| AIMTo investigate the protective effects of different polarity alkaloid from Noble dendrobium and syringic acid on H202 induced lens damage of human, then for the development of drugs which have the effect of anti-cataract providing earlier studies.METHODS1. Extracting the Dendrobium nobile's powder by ethanol extraction, falling film concentration, acidification, salting, extraction; washing, recycling the Chloroform and finally the fat-soluble alkaloids was be obtained. The weak polar alkaloid can be obtained by acidification, extracting and recycling the Chloroform. The acidic water layer which was acidified and concentrated was extracted with chloroform and alkalized. Then Low-polar alkaloids was be received. After alkaline the water layer extracted with n-butanol, saturation water washed and hydration. then water-soluble alkaloids was be obtained.2. Mapping the growth curve of Lens epithelial cell. Human lens epithelial cell was cultured in DMEM serum medium with 10% FBS, Placed in 37℃,5%C02 incubator culture. General digest. The density of inoculated cell was 5×104/mL in every Costar 21 bottles of cells were inoculated, cells are counted every 24 hours, the average cells number were calculated 3 random sampling bottles every time.This will be sustained seven days. According to cells number calculating results mapped the growth curve with the number of unit cells (cells/mL) for the vertical and time as abscissa.3. The H202 and HLEC were incubated as oxidative damage model. At the same time, the different polarity alkaloid were added sustaining 24h. The proliferation of HLEC was examined with MTT and apoptosis was detected by FCM.4. The H2O2 and HLEC were incubated as oxidative damage model. Pirenoxine,Fat-soluble alkaloids and different concentrations of syringic acid were allo-cated to oxidative Damage cells after sustained 24h. The proliferation of HLEC was examined with MTT and apoptosis was detected by FCM. And compared the role of pirenoxine,Fat-soluble alkaloids and syringic acid on cell proliferation and cell cycle of oxidative Damage cells.RESULTS1. Different polar alkaloids of Dendrobium nobile was be separated. According to the experiments of single factor, A few main factors that affected the extra-cting rate were determined. Finally, the main factors which affect the extra-ction rate are as follows:concentrations of extraction solvent, extracting time, extracting times.2. The growth curve of human lensepithelial cell was "S" type.3. The proliferation of HLEC in H202 group were declined. The group Of Fat-soluble alkaloids and Water-soluble alkaloids (25ug. L-1) were significantly increased compared to H2O2-induced group. The group Of Fat-soluble alkaloids could significantly reduced the apoptosis on H202-induced. (p<0.01). piren-oxine and Fat-soluble alkaloids had a similar effect, and there was no signica-nt difference between them(P>0.05).4. The proliferation of HLEC in H202 group were declined. syringic acid (0.2mg/ml)Could promote the proliferation of HLEC which was oxidative damaged It had a similar effect to pirenoxine and Fat-soluble alkaloids, they were no significant difference (P>0.05). syringic acid (0.2mg/ml) was the most effective for anti-oxidative-damage, syringic acid(0.8mg/ml)had Cytotoxicity. syringic acid,pirenoxine and Fat-soluble alkaloids can regulate Cell cycle, and there was no significant difference between them(P>0.05).CONCLUSIONThe result shows that the best extraction method is that 80% alcohol extracting, continued for 3 hours extracting 3 times. the growth curve of human lensepithe-lial cell is "S" type。Syringic acid and Fat-soluble alkaloids Promoted cell proliferation, Fat-soluble alkaloids can inhibit apoptosis, syringic acid (0.2mg/ml) is the most effective in anti-oxidative-damage, syringic acid (0.8mg/ml)has Cytotoxicity. Syringic acid can regulate Cell cycle. pirenoxine and Fat-soluble alkaloids have the same effect. |
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