HPLC-一测多评法同时测定大青叶中6种抗肿瘤活性成分的含量
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摘要
目的:建立同时测定大青叶中6种抗肿瘤活性成分含量的方法。方法:采用高效液相色谱法。色谱柱为Waters XTERRA?MS C18,流动相为甲醇-0.1%甲酸水溶液(梯度洗脱),流速为1.0 mL/min,柱温为35℃,检测波长为254 nm,进样量为10μL。以木犀草苷为内标,分别计算其与尿苷、腺苷、金丝桃苷、7-甲氧基香豆素、芹菜素的相对校正因子(RCF),通过RCF计算大青叶中上述5种抗肿瘤活性成分的含量,同时采用外标法测定这6种成分的含量,比较一测多评法和外标法的含量测定结果。结果:尿苷、腺苷、木犀草苷、金丝桃苷、7-甲氧基香豆素、芹菜素进样量检测线性范围分别为0.122 6~4.902、0.063 4~2.534、0.032 7~1.309、0.038 2~1.527、0.032 3~1.291、0.052 5~2.098μg(r≥0.999 1);检测限分别为3.06、1.58、0.82、0.96、0.81、1.31μg/mL,定量限分别为10.21、5.28、2.73、3.18、2.69、4.37μg/m L;精密度、稳定性(24 h)、重复性试验的RSD<2%(n=6);加样回收率为96.45%~99.14%(RSD为0.8%~1.6%,n=6)。尿苷、腺苷、金丝桃苷、7-甲氧基香豆素、芹菜素的RCF分别为0.197、0.413、0.732、0.825、0.587。在不同试验条件下,RCF重现性良好。采用一测多评法和外标法的含量测定结果比较,差异无统计学意义(P>0.05)。结论:该方法操作简单快捷、结果准确可靠,可用于大青叶中尿苷、腺苷、木犀草苷、金丝桃苷、7-甲氧基香豆素和芹菜素6种抗肿瘤活性成分含量的同时测定。
OBJECTIVE:To establish a method for simultaneous determination of 6 antitumor active constituents in Folium isatidis. METHODS:HPLC method was adopted. The determination was performed on Waters XTERRA?MS C18 column with mobile phase consisted of methanol-0.1% formic acid(gradient elution)at the flow rate of 1.0 mL/min. The column temperature was 35 ℃,and detection wavelength was 254 nm. The injection volume was 10 μL. Using luteolin as internal standard,relative correction factors(RCF) of uridine,adenosine,hyperin,7-methoxycoumarin and apigenin were calculated respectively. The contents of above 5 antitumor active components in F. isatidis were calculated by RCF. The contents of 6 components were determined by external standard method(ESM). The results of content determination by QAMS were compared with ESM.RESULTS:The linear range was 0.122 6-4.90 2 μg for uridine,0.063 4-2.534 μg for adenosine,0.032 7-1.309 μg for luteolin,0.038 2-1.527 μg for hyperin,0.032 3-1.291 μg for 7-methoxycoumarin,0.052 5-2.098 μg for apigenin(r≥0.999 1). The limits of detection were 3.06,1.58,0.82,0.96,0.81,1.31 μg/mL,respectively. The limits of quantification were 10.21,5.28,2.73,3.18,2.69,4.37 μg/mL. RSDs of precision,stability(24 h)and reproducibility tests were less than 2%(n=6). The average recoveries were 96.45%-99.14%(RSDs were 0.8%-1.6%,n=6). RCFs of uridine,adenosine,hyperin,7-methoxycoumarin and apigenin were 0.197,0.413,0.732,0.825 and 0.587,respectively. RCFs repeatability was perfect under different experiment conditions.There was no significant difference between the quantitative results of the QAMS and ESM(P>0.05). CONCLUSIONS:QAMS method is simple,fast,accurate and feasible,and can be used for simultaneous determination of uridine,adenosine,luteolin,hyperin,7-methoxycoumarin and apigenin in F. isatidis.
OBJECTIVE:To establish a method for simultaneous determination of 6 antitumor active constituents in Folium isatidis. METHODS:HPLC method was adopted. The determination was performed on Waters XTERRA?MS C18 column with mobile phase consisted of methanol-0.1% formic acid(gradient elution)at the flow rate of 1.0 mL/min. The column temperature was 35 ℃,and detection wavelength was 254 nm. The injection volume was 10 μL. Using luteolin as internal standard,relative correction factors(RCF) of uridine,adenosine,hyperin,7-methoxycoumarin and apigenin were calculated respectively. The contents of above 5 antitumor active components in F. isatidis were calculated by RCF. The contents of 6 components were determined by external standard method(ESM). The results of content determination by QAMS were compared with ESM.RESULTS:The linear range was 0.122 6-4.90 2 μg for uridine,0.063 4-2.534 μg for adenosine,0.032 7-1.309 μg for luteolin,0.038 2-1.527 μg for hyperin,0.032 3-1.291 μg for 7-methoxycoumarin,0.052 5-2.098 μg for apigenin(r≥0.999 1). The limits of detection were 3.06,1.58,0.82,0.96,0.81,1.31 μg/mL,respectively. The limits of quantification were 10.21,5.28,2.73,3.18,2.69,4.37 μg/mL. RSDs of precision,stability(24 h)and reproducibility tests were less than 2%(n=6). The average recoveries were 96.45%-99.14%(RSDs were 0.8%-1.6%,n=6). RCFs of uridine,adenosine,hyperin,7-methoxycoumarin and apigenin were 0.197,0.413,0.732,0.825 and 0.587,respectively. RCFs repeatability was perfect under different experiment conditions.There was no significant difference between the quantitative results of the QAMS and ESM(P>0.05). CONCLUSIONS:QAMS method is simple,fast,accurate and feasible,and can be used for simultaneous determination of uridine,adenosine,luteolin,hyperin,7-methoxycoumarin and apigenin in F. isatidis.
