Ji Xin, Liu Xiao-Qian, Gao Lu, Xiao Su-Ping, Liang Yao-Hua, Li Chun, Wang Zhi-Min
National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700,China.
Xiuzheng Pharmaceutical Group Co.,Ltd. Changchun 130012,China.
Zhongguo Zhong Yao Za Zhi. 2021 Mar;46(5):1168-1178. doi: 10.19540/j.cnki.cjcmm.20200706.204.
Triterpenoids are one of the most active constituents in Ligustri Lucidi Fructus, but only oleanolic acid has been mostly studied. In recent years, a growing number of studies have shown that other triterpenes from Ligustri Lucidi Fructus also have various biological activities, so it is necessary to build up a detailed profile of the triterpenoids in Ligustri Lucidi Fructus. The chromatographic separation was performed on a C_(18) column(4.6 mm×250 mm, 5 μm) with mobile phase of methanol-acetonitrile-0.2% formic acid for gradient elution. The detection wavelength was set at 210 nm, with a flow rate of 0.5 mL·min~(-1), and the column temperature of 25 ℃. The HPLC fingerprint of triterpenoids in Ligustri Lucidi Fructus was built by testing 21 batches of samples from different sources. The structures of the total 15 common chromatographic peaks were elucidated with UHPLC-ESI-Orbitrap-MS/MS technique and six of them were identified as tormentic acid, pomolic acid, maslinic acid, botulin, oleanolic acid and ursolic acid by comparison to the reference substances. Under the same chromatographic condition, four main triterpenes(podocarboxylic acid, hawthorn acid, oleanolic acid and ursolic acid) were quantified and the results of system adaptability and methodology investigation all met the requirements of content determination. Meanwhile, with oleanolic acid(A) as the internal reference substance, quantitative analysis of multi-components by single marker(QAMS) method was used to analyze the above four components. The relative correction factor of oleanolic acid(B), hawthorn acid(C) and ursolic acid(D) to oleanolic acid was f_(B/A)=1.12, f_(C/A)=1.02 and f_(D/A)=0.88, respectively, and the relative retention values of these three to oleanolic acid was RRV_(B/A)=0.46, RRV_(C/A)=0.70 and RRV_(D/A)=1.03, respectively. The contents determined by two methods were similar. In conclusion, the method built in this paper is proved to be simple, reliable and specific for the simultaneous qualitative and quantitative analysis of the triterpenoids in Ligustri Lucidi Fructus, which can lay foundation for further assays of the triterpenoids in Ligustri Lucidi Fructus and the relative products.
三萜类化合物是女贞子中活性最强的成分之一,但目前仅对齐墩果酸进行了较多研究。近年来,越来越多的研究表明,女贞子中的其他三萜类化合物也具有多种生物活性,因此有必要建立女贞子中三萜类化合物的详细图谱。采用C18色谱柱(4.6 mm×250 mm,5 μm)进行色谱分离,流动相为甲醇 - 乙腈 - 0.2%甲酸,进行梯度洗脱。检测波长设定为210 nm,流速为0.5 mL·min-1,柱温为25℃。通过检测21批不同来源的样品,建立了女贞子中三萜类化合物的HPLC指纹图谱。采用UHPLC - ESI - Orbitrap - MS/MS技术对15个共有色谱峰的结构进行了解析,其中6个通过与对照品比较,鉴定为 tormentic acid、pomolic acid、maslinic acid、botulin、齐墩果酸和熊果酸。在相同色谱条件下,对4种主要三萜类化合物(坡模酸、山楂酸、齐墩果酸和熊果酸)进行了定量分析,系统适应性和方法学考察结果均符合含量测定要求。同时,以齐墩果酸(A)为内参物,采用一测多评法(QAMS)对上述4种成分进行了分析。齐墩果酸(B)、山楂酸(C)和熊果酸(D)对齐墩果酸的相对校正因子分别为fB/A = 1.12、fC/A = 1.02和fD/A = 0.88,这3种成分对齐墩果酸的相对保留值分别为RRVB/A = 0.46、RRVC/A = 0.70和RRVD/A = 1.03。两种方法测定的含量相近。综上所述,本文建立的方法被证明简单、可靠且专属性强,可用于女贞子中三萜类化合物的同时定性和定量分析,可为进一步研究女贞子及其相关产品中的三萜类化合物奠定基础。