川芎总酚酸提取工艺研究的研究医学毕业论文.doc

本科毕业论文（设计）论 文 题 目： 川芎总酚酸提取工艺研究 目 录 摘要IAbstractII前言11. 材料与方法11.1 仪器11.2 材料21.3 提取方法21.4 紫外分光光度法测定酚酸含量21.4.1 对照品溶液配制21.4.2 供试品溶液配制21.4.3 最大波长选择21.4.4 标准曲线绘制41.4.5 精密度试验51.4.6 稳定性试验51.4.7 重复性试验51.4.8 加样回收试验61.4.9 样品测定62. 结果62.1 均匀设计优化提取工艺62.2 因素水平设置62.3 实验安排及结果72.4工艺筛选82.5 优选工艺条件验证试验83. 结论94. 讨论9参考文献10译文原文11致谢24 川芎总酚酸提取工艺研究摘 要 目的：寻找川芎总酚酸提取优化方法。方法：选用L9(34)进行均匀设计实验对阿魏酸和总酚酸提取工艺进行优选，以阿魏酸含量和总酚酸含量为指标，考察乙醇浓度、液固比、提取时间及提取次数对总酚酸和阿魏酸提取的影响。结果：采用80%乙醇、液固比为12倍量、提取时间为2h、提取3次，川芎中的阿魏酸及总酚酸，可以获得较高的提取效率。结论：均匀设计法是优化多因素与多水平实验的有效方法，该优化工艺阿魏酸和总酚酸得率较高，稳定性好，适合于工业生产。关键词：川芎；总酚酸；均匀设计；紫外分光光度法Optimization of Extracting Technology of total phenolic acid of Ligusticum Chuanxiong Hort. Abstract Objective: To optimize an extraction method of total phenolic acid of Ligusticum Chuanxiong HortMethods: The extracting conditions was optimized bg using L9(34) homogeneous design. With the content of ferulic acid and total phenolic acid as the observation index. The effects of ethanol concentration, liquid-solid ratio, extrac- tiontime and extraction times were investigated. Results: The more effective way to extract phenolic acid from Ligustlcum chuanxiong Hort was extracting with the liquid solid ratio for 12 times amount of 80% alcohol for two hours, refluxing and extrac- tion for 3 times. Conclusion: The extraction method showed greater effects than others on the extraction efficiency of ferulic acid and total phenolic aci content. It is available for industrial productionKey words: Ligusticum chuanxiong Hort.; Total phenolic acid; Homogeneous design; UV spectrophotometric method前 言川芎为伞形科植物川芎(Ligusticum chuanxiong Hort.)的干燥根茎，气浓香，味苦、辛，是常用中药，始载于神农本草经，列为中品，被誉为“血中气药”。川芎及川芎为主的制剂对冠心病心绞痛有很好的疗效，通过配伍，可用于坐骨神经痛、末梢神经炎、缺血性脑病等疾病的治疗。另外，川芎可代茶饮美容、制成耳枕能辅助治疗高血压。川芎含生物碱、 酚类、内酯类以及中性油类等多种成分1。由于化学成分复杂，有效成分药理活性表现出多种作用，主要为：抑制血小板激活，防治脑缺血，保护血脑屏障，改善血液流变性，抗脂氧化作用，对心血管系统作用，清除氧自由基、防治再灌注损伤作用，抗过敏、抗血管炎症作用，减轻肺水肿，防治急性呼吸窘迫综合征和急性肺损伤，抗炎和抗肿瘤等活性，同时对心、肝、肺、肾、血液系统均无损害2，临床主要用于治疗脑血管疾病、肺心病、慢性肾病与肝病、神经性疼痛等。川芎对呼吸系统、骨髓造血、泌尿和消化系统、心脑血管系统、胃功能有一定的作用和影响，并具有解痉、抑制血小板凝集、抗癌、抗自由基作用1,3-6。阿魏酸、咖啡酸、原儿茶酸、亚油酸、瑟丹酸、大黄酚、川芎酚、香草酸及对羟基苯甲酸等酚酸类成分具有抗血栓、降血脂、防治冠心病等药理活性7。目前国内外关于川芎的研究较为广泛和深入，一些以川芎有效成分为原料制成的药物在临床上具有显著的疗效。川芎价格低廉，作用广泛，对川芎酚酸生物活性、中药配伍、结构改造等方面的研究，为总酚酸提供广泛的应用前景；阿魏酸和总酚酸的含量和稳定性，是研究酚酸类药理活性的前提。