The Dynamic Changes And Extraction-purification Technology Of Ilexsaponin A1 From Ilex Pubescens Leaves

Objective:The traditional southern Chinese medicine,Ilex pubescens,is commonly used to dissipating heat,promoting blood circulation,cooling the blood and reducing swelling.It's effective to treat coronary heart disease,cerebral thrombosis and thrombus vasculitis.According to the report,the root had been studied in detail,while the leaves of Ilex pubescens reported less in the field of chemical composition and pharmacology.The traditional medicinal part is wild Ilex pubescens root,but this plant is slow-growing,while the leaf is rich in resources and renewable.With the increasing use of clinical application,the shortage of Ilex pubescens root resource is getting serious.Research findings indicate the roots and the aerial parts of Ilex pubescens are similar in chemical composition and pharmacological activity,such as increasing coronary artery flux of the rat left-body heart to against myocardial ischemia,so the leaves of Ilex pubescens have potential value in the treatment of diseases.In order to utilize the leaves of Ilex pubescens rationally,our study investigated the variation of Ilexsaponin A1 in different months to determine the optimal harvesting time,and we studied the extraction and refinement process of total saponins to provide references for the exploition of relevant drugs.Methods:1.The dynamic changes of ilexsaponin A1 content in different months.To establish a suitable HPLC method for determination of Ilexsaponin A1,and collected the leaves of Ilex pubescens in different months,the content of ilexsaponin A1 was determined to summarize the dynamic changes.2.The extraction process study of Ilexsaponin A1 from Ilex,pubescens leaves.With the retention rate of Ilexsaponin A1 as the indicator,we determined the extraction process and optimized technology parameters,and then we investigated the thermal stability of concentrated solution.3.The refining process study of total saponins from Ilex pubescens leaves.To establish a method for determination of total saponins,and optimized suitable technology parameters of refinement.We carried out verification experiments for refining process,and investigated the influence of different drying methods on the indicator of refined liquor.4.The study of total saponins ' pilot production.On the basis of test in laboratory technology,we carried out pilot production and reserved sample pilot plant.Next,we determined the content of Ilexsaponin A1 and total saponins to verify the feasibility of process parameters in pilot scale production,Results:1.The content of Ilexsaponin A1 had regional difference and seasonal difference.It is low in January to March and increase gradually in April,and kept higher levels from June to August,and reduced gradually in September,while the content in Guangzhou had second peak in December.Considering the content and output of leaves,the optimal harvest time is June to August.2.The optimized extraction process was adding 9,6 times amount of 60%ethanol into materials and refluxing for 2 times,each time kept for half hour.The centrifugal process is:Concentrated the ethanol from extraction of leaves until containing a small amount of ethanol(ethanol content is between 10%?20%),and then added water to dilute concentrated solution(0.1g/ml),centrifuged the solution(1000rpm*5min),poured out the supernatant and centrifuged again with a little water,at last we combined the supernatant.The results of the stability experiment indicate the concentrate after heated under 60??80??100?for five hours,the retention rate of Ilexsaponin A1 was 99.5%,96.5%,87.1%;the retention of total saponins was 90.4%,83.9%,75.7%.The content of indicators reduced shaply under 100?,so the concentrated temperature should be under 80?.3.The refining process study of total saponins was poured out the supernatant on AB-8 macroporous adsorption resin column,with the adsorption capacity of total saponins is 335.48mg/g dry resin.Next,we washed resin column with water,and eluted with 4 times elution volume 30%,70%ethanol respectively and collected the 70%ethanol eluent.The verification results showed that the retention rate of total saponins and ilexsaponin A were 52.6%,98.5%.The content of indicators in solids were 95.1%,38.1%.The result showed that the refining process was effective to enrich total saponins.The result of drying progress showed that the retention rate of decompression drying and spray drying was higher than 85%,95%,while drying under normal pressure loss more content,so the optimized drying method was decompression drying or spray drying.4.The result of pilot test showed that the retention rate of total saponins in extraction process was 87.2%,84.1%in concentration process,47.6%in refining process and 41.2%in drying process;The retention rate of Ilexsaponin A1 in extraction process was 72.2%,70.6%in concentration process,67.7%in refining process and 65.0%in drying process.The content of total saponins and Ilexsaponin A1 in solids were 89.6%,38.0%.The result conformed to the needs of follow-up research.Conclusion:This study investigated the dynamic changes of Ilexsaponin A1 content in different months to determine the optimal harvest time,and we optimized extraction and refinement process parameters for total saponins of Ilex pubescens leaves,we also optimized the drying methods of refining liquor.Then we verified experiments by conducting pilot plant test.At last we got total saponins with high amounts of Ilexsaponin A1.This study provides reference for the development of Ilex pubescens leaves resource.