Study On The Effect Components Of Buyang Huanwu Decoction On Blood Circulation And Activating Blood By Erythrocyte Membrane Chromatography

Buyang Huanwu Decoction（BYHWD）which consists of seven ingredients of astragalus,angelica,chuanxiong,radix paeoniae rubra,peach kernel,safflower and earthworm has a good overall effect at the time of clinical ischemic stroke precursors,acute phase,convalescence and sequelae.This prescription has the effect of Qi promoting,blood circulation and activating blood.The aim of this study was to screen the target components of BYHWD interacted with erythrocyte membrane.The biomembrane solid phase chromatography,which can be used for high throughput screening of the complex characteristics of traditional Chinese medicine,is used to screen the target components,and then verify the pharmacological activity of the selected components accordingly.And to consolidate the theoretical basis of"blood circulation and activating blood"pharmacodynamics level of BYHWD as well as to better guide the preparation of prescription.Objective:High-throughput erythrocyte membrane chromatography was used to screen the target components of BYHWD interacted with erythrocyte membrane,and to identify effect compounds and then explore what kids of medicinal materials they belong to.Furthermore,to verify biological activity of erythrocyte membrane of effect compounds.This paper is for the purpose of consolidating the theoretical basis of"blood circulation and activating blood"pharmacodynamics level of BYHWD as well as discovering bioactive constituents of BYHWD.Methods:1.The pretreatment methods of BYHWD were established by screening differentSPE fillers and eluting processes by erythrocyte membrane solid-phase chromatography,and the method of erythrocyte membrane solid phase chromatography was established through the combination and dissociation of effect compounds of BYHWD interacted with erythrocyte membrane suspension.2.Combined erythrocyte membrane solid phase chromatography with UPLC-MS/MS,the effect components of BYHWD interacted with erythrocyte membrane were identificated with literature database and standard control.After obtaining the molecular information,the components were compared with the molecular information of the extracts of single medicinal herbs to determine the ownership of the medicinal materials.3.The effect of active compounds of BYHWD on the content of Na⁺-K⁺-ATPase,sialic acid,sulfhydryl,superoxide dismutase,malondialdehyde and cholesterol in erythrocyte membrane was studied.Furthermore,to discusses the impact of active compounds of BYHWD interacted with erythrocyte membrane in"blood circulation and activating blood".Results:1.The sample was enriched by C18-N solid phase extraction（SPE）column through the optimization of filler types and eluting method.The eluting step was as follows:chromatograph methanol and deionized water was used to balance SPE column first,and then washed with deionized water after the samples had been loaded,stood at room temperature for 30s,eluted with 30%acetonitrile aqueous solution and purified with nitrogen.Through the investigation of the recovery of C18-N SPE cartridges,it was found that there were 13 absorption peaks with a recovery of more than 30%compared with those before the column,which can be applied to the enrichment of dissociation solution in the subsequent erythrocyte membrane solid phase chromatography set.The number of unbound ingredients for washing and the amount of the test solution is optimized.Finally 5 m L BYHWD solution for the test solution was added to 5 m L of erythrocyte membrane suspension,and the number for washing non-specific binding components were determined to be 4 times.2.13 compounds were initially identified in the dissociation solution by comparisonwith the database.Among them,the ion information of the 13 compounds is detected in the positive ion mode.The formulas were C₁₆H₁₂O₅,C₂₃H₂₈O₁₁,C₂₇H₃₀O₁₇,C22H22O10,C48H78O18,C17H16O5,C24H24O11,C23H26O10,C23H28O10,C47H78O19,C₂₅H₂₈O₁₁,C₄₃H₇₀O₁₅ and C₄₅H₇₂O₁₆.After compared with LC-MS/MS Spectrum of standars,the compounds of BYHWD interacted with erythrocyte membrane was finally determined to calycosin,albiflorin,6-hydroxykaempferol-di-O-glucoside,calycosin-7-O-β-D-glucoside and soyasaponin Bb.3.After analyzing and comparing the fragment information obtained from the effect compounds of BYHWD with single herbal medicine extracts,the compounds C₁₆H₁₂O₅,C₂₄H₂₄O₁₁,C₂₃H₂₈O₁₀ and C₂₃H₂₆O₁₀ were derived from Astragalus medicinal