| Xianggui Huazhuo capsules is a prescription for the clinical application of the First Hospital of Hebei Medical University, years of experience side by cinnamon, earth rhubarb, patchouli. Its efficacy are considered to be aromatic Xingpi, clear dampness cloud of power. After attending various diseases due to remain entirely evil, spleen and internal injuries caused by long loose stool or mucus, bowel distension, anorexia or fever, greasy tongue coating, slippery or see yellowish or white, pulse slow; Mainly it is used to chronic enteritis,fungal enteritis caused by spleen wet storm treatment. There are some reports about its preparation process, quality control and toxicology. The major Chemical compositions in the Xianggui Huazhuo capsule are cinnamic aldehyde and patchouli alcohol. The studies are about the concentration of cinnamic aldehyde, the concentration of cinnamic acid and tissue distribution in the blood. Though the studies we can expect to improve the effective and scientific nature, rationality of the caplues on the clinical application.This paper reported the research of the Xianggui Huazhuo capsules by HPLC, and GC-MS methods. We established the concentration of cinnamic aldehyde, the concentration of cinnamic acid and tissue distribution in the blood of Xianggui Huazhuo capsules.In this study, we combined natural medicine chemistry, analytical chemistry, and pharmacy to study the concentration of cinnamic aldehyde, the concentration of cinnamic acid and tissue distribution. The studies can lead a meaningful exploration for effective and scientific nature, rationality of the caplues on the clinical application. Part one Determination of cinnamic aldehyde content in XiangguiHuazhuo capsules by GC-MSObjective: To establish a method for determining cinnamic aldehyde content in Xianggui Huazhuo capsules by GC-MS.Methods:1 GC-MS: HP-5 capillary column(30 m×0.25 mm,0.25 μm); the carrier gas was Helium; the flow rate was 1.0 m L·min-1 and the split ratio was 50:1; a programmed temperature was employed, feed injector temperature was 230 ℃; Injection volume was 1.0 μL. Electron impact(EI) was 230 ℃and electron energy was 70 e V; column pressure was 65.072 k Pa with the quadrupole mass analyzer.2 Methodology: The system suitability, specificity, linearity, limit of de- tection and limit of quantification, precision, repeatability, stability and recovery of the method were determined.3 Determination of cinnam aldehyde content: 3 batches of sample solut- ion were determined, and the data was processed by standard curve method to calculate the content of cinnamic aldehyde.Results:1 Methodology: The cinnamic aldehyde was separationed well with the other ingredients, and the oretical plate number > 50 000; the good linear relationships of cinnamic aldehyde(0.02~4.00 mg·m L-1) were observed. The linear regression equation was Y=281 786X+2E+06, and the correlation coefficient was 0. 999 4. the lowest detection limit was 0.75 μg·m L-1, and 2.51 μg·m L-1 as the limit of quantification. The RSD of the precision test was 1.64%, indicating that the method with good precision. The sample solution of Cinnam aldehyde was stable within 24 h with the RSD of the stability test being 1.76%. The RSD of the repeated test was 1.81%, and the average recovery was 96.2%, RSD was 2.11%.2 Determination of the content: The content of cinnam aldehyde in Xia- nggui Huazhuo capsules was determinated by standard curve method. The content of cinnam aldehyde wasn`t less than 0.48 mg in a capsule with the total weight of the capsule being 0.39 g.Conclusion: The determination of the cinnamic aldehyde content in Xianggui Huazhuo capsules was by GC-MS. The method is simple, accurate and suitable for content determination of cinnamic aldehyde. Part two Pharmacokinetic studies of cinnamic aldehyde in XiangguiHuazhuo capsules in ratObjective: The experiment of studing the drug concentration in the rat plasma after oral administration of Xianggui Huazhuo capsule was made by HPLC. The appropriate processing method was carried to analysis the data, to improve the rationality and the validity of the clinical therapeutic effect.Method:1 Optimization of the chromatographic conditions: The Chromatographic conditions such as mobile phase, wavelength and column temperature were tested for choosing the best one.2 Methodology: The system suitability, specificity, linearity, limit of de- tection and limit of quantification, precision, repeatability, stability and recovery of the method were determined.3 Determination of cinnamic acid in rat plasma: The drug concentration in the rat plasma was determinated, the plasma concentration-time curve of cinnamic acid was made to conculate the pharmacokinetic parameters.Results:1 Optimization of the chromatographic conditions: The column was Dia- monsil TM C18(250 mm×4.6 mm, 5 μm). The mobile phase was consisted by methanol-acetonitrile-aqueous acetic acid(0.1%). The detection wavelength was set at 270 nm, the flow rate was 1.0 m L·min-1 and the column temperature was room temperature. and the sample injection volume was 20 μl. The sensitivity was 1.0 AUFS.2 Methodology: The theoretical plate number was not less than 8000, and the degree of separation of the cinnamic acid peak with the other peaks was not greater than 1.5. There was no inference to the analysis in the blank plasma. The linear range of cinnamic acid was 0.2 to 160 μg·m L-1(r = 0.999 4) with the regression equation Y = 27.086 