Study On The Preparation Process Of Compound Partridge Oral Liquid And Its Molecular Mechanism Against Ischemic Nerve Injury

Object:Ischemic nerve injury has a high incidence,serious can cause death and seriously threaten human health.While the ideal clinical treatment and medication are still lack in the clinical,it is significant to study relevant innovative drugs.Zhegu prescription,the traditional national medicine of Li ethnic,consists of four herbs of Li ethnic.It has the functions of strengthening brain and promoting intelligence and detoxifying heat,which has a long history of application in Li ethnic regions of Hainan province.Previous studies have shown that Zhegu prescription has a neuroprotective effect and promotes the proliferation of neural stem cells(NSCs)after brain injury,and has a good application prospects,but relevant studies are not sufficient.In this study,the traditional small compound prescription of Li ethnic was developed into oral liquid with convenient and controllable quality by modern technology,and its pharmacological function was clarified.On the one hand,the topic is helpful to the inheritance and development of traditional ethnic medicine,on the other hand,it is helpful to improve the economic value and social value of traditional ethnic medicines.The aim of this study is to prepare the traditional national medicine Zhegu prescription into compound Zhegu oral liquid,solve the clarification problem of oral liquid,and establish the quality control standard of compound partridge oral liquid.the pharmacological action and molecular mechanism of compound Zhegu oral solution in the treatment of ischemic nerve injury were studied systematically in vivo and in vitro.Method:1.Study on preparation technology of compound Zhegu oral solution:according to the physical and chemical properties of its effective components and the traditional usage,with the purified water to the partridge leaves and other 3 herbs,examine with purified water volume,decocting time and decocting number's influence on the content of active ingredients,according to the enterprise the cost of production and active ingredient content to determine the best extraction process.2.In order to solve the problem of precipitation caused by long-term oral liquid storage,according to the main reason for the precipitation of chitosan as oral liquid clarifying agent,with gallic acid content in the oral liquid,sedimentation ratio and absorbance as evaluation index,dosage of chitosan,solution pH,flocculation temperature,mixing time on the single factor investigation;According to the results of single-factor experiment,choose the solution pH value(factor A),flocculation temperature(factor B)and chitosan dosage(factor C)as the inspection factors,the content of gallic acid,sedimentation ratio and absorbance were used as evaluation indicators to conduct L9(34)orthogonal experiment to comprehensively select the best clarification process conditions for compound Zhegu oral solution.3.In the study of quality standards,using high-performance liquid chromatography(HPLC)to determinate the content of ellagic acid,gallic acid and baicalin,the main components in compound Zhegu oral solution,and completed the precision experiments,stability experiments,repetitive experiments,sample recovery experiments,etc.establish the quality standard of the compound Zhegu oral solution.4.Pharmacological study of compound Zhegu oral solution against ischemic nerve injury.Healthy male 6-week-old KM mice were randomly divided into sham-operated group,model group,positive drug group,high-,medium-,and low-dose groups of compound Zhegu oral solution.Cerebral ischemic injury model was established by bilateral common carotid artery occlusion(BCCAO)in mice.After 30 min ischemia,bilateral common carotid arteries were recirculated to produce ischemia-reperfusion injury.The positive drug group was given chitosan-treated ginsenoside Rgl aqueous solution(0.03 g·kg-1·d-1),the high,middle,and low dose groups received compound Zhegu oral solution(5.10,1.70,and 0.57 g·kg-1·d-1),Sham-operated group and model group given the purified water(1 mL/0.1 kg).The neurological function scores were performed on the 4th,7th,and 10th day after administration,respectively.Then the brains of each group of mice were subjected to paraffin embedding and sectioning,using HE staining to observe the pathological conditions of the brain.Immunohistochemical technique was used to observe the expression of nestin in mouse cerebral cortex and SGZ region NSCs.5.To study the effect of compound Zhegu oral solution on the proliferation of isolated NSCs and the protein content of key nodes in Wnt pathway.NSCs were used for creating Oxygen-glucose deprivation/reperfusion(OGD/R)model,and the biological characteristics of NSCs were identified by immunocytochemistry.Cells were divided into control group,model group,positive drug group,and high-,medium-,and low-dose groups of compound Zhegu oral solution.The cells in the control group were always cultured in the culture medium treated with chitosan,the model group was given chitosan-treated normal medium solution,and the positive group was given Chitosan-treated medium solution containing ginsenoside Rgl(0.01 mg/mL),the high-,medium-,and low-dose groups were administered with medium solution containing compound Zhegu oral solution,cells in each group were cultured for 48 h.CCK-8 assay was used to observe the cell viability.The ability of compound Zhegu oral solution to promote the proliferation of NSCs was compared.The protein of each group was extracted 48 h after administration,and Western Blot analyzing the protein content of ?