引文
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[14]王智民,钱忠直,张启伟,等.一测多评法建立的技术指南[J].中国中药杂志,2011,36(6):657-658.
[15]刘洋,李明花,邢向红.核苷类抗肿瘤药物研究进展[J].中国新药杂志,2012,21(21):2493-2498.
[16]田亭,罗和生,周宇,等.腺苷受体在消化系统肿瘤中作用的研究现状[J].胃肠病学和肝病学杂志,2016,25(10):1198-1201.
[17]王婷婷,王少康,黄桂玲,等.木犀草苷对人食管鳞状癌Eca109细胞生长的抑制作用及其机制[J].中草药,2013,44(12):1604-1609.
[18]孙时华,姜荣华,祝海燕,等.金丝桃苷体外抗胃癌作用及其机制研究[J].中国临床药理学与治疗学,2014,19(9):979-983.
[19]李林虎,陈莉,夏玉凤.抗肿瘤香豆素类化合物的研究进展[J].中国药科大学学报,2013,44(4):374-379.
[20]黄好武,黄欢文,姜一平,等.芹菜素对肺癌细胞的抗增殖作用和抗肿瘤药物的增敏作用[J].中国药业,2017,26(12):20-22.
[2]李雪虎,梁剑平,陆锡宏.大青叶的化学成分的研究[J].时珍国医国药,2011,22(9):2144-2145.
[3]姬志强,李永丽,张永飞,等.大青叶药材化学成分研究[J].中国药师,2013,16(10):1467-1469.
[4]李宝国,李峰.大青叶中18种无机元素的含量测定[J].中国现代药物应用,2009,3(18):1-2.
[5]吴启南,王立新,蒋小文,等.大青叶中有机酸药理作用研究[J].南京中医药大学学报,2008,24(3):187-189.
[6]简晓顺,尹一子,陈健清,等.大青叶乙醇提取液含药血清体外抗肿瘤活性研究[J].中药材,2013,36(4):633-635.
[7]张海涛,刘祺凤,田璐阳,等.超声波辅助提取大青叶中总黄酮及其抗氧化性研究[J].湖北农业科学,2014,53(9):2124-2126.
[8]高博,张建明,王宇平,等.大青叶水溶性提取物对登革病毒抑制作用及机制探讨[J].海峡药学,2013,25(7):205-207.
[9]何兵,刘艳,杨世艳,等.HPLC一测多评法同时测定双青咽喉片中10种成分[J].中草药,2013,44(8):974-981.
[10]何兵,刘艳,李春红,等.多指标定量指纹图谱在中药金银花质量评价中的应用[J].中国药学杂志,2015,50(14):1237-1242.
[11]梁文仪,袁永兵,陈文静,等.一测多评技术在3种丹参制剂质量控制中的可行性分析[J].中草药,2016,47(23):4179-4185.
[12]常志惠,胡军华,万亚菲,等.一测多评法同时测定保肾乙丸中5种蒽醌类成分的含量[J].中国药房,2016,27(36):5156-5159.
[13]刘慧妍,沈国滨.一测多评法同时测定野菊花中7种成分[J].中草药,2017,48(10):2012-2017.
[14]王智民,钱忠直,张启伟,等.一测多评法建立的技术指南[J].中国中药杂志,2011,36(6):657-658.
[15]刘洋,李明花,邢向红.核苷类抗肿瘤药物研究进展[J].中国新药杂志,2012,21(21):2493-2498.
[16]田亭,罗和生,周宇,等.腺苷受体在消化系统肿瘤中作用的研究现状[J].胃肠病学和肝病学杂志,2016,25(10):1198-1201.
[17]王婷婷,王少康,黄桂玲,等.木犀草苷对人食管鳞状癌Eca109细胞生长的抑制作用及其机制[J].中草药,2013,44(12):1604-1609.
[18]孙时华,姜荣华,祝海燕,等.金丝桃苷体外抗胃癌作用及其机制研究[J].中国临床药理学与治疗学,2014,19(9):979-983.
[19]李林虎,陈莉,夏玉凤.抗肿瘤香豆素类化合物的研究进展[J].中国药科大学学报,2013,44(4):374-379.
[20]黄好武,黄欢文,姜一平,等.芹菜素对肺癌细胞的抗增殖作用和抗肿瘤药物的增敏作用[J].中国药业,2017,26(12):20-22.