X + 0.503. The lower limit of quantification was for 0.2 μg·m L-1, with the lower limit of detection for 0.15 μg·m L-1. The RSD of the precision test were 2.87%,1.70%,0.48%, indicating that the method with good precision. The sample solution of Cinnam aldehyde was stable with the RSD of the stability test being 2.4%,0.25%,1.01%, and the average recovery was in the specified range.3 Determination of cinnamic acid in rat plasma: The plasma concentratei- on-time curve of cinnamic acid conformed to two- compartment open model. The pharmacokinetic parameters were as follows: tmax, t1/2β: 15.6 min, 11.077 min.Conclusion: In this paper, the method of HPLC, with cinnamic acid as the reference peak, the screened chromatographic conditions were used for the study of pharmacokinetics in the rat. The results were suitable for the requirements of determination of biological sample. Cinnamic aldehyde was metabolized to cinnamic acid in vivo quickly, about half an hour reaching the max plasma drug concentration, and decreased with time quickly. The plasma concentration-time curve of cinnamic acid conformed to two-compartment open model, with the short distribution and elimination half-life. It was conformed that the absorption and distribution were rapid.The method is simple, rapid, sensitive to determinate the plasma concentration of cinnamic acid after oral Xianggui Huazhuo capsules. Part three The drug concentration studies of cinnamic aldehyde inXianggui Huazhuo capsules in rat tissuesObjective: The experiment of studing the drug concentration in the rat tissues after oral administration of Xianggui Huazhuo capsule was made by HPLC. The appropriate processing method was carried to analysis the data, to improve the rationality and the validity of the clinical therapeutic effect.Method:1 Optimization of the chromatographic conditions: The Chromatographic conditions such as mobile phase, wavelength and column temperature were tested for choosing the best one.2 Methodology: The system suitability, specificity, linearity, limit of det ection and limit of quantification, precision, repeatability, stability and recovery of the method were determined.3 Determination of cinnamic acid in rat tissues: The distributions of cin- namic acid in the liver, kidney, heart, lung, spleen and stomach of the rat were determination to analysis the process after the cinnamic aldehyde metabolizing the cinnamic acid.Results:1 Optimization of the chromatographic conditions: The column was Dia- monsil TM C18(250 × 4.6 mm, 5 μm). The mobile phase was consisted by methanol-acetonitrile-aqueous acetic acid(0.1%). The detection wavelength was set at 270 nm, the flow rate was 1.0 m L·min-1 and the column temperature was room temperature. and the sample injection volume was 20 μl. The sensitivity was 1.0 AUFS.2 Methodology: The theoretical plate number was not less than 8000, and the degree of separation of the cinnamic acid peak with the other peaks was not greater than 1.5 in the tissues. There was no inference to the analysis in the blank tissues. The linear range of cinnamic acid was 0.4 to 600 μg·m L-1. The regression equation in the tissues were as follows, plasma: Y = 27.086 X + 0.503, r = 0. 999 4; liver: Y =0.428 9 X- 0.241,r = 0. 999 9; kidney: 0.496 3 X + 0.382 3, r = 0. 999 6; heart: Y = 0.737 X + 0.7353, r = 0. 999 9; lung: Y = 0.728 4 X + 0.388 6, r = 0. 999 9; spleen: 0.7115 X + 0.4163, r = 0. 999 7; stomch: Y =0.706 1 X + 0.132 2, r = 0. 999 5. The lower limit of quantification was for 0.25 μg·m L-1, with the lower limit of detection for 0.15 μg·m L-1. The RSD of the precision test at low, middle and high levels were as follows, plasma: 2.87%, 1.70%, 0.48%; liver: 2.54%, 1.46%, 1.02%; kidney: 2.35%, 1.69%, 1.21%; heart: 1.28%, 2.43%, 2.18%; lung: 2.50%, 1.70%, 2.01%; spleen: 1.19%, 1.84%, 0.46%; stomch: 2.36%, 2.46%, 1.27%. The RSD of the stability test at low, middle and high levels were as follows, plasma: 2.4%, 0.25%, 1.01%; liver: 1.48%, 0.83%, 1.87%; kidney: 2.04%, 1.94%, 1.45%; heart: 1.15%, 1.94%, 2.01%; lung: 2.08%, 1.86%, 1.93%; spleen: 2.52%, 1.97%, 1.20%; stomch: 2.25%, 2.25%, 1.46%. The average recoveries at low, middle and high levels were as follows, plasma: 99.96%, 99.63%, 106.55%; liver: 86.74%, 97.07%, 101.92%; kidney: 87.22%, 103.45%, 97.93%; heart: 99.68%, 94.17%, 97.72%; lung: 90.46%, 92.39%, 95.37%; spleen: 87.63%, 100.52%, 101.29%; stomch: 87.85%, 103.22%, 98.01%.3 Determination of cinnamic acid in rat tissues: The cinnamic acid wasn`t detected in brain, but could be detected in the liver, kidney, heart, lung, spleen and stomach. The content distribution in different tissues were as follows: stomach>lung>liver>kidney>spleen>heart.Conclusion: Cinnamic aldehyde was metabolized to cinnamic acid in vivo, decreased with time quickly, eliminated completely in 5 hours. It was descripted that the cinnamic acid was absorpted and distributed rapidly in vivo, and it can reach the effective concentration quickly through extravascular administration. The metabolism of Cinnamic acid in the heart samples was conformed faster than the other organizations, and the metabolism in the lung was the longest. In this paper, the method of HPLC, with cinnamic acid as the reference peak, the screened chromatographic conditions were used for the study of pharmacokinetics in the rat. The results were suitable for the requirements of determination of biological sample. |
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