Message Board

Respected readers, authors and reviewers, you can add comments to this page on any questions about the contribution, review,        editing and publication of this journal. We will give you an answer as soon as possible. Thank you for your support!

Name
E-mail
Phone
Title
Content
Verification Code
x Close Forever Close
Volume 41 Issue 6
Jun.  2023
Turn off MathJax
Article Contents
Article Navigation >  Journal of Pharmaceutical Practice and Service  > 2023  >  41(6): 358-365

DAI Yuanyuan, MA Minghua, XU Xiaohong, WANG Xiaohe, ZHANG Ruoxi, XU Zhiru, NIAN Hua. In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 358-365. doi: 10.12206/j.issn.2097-2024.202111008
Citation: DAI Yuanyuan, MA Minghua, XU Xiaohong, WANG Xiaohe, ZHANG Ruoxi, XU Zhiru, NIAN Hua. In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 358-365. doi: 10.12206/j.issn.2097-2024.202111008
shu

In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS

doi: 10.12206/j.issn.2097-2024.202111008
  • 1.

    Yueyang hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Tradional Chinese Medcine, Shanghai 200437 China

  • 2.

    Department of Pharmacy, Yangpu Hospital, Tongji University, Shanghai 200090 China

  • 3.

    The Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 201306 China

  • 4.

    State Key Laboratory of New Drug & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203 China

  • 5.

