葛根芩连汤中14种功效成分在大鼠体内的组织分布
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摘要
目的:研究葛根芩连汤(GQT)功效成分在大鼠体内的组织分布特征,通过方药功效成分在体内各组织中的靶向性揭示其作用机制。方法:大鼠给予GQT后不同时间点取组织(小肠、大肠、肝、心、脾、肺、肾),样品处理后采用HPLC-MS/MS分析14种功效成分在各组织匀浆液中的含量,Agilent ZORBAX SB-C_(18)色谱柱(2. 1 mm×100 mm,3. 5μm),流动相0. 1%甲酸水溶液-乙腈梯度洗脱,流速0. 4 m L·min~(-1),电喷雾离子源,正离子模式检测,选择柚皮苷作为内标物。结果:建立了同时测定组织匀浆液中14种GQT主要功效成分的HPLC-MS/MS分析方法,该方法的专属性、标准曲线、回收率、基质效应、精密度、准确度和稳定性良好。在小肠组织中,异黄酮类主要成分葛根素、大豆苷元和3’-羟基葛根素的药时曲线下面积(AUC_(0-10 h))分别为22 174. 9,3 882. 5,15 893. 1 h·mg·L~(-1),黄酮类主要成分黄芩素、汉黄芩素、汉黄芩苷和黄芩苷的AUC_(0-10 h)分别为15 423. 6,15 408. 4,7 017. 3,3 697. 7 h·mg·L~(-1),生物碱类主要成分小檗碱的AUC_(0-10 h)=2 544. 0 h·mg·L~(-1),这些成分在小肠组织的分布水平显著高于其他组织。结论:GQT的功效成分群主要分布于肠道组织,推测其抗炎、止泻的药理作用可能与其成分在肠道组织的作用趋向相关。
Objective: To investigate the tissue distribution of major bioactive components from Gegen qinlian Tang( GQT) in rats,and to reveal the mechanism for the efficacy of GQT by the tissue targeting of its bioactive ingredients in vivo. Method: After oral administration of GQT in rats,tissues were collected at different time points, including small and large intestine, liver, heart, spleen, lung, and kidney. Samples were prepared for determination of 14 bioactive components of GQT in tissue homogenate by HPLC-MS/MS. The chromatography separation was performed on an Agilent ZORBAX SB-C_(18) column( 2. 1 mm × 100 mm,3. 5 μm)with acetonitrile-0. 1% formic acid in water for gradient elution. Electrospray ionization( ESI) was applied and operated in the positive ion mode. Meanwhile, naringin was used as the internal standard for determining.Result: HPLC-MS/MS for simultaneous determination of 14 components from GQT in tissue homogenate was developed and validated by specificity, calibration curves, recovery test, matrix effect, precision, accuracy,and stability. In the small intestine,the the area under the curve( AUC_(0-10 h)) of major isoflavonoids( puerarin,3’-hydroxypuerarin,and daidzein) were 22 174. 9,15 893. 1,3 882. 5 h·mg·L~(-1),major flavonoids( baicalein,wogonin,wogonoside,and baicalin) were 15 423. 6,15 408. 4,7 017. 3,3 697. 7 h·mg·L~(-1),and major alkaloid( berberine) was 2 544. 0 h·mg·L~(-1),respectively. The distribution of these ingredients in the small intestine was significantly higher than that in other tissues. Conclusion: The effective ingredients of GQT are mainly distributed in the intestinal tissues,which speculates that the anti-inflammatory and antidiarrheal activities of GQT may be related to its targeting in the intestine.
Objective: To investigate the tissue distribution of major bioactive components from Gegen qinlian Tang( GQT) in rats,and to reveal the mechanism for the efficacy of GQT by the tissue targeting of its bioactive ingredients in vivo. Method: After oral administration of GQT in rats,tissues were collected at different time points, including small and large intestine, liver, heart, spleen, lung, and kidney. Samples were prepared for determination of 14 bioactive components of GQT in tissue homogenate by HPLC-MS/MS. The chromatography separation was performed on an Agilent ZORBAX SB-C_(18) column( 2. 1 mm × 100 mm,3. 5 μm)with acetonitrile-0. 1% formic acid in water for gradient elution. Electrospray ionization( ESI) was applied and operated in the positive ion mode. Meanwhile, naringin was used as the internal standard for determining.Result: HPLC-MS/MS for simultaneous determination of 14 components from GQT in tissue homogenate was developed and validated by specificity, calibration curves, recovery test, matrix effect, precision, accuracy,and stability. In the small intestine,the the area under the curve( AUC_(0-10 h)) of major isoflavonoids( puerarin,3’-hydroxypuerarin,and daidzein) were 22 174. 9,15 893. 1,3 882. 5 h·mg·L~(-1),major flavonoids( baicalein,wogonin,wogonoside,and baicalin) were 15 423. 6,15 408. 4,7 017. 3,3 697. 7 h·mg·L~(-1),and major alkaloid( berberine) was 2 544. 0 h·mg·L~(-1),respectively. The distribution of these ingredients in the small intestine was significantly higher than that in other tissues. Conclusion: The effective ingredients of GQT are mainly distributed in the intestinal tissues,which speculates that the anti-inflammatory and antidiarrheal activities of GQT may be related to its targeting in the intestine.
