Studies On The Quality Control Standard Of Yi Gankang Granules

Yi Gankang Granules is a new compound preparation consisted of 9 herbs and the herbs are Radix Salviae Miltiorrhiae, Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Atractylodis Macrocephalae, Poria cocos, corium stomachium galli, magnolia bark and radix bupleuri. It has been used for the treatment of chronic hepatitis and hepatic fibrosis.In the present study, a series of thin layer chromatography methods for identification of salvia miltiorrhiza, Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Atractylodis Macrocephalae, and radix bupleuri in the compound preparation of Yi Gankang Granules were established, and reversed phase high performance liquid chromatography methods for determination of tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣand paeoniflorin were developed, separately. It was shown that the methods were simple, accurate, sensitive and stable. They can be used for the quality control of Yi Gankang Granules. A simultaneous determination method with RP-HPLC for of the five chemical components in the Yi Gankang Granules was established. A HPLC fingerprint of the Yi Gankang Granules at different production batches was established. The chemical characteristic of Yi Gankang Granules was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The serum fingerprint was investigated in rat after administrated Yi Gankang Granules suspension orally, so as to illuminate the therapeutical basis of Yi Gankang Granules preliminary. In the present study, a method for global assessment the quality of Yi Gankang Granules was established and was used for a guarantee of quality of the the compound preparation of Yi Gankang Granules.Part oneStudies on the methods to identify the Yi Gankang Granules by TLCObjective: To establish a series of TLC methods to identify traditional Chinese medicinal herb in Yi Gankang Granules in order to provide one guideline for product quality control.Methods: (1) Choose items for identification. (2) Choose the control articles for identification. (3) Optimization of the extraction method: different extraction methods, extraction solvents of different solvent composition and extration time were tested, for optimization of the effective extraction of test components from Yi Gankang Granules, further more avoidance interference. (4) Optimization of the thin layer chromatographic conditions: the thin layer chromatographic conditions such as static phase, composition and proportion of developing agent were tested for optimization of the chromatographic conditions. (5) Select appropriate colouration or examine conditions in order to make out the speckle clearly. (6) The test of selectivity: negative control samples were analyzed to evaluate the selectivity of the developed method.Results: (1) List the items of identification of salvia miltiorrhiza, Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Atractylodis Macrocephalae, and radix bupleuri into the quality standard of Yi Gankang Granules. Choose reference substance such as tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣpaeoniflorin, saikoside A; reference medicinal herb such as Radix Angelicae Sinensis and Rhizoma Atractylodis Macrocephalae the control articles for identification. We didn't list the items of identification of Poria cocos, corium stomachium galli and magnolia bark into the quality standard of Yi Gankang Granules, because there was not any characteristic speckle in thin layer chromatogram during attempt. (2) The established method was applied for identification of salvia miltiorrhiza, Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Atractylodis Macrocephalae, and radix bupleuri in Yi Gankang Granules. The result showed that the spots of the test solution were in the same position as those of the reference solution, without the interference of other chemical components originated from the coexisting herbs.Conclusion: The developed methods were proved to be accurate, sensitive and convenient, and can be used for the identification of Yi Gankang Granules, which provided some guideline for qulity control.Part twoStudies on the methods to determination of Yi Gankang Granules by HPLCObjective: To establish a series of reversed phase high performance liquid chromatography methods for determination of the key chemical components in Yi Gankang Granules,for ensure the safety, efficiency and qulity controllability of the product.Methods: (1) Choose items for determination. (2) Select the effective extraction of test components from Yi Gankang Granules. (2) Choose appropriate chromatographic column, and adjust the formulation, proportion flow rate in order to separate the peaks of test components well. (3) Validated the methodology. (4) The established methods were applied for determination of tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣpaeoniflorin in Yi Gankang Granules of different production batches.Results: (1) List the items of determination of tanshinone Ⅱ-A, salvianolic acid B, astragalosideⅣ, paeoniflorin into the quality standard of Yi Gankang Granules, which is grounded upon the prescription, technology and studies material base of traditional Chinese herbal. (2) Optimization of the extraction method. For determination tanshinoneⅡ-A and paeoniflorin, Yi Gankang Granules was extracted with methanol by a sonifier for 20 min. For determination salvianolic acid B, the preparation was extracted with water by a sonifier in icewater bath for 20 min. There were a series of steps to prepare sample solution for determination astragalosideⅣ, such as extraction with methanol by a sonifier, abstraction with equilibrium n-butanol, ablution with ammoniae aqua, elution with 70% ethanol though a D101 macroporous resin. (3) The chromatographic condition: The HPLC system was performed on a C18 analytical column. The mobile phases for determination of tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣand paeoniflorin were methanol-water (80:20), acetonitrile- methanol-0.08% methanoic acid (37:111:252), acetonitrile-water (33:67) and acetonitrile-0.05%phosphoric acid (18:82), separately. The detector for determination of tanshinoneⅡ-A, salvianolic acid B and paeoniflorin was UV, and the wavelengthes were set at 270 nm, 286 nm, 230 nm separately. The detector for determination of astragalosideⅣwas ELSD, under the conditions: The temperature of the diift tube was 80℃, and the carrier gas was nitrogen with follow rate of 25 psi. (4) Validated the methodology: The calibration curves of tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣand paeoniflorin showed good linearity at the ranges of 0.1334-2.668μg, 0.104-2.08 mg, 0.6930-13.86μg and 2.00-40.1 mg and the average recoveries were100.9%,100.5%,98.0%,101.7%, with