Study On Extraction And Purification Technology Of Ilex Pubescens Hook. Et Arn.

The traditional Chinese medicine,Ilex Pubescens,dry roots of Ilex pubescens Hook.et Arn.,has the function of dissipating heat and detoxifying,activating the blood and invigorating the pulse,detumescence,and is a kind of drug for treating on analgesic coronary heart disease,angina pectoris vasculitis.According to other literatures,the major components of Ilex Pubescens include triterpenoids,phenl propanoids,Iridoids,quinones and Phytosterol,etc.The pharmacological study of Ilex Pubescens is focus on Ilexonin-A which is the sodium salts of pentacyclic triterpenoid estered by succinic acid.The study on preparation technology mostly adopt the methods of water extracting-alcohol precipitating or acid sediment and alkaline dissolving after water extracting-alcohol precipitating,but the evaluation on traditional technology and new technology of Ilex Pubescens have not been reported.In this study,in order to improve the drug loading and clinical efficacy,the HPLC fingerprint analytical method was established and was applicated to quality and process control;Macroporous adsorption resin technology was introduced to the Extraction and Purification Technology,which reserved the chemicals represented by the characteristic peak of HPLC chromatography,and reduced the yield of solids,at the same time,overcame the disadvantages of the loss of chemicals in the traditional technology process in order to reach the aim of enrichment and purification.The main results are shown as following: 1,Establishment of the analytical method of HPLC fingerprint chromatographThe analytical method of RP-HPLC fingerprint chromatography for Ilex Pubescens was established.The chromatography condition was determined by choosing the best condition from different detect wave length,mobile phase,elution gradient,column temperature and flow rate.The methodology was investigated by the precision of apparatus,stability of samples and reproducibility of analytical method,and similarity of HPLC fingerprint chromatography on 11 batches Ilex Pubescens crude drugs saled in market was evaluated.2,Study on Wateror alcohol Extracting TechnologyThe absorbance combined with retention rate of characteristic peak in HPLC fingerprint were used as indexes,the extraction solvent and extract times were investigated by single factor experiment.The absorbance was used as index, the extraction process was studied by orthogonal experiment of three factors at four levels.The retention rate of of characteristic peak in HPLC fingerprint was used as index,the study on selected technology compared with traditional water extracting technology was carried out.The results show that the retention rate of the alcohol extraction technology is 1.6 time than that of water extraction technology taken retention rate of characteristic peak in HPLC fingerprint as index,the optimized alcohol extraction process was as follow:the concentration of ethanol 70%,ratio of material to liquid 1:6, heating reflux extraction time 1h,2 times.3,Evaluation on the traditional technology by the retention rate of characteristic peak in HPLC fingerprint chromatographyTaken retention rate of characteristic peak in HPLC fingerprint as index, the proess of acid sediment and alkaline dissolving after water extracting-alcohol precipitating was investigated according the preparation technology about Ilex Pubescens injection in ministry standard of Chinese Herbs;the influence of alcohol concentration on the retention rate of characteristic peak in HPLC fingerprint in the water extracting-alcohol precipitating was investigated.The results were shown as follow:the influence of acid sediment on the retention rate of characteristic peak representatives as the weak polar constituent and medium polar constituent was greater,the chemical represented by peak 7 was completely lost,but alkaline dissolving has no influence on the retention rate of characteristic peak.The alcohol precipitating was beneficial to the reducing the yield of solids,and the optimized concentration is 70~80%.4,Study on the macroporous adsorption resin purification technology for Ilex PubescensThe absorbance combined with retention rate of characteristic peak in HPLC fingerprint were used as indexes,dynamics absorption and elution process of AB-8 macroporous resin were investigated,and the saturation,elution solvent and amount of solvent were determined.The results showed that the saturation of Ilex Pubescens is 480mg(crude drug)/g(dry resin),in this saturation,the characteristic peak of elution has no obvious leakage.The elution solvent was determined as 4 times column volume of 70%alcohol.The resultof verification showed that the HPLC fingerprint chromatograph between sample solution and alcohol elution solutin was basically identical,the chromatography information pre-and post purification was the same through the TLC chromatography.The yield of solids decreased from 14%to 8%.5,Preliminary pharmacodynamic test of macroporous adsorption resin purification for Ilex Pubescens The test on anti-platelet aggregation and hypoxia-resistance were investigated,the results showed that the macroporous adsorption resin purification for Ilex Pubescens had the activities of anti-platelet aggregation and hypoxia-resistance.6,Study on the concentration and drying technology of Ilex Pubescens extractionAccording to production practice of concentration,drying technology in actual preparation process,the influence of heating temperature and time on the stability of the chemicals represented by characteristic peak in the HPLC chromatography of Ilex Pubescenswas studied.The result was shown as follow: The influences of heating temperature and time on the chemicals represented by peak 5(28 min),peak 6(30min)and peak 7(47min)were significant,especially heating temperature was 100℃and heating time was 6h,the average retention rate of them was only 3%.The result showed the lower temperature and shorter time of heating was beneficial to the retention for the chemicals in the process of Ilex Pubescens preparation.So,the concentration,drying technology of Ilex Pubescens should adopt low temperature and short time process,such as vacuum concentration,vacuum drying etc.