| ObjectiveA fingerprint and Content Determination methods were established for the quality control of Buyang Huanwu Decoction.On the basis of separation of high purity brain microvascular endothelial cells,Establishment of brain microvascular endothelial cell membrane for Screening of efficacy ingredients of "Promote angiogenesis" in Buyang Huanwu Decoction on the basis of high purity brain microvascular endothelial cells were isolated and cultured,then research on the structural identification,pharmacological validation and mechanism of ingredients binding to the cell membrane.Methods1.Establishment of fingerprint and content determination method of buyang huanwu decoctionThe method of reverse phase high performance liquid chromatography(RP-HPLC)was used to establish the fingerprint method of Buyang Huanwu Decoction.At the same time,the contents of calycosin-7-O-?-D-glucoside,ferulic acid,formononin-7-O-?-D-glucoside and hydroxy safflower yellow A in the samples of Buyang Huanwu Decoction were measured for the quality control of the sample of buyang huanwu decoction.2.Isolation and identification of primary brain microvascular endothelial cellsIn this paper,the primary brain microvascular endothelial cells were isolated from the brain tissue of 10-day-old rats refer to the method reported,and the purity of the cells was examined by cell moiphology and imnunofluorescence staining.3.Establishment of brain microvascular endothelial cell membrane chromatographyThe samples were pretreated by solid phase extraction,and the effects of different packing SPE columns and different elution programs on the recovery of common peaks in the samples of Buyang Huanwu Decoction were investigated.Futher,the most appropriate concentration of administration and non-specific binding component washing times were inspected to establish the brain microvascular endothelial cell lembrane chromatography.The components of the dissociation solution obtained by cell membrane solid phase chromatography were identified by LC-MS.The MS information obtained was matched with the literature to determine the binding composition of compounds.The protective effect of six binding components on brain microvascular endothelial cells induced by glucose deprivation and reperfusion model was verified by CCK-8.4.Effects of active ingredients on the expression of VEGF and bFGF in Oxygen glucose deprivation reperfusion model cells.The effects of the five active ingredients on the expression of VEGF and bFGF in the model of glucose deprivation and reperfusion were determined by ELISA.The mechanism of the active constituents on angiogenesis was explored.Results1.Fingerprint of Buyang Huanwu Decoction injection and Content determinationThe method of fingerprinting has good specificity,precision and stability.The similarity degree is used to evaluate the similarity of the 10 batches of injection.The similarity results show that the similarity of the 10 batches of samples was higher than 0.9,which can be used for the quality control of the injection sample.The results showed that the content of each components in the 10 batches of injection was as follows:the content of calycosin-7-O-?-D--glucoside was 90.180?114.799?g/mL,the content of ferulic acid was 25.637?42.973?g/mL,the content of formononin-7-O-?-D-glucoside was 12.193?19.845?g/mL,and the content of hydroxy safflower yellow A was 132.097?181.284?g/mL.2.Isolation and identification of primary brain microvascular endothelial cellsThe isolated primary brain microvascular endothelial cells were observed presenting a long spindle shapedunder inverted microscope,and the cells were fused with "vortex" and typical "pavement" characteristics of endothelial cells.The purity was 95%by immunofluorescence staining.3.Brain microvascular endothelial cell membrane chromatographyThe final experiment was carried out to select the STRATA-X type solid-phase column,and the elution method was:the ultra-pure water was used to activate the SPE column after equilibrated by methanol,the 50%acetonitrile solution was used to elute the adsorbed component after ultra-pure water washed.The method established is simple and has a high recovery rate for each common peak,which can be used for the purification and enrichment of washing solution and cell dissociation solution obtained in cell membrane chromatography.The results showed that the concentration of the optimal concentration of administration was 55mg/mL and the number of times of washing was 5.The contents of the six components were determined by method.established.Further analysis of the results of mass spectrometly showed that the six binding components were 6-hydroxy kaempferol-3,6-di-O-glucoside,paeoniflorin,calycosin-7-O-?-D-glucoside,gallonylpaeoniflorin,7-2,-hydroxy-3',4'-dimethoxy-isoflavan and formononin-7-O-?-D-glucoside.Pharmacological validation showed that,the cell viability of the model group was significantly lower than the normal group(P<0.01),suggesting that the glucose-deprivation and reperfusion caused significant damage to the brain microvascular endothelial cells.Namely,the OGD/R model was successfully established;the concentration of calycosin-7-O-?-D-glucoside in the range of 1-20?g/mL,can significantly enhance the cell activity of glucose-deprived reperfusion model cells(P<0.01);the concentration of paeoniflorin in the range of 1-40 ?g/mL,can significantly enhance the cell activity of glucose-deprived reperfusion model cells(P<0.01);the concentration of fomononetin-7-O-?-D-glucoside in the range of 0.1-20?g/mL,can significantly enhance the cell activity of glucose-deprived reperfusion model cells(P<0.01);The concentration of galloylpaeoniflorin in the range of 1-20 ?g/mL,can significantly enhance the cell activity of glucose-deprived reperfusion model cells(P<0.05,P<0.01);The concentration of(3R)-7,2'-hydroxy-3',4'-dimethoxy-isoflavan in the range of 0.5-40?g/mL,can significantly enhance the cell activity of glucose-deprived reperfiision model cells(P<0.05 or P<0.01).As for 6-hydroxy kaempferol-3,6-di-O-glucoside,it not only had no protective effect on glucose-deprivation and reperfiision injury,but also showed a significant inhibitory effect on the cell activity at overdose concentrations(20,40?g/mL)4.Effects of active ingredients on the expression of VEGF and bFGF in OGD/R model cellsCompared with the normal group,the VEGF level in the model group was significantly up-regulated(P<0.01);VEGF was significantly increased after treated with calycosin-7-O-?-D-glucoside,paeoniflorin,formononetin-7-O-?-D-glucoside and galloylpaeoniflorin at different concentration setted(P<0.01 or P<0.05).As for 7,2'-hydroxy-3',4'-dimethoxy-isoflavan,the expression of VEGF in the low and high concentration group were not significantly higher than the model group.But the expression of VEGF was significantly increased in high concentration group compared with the model group(P<0.05).Compared with the model grroup,the expression of bFGF was significantly increased after treated by calycosin-7-O-?-D-glucoside,paeoniflorin,formononetin-7-O-?-D-glucoside and galloylpaeoniflorin at different concentration setted(P<0.01 or P<0.05).As for 7,2'-hydroxy-3',4'-dimethoxy-isoflavan,the expression of bFGF in the low concentration group were not significantly higher than the model group.But the expression of bFGF was significantly increased in the medium and high concentration group compared with the model group(P<0.05).ConclusionThe purity of the primary brain microvascular endothelium was higher and could be used for the cell membrane chromatography.The microvascular endothelial cell membrane chromatography established was simple and accurate.Chromatography screening out the six binding components.Futher,paeoniflorin,calycosin-7-O-?-D-glucoside,gallonylpaeoniflorin,7-2'-hydroxy-3',4'-dimethoxy-isoflavan and formononin-7-O-?-D-glucoside were confirmed to have the effect on the protection of oxygen glucose deprivation reperfusion injury brain microvascular endothelial cells in the appropriate concentration.All of the five active ingredients can increase the level of VEGF and bFGF in different degrees,suggesting that increasing the expression of VEGF and bFGF were important mechanisms of promoting angiogenesis for the five active components. |
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