| Objective:Baqi Lingmao formula was composed of 10 herbal medicines(roots of Morinda officinalis How;leaf of Epimedium brevicornu Maxim;root of Astragalus membranaceus(Fisch)Bge;root of Rehmannia glutinosa(Gaetn.)Libosch.ex Fisch.et Mey;root of Atractylodis macrocephalae Koidz;root of Ranunculi ternati Thunb;fruiting body of Ganoderma lucidum(Curtis:Fr.)P.Karst;root of Salvia miltiorrhiza Bunge;root of Sophorae flavescentis Ait and peel of Citri reticulatae Blanco),it’s decoction was used in the treatment of Hepatitis B.Since there is no relevant research on the quality control of this prescription,this study mainly studies the qualitative and quantitative analysis methods for the flavor of the the Baqi Lingmao formula,and establishes the quality evaluation system of of the Baqi Lingmao formula,in order to provide guarantee for the efficacy and safety of Baqi Lingmao formula in clinical application,and lay a foundation for the quality standard research of granules in the future.Methods:1.The fingerprint spectrum of 10 batches of the decoction of Baqi Lingmao formula were characterized by HPLC.Calculating the similarity of those fingerprint spectrum by a similarity software in order to establish the characteristic mode of HPLC fingerprint.Identifying common peaks and recognizing those peaks.2.As the feature components in Morinda officinalis How and Ganoderma lucidum,TLC has been used to identify the two medicines qualitatively at the same time and TLC conditions.3.HPLC-DAD method was used to establish the component content of the five components in Baqi Lingmao formula(such as Narirutin,Hesperidin,lithospermic acid B,Epmedin B and Icariin)at the same time.4.Using HPLC-ELSD method,the content determination method of Nystose in the Baqi Lingmao formula was established.Results:1.Established a HPLC method of fingerprint.Chromatographic conditions:chromatographic column: CAPCELL PAK AQ C18(4.6 × 250 mm,5 μm);mobile phase:acetonitrile(A)-0.1% phosphoric acid(B),0～10 min,5%→10% A;10～20 min,10%→20% A;20～60 min,20%→30% A;60～62 min,30%→40% A;62～80 min,40%→60% A;detection wavelength: 280 nm,;column temperature: 30℃;flow rate:1.0 m L/min.There should be 25 common peaks in the fingerprint.The similarity of 10 batches of Baqi Lingmao formula was analyzed,and the similarity was between 0.963 and 0.989.The common peaks were assigned,and 12 of the chromatographic peaks were identified,which were Salvianic acid A sodium,Chlorogenic acid,p-hydroxy-cinnamic acid,Calycosin-7-glucoside,Narirutin,Hesperidin,Rosmarinic acid,Salvianolic acid B,Epimedin A,Epimedin B,Epimedin C,Icariin.2.The TLC identification method has been established.Chromatographic conditions:the preparation method of the sample is methanol ultrasound extraction;mobile phase:trichloromethane and anhydrous ethanol(7:1,v/v);color-developing agent: 10%alcoholic solution of sulfuric acid.After heating 2 min at 105℃,the samples were observed under the UV light at 366 nm.It was seen two spot features blue and green fluorescent for Morinda officinalis How and one spot feature red fluorescent for Ganoderma lucidum.The results shown that this TLC identification method has good stability and reproducibility.3.The determination of the five components of Narirutin,Hesperidin,Salvianolic acid B,Epimedin B and Icariin in Baqi Lingmao formula was established by switching wavelengths by HPLC-DAD.The chromatographic conditions are: chromatographic column: CAPCELL PAK AQ C18(4.6 × 250 mm,5 μm);mobile phase: acetonitrile(A)-0.1% phosphoric acid(B),0～15 min,20%→20% A;15～20 min,20%→23% A;23～25 min,23%→27% A;25～45 min,27%→30% A,0～23 min,284 nm;23～35 min,286nm;35～45 min,Detection wavelength: 0～23 min,284 nm;23～35 min,286 nm;35～45 min,270 nm;column temperature: 30℃;flow rate: 1.0 m L/min;The linear relationship of Narirutin,Hesperidin,Salvianolic acid B,Epimedin B and Icariin are good in the range of 5.5～54.5 μg/m L,19.8～197.8 μg/m L,40.9～408.6 μg/m L,5.6～56.2 μg/m L,5.7～57.1 μg/m L,and the average recoveries are: 102.03%,101.12%,101.13%,100.91%,100.12%;the average content of 10 batches of Baqi Lingmao formula of Narirutin,Hesperidin,Salvianolic acid B,Epimedin B and Icariin were 1.3 μg/mg,2.2 μg/mg,5.0μg/mg,0.9 μg/mg,0.8 μg/mg.The HPLC method calculates the content of Narirutin,Hesperidin,Salvianolic acid B,Epimedin B and Icariin,according to the dry product of Baqi Lingmao formula contains at least 1.04 μg/mg of Narirutin,1.76 μg/mg of Hesperidin,4.00 μg/mg of Salvianolic acid B,0.72 μg/mg of Epimedin B,and 0.64 μg/mg of Icariin.4.The content determination of Nystose by HPLC-ELSD method has been established.The chromatographic conditions are: chromatographic column: Thermo Syncronis C18(4.6 × 250 mm,5 μm);mobile phase: water-methanol(97:3);column temperature: 30℃;flow rate: 0.8 m L/min;temperature in drift tube: 80℃。The result indicates that Nystose has a good linear relastionsip at 101.8-1018ug/m L and the average recovery rate is 101.22%.The average content of Nystose in the 10 batches Baqi Lingmao Formula decoction is 15.2 ug/m L,it is specified that the content of Nystose of Baqi Lingmao formula shall not be less than 12.2 μg/mg.Conclusion:The established HPLC fingerprint method and TLC method are simple and stable,with good repeatability,which provides an effective means for ensuring the stable and uniform quality of Baqi Lingmao formula.The established HPLC-DAD content determination method can quantitatively analyze the five components of Narirutin,Hesperidin,Salvianolic acid B,Epimedin B and Icariin in the formula,the HPLC-ELSD determination method can quantitatively analysis the content of Nystose in the formula;the two content measurement methods established are accurate and reliable,with good stability,with the established fingerprint map,thin layer map method from the qualitative and quantitative aspects of the quality control system of Baqi Lingmao formula,which enhances the controbility of Baqi Lingmao formula. |
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