| Huangzhi Yishen Capsule is composed of Astragalus membranaceus,leech,medlar,yam,coix seed,Scrophularia,Glehnia,Eclipta,Achyranthes bidentata,plantain seed,Ziheche,eucommia,Panax notoginseng,Leonurus,cicada slough and other 15 ingredients.It is used for mild and moderate chronic primary common nephritis with deficiency of both qi and yin or with blood stasis and dampness.The improvement of quality standard of Huangzhi Yishen Capsule was studied,including inspection,microscopic identification,TLC identification,content determination and fingerprint.According to the pharmaceutical process provided by the manufacturer,the content of preparation method in the standard is supplemented.The microscopic identification method of Huangzhi Yishen Capsule was revised,and the microscopic characteristics in the original standard were identified as Panax notoginseng,leech and Ziheche.The TLC identification method of Huangzhi Yishen Capsule was revised,and the TLC qualitative identification of Achyranthes bidentata and Plantago asiatica in the preparation was added.The control drug and reference substance were used to carry out the control test,and the extraction method and development conditions of TLC identification of Lycium barbarum in the original standard were optimized.Through the methodological study,the newly established TLC identification method has good specificity and durability,and the corresponding characteristics of medicinal materials can be detected in 16 batches of samples.The general inspection items such as volume difference,water content and extract of the preparation were investigated,and the results were in line with the provisions of general principles in part Ⅳ of 2015 edition of Chinese Pharmacopoeia.Referring to the limit of heavy metals and harmful elements under Astragalus membranaceus in 2015 edition of Chinese Pharmacopoeia,lead,arsenic,cadmium,copper and mercury in Huangzhi Yishen Capsule were detected by inductively coupled plasma mass spectrometry(ICP-MS).Results all the elements in 16 batches of samples were far lower than the standard requirements.HPLC-UV method was established for the simultaneous determination of four flavonoids in Huangzhi Yishen Capsule.C18 column(250 mm×4.6 mm,5 μm)was used with acetonitrile(A):water(B)as mobile phase,gradient elution(0-8 min,16%;8-32 min,16%-28%;32-50 min,28-55%A);flow rate was 1 mL·min-1;detection wavelength was 248 nm.The number of theoretical plates should not be less than 10000 according to the glucoside peak of pistil isoflavone.The average recovery of sample addition is 90.89%-100.36%,rsdl.16-1.67%(n=6).The average contents of four flavonoids in 16 batches of samples were 0.128 mg·g-1,0.0604 mg·g-1,0.174 mg·g-1,0.0673 mg·g-1.The total contents ranged from 0.297mg·g-1-0.525 mg·g-1,and the average content was 0.428 mg·g-1.HPLC-ELSD method was established for the determination of astragaloside in huangzhiyishen capsule.C18 column(250 mm,5μm);acetonitrile water(33:67,v/v)as mobile phase,elution with equal degree;flow rate of 1 mL.min-1;theoretical plate number calculation shall not be less than 10000.The results of the methodology showed that the linear relationship was good,the specificity,precision,stability,repeatability and durability were all good;the average recovery rate of sample addition was 97.84%(RSD=1.08%),and the content of astragaloside in 16 samples was 0.718-1.284 mg·g-1,and the average content was 1.062 mg·g-1.UPLC-MS/MS method was established for the determination of progesterone in Huangzhi Yishen capsules by.Acquity UPLC HSS T3(100 mm×2.1 mm,1.8 μm)column was used.Acetonitrile:0.1%formic acid water(65:35,V/V)was used as mobile phase.The flow rate was 0.3 mL.min-1.The results showed that the linear relationship was good,the specificity,precision,stability and repeatability were good;the average recovery was 103.81%(RSD=2.84%);the content of progesterone in 16 batches ranged from 5.223 to 15.162 ng·g-1,the average content was 9.418 ng·g-1.HPLC-UV method was used to establish the fingerprint of Huangzhi Yishen Capsule.Combined with chemical pattern recognition technology,the fingerprint data were analyzed by cluster analysis(HCA),principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA).The chromatographic column was waters XBridge Shield RP18(250 mm×4.6 mm,5 μm),with acetonitrile-0.05%phosphoric acid aqueous solution as mobile phase,gradient elution,flow rate of 1.0 mL·min-1,column temperature of 30℃,detection wavelength of 210 nm.A total of 24 common peaks were identified in 16 batches of samples,and the similarity evaluation was greater than 0.9.The common peaks were qualitatively analyzed by HPLC-Q-TOF-MS/MS,and they were classified into seven drugs.The samples were divided into three categories by HCA and PCA identification methods.The PLS-DA screening included 2’-hydroxy-3’,4’-dimethoxy-isoflavan-glucoside,Ginsenoside Rg1,calycosin-glucopyranoside,7,2’-dihydroxy-3’,4’-dimethoxy-isoflavan,pinoresinol diglucoside,calycosin,formononetin and chlorogenic acid. |
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