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Background : D. lablab is tropical vine grown as a garden annual belonging to Leguminosae, which is used in oriental medicine for coldness like a cooling disease, diarrhea and abdominal pain etc. when it is hot weather in summer and plenty of rainy seasons. In previous study, D. lablab showed an inhibitory activity for trypsin. Reactive oxygen species (ROS) was produced by trypsin via a second protease-activated receptor. Therefore, it is considered that there are substances having antioxidant activity in D. lablab.
Methods and Results : The seed of dried D. lablab was extracted hot water, and then partitioned with Et2O, EtOAc, n-butanol and aqueous. The aqueous layer was separated from silica gel and Diaion HP-20 liquid chromatography and HPLC-prep, resulting in confirmed two compounds. The two compounds was identified to C14H14N2O4 and stachyose from ESI-MS and 1H- and 13C-NMR. The two compounds was performed the intracellular ROS measurement and the expression of catalase and Cu/Zn SOD. The intracelluar ROS measurement was detected using DCF-DA in LPS-treated RAW264.7 cell. The results were shown that two compounds inhibited intracellular ROS levels to dose-dependent. And the expression of genes was detected quantitative real-time PCR in LPS-trated RAW264.7 cell. Although the two compounds were not significantly different towards the Cu/Zn SOD expression level, the expression level of catalase genes were indicated an increase rate of about 347% in C14H14N2O4 and about 242% in stachyose at 50 ㎍/㎖ compared with LPS-not treated cells.
Conclusion : From the above results, both compounds significantly shown antioxidant activity in dose-dependent by examining the amount of intracellular ROS and the expression levels of SOD and catalase enzymes. As screening ROS inhibition of C14H14N2O4 and stachyose in vitro, they may be a good candidate for regulating the progression of human oxidant stress diseases and warrants further studies.
