| BACKGROUNDAnweiyang Capsule,which is the five new drugs that made of total flavoniods,is the national medicine to protect species,varieties of national health insurance and the competitive products in Jiuhui Pharmaceutical Co.LTD.The drug won the "National New Product","Guangdong Province outstanding new products","Guangdong Provincial Science and Technology Progress Award","Huizhou City of Science and Technology Progress Award" title,and was the first one to get national Development and Reform Commission supports Chinese high-tech industrialization demonstration project in Guangdong Province,(project name is " An weiyang Capsule and licorice flavonoids high-tech industrialization demonstration project").In addition,this drug won "SME Technology innovation Fund." that was from national Science and Technology in 2000.Pharmacodynamic studies have shown that the product can effectively inhibit the secretion of gastric juice and pepsin,inhibit gastric free acid and total acid,lower acid concentration,repair gastric and duodenal ulcers mucosal and promote healing of ulcers,which proved that this product have the exact anti-ulcer effect on a variety of experimental ulcer models.At the same time,clinical studies have shown that total effective of Anweiyang Capsule on chronic superficial gastritis rate was 91.83%.The raw material medicine of An weiyang Capsule is total flavonoids of licorice,there exists some shortcomings such as too large oral dosage,poor solubility,low bioavailability and so on.In terms of the extraction process,using acid-alkali method to extract the total flavonoids in licorice redidues is too much complex,multi-link and unstable quality.In terms of quality standards,gravimetric analysis method is still used to determine the quality of An weiyang,this method can not reflect the overall quality of An weiyang accurately and can not be in line with international quality standards.Meanwhile,as a famous Chinese medicine,An weiyang has not been fully demonstrate the connotation of proprietary Chinese medicine,and the lack of protection of intellectual property rights.PURPOSEIn order to control inherent quality of Anweiyang Capsule accurately and comprehensively,this article use the licorice residues as raw material which have been extracted licorice acid.It is necessary to study quality control of licorice residue systematically,this study has significance for the further development and revise the quality standards.In this study,enzymic method combined with the ethanol extraction was used for the extraction,and macroporous resins were used for identification and pharmacodynamics of total flavonoids of licorice residue in An weiyang Capsule were investigated.Meanwhile,the HPLC fingerprint of total flavonoids of licorice in An weiyang Capsule from three spicies was established for the effective and reasonable exploitation and evaluation of An weiyang Capsule.METHOD1.Study on Qualification of Total Flavonoids in Anweiyang by Ultraviolet SpectrophotometryTo select the appropriate standard for determination of total flavonoids in An weiyang Capsule,this study used 5 reference substances that including liquiritin,liquiritigenin,licochalcone A,naringin and rutin to compare the largest absorption wave length by wave length scanning of ultraviolet spectrophotometry.2.Study on Quality Standard of Raw Meterial Medicine of AnweiyangOn the basis of licorice in the Chinese pharmacopoeia 2010 edition to formulate the quality standard of licorice residue which was the raw material medicine of An weiyang Capsule.In this study,the content of moisture,total ash and acid-insoluble ash were detected.Taking glycyrrhizic acid ammonium salt as control substances and taking Glycyrrhiza uralensis Fisch.,Glycyrrhiza inflate Bat as the control herbs,the medicinal materials of licorice residue is indentified by TLC.And the content of glycyrrhizic acid ammonium salt and liquiritin were determined by HPLC,the precision,stability,specificity and recovery of method were studied.At the same time,the content of total flavonoids were determined by ultraviolet spectrophotometry.3.Study on Extraction Process Optimization of AnweiyangTo optimize extracting process of total flavonoids from licorice residue in An Weiyang by enzymatic hydrolysis assisted ethanol extraction,With content of total flavonoids as index,effects of 5 factors including kinds of enzyme,enzyme concentration,pH,temperature and time on enzymatic technology and effects of 3 factors including the concentration of ethanol,extraction temperature and extraction time on ethanol extraction technology was investigated by single factor test and orthogonal test.4.Study on Separation and Purifieation Optimization of Anweiyang with Macroporous ResinTo study the purification conditions with macroporous resin in licorice residue,with licorice flavonoid extract as the research object,and the extract yield and purity of licorice flavonoids as the index.Using the static adsorotion