    Shanghai International Medical Center, Shanghai 200120 China

  • Received Date: 2021-11-02
  • Rev Recd Date: 2022-03-27
    • Available Online: 2023-07-14
  • Publish Date: 2023-06-25
  •   Objective   To establish a method and study the pharmacokinetics for concentration determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS.   Methods  The mobile phase was methanol-water (0.1% formic acid) system under the positive ion mode of C18 chromatographic column, gradient elution was adopted, and the flow rate was 0.3 ml/min. In the negative ion mode, the mobile phase was acetonitrile-water (0.1% formic acid) system, gradient elution, with a flow rate of 0.4 ml/min. Caffeic acid, rosmarinic acid, syringic acid, rutin in positive ion mode and Atractylodes lactone II and Atractylodes lactone III in negative ion mode were respectively determined. Normal rats were intragastrically given Xiakucao Xiaoliu Mixture 7.8 ml/kg, and blood was taken from the orbit at different time points after the administration. The blood concentration was determined by the validated LC-MS/MS method and the non-standard DAS2.0 software was used. The pharmacokinetic parameters of rats after administration were calculated by the compartment model.   Results   The pharmacokinetic parameters belonged to non atrioventricular model. The pharmacokinetic characteristics of the four main anti-cancer active ingredients of Caffeic acid, Rosmarinic acid, Syringic acid and Atractylodes Ⅲ in rats after administration of Xiakucao Xiaoliu Mixture were significantly different from those reported in the literature after the administration of monomers.   Conclusion  The established method was simple, accurate and sensitive, which could be suitable for the content determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma, which would be a valuable information for the study on main anticancer active substances.
  • [1] 马明华, 汪晓河, 戴媛媛, 等. 夏枯草消瘤合剂对lewis肺癌小鼠的药效作用[J]. 药学实践杂志, 2020, 38(1):57-62.
    [2] 贺天临, 谢国群, 陈洁, 等. 夏枯草消瘤合剂联合化疗治疗中晚期非小细胞肺癌30例[J]. 上海中医药大学学报, 2015, 29(5):29-32. doi:  10.16306/j.1008-861x.2015.05.007
    [3] 戴媛媛, 汪晓河, 马明华, 等. 高效液相-高分辨飞行时间质谱对复方夏枯草消瘤方化学成分的鉴别[J]. 药学实践杂志, 2020, 38(2):138-142,155.
    [4] MIN J, SHEN H, XI W, et al. Synergistic anticancer activity of combined use of caffeic acid with paclitaxel enhances apoptosis of non-small-cell lung cancer H1299 cells in vivo and in vitro[J]. Cell Physiol Biochem,2018,48(4):1433-1442. doi:  10.1159/000492253
    [5] LI Y, JIANG F, CHEN L J, et al. Blockage of TGFβ-SMAD2 by demethylation-activated miR-148a is involved in caffeic acid-induced inhibition of cancer stem cell-like properties in vitro and in vivo[J]. FEBS Open Bio,2015,5(1):466-475. doi:  10.1016/j.fob.2015.05.009
    [6] TYSZKA-CZOCHARA M, BUKOWSKA-STRAKOVA K, KOCEMBA-PILARCZYK K A, et al. Caffeic acid targets AMPK signaling and regulates tricarboxylic acid cycle anaplerosis while metformin downregulates HIF-1α-induced glycolytic enzymes in human cervical squamous cell carcinoma lines[J]. Nutrients,2018,10(7):841-862. doi:  10.3390/nu10070841
    [7] LIAO X Z, GAO Y, SUN L L, et al. Rosmarinic acid reverses non-small cell lung cancer cisplatin resistance by activating the MAPK signaling pathway[J]. Phytother Res,2020,34(5):1142-1153. doi:  10.1002/ptr.6584
    [8] XU Y, HAN S, LEI K, et al. Anti-Warburg effect of rosmarinic acid via miR-155 in colorectal carcinoma cells[J]. Eur J Cancer Prev,2016,25(6):481-489. doi:  10.1097/CEJ.0000000000000205
    [9] 周勇, 王帅, 华银槐, 等. 迷迭香酸调节PD-1/B7-H1(PD-L1)信号通路对非小细胞肺癌细胞增殖的抑制研究[J]. 中华中医药学刊, 2020, 38(6):147-151,272. doi:  10.13193/j.issn.1673-7717.2020.06.035
    [10] ZHANG L, LIU R, NIU W. Phytochemical and antiproliferative activity of proso millet[J]. PLoS One,2014,9(8):e104058. doi:  10.1371/journal.pone.0104058
    [11] CHEEMANAPALLI S, ANURADHA C M, MADHUSUDHANA P, et al. Exploring the binding affinity of novel syringic acid analogues and critical determinants of selectivity as potent proteasome inhibitors[J]. Anti Cancer Agents Med Chem,2016,16(11):1496-1510. doi:  10.2174/1871520616666160513131928
    [12] KARTHIK G, ANGAPPAN M, VIJAYAKUMAR A, et al. Syringic acid exerts antiangiogenic activity by downregulation of VEGF in zebrafish embryos[J]. Biomed Prev Nutr,2014,4(2):203-208. doi:  10.1016/j.bionut.2014.01.007
    [13] 孔祥虎, 李志欣, 房丽君, 等. 芦丁对人肺癌A549/DDP细胞耐药性的逆转作用及其机制[J]. 中国免疫学杂志, 2019, 35(19):2332-2336. doi:  10.3969/j.issn.1000-484X.2019.19.006
    [14] KHAN F, PANDEY P, UPADHYAY T K, et al. Anti-cancerous effect of rutin against HPV-C33A cervical cancer cells via G0/G1 cell cycle arrest and apoptotic induction[J]. Endocr Metab Immune Disord Drug Targets,2020,20(3):409-418. doi:  10.2174/1871530319666190806122257
    [15] SALEH A, ELFAYOUMI H M, YOUNS M, et al. Rutin and orlistat produce antitumor effects via antioxidant and apoptotic actions[J]. Naunyn Schmiedebergs Arch Pharmacol,2019,392(2):165-175. doi:  10.1007/s00210-018-1579-0
    [16] TIAN S, YU H D. Atractylenolide II inhibits proliferation, motility and induces apoptosis in human gastric carcinoma cell lines HGC-27 and AGS[J]. Molecules,2017,22(11):1886. doi:  10.3390/molecules22111886
    [17] WANG J, NASSER M, ADLAT S, et al. Atractylenolide II induces apoptosis of prostate cancer cells through regulation of AR and JAK2/STAT3 signaling pathways[J]. Molecules,2018,23(12):3298. doi:  10.3390/molecules23123298
    [18] ZHANG R, WANG Z, YU Q, et al. Atractylenolide II reverses the influence of lncRNA XIST/miR-30a-5p/ROR1 axis on chemo-resistance of colorectal cancer cells[J]. J Cell Mol Med,2019,23(5):3151-3165. doi:  10.1111/jcmm.14148
    [19] JI G Q, CHEN R Q, WANG L. Anti-inflammatory activity of atractylenolide III through inhibition of nuclear factor-κB and mitogen-activated protein kinase pathways in mouse macrophages[J]. Immunopharmacol Immunotoxicol,2016,38(2):98-102. doi:  10.3109/08923973.2015.1122617
    [20] YOOU M S, NAM S Y, JIN M H, et al. Ameliorative effect of atractylenolide III in the mast cell proliferation induced by TSLP[J]. Food Chem Toxicol, 2017, 106(pt a): 78-85.
    [21] KONISHI Y, HITOMI Y, YOSHIDA M, et al. Pharmacokinetic study of caffeic and rosmarinic acids in rats after oral administration[J]. J Agric Food Chem,2005,53(12):4740-4746. doi:  10.1021/jf0478307
    [22] 郭少波, 徐露露, 蒋丽娟, 等. 迷迭香酸的大鼠体内代谢产物及代谢途径分析[J]. 中国中药杂志, 2019, 44(21):4704-4712. doi:  10.19540/j.cnki.cjcmm.20190314.001
    [23] WANG R, WANG G, HAO H, et al. Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2006,831(1-2):36-41. doi:  10.1016/j.jchromb.2005.11.026
    [24] WAXMAN D J. Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme[J]. J Biol Chem,1984,259(24):15481-15490. doi:  10.1016/S0021-9258(17)42574-9
    [25] WAXMAN D J, DANNAN G A, GUENGERICH F P. Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes[J]. Biochemistry,1985,24(16):4409-4417. doi:  10.1021/bi00337a023
    [26] 赵阳, 柳晓泉, 钱之玉, 等. 药物代谢的性别差异[J]. 药学进展, 2001, 25(5):289-293. doi:  10.3969/j.issn.1001-5094.2001.05.008
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(4)  / Tables(7)

