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       【摘要】 
       目的研究大鼠灌胃给予当归补血汤后阿魏酸在体内的组织分布特性。方法采用高效液相色谱法；色谱柱为Kromasil C18（4.6 mm×150 mm,5 μm）；流动相为0.2%甲醇乙腈-0.2%磷酸0.2%三乙胺水溶液（18：82）；检测波长322 nm；柱温40℃；流速0.8 ml/min。结果各组织中的阿魏酸在10～400 ng范围内与峰面积呈良好的线性关系；回收率平均为87.97%±1.95%；日内和日间的RSD分别1.92% ～3.87% 和3.21%～6.39% 之间。阿魏酸在各组织器官中浓度差异较大。结论大鼠灌胃给予当归补血汤后阿魏酸主要在肝脏和肾脏分布，阿魏酸不能通过血脑屏障。
       【关键词】  当归补血汤； 阿魏酸； 反相-高效液相色谱； 组织分布； 大鼠
       Tissue Distribution of Ferulic Acid after Oral Administration of Tangkuei Blood-Supplementing Decoction to Rats
       WANG Yonglu, WANG Liyao,  CHENG Guoguang, ZHANG Dongxu, WEI Ping
       (1. Nanjing University of Technology, Nanjing, 210009, China;. China Pharmacological University, Nanjing, 210009, China)
       Abstract:ObjectiveTo investigate the tissue distribution of ferulic acid in rats after oral administration of Tangkuei blood-supplementing decoction.MethodsHPLC was used；Column: Kromasil C18（150mm×4.6mm ，5μm）; mobile phase: 0.2%methanol acetonitrile-0.2% phosphoric acid 0.2%triethylamine solution（18：82）; detection wavelength was at 322nm; temperature was at 40℃; flow velocity was 0.8ml/min.ResultsThe linear ranges of ferulic acid in the heart, liver, spleen, lung and kidney were 10～400ng. The recovery of  ferulic acid was 87.97%±1.95% on average . The intra-day and inter-day RSD were 1.92%～3.87% and 3.21%～6.39%, respectively. The concentrations of ferulic acid were very different in organs. ConclusionThe major distribution organs of ferulic acid in rats are liver and kidney. Ferulic acid can not pass though the blood-barrier.
       Key words:Tangkuei blood-Supplementing decoction ;   Ferulic acid;   Tissue distribution；  Rats
       
       Tangkuei Blood-Supplementing Decoction has been widely used for tonifying blood and treating anemia, female irregular menstruation and amenorrhoea in the traditional Chinese medicine for hundreds of years. Previous studies have demonstrated that Tangkuei Blood-Supplementing Decoction can inhibit lipid peroxidation, maintain activities of SOD and GSH, enhance cell ability against oxidation, improve immune function, and lessen ischemia/reperfusion injury in the heart and the brain［1］. Ferulic acid, one of the important components of Tangkuei Blood-Supplementing Decoction［2］, diminished the production of H2O2 in hepatocytes and protected membrane lipid after ischemia/reperfusion. It also plays a role of dilating blood vessel［3］, inhibiting platelet aggregation, inhibiting inflammatory reaction, anti-oxydation and cleaning free radical, acting on eliminating and inhibiting. In the present study, the tissue distribution of ferulic acid in rats was investigated by high-performance liquid chromatography after oral administration of Tangkuei blood-supplementing decoction.
       1  Material and methods
       1.1  Apparatus and reagentsThe DIONEX HPLC system consisted of a P680 quaternary pump and a UVD170U variable wavelength detector. The data were collected and processed with a CHROMELEON (Version 6.60) chromatographic workstation. HT-230A thermostated column compartment was bought from Heng-ao Co. (Tianjin, China).
         
       Ferulic acid (FA) was provided by National Institute for the Control of Pharmaceutical and Biological Products (batch number: 0773-9910; for use of determination).The ultrapure water and HPLC-grade solvents, including methanol and acetonitrile were bought from Wan-qing Co. (Nanjing, China). All other chemicals were of analytical grade. Astragali radix and angelicae radix were bought from Nanjing Pharmaceutical Co. (Nanjing, China).
       1.2  AnimalsSprague-Dawley rats aged 6-8 weeks (provided by the Department of Laboratory Animals of Dongnan University, Nanjing China) weighing 160-250 g were used in the experiments.
       1.3  Chromatographic conditionColumn: Kromasil C18（150 mm×4.6 mm，5 μm）; mobile phase: 0.2% methanol acetonitrile-0.2% phosphoric acid 0.2% triethylamine solution（18：82）; detection wavelength was at 322 nm; temperature:40℃; flow velocity:0.8 ml/min; the injected volume:20 μl.
       1.4  Extraction process of the decoctionAccording to the recipe, angelicae radix 25 g and astragali radix 175 g were weighed, and decocted together. The extraction processes were repeated twice. The added water was 2 400 milliliter and the decocted time was 2 hours. Then the resulting solution was collected and concentrated. Finally 1g primary herbs were contained in 1ml.
       1.5  Sample collectionAfter a day of fast, the rats were given orally the decoction of Tangkuei blood-supplementing at the dose of 1.5 ml/100 g. Twenty minutes after the administration, the rats were decollated quickly and the heart, liver, spleen, lung, kidney and brain were dissected out and put into normal saline solution to remove the blood. The water was removed with filter paper, and then the tissue samples were weighed for wet weight and homogenized in saline solution, respectively. The supernatant was collected and centrifuged , which was stored at -20℃.
       1.6  Sample preparationThe procedures for analyzing the frozen tissue samples consisted of the following steps. The frozen tissue samples were thawed at 25℃ and were mixed with 500 μl HCl(0.1mol/L) and 6ml ethyl acetate. The resulting mixture was vortexed for an additional 1 min. The samples were centrifuged for 20 min at 3 000 r/min.The supernatant liquor about 5 ml was transfered to a suitable tube and evaporated to dryness under a stream of nitrogen gas at 40℃. After 5 μl methanolic acid was added, the residue was dissolved in 0.5ml of mobile phase. After filtration by millipore filter of 0.22μm, 20 μl of solution was injected into the chromatographic system, and the chromatograms were recorded.
       1.7  Method validation experiment
       1.7.1  Linear relationThe stock solution of ferulic acid was prepared by dissolving 1.85 mg ferulic acid in 25 ml methanol and 250 μl methanolic acid was added. Calibration samples of ferulic acid were prepared from the stock solution by appropriate dilution with absolute ethanol..
       
