HPLC法同时测定不同生长年限不同部位杜仲中5种苯丙素类成分
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摘要
目的:建立高效液相色谱法同时测定不同生长年限、不同部位杜仲中的5种苯丙素类成分含量。方法:采用Hi Q Sil C18色谱柱(250 mm×4.6 mm,5?μm),以0.2%甲酸(A)-乙腈(B)为流动相,梯度洗脱:0～12 min,4%～12%B;12～35 min,12%～14%B,流速1.0 mL/min,检测波长277 nm,柱温25℃,进样量5?μL。结果:绿原酸、咖啡酸、原儿茶酸、松柏苷、紫丁香苷5种成分分别在0.04～1 mg/m L(R~2=0.999 7)、0.001 6～0.029 mg/m L(R2=0.9940)、0.001 6～0.04 mg/mL(R2=0.998 9)、0.008～0.04 mg/mL(R2=0.998 7)、0.013～0.029 mg/mL(R2=0.998 6)范围内线性关系良好。不同添加水平加标回收率为94.05%～107.02%,其相对标准偏差在0.95%～4.11%之间。结论:建立方法适合方法学验证要求,可用来对杜仲叶、杜仲干皮中5种苯丙素类成分(绿原酸、咖啡酸、原儿茶酸、松柏苷、紫丁香苷)进行含量测定。不同生长年限杜仲叶中的5种苯丙素类活性成分总量高于杜仲干皮。因此,在制备含杜仲保健食品时,可考虑应用杜仲叶作为杜仲干皮的替代物。
Objective: To establish a high performance liquid chromatography(HPLC) method for the determination of five phenylpropanoids in Eucommia ulmoides Oliv. from different ages and parts. Methods: The samples were separated on HiQ Sil C18 column(250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of 0.2% formic acid(A) and acetonitrile(B) with gradient elution(4%–12% B between 0 and 12 min; 12%–14% B between 12 and 35 min) at a flow rate of 1.0 mL/min and detected at 277 nm, and the column temperature was kept at 25 ℃. The injection volume was 5 μL. Results: The method had good linearity in the ranges of 0.04–1 mg/m L(R2 = 0.999 7) for chlorogenic acid, 0.001 6–0.029 mg/m L(R2 = 0.994 0)for caffeic acid, 0.001 6–0.04 mg/mL(R2 = 0.998 9) for protocatechuic acid, 0.008–0.04 mg/mL(R2 = 0.998 7) for coniferin, and 0.013–0.029 mg/mL(R2 = 0.998 6) for syringoside. The average recoveries of spiked samples ranged from94.05%–107.02%, with relative standard deviations(RSDs) in the range of 0.95%–4.11%. Conclusion: This method met the requirements of methodological validation, and it could be used for the determination of chlorogenic acid, caffeic acid, protocatechuic acid, coniferin, and syringoside in different parts of E. ulmoides Oliv. The total content of five phenylpropanoid compounds in leaves was higher than in barks, suggesting that E. ulmoides Oliv. leaves can be considered an alternative to its barks in functional foods.
Objective: To establish a high performance liquid chromatography(HPLC) method for the determination of five phenylpropanoids in Eucommia ulmoides Oliv. from different ages and parts. Methods: The samples were separated on HiQ Sil C18 column(250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of 0.2% formic acid(A) and acetonitrile(B) with gradient elution(4%–12% B between 0 and 12 min; 12%–14% B between 12 and 35 min) at a flow rate of 1.0 mL/min and detected at 277 nm, and the column temperature was kept at 25 ℃. The injection volume was 5 μL. Results: The method had good linearity in the ranges of 0.04–1 mg/m L(R2 = 0.999 7) for chlorogenic acid, 0.001 6–0.029 mg/m L(R2 = 0.994 0)for caffeic acid, 0.001 6–0.04 mg/mL(R2 = 0.998 9) for protocatechuic acid, 0.008–0.04 mg/mL(R2 = 0.998 7) for coniferin, and 0.013–0.029 mg/mL(R2 = 0.998 6) for syringoside. The average recoveries of spiked samples ranged from94.05%–107.02%, with relative standard deviations(RSDs) in the range of 0.95%–4.11%. Conclusion: This method met the requirements of methodological validation, and it could be used for the determination of chlorogenic acid, caffeic acid, protocatechuic acid, coniferin, and syringoside in different parts of E. ulmoides Oliv. The total content of five phenylpropanoid compounds in leaves was higher than in barks, suggesting that E. ulmoides Oliv. leaves can be considered an alternative to its barks in functional foods.
引文
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[7]周云雷,郭婕,王志宏,等.H P L C法同时测定矮林杜仲叶中6种成分含量[J].中药材,2015,38(3):540-543.DOI:10.13863/j.issn1001-4454.2015.03.029.
[8]魏薇,王建刚.HPLC测定不同树龄杜仲叶中黄酮类成分的含量[J].安徽农业科学,2011,39(18):10779-10781.DOI:10.3969/j.issn.0517-6611.2011.18.020.
[9]陈晓青.液相色谱方法用于复杂体系的分离分析研究[D].长沙:中南大学,2006.
