The Study On Purification Technology And Anti-Myocardial Hypoxia Of Effective Substances In Shengmaiyin

Coronary Atherosclerotic Heart Disease（CAHD）refering to one of heart diseases is caused by myocardial ischemia and hypoxia or necrosis.This is caused by vascular cavity stenosis or occlusion because of coronary atherosclerosis as well as the change of Coronary artery function.CAHD,also known as ischemic heart disease,is called Coronary Heart Disease（CHD）for short.The pathogenesis of CHD is heart pulse impassability which caused by Blood stasis,Turbid phlegm and Qi stagnation that are the result of viscera dysfunction for a long time considered by Traditional Chinese Medicine.Asthenia of the viscera is the principal of CHD and Blood stasis,Turbid phlegm and Qi stagnation are the subordinate.“Deficiency in origin and excess in superficiality”.Therefore,tonifying Qi,nourishing Yin,promoting blood circulation and removing blood stasis are the keys to CHD’s treatment.In clinic,Fufang danshen which takes temporary solution by promoting the circulation of Qi,eliminating blood stasis and relieving pain and Shengmai which effects a permanent cure by tonifying Qi,nourishing yin and promoting the secretion of saliva or body fluid are usually combined to reach the point that tackling both the root causes as well as the manifestations.As a part of“The Study on Combined prescription Preparation of Fufang danshen and Shengmai”,this topic established the method of determinating effective substances in Shengmaiyin and optimized purification technological parameters for the effective substances in Shengmaiyin with macroporous resin and studied the anti-myocardial hypoxia of the effective substances in Shengmaiyin enriched by macroporous resin.So this topic lays a foundation for further preparation modernization research.Part one Establishing the method of determinating effective substances in Shengmaiyin.Objective:To establish the method of determinating effective substances in Shengmaiyin.Methods:（1）The colorimetric method was used to determinate the contents of Ginsenosides in Shengmaiyin.（2）The colorimetric method was used to determinate the contents of total saponins of Ophiopogon in Shengmaiyin.（3）The Rp-HPLC method was used to determinate the contents of Schizandrin,Deoxyschizandrin and Schizandrin in Shengmaiyin.Results:（1）With ginsenoside Re as the reference substance,5%vanillin-glacial acetic acid method was adopted to determine the contents of Ginsenosides in Shengmaiyin.The detection wavelength was 545nm.The linearity range was0.0752⁰.2006mg（r=0.9993）.The average recovery rate（n=6）was 100.64%and the RSD was 2.88%.The sample was stable after colorated for 30 minutes and the RSD was 2.90%.（2）After subtracting the concents of Ginsenosides from the concents of total saponins in Shengmaiyin,the contents of total saponins of Ophiopogon in Shengmaiyin which metered by Ruscogenin was calculated.The concents of total saponins in Shengmaiyin was determined by perchloric acid colorimetric method.The detection wavelength was 386nm.The linearity range was 0.1881⁰.8151mg（r=0.9996）.The average recovery rate（n=6）was 101.33%and the RSD was 2.01%.The sample was stable after colorated for 30 minutes and the RSD was 0.50%.The formula for calculating the contents of total saponins in Ophiopogon was C=（A-1.82B）/4.16.In the formula,A refers to the contents of total saponins in Shengmaiyin determined by perchloric acid colorimetric method with ginsenoside Re as the reference substance,B refers to the contents of Ginsenosides in Shengmaiyin determined by vanillin-glacial acetic acid method,C refers to the contents of total saponins in Ophiopogon in Shengmaiyin metered by Ruscogenin.（3）The chromatographic condition for Schizandrin,Deoxyschizandrin and Schizandrin were as follows.The chromatographic column was Discovery C₁₈（25cm×4.6cm,5μm）.Methanlo-water（70:30）was used as the mobile phase and the flow rate was 1.0mL·min^-1,the temperature was 30℃.The analytes were detected at250nm.The injection volume was 10μL.The linearity range for Schizandrin,Deoxyschizandrin and Schizandrin were 0.266³.724μg（r=1.0000）,0.120¹.680μg（r=1.0000）and 0.218³.052μg（r=1.0000）for separately.The average recovery rates were 105.10%,96.25%,96.79%and the RSD were0.424%,1.42%,1.12%for separately.Conclusion:This experiment established colorimetric method for analyzing the contents of Ginsenosides and total saponins of Ophiopogon in Shengmaiyin and Rp-HPLC method for analyzing the contents of Schizandrin,Deoxyschizandrin and Schizandrin in Shengmaiyin.By methodological investigation,these methods were sensitive,specific,accurate and reliable.Part two The macroporous resin purification process of effective substances from ShengmaiyinObjective:To optimize purification technological parameters for the effective substances in Shengmaiyin with macroporous resin.Methods:（1）The best type of resin was optimized by investigating adsorption capacity and desorption rate of active constituents in Shengmaiyin with different macroporous resins.（2）Purification technological parameters for the active constituents was optimized by investigating the max adsorption quantity,the concentration and adsorption velocity of sample,the concentration of eluant,the dosage of eluent and so on.Results:（1）We have optimized D101 resin for further experiment of dynamic adsorption and elution effect.（2）The max adsorption quantity for D101 resin was 1:9.3.The concentration of sample was 0.1g·mL^-1 metered by medicinal materials.The adsorption velocity of sample was 4BV·h^-1.The concentration of eluant was 95%ethanol and the dosage of eluent was 8.5BV.The transfer rate for Ginsenosides,total saponins in Ophiopogon,Schizandrin,Deoxyschizandrin and Schizandrin were 86.53%,81.01%,77.96%,65.52%and 45.37%for separately.The contents of each active ingredient in extract enriched by macroporous resin were 30.06%,7.43%,6.22%,0.93%and1.65%for separately.Conclusion:（1）D101 resin optimized in this experiment had the high adsorption capacity and desorption rate of active constituents in Shengmaiyin.This experiment had solved the purification problem of each active ingredient used only one resin.Each active ingredient had a high transfer rate.The method had simple operation steps.（2）This kind of resin was common used and can meet the needs of production.Part three Anti-myocardial hypoxia study of the extract from Shengmaiyin enriched by macroporous resinObjective:To study on anti-myocardial hypoxia effects of the extract from Shengmaiyin enriched by macroporous resinMethods:Using the myocardial ischemia model of mice caused by isoprenaline to study anti-myocardial hypoxia effects of the extract from Shengmaiyin enriched by macroporous resin.Results:The extract from Shengmaiyin enriched by macroporous resin could prolong the survival time of mice obviously.The protection rates of medium and high dosage groups were 40.06%and 73.02%.Conclusion:This experiment studied the pharmacodynamics of the extract from Shengmaiyin enriched by macroporous resin.The results showed that the extract from Shengmaiyin enriched by macroporous resin could prolong the survival time of mice obviously and it had the effect of anti-myocardial hypoxia distinctly.