HPLC法测定紫花地丁中香豆素和黄酮的量
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摘要
以紫花地丁为材料,建立了同时测定紫花地丁中6种香豆素和黄酮含量的检测方法。结果表明:高效液相色谱(HPLC)选用色谱柱Inertsil ODS-3 C18柱(5μm, 250 mm×4.6 mm);流动相为A (乙腈)和B (15 mmol/L醋酸铵溶液)。梯度洗脱方法, 0～10 min,流动相A 10%～25%; 10～20 min,流动相A 25%～60%,紫外检测波长290 nm,柱温30℃,流速1.0 mL/min。6个化合物均在15 min内实现基线分离;线性关系良好(R2>0.998);添加高、中、低浓度的对照品,6个化合物的平均回收率在91.18%～99.92%之间,相对标准偏差均在1.06%～2.14%之间(N=6)。该方法简便、准确、重复性好。对紫花地丁全株和花器官中6种成分含量测定结果表明:全株中秦皮甲素、秦皮乙素、芦丁、木犀草苷、木犀草素、柚皮素的含量分别为2 406, 2 961, 887, 1 371, 382和165 mg/kg,花器官中含量分别为4 345, 5 665, 587, 871,582和465 mg/kg。
A high performance liquid chromatography(HPLC) method for simultaneously determining the content of esculin hydrate, esculetin, rutin, luteolin, luteolin and naringin in Viola yedoensis was established. The chromatographic separation was performed on a Shimadzu LC-20 AT Inertsil ODS-3 C18(5 μm, 250 mm×4.6 mm). The mobile phase consisted of acetonitrile(A) and 15 mmol/L ammonium acetate in water(B) with linear gradient elution(0-10 min, 10% A-25% A, 10-20 min, 25%A-60% A). The flow rate was 1.0 mL/min. The column temperature was 30 ℃, and the detection wavelength was 290 nm.Six components were separated to baseline. Each component had a wide linear range and a good linear relationship. Though adding high, medium and low concentrations of reference, the average recoveries were in the range 91.18% to 99.92% with the relative standard deviations ranging from 1.06% to 2.14%(N=6). The developed HPLC method was reproducible, accurate and efficient. The determination results showed that the contents of esculin hydrate, esculetin, rutin, luteolin, luteolin and naringenin in whole plant were 2 406, 2 961, 887, 1 371, 382 and 165 mg/kg, and the contents of the six components in flowers were 4 345,5 665, 587, 871, 582 and 465 mg/kg.
A high performance liquid chromatography(HPLC) method for simultaneously determining the content of esculin hydrate, esculetin, rutin, luteolin, luteolin and naringin in Viola yedoensis was established. The chromatographic separation was performed on a Shimadzu LC-20 AT Inertsil ODS-3 C18(5 μm, 250 mm×4.6 mm). The mobile phase consisted of acetonitrile(A) and 15 mmol/L ammonium acetate in water(B) with linear gradient elution(0-10 min, 10% A-25% A, 10-20 min, 25%A-60% A). The flow rate was 1.0 mL/min. The column temperature was 30 ℃, and the detection wavelength was 290 nm.Six components were separated to baseline. Each component had a wide linear range and a good linear relationship. Though adding high, medium and low concentrations of reference, the average recoveries were in the range 91.18% to 99.92% with the relative standard deviations ranging from 1.06% to 2.14%(N=6). The developed HPLC method was reproducible, accurate and efficient. The determination results showed that the contents of esculin hydrate, esculetin, rutin, luteolin, luteolin and naringenin in whole plant were 2 406, 2 961, 887, 1 371, 382 and 165 mg/kg, and the contents of the six components in flowers were 4 345,5 665, 587, 871, 582 and 465 mg/kg.
引文
[1]国家药典委员会.中华人民共和国药典一部[S].北京:中国医药科技出版社,2015:337.
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[12]陈胡兰,董小萍,张梅,等.紫花地丁化学成分研究[J].中草药,2010,41(6):874-877.
[13]高意,周光明,张彩虹,等.高效液相色谱法同时测定紫花地丁中6种活性成分[J].食品科学,2016,37(6):101-105.