[10]曹宇.杜仲盐炙的化学成分和质量研究[D].沈阳:辽宁中医药大学,2009.
[11]何峰,王永林,郑林,等.UPLC-PDA-ESI-MS分析杜仲中化学成分[J].中国实验方剂学杂志,2014,20(3):59-62.DOI:10.11653/syfj2014030059.
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[14]褚洪标,张志华,陈冬,等.大王马先蒿苯丙素类含量测定及有效部位抗疲劳活性研究[J].天然产物研究与开发,2016(5):754-760.DOI:10.16333/j.1001-6880.2016.5.021.
[15]CHEN X J,ZHOU C S,WEI W.The comparison between different extractions of chlorogenic acid and flavonoid in Eucommia ulmoides Olive[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(1):38-40.DOI:10.3969/j.issn.1005-1678.2006.01.012.
[16]陈乃炽,汪洪武,刘艳清,等.杜仲叶中绿原酸的提取与含量测定[J].经济林研究,2001(2):59-61.DOI:10.14067/j.cnki.1003-8981.2001.02.021.
[17]陈海莉.杜仲叶提取物制备工艺及质量标准研究[D].开封:河南大学,2014.
[18]许先猛,董文宾.杜仲叶总黄酮的超声提取工艺[J].食品研究与开发,2010,31(12):15-18.DOI:10.3969/j.issn.1005-6521.2010.12.005.
[19]齐惠丽,赵广荣,李东.杜仲叶中绿原酸的超声提取工艺研究[J].西北药学杂志,2 0 0 7,2 2(6):3 0 0-3 0 1.D O I:1 0.3 9 6 9/j.issn.1004-2407.2007.06.008.
[20]龙文静,张盛,袁玲,等.反相高效液相色谱法同时测定咖啡豆中的6种酚酸类化合物[J].色谱,2011,29(5):439-442.DOI:10.3724/SP.J.1123.2011.00439.
[21]马伟,曾里,程健,等.HPLC法测定杜仲茶中车叶草苷含量的研究[J].食品与发酵科技,2016,52(3):93-96.DOI:10.3969/j.issn.1674-506X.2016.03-022.
[22]项丽玲,温亚娟,苗明三.杜仲叶的化学、药理及临床应用分析[J].中医学报,2017,32(1):99-102.DOI:10.16368/j.issn.1674-8999.2017.01.024.
[23]姜凌宇,姜月华,郭金昊,等.杜仲治疗高血压研究进展[J].山东中医杂志,2017(3):249-252.DOI:10.16295/j.cnki.0257-358x.2017.03.023.
[24]潘梓烨,常念伟,周梦鸽,等.桑菊饮抗炎活性成分筛选与单体验证[J].中草药,2016,47(8):1289-1296.DOI:10.7501/j.issn.0253-2670.2016.08.007.
[25]刘静.忍冬藤注射液制剂的研究[D].长春:吉林大学,2013.
[26]骆成尧.绿原酸与咖啡酸体外代谢机制及相互作用研究[D].南昌:南昌大学,2012.
[27]宋亚玲,王红梅,倪付勇,等.金银花中酚酸类成分及其抗炎活性研究[J].中草药,2015,46(4):490-495.DOI:10.7501/j.issn.0253-2670.2015.04.006.
[28]周英,李敏,杨付梅,等.绿原酸、咖啡酸、阿魏酸对微血管内皮细胞的损伤作用[J].中国药理学通报,2016,32(10):1474-1475.DOI:10.3969/j.issn.1001-1978.2016.10.027.
[29]戚向阳,陈维军,张声华.反相高效液相色谱法测定杜仲中京尼平苷、京尼平苷酸及绿原酸的含量[J].药物分析杂志,2000(1):22-24.DOI:10.1007/s11769-000-0028-3.
[30]薛晓丽.高效液相色谱法测定金银花中绿原酸的含量[J].江苏农业科学,2010(2):296-297.DOI:10.3969/j.issn.1002-1302.2010.02.122.
[2]王丽楠,杨美华.不同部位、不同生长年限杜仲有效成分的含量变化(摘要)[C]//全国天然药物资源学术研讨会.2008.
[3]余椿生,常效琳.杜仲大面积环状剥皮后树皮再生的解剖学初步观察[J].中药材科技,1979(4):15-19.DOI:10.13863/j.issn1001-4454.1979.04.004.
[4]ZHU M J,CAI C F,TAN N H,et al.Anti-fatigue effects of extracts from Pedicularis densispica Franch on exercise mice[J].Natural Product Research and Development,2012,24:1545-1548.DOI:10.16333/j.1001-6880.2012.11.009.
[5]CAI R L,YANG M H,SHI Y,et al.Antifatigue activity of phenylethanoid-rich extract from Cistanche deserticola[J].Phytotherapy Research,2010,24(2):313-315.DOI:10.1002/ptr.2927.
[6]王娟娟,秦雪梅,高晓霞,等.杜仲化学成分、药理活性和质量控制现状研究进展[J].中草药,2017,48(15):3228-3237.DOI:10.7501/j.issn.0253-2670.2017.15.031.
