以木通皂苷为例探讨中药中总三萜皂苷含量测定方法
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摘要
由于缺乏对照品,中药中三萜皂苷类成分的含量通常以比色法测定总皂苷含量来表征。但是,比色法专属性差,测定结果不够准确。因此,该文以木通皂苷为例探讨总三萜皂苷含量测定方法。采用HPLC测定木通药材酸水解产物中3种主要皂苷元阿江榄仁酸、常春藤皂苷元和齐墩果酸的含量。参考2015年版《中国药典》中银杏叶总黄酮醇苷的测定思路,以上述3种皂苷元含量之和乘以转换系数作为木通总三萜皂苷含量。LC-MS/MS分析结果显示mutongsaponin C和saponin PJI是木通中含有的2个最主要的三萜皂苷类化合物,且二者相对分子质量相同。因此,mutongsaponin C的相对分子质量与阿江榄仁酸、常春藤皂苷元和齐墩果酸相对分子质量的比值的均值被定义为转换系数。HPLC方法中采用ACE Excel 3 C18-AR色谱柱(4. 6 mm×150 mm,3μm)进行分离,甲醇-水(含有0. 04%冰醋酸和0. 02%三乙胺)为流动相,梯度洗脱,检测波长210 nm,流速0. 5 m L·min-1。结果显示阿江榄仁酸、齐墩果酸和常春藤皂苷元分别在1. 053～16. 84,0. 200～3. 200,1. 515～24. 24μg线性关系良好,平均加样回收率分别为97. 90%,97. 50%,100. 5%,RSD为2. 0%,2. 9%,2. 9%。方法学考察结果均符合含量测定要求。mutongsaponin C对3种皂苷元的转换系数的均值为2. 31。该方法简便,可靠,可用于木通药材中总三萜皂苷的含量测定,该思路可用于其他中药中总三萜皂苷的测定。
Due to lack of reference substances,the content of triterpenoid saponins in traditional Chinese medicines is usually characterized by colorimetric determination of total saponins. However,the specificity of colorimetric method is poor,and the determination result is not accurate enough. So,in this paper,the content determination method of total triterpenoid saponins was studied by taking Akebiae Caulis saponins as an example. The contents of three main saponin aglycones,including arjunolic acid,hederagenin and oleanolic acid,were determined by HPLC method. Referring to the content determination method of total flavonol glycosides in Ginkgo biloba leaves in the 2015 edition of Chinese Pharmacopoeia,the content of Akebiae Caulis saponins was obtained by multiplying the total content of the three above-mentioned aglycones with conversion coefficient. LC-MS/MS analysis results showed that mutongsaponin C and aponin PJIwere the two main triterpene saponins in Akebiae Caulis,and they shared the same molecular formula. So,the average value of the ratios of the molecular weight between mutongsaponin C and the three aglycones was defined as the conversion coefficient.The three aglycones were separated on an ACE Excel 3 C18-AR column( 4. 6 mm×150 mm,3 μm),and methanol-water( containing0. 04% glacial acetic acid and 0. 02% triethylamine) was used as mobile phase with gradient elution. The detection wavelength was set at 210 nm,and the flow rate was 0. 5 m L·min-1. The results showed that there was a good linearity among the ranges of 1. 053-16. 84,0. 200-3. 200 and 1. 515-24. 24 μg for arjunolic acid,hederagenin and oleanolic acid,respectively. Their average recoveries were97. 90%,97. 50% and 100. 5%,with RSD of 2. 0%,2. 9% and 2. 9%,respectively. The results of methodological investigation met the requirements of content determination. The conversion coefficient was 2. 31. This method is simple and reliable,and can be used for the determination of total triterpenoid saponins in Akebiae Caulis. The assay strategy can be used for the determination of total triterpenoid saponins in other traditional Chinese medicines.