Get Citation
PDF
XML

Article Metrics

Article views(5526) PDF downloads(14) Cited by()

Related
Proportional views

In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS

doi: 10.12206/j.issn.2097-2024.202111008
  • 1. Yueyang hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Tradional Chinese Medcine, Shanghai 200437 China
  • 2. Department of Pharmacy, Yangpu Hospital, Tongji University, Shanghai 200090 China
  • 3. The Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 201306 China
  • 4. State Key Laboratory of New Drug & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203 China
  • 5. Shanghai International Medical Center, Shanghai 200120 China
  • Received Date: 2021-11-02
  • Rev Recd Date: 2022-03-27
  • Available Online: 2023-07-14
  • Publish Date: 2023-06-25

Abstract:   Objective   To establish a method and study the pharmacokinetics for concentration determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS.   Methods  The mobile phase was methanol-water (0.1% formic acid) system under the positive ion mode of C18 chromatographic column, gradient elution was adopted, and the flow rate was 0.3 ml/min. In the negative ion mode, the mobile phase was acetonitrile-water (0.1% formic acid) system, gradient elution, with a flow rate of 0.4 ml/min. Caffeic acid, rosmarinic acid, syringic acid, rutin in positive ion mode and Atractylodes lactone II and Atractylodes lactone III in negative ion mode were respectively determined. Normal rats were intragastrically given Xiakucao Xiaoliu Mixture 7.8 ml/kg, and blood was taken from the orbit at different time points after the administration. The blood concentration was determined by the validated LC-MS/MS method and the non-standard DAS2.0 software was used. The pharmacokinetic parameters of rats after administration were calculated by the compartment model.   Results   The pharmacokinetic parameters belonged to non atrioventricular model. The pharmacokinetic characteristics of the four main anti-cancer active ingredients of Caffeic acid, Rosmarinic acid, Syringic acid and Atractylodes Ⅲ in rats after administration of Xiakucao Xiaoliu Mixture were significantly different from those reported in the literature after the administration of monomers.   Conclusion  The established method was simple, accurate and sensitive, which could be suitable for the content determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma, which would be a valuable information for the study on main anticancer active substances.

DAI Yuanyuan, MA Minghua, XU Xiaohong, WANG Xiaohe, ZHANG Ruoxi, XU Zhiru, NIAN Hua. In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 358-365. doi: 10.12206/j.issn.2097-2024.202111008
Citation: DAI Yuanyuan, MA Minghua, XU Xiaohong, WANG Xiaohe, ZHANG Ruoxi, XU Zhiru, NIAN Hua. In vivo pharmacokinetic study on determination of effective components in Xiakucao Xiaoliu mixture in Normal Rat Plasma By LC-MS/MS[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 358-365. doi: 10.12206/j.issn.2097-2024.202111008
shu