method for screening optimum macroporons resin to be purification of total flavonoids in licorice residue and using dynamic adsorption method to study optimum macroporous resin conditions of purification of total flavonoids in licorice residue.5.Study on Fingerprint of Total Flavonoids of Licorice in AnweiyangTo set up a method for total flavonoids of licorice in Anweiyang Capsule fingerprint detection by the multi-wavelength switching technique in order to acquire more information about total flavonoids.RP-HPLC was used to establish fingerprints with C-18 column and the mobile phase was 0.1%formic acid solution(A)-acetonitrile(B)with gradient elution at a flow rate of 0.8mL·min-1,the detection wavelength:0?22min:276nm;22?27min:368nm;27?37min:250nm;37?50min:368nm;50?64min:276nm;64?74min:250nm;74?90min:360min.RESULTS1.Study on Qualification of Total Flavonoids in Anweiyang by Ultraviolet SpectrophotometryReference substance Liquiritin and samples processed by alkali liquor had the largest absorption at the 337nm wavelength,while reference substance liquiritigenin and reference substance naringin had the largest absorption at about 440nm wavelength,licochalcone A had the largest absorption at about 410nm wavelength.And reference substance lutin by Al2(NO3)3-NaOH-NaNO2 colorimerty method had the largest absorption at the 510nm wavelength while samples processed at the 363nm.And the methodological evaluation showed that reference substance lquiritin are the most suitable for the total flavonoids in Anweiyang Capsule and this method had a good repeatability and reproducibility2.Study on Quality Standard of Raw Meterial Medicine of AnweiyangCombined with "Chinese Pharmacopoeia" 2010 edition about licorice,thin layer identification,content determination and general routine inspection program were investigated.In TLC,glycyrrhizic acid ammonium salt as the reference substances,licorice and Glycyrrhiza inflate as the reference drug,using silica gel G plate of 1%sodium hydroxide solution prepared,ethyl acetate-formic acid-acetic acid-water(15:1:1:2)as the developing solvent,ethanol solution of 10%sulfuric acid as the reagent,heated at 105? to clear spot color and viewed at 365nm.The TLC indicated that three identified herbs could show the same color pot as the control herbs or contrast in the same position.Moisture,total ash,acid insoluble ash were 7.66%,7.55%and 2.17%in licorice residue respectively.Quantitative determination included the determination of total flavoniods by UV and the content determination of liquiritin and glycyrrhizic acid ammonium salt by HPLC.The determination of total flavoniods by UV was used liquiritin as reference substance,10%potassium hydroxide solution as chromogenic agent,the detective wavelength was 337nm,the content of total flavoniods was determined for five samples.The result showed the linear correlation was y=5.462x-0.0083(r=0.9997)and the average recovery was 100.74%(RSD=1.38%),the average content of total flavoniods in Licorice residue was 0.18%.This method was simple,reproducible,accurate comparing the usual method.The content determination of liquiritin and glycyrrhizic acid ammonium salt by HPLC.The sample was extracted by ultrasonic method,which the reagent was 70%ethanol,RP-HPLC was used to establish with C-18 column and the mobile phase was 0.05%phosphoric acid solution(A)-acetonitrile(B)with gradient elution at a flow rate of 0.7mL·min-1,the detection wavelength was 237nm.The result showed that the linear correlation of liquiritin and glycyrrhizic acid ammonium salt were y=14314x+31.44,(r=0.9992)and y=3892.9x+21.473,(r=1),respectively.The detection limits of liquiritin and glycyrrhizic acid ammonium salt were 0.222?1.11mg·ml/and 0.36?2.16mg·mL-1,respectively.The average recovery of liquiritin and glycyrrhizic acid ammonium salt were 100.74%(RSD=1.38%)and 99.34%(RSD=2.50%),respectively.The average content of liquiritin and glycyrrhizic acid ammonium salt in licorice residue were 0.019%and 0.57%,separately.3.Study on Extraction Process optimization of AnweiyangThe licorice residues,the raw material of Anweiyang Capsule were treated by means of cellulase before extraction by refluxing ethanol.Single factor tested to determine the impact of the extraction rate of kinds of enzyme,enzyme concentration,pH,temperature and time on enzymatic technology and effects of 3 factors including the concentration of ethanol,extraction temperature and extraction time on ethanol extraction technology.And on this basis to L9(43)orthogonal test,respectively.The result revealed that optimum conditions as follows:cellulase enzyme dosage was 1g,the amount of water was ten times,the optimum temperature was 50?,and the extraction time 90min.After enzymatic hydrolysis,optimum conditions of ethanol extraction as follows:80%ethanol concentration,the refluxing temperature was 80? with the extraction time 90min.Compared with traditional extracting technology by ultraphonic,extraction yield of