| Objective:Research and improve the quality standard of Fengshigutong Capsules（FSGTC）,and study the law of quantity value transfer in the production process of FSGTC.Method:1 Quality control research:（1）The TLC identification of Ephedra herba（EH）,Aconiti Radix Cocta（ARC）and Aconiti Kusnezoffii Radix Cocta（AKC）was conducted by using n-hexane-chloroform-ethyl acetate-methanol（4:7:6:2）as the developing agent and iodine vapor as the developer;TLC identification of Glycyrrhizae Radix Et Rhizoma（GRER）was conducted by using ethyl acetate-formic acid-glacial acetic acid-water（15:1:1:2）as the developing agent,and 10%sulfuric acid ethanol solution as the developer.The TLC identification of Chaenomelis Fructus（CF）was conducted by using TLC-bioautography of chloroform-ethyl acetate-acetone-formic acid（8:2:2:0.4）as the developing agent,2,2-biphenyl-1-picrylhydrazino（DPPH）anhydrous ethanol solution as the developer.（2）Fingerprints of different batches FSGTC were established by HPLC.HPLC analysis was performed with acetonitrile（A）-0.2%phosphoric acid aqueous solution（B）as the mobile phase for gradient elution at a flow rate of 1.0 m L·min-1.The wavelength switching technology was used 210nm（0-20 min）,310 nm（20-36 min）,235nm（36-60 min）,the column temperature was 30℃,and the injection volume was 10μL.The similarity was calculated by the chromatographic fingerprint similarity evaluation system of traditional Chinese medicine（2012A）,and PCA,PLS-DA and HCA were analyzed by simca13.0 software.（3）Content determination study:HPLC technology was used to establish a method for the simultaneous determination of BMA,benzoylaconine（BAC）and benzoylhypaconine（BHA）by using ZORBAX SB C18 column（250 mm×4.6 mm,5μm）as the chromatographic column,acetonitrile-tetrahydrofuran（25:15）（phase A）-0.1 mol/L ammonium acetate solution（0.5 m L of glacial acetic acid per 1000 m L）（phase B）as mobile phase for gradient elution.The flow rate was 1.0 ml·min-1.The detection wavelength was set at 235 nm,and the column temperature was run at 30℃.The content of BMA,BAC and BHC in 21 batches of FSGTC were determined.A method for determining the content of Glycyrrhizic acid（GA）was established by using TSKge L ODS-100S C18 column（250 mm×4.6 mm,5μm）as the chromatographic column,methanol-water-glacial acetic acid-triethylamine（62:38:1:0.3）as mobile phase.The detection wavelength is set at 250 nm,and column temperature is 30°C.The sample volume is 20μL.The content of GA in 21 batches of FSGTC was determined.Using chloroform-ethyl acetate-acetone-formic acid（8:2:2:0.4）as the developing agent,unfold and dry,inspect at 254 nm,scan at 300 nm to determine the content;the thin plate was soaked with DPPH absolute ethanol solution,dried,and scanned at 535 nm to determine the content.TLC-bioautography scanning method for determining the content of protocatechuic acid was established.2 Study on the Law of Value Transmission（LVT）:（1）Based on the quantitative measurement method,explore the LVT of the main index components in the production process.Taking the three monoester alkaloids,GA,EH and PH in FSGTC as inspection indicators,the transfer rate in each step of the production process was calculated,and the LVT in the production process of FSGTC was analyzed.（2）Exploring the stability of the production process based on fingerprints,determining the chemical components in each step of the FSGTC production process,and exploring the stability of the FSGTC production process through the similarity.Result:1 Quality control study:（1）The two TLC methods established have clear spots,moderate Rf values,and good resolution. TLC-bioautography achieves the purpose of identifying and characterizing antioxidant active ingredients in Chaenomelis Fructus.（2）the HPLC fingerprint of FSGTC was established,31 common peaks were marked.the similarity calculation results were ranged from 0.957 to 0.985.Through literature review and comparison of reference materials,10 components of protocatechuic acid,EH,PH,chlorogenic acid,liquiritin apioside,BMA,liquiritigenin,liquiritin,isoliquiritin and GA were identified.PCA and PLS-DA analysis showed that different batches of FSGTC had the same composition,but the peak areas of 31 common components were different.