Vasodilatory Effects And Underlying Mechanisms Of The Active Ingredients From Gastrodia

Epidemiologically, ischemic cerebrovascular diseases (ICVD) oftencause insufficient blood flow to part or all of the brain with high mortality andmorbidity. The principle of treatment is to restore the blood and oxygen supply, tosuppress inflammation and to maintain the integrity of the structure and function ofthe nervous. The early recovery of blood and oxygen can reduce damages of braininuduced by ICVD, and the vasodilatation generated by endothelium-derivedvasodilators provide considerable contribute to the restoration of blood and oxygensuply. Our preliminary study shows that Gastrodia extracts can protect cerebralischemia-reperfusion injury in rats. Meanwhile, the ethyl acetate extracts of Gastrodiacontain soluble phenolic compounds and significantly relax the rat aortic rings. Theunderlying mechanisms involves the inhibition of the extracellular calcium influx.The finding indicates that the vasodilatory effect induced by Gastrodia may playimportant role in reducing cerebral ischemic injuries. On the basis of our preliminarywork, the present study investigate the endothelium-dependent and-independentvasodilatations of isolated rat aortic rings induced by the active ingredients fromGastrodia. Meanwhile, the mechanisms involved were examined.The study was divided into the following five parts:1. Extraction and Separation of GastrodiaObjective: To extract and separate different polarity parts of GastrodiaMethod: Using reflux with ethanol, rotary evaporator to dry Gastrodia (500g),getting the total extract, Separatory funnel was used to extract ethyl acetate extractsand aqueous extracts.2. Selecting vasodilation of the different polar of Gastrodia fractions in rat aorticrings in vitroObjective: To filter out the active site of Gastrodia has vasodilatory, and lay thefoundation for further study. Method:Using the isolated perfused vascular surgery, to observe the vasodilatory effects on endothelial-intact and vascular endothelial-removal of different concentrations of Gastrodia ethanol extract, ethyl extrac, aqueous extract under the pre-contracted situation of phenylephrine (PE,10μM) and potassium chloride (Kcl,60mM).Results:(1) In endothelium-intact and-denuded aortic rings the ethanol extract of Gastrodia showed concentration-dependent vasodilatory effects.(2) In endothelium-intact and denuded aortic rings in different concentrations of ethyl acetate extract had concentration-dependent relaxation effect by PE and KC1pre-contracted, under PE pre-contraction, when the concentration at0.75,1.5,3mg/ml, group endothelium-intact relaxation rate is slightly stronger than the endothelium-denuded group, but the difference between the two groups was not statistical significantly (P>0.05); when the concentration increased to6,9mg/ml endothelium intact group is stronger than the endothelium relaxation rate of the group, the difference between the two groups was statistical significantly (P<0.05).(3) Under PE pre-contraction, the diastolic rate of intact endothelium ring of maximum concentration (9mg/ml) aqueous extract was20%-30%, there is no need to do the next step separation and screen.(4) Ethyl acetate extracts were the main active site of vasodilatation, using silica gel column chromatography to further extrication and separation, obtaining five more phenolic compounds:Ⅱ,Ⅲ,Ⅳ,Ⅵ,Ⅷ.Conclusion:The ethyl acetate extract of Gastrodia elata were the main active site of vasodilatory ring3. Research of the relation of vascular endothelial activity screening and phenolic constituents of Gastrodia diastolicObjective:To filter out the vasodilator activity ingredients from Gastrodia phenolic compounds, and clear the role of the endothelium relaxation correlation.Methods:Using the isolated perfused vascular surgery, in PE pre-contraction, the effects of different concentrations of Ⅱ, Ⅲ, Ⅳ,Ⅵ,Ⅷ endothelial integrity and relaxation of vascular endothelial remove rings, relaxing effect screened more than50%of the active ingredient, dose-response curves plotted to calculate IC50, and vasodilation observed correlation with vascular endothelium.Results:(1)The relaxant effect of Ⅳ,Ⅷ,Ⅵ,Ⅲ,Ⅱ(0.3、0.03、0.003mg/ml) on vascular endothelium intact and denuded rings weakened Successively. Compared with each other, there has significant function, when the concentration was0.3mg/ml, Ⅵ, Ⅷ, Ⅳ, Ⅲ, Ⅱ; which Ⅱ, Ⅲ,Ⅳ, Ⅷ have endothelium-dependent relaxation effect (P<0.05);Ⅵ has endothelium-independent relaxation effect (P>0.05).