因此，我们对总酚酸的提取方法进行了较为深入的研究。均匀设计是一种以少量实验处理多因素多水平的方法，近来得到广泛的应用。该种设计具有方便、迅速、准确、减少实验次数、节省实验时间和费用等优点。刘旭等用正交试验比较了热浸法、超声法、渗漉法、热回流法四种提取工艺，以阿魏酸含量为考察指标，得出热浸法是比较好的方案8。本实验采用均匀设计法安排实验，选取乙醇浓度、回流时间、乙醇倍量和提取次数4个因素，各取3个水平，采用热回流法，优选出川芎总酚酸较为合理的提取工艺，并对其结论进行验证，该研究为川芎总酚酸的提取分离纯化奠定基础，为生产工艺参数的选择提供一定的科学数据。1. 材料与方法1.1 仪器14目标准筛，孔径1.43 mm（浙江上海市道墟张兴纱筛厂）；SHZ- III型循环水真空泵（上海亚荣生化仪器厂）；BUCHI Rotavapor R-200旋转蒸发仪（上海亚荣生化仪器厂）；DK-98-I电子恒温水浴锅（天津泰斯特）；DZF-2型真空干燥箱（北京市永光明医疗仪器厂）；SG5200HPT超声波（上海冠特超声仪器有限公司）；SZ-93自动双重纯水蒸馏器（上海亚荣生化仪器厂）；TU1901型双光束紫外-可见分光光度计（北京普析通用仪器有限责任公司）；HZT-B100电子天平（d=0.1，福州华志科学仪器有限公司）；FA1004N电子天平（d=0.0001，上海精密科学仪器有限公司天平仪器厂）。1.2 材料甲醇（分析纯AR，上海振兴化工一厂）；十二烷基硫酸钠（化学纯CP，湖南湘中化学试剂开发中心出品）；六水三氯化铁（分析纯AR，天津市大茂化学试剂厂）；铁氰化钾（分析纯AR，天津致远化学试剂有限公司）；盐酸（分析纯AR，成都市科龙化工试剂厂）；阿魏酸对照品（99.6%，批号：110773-201012，中国药品生物制品检定所）；川芎购于（芝林大药房），经遵义医学院药学院生药学教研室杨建文副教授鉴定为：伞形科植物川芎的干燥根茎。1.3 提取方法 取干燥粉碎并过14目筛川芎，精密称取2.0 g川芎于25 mL圆底烧瓶中，按均匀设计所设计的各实验条件加入一定浓度、一定体积的乙醇，浸泡1 h；回流提取按设计（见因素水平设置表9）的次数和时间，趁热抽滤、合并滤液、减压回收乙醇至膏状、真空干燥、计算得膏率；以紫外分光光度法测定阿魏酸及总酚酸的含量。1.4 紫外分光光度法测定酚酸含量目前国内关于总酚酸含量的测定报道，主要采用铁氰化钾-三氯化铁等试剂显色，再以紫外分光光度法进行酚酸含量测定，如李文兰等9就通过该方法对川芎总酚酸含量进行了测定。1.4.1 对照品溶液配制 精密称取阿魏酸对照品2.3 mg，置于25 mL容量瓶中，加甲醇溶解并定容，即得阿魏酸0.092 mgmL1阿魏酸溶液。1.4.2 供试品溶液配制取川芎浓缩干燥恒重膏体适量，精密称定，置于25 mL容量瓶中，用少许甲醇超声溶解，定容至刻度。摇匀，作为样品液。1.4.3 最大波长选择 A. 阿魏酸最大波长选择 分别精密移取对照品溶液、供试品溶液1 mL于10 mL容量瓶中，用甲醇定容。以甲醇为空白，用紫外分光光度法，在200-800 nm之间扫描，结果表明：对照品溶液和供试品溶液在314 nm处有较大吸收，故选择314 nm为测定波长（见图1、2）。图1 对照品溶液光谱扫描Fig.1 Spectral scanning control solution图2 供试品溶液光谱扫描Fig.2 Spectral scanning the test solution B. 总酚酸最大波长选择 分别精密移取对照品溶液、供试品溶液1 mL于10 mL容量瓶中，分别加甲醇1 mL，0.3%十二烷基硫酸钠0.8 mL，0.6%六水三氯化铁-0.9%铁氰化钾（体积比为1 : 0.9）的混合液0.4 mL，混匀，在暗室放置5 min，加0.1 mol/L盐酸定容，置于暗室20 min，以不加供试品溶液为空白，用紫外分光光度法，在200-900 nm之间扫描，结果表明：对照品溶液和供试品溶液均在734 nm处有最大吸收（见图3），故选择734 nm为测定波长。图3 对照品溶液和供试品溶液最大波长扫描Fig.3 Control solution and the test solution maximum wavelength scanning1.4.4 标准曲线绘制 A. 阿魏酸标准曲线 精密吸取阿魏酸对照品溶液（0.0092 mg/mL）0.1、0.2、0.4、0.6、0.8、1.0 mL，注入10 mL容量瓶中，加甲醇定容，摇匀。以甲醇为空白，在314 nm波长下测定吸光度。以阿魏酸质量体积浓度为横坐标，吸光度为纵坐标，得回归方程为Y=88.594X+0.0126，R2=0.9994，结果见表1、图4。表1 阿魏酸浓度与吸光度Table 1 Consistency and absorbency of ferulic acid 浓度C（mg/mL）0.000920.00184 0.00368 0.00552 0.00736 0.0092吸光度A 0.096 0.1830.343 0.509 0.662 0.822图4 阿魏酸标准曲线Fig.4 Ferulic acid standard curve B. 