液质联用技术追踪生物碱类成分在川乌茎叶生长周期的动态变化规律
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摘要
目的研究川乌茎、叶中生物碱类成分在不同生长期的含量变化。方法采用Phenomenex C18色谱柱(150 mm×4.6 mm,5μm),流动相为0.1%甲酸水溶液(A)-乙腈(B),质谱采用ESP多反应监测(multiple reaction monitoring,MRM)模式扫描;建立HPLC-MS/MS方法同时测定川乌茎叶中乌头碱、新乌头碱、印乌头碱、次乌头碱、苯甲酰乌头原碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱、乌头原碱、尼奥林、附子灵、宋果灵、塔拉乌头胺、去甲乌药碱、去甲猪毛菜碱14种生物碱的含量。结合主成分分析(PCA)追踪川乌茎叶在生长过程中不同生物碱的含量变化规律。结果 14种生物碱在各自浓度范围内线性关系良好(r~2>0.990 0),定量限为2.27～18.27 ng/mL,平均回收率在94.73%～104.50%。结果表明,川乌茎叶中均含有相当量的生物碱,且茎中生物碱总量高于叶,茎在5、6、7、8月的生物碱总量分别为0.087 1%、0.182 8%、0.141 0%、0.199 4%,呈现波浪形上升趋势,8月份到达峰值;叶在5、6、7、8月生物碱总量分别为0.074 7%、0.075 9%、0.0814%、0.0589%,呈现出先上升后下降的变化趋势,叶中生物碱的总量7月最高。结论 PCA发现茎、叶中生物碱含量在不同时期表现出不同的变化趋势。茎中生物碱在采收期达到峰值,生物碱含量可观,具有成为新药用资源的潜力与开发价值。
Objective To investigate the content changes of alkaloids in stems and leaves of Aconiti Radix at different growth period. Methods The Phenomenex C18 column(150 mm × 4.6 mm, 5 μm) was used and 0.1% formic acid aqueous solution-acetonitrile was selected as mobile phase; The mass spectrum was scanned by ESI+ multiple reaction monitoring(MRM) mode. The HPLC-MS/MS method was established for the simultaneous determination of aconitine, mesaconitine, hypaconitine, indaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconitine, aconine, fuziline, neoline, talatisamine, songorine, higenamine, and salsoline in the stems and leaves of Aconite Radix. Combined with principal component analysis(PCA), the transfer rule of various alkaoids was tracked in the growth cycle of Aconiti Radix. Results Methodological validation results showed that the linear range of the 14 compounds was good(r~2 > 0.990 0). The limit of quantification was 2.27—18.27 ng/mL, and the average recovery was 94.73%—104.50%. The results showed that there was considerable amounts of alkaloids in stems and leaves of Aconiti Radix, and the total contents of alkaloids in stems were higher than those in leaves. In May, June, July, and August, the total content of alkaloids in stems was 0.087 1%, 0.182 8%, 0.141 0%, and 0.199 4% respectively, which showed a wave-like upward trend. The total content of alkaloids in leaves was 0.074 7%, 0.075 9%, 0.081 4%, and 0.058 9% respectively, which showed a trend of rising first and then decreasing, and the total content of alkaloids in leaves was highest in July. Conclusion PCA found that the alkaloids content in stems and leaves showed different variation trend in the different period. The considerable alkaloids in stems reached the peak value at the harvest time. The amount of alkaloids is considerable, which has the potential and development value of becoming new medicinal resources.
Objective To investigate the content changes of alkaloids in stems and leaves of Aconiti Radix at different growth period. Methods The Phenomenex C18 column(150 mm × 4.6 mm, 5 μm) was used and 0.1% formic acid aqueous solution-acetonitrile was selected as mobile phase; The mass spectrum was scanned by ESI+ multiple reaction monitoring(MRM) mode. The HPLC-MS/MS method was established for the simultaneous determination of aconitine, mesaconitine, hypaconitine, indaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconitine, aconine, fuziline, neoline, talatisamine, songorine, higenamine, and salsoline in the stems and leaves of Aconite Radix. Combined with principal component analysis(PCA), the transfer rule of various alkaoids was tracked in the growth cycle of Aconiti Radix. Results Methodological validation results showed that the linear range of the 14 compounds was good(r~2 > 0.990 0). The limit of quantification was 2.27—18.27 ng/mL, and the average recovery was 94.73%—104.50%. The results showed that there was considerable amounts of alkaloids in stems and leaves of Aconiti Radix, and the total contents of alkaloids in stems were higher than those in leaves. In May, June, July, and August, the total content of alkaloids in stems was 0.087 1%, 0.182 8%, 0.141 0%, and 0.199 4% respectively, which showed a wave-like upward trend. The total content of alkaloids in leaves was 0.074 7%, 0.075 9%, 0.081 4%, and 0.058 9% respectively, which showed a trend of rising first and then decreasing, and the total content of alkaloids in leaves was highest in July. Conclusion PCA found that the alkaloids content in stems and leaves showed different variation trend in the different period. The considerable alkaloids in stems reached the peak value at the harvest time. The amount of alkaloids is considerable, which has the potential and development value of becoming new medicinal resources.