[14]陈胡兰,汤沛然,张梅,等.紫花地丁药材及其混淆品的HPLC指纹图谱研究[J].中国药学杂志,2010,45(18)1372-1375.
[15]孟繁姝,刘权友,高东立,等.HPLC法测定紫花地丁中槲皮素、芹菜素、木犀草素含量[J].中国药品标准,2011,12(1):48-49.
[16]刘旬.紫花地丁中水溶性化学成分的研究[D].长春:吉林大学,2009.
[17]王晶.秦皮甲素对肺癌小鼠抑瘤作用的研究[J].中成药,2014,36(2):249-252.
[18]周桂芬,陈素红,吕圭源,等.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].中国中药杂志,2013,38(4):520-523.
[19]杨欢,程笑,王月华,等.秦皮甲素对角叉菜胶致小鼠足肿胀及炎症的作用[J].中国新药杂志,2016,25(20):2319-2322.
[20]杨欢,程笑,张惠芳,等.秦皮甲素对D-半乳糖诱导大鼠晶状体损伤的作用[J].中国药学杂志,2016,51(19):1666-1670.
[2]毛晓霞,苗光新,于海龙,等.紫花地丁的研究进展[J].承德医学院学报,2010,27(3):302-305.
[3]OSHIMA N,NARUKAWA Y,TAKEDA T,et al.Collagenase inhibitors from Viola yedoensis[J].Journal of Natural Medicines2013,67(1):240-245.
[4]DU D S,CHENG Z H,CHEN D F,et al.Anti-complement sesquiterpenes from Viola yedoensis[J].Fitoterapia,2015,3(101):73-79.
[5]ZENG H R,WANG B,ZHAO Z H,et al.Effects of Viola yedoensis makino anti-itching compound on degranulation and cytokine[J].Journal of Ethnopharmacology,2016,08(189):132-138.
[6]黄美娥,卓儒洞,唐莉.紫花地丁乙醇提取物的抗氧化性研究[J].食品科技,2007,32(2):151-154.
[7]LI W,XIE J Y,LI H,et al.Viola yedoensis liposoluble fraction ameliorates lipopolysaccharide-induced acute lung injury in mice[J].American Journal of Chinese Medicine,2012,40(5):1007-1018.
[8]王志红.紫花地丁汤治疗盆腔炎84例临床疗效研究[J].中药药理与临床,2015,31(3):156-157.
[9]陈胡兰.紫花地丁化学成分及质量控制研究[D].成都:成都中医药大学,2009.
[10]OSHIMA N,NARUKAWA Y,TAKEDA T,et al.Collagenase inhibitors from Viola yedoensis[J].Journal of Natural Medicines,2013,67(1):240-245.
[11]曹捷.紫花地丁黄酮苷类成分的定性定量研究[D].上海:复旦大学,2013.
[12]陈胡兰,董小萍,张梅,等.紫花地丁化学成分研究[J].中草药,2010,41(6):874-877.
[13]高意,周光明,张彩虹,等.高效液相色谱法同时测定紫花地丁中6种活性成分[J].食品科学,2016,37(6):101-105.
[14]陈胡兰,汤沛然,张梅,等.紫花地丁药材及其混淆品的HPLC指纹图谱研究[J].中国药学杂志,2010,45(18)1372-1375.
[15]孟繁姝,刘权友,高东立,等.HPLC法测定紫花地丁中槲皮素、芹菜素、木犀草素含量[J].中国药品标准,2011,12(1):48-49.
[16]刘旬.紫花地丁中水溶性化学成分的研究[D].长春:吉林大学,2009.
[17]王晶.秦皮甲素对肺癌小鼠抑瘤作用的研究[J].中成药,2014,36(2):249-252.
[18]周桂芬,陈素红,吕圭源,等.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].中国中药杂志,2013,38(4):520-523.
[19]杨欢,程笑,王月华,等.秦皮甲素对角叉菜胶致小鼠足肿胀及炎症的作用[J].中国新药杂志,2016,25(20):2319-2322.
[20]杨欢,程笑,张惠芳,等.秦皮甲素对D-半乳糖诱导大鼠晶状体损伤的作用[J].中国药学杂志,2016,51(19):1666-1670.