HPLC determination of active diterpene lactones from Andrographis paniculata Nees planted in various seasons and regions in Thailand

A quantitative determination of four known diterpene lactone compounds : dehydroandroghapholide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide-19-β-D glucoside (C-5) from the leaves of Andrographis paniculata, Nees. has been studied by using a reversed phase high performance liquid chromatographic technique. The system comprised with a µ-bondapak C 18 column as stationary phase, a 42:58 mixture of methanol : water as mobile phase (flow rate 2 ml/min) and a UV-detector at wavelength of 255 and 220 nm. The relative retention time of each diterpene lactone were found to be 0.22 (C-3), 0.45 (C-5), 0.75 (C-2) and 1.00 (C-4). The linear standard curve of each compound was obtained in the following concentration range; C-2 (0.10-0.97 mg/ml, correlation coefficient (r) = 0.9990 and 0.9709), C-3 (0.10-0.42 mg/ml, r = 0.9998 and 0.9925), C-4 (0.10-0.30 mg/ml, r = 0.9998 and 0.9886) and C-5 (0.10-0.20 mg/ml, r = 0.9991 and 0.9917). The method was found to be highly accurate with low relative standard deviation as follows: 0.89% (C-2), 0.80% (C-3), 1.27% (C-4) and 1.67% (C-5). The contents of the four diterpene lactones in the sample of Andrographis paniculata collected from various regions in Thailand determined in terms of average percentage content from northern, southern, central & east and northeastern region, as follows: C-2 = 2.13 ± 0.96, 1.39 ± 0.32, 1.52 ± 1.00 and 1.81 ± 0.76, respectively; C-3 = 2.76 ± 0.82, 4.39 ± 1.02, 3.55 ± 1.69 and 3.24 ± 1.12, respectively; C-4 = 1.89 ± 0.44, 1.63 ± 0.37, 1.42 ± 0.59 and 1.57 ± 0.80, respectively; C-5 = 1.82 ± 1.07, 0.71 ± 0.54, 0.73 ± 0.4 and 1.07 ± 1.11, respectively. The contents of four diterpene lactones from the samples collected every month in one year-cycle were found in terms of the highest content and the lowest content as follows: C-2 = 7.30 ± 0.48% w/w (April), and 0.61 ± 0.58% w/w (December); C-3 = 6.02 ± 0.47% w/w (November) and 0.82 ± 0.86% w/w (February); C-4 = 2.02 ± 0.35% w/w (December) and 0.61 ± 0.58% w/w (April); C-5 = 3.81 ± 0.39% w/w (March) while C-5 content was too low to be detected (October and November). Moreover, the total diterpene lactone content in the leaves of Andrographis paniculata was found to be highest at 11.81% w/w in April and lowest at 7.68% w/w in October.