    HTML

  • 夏枯草消瘤合剂源于著名中医肿瘤学专家钱伯文教授的经验方，是上海中医药大学附属岳阳中西医结合医院用于肿瘤治疗的特色院内制剂（批准文号：沪药制字Z05050340），已在临床应用30余年，对于肺癌、肝癌、甲状腺癌等癌症治疗效果显著，现已成为医院肿瘤内科的一线用药。该方由夏枯草、生地黄、牡蛎、煅牡蛎、莪术、麸炒白术、麸炒苍术等七味药组成，具有化痰、散结、解毒的功效[1-2]。

    课题组前期采用高效液相色谱-飞行时间串联质谱（HPLC-TOF/MS）对夏枯草消瘤合剂中的37种主要药效成分进行了定性鉴别[3]。通过文献检索，明确夏枯草消瘤合剂中主要含有咖啡酸[4-6]、迷迭香酸[7-9]、丁香酸[10-12]、芦丁[13-15]、白术内酯Ⅱ[16-18]、白术内酯Ⅲ[19-20]等多种具有抗肺癌药理作用的活性成分。尽管夏枯草消瘤合剂临床疗效显著，抗癌活性成分明确，但作用机制尚不明确，因此，对夏枯草消瘤合剂开展体内药动学研究十分有必要。

    笔者采用超高效液相色谱-串联质谱（UPLC-MS/MS）分析技术，利用正常大鼠，经口灌胃给药，对夏枯草消瘤合剂中6种主要的抗癌活性成分进行体内药动学研究，为其临床合理用药评价提供必要的科学支持。

    • 1.   材料

      1.1.   仪器

    • Xevo TQ-S超高效液相色谱-串联质谱（UPLC-MS/MS）；I-CLASS UPLC液相色谱（Waters ）；CP225D型电子天平（德国赛多利斯公司）；Heraeus Fresco 21小型高速冷冻离心机（赛默飞）。

      • 1.2.   试剂与药品

    • 夏枯草消瘤合剂（上海中医药大学附属岳阳中西医结合医院）；咖啡酸标准品（批号111871-201706，含量以99.7%计）、迷迭香酸标准品（批号110885-201703，含量以98.1%计）、芦丁标准品（批号100080-201811，含量以91.7%计）、白术内酯Ⅱ标准品（批号111976-201501，含量以99.9%计）、白术内酯Ⅲ标准品（批号111978-201501，含量以99.9%计），以上标准品均由中国食品药品检定研究院提供；丁香酸标准品（批号180919，北京北纳创联生物技术研究院，含量以97%计）；内标对照品甲苯磺丁脲标准品（99.7%）、卡利拉嗪标准品（99.5%）均由实验室提供。

      • 1.3.   动物

    • 健康SD大鼠6只，雌雄各半，每只体重约250 g[动物使用许可证：SYXK（沪）2019-0027]。

    2.   方法与结果

      2.1.   色谱及质谱检测条件

    • 色谱柱为 ACQUITY UPLC BEH C18 (2.1 mm×100 mm，1.7 μm)。正离子模式流动相为甲醇-水（0.1% 甲酸）；流速为 0.3 ml/min；进样量为2 μl；柱温为 40 ℃，内标为卡里拉嗪。负离子模式流动相为乙腈-水（0.1% 甲酸）；流速为0.4 ml/min；进样量为5 μl；柱温为50 ℃，内标为甲磺酸丁脲。离子源为电喷雾离子化源 (ESI)；源温度为120 ℃，雾化气体温度为 500 ℃；毛细管电压为3.2 kV；扫描方式为多反应监测(MRM)。待测物与内标的质谱检测参数见表1。

      化合物检测方式质谱扫描锥孔电压
      （P/V）      		碰撞电压

      （P /V）
      母离子碎片离子
      咖啡酸ESI-178.94135.0428.0016.00
      迷迭香酸ESI-359.02161.0329.0014.00
      丁香酸ESI-196.95181.9615.0012.00
      芦丁ESI-609.09299.9839.0032.00
      甲苯磺丁脲(IS)ESI-268.94169.8443.0018.00
      白术内酯ⅡESI+233.07187.1332.0016.00
      白术内酯ⅢESI+293.1163.0955.0018.00
      卡利拉嗪(IS)ESI+427.26382.0425.0024.00

