高速逆流色谱法制备罗汉果根中葫芦素类化合物

doi:10.3724/SP.J.1123.2021.07010

摘要/Abstract

摘要：

葫芦素作为四环三萜类化合物广泛存在于葫芦科植物中,但其含量较低、结构相似,采用常规的柱层析分离法较难得到大量、高纯度的单体化合物,导致其活性的研究与应用受到限制。研究采用高速逆流色谱法(HSCCC),建立了一种从罗汉果根提取物中制备葫芦素类化合物的方法。罗汉果根乙醇提取物经HPD-100大孔树脂、MCI、RP-C18柱层析分离后获得葫芦素粗品。通过基于薄层色谱的溶剂选择法(GUESS)对液-液萃取与高速逆流色谱的溶剂体系进行快速筛选,确定采用正己烷-乙酸乙酯-甲醇-水(3∶7∶3∶7, v/v/v/v)萃取葫芦素粗品,有机层萃取物再以正己烷-乙酸乙酯-甲醇-水(4∶6∶5∶5, v/v/v/v)进行HSCCC分离,以上相作为固定相,下相作为流动相,流速2.0 mL/min,主机转速860 r/min,进样量280 mg,一次分离即可获得5种葫芦素类化合物,经高效液相色谱检测其纯度分别为97.0%、95.4%、96.3%、91.6%和95.3%,并通过核磁共振技术、高分辨质谱技术及文献资料对比对各化合物的结构进行鉴定。经鉴定,5种化合物分别为葫芦素Q1、23,24-二氢葫芦素F-25-乙酸酯、葫芦素B、23,24-二氢葫芦素B和二氢异葫芦素B-25乙酸酯。该方法操作简单、快速,分离效果好,可作为规模化制备葫芦素类化合物的一种新方法。

关键词: 高速逆流色谱, 高效液相色谱, 制备, 葫芦素, 罗汉果根

Abstract:

Siraitia grosvenorii (Swingle) C. Jeffrey, belonging to the family Cucurbitaceae, is a natural sweetener. The roots of this plant are used in folk medicine for the treatment of rheumatoid arthritis. Cucurbitacins play an important role in the resistance of this plant to insects and adversity, and have anti-inflammatory, anti-tumor, and other biological activities. They usually exist as a variety of similar structures in Cucurbitaceae plants. Separation of a large amount of high-purity monomer compounds by the conventional separation method based on column chromatography is difficult, which limits the research and application of their activities. Therefore, we chose a new method for this separation. High-speed countercurrent chromatography (HSCCC) is a liquid-liquid chromatographic technique characterized by high recovery and reproducibility, and is considered a very effective method for the separation of natural compounds present in various plant extracts. An appropriate solvent system is the key for efficient separation, but its selection is tedious, which hampers the wider implementation of HSCCC in chemical research involving preparative separations. In this study, based on the general estimation strategy by using the TLC solvent system (GUESS), the corresponding relationship between the partition distribution coefficient (K value) and the TLC retention factor (R_f value) of the compounds was established by the partition experiment. The R_f value and separation coefficient α were calculated using the water-saturated organic phase as the expansion agent, which could minimize the number of countercurrent separation experiments required in solvent system selection. In this study, HSCCC was used to establish an efficient method for the extraction of cucurbitacins from the root extract of Siraitia grosvenorii. A fraction rich in cucurbitacins was obtained from the ethanol extract of Siraitia grosvenorii roots after separation by column chromatography on HPD-100, MCI, and C18 columns. Six types of solvent systems with different compositions were investigated using the GUESS method. The results showed that employing the solvent system of n-hexane-ethyl acetate-methanol-water (3∶7∶3∶7, v/v/v/v) to partition the cucurbitacin fraction could remove a large number of impurities. The components retained in the upper phase in the partition experiment were subsequently purified by HSCCC. The favorable solvent system for HSCCC was n-hexane-ethyl acetate-methanol-water (4∶6∶5∶5, v/v/v/v), while the upper and lower phases were selected as the stationary and mobile phases, respectively, with a flow rate of 2.0 mL/min, a rotation speed of 860 r/min, and an injected sample weight of 280 mg. Five cucurbitacin compounds were obtained by one-time separation. The weights of the five compounds were 14.73, 8.82, 30.74, 5.03, and 3.81 mg. The purities of these compounds were 97.0%, 95.4%, 96.3%, 91.6%, and 95.3%, respectively. Their structures were identified as cucurbitacin Q1, 23,24-dihydrocucurbitacin F-25-acetate, cucurbitacin B, 23,24-dihydrocucurbitacin B, and dihydroisocucurbitacin B-25-acetate by¹H-NMR and ¹³C-NMR spectroscopies, along with comparison with the literature. This study demonstrated how GUESS guidance accelerates the selection of HSCCC solvent systems, simplifies the workflow, and it provides an efficient preparative method for the separation of chemical constituents from the Siraitia grosvenorii roots, which can also be used as a new method for the large-scale preparation of cucurbitacin compounds.

Key words: high speed countercurrent chromatography (HSCCC), high performance liquid chromatography (HPLC), cucurbitacin, preparation, Siraitia grosvenorii roots

中图分类号:

O658

孙嘉忆, 孙佳祺, 李和平, 颜小捷, 李典鹏, 卢凤来. 高速逆流色谱法制备罗汉果根中葫芦素类化合物[J]. 色谱, 2022, 40(4): 364-371.

SUN Jiayi, SUN Jiaqi, LI Heping, YAN Xiaojie, LI Dianpeng, LU Fenglai. Preparation of cucurbitacin compounds in Siraitia grosvenorii roots by high speed countercurrent chromatography[J]. Chinese Journal of Chromatography, 2022, 40(4): 364-371.

图/表 5

图1 葫芦素粗品萃取前后的HPLC色谱图

Fig. 1 HPLC chromatograms of crude cucurbitacins before and after partition a. before partition; b. supper phase extracted by n-hexane-ethyl acetate-methanol-water (3:7:3:7, v/v/v/v); c. lower phase extracted by n-hexane-ethyl acetate-methanol-water (3:7:3:7, v/v/v/v).

表1 化合物在不同溶剂体系中的Rf和α

Table 1 Retention factor (Rf) and separation factor (α) values of the compounds in different solvent systems

H:E:M:W	R_f					α₁	α₂	α₃	α₄
H:E:M:W	No. 1	No. 2	No. 3	No. 4	No. 5	α₁	α₂	α₃	α₄
5:5:6:4	0.04	0.10	0.20	0.26	0.28	2.50	2.00	1.30	1.08
5:5:5:5	0.16	0.24	0.42	0.50	0.54	1.50	1.75	1.19	1.08
4:6:6:4	0.18	0.24	0.42	0.48	0.54	1.33	1.75	1.14	1.13
4:6:5:5	0.22	0.30	0.49	0.60	0.64	1.36	1.63	1.22	1.07
4:6:4:6	0.31	0.38	0.58	0.66	0.70	1.23	1.53	1.14	1.06
3:7:3:7	0.58	0.64	0.80	0.84	0.88	1.10	1.25	1.05	1.05

图2 罗汉果根葫芦素类化合物的高速逆流色谱图

Fig. 2 High speed countercurrent chromatogram (HSCCC) of cucurbitacin compounds in Siraitia grosvenorii roots

图3 化合物1~5的HPLC色谱图

Fig. 3 HPLC chromatograms of compounds 1-5

图4 化合物1~5的结构式

Fig. 4 Chemical structures of compounds 1-5

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