引文
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[11]杨连荣,张丽杰,姚姣姣,等.白头翁皂苷B4在正常大鼠和溃疡性结肠炎大鼠体内组织分布情况的比较[J].中国实验方剂学杂志,2017,23(21):93-97.
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[13] KANG M J,Ko G S,Oh D G,et al. Role of metabolism by intestinal microbiota in pharmacokinetics of oral baicalin[J]. Arch Pharm Res,2014,37(3):371-378.
[14]钟凌云,马冰洁,叶喜德,等.葛根主要药效成分止泻作用研究[J].世界科学技术—中医药现代化,2015,17(1):109-113.
[15]苏妙贤,李药兰,周艳晖,等.瑶药“野葛花”的抗病毒和抑菌活性[J].暨南大学学报:自然科学与医学版,2006,27(2):204-208.
[16] Tsai C C,LIN M T,WANG J J,et al. The antipyretic effects of baicalin in lipopolysaccharide-evoked fever in rabbits[J]. Neuropharmacology,2006,51(4):709-717.
[17]王利红,唐文照,辛义周.黄连中生物碱成分及药理作用研究进展[J].山东中医药大学学报,2015,39(4):389-392.
[2]凌霄,项煜强,陈飞龙,等.西藏小型猪大肠埃希菌感染性腹泻模型的建立及葛根芩连汤对其作用分析[J].中国实验方剂学杂志,2018,24(3):125-131.
[3] WANG Q,SONG W,QIAO X,et al. Simultaneous quantification of 50 bioactive compounds of the traditional Chinese medicine formula Gegen-Qinlian decoction using ultra-high performance liquid chromatography coupled with tandem mass spectrometry[J]. J Chromatogr A,2016,1454:15-25.
[4] LING X,XIANG Y,TANG Q,et al. Comparative pharmacokinetics of eight major bioactive components in normal and bacterial diarrhea mini-pigs after oral administration of Gegen Qinlian Decoction[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2017,1044-1045:132-141.
[5] YAO X J,YIN J A,XIA Y F,et al. Puerarin exerts antipyretic effect on lipopolysaccharide-induced fever in rats involving inhibition of pyrogen production from macrophages[J]. J Ethnopharmacol,2012,141(1):322-330.
[6] Lee W,Ku S K,Bae J S. Anti-inflammatory effects of baicalin,baicalein,and wogonin in vitro and in vivo[J].Inflammation,2015,38(1):110-125.
[7] LIU C S,ZHENG Y R,ZHANG Y F,et al. Research progress on berberine with a special focus on its oral bioavailability[J]. Fitoterapia,2016,109:274-282.
[8]张玉龙,王梦月,杨静玉,等.炙甘草化学成分及药理作用研究进展[J].上海中医药大学学报,2015,29(3):99-102.
[9] REN M,SONG S,LIANG D,et al. Comparative tissue distribution and excretion study of alkaloids from Herba Ephedrae-Radix Aconiti Lateralis extracts in rats[J]. J Pharm Biomed Anal,2017,134:137-142.
[10]冯果,吴增光,何新,等. UPLC-MS/MS分析瓜蒌子中3,29-二苯甲酰基栝楼仁三醇在大鼠体内的组织分布情况[J].中国实验方剂学杂志,2017,23(21):86-92.
[11]杨连荣,张丽杰,姚姣姣,等.白头翁皂苷B4在正常大鼠和溃疡性结肠炎大鼠体内组织分布情况的比较[J].中国实验方剂学杂志,2017,23(21):93-97.
[12] ZHANG Y,YUAN J,ZHANG Y,et al. LC-MS/MS analysis of Gegen Qinlian Decoction and its pharmacokinetics after oral administration to rats[J].Biomed Chromatogr,2015,29(4):485-495.
[13] KANG M J,Ko G S,Oh D G,et al. Role of metabolism by intestinal microbiota in pharmacokinetics of oral baicalin[J]. Arch Pharm Res,2014,37(3):371-378.
[14]钟凌云,马冰洁,叶喜德,等.葛根主要药效成分止泻作用研究[J].世界科学技术—中医药现代化,2015,17(1):109-113.
[15]苏妙贤,李药兰,周艳晖,等.瑶药“野葛花”的抗病毒和抑菌活性[J].暨南大学学报:自然科学与医学版,2006,27(2):204-208.
[16] Tsai C C,LIN M T,WANG J J,et al. The antipyretic effects of baicalin in lipopolysaccharide-evoked fever in rabbits[J]. Neuropharmacology,2006,51(4):709-717.
[17]王利红,唐文照,辛义周.黄连中生物碱成分及药理作用研究进展[J].山东中医药大学学报,2015,39(4):389-392.