总酚酸标准曲线 精密吸取阿魏酸对照品溶液（0.0092 mg/mL）0.1、0.2、0.4、0.6、0.8、1.0 mL，注入10 mL容量瓶中，加甲醇至2 mL，加0.3%十二烷基硫酸钠0.8 mL，0.6%三氯化铁-0.9%铁氰化钾（体积比为1 : 0.9）的混合液0.4 mL，混匀，在暗处放置5 min，加0.1 mol/L盐酸溶液至刻度，摇匀，置于暗处20 min，以显色剂为空白，在734 nm波长下测定吸光度。以阿魏酸质量体积浓度为横坐标，吸光度为纵坐标得回归方程为Y=156.77X+0.0384，R2=0.9982。结果表明：阿魏酸在0.9-9 g/mL范围内呈良好线关系，见表2和图5。表2 总酚酸浓度与吸光度Table 2 Consistency and absorbency of total phenolic acid浓度C（mg/mL）0.000920.00184 0.00368 0.00552 0.00736 0.0092吸光度A 0.1880 0.36130.61530.9188 1.1930 1.4638图5 总酚酸标准曲线Fig.5 Standard curve of total phenolic acid1.4.5 精密度试验 取同一份对照品溶液，重复测定6次，RSD%为0.639%，结果见表3，表明仪器精密度良好。表3 精密度试验结果Table 3 Precision of test resultN123456RSD%吸光度0.40050.40630.40670.40670.40710.40720.6391.4.6 稳定性试验 取同一样品溶液，分别于0、10、20、30、40、50、60 min测定吸收度，以阿魏酸计算总酚酸含量（见表4），结果表明：样品在2050 min内RSD%为2.72%，在10 min以内和50 min以后，吸光度差异较大，故样品溶液在2050 min内稳定。 表4 稳定性试验结果Table 4 Stability Test Results 时间t (min)0102030405060吸收度0.73550.80660.84200.86620.88310.89720.91101.4.7 重复性试验取同一样品5份，每份约17 mg，精密称定，按照“供试品溶液制备”项下操作，测定，样品中阿魏酸平均含量为0.744%，RSD为1.99%；总酚酸平均含量为2.079%，RSD为2.06%。结果表明，供试品重复性良好（见表5、6）。表5 阿魏酸重复性试验结果Table 5 Ferulic acid reproducible results样品号称样量（mg）阿魏酸的含量（%）阿魏酸平均含量（%）RSD(%)117.20.7540.7441.99217.10.726317.20.759417.10.731517.20.752表6 总酚酸重复性试验结果Table 6 Total phenolic acid reproducible results样品号称样量（mg）总酚酸含量（%）总酚酸平均含量（%）RSD(%)117.22.1332.0792.06217.12.072317.22.081417.12.015517.22.0961.4.8 加样回收试验 取已知阿魏酸和总酚酸含量的样品6份，每份约18 mg，精密称定，分别精密加入阿魏酸对照品溶液（0.092 mg/mL）0.1 mL，按照“供试品溶液制备”项下操作，测定吸光度，测得阿魏酸平均回收率为98.33%，RSD为0.75%（见表7）；总酚酸平均回收率为98.06%，RSD为0.10%（见表8）。表7 阿魏酸加样回收率试验Table 7 Ferulic acid recovery rate test编号称样量(mg)样品含量(mg)对照品加入量(mg) 实测含量(mg)回收率(%) 平均值(%) RSD(%)118.3 0.005272 0.0143298.34217.80.0052320.01424 98.703 18.10.005060 0.0092 0.01408 98.74 98.33 0.754 17.9 0.005040 0.01407 98.81517.9 0.005052 0.01409 97.04表8 总酚酸加样回收率试验Table 8 total phenolic acid recovery rate test编号称样量(mg)样品含量(mg)对照品加入量(mg) 实测含量(mg)回收率(%) 平均值(%) RSD(%)118.3 0.014640 0.0234198.20217.80.014096 0.0228698.13318.10.016144 0.0092 0.02484 98.01 98.06 0.10417.9 0.015336 0.02405 98.02517.9 0.015340 0.02404 97.961.4.9 样品测定 川芎提取液减压浓缩至膏状，真空干燥至恒重，精密称定，计算浸膏得率。取适量恒重膏体，精密称定，于25 mL容量瓶内，用少量甲醇超声溶解，定容，取适量的样品溶液于10 mL容量瓶内，按1.4.3（A和B）项操作，并以阿魏酸含量计总酚酸含量。