       Calibration samples at 10,25,50,100,200 and 400 ng/ml for ferulic acid were prepared by adding the appropriate calibration solution to 1.0ml of blank rat tissue homogenates. The samples were processed and analyzed by the methods described above. The calibration curve was established by plotting the peak areas against the concentrations of ferulic acid.
       1.7.2  The limit of detection (LOD)The limit of detection was measured through diluting the calibration samples to a certain degree resulting S/N=3.
       1.7.3  RecoveriesJust like the process for preparation of calibration curve, 508.22 ng/ml，101.60 ng/ml and 12.56 ng/ml of tissue homogenates samples were prepared to assess the recoveries. The recoveries were calculated by using the ratio of the detected peak areas to that of ferulic acid added.
       1.7.4  PrecisionInjecting continuously the tissue samples of 508.22 ng/ml，101.60 ng/ml and 12.56 ng/ml for five times and measuring the peak areas of ferulic acid.
       1.8  Quantitation The concentrations of ferulic acid in tissue sample were quantified from the calibration curve according to the peak areas.
       2  Results
       2.1  Performance of the HPLC systemRepresentative chromatograms are shown in Figure 1. The retention time of ferulic acid was about 9.3±0.7 min; No interference was observed. Ferulic acid can not be detected in cerebrum.
       2.2  Linearity and LODThe peak area (A) and concentration of ferulic acid (C) were subjected to regression analysis to calculate the calibration equation and correlation coefficient. The calibration equations of ferulic acid in each organ were shown in Table 1.The results showed an excellent correlation between the peak area and the concentration of ferulic acid. The limit of detection of the assay was calculated to be 9.8ng/ml by the ratio of signal to noise 3:1.
       2.3  Recoveries and precisionThe results are given in Table 2. The recoveries were over 86%. The average recoveries(87.97%±1.95%) were high enough to reach the normal analytical criterion. The intra-day RSD ranged from 1.92% to 3.87%. The inter day RSD ranged from 3.21% to 6.39%.
       2.4  The concentrations of ferulic acid in each organThe concentrations of ferulic acid (ng/g) in such major organs as heart, liver, spleen, lung and kidney are shown in Figure 2.
       3  Discussion
        Ferulic acid absorbed quickly in rats［4］ and rabbits［5］. The Tmax ranged from ten to twenty minutes. The sample collection time was decided to be twenty minutes, in which the concentrations of organ sample were the highest according to the preliminary experiment. Figure 2 illustrated the distribution of ferulic acid in different rat organs. The concentrations of ferulic acid were very different in organs:
       Cliver>Ckidney>Clung>Cheart>Cspleen>Ccerebrum at 15min after administration of Tangkuei blood-supplementing decoction. Ferulic acid could not be detected in rat brain, one possible reason was that ferulic acid could not pass though the blood-barrier and thus could not act on the central nervous system. The concentration of ferulic acid in heart was lower too, just as one-tenth of which in liver. This study clarifies that the major distribution organs of ferulic acid in rats are liver and kidney, where the blood flow rate is faster than other organs. Although Tangkuei Blood-Supplementing Decoction is efficient in lessening ischemia/reperfusion injury in heart and brain, the experiment result illustrated that the major site of ferulic acid may not the heart and brain.Table1  Linear equations and correlation coefficients of ferulic acid in each organ（略）Table2  Recovery and precision of the HPLC method to determine Ferulic Acid in rat tissues（略）
       【参考文献】
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       ［2］Lu Xin-hua, Zhang Jin-juan, Liang hong, et al. Chemical Constituents of Angelica sinensis［J］. Journal of Chinese Pharmaceutical Sciences, 2004, 13(1):1.
       
       ［3］CAI Qi, LI Xiao-mei, WANG Hai-yan. Astragali and Angelica protect the kidney against ischemia and reperfusion injury and accelerate recovery［J］. Chinese Medical Journal, 2001, 114(2):119.
       
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       ［5］SONG Jin-chun, LI Yu-qin, WANG Ren-song, et al. Comparison of the pharmacokinetics character of ferulic acid to Shenghuatang in rabbits by HPLC ［J］. Chin Hosp Pharm Journal, 2005, 25(3):213