| Shanzhuang lipid-lowering tablets are an over-the-counter fufang preparation consisted of 3 herbs and the herbs are cassia seed, hawthorn, lotus leaf. It is provided with the effect of heat clearing, activate blood, relaxing bowels, It has been used for the treatment of hypertension,hyperlipidemia caused by phlegm stasis and the prevention of atherosclerosis. Quality control of drug is one of its basic attributes and the basement of security and validity. Quality control standard is vital to drug quality, and it is also embodiment for quality control. In order to control quality of Shanzhuang lipid-lowering tablets, the quality standard of it was studied.A reversed phase high performance liquid chromatography and UV spectrophotometry method for simultaneous determination of major chemical components in the fufang preparation of Shanzhuang lipid-lowering tablets was developed. High performance chromatographic fingerprints of the Shanzhuang lipid-lowering tablets at different production batches were constructed. The chemical characteristic of Shanzhuang lipid-lowering tablets 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 methods were precise and accurate and the methods can be effectively applied for the quanlity control and evaluation of Shanzhuang lipid-lowering tablets at different production batches. 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 Shanzhuang lipid-lowering tablets. Part oneStudy on quality standards of Shanzhuang lipid-lowering tablesObjective: 1.To establish methods for identifying cassia seed,lotus leaf and total flavonoids by TLC; To develop methods for determining the contents of aurantio– obtusifolin,emodin,chrysophanol and nuciferine in Shanzhuang lipid-lowering tablets by HPLC. To develop a method to determine the contents of total flavonoids in Shanzhuang lipid-lowering tablets by UV spectrophotometry.Methods: 1 Identification:⑴Iidentification of aurantio-obtusifolin,emodin,chrysophanol: silica gel G was used as the coating material and petroleum ether( (60-90℃)- ethyl acetate - formic acid(15:5:1)as the mobile phase, examining under ultravioletlight(365nm);⑵Identification of quercetin: silica gel G was used as the coating material and Hexane - ethyl acetate - formic acid (15:12:1) as the mobile phase,examining under ultravioletlight(365nm);⑶Identification of nuciferine, silica gel GF254 was used as the coating material and the under solution of chloroform-acetoacetate-methanol-water(4:4:2:1)as the mobile phase, examining under ultravioletlight(254nm). 2 Determination of the contents of active substances.⑴Chromatogram condition: choose appropriate stationary phase and adjust the composition, proportion and flow rate of mobile phase to separate the peak well and meet the requirements of number of theoretical plates. UV spectrophotometry: choose a suitable coloration method and the wavelengths of maximalab sorption.⑵The investigation of linear range of HPLC: a series of the reference solutions were prepared and the regression equation was obtained with the contents of reference as abscissa and the peak area as ordinate; The investigation of UV spectrophotometry: the contents of reference as abscissa and the absorbability as ordinate.⑶The investigation of technology: investigate the precision, reproducibility, stability and recovery.⑷Determination of the sample: take five batch of samples and prepare for the test solution, determine the content of actives ubstance in samples by HPLC or UV spectrophotometry .Results: 1 Identification : aurantio-obtusifolin,emodin,chrysophanol,quercetin,nuciferine all had clear map of TLC. Sample solutions showed the same color of dots in the corresponding position with reference solutions, and negative control solutions showed no interference. 2 Determination of the contents :⑴aurantio- obtusifolin,emodin,chrysophanol chromatographic conditions: DiamonsilTM C18 (150mm x 4.6mm,5um) column; The mobile phase consisted of methanol (B) and 0.1% aqueous phosphoric acid (v/v, D): 0~5 min, 65%~70% B; 5~25 min, 70%~85% B; flow rate 1.0ml·min-1; column temperature: 35℃; The detection wavelength was set at 286 nm. Aaurantio -obtusifolin,emodin,chrysophanol Regression equation: y=221.9x-32.05 ( r=0.9999,n=7 ) ; y=123.4x+9.979 ( r=0.9998,n=7 ) ; y=67.16x+2.650(r=0.9998,n=7); and showed good linear relationship in 0.946~11.3mg·L-1,0.137~1.65 mg·L-1 and 0.153~1.83 mg·L-1. The RSD of the precision were 0.38%,0.84%,2.7%, respectively, reproducibility were 0.12%,0.57%,0.97%, respectively. The sample was stabile in twenty-four -hour period, RSD were 0.76%,0.63% and 0.73% respectively. The average recovery were 99.5%,99.6%,98.9%, and RSD were 0.61%,0.13% and 0.18% respectively. The content of aurantio– obtusifolin in each batch was not less than 50.6μg·g-1, the content of emodin in each batch was not less than 7.20μg·g-1, the content of chrysophanol in each batch was not less thanμg·g-1.⑵Nuciferine chromatographic conditions: DiamonsilTM C18 (150 mm x 4.6 mm,5μm) column; mobile phase: acetomitrile-water-ethylamine(45:55:0.5) and adjusted to pH6 with acetic acid; flow rate 1.0 ml·min-1; column temperature: room temperature; The detection wavelength was set at 270 nm. Regression equation: y=1.04×105x-5.30×104 (r=0.9999), and showed good linear relationship in11.0～44.0μg·ml-1. The RSD of the precision and reproducibility were 0.61%,1.4%, respectively. The sample was stabile in eight-hour and the RSD was 0.06%. The average recovery rate and RSD were 98.4% and 0.25%. The content of nuciferine in each batch was not less than 19.5μg·g-1.