RSD of 2.5%, 2.3%, 1.2%, 1.1% respectively. The reproducibilities of thses methods were good with RSD of 2.3%, 1.5%, 1.6% and 2.9%.Conclusion: A series of reversed phase high performance liquid chromatography methods for the determination of tanshinoneⅡ-A, salvianolic acid B, astragalosideⅣand paeoniflorin in the compound preparation of Yi Gankang Granules were established. The developed methods were sensitive, precise, accurate and selective and can be applied for determination of the key chemical components in Yi Gankang Granules for efficient quality control.Part threeSimultaneous determination of the major chemical components in Yi Gankang Granules by HPLCObjective: To establish a reversed phase high performance liquid chromatography method for simultaneous determination of the major chemical components in the compound preparation of Yi Gankang Granules. The method can be applied for determination of different production batches for quality control and evaluation quality of the product. Methods: (1) Different extraction methods, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy from Yi Gankang Granules. (2) Choose appropriate chromatographic column, and adjust the formulation, proportion flow rate in order to separate the peaks of test components well. (3) System suitability test and validity check of the methodology.Results: The simultaneous determination of the five chemical components in Yi Gankang Granules: (1) Optimization of the extraction method. Extract Yi Gankang Granules by a sonifier, with water first, with 50% methanol subsequently for 10 min everytime. (2) The chromatographic condition: A Restek PinnacleⅡC18 column (250 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of 0.1% aqueous phosphoric acid (A) and acetonitrile - methanol (50:50, B). The gradient program for quantitative analysis was: 0 ~5 min 1%B; 8 min 5%B; 30 min 35%B; 40 min 45%B; 50 min 80%B; 60 min 90%B. The detector for determination was UV-DAD, and the wavelengthes were set at 230 nm for paeoniflorin, and at 270nm for gallic acid, danshensu, salvianolic acid B and tanshinoneⅡ-A, simultaneously. (3) The calibration curves of galic acid, tanshinol, paeoniflorin, salvianolic acid B and tanshinoneⅡ-A showed good linearity at the ranges of 31.40-376.8, 452.7-5659.2, 201.6-2520, 1657-20710 and 54.33-679.2μg. The average recoveries were 102.5%, 104.6%, 99.4%, 105.0%, 102.9% respectively, with RSD not more than 3.5%.Conclusion: The reversed phase high performance liquid chromatography method for the simultaneous determination of the major chemical components in the compound preparation of Yi Gankang Granules was established. The DAD detector was utilized to monitor at double wavelengthes simultaneously, and the detection sensitivity was greatly improved by using this detection method. This method is sensitive, quick, precise, accurate, and can be used to simultaneous determination of the major chemical components in Yi Gankang Granules for efficient quality control.Part fourStudies on the fingerprinting analysis of Yi Gankang GranulesObjective: To establish a reversed phase high performance liquid chromatography method for the fingerprinting analysis of Yi Gankang Granules. The HPLC fingerprints of the compound preparation of Yi Gankang Granules was compared with the fingerprints of the constituent herbs using the established method, to find out which constituent herb is the chemical characteristic of the preparation derived from. The serum fingerprints were investigated in rats after administrated Yi Gankang Granules suspension orally, so as to illuminate the therapeutical basis of Yi Gankang Granules preliminary.Methods: 1. Studies on the fingerprinting analysis of Yi Gankang Granules: (1) Optimization of the chromatographic condition and sample preparation methods. (2) Validity check of the methodology. (3) The established method was applied for construction of the chromatographic fingerprints of Yi Gankang Granules at different production batches. (4) The similarity of Yi Gankang Granules fingerprints was analyzed with computer by the professional software. 2 Analysis was carried out for the chromatographic fingerprints of the constituent herbs of Yi Gankang Granules by the established methods. Search for the constituent herbs, which the characteristics of the preparation derive from. 3 The rat serum fingerprints after administrated Yi Gankang Granules suspension orally were analyzed.Results: 1. Studies on the fingerprinting analysis of Yi Gankang Granules: (1) Optimization of the extraction method. Extract Yi Gankang Granules by a sonifier, with water first, with 50% methanol subsequently for 10 min every time. (2) The chromatographic condition: The separation was performed on a C18 analytical column with a gradient mobile phase consisting of 0.1% phosphoric acid (A) and acetonitrile (B) at the flow rate of 1.0 mL/min. The gradient program for quantitative analysis was: 0 min 2%B; 10 min 10%B; 25~30 min 25%B; 55 min 75%B.The detection wavelength was set at 270 nm and the temperature of column was kept at 30℃. (3) Under the above conditions, the peaks of Yi Gankang Granules fingerprints were separated well with high precision and good repeatability, which was shown that the method satisfied all requirements of establishment chromatographic fingerprints. (4) 10 production batches of Yi Gankang Granules were determined and 21 common peaks of chromatogram were detected. The similarities of 10 batches were between 0.961 and 0.993. 2. Analysis was carried out for the chromatographic fingerprints of the constituent herbs of Yi Gankang Granules by the established methods. It was shown that the characteristics of the preparation derive from salvia miltiorrhiza, Radix Astragali and Radix Paeoniae Rubra. 3. 10 batches of the rat serum containing Yi Gankang Granules were determined, and 10 common peaks were signed, with 8 of them were the control serum inherent ones, and 2 were metabolites.Conclusion: The reversed phase high performance liquid chromatography method for construction of the chromatographic fingerprints of Yi Gankang Granules was established. It was shown that the method is precise, accurate, and repeatable, which provided significant bases on quality control of Yi Gankang Granules efficiently. The HPLC fingerprints of the compound preparation of Yi Gankang Granules was compared with the fingerprints of the constituent herbs using the established method, and it was shown that the chemical characteristic of the preparation derived from salvia miltiorrhiza, Radix Astragali and Radix Paeoniae Rubra. The serum fingerprints were investigated in rats after administrated Yi Gankang Granules suspension orally. It illuminated the therapeutical basis of Yi Gankang Granules preliminary.