| Objective:Lichong Shengsui Yin is a kind of complex Chinese patent medicine,composed of eight herbs epimedium,astragalus,ginseng,pilose antler,leech,zedoary,rhizoma sparganii,fritillaria thunbergii.The medicine has a significant effect on the treatment of ovarian cancer.Based on the previous research work of LCSS Y work team,our research identified flavonoids,saponins,polysaccharides,proteins were effective ingredients,established a analysis method for the Lichong Shengsui Yin determination of multicomponent,got the best technics parameters by using multiple indexes orthogonal test,determinated the content of representative effective ingredients.Method:Supercritical fluid extraction technology combined with alcohol extraction method and water extraction method were applied in this research to prepare Lichong Shengsui Yin.①Firstly,the content of flavonoids,saponins,polysaccharides,proteins were measured by spectrophotometry.The content of flavonoids in ethanol extract was determined by AlCl3 color reaction method at 415nm.The content of saponins in ethanol extract was determined by the vanillin acetic acid perchloric acid colorimetric method at 544nm.The content of polysaccharides in water extract was determined by the phenol sulfuric acid method at 489nm.The content of proteins in water extract was determined by the coomassie brilliant blue G-250 method at 595nm.②Then,the HPLC method was used to analyse the flavonoids,saponins,and the conditions of chromatography was optimized.The chromatographic conditions are as follows:Dikma Diamonsil-C18 column(250mm×4.6mm,5μm)with the temperature of 30℃,acetonitrile-water containing 0.1%phosphoric acid as mobile phase with the flow rate of 1mL/min.The sample size was 20μL,the flavonoids’wavelength of detection was 270nm,the saponins’ wavelength of detection was 203nm.The reference solutions of calycosin glycoside,icariin,baohuoside I,ginsenoside Rg1,ginsenoside Re,ginsenoside Rbl,ginsenoside Rd and astragaloside were used to draw standard curves.Then,the method’s linear relation,precision,repeatability,recovery rate and stability were examined.③Finally,combined with content determination analytical method and orthogonal experiment,we got the optimum technology of alcohol extraction method and water extraction method.The average content of flavonoids,calycosin glycoside,icariin,baohuoside I,saponins,ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1,ginsenoside Rd and astragaloside in Lichong Shengsui Yin were used as evaluating indicator,the ethanol concentration,extraction duration,extraction temperature,menstruum dosage were used as necessary factors,we got the optimum extraction conditions of alcohol extraction method.The average content of polysaccharide and water soluble protein in Lichong Shengsui Yin were used as evaluating indicator,the amount of water,soaking time,extraction duration,extraction times were used as necessary factors,we got the optimum extraction conditions of water extraction method.Conclusion:①The linearity range of flavonoids was 0.01～0.1 g·L-1,the average recovery rate was 100.5%,the RSD was 1.61%.The linearity range of saponins was 0.02～0.15g·L-1,the average recovery rate was 99.82%,the RSD was 1.15%.The linearity range of polysaccharide was 0.01～0.1 g·L-1,the average recovery rate was 99.35%,the RSD was 1.41%.The linearity range of water soluble protein was 0.1～1.0g·L-1,the average recovery rate was100.2%,the RSD was 1.89%.②The linearity range of calycosin glycoside,icariin and baohuoside I were 0.1～0.2 g·L-1,0.1～1.0 g·L-1 and 0.01～0.1g·L-1 respectively,the average recovery rate were 100.02%,99.64%and 99.59%respectively,the RSD were 1.83%,1.61%and 1.58%respectively.The linearity range of ginsenoside Rgl,ginsenoside Re,ginsenoside Rb1,ginsenoside Rd and astragaloside were 0.05～0.2,0.1～1,0.1～1,0.1～1 and 0.1～1g·L-1 respectively,the average recovery rate were 99.15%,99.48%,99.69%,99.26%and 100.01%respectively,the RSD were1.63%,1.45%,1.38%,1.22%and 1.85%respectively.③The optimum extraction conditions of alcohol extraction method was 1.5 hours reflux extraction at 80℃ after soaking 2 hours with 10 times of 60%alcohol and extracting twice.The average content of flavonoids,calycosin glycoside,icariin,baohuoside I,saponins,ginsenoside Rgl,ginsenoside Re,ginsenoside Rb1,ginsenoside Rd and astragaloside in Lichong Shengsui Yin were 2.29%,0.091%,0.26%,0.025%,1.68%,0.081%,0.16%,0.22%,0.22%and 0.12%.The optimum extraction conditions of water extraction method was 2 hours reflux extraction at 100℃ after soaking 1 hour with 10 times of water and extracting three times.The average content of polysaccharide and water soluble protein in Lichong Shengsui Yin were 8.53%and 0.35%.Conclusion:①The content of flavonoids,saponins,polysaccharides and proteins were measured by spectrophotometry,the results showed that precision,stability and recovery rate were all satisfactory.②The content of calycosin glycoside,icariin,baohuoside I,ginsenoside Rg1,ginsenoside Re,ginsenoside Rbl,ginsenoside Rd and astragaloside were measured by spectrophotometry,the results showed that precision,repeatability,recovery rate and stability were all satisfactory.③The results showed that the extraction rate and feasibility of the optimized alcohol extraction method and water extraction method were all satisfactory.In this paper,we provided a theoretical basis for the standard techniques selection and quality control of Lichong Shengsui Yin,could be used to ensure the clinical curative effect. |
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