioxidant, and Anti-Inflammatory Activities of Polyherbal Formulation (Ammarit-Osot) Extracts

This study was carried out to develop a TLC method for quantifying one of bioactive, piperine, from polyherbal formulation (Ammarit-Osot) extracts. The extracts were prepared by different solvents such as methanol (ME) and water (WE). The mobile phase of toluene:acetone:formic acid (7:2:1, v/v/v) was used for separation. The calibration curve showed a good linearity (r2 = 0.9972) in the range of 50 – 500 ng/spot. This assay was assessed by intra-and interday precision (RSD 0.25 – 2.49%), and accuracy (101.05 – 101.56 %). The piperine content was found to be 23.06 ± 0.06 and 0.44 ± 0.03% w/w for ME and WE, respectively. Both ME and WE demonstrated significant activity in DPPH and FRAP assays, indicating their antioxidant potential. The TPC of ME and WE were 53.40 ± 2.21 and 101.18 ± 0.46 (mg GAE/g extract), respectively. The TFC of ME and WE were 43.35 ± 3.94 and 25.31 ± 1.74 (mg catechin/g extract), respectively. In addition, the cytotoxicity of extracts on RAW264.7 was investigated by MTT assay. The ME and piperine showed IC50 with 162.40 ± 7.84 and 115.68 ± 11.96 µg/mL, respectively. While the WE exhibited low cytotoxicity (IC50 > 1,000 µg/mL), the anti-inflammatory activity was assessed by LPS-stimulated NO overproduction. The ME and piperine displayed the ability to inhibit NO production, but WE not inhibited. The results indicated that the different solvent extracts of Ammarit-Osot have the potential to be antioxidant, anti-inflammatory and immune-stimulate agent.