引文
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[2]张定堃,韩雪,李瑞煜,等.UPLC-Q-TOF-MS分析不同产地泥附子化学成分的差异[J].中国中药杂志,2016,41(3):463-469.
[3]王玉,涂胜豪.乌头汤加味治疗类风湿关节炎临床研究[J].中医学报,2017,32(9):1716-1719.
[4]赵志浩,张定堃,吴明权,等.基于大鼠室性早搏心脏毒性的附子质量生物评价方法研究[J].中国中药杂志,2016,41(20):3814-3820.
[5]白云飞,林志健,张冰,等.祛风湿类中药饮片安全问题分析与用药警戒思考[J].中华中医药杂志,2016,31(10):4117-4123.
[6]郑亚秋,耿胜男,孟明静,等.川乌对小鼠二阶段皮肤乳头状瘤的抑制作用[J].河南大学学报:医学版,2016,35(1):5-8.
[7]王晓丽,李丽静,李玉梅,等.附子与人参不同配伍对心肌细胞的减毒作用[J].中国实验方剂学杂志,2015,21(11):153-158.
[8]Duc L V,Thanh T B,Huu T N,et al.Alkaloids isolated in the roots of aconitum carmichaeli debx growing in Vietnam[J].Inter J Pharm Phytochem Res,2015,7(3):576-578.
[9]欧水平.基于“一体多用”的乌头新药用部位及制剂研究--川乌头茎叶治疗类风湿性关节炎的成药性研究[D].成都:成都中医药大学,2013.
[10]吴征镒,陈心启.中国植物志(第1卷)[M].北京:科学出版社,2004.
[11]李凤,陈昌霞.川乌茎叶中生物碱提取及含量测定[J].黑龙江科技信息,2007(16):39-39.
[12]欧水平,郑琴,王森,等.乌头各组织部位生物碱类成分动态差异性研究[J].中国实验方剂学杂志,2012,18(13):83-87.
[13]李梦然,曲玮,梁敬钰.乌头属化学成分和药理作用研究进展[J].海峡药学,2010,22(4):1-6.
[14]邢俊鹏,林娜,刘志强,等.超高效液相色谱-质谱结合主成分分析方法研究制川乌配伍前后的肠内菌代谢差异[J].分析化学,2014,42(11):1646-1650.
[15]马建烈.四川道地药材附子栽培技术[J].四川农业科技,2016(3):12-14.
[16]刘振启.走进四川布拖附子GAP种植生产基地[J].首都医药,2012,19(5):39.
[17]杨华生,黎晓丽,闻丽珍,等.基于经皮给药途径研究白芍对制川乌6个酯型生物碱组织分布的影响[J].药物分析杂志,2017,37(6):1029-1037.
[18]熊茜,潘媛,张婷,等.HPLC-MS/MS多组分定量测定结合化学计量学研究市售小金丸的质量一致性[J].中草药,2017,48(11):2189-2196.
[19]王蕾,张先灵,王鑫,等.UPLC-MS法对生川乌、生附片化学成分的比较研究[J].药物分析杂志,2017,37(9):1640-1647.
[20]中国药典[S].一部.2015.
[21]李燕,李芸霞,龚小红,等.附子灵和尼奥宁的药理研究进展[J].中药与临床,2014,5(3):56-58.
[22]Guo S,Li P,Fu B,et al.Systems-biology dissection of mechanisms and chemical basis of herbal formula in treating chronic myocardial ischemia[J].Pharm Res,2016,114:196-208.
[23]邵财,张浩,张亚玉.草乌适宜采收期的初步研究[J].时珍国医国药,2016,27(2):470-472.