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       【关键词】  Zishenligan,Granule；,,Paeoniflorin；,,Reversed-Phase,High,Performance,Liquid,Chromatography；,,Quantitative,Analysis
       (Jiangsu Provincial Center for Research and Development of Marine Drugs, Nanjing University of Traditional Chinese Medicine, Jiangsu Nanjing 210029, China )
       Abstract:ObjectiveTo establish a RP-HPLC method for determination of the content of Paeoniflorin in Zishenligan Granule. MethodsThe analysis was carried out by using a Kromasil C18 analytical column with methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (32:68, v/v) as mobile phase. The UV detection wavelength was 230nm and the flow rate was 1ml・min-1. ResultsUnder the chromatographic conditions used, there was no interference found in the position of Paeoniflorin peak. Paeoniflorin had fine linear responses (r=0.999 9) in the concentration range of 24.28～121.40 μg・ml-1, the average recovery of Paeoniflorin in Zishenligan Granule was 98.32% with the relative standard deviation of 1.34% (n=9). ConclusionThe method employed in this analysis is convenient with good repetition and can be used as a reliable tool for quality control in the production of Zishenligan Granule. 
       Key words:Zishenligan Granule；  Paeoniflorin；  Reversed-Phase High Performance Liquid Chromatography；  Quantitative Analysis
       Zishenligan Granule is a pure formulated preparation of traditional Chinese medicine (TCM) made of crude herbal materials Rhizoma Curcumae, Radix Paeoniae Rubra, Radix Angelicae Sinensis, Radix Notoginseng, Cordyceps and Fructus Forsythiae etc. by the methods of extraction, refinement and dry granulation developed in our research center. Zishenligan Granule has the following functions: softening stiffness and resolving mass, dissolving ecchymosis and detoxicating，supplementing Qi and nourishing Yin. It can be mainly used to treat patients with chronic hepatitis B and fiberized liver in clinical application. Paeoniflorin is the major active constituent of Radix Paeoniae Rubra and also one of the main effective contents in Zishenligan Granule. The Pharmacopoeia of China requires the content of Paeoniflorin in Radix Paeoniae Rubra to be no less than 1.8%［1］. Paeoniflorin is a water-soluble compound first isolated from the root of Paeonia lactiflora and has been reported to exhibit immunoregulating activity and the function of protecting liver［2］. The chemical structure of Paeoniflorin is shown in Figure 1.
       Fig 1  The chemical structure of PaeoniflorinFor the determination of the content of Paeoniflorin, the major analytical methods reported in the literatures mostly are TLC, HPLC and CE［3~5］. （略）
       Since HPLC has the advantage of convenient and good repetition, thus in the study of quality control of Zishenligan Granule, we choose the index of Paeoniflorin as quality control criteria and establish a RP-HPLC method for determining the content of Paeoniflorin in Zishenligan Granule. The analytical results are satisfying and will provide a quantitative evaluation for quality control in the production of Zishenligan Granule.
       　　1  Apparatus and chemicals
       Chromatographic analysis was performed on a Model 515 high performance liquid chromatograph (Waters, USA) equipped with a Model 2487 dual wavelength UV detector and a Model 717 auto-sampler connected to a Millennium 32 data processing system. A Model BP211D electronic balance (Sartorius, Germany) was used for weighing of samples.Paeoniflorin (Batch number: 110736-200423) was supplied from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, P.R. China) and its purity was large than 98.5% by the identification from RP-HPLC. Zishenligan Granule (Batch number: 040912, 040919, 040926) was researched and developed in our research center. All reagents and chemicals used for the preparation and for the RP-HPLC analysis were of chromatographic grade (methanol) and analytical grade (potassium dihydrogen phosphate). The water used for the preparation and for the RP-HPLC analysis was freshly deionized and redistilled.
       　　2  Experimental methods and results
       2.1  Chromatographic conditions and systematic compatibility testSeparations were obtained on a Kromasil C18 analytical column（250 mm×4.6 mm，φ5 μm）(Hanbon Sci. & Tech.，P.R. China) with methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (32:68, v/v) as mobile phase. The UV detection wavelength was 230nm and the flow rate was 1ml・min-1. The column temperature was 30℃ and injection volume was 10μl. Under the chromatographic conditions used, the number of theoretical plates calculated on the peak of Paeoniflorin was no less than 3000.
       2.2  The preparation of standard solution, sample solution and blank solution
       2.2.1  The preparation of standard solutionA suitable amount of Paeoniflorin was accurately weighed and dispersed in methanol, mixed, a 72μg per milliliter standard solution was obtained.
       2.2.2  The preparation of sample solutionAbout 0.3g of the sample (comminuted powder) was accurately weighed and dispersed in 20ml of methanol, extracted ultrasonically for 30min. After cooling, methanol was added to this mixture till the total volume 25ml, mixed, the filtrate was obtained as a sample solution by filtration.
       2.2.3  The preparation of blank solutionAccording to the proportion of prescription, other crude herbal materials in the prescription were weighed in the suitable amounts except Radix Paeoniae Rubra. Using preparation technology of Zishenligan Granule and preparation method of sample solution, a blank solution was obtained.
