Anti-thrombus Function And Mechanism Of Phenolic Compounds Extracted From Gastrodia Elata Blume

Cerebral infarction is due to regional cerebral blood disorder resulting in acuteischemic brain tissue, cerebral hypoxia and then brain necrosis, encephalomalacia.Thrombosis is one of the important causes of cerebral infarction. Our previousresearches show, ethyl acetate extracts from Gastrodia elata Blume. have significantcerebral protective effects on cerebral ischemic injury animal model, also have stronganti-thrombus function and anti-paltelet aggregation. Prompt that ethyl acetateextracts from Gastrodia elata Blume. possible through anti-thrombus to achieve thepurpose of the prevention of cerebral infarction occurred. In subsequent studies foundthat cerebral protective effects′material basis of Gastrodia are a group of phenoliccompounds from ethyl acetate extracts, among which, different compounds mightthrough anti-oxidant damage or protecting the blood-brain barrier to protect brain,prompt that cerebral protective effects of Gastrodia may be the combined effects ofmultiple chemical compounds which work on different aspects of cerebral ischemicinjury. In order to further ascertain the characteristics of cerebral protectionmechanism of those ingredients, combine with previous studies, phenolic compoundsB, C, D extracted from Gastrodia ethyl acetate extracts which have much srongeranti-platelet aggregation in vitro as representative are used to study their effect andmechanism from the perspective of anti-thrombotic in this research.1.The Research of Anti-thrombus Function of Phenolic Compounds B, C, DExtracted from Gastrodia elata Blume.Objective: To study the anti-thrombus activity of Phenolic Compounds B, C, Dextracted from Gastrodia elata Blume. Methods: We used inferior vena cava ligation method to cause venousthrombosis and the injection of thrombosis inducers via intracarotid artery method, inorder to investigate the anti-thrombus function of Gastrodia phenolic compound B, C,D.Results: Phenolic compounds B, C, D(20mg/kg﹑10mg/kg) all could inhibitthe venous thrombosis and reduce the wet weight of thrombus significantly. Thosethree compounds also could inhibit cerebral thrombosis significantly.Conclusions: Phenolic compounds B, C, D could inhibit venous thrombosis andcerebral thrombosis in rats significantly.2. The Research of Anti-thrombus Mechanism of Phenolic Compounds B, C, DExtracted from Gastrodia elata Blume.Objective: To investigate the anti-thrombus mechanism of phenolic compoundsB, C, D from the perspective of anti-platelet aggregationMethods:(1) According to Bron′s, ADP, AA and PAF were used as inducers to study theanti-platelet aggregation function of compounds B, C, D extracted from Gastrodiaelata Blume in vitro.(2) The concentrations of intracellular calcium([Ca2+]i) of platelet was measuredby fluorescent probe to explore whether the phenolic compounds from Gastrodia elataBlume could inhibit the platelet cell intracellular calcium releasing and the plateletcell extracellular calcium flowing into.(3) The activity of cyclic AMP was tested by Elisa Assay kits. The contents ofcAMP was calculated to evaluate the effect of compounds B, C, D.(4) To investigate whether phenolic ingredients C, D have the inhibition of theexpression of P2Y12，We used Western blot to test the effect of phenolic ingredients onexpression of P2Y12.Results:(1) Phenolic compounds have selective antagonism on platelet aggregationthrough three ways of ADP, AA, PAF, through vitro screening method showing:phenolic compound B had the strongest anti-platelet aggregation effect against way of AA, for ADP, only at high concentration had a significant anti-platelet aggregation function; Phenoliccompound C and D had the strongest anti-platelet aggregation effect against way of ADP;Those three compounds had no significant anti-platelet aggregation function induced by PAF atexperimental concentrations.(2) Phenolic compound B had a significant function in inhibiting of the rabbitplatelet cell intracellular calcium releasing and extracellular calcium entry induced byAA(P<0.01; P<0.05). Phenolic compound C had a significant effect in inhibiting ofthe rabbit platelet cell intracellular calcium releasing and extracellular calcium entryinduced by ADP(P<0.01; P<0.05); At experimental concentrations, phenoliccompound D had no significant function in inhibiting of the rabbit platelet cellintracellular calcium releasing and extracellular calcium flowing into induced byADP(P>0.05).(3) Phenolic compound B(0.4mg/mL、0.1mg/mL)、compound C(0.4mg/mL、0.1mg/mL)、compound D(0.4mg/mL、0.1mg/mL) all could increase contents ofcAMP which came from platelet rich plasma (P<0.01；P<0.05).(4) Phenolic compound C(20mg/kg、10mg/kg)、compound D(20mg/kg、10mg/kg) both could inhibit the expression of receptor-P2Y12significantly(P<0.05); Atexperimental concentrations, phenolic compound B had no significant function ininhibiting the expression of receptor-P2Y12(P>0.05).Conclusions:(1) Phenolic compound B could inhibit experimental thrombus significantly, itsfunction was mainly by inhibiting the rabbit platelet cell intracellular calciumreleasing and extracellular calcium entry and increasing platelet cAMP levels againstplatelet aggregation to prevent thrombosis.(2) Phenolic compound C could inhibit experimental thrombus significantly, itsfunction was mainly by inhibiting the rabbit platelet cell intracellular calciumreleasing and extracellular calcium entry and inhibiting the expression ofreceptor-P2Y12and increasing platelet cAMP levels against platelet aggregation toprevent thrombosis.(3) Phenolic compound D could inhibit experimental thrombus significantly, its function was mainly by inhibiting the expression of receptor-P2Y12and increasingplatelet cAMP levels against platelet aggregation to prevent thrombosis.3. The Research of Acute toxicity of Phenolic Compounds B, C,D Extracted fromGastrodia elata Blume.Objective: In order to find out the situation of three ingredients′acute toxicity,the acute toxicity of phenolic compounds B, C, D from Gastrodia by oraladministration in mice were investigatedMethods: We observed the acute toxicity of mice after phenolic compoundsextracted from Gastrodia elata Blume being given through the mouth to evaluate thesafety of phenolic ingredient B, C, D preliminarily.Results: The lethal dose50(LD50) phenolic compound B given through themouth was3.45g/kg; The LD50of phenolic compound C was1.34g/kg; The LD50ofphenolic compound D was1.23g/kg.Conclusions: Phenolic compound B, C, D all had acute toxicity at differentdegrees.