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KMID : 0613820130230081025
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Journal of Life Science
2013 Volume.23 No. 8 p.1025 ~ p.1031
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Simultaneous HPLC Determination of Marker Compounds for the Standardization of Hedyotis diffusa
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Bang Han-Yeol
Yang Eun-Ju
Kim Jeong-Ah
Song Kyung-Sik
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Abstract
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From a 95% ethanolic extract of H. diffusa, four marker compounds (HD1~HD4) were isolated, which were relatively unique and exist in comparably high contents. The structures of marker compounds were identified as digitolutein (1), 2-hydroxy-3-methylanthraquinone (2), (E/Z)-6-O-p-coumaroyl scandoside methyl ester (4:1 mixture) (3), and (E/Z)-6-O-p-methoxycinnamoyl scandoside methyl ester (4:1 mixture) (4), respectively, on the basis of ^13C and ¹H-NMR analyses. The calibration curves of marker compounds showed high linearity, as their correlation coefficient (R²) were in the range of 0.9991~0.9999. In addition, the limit of detection (LOD) and the limit of quantification (LOQ) were 0.03~0.07μg/ml and 0.099~0.231μg/ml, respectively. The intra-day/inter-day precision and accuracy were 0.23~2.00%/0.25~1.16% and 94.60~108.44%/94.73-110.23%, respectively. The optimal HPLC conditions for the simultaneous quantification of HD1~HD4 were as follows: stationary phase; Merck Chromolith RP-18e (100×4.6mm,5㎛), column temp.; room temperature, UV detection at 280 nm, flow rate; 2.0 ml/min, injection volume; 10μl, mobile phase; start with the mixture of 80% solvent A (H₂O containing 0.5% acetic acid) and 20% solvent B (methanol containing 0.5% acetic acid) and gradually decrease solvent A to 40% in 9 min., then retain this condition to 18 min. Under the HPLC condition, the four marker compounds 1~4 were successfully separated without any interference of other constituents. The results obtained in this study are expected to be helpful for the development of nutraceutics and natural medicines and for the quality control of this plant.
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KEYWORD
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Anthraquinone, Hedyotis diffusa, HPLC quantification, iridoid, simultaneous analysis
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