-catenin and CyclinDl in Wnt signaling pathway to investigate the molecular mechanism of compound Zhegu oral solution in promoting the proliferation of NSCs.Results:1.In the study of the extraction process of compound Zhegu oral solution,measured the content of gallic acid,ellagic acid(expressed by the peak area ratio)as evaluation index by HPLC technique.Examining purified water volume,decocting time,and decocting number,among the three factors,purified water volume has significant influence on the results.The decocting number has little influence.Comprehensively considering the cost,determining the optimal extraction process for compound Zhegu oral solution was 10 times purified water,decocting for 90 min,and decocting only once.2.In the study of clarification process of compound Zhegu oral solution,the single factor investigation results showed that the gallic acid content is higher and the precipitation is larger when the amount of chitosan was between 1%and 3%(v/v),the pH was between 3?5,and the flocculation temperature ranged from 60 ? to 80 ?,While the stirring time has little influence,and the stirring was selected for 15 min.L9(34)orthogonal experimental results show that the optimal clarification process conditions for compound Zhegu oral solution is A2B2C2.That is,the amount of chitosan is 2%(v/v),the pH is 4 and the temperature is 70 ?.The amount of chitosan had significant difference in the content of gallic acid in the oral solution(P<0.05).3.In the study on the quality standards of compound Zhegu oral solution,the results showed that the content of ellagic acid was 5.14?5.29 ?g/mL,the acid content was 6.57?6.68 ?g/mL and the baicalin content was 0.91?0.97 ?g/mL.The recoveries of ellagic acid,gallic acid and baicalin were 98.61%(RSD=0.95),100.09%(RSD=1.27),and 98.88%(RSD=0.89),respectively.The results show that this method can be used to control the quality of compound Zhegu oral solution.4.Pharmacological study of compound Zhegu oral solution against ischemic nerve injury.The results showed that each group of compound Zhegu oral solution administration can improve the neurological dysfunction in BCCAO mice,and there is a significant difference compared with the model group(P<0.05).HE staining showed that compound Zhegu oral solution could significantly improve the loss of neurons in the cerebral cortex and SGZ region caused by BCCAO.On the 10 th day after administration,compared with model group,the expression of Nestin protein in the cortex and SGZ area of compound Zhegu oral solution administration group increased obviously.The results showed that compound Zhegu oral solution had anti-ischemic nerve injury pharmacological effects.5.To study the effect of compound Zhegu oral solution on the proliferation of isolated NSCs and the protein content of key nodes in Wnt pathway.NSCs were cultured in vitro and administered 48 hours after OGD/R.The results of CCK-8 assay showed that compared with the model group,there was a significant difference in cell viability of compound Zhegu oral solution group(P<0.05),indicating that compound Zhegu oral solution has the effect of promoting proliferation of NSCs.Western blot experimental results show that compared with the model group,the content of ?-catenin and CyclinD1 protein in the drug-administered group is significantly increased(P<0.05).It was shown that compound Zhegu oral solution promotes the proliferation of NSCs and it's related to the activation of Wnt signaling pathway.Conclusion:This study has perfected the extraction process and clarification process of compound Zhegu oral solution,and established the quality control standard of compound Zhegu oral solution,and measured the content of ellagic acid,gallic acid and baicalin in the oral liquid.In this experiment,the protective effect of compound Zhegu oral solution on ischemic nerve injury was found at the overall level and cellular level,and this effect was related to the regulation of Wnt signaling pathway and promotion of endogenous NSCs proliferation.On the one hand,the study provides experimental data for the establishment of the extraction process,clarification process,and quality control standards for compound Zhegu oral solution.On the other hand,it provides experimental basis for the study of the molecular mechanism of compound Zhegu oral solution against ischemic nerve injury,and provedes data for the subsequent development and research.