(2) the IC50of the active ingredients relaxation rates Over50%of are:Ⅲ IC50=1.5mM,Ⅳ IC50=0.61mM,Ⅵ IC50=0.46mM,Ⅷ IC50=2.91μM.Conclusion:Ⅲ, Ⅳ,Ⅵ,Ⅷ isolated from the phenolic compounds Gastrodia ethyl acetate extracts as the main active ingredient on Gastrodia vasodilation rings, among which Ⅲ,Ⅳ,Ⅷ were endothelium-dependent relaxation, Ⅵ was endothelium-independent relaxation.4. Gastrodia vasodilator active ingredient Ⅲ,Ⅳ, Ⅷ endothelium-dependent relaxation mechanismObjective:To study the endothelium-dependent relaxation mechanism of active ingredient of Gastrodia vasodilation Ⅲ,Ⅳ,ⅧMethods:(1) Using the isolated perfused vascular surgery, add L-NAME (nitric oxide synthase inhibitor,100μM), MB (soluble guanylyl cyclase inhibitor,10μM), Indo after (cyclooxygenase inhibitor,10μM) were incubated in PE pre-contraction, adding Ⅲ,Ⅳ, Ⅷ cumulatively, observe relaxant effect on vascular ring.(2) Ⅲ, Ⅳ, Ⅷ of endothelial NO release was observed by cultured endothelial cells①Cell counting method was used to describe endothelial cell growth curve, the2×104cells/ml were seeded in18flasks, counting after24h,36h,48h,60h,72h,84h,96h,108h respectively; Using MTT method to observe the impact of Ⅲ, Ⅳ, Ⅷ on normal endothelial cells.②Determine the release of nitric oxide (NO) in endothelial and use ELIAS to detective the activity of nitric oxide synthase (eNOS) in endothelial.Results:(1)Ⅷ, Ⅳ, Ⅲ after the incubation Of three blockers Indo, MB, L-NAME, the relaxant effect on vascular rings decreased significantly, compared with normal group, there was statistical significantly (P<0.01).(2) The effect of Ⅷ, Ⅳ, Ⅲ on normal rat aortic ECs of NO Ⅷ, Ⅳ, Ⅲ(1,3,10μM) to promote the release of NO in normal ECs (P<0.01); the activity of eNOS tended to increase, but has no significant effects (P>0.05).Conclusion:The endothelium-dependent relaxation mechanism of Gastrodia vasodilator active ingredient Ⅲ,Ⅳ,Ⅷ by NO-sGC-cGMP pathway, mediated by cyclooxygenase pathway, and had relationship with promoting the release of NO on endothelial cells.5. Research the endothelium-independent relaxation mechanism of Gastrodia vasodilator active ingredient ⅥObjective:To study the vasodilation endothelium-independent relaxation mechanism of Gastrodia active ingredients ⅥMethods:(1) The endothelium-removal rings were incubated with Calcium-free solution, to observe the contraction effects of different concentrations Ⅵ induced by PE (10-5M), then give CaCl2(30mM) on stable state, observe the external calcium inflowing and calcium releasing effect of compound Ⅵ.(2) The endothelium-removal rings were incubated with Calcium-free solution, after incubating heparin (Heparin, inositol triphosphate receptor blocker), caffeine (CF, RYR agonist), phorbolester (PD, protein kinase C receptor agonist), observe the relaxant effect of different concentrations Ⅵ on vascular ring.Results:(1) Different concentrations of Ⅵ did not block the flow of calcium in outer (P>0.05); Different concentrations of Ⅵ could block the calcium releasing (P<0.01).(2) Different concentrations of Ⅵ have strong relaxant effects pretreated by Heparin, PD (P<0.01);Different concentrations of Ⅵ have no significant relaxant effects on vascular rings pretreated by CF obvious (P>0.05)Conclusion:The Gastrodia vasodilator relaxation mechanism of active ingredient Ⅵ had relationship with blocking calcium releasing mediated by inositol triphosphate receptors and protein kinase receptor...