Study On Preparation Of Xuebitong Capsule And Its Therapeutic Mechanism On Thromboangiitis Obliterans

BackgroundThromboangiitis Obliterans,also known as Buerger’s disease.It is a common peripheral vascular disease.The causes of Thromboangiitis Obliterans are complex and varied,including cold and wet living conditions,smoking,infections,autoimmune disorders and genetics.A variety of factors lead to endothelial dysfunction which induces intravascular thrombosis and vasculitis.As a result,it eventually leads to Thromboangiitis Obliterans disease.The main clinical manifestations of Thromboangiitis Obliterans are cold and blue limbs,zombie,clonus and pain,claudication in severe cases and even gangrene.Currently,there are two types of treatment for Thromboangiitis Obliterans,such as surgical and conservative methods.Because surgical treatment causes great pain to patients,and surgical treatment is not ideal for the recovery and prognosis of the disease,the clinical drug therapy is usually the main treatment.The research shows that Xuebitong has the functions of dispelling wind and dredging collaterals,breaking blood and removing blood stasis,stopping wind and stopping spasm.Xuebitong has some therapeutic effect on thromboangiitis Obliterans,but its mechanism is not clear.PurposesThis study will optimize the preparation of Xuebitong capsule,explore the therapeutic effect of Xuebitong capsule on Thromboangiitis Obliterans in animal model,and further clarify the mechanism of Xuebitong capsule.MethodsIn this paper,the 9 kinds of effective components in traditional Chinese medicines,such as Radix astragali,Salviae miltiorrhizae bge,Radix paeoniae rubra,Centipede,Pberetima,Eupolyphaga sinensis,Caulis trachelospermi,Exocarpium citri reticulatae and Uncaria rhynchophylla,were identified by Column chromatography,Separation chromatography,thin layer chromatography and High-performance liquid chromatography chromatography.At the same time,the best extraction process was determined by orthogonal test.The best preparation technology of Xuebitong capsule was determined by the study of granulating excipients,the ratio of extract powder and excipients,the determination of bulk density,quality,moisture detection and disintegration time.Apoptosis was induced by H₂O₂,and the effect of Rat Serum Containing Xuebitong on cell survival rate was detected by colorimetric method.At the same time,the concentrations of ROS,LDH,GSH,SOD,MDA and NO were measured.The expression of eIF2α,IRE1α,cleaved caspase-3 was detected by Western Blot.To establish a rat Thromboangiitis obliterans model,the effects of Xuebitong on platelet adhesion,platelet aggregation and thrombus formation in vitro were examined.The effects of Xuebitong on serum levels of IL-6,CRP,ET-1,TXB₂ and6-Keto-PGF1αin rats were determined by Elisa.Results80%ethanol was extracted twice for 1 h each time.This is the best extraction process of Xuebitong.The ratio of excipients to medicinal materials is 4:6,which can be used to prepare Xuebitong capsule in accordance with the requirements of 2015board Chinese PHARMACOPOEIA.Compared with H₂O₂ Injury Control Group:（1）Xuebitong Group had protective effect on HUVEC.（2）Xuebitong group can effectively reduce Ros concentration in HUVEC cells.（3）In HUVEC cells,rat serum containing xuebitong could inhibit the apoptosis induced by H₂O₂ and decrease the gene expression of cleaved caspase-3,PARP,IRE-1αand eIF-2α.（4）The GSH concentration in HUVEC was maintained effectively in Xuebitong group.（5）In Xuebitong Group,the LDH concentration in HUVEC cells was decreased effectively.（6）Xuebitong group can effectively reduce the concentration of MDA in HUVEC cells.（7）Xuebitong group could effectively maintain NO concentration in HUVEC cells.（8）Xuebitong group can effectively maintain SOD concentration in HUVEC.Compared with the Model Group:（1）the pathological grade of rats in the high and low dose groups of Xuebitong was lower;（2）the maximum platelet aggregation time was significantly decreased in the high and low dose groups of Xuebitong;（3）the high and low dose groups of Xuebitong significantly decreased the platelet adhesion rate;（4）the high and low dose groups of Xuebitong significantly inhibited the formation of thrombus in vitro;（5）the content of TXB₂ and the ratio of TXB₂ to6-Keto-PGF_1αin serum of rats in high and low dose groups of Xuebitong could be reduced,and the content of 6-Keto-PGF_1αin serum of rats could be increased;（6）The expressions of IL-6,CRP and ET-1 in serum were significantly decreased in the high and low dose groups of Xuebitong.InnovativenessAfter examination,the Chinese Medicine Prescription Xuebitong,which is obtained by optimizing the extraction method,contains all the effective components in the prescription.Part of the active ingredient content was increased.The quality of Xuebitong capsule meets the requirements of capsule drugs in Pharmacopoeia 2015edition.Xuebitong,a traditional Chinese medicine prescription,can effectively inhibit platelet aggregation,adhesion and thrombosis.Xuebitong has thrombolytic and anti-oxidation effects,and can regulate the active substances in blood vessels.Xuebitong has protective effect on Endothelium,which may be related to inhibition of apoptosis.