⑶T he determination of total flavonoids: The maximal absorption wavelength was 375nm. Regression equation A=8.22×10-2C+3.34×10-3, r=0.9992, and quercetin showed good linear relationship in 2.79~8.72 mg·L-1. The RSD of the precision and reproducibility were 0.14%,1.3%, respectively. The sample was stabile in one-hour period, and the RSD was 0.73%. The average recovery rate and RSD were 96.1% and 0.085%. The content of quercetin in each batch was not less than 0.653 mg·g-1.Conclusion: It is the first time to establish the TLC methods for identifying cassia seed,lotus leaf and total flavonoids of Shanzhuang lipid-lowering tablets, and the methods had good specificity and clear spots, which can be used for the accurate identification of the traditional Chinese medicine in preparation; It is the first time to establish HPLC and UV spectrophotometry methods, and they were stable, accurate, specific. They can effectively control the quality of the preparation. Part two Study on Fingerprint of Shanzhuang lipid-lowering tabletsObjective: To establish a reversed phase high performance liquid chromatography method for the fingerprinting analysis of Shanzhuang lipid-lowering tablets; get the control chromatogram and the fingerprints of Shanzhuang lipid-lowering tablets collected from different production batches were compared so as to establish a sensitive and specific method for controlling the quality of Shanzhuang lipid-lowering tablets.Methods: (1) Extraction: different extraction method and extration time were tested for optimization of the extraction efficacy of Shanzhuang lipid-lowering tablets. The most efficacy extration method was selected for the extraction of the major chemical components of Shanzhuang lipid-lowering tablets. (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 aurantio– obtusifolin 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 stability: the test of stability was tested by determining the same sample solution stored at room temperature at different time of 0, 2, 4, 8, 12 , 24 h. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the stability. (6) The test of reproducibility: reproducibility was evaluated by analyzing six replications prepared from the sample within a day. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the reproducibility. (7) The established method was applied for construction of the chromatographic figerprints of Shanzhuang lipid-lowering tablets at different production batches, and the analysis results of similarities of the HPLC fingerprints of Shanzhuang lipid-lowering tablets can be applied for qualtity evaluation of Shanzhuang lipid-lowering tablets at different production batches.Results: The fingerprinting analysis of Shanzhuang lipid-lowering tablets (1)The extraction method: The method of supersonic wave-extraction with chloroform and 2.5mol·L-1 sulfate for 60 mins was simple, quick and stable. (2)The chromatographic condition: A DiamonsilTM C18 column (150×4.6mm,5μm)was used throughout. The mobile phase consisted of methanol (B) and 0.1% aqueous phosphoric acid (v/v, D). The gradient program for fingerprinting analysis was: 0~20 min, 6%~45% B; 20~35 min, 45%~65% B; 35~55min, 65%~90% B. The detection wavelength was set at 254 nm. (3) System suitability test: Under the above conditions, the peak of aurantio– obtusifolin was separated well with the resolution of more than 1.5 and about 5000 of theoretical plate number. (4) Precision: The precision of sample was good and the RSD of the relative retention times and the relative peak areas were between 0.012%~0.11% and between 1.4%~2.7%, respectively. (5) Stability: the RSD% of the retention times and the peak areas for the tests of sample were between 0.14%~2.1% and between 1.4%~2.7%, respectively. The sample solution was stable within 24 hours. (6) Reproducibility: the RSD% of the retention times and the peak areas for the tests of sample were between 0.099%~1.9%and between 0.8%~2.7%. (7) The established method was applied for the construction of the HPLC fingerprints of Shanzhuang lipid-lowering tablets at different production batches, get 17 relative fingerprint chromatograms, the results were analyzed for quality evaluation, similarity analysis was performed and the results could significantly reflect the quality of Shanzhuang lipid-lowering tablets from different bacthes.Conclusion: It is the first time to establish the RP-HPLC fingerprint Chromatogram of Shanzhuang lipid-lowering tablets,get reference fingerprint Chromatogram, compare the fingerprint of 10 different production batches, and study their difference with similarity. The most efficacy extration method was selected, establish a better fingerprint chromatogram by choosing appropriate column and adjust different formulation and column temperature, get 17 relative fingerprint chromatograms, the results were analyzed for quality evaluation. The results of the simultaneous determination could provide sufficient information for efficient quality control and evaluation of Shanzhuang lipid-lowering tablets.
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