2. 结果2.1 均匀设计优化提取工艺中药有效成分提取是中药生产过程中的关键环节，提取工艺首先要考虑有效成分的收率，同时还要考虑周期、可控性、经济实用性及产品质量，因此合理设计提取工艺条件至关重要。在确定溶媒的基础上，对影响有效成分提取率的主要因素进行研究。2.2 因素水平设置溶剂法提取中影响提取效率的因素主要有溶媒浓度、回流时间、溶媒用量和提取次数等。根据实际生产需要，本研究对醇浓度、回流时间、溶媒用量和提取次数4个因素，进行均匀设计，因素水平设置见表9。表9 因素-水平Table 9 Factors - the level水 平因 素A提取时间(h)B提取次数C溶媒用量(倍)D醇浓度(%)10.5187021.52108032.0312902.3 实验安排及结果 考察影响提取率的主要因素：乙醇浓度、乙醇用量、提取时间和次数，选取L9(34)进行试验。称取过筛的川芎药材2.0 g，按表10安排试验，以阿魏酸含量和总酚酸的含量为考察指标，试验方案及结果分析见表10、11、12。表10 均匀设计实验安排及结果Table 10 Experimental arrangements and results of Homogeneous design test试验号因 素浸膏得率（%）阿魏酸提取率（%）总酚酸的含量（%）ABCD1111121.140.7051.6232122224.070.7651.3733133324.680.8581.9724212321.030.6891.4925223123.840.7182.0706231221.640.7761.6477313224.010.8301.7758321319.970.6851.4959332125.830.7552.003均值10.7660.7410.7220.733均值20.7280.7230.7430.790均值30.7630.8030.8020.744均值1.6561.6301.5881.899均值1.7361.6461.6231.598均值1.7581.8741.9391.653极差0.0480.0800.0800.057极差0.1020.2440.3510.301 表11 影响川芎药材阿魏酸提取得方差分析Table 11 Affect the extraction of ferulic acid in Chuanxiong analysis of variance来源离均差平方和自由度方差F值F临界值A0.00420.0020.5164.46B0.01120.0061.4194.46C0.01020.0051.2904.46D0.00620.0030.7744.46误差0.038以上图表均值分析知，因素A：132，因素B：312，因素C：321，因素D：231；通过方差分析，得出影响阿魏酸提取率的因素为：BCDA；在所考察的范围内影响最大的是提取次数，其次是溶媒用量和醇浓度，而影响最小提取时间。综合阿魏酸各因素各水平的含量和均值大小，得各因素最佳组合为A1B3C3D2。表12 影响川芎药材总酚酸提取得方差分析Table 12 Affect the extraction of total phenolic acid in Chuanxiong analysis of variance来源离均差平方和自由度方差F值F临界值A0.01720.0090.1344.460B0.11220.0560.8844.460C0.22420.1121.7674.460D0.15420.0771.2154.460误差0.518 以上图表均值分析表明，因素A：321；因素B：321；因素C：321 ；因素D：132；通过方差分析，影响总酚酸提取的因素CDBA；在所考察的范围内影响最大的是溶媒用量，其次为醇浓度和提取次数，提取时间则影响最小。综合总酚酸各因素水平下的含量和均值大小，知各因素的最佳组合为A3B3C3D1。2.4工艺筛选以阿魏酸和总酚酸含量为指标，两种最佳工艺提取次数和溶媒用量相同，提取时间以多者为计，综合筛选得川芎的提取工艺为：80%乙醇，液固比为12倍，提取时间为2 h，提取3次。2.5 优选工艺条件验证试验按上述筛选出的川芎总酚酸提取最佳工艺进行验证试验：取川芎药材2.0 g，以12倍量的80%乙醇80C回流提取3次，每次120 min。回收乙醇，50C真空干燥。平行实验3个，结果见表13。表13 验证试验结果Table 13 Validation test results试验号 浸膏得率(%)阿魏酸含量(%) 总酚酸含量(%)124.06 0.909 2.051224.08 0.846 2.047324.07 0.836 1.929平均含量（%）24.07 0.863 2.093 以阿魏酸和总酚酸含量为评价指标，采用4因素3水平进行试验，研究提取工艺条件，确定川芎酚酸类成分的提取工艺条件为：80%乙醇、液固比为12倍量、提取时间为2 h、提取3次。在该工艺条件下，提取浸膏中阿魏酸含量达0.8%以上、总酚酸含量达2%以上，为川芎总酚酸分离纯化奠定基础，为研究川芎酚酸类药理作用、为以川芎酚酸类为主要有效成分的制剂、为以川芎为原料的药物进行质量控制等提供科学依据。3. 