| Objective To study multi-source normalized characteristics and effects of major metabolites on ischemic stroke of breviscapine and scutellarin as the representative of scutellarin group in Erigeron breviscapus in rats,and search for the real active form of Erigeron breviscapus in vivo.Method The plasma,urine and feces of rats were collected after intragastric administration of breviscapine and scutellarein.The metabolites were identified by UPLC-qTOFMS,and then the corresponding metabolites were isolated from urine samples,and the multi-source normalized characteristics of the metabolism of scutellarin group in rats was determined by means of nuclear magnetic resonance(NMR).High performance liquid chromatography(HPLC)was used to analyze the distribution of scutellarin group in different tissues of rats,especially prototypes and metabolites in target organs such as brain and liver.Antioxidant,anti-inflammatory,anticoagulant and other experiments in vitro which have been reported,are closely related to effects of Erigeron on ischemic stroke,and amount of compound less needed,were carried out to evaluate the effects of key metabolites on the anti ischemic stroke,while contrast the effects of prototypes with metabolites.Result Scutellarein group in Erigeron breviscapus can produce common metabolites:scutellarein and hispidulin,which are the main forms of the stomach,small intestine,liver,kidney and brain.The common metabolite--scutellarein has good antioxidant capacity,the IC50 value of scavenging DPPH free radical is 0.171mM,the IC50 value of scavenging ABTS is 0.049 mM,and similar to the effect of the prototype breviscapine(the IC50 value of DPPH is 0.171 mM,the IC50 value of ABTS is 0.049 mM),and hispidulin had no significant antioxidant capacity.The IC50 value of expression of scutellarein on IL-6 is 44.25 μM,while hispidulin is 35.46μM,and similar to the effect of the prototype breviscapine(the IC50 value of expression of IL-6 is 28.90μM);the IC50 value of expression of scutellarein on TGF-β is 25.17μM,while hispidulin is 23.95μM,and similar to the effect of the prototype breviscapine(the IC50 value of expression of TGF-β is 14.59μM);the IC50 value of expression of scutellarein on NF-κB is 248.23μM,while hispidulin is 158.71μM,and the effect intensity was weaker compared with the prototype breviscapine(the IC50 value of expression of breviscapine on NF-κB is 53.45μM).The expression of scutellarein on Wnt3a signaling pathway is dose dependent(20.0μM,the strength is 5.6 times to that of prototype),while hispidulin is 9.88μM,and the effect intensity was better than that of breviscapine,the results showed that the activity of metabolites in up-regulation of Wnt3a signaling pathway was significantly higher than that of the prototype breviscapine,the above data show that the activity of expression of common metabolites on IL-6 and TGF-β activity is similar to that of the prototype breviscapine,the expression of common metabolites on NF-κB was weaker compared with the prototype breviscapine,and the activity of expression of common metabolites on neural repair related Wnt3a signaling pathway was higher than that of the prototype breviscapine.The common metabolites of scutellarein had better anticoagulant effect,scutellarein in low concentration(0.5mg/ml)can significantly extend the value of PT(12.35 ±0.31,p<0.01,compared with blank control),the effect of it is higher than that of the prototype breviscapine in same low concentration(11.55 ± 0.21,p>0.05,compared with blank control);scutellarein in low concentration(0.5mg/ml)can significantly extend the value of TT(53.60±0.56,p<0.01,compared with blank control),the effect of it is higher than that of the prototype breviscapine in same low concentration(49.30±0.36,p<0.05,compared with blank control);scutellarein in low concentration(0.5mg/ml)could not significantly increase the value of APTT(33.38±1.06,p>0.05,compared with blank control),and the prototype breviscapine in same low concentration could also not significantly increase the APTT value(32.78±0.15,p>0.05,compared with blank control);scutellarein in low concentration(0.5mg/ml)can significantly decrease the value of FIB(0.62±0.05,p<0.01,compared with blank control),the effect of it is similar to that of the prototype breviscapine in same low concentration(0.66±0.05,p<0.01,compared with blank control);scutellarein in high concentration(1.Omg/ml)have significant effect on the four indexes of coagulation(p<0.01,compared with blank control),the effect of it is similar to that of the prototype breviscapine in same high concentration(p<0.01,compared with blank control).Conclusion The compound of scutellarein group in Erigeron breviscapus has the characteristics of multicompounds producing same metabolites;breviscapus,scutellarein and hispidulin had a good anti-inflammatory effect;breviscapus and scutellarein had great antioxidant and anticoagulant effect.The active form of Erigeron breviscapus can be the prototype compounds and metabolites. |
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