      • 2.2.   对照品溶液的配制

    • 精密称取咖啡酸、迷迭香酸、芦丁、丁香酸、白术内酯Ⅱ、白术内酯Ⅲ标准品适量，甲醇溶解配制1 mg/ml的对照品储备液。前4种储备液按比例混合后使用50%甲醇稀释，得到混标对照品溶液a；另取白术内酯Ⅱ、白术内酯Ⅲ储备液按比例混合后使用50% 甲醇稀释，得到混标对照品溶液b。

      • 2.3.   内标物溶液的配制

    • 分别精密称取甲苯磺丁脲、卡利拉嗪标准品，甲醇溶解配制1 mg/ml的对照品储备液，并用80%甲醇稀释至20 ng/ml及100 ng/ml，备用。

      • 2.4.   工作曲线和质控样品的制备

    • 取“2.2”项下混标对照品溶液a 5 μl、大鼠空白血浆50 μl，混匀得到不同浓度的工作曲线样本。加入内标溶液 5 μl、1 mol/L盐酸30 μl，混匀后加入400 μl乙酸乙酯，震荡10 min后离心(13 000 r/min，10 min，4 ℃)，取360 μl上清液，40 ℃条件氮气吹干。用50 μl 50%甲醇复溶，离心(13 000 r/min，3 min，4 ℃)，取上清即得负离子质控样本。正离子质控样本制备则取“2.2”项下混标对照品溶液b 5 μl，操作同前，在加入内标溶液后，加入5‰氢氧化钠溶液30 μl 后，同负离子质控样品操作，即得。对照品工作曲线和质控样品浓度见表2。

      待测物线性范围（ng/ml）质控样品浓度（ng/ml）
      低中高
      咖啡酸2~400540320
      迷迭香酸1.5~3003.7530240
      芦丁2.5~5006.2550400
      丁香酸5~100012.5100800
      白术内酯Ⅱ1.5~240324192
      白术内酯Ⅲ3~480648384

      • 2.5.   血浆样品处理

    • 精密吸取大鼠血浆样本50 μl，加入内标溶液5 μl、50%甲醇溶液5 μl后，按“2.4”项下处理，取上清液进行分析。

      • 2.6.   专属性研究

    • 空白大鼠血浆、定量下限样品及给药后血浆样品中，咖啡酸、迷迭香酸、丁香酸、芦丁（负离子模式）的色谱图见图1，白术内酯Ⅱ、白术内酯Ⅲ（正离子模式）的色谱图见图2，内标甲苯磺丁脲、卡利拉嗪的色谱图见图3。实验结果可见，大鼠血浆中的内源性物质均不干扰各待测成分以及内标的测定。

      • 2.7.   标准曲线及线性范围

    • 按“2.4”项下制备系列标准血浆样品溶液，每个浓度平行3份。以各待测物峰面积与内标峰面积的比值 (AS/AIS，Y) 对相应的浓度 (C，X) 进行加权回归（权重为1/X2），得到各待测物在大鼠血浆中的标准曲线数据，见表3。相关系数r>0.9983，各待测物在浓度范围内呈线性，且线性关系良好。

      待测物线性范围（ng/ml）标准曲线相关系数r
      咖啡酸2~400Y=7.31×10−4+1.28×10−3X0.9987
      迷迭香酸1.5~300Y=-4.79×10−4+8.11×10−4X0.9988
      芦丁2.5~500Y=5.96×10−4+8.98×10−5X0.9987
      丁香酸5~1000Y=1.13×10−4+3.55×10−5X0.9984
      白术内酯Ⅱ1.5~240Y=8.62×10−3+1.52×10−2X0.9983
      白术内酯Ⅲ3~480Y=2.05×10−5+1.98×10−3X0.9983

      • 2.8.   准确度和精密度

    • 按照“2.4”项下方法配制定量下限、低、中及高 4 个浓度的大鼠血浆质控样品各 5 份，连续测定3批。精密度与准确度结果见表4。

      成分理论浓度
      （ng/ml）      		日内（n=5）日间（n=5）
      RSD
      （%）      		准确度
      （%）      		RSD
      （%）      		准确度
      （%）
      咖啡酸26.780.816.695.6
      58.487.112.894.9
      407.599.08.897.3
      3207.996.76.496.3
      迷迭香酸1.512.0118.79117.3
      3.753.694.4996.4
      3012.094.510.796.3
      24011.0100.76.9102.3
      丁香酸56.1111.413.8105.2
      12.58.887.31396.9
      1007.393.512.594.7
      80011.6103.49.8105.3
      芦丁2.511.387.212.291.8
      6.253.493.912.695.4
      509.098.611.392.1
      4008.0102.09.996.9
      白术内酯Ⅱ1.511.995.712.5100.9
      39.3100.912.4101.3
      2410.997.811.899.8
      1928.799.78.993.9
      白术内酯Ⅲ314.895.915.4101.4
      612.4108.012.8105.9
      488.2102.97.7100.2
      3843.5103.19.299.1