[7]周云雷,郭婕,王志宏,等.H P L C法同时测定矮林杜仲叶中6种成分含量[J].中药材,2015,38(3):540-543.DOI:10.13863/j.issn1001-4454.2015.03.029.
[8]魏薇,王建刚.HPLC测定不同树龄杜仲叶中黄酮类成分的含量[J].安徽农业科学,2011,39(18):10779-10781.DOI:10.3969/j.issn.0517-6611.2011.18.020.
[9]陈晓青.液相色谱方法用于复杂体系的分离分析研究[D].长沙:中南大学,2006.
[10]曹宇.杜仲盐炙的化学成分和质量研究[D].沈阳:辽宁中医药大学,2009.
[11]何峰,王永林,郑林,等.UPLC-PDA-ESI-MS分析杜仲中化学成分[J].中国实验方剂学杂志,2014,20(3):59-62.DOI:10.11653/syfj2014030059.
[12]LUO X,MA M,CHEN B,et al.Analysis of nine bioactive compounds in Eucommia ulmoides Oliv.and their preparation by HPLC-photodiode array detection and mass spectrometry[J].Journal of Liquid Chromatography&Related Technologies,2004,27(1):63-81.DOI:10.1081/JLC-120027086.
[13]YUAN P F,MA Y J,SU D,et al.Quantification of seven phenylpropanoid compouds in Chinese cinnamomi crotex and ramulus by HPLC[J].Journal of Chinese Pharmaceutical Sciences,2015(9):591-599.DOI:10.5246/jcps.2015.09.075.
[14]褚洪标,张志华,陈冬,等.大王马先蒿苯丙素类含量测定及有效部位抗疲劳活性研究[J].天然产物研究与开发,2016(5):754-760.DOI:10.16333/j.1001-6880.2016.5.021.
[15]CHEN X J,ZHOU C S,WEI W.The comparison between different extractions of chlorogenic acid and flavonoid in Eucommia ulmoides Olive[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(1):38-40.DOI:10.3969/j.issn.1005-1678.2006.01.012.
[16]陈乃炽,汪洪武,刘艳清,等.杜仲叶中绿原酸的提取与含量测定[J].经济林研究,2001(2):59-61.DOI:10.14067/j.cnki.1003-8981.2001.02.021.
[17]陈海莉.杜仲叶提取物制备工艺及质量标准研究[D].开封:河南大学,2014.
[18]许先猛,董文宾.杜仲叶总黄酮的超声提取工艺[J].食品研究与开发,2010,31(12):15-18.DOI:10.3969/j.issn.1005-6521.2010.12.005.
[19]齐惠丽,赵广荣,李东.杜仲叶中绿原酸的超声提取工艺研究[J].西北药学杂志,2 0 0 7,2 2(6):3 0 0-3 0 1.D O I:1 0.3 9 6 9/j.issn.1004-2407.2007.06.008.
[20]龙文静,张盛,袁玲,等.反相高效液相色谱法同时测定咖啡豆中的6种酚酸类化合物[J].色谱,2011,29(5):439-442.DOI:10.3724/SP.J.1123.2011.00439.
[21]马伟,曾里,程健,等.HPLC法测定杜仲茶中车叶草苷含量的研究[J].食品与发酵科技,2016,52(3):93-96.DOI:10.3969/j.issn.1674-506X.2016.03-022.
[22]项丽玲,温亚娟,苗明三.杜仲叶的化学、药理及临床应用分析[J].中医学报,2017,32(1):99-102.DOI:10.16368/j.issn.1674-8999.2017.01.024.
[23]姜凌宇,姜月华,郭金昊,等.杜仲治疗高血压研究进展[J].山东中医杂志,2017(3):249-252.DOI:10.16295/j.cnki.0257-358x.2017.03.023.
[24]潘梓烨,常念伟,周梦鸽,等.桑菊饮抗炎活性成分筛选与单体验证[J].中草药,2016,47(8):1289-1296.DOI:10.7501/j.issn.0253-2670.2016.08.007.
[25]刘静.忍冬藤注射液制剂的研究[D].长春:吉林大学,2013.
[26]骆成尧.绿原酸与咖啡酸体外代谢机制及相互作用研究[D].南昌:南昌大学,2012.
[27]宋亚玲,王红梅,倪付勇,等.金银花中酚酸类成分及其抗炎活性研究[J].中草药,2015,46(4):490-495.DOI:10.7501/j.issn.0253-2670.2015.04.006.
[28]周英,李敏,杨付梅,等.绿原酸、咖啡酸、阿魏酸对微血管内皮细胞的损伤作用[J].中国药理学通报,2016,32(10):1474-1475.DOI:10.3969/j.issn.1001-1978.2016.10.027.
[29]戚向阳,陈维军,张声华.反相高效液相色谱法测定杜仲中京尼平苷、京尼平苷酸及绿原酸的含量[J].药物分析杂志,2000(1):22-24.DOI:10.1007/s11769-000-0028-3.
[30]薛晓丽.高效液相色谱法测定金银花中绿原酸的含量[J].江苏农业科学,2010(2):296-297.DOI:10.3969/j.issn.1002-1302.2010.02.122.
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