total flavonoids was increased by 143.09%by using this enzyme-based extraction technology.4.Study on Separation and Purification of Total Flavonoids of Licorice in An Weiyang Capsule with Macroporous ResinTo study the purification conditions with macroporous resin in licorice residue.Using the static adsorption method for screening optimum macroporons resin to be purification of total flavonoids of licorice in An weiyang Capsule,using dynamic adsorption method to study optimum macroporous resin conditions of purification of total flavonoids of licorice in An weiyang Capsule.The results showed that the macroporous resin adsorption of AB-8 on total flavonoids of licorice in An weiyang Capsule was the best,the best purification conditions were:the height-to-diameter ratio was 1:10,the sample concentration was 2.0mg·mL-1 with pH 5.0,volume was 50mL,sample liquid rate was 1.0 mL·min-1,the best condition of desorption were by distilled water 40mL before 70%ethanol solution was used with pH 10.0,and the column volume was 100 mL with 1.0mL·min-1 rate elution.As a result.The purity of total flavonoids could reach about 36.83%and the yield was 4.98%,which means that the AB-8 macroporous adorsption resin show better comprehensive adsorption property and can be used to separate and purify the flavonoids from licorice residue.5.Studies on Fingerprint of Total Flavonoids of Licorice in AnweiyangTo set up a method for total flavonoids of licorice in An weiyang Capsule fingerprint detection by the multi-wavelength switching technique in order to acquire more information about total flavonoids of licorice in An weiyang Capsule.Under grads eluting condition,ten batches of samples from different producing area and different producing time were determined by the software of Similarity Evaluation System for Chromatographic Fingerprint of TCM(Version 2004A).The result showed that the 21 mutual characteristic peaks were detected for ten batches of samples.The result showed that the reference substance included liquiritin,liquiritigenin,isoliquiritin,isoliquiritigenin and licochalcone A were identified,while the reference substance of glycyrrhizic acid ammonium salt was not detected which meant this reference substance may not exist or had lowest content resulted in the instrument can not detect.And the similarity was between 0.796?1,which indicated the samples existed difference.By establishing the finger print on total flavonoids of licorice in Anweiyang Capsule,the genuine or fake from the medicinal material was differentiated.In a word,a new method controlling the quality of An weiyang Capsule was established.CONCLUSION1.The reference glycyrrhizin was applied to measured the contents of total flavonoids of licorice in Anweiyang by UV spectrophotometry.The methodological evaluation showed that reference substance liquiritin are the most suitable for the total flavonoids of licorice in Anweiyang and this method had a good repeatability and reproducibility.2.The quality standard of flavonoids in licorice residue was refered to "Chinese Pharmacopoeia" 2010 edition about licorice,thin layer identification,content determination and general routine inspection program were investigated.Meanwhile,on the basis of the original determination of licorice,the content of total flavonoids was added to reflect the quality of Anweiyang better.3.The licorice residue,the raw material medicine of Anweiyang,was treated by means of cellulase before extraction by refluxing ethanol.Single factor test and orthogonal test were adopted to optimize the extraction.Compared with traditional extracting technoligy by ultraphonic,extraction yield of total flavonoids was increased by 143.09%by using this enzyme-based extraction technology,which revealed that using enzyme-assisted extraction of flavonoids in licorice residue can significantly increase the extraction rate of total flavonoids,at the same time,the method is simple,feasible and low cost.4.To study the purification conditions with macroporous resin in licorice residue.Using the static adsorption method for screening optimum macroporons resin to be purification of total flavonoids in licorice residue,using dynamic adsorption method to study optimum macroporous resin conditions of purification of total flavonoids in licorice residue.The results showed that the AB-8 macroporous adorsption resin show better comprehensive adsorption property and can be used to separate and purify the flavonoids from licorice residue.5.To set up a method for total flavonoids of licorice in Anweiyang fingerprint detection by the multi-wavelength switching technique in order to acquire more information about total flavonoids of licorice in An weiyang Capsule.By establishing the finger print on flavonoids of licorice residue,the genuine or fake from the medicinal material was differentiated.In a word,a new method controlling the quality of Anweiyang was established. |
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