      • 2.9.   提取回收率

    • 按照“2.4”项下方法配制低、高两个浓度的大鼠血浆质控样品，平行制备6份，作为基质样品。取大鼠空白血浆50 μl，按“2.4”项下操作至复溶时加入混标溶液 5 μl、内标溶液5 μl、50% 甲醇40 μl混匀并离心取上清液，作为提取基质样品。通过基质样品峰面积与提取基质样品峰面积的比值求得方法的回收率，结果见表5，回收率在50%~110%，RSD小于15%，待测成分提取回收率良好。

      成分提取回收率（%）RSD（%）
      咖啡酸75.16±0.045.5
      75.36±0.0911.9
      迷迭香酸76.56±0.056.3
      75.07±0.079.4
      芦丁60.62±0.0812.7
      60.62±0.059.0
      丁香酸103.9±0.076.3
      92.97±0.1314.1
      白术内酯Ⅱ53.48±0.0712.5
      50.04±0.047.5
      白术内酯Ⅲ58.47±0.069.9
      62.54±0.069.1
      卡利拉嗪(IS)74.47±0.079.8
      甲苯磺丁脲(IS)98.77±0.054.6

      • 2.10.   稳定性考察

    • 按照“2.4”项下方法配制低、高两个浓度的大鼠血浆质控样品均平行制备 5份，根据当日随行的标准曲线计算样品浓度，考察处理前放置 4 h、冻融循环1次（−80 ℃）、放置1周(−80 ℃)、处理后放置 4 h和进样器内(4 ℃)放置 24 h的稳定性，结果准确度在85%~115%范围内，RSD小于15%，在上述条件下样品稳定。

      • 2.11.   稳基质效应考察

    • 取对照品工作溶液 5 μl、内标溶液5 μl、50% 甲醇40 μl 混匀后，作为无基质样品进行 UPLC-MS/MS分析。按照“2.4”项下方法制备提取基质样品。低、高两个浓度样品均平行制备6份。

      通过提取基质样品中待测物峰面积与无基质样品待测物峰面积的比值比上提取基质样品中内标的峰面积与无基质样品中内标峰面积的比值即为基质效应，结果见表6，白术内酯Ⅲ基质效应较高，但高、低浓度对基质效应影响较为一致，不影响测量结果的准确性。基质效应RSD小于15%，样品中基质对方法无影响。

      成分基质效应（%）RSD（%）
      咖啡酸113.2±0.1110.0
      112.0±0.108.6
      迷迭香酸104.8±0.087.7
      108.6±0.043.8
      芦丁109.0±0.109.2
      104.1±0.065.9
      丁香酸114.5±0.1210.5
      110.0±0.109.4
      白术内酯Ⅱ99.7±0.109.9
      107.8±0.1514.0
      白术内酯Ⅲ166.7±0.0813.7
      169.8±0.0411.5

      • 2.12.   大鼠体内药动学

    • SD大鼠6只，采取灌胃给药7.8 ml/kg，给药后分别于0、10、20、30、45 min，以及 1、2、3、4、6、8、10 h 眼眶采全血约100 μl（1% 肝素抗凝），低温(4 ℃) 离心 8000 r/min，8 min，分别取50 μl 上清液于对应离心管中，−80 ℃低温备用。

      大鼠经口灌胃夏枯草消瘤合剂后，各待测物在大鼠体内的平均血药浓度-时间曲线见图4，采用 DAS2.0 软件的非房室模型计算分析大鼠给药后的药动学参数，详见表7。其中芦丁在大鼠体内血药浓度低于定量下限，白术内酯Ⅱ在生物样品中未被检测出，故未能进行计算。

