Protective Effect Of Chemical Constituents In Fengliao Changweikang On Intestinal Epithelial Model

ObjectiveIn order to illuminate pharmacodynamic activity material basis of Fengliao Changweikang and explore its pharmacological activity and potential practical value,along with revealing its clinical mechanism of action and promoting its development and application,our studies focus on the protective effect of total alkaloids,total flavonoids and rutin from Fengliao Changweikang on intestinal epithelium tight junction established with Caco-2 Cells.MethodsTo extract the original medicine materials by water reflux method and to purify water extraction preliminarily by alcohol sedimentation;and then separation and purification of total alkaloids and total flavonoids were accomplished by solvent extraction method and macroporous resin column chromatography,respectively.MTT was exploited to evaluate the cytotoxicity of total alkaloids,total flavonoids and rutin to Caco-2 and IC50 values were obtained.And then intestinal epithelium tight junction was established with Caco-2 cells,the validation of which was confirmed by transepithelial electrical resistance(TEER)and the apparent permeability coefficient(Papp)of marker compound fluorescein.Next,intestinal epithelium tight junction was disrupted by interferon-?(IFN-?)and tumor necrosis factor-?(TNF-?),i.e.100U/mL IFN-? was added till 19h firstly and then lOng/mL TNF-? was added till 48h.Finally,the protective effect of total alkaloids,total flavonoids and rutin on the disruption of tight junction was investigated.In a similar way,the integrity of tight junction was evaluated by TEER and Papp of fluorescein.ResultsMTT trials showed that the IC50 of total alkaloids to Caco-2 was 550?g/mL;total flavonoids and rutin showed no cytotocxicity to Caco-2,but instead,promoted the proliferation.After being delt with TNF-a and INF-y,TEER of Caco-2 monolayer dropped significantly and Papp increased remarkably.After being delt with total alkaloids 24,48h,though TEER dropped and Papp increased as well,compared with TNF-a and INF-? group,total alkaloids alleviated the decline of TEER and the raise of Papp(P<0.05).However,after being delt with total flavonoids and rutin,compared with TNF-? and INF-? group,TEER and Papp of total flavonoids group and rutin group had no significant amelioration(P>0.05).ConclusionTotal alkaloids of Fengliao Changweikang possess some certain protective effect on disrupted tight junction by TNF-a and INF-y;total flavonoids and rutin showed no significant protective effect.The clinical mechnism of Fengliao Changweikang may be the protective effect of total alkaloids on intestinal epithelium tight junction.