Due to lack of reference substances,the content of triterpenoid saponins in traditional Chinese medicines is usually characterized by colorimetric determination of total saponins. However,the specificity of colorimetric method is poor,and the determination result is not accurate enough. So,in this paper,the content determination method of total triterpenoid saponins was studied by taking Akebiae Caulis saponins as an example. The contents of three main saponin aglycones,including arjunolic acid,hederagenin and oleanolic acid,were determined by HPLC method. Referring to the content determination method of total flavonol glycosides in Ginkgo biloba leaves in the 2015 edition of Chinese Pharmacopoeia,the content of Akebiae Caulis saponins was obtained by multiplying the total content of the three above-mentioned aglycones with conversion coefficient. LC-MS/MS analysis results showed that mutongsaponin C and aponin PJIwere the two main triterpene saponins in Akebiae Caulis,and they shared the same molecular formula. So,the average value of the ratios of the molecular weight between mutongsaponin C and the three aglycones was defined as the conversion coefficient.The three aglycones were separated on an ACE Excel 3 C18-AR column( 4. 6 mm×150 mm,3 μm),and methanol-water( containing0. 04% glacial acetic acid and 0. 02% triethylamine) was used as mobile phase with gradient elution. The detection wavelength was set at 210 nm,and the flow rate was 0. 5 m L·min-1. The results showed that there was a good linearity among the ranges of 1. 053-16. 84,0. 200-3. 200 and 1. 515-24. 24 μg for arjunolic acid,hederagenin and oleanolic acid,respectively. Their average recoveries were97. 90%,97. 50% and 100. 5%,with RSD of 2. 0%,2. 9% and 2. 9%,respectively. The results of methodological investigation met the requirements of content determination. The conversion coefficient was 2. 31. This method is simple and reliable,and can be used for the determination of total triterpenoid saponins in Akebiae Caulis. The assay strategy can be used for the determination of total triterpenoid saponins in other traditional Chinese medicines.
引文
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[14] Ling Y,Zhang Q,Zhu D D,et al. Identification and characterization of the major chemical constituents in Fructus Akebiae by high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time-of-flight mass spectrometry[J]. J Chromatogr Sci,2016,54(2):148.
[15]高伟,李遵,段伟华,等.比色法测定木通属植物总皂苷含量的研究[J].中南林业科技大学学报,2015,35(10):134.
[2]中国药典.一部[S]. 2015.
[3] Mitiiti F,Hideji I,Ypshihiro K,et al. The study on the constituents of clematis and Akebia spp. II. on the saponins isolated from the stem of Akebia quinata Decne[J]. Yaku-Aaku Zasshi,1974,94(2):189.
[4]郭林新.三叶木通化学成分及生物活性研究[D].西安:陕西科技大学,2017.
[5]马红梅,张伯礼.不同科属木通比较[J].中国中药杂志,2002,27(6):15.
[6] Mimaki Y,Kuroda M,Yokosuka A,et al. Triterpenes and triterpene saponins from the stems of Akebia trifoliate[J]. Chem Pharm Bull,2003,51(8):960.
[7] Gao H M,Wang Z M. Triterpenoid saponins and phenylethanoid glycosides from stem of Akebia trifoliata var. australis[J]. Phytochemistry,2006,67(24):2697.
[8] Mimaki Y,Doi S,Kuroda M,et al. Triterpene glycosides from the stems of Akebia quinata[J]. Chem Pharm Bull,2007,55(9):1319.
[9] Choi J,Jung H J,Lee K T,et al. Antinociceptive and anti-inflammatory effects of the saponin and sapogenins obtained from the stem of Akebia quinata[J]. J Med Food,2005,8(1):78.
[10] Chen Y F,Roan H Y,Li C K,et al. Relationship between antioxidant and antiglycation ability of saponins,polyphenols,and polysaccharides in Chinese herbal medicines used to treat diabetes[J]. J Med Plants Res,2011,5(11):2322.
[11]高慧敏.白木通化学和木通质量研究[D].北京:中国中医科学院,2003.
[12]张雪松.木通药理活性成分及质量控制的研究[D].长春:吉林大学,2010.
[13]孟月华,黄何松,余黄鹏,等.液质联用技术鉴定预知子提取物中的主要化学成分[J].中草药,2013,44(12):1562.
[14] Ling Y,Zhang Q,Zhu D D,et al. Identification and characterization of the major chemical constituents in Fructus Akebiae by high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time-of-flight mass spectrometry[J]. J Chromatogr Sci,2016,54(2):148.
[15]高伟,李遵,段伟华,等.比色法测定木通属植物总皂苷含量的研究[J].中南林业科技大学学报,2015,35(10):134.