      参数咖啡酸迷迭香酸丁香酸白术内酯Ⅲ
      雌性雄性雌性雄性雌性雄性雌性雄性
      AUC(0-t)（mg·h/L）384.1±2.946**156.6±3.658203.6±9.177**67.57±2.932151.5±9.637115.8±18.63114.0±2.869**10.32±3.749
      AUC(0-∞)（mg·h/L）395.5±7.342**161.8±4.451230.7±32.08*73.84±7.545167.0±11.45144.8±11.22125.3±5.275**12.33±2.454
      MRT(0-t)（t/h）1.596± 0.050.997±0.0522.265±0.1361.339±0.0561.219±0.0201.846±0.6161.959±0.2120.573±0.178
      MRT(0-∞)（t/h）1.771± 0.1221.110±0.053.285± 1.2982.205±0.7761.634±0.0673.082±0.6052.707±0.2530.890±0.213
      t1/2z（t/h）1.041 ±0.0940.765± 0.0602.160±1.0052.071±1.3341.199±0.0972.137±0.4021.797±0.346*0.426±0.040
      tmax（t/h）1**0.330.330.330.33*0.1670.330.33
      CLz/F（L/h）0.008 ±0.001**0.018± 0.0010.013±0.002**0.041±0.0040.018±0.0010.021±0.0020.024±0.001*0.249±0.045
      cmax（mg/L）208.2 ±6.721**136.0 ±3.798129.9±2.462**82.46±1.373125.057±3.101*85.510±13.59769.90±5.080**16.23±1.411
      *P<0.05, **P<0.01，与雄性大鼠组比较。
    3.   讨论

    • 本次实验采用单次灌胃给予夏枯草消瘤合剂，给药后不同时间点眼眶取血，利用LC-MS/MS检测合剂中主要药效成分（包括咖啡酸、迷迭香酸、丁香酸、白术内脂Ⅲ等）的血药浓度，并绘制“药-时曲线”，结果呈现出典型的血管外给药特征。其中，咖啡酸和迷迭香酸在大鼠体内代谢的半衰期及达峰时间较两种成分单独代谢均显著延长[21]，表明复方配伍可以减缓咖啡酸和迷迭香酸在大鼠体内代谢速度，延长两者在体内发挥作用的时间，起到了增加疗效的作用。其中，咖啡酸为迷迭香酸的代谢产物[22]，合剂中咖啡酸半衰期及达峰时间的延长可能与迷迭香酸等酚酸类成分的代谢相关，迷迭香酸的代谢造成了体内咖啡酸的总量增加，间接造成了咖啡酸“在体内拥有更长的滞留时间及进入血液循环更高的药物浓度”的表象。此外，丁香酸的药时曲线出现双峰，而此前未有文献报道此类情况，原因有待进一步研究。白术内酯III在复方中代谢与其单独代谢时相比达峰时间明显缩短[23]，可能是复方中的成分影响体内代谢酶活性，进而促进了白术内酯III的代谢。

      本研究结果显示，合剂中各成分的代谢均存在显著性别差异。咖啡酸在雌性与雄性大鼠体内暴露的AUC值具有2倍的差异，且雌性相较于雄性到达tmax（峰浓度时间）有30 min延迟，提示雌雄个体间药物进入血液循环的相对量及达峰时间存在明显的差异。同样，白术内酯Ⅲ的性别差异也极为明显，AUC存在约10倍的差异，MRT、t1/2、cmax存在4倍差异，提示白术内酯Ⅲ在雌性体内仍发挥药效的同时在雄性大鼠体内早已被清除。现有研究证实[24-25]，大鼠药动学的性别差异主要原因是雌性与雄性大鼠肝中CYP450酶的种类差异。大鼠与人类的CYP450酶系在酶的组成和活性上亦存在明显差异[26]。

      在夏枯草消瘤合剂用于临床治疗非小细胞肺癌的合理用药方案制订、与化疗药物等相互作用评价、药物本身体内代谢过程监测及根据患者性别不同的个体差异化给药方案调整等过程中，本次实验的研究结果将为中药临床药动学方面的评估提供科学依据。

Reference (26)

Catalog

    版权所有：《药学实践与服务》编辑委员会

    Supervisor & Sponsors: Address: No 325 Guohe Road, ShanghaiPost Code: 200433

    Tel: （021）81871324,81871397 E-mail: yxsjzzs@163.com

    网站备案号 沪ICP备05003363号-1

    Supported by: Beijing Renhe Information Technology Co., Ltd.  

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return