materials;the compounds C₂₃H₂₆O₁₀,C₂₃H₂₈O₁₁ were derived from Radix Paeoniae Rubra medicine;compound C₁₆H₁₂O₅ was derived from Chuanxiong medicine;compound C₂₇H₃₀O₁₇ was derived from Safflower herbs;compound C₂₂H₂₂O₁₀ was derived from Peach Kernels;no compounds were found in the test solution of Angelica Sinensis and Earthworm.Among them,the compounds C₂₃H₂₆O₁₀ and C₂₃H₂₈O₁₀ are both contained in Radix Astragali and Radix Paeoniae Rubra,and the compound C₁₆H₁₂O₅ is contained in Radix Astragali and Chuanxiong Rhizoma.4.Effect of active compounds on Na⁺-K⁺-ATPase activity on erythrocyte membrane:The effect of 6-hydroxykaempferol-di-O-glucoside and calycosin-7-O-β-D-glucoside on the activity of Na+-K+-ATPase on the erythrocyte membrane was slightly decreased in the concentration range of 0.1～10μg·m L^-1.Both calycosin and albiflorin had dual regulatory effects on the activity of Na⁺-K⁺-ATPase.Soyasaponin Bb showed a significant increase in the activity of Na⁺-K⁺-ATPase in the concentration range of0.1～10μg·m L^-1.Effect of active compounds on the sialic acid（SA）content of erythrocyte membrane:In the concentration range of 0.1～10μg·m L^-1,the constituents of calycosin,albiflorin,6-hydroxykaempferol-di-O-glucoside and calycosin-7-O-β-D-glucoside could increase the content of SA to some extent.As for the albiflorin,group of 0.1 and1.0μg·m L^-1 had a significant effect on the content of SA compared with the group of10.0μg·m L^-1.soyasaponin Bb showed an inverse trend with the increase of drug concentration.Effect of active compounds on the sulfydryl content of erythrocyte membrane:In the concentration range of 0.1～10μg·m L^-1,the constituents of calycosin,paeoniflorin,6-hydroxy-kaempferol-O-glucoside,calycosin and soyasaponin Bb can significantly increase the total sulfydryl content of erythrocyte membrane.Effect of active compounds on total superoxide dismutase（T-SOD）activity of erythrocyte membrane:In the concentration range of 0.1～10μg·m L^-1,both calycosin and calycosin-7-O-β-D-glucoside significantly increased T-SOD activity of erythrocyte membrane.Albiflorin significantly increased the activity of T-SOD only at the concentration of 1.0μg·m L^-1.6-hydroxykaempferol-di-O-glucoside significantly increased the activity of T-SOD in the concentration range of 0.5～10.0μg·m L^-1.As the concentration of soyasaponin Bb increased,the activity of T-SOD in erythrocyte membrane increased accordingly.Effect of active compounds on Malondialdehyde（MDA）content of erythrocyte membrane:There was no difference in the content of MDA of erythrocyte membrane at the concentration of 0.1,0.5 and 10.0μg·m L^-1.The effect of 6-hydroxykaempferol-di-O-glucoside on the MDA content of erythrocyte membrane in the concentration range of 0.1～1.0μg·m L^-1 was not obtained.Calycosin-7-O-β-D-glucoside in the concentration range of 0.1～10.0μg·m L^-1 showed a decrease of MDA content in rat erythrocyte membrane,the results were no significant difference compared with the control group.No results were found in each group of Albiflorin and soyasaponin Bb.Effect of active compounds on the total cholesterol（T-CHO）content in erythrocyte membrane:In the concentration range of 0.1～10μg·m L^-1,the constituents of calycosin,6-hydroxykaempferol-di-O-glucoside and soyasaponin Bb showed a decrease of T-CHO content of erythrocyte membrane,and both calycosin and soyasaponin Bb showed a significant decrease.The results of albiflorin and calycosin-7-O-β-D-glucoside at various concentrations showed no significant difference compared with the control group.Conclusion:By using the method of combining erythrocyte membrane solid-phase chromatography and high-sensitive liquid chromatography-mass spectrometry to qualitatively analyze the effect compounds of BYHWD interacted with erythrocyte membrane,a total of 13 compounds were found to be bound to the erythrocyte membrane.Among them,the constituents of calycosin,albiflorin,6-hydroxykaempferol-di-O-glucoside,calycosin-7-O-β-D-glucoside and soyasaponin Bb were identified through comparison to standard preparation.And furthermore,the impacts of effect compounds on biological indicators of erythrocyte membrane were explored.The results indicated five kinds of effect components except soyasaponin Bb play a role in different aspects in“blood circulation and activating blood”.It revealed that material basis of the function of“blood circulation and activating blood”in BYHWD may be calycosin,albiflorin,6-hydroxykaempferol-di-O-glucoside and calycosin-7-O-β-D-glucoside.