结论(1) 均匀设计法是多因素与多水平实验的一种实验方法，能减少实验次数，缩短实验时间，定量的预测优化条件和结果。该方法具有方便、适用、迅速、预测性好等特点，是优化提取工艺一个非常有效的工具。(2) 本实验以乙醇为提取溶剂，寻找川芎酚酸类成分提取优化工艺。以阿魏酸和总酚酸含量作为考察指标，选取4因素3水平进行均匀设计，对提取工艺条件进行优化，确定了川芎酚酸类成分的提取工艺条件为：80%乙醇，液固比为12倍量，提取时间为2 h，提取3次。在该工艺条件下，阿魏酸含量为0.863%，总酚酸含量为2.093%。4. 讨论(1) 阿魏酸性质不稳定，在80C以上就会发生分解反应，其酚酸类在高温下也不稳定，因此在提取、浓缩及干燥的过程中须严格控制温度，尽量在低温下进行操作。阿魏酸对光也不稳定，溶液久置后会发生化学变化，故样品液和对照品溶液应避光保存。如果在对照品溶液中加入少量的酸，可使溶液稳定性增加8。(2) 本实验采用铁氰化钾-三氯化铁等试剂对酚酸类显色后，以紫外分光光度法进行酚酸含量测定。温度、反应时间和放置时间对酚酸类溶液稳定性有明显的影响，须对空白液、对照品溶液、样品溶液加入各种试剂和测定时间严格控制，应保证最大波长扫描和含量测定在同一台仪器上操作。 (3) 实验结果表明，乙醇浓度、提取时间、乙醇用量、提取次数在川芎总酚酸提取过程中，均有影响。根据实验及实际情况，最终确定川芎提取工艺：乙醇浓度80%、溶媒为12倍、提取时间2 h、提取次数为3次。该工艺设备简单、成本较低、可行性好、所得浸膏阿魏酸和总酚酸含量较高，为川芎酚酸类成分的后续研究提供原料。参考文献 1 王文祥, 顾明, 蒋小刚, 等. 川芎化学成分研究J. 中草药, 2002, 33(1): 4-5. 2 祁淑玲. 川芎的研究及应用进展J. 天津药学, 2005, 17(1): 61-64.3 舒冰, 周重建, 马迎辉, 等. 中药川芎中有效成分的药理研究进展J. 中国药理学通报, 2006, 22(9): 1043-1047.4 李秋怡, 干国平, 刘焱文. 川芎化学成分及药理研究进展J. 时珍国医国药, 2006, 17(7): 1298-1299.5 包旭. 中国川芎近年来药理研究进展J. 四川生理科学杂志, 2000, 22(2): 15-16.6 肖永庆, 李丽, 游小琳, 等. 川芎化学成分研究J. 中国中药杂志, 2002, 27(7): 519-522. 7 欧仕益, 包惠燕, 蓝志东. 阿魏酸及其衍生物的药理作用研究进展J. 中药材, 2001, 24(3): 220-221.8 刘旭, 杨雪梅, 徐江平, 等. 正交试验对川芎提取工艺的筛选研究J. 广东药学, 2003, 13(6): 3-5.9 李文兰, 范玉奇, 季宇彬, 等. 均匀设计法优选川芎中阿魏酸和总酚酸的提取工艺J. 中 药与天然药, 2007, 24(3): 198-201. 译文原文 RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning LyophilizerXiaorui Guo, Xiaohui Chen, Weiming Cheng, Kaiyu Yang, Yongfen Ma, Kaishun BiSchool of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, Liaoning Province,Peoples Republic of China; E-Mail: Received: 28 November 2007 / Revised: 14 February 2008/ Accepted: 29 February 2008Online publication: 18 April 2008AbstractA sensitive, simple, and accurate method for determination and phmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C18 column (2504.6 mm i.d. with 5 m particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were liear over the range 5.780-5780 ngmL-1 for ferulic acid and 1.740-348.0 ngmL-1 for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ngmL-1 for ferulic acid and 1.740 ngmL-1 for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.KeywordsColumn liquid chromatography; Pharmacokinetic study; Ferulic acid and isoferulic acid; Guanxinning LyophilizerIntroductionTraditional