| Kangnaoshuai capsule (KNS) is an over-the-counter fufang preparation consisted of 19 herbs and the herbs are Radix Polygoni Multiflori, Radix rehmanniae preparata, Fructus lycii, Radix Ginseng, Radix Astragali, Rhizoma Dioscoreae, Radix Salviae Miltiorrhiae, Radix Paeoniae Alba, Rhizome Acori Talarinowii, radix polygalae, Semen ziziphi spinosae, Rhizoma Cyperi, Radix Scutellariae, Radix codonopsis, Indian bread with host wood, Radix ophiopogonis, Radix Puerariae, Flos Chrysanthemi. It has been used for the treatment of neurolysis, insomnia and dreamful sleep, neurastheria, memory deterioration. It has also been reported to be beneficial in the treatments for age-related dementia, encephalatrophy, cerebral infarction, cerebral hypoplasia, sequelae of cerebral thrombosis, brain injuries caused by stroke, cerebral trauma and encephalitis.A reversed phase high performance liquid chromatography method for simultaneous determination of the seven chemical components in the fufang preparation of kangnaoshuai capsule was developed. High performance chromatographic fingerprints of the constituent herbs and kangnaoshuai capsule at different production batches and different manufactures were constructed. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the method can be effectively applied for the quanlity control and evaluation of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule. Part oneSimultaneous determination of the major chemical components in kangnaoshuai capsule by HPLCObjective: To establish a reversed phase high performance liquid chromatography method for simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule. The established method can be applied for determination of the key chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the quantitative analyses were compared for quality control and evaluation of Kangnaoshuai capsule.Methods: (1) Optimization of the extraction method: different extraction methods, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution of the major chemical components and the theoretical plate number the peak of baicalin was calculated. (4) A series of seven calibration solutions were prepared by diluting the stock standard solution to appropriate concentrations for construction of the regression equations. The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration. (5) The test of LOD and LOQ: the limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise ratio of about 3 and 10 by analyzing the diluted standard solution. (6) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the standard solution. The retention times and the peak areas of the seven chemical components were analyzed for evaluation of the precision. (7) The test of the intra-day and inter-day precision: intra-day precision was evaluated by analyzing six replications prepared from the KNS sample within a day. Duplicate copies of the KNS sample were prepared within a day for the consecutive three days, and inter-day precision was evaluated by analyzing the six sample solutions of three days. (8) The test of stability: stability was tested by determining the same stock standard solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. (9) The test of recovery: Accurately weighted amounts of each reference compound at three levels were mixed with a fixed amount of the contents of KNS sample. Three replications at each level were extracted and analyzed to evaluate the accuracy of the developed method. (10) The test of selectivity: negative control samples were analyzed to evaluate the selectivity of the developed method. The results were compared to confirm that the chemical components were determined without the interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the seven chemical components in kangnaoshuai capsule of different production batches and different manufactures.Results: The simultaneous determination of the seven chemical components in kangnaoshuai capsule (1) The extraction method: An accurately weighted dried powder of the contents of KNS capsule was extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method of the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for quantitative analysis was: 0~15 min, 12%~20% A; 15~30 min, 20%~45% A; 30~40 min, 45%~75% A; 40~50 min, 75%~100% A, 50~55 min, 100%~12% A. The detection wavelength was programmed as: 0.00~6.10 min, 320 nm; 6.10~8.50 min, 248 nm; 8.50~15.00 min, 230 nm; 15.00~25.00 min, 320 nm; 25.00~27.10 min, 275 nm; 27.10~32.50 min, 286 nm; 32.50~50.00 min, 275 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration for construction of the regression equations. All calibration curves showed good linearity within the test ranges. (5) The limits of LOD and LOQ: The limits of LOQ of the seven chemical components were 4.90,16.40,13.30,4.26,1.98,3.88,1.04 ng/ml;The limits of LOD of the seven chemical components were 19.58,65.68,6.65,17.03,7.92,7.76,2.08 ng/ml. (6) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.24; The RSD% of the peak areas for the tests of injection precision was not more than 0.85. (7) Intra-day and inter-day precision: the RSD% of the retention times for the tests of intra-day precision was not more than 0.37, the RSD% of the peak areas for the tests of intra-day precision was not more than 3.03;the RSD% of the retention times for the tests of inter-day precision was not more than 0.46, the RSD% of the peak areas for the tests of inter-day precision was not more than 2.94. (8) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (9) Recovery test: the results of the recovery test at three levels were from 90.0%~108.3% and the RSD% were not more than 4.72. (10) Selectivity: negative control samples were analyzed and the results were compared with that of the sample preparation. The results showed that the chemical components were determined without the interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The content of each chemical component was calculated according to the established calibration curves.Conclusion: The reversed phase high performance liquid chromatography method for the simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule was established. The UV detector was programmed to monitor at each component's maximum absorption wavelength by varying the detection wavelength during a single elution process and the detection sensitivity was greatly improved by using this detection method. The developed methods was sensitive, precise, accurate and selective and can be applied for simultaneous determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the simultaneous determination could provide sufficient information for efficient quality control and evaluation of kangnaoshuai capsule.Part twoStudies on the fingerprinting analysis of kangnaoshuai capsuleObjective: To establish a reversed phase high performance liquid chromatography method for the fingerprinting analysis of kangnaoshuai capsule. The established method can be applied for fingerprinting analysis of kangnaoshuai capsule at different production batches and different manufactures. The established method can also be applied for the construction of the HPLC fingerprints of the constituent herbs of kangnaoshuai capsule. The results of the fingerprinting analyses were compared for quality control and evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components.Methods: (1) Optimization of the extraction method: different extraction method, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution and the theoretical plate number the peak of baicalin was calculated. (4) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the same sample solution. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the precision. (5) The test of reproducibility: reproducibility was evaluated by analyzing six replications prepared from the KNS sample within a day. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the reproducibility. (6) The test of stability: the test of stability was tested by determining the same sample solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the stability. (7) The established method was applied for construction of the chromatographic figerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can be applied for qualtity evaluation of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule, the constituent herbs of kangnaoshuai capsule. The HPLC fingerprints of kangnaoshuai capsule were compared with that of the constituent herbs to identify the sub-chemical characteristic of the constituent herbs.Results: The fingerprinting analysis of kangnaoshuai capsule (1) The extraction method: An accurately weighted powder of the contents of KNS capsule and the constituent herbs were extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method for the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for fingerprinting analysis was: 0~30 min, 3%~30% A; 30~40 min, 30%~65% A; 40~50 min, 65%~100% A; 50~55 min, 100%~3% A. The detection wavelength was set at 200 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.07; The RSD% of the peak areas for the tests of injection precision was not more than 1.59. (5) Reproducibility: the RSD% of the retention times for the tests of injection precision was not more than 1.46; The RSD% of the peak areas for the tests of injection precision was not more than 3.96. (6) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (7) The established method was applied for the construction of the HPLC fingerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. The results were analyzed for quality evaluation. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out on the chromatographic figerprints of kangnaoshuai capsule, and the constituent herbs. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified.Conclusion: The reversed phase high performance liquid chromatography method for construction of the chromatographic fingerprints of kangnaoshuai capsule and the constituent herbs was established. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule and the constituent herbs. Analysis of the chromatographic fingerprints of kangnaoshuai capsule at different production batches and different manufactures was carried out. The results of the fingerprinting analyses were analyzed and compared by the professional software recommended by State Food and Drug Administration of China for the quality evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the analysis result can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule.
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