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This research was a quantity determination of total lactones content in Andrographis paniculata leaves using spectrophotometry technique. In addition, the lactones isolation procedures for standard compound of quantity analysis were also studied. The crude acetone extract was separated by column chromatography and elucidated by 1H NMR spectroscopy technique. The isolated structures were 14-deoxy-11,12-didehydroandrographolide and andrographolide. Then the isolated andrographolide was used as a standard compound for total lactones determination by using colorimetric method with Kedde’s reagent with maximum absorption wavelength (lmax) at 541 nm. The method validation was reliable in the terms of specificity, linearity and a range correlation coefficient (R2) of 0.9969 at the concentration ranges of 5-20 %w/w and precision (% RSD of 1.01). Moreover, the detection limit (LOD) and the quantitation limit (LOQ) were 0.0089 and 0.0211 mg/mL, respectively, which complied with the acceptance criteria in the validation protocol. The total lactones content, calculated as andrographolide, determined by this method was 8.14±0.13 % (n = 3), and by the official method, Thai Herbal Pharmacopoeia, was 7.86±0.26 % (n = 3). The results of the two methods do not differ significantly at p < 0.05.
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