Chinese medicines (TCMs) are natural therapeutic agents used in accordance with the guiding theory of traditional Chinese medical science. TCMs are used mostly in combinations in China and made into preparations for easy and efficient use. Guanxinning injection, which was recorded in a ministerial standard 1, is a clinical medicine commonly used in China for the treatment of coronary artery disease and angina pectoris 2-4. It is consisting of two well- known Chinese TCMs Salvia miltiorrhiza Bge. (SMB) and Ligusticum chuanxiong Hort. (LCH). In order to improve the stability of Guanxinning liquid injection, the injection was made into lyophilizer. The main chemical components of Guanxinning Lyophilizer are phenolic acids, which play an important role in its potency 5-15. Ferulic acid (FA) and isoferulic acid (IFA) were reported to be active for the treatment of coronary heart disease, angina pectoris, heart-stroke and cardiovascular disease 6,7. FA has been used as a marker for in-vitro monitoring of the quality of Guanxinning injection by thin-layer chromatography 1. Pharmacokinetic studies of active ingredients in TCMs and traditional Chinese medicinal preparations (TCMPs) will greatly help in illustrating their action mechanisms and in promoting the development of TCM and TCMP. Currently, RP-LC methods have been employed for the determination of FA in crude drugs and TCMPs 16-18, but there was no report on pharmacokinetic studies of IFA. As far as we are aware, no report is available on pharmacokinetic studies of Guanxinning injection. In order to support preclinical pharmacokinetic studies requiring quantitation of FA and IFA, a sensitive, simple and accurate method for the determination and pharmacoki- netic study of FA and IFA in rat plasma after oral administration of Guanxinning Lyophilizer was developed.Experimental Chemicals and ReagentsFerulic acid was purchased from the National Institute for Control of Biological and Pharmaceutical Products (Beijing, China). Isoferulic acid was purchased from Acros Oranics (New Jersey,USA). Coumarin, used as internal standard (IS), was purchased from Sigma (St Louis, USA). Methanol and formic acid were of LC