| Objective:Semen aesculi is the dry mature seed of Aesculus chinensis Bge.,Aesculus chinensis Bge.var.chekiangensis(Hu et Fang)Fang or Aesculus wilsonii Rehd,which can cure stomach pain and dredge the liver.It is often used in the treatment of stagnation qi in the liver and stomach,epigastric pain,chest fullness and so on.Aescin is the main active ingredient of extracts from semen aesculi,having pharmacological effects on anti-inflammatory swelling,antioxidation,reducing the nerve injury and antitumor function.The chemical composition in semen aesculi is complex and this experiment was conducted on 10 batches of semen aesculi materials from Shanxi,Henan,Zhejiang,Hubei and Guangxi different regions in order to better control its quality.We established the fingerprint and carried out LC-MS analysis,providing a reference and basis for the quality control of the semen aesculi materials.In addition,the method of organic reagent was mostly used in the extraction and separation of aescin at present,which is in the stage of laboratory exploration.In this study,the macroporous resin process was used to enrich and purify for the main effective components of aescin parts(β-aescin,namely,escin Ia and escin Ib),to obtain high content(more than 50%)of saponins intermediate,better for the life production services.Methods:1 In this experiment,HPLC-PDA technique was used to research on the fingerprint of semen aesculi extract of 70%ethanol concentration.Firstly,the single factor investigation was conducted on the column temperature,flow phase,wavelength,extraction solvent concentration factor and so on.On this basis,the experimental conditions of the fingerprint were determined and the fingerprint of semen aesculi is established.According to the software of“Chinese medicine chromatographic fingerprint similarity evaluation system A version "published by the national pharmacopoeia committee,the standard fingerprint map of semen aesculi was generated to determine the common peak,and the relative reserve time and relative peak area were calculated as the reference peak of escin Ia.2 HPLC-MS technology was used to detect and analyze the ethanol extract of the semen aesculi materials in the condition of fingerprint chromatographic condition.By analyzing the fragmentation regularities of mass spectra and comparing with relevant literature,the chemical composition was speculated which could be used to study the chemical material basis of the semen aesculi materials.3 Taking the extraction rate of aescin as the index,considering the paste rate as well,we carried out investigation on single factors such as the extraction time(ci h.c2 h.c3h、c4h、C5h),ethanol concentration(a1%、a2%、a3%、a4%、as%),the extraction times(di、d2、d3、d4、d5),and material liquid ratio(b1 times,b2 times,b3 times,b4 times,bs times)and so on.Based on the single factor investigation,we selected appropriate factors level to do L9(34)orthogonal test and established the best extraction technology with the reference index.4 Firstly,the adsorption and desorption rate were selected as the indexes to optimize the suitable macroporous adsorption resin for aescin purified and enriched.Then the content and the transfer rate of escin la and escin Ib were investigated as index to optimize single factor of macroporous resin purified technology,studing on the sample concentration(ei g/mL、e2 g/mL.e3g/mL、e4g/mL、esg/mL),the sample flowrate(f1BV/h、f2BV/h、f3BV/h、f4BV/h、f5BV/h),alcohol washing concentration(ai%、a2%、a3%、a4%、as%),alcohol washing flow velocity(fi BV/h、f2BV/h、f3BVh、f4V/Vh、fsBV/h)and diameter ratio(g1、g2、g3)level.In the next step,L9(34)orthogonal purification process test was taked and the stable purification process was obtained.5 In this paper,the optimum extraction and purification process was used to study escin la and escin lb content from 10 batches of intermediate samples(10 batches of medicinal materials from different producing areas).Primarily determined the rationality of the intermediate produced method by this process.Results:1 There were 13 common chromatographic peaks in the 10 batches of the semen aesculi materials,which were identified as escin la,escin lb,isoescin Ia,and isoescin Ib by the standard product.The common peak relative retention time RSD was relatively small,while RSD of relative peak area was larger.In the 10 batches of semen aesculi materials,most similarity was above 0.9 and the similarity degree of the three batches of Chinese medicinal materials was larger,less than 0.9.The chemical composition of semen aesculi materials is similar in different producing areas,but there’s a difference in content.2 Through the analysis of HPLC-MS technology,the regularity of mass spectra cracking and comparison with relevant literature,15 chemical constituents were identified.3 The optimal extraction process of escin from the semen aesculi was to take the appropriate amount of the medicinal herbs,and add b2 times the amount of a4%ethanol to soak for ci h.Then through heating reflux method to extract,we extract d2 times,and the extract time was c1 h for each time.Finally,filter and combine d2 extracting solution,then get it.4 The optimal enrichment process of escin from the semen aesculi was using C macroporous resin to purify the escin parts.The sample concentration was es g/mL,the sample flow rate of fi BV/h,the ethanol elution concentration of a3%concentration ethanol,and the flow rate of ethanol elution was f2 BV/h with the ratio of resin column diameter of g2.It mainly collected a3%ethanol elution liquid from the 3/4 column volume to the front of 3/4 of the third column.5 In this paper,the optimum extraction and purification process was used to study escin la and escin Ib content from 10 batches of intermediate samples(10 batches of semen aesculi materials from different producing areas).And preliminarily the rationality of the intermediate method is determined by this process.Conclusion:The fingerprint analysis method is of good precision,stability and reproducibility.And the similarity degree of semen aesculi materials from different regions is different.The similarity degree of five batches semen aesculi materials from Hanzhong in Shanxi province are similar to those in Henan province,and the similarity degree is above 0.9.While the similarity was lower in Zhejiang,Hubei,and Guangxi provinces,indicating that the content of the chemical composition of semen aesculi is relatively correlated with the source of origin.Combined with LC-MS analysis,it can provide a theoretical reference for the quality control research of semen aesculi.The extraction and purification process of escin parts is simple and easy,which is high in content and transfer rate.It has good repeatability and stability and is suitable for industrial production. |
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