Comparative Studies On The Interaction Between Flavonoids And Phosphodiesterase 4 And 5-lipoxygenase

Phosphodiesterase 4(PDE 4) and 5- lipoxygenase(5-LOX) are important targets, which are closely related with human inflammatory autoimmune disease. In vitro enzyme inhibition assay and ultrafiltration liquid chromatography coupled to electrospray ionization tandem mass spectrometry(ultrafiltration LC-ESI-MSn) were combined to screen both PDE 4 and 5-lipoxygenase(5-LOX) inhibitors from six flavonois i.e. sec-O-glucosylhamaudol, astilbin, rutin, isoquercitrin, astragalin and quercetin, respectively.In vitro PDE 4 inhibition assay, cyclic AMP(c AMP) was used as substrate; phosphodiesterase 4 optimum reaction condition was optimized by changing the reaction time, reaction temperature, p H buffer solution and substrate concentration, respectively. The reaction time was then set at 50 min, reaction temperature was 33℃, p H buffer solution was 7.4 and the substrate concentration is 80 μM, respectively. The value of michaelis constant(Km) and maximum reaction rate(Vm) were achieved at 9.78 μM and 2.44 μM·S-1 under this condition. Astilbin showed the highest PDE4 activity with an IC50 of 22.89 μM, followed by sec-O-glucosylhamaudol, rutin, isoquercitrin, astragalin with IC50 s of 42.04 μM, 43.30 μM, 75.30 μM, 93.00 μM, respectively. Astilbin had the highest binding affinity to PDE-4, its enhancement factor is 29.05, followed by sec-O-glucosylhamaudol, rutin, isoquercitrin, astragalin with enhancement factor were 27.59, 20.57, 15.64 and 8.52, respectively. However, quercetin didn’t show inhibitory activity. The results of ultrafiltration LC-MSn were consistent with the in vitro inhibition results.In vitro 5-lipoxygenase inhibition assays and ultrafiltration LC-MSn, arachidonic acid were used as substrate, six flavonois was studies in 5-lipoxygenase inhibition, the result show that sec-O-Glucosylhamaudol is the best inhabitor, the value of IC50 is 7.45 μM, fllowed by astilbin, rutin, astragalin, quercetin and isoquercitrin, the value of IC50 is 8.45 μM, 44.18 μM, 94.17 μM, 172.49 μM, 468.45 μM, respectively; the result of ultrafiltration LC-MSn show that the sec-O-Glucosylhamaudol is the best one to combine 5-lipoxygenase, the combination rate is 65.63, followed by astilbin, rutin, astragalin, quercetin and isoquercitrin, the value is 49.24, 14.23, 13.44, 8.90 and 5.20, respectively; The results of ultrafiltration LC-MSn were consistent with the in vitro inhibition results.Since PDE 4 and 5-LOX have different mechanism action, same compound may show different inhibitory effect. However, astilbin and sec-O-glucosylhamaudol showed stronger inhibitory enzyme effect in both PDE 4 and 5-LOX system, which are considered as the double enzyme inhibitors. Evaluation of anti-inflammatory activity of cells was then developed for further research on the anti-inflammatory mechanism of astilbin and sec-O-glucosylhamaudol. The results indicate that astilbin and sec-O-alucosylhamaudol have very good protection effect on damage RAW 264.7 cells induced by lipopolysaccharide. Both of them have anti-inflammatory immune effect by reducing TNF-α, IL- 6 gene expression level in RAW 264.7 cells.