甜瓜蒂中主要四环三萜结构鉴定及葫芦素B,葫芦素D含量测定

Structure Identification of Main Tetracyclic Triterpenoids and Simultaneous Determination of Cucurbitacin B and D in Pedicellus Melo

目的:阐明甜瓜蒂中主要四环三萜类成分化学模式,建立HPLC测定甜瓜蒂药材中葫芦素B和葫芦素D含量的方法。方法:采用UPLC-ESI-Orbitrap-MS/MS技术分析甜瓜蒂中主要的四环三萜类成分,Eclipse XDB-C_(18)色谱柱(4.6 mm×150 mm,5μm),以0.1%甲酸-乙腈溶液为流动相梯度洗脱;采用RP-HPLC法测定7批甜瓜蒂药材中葫芦素B和葫芦素D含量,Inert Sustain C_(18)色谱柱(4.6 mm×250 mm,5μm),以0.1%冰乙酸溶液(含庚烷磺酸钠1 g·L^(-1))-乙腈为流动相,检测波长228 nm。结果:在化学模式定性分析中,共发现并初步鉴定出甜瓜蒂中主要的8种四环三萜类成分,包括雪胆素甲,异雪胆素甲,葫芦素I,葫芦素D,葫芦素B及25-脱乙酰基葫芦素B的3个同分异构体;在定量分析中,葫芦素B,葫芦素D与有关物质分离度均符合要求,其质量浓度分别在49.5~297.0 mg·L^(-1)(r=0.999 99),10.3~61.8 mg·L^(-1)(r=0.999 9)与峰面积呈现良好的线性关系,平均回收率分别为99.18%,RSD 1.0%;99.13%,RSD 1.1%。结论:该研究基本阐明了甜瓜蒂中主要的四环三萜类成分,并建立了用于定量的HPLC方法,准确度高,重复性好,可用于甜瓜瓜蒂药材中葫芦素B,葫芦素D的含量测定。

Objective： To clarify the chemical pattern of major tetracyclic triterpenoids and establish an HPLC method for determination of cucurbitacin B and D in Pedicellus Melo. Method ： UPLC-ESI-Orbitrap-MS/MS was used to analyze the major tetracyclic triterpenoids on Agilent Eclipse XDB-Cls （4.6 mm ×150 mm, 5 μm） , with 0. 1% formic acid solution-acetonitrile as the mobile phase for gradient elution at a flow rate of 0.5 mL·min^-1 RP-HPLC was performed for the determination of cucurbitacin B and D in Pedicellus Melo on Inert Sustain CIS （4.6 mm × 250 mm, 5 μm） , with 0. 1% glacial acetic acid solution-acetonitrile as the mobile phase for gradient elution at a flow rate of 1.0 mL.min-1. Result： In the qualitative analysis of chemical pattern, 8 major tetracyclic triterpenoids were found and firstly identified in Pedicellus Melo, including cucurbitacin II a, isocucurbitacin Ⅱa, cucurbitacin I, cucurbitacin D, cucurbitacin B, and three isomers of deacetylatecucurbitacin B. In quantitative analysis, the separation degree of cucurbitacin B, D and related substances met the requirement, and the calibration curve showed good linearity for cucurbitacin B and D in the concentration range of 49.5-297.0 mg·L^-1 （r =0. 999 99） and 10.3-61.8 mg·L^-1 （r = 0. 999 9）. The average recoveries of cucurbitacin B and D were 99. 18% （RSD 1.0% ） and 99.13% （RSD 1.1% ） respectively. Conclusion： The major tetracyclic triterpenoids were reported for the first time in Pedicellus Melo, and the rapid and reproducible HPLC method was established, which was suitable for the determination of cucurbitacin B and D in Pedicellus Melo.