（3）Establish a HPLC method for the simultaneous determination of BMC,BAC and BHC in FSGTC.The contents of BMA,BAC and BHA were linear within 2.200-280.0,2.370-75.76,3.180-101.6μg·m L-1,respectively,with good correlation coefficient（>0.9999）.The average recovery rates were92%-107%with RSD within 2.85%-5.62%for BMA,BAC and BHA.The contents of BMA,BAC and BHA in 21 batches of samples are 35.00-80.15,6.92-13.57,9.94-17.99μg·capsule-1.Establish a HPLC method for the determination of GA.The linear range of GA is 6.100-97.58μg·m L-1 （r=0.9998）.The average recovery rate was 100.9%with RSD of 1.95%.The content of GA in 21 samples was within 1.70-2.21 mg·capsule-1.A method for the determination of protocatechuic acid in thin-layer-bioautographic scanning was established.The results of the linear relationship investigation showed that the protocatechuic acid was 77.25-741.8μg（r=0.9994）with a good linear relationship with the peak area under 254 nm inspection.After DPPH color development,protocatechuic acid showed a good linear relationship with the peak area between 38.50-618.0μg（r=0.9983）2 Study on the law of quantity transfer:（1）The average migration rate of the sum of three monoester alkaloids,medicinal materials→after granulation is 31.84%,after granulation→dry total mixture is 83.63%,dry total mixture→finished product is 98.45%,and the total average transfer rate of medicinal materials→finished product is 26.12%.The average transfer rate of glycyrrhizic acid,medicinal materials→after granulation is 98.22%,after granulation→dry total mixture is 92.80%,dry total mixture→finished product is 102.9%,and the total average transfer rate of medicinal material→finished product is 93.85%.The average transfer rate of the sum of ephedrine hydrochloride and pseudoephedrine hydrochloride,medicinal materials→first decoction is 88.35%,medicinal material→second decoction is 39.43%,medicinal material→thick ointment is 69.34%,thick ointment→after granulation is 93.65%,and after granulation→dry total mixture is 92.10%,dry total mixture→finished product is 103.1%,and the total average transfer rate of medicinal materials→finished product is 61.49%.The transfer rate of glycyrrhizic acid is relatively stable throughout the production process.The transfer rate of the sum of the three monoester alkaloids is lower from medicinal material to after granulation,and the transfer rate of the sum of ephedrine hydrochloride and pseudoephedrine hydrochloride is lower from medicinal material to thick ointment.However,the transfer rates of the three monoester alkaloids,glycyrrhizic acid,ephedrine hydrochloride and pseudoephedrine hydrochloride in different batches in each stage of the production process are in line with"the acceptable range of transfer rate is the mean plus or minus 3 times SD（x±3SD）or（70%-130%of the average value）”requirements,indicate that the batch-to-batch quality of the production process is relatively stable.（2）The results of HPLC fingerprint analysis showed that except hydroxysafflor yellow A,the differences of other major substance groups were small.Conclusion:1 The method for identification of EH,ARC,AKC,and GRER is simple,with good separation between spots and clear bands,it is suitable for qualitative identification of EH,ARC,AKC and GRER in FSGTC.The established TLC-bioautographic identification method for CF has good separation between spots,clear bands,and high sensitivity,which gives play to the advantages of the TLC-bioautographic technology for qualitative identification and activity characterization.2 The HPLC fingerprint spectrum has high precision and good stability,and is suitable for the overall comprehensive evaluation of the quality consistency of FSGTC between batches.4 The HPLC content determination method has high precision and good stability,and can be used for the determination of BMA,BAC,BHA and GA in FSGTC.The TLC-bioautography scanning method realizes the quantitative determination of protocatechuic acid.5The LVT in production process was explored based on quantitative determination and fingerprint.To achieve the purpose of improving the quality standard of FSGTC as a whole,and to provide the basis for clinical application. |
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