       2.3  Interference test of blank solution10μl of standard solution, sample solution and blank solution were injected respectively into the RP-HPLC column under the chromatographic conditions described above. The results (Figure 2) showed that there was a same peak of Paeoniflorin appeared in the same retention time between standard solution and sample solution, whereas there was no peak of Paeoniflorin appeared in this retention time in blank solution, thus there was no interference for the determination of the content of Paeoniflorin in sample solution.
       achromatogram of the standard solution
       b chromatogram of the sample solution
       cchromatogram of the blank solution
       1. Paeoniflorin                     
       Fig 2  Typical chromatograms for the determination of the content of Paeoniflorin  （略）   
       2.4  Determination of the content of Paeoniflorin in Zishenligan Granule
       2.4.1  Calibration curve and linear rangeA bout 12.14mg of Paeoniflorin was accurately weighed and dispersed in 100ml of methanol, mixed, a 121.40μg per milliliter standard stock solution was obtained. Then 2.0,4.0,6.0,8.0,10.0ml of this standard stock solution were taken respectively in five 10ml volumetric flasks, diluted with methanol to a constant-volume of 10ml, mixed, a series of standard solutions were obtained. Under the chromatographic conditions described above, 10μl of each standard solution was taken for measuring and the chromatograms were recorded. After regression analysis of linearity , the calibration curve was described by regression equation A=11 808.87C+4 583.17，r=0.999 9, where A was the peak area of Paeoniflorin (as longitudinal coordinate), C was the concentration of Paeoniflorin (as horizontal coordinate), and r was the correlation coefficient. The results showed that the peak area of Paeoniflorin and the concentration of Paeoniflorin had fine linear responses in the concentration range of 24.28～121.40μg・ml-1.
       2.4.2  Chromatographic test of precision10μl of a same concentration of Paeoniflorin standard solution was taken to continuously analyze for six times, the RSD of peak area of Paeoniflorin was 0.84%. The results showed that the chromatographic method was quite precise.
       2.4.3  Chromatographic test of stabilityAccording to the method for the determination of the content of Paeoniflorin, a sample of the same batch number was accurately weighed and the sample solution was prepared, and 10μl of this solution was measured in 0, 2, 4, 6, 8, 10, 12h within a day, the RSD of peak area of Paeoniflorin was 1.45%. The results showed that the sample solution was stable within 12h.
       2.4.4  Chromatographic test of reproducibilitySix of 0.3g of sample (comminuted powder, Batch number: 040919) were accurately weighed, and the content of Paeoniflorin was determined after treated like the method of determination of the content of sample, the average content of Paeoniflorin was 5.06 mg・g-1, and the RSD was 1.56%. The results showed that the chromatographic method had a good reproducibility.
       2.4.5  Recovery of the chromatographic methodNine of 0.15g of samples (comminuted powder, Batch number: 040919) were accurately weighed and put into nine of 25ml volumetric flasks respectively. Then three of 5, 6.2, 7.4ml of the standard stock solution of Paeoniflorin (121.40μg per milliliter) and three of 15, 13.8, 12.6 ml of methanol were added respectively, extracted ultrasonically for 30min. After cooling, these mixtures were diluted with methanol to a constant-volume of 25ml, mixed, the filtrates were obtained by filtration, 10μl of each filtrate was taken for measuring and the recovery was calculated, the average recovery of Paeoniflorin was 98.32% and the RSD was 1.34% (n=9). The results were shown in Table 1.
       Tab 1  Recovery test of Paeoniflorin（略）
       2.4.6  Determination of the content of sampleThree of 0.3g of samples (comminuted powder, Batch number: 040912, 040919, 040926) were accurately weighed, and treated like the item “2.2.2”, nine of sample solutions were obtained. 10μl of each sample solution was taken for measuring and the content of C23H28O11 was calculated based on calibration curve. The results were shown in Table 2.
       Tab 2  Result of sample analysis Batch（略）
       　　3  Discussion
       3.1  Selection of detection wavelengthThe UV spectrum showed that Paeoniflorin had obvious absorbent peak near the wavelength 230nm after UV-scanning, thus 230nm was selected as detection wavelength.
       3.2  Selection of chromatographic column and mobile phaseThe experiments compared the chromatographic behavior of the chromatographic columns YWG C18, Kromasil C18, Kromatorex C18, Lichrospher C18 and the mobile phases methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (40:60, 32:68, 24:76). The results showed that under the conditions of the chromatographic columns YWG C18, Kromatorex C18, Lichrospher C18 and the mobile phases methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (40:60, 24:76), there were not complete baseline separations among Paeoniflorin and other components, whereas there was a complete baseline separation among Paeoniflorin and other components and also a symmetric chromatographic peak by using the chromatographic column Kromasil C18 and the mobile phase methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (32:68). Thus the chromatographic column Kromasil C18 and the mobile phase methanol and 0.05mol・L-1 potassium dihydrogen phosphate solution (32:68) were selected as the best chromatographic conditions due to their better degrees of resolution.
       3.3  Selection of the methods of sample pre-treatmentThe experiments investigated three kinds of extraction methods ultrasonic extraction, refluxing extraction and Soxhlet’s extraction. The results showed that the sample solution treated by ultrasonic extraction had minimum impurities that disturbed the determination of the Content of Paeoniflorin, and the effect of extraction was the best. On further investigation to the time of ultrasonic extraction, we found that Paeoniflorin could be extracted at the maximum after the sample being extracted ultrasonically for 30min. In addition, the experiments compared the effect of different extraction solvents (methanol, mobile phase and redistilled water) and different number of extractions on the results, which revealed that methanol as extraction solvent would give better separation effect and Paeoniflorin would be extracted completely from the sample only one time.
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