grade. Double distilled water was used throughout the work. All dilutions were performed in standard volumetric flasks.InstrumentationThe LC system consisted of a Shimadzu LC-10ATVP chromatograph, a SPDM10AVP detector, and a column oven (Shimadzu Corporation, Japan).Chromatographic ConditionsThe mobile phase consisted of water (1%, v/v formic acid)-methanol (33:67, v/v), filtered through a 0.45 m Nylon membrane filter under vacuum and degassed prior to use. The analysis was run at a flow rate of 1.0 mLmin1. The detector was set at a wavelength of 320nm. The chromatographic separation of the analytes was achieved at 35oC using Kromasil C18 (2504.6 mm i.d. with 5m particle size) analytical column from Zhonghuida Scientific Instrument Co. (Dalian, China) fitted with a guard C18 column (5 m). Data were acquired and processed with a Class-VP Ver. 6.0 chromatography data system.Preparation of Stock and Working Standard SolutionsBy dissolving different accurately weighed amounts of standards in solution of mobile phase the stock solutions were gained as follows: FA 57.8 gmL1, IFA 3.48 gmL1, and the IS 4.88 gmL1. Different volumes of each stock solution were transferred into volumetric flasks and then diluted to volume to make working standard solutions with the mobile phase.Preparation of Calibration StandardsTo 200 L of blank rat plasma, 20 L of the standard working solutions were added to yield final respective concentration ranges at: 5.780-5780 ngmL1 for FA in plasma, and 1.740-348.0 ngmL1 for IFA in plasma. Quality control (QC) samples (FA 11.56, 1156, 4624 ngmL1, IFA 3.480, 34.80, 278.4 ngmL1) were independently prepared in the same manner.Sample Extraction ProcedureTo 200 L plasma 50L IS solution and 600 L methanol was added. Each tube was mixed thoroughly by vortex mixing for 3 min and then centrifugated at 10464g for 5 min. The supernatant liquid was transferred into a clean labeled test tube and evaporated to dryness at 30 oC under a stream of nitrogen. The residual was reconstituted in 100 L mobile phase with vortexing for 1 min. After centrifugated at 10464g for 5 min, a 20 L aliquot was injected into the chromatographic column.Application to Pharmacokinetic Study in R