Preparation of cucurbitacin compounds in Siraitia grosvenorii roots by high speed countercurrent chromatography

doi:10.3724/SP.J.1123.2021.07010

Abstract

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

CLC Number:

O658

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.

Figures/Tables 5

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).

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

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

Fig. 3 HPLC chromatograms of compounds 1-5

Fig. 4 Chemical structures of compounds 1-5

References

[1]	Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China, Part 1. Beijing: China Medical Science Press, 2015
[1]	国家药典委员会. 中华人民共和国药典, 一部. 北京: 中国医药科技出版社, 2015
[2]	Chen Q B, Tang G M, Xu Q, et al. Chemical World, 2003, 58(1): 21
[2]	陈全斌, 汤桂梅, 徐庆, 等. 化学世界, 2003, 58(1): 21
[3]	Li D P, Mona E A, Tsuyoshi I, et al. Chem Pharm Bull, 2009, 57(8): 870
[4]	Wang X F, Lu W J, Chen J Y, et al. Chinese Herbal Medicines, 1996, 9(25): 515
[4]	王雪芬, 卢文杰, 陈家源, 等. 中草药, 1996, 9(25): 515
[5]	Lan Q, Jin C Z, Mo Y W. Northern Horticulture, 2018, (10): 144
[5]	蓝群, 金晨钟, 莫亿伟. 北方园艺, 2018, (10): 144
[6]	Si J Y, Chen D H, Shen L G, et al. Acta Pharmaceutica Sinica, 1999(12): 918
[6]	斯建勇, 陈迪华, 沈连钢, 等. 药学学报, 1999(12): 918
[7]	Hu Z Y, Lu F L, Zhao L C, et al. Journal of Jilin Agricultural University, 2020, doi: 10.13327/j.jjlau.2020.5596
[7]	扈芷怡, 卢凤来, 赵立春, 等. 吉林农业大学学报, 2020, doi: 10.13327/j.jjlau.2020.5596
[8]	Garg S, Kaul Sunil C, Wadhwa R. Int J Oncol, 2018, 52(1): 19
[9]	Jiang J Z, Xu C, Wang C Y, et al. Chinese Journal of Pathophysiology, 2020, 36(5): 886
[9]	姜京植, 徐畅, 王重阳, 等. 中国病理生理杂志, 2020, 36(5): 886
[10]	Zhang X Q, Tian L P, Zhang Y H, et al. Food Research and Development, 2018, 39(16): 164
[10]	张晓倩, 田丽萍, 张义晖, 等. 食品研究与开发, 2018, 39(16): 164
[11]	Jevtic B, Djedovic N, Stanisavljevic S, et al. J Agric Food Chem, 2016, 64(24): 4900
[12]	Cao X L. High-Speed Counter-Current Chromatography and its Application. Beijing: Chemical Industry Press, 2005: 41
[12]	曹学丽. 高速逆流色谱分离技术与应用. 北京: 化学工业出版社, 2005: 41
[13]	Peng S T, Liu Z L, Song Z Q, et al. Journal of Basic Chinese Medicine, 2021, 27(5): 821
[13]	彭诗涛, 刘振丽, 宋志前, 等. 中国中医基础医学杂志, 2021, 27(5): 821
[14]	Shi M. Yunnan Chemical Technology, 2019, 46(8): 113
[14]	施敏. 云南化工, 2019, 46(8): 113
[15]	Liang Y H, Xu W L, Hu R X, et al. Chinese Traditional Patent Medicine, 2020, 42(8): 2125
[15]	梁元昊, 徐文丽, 胡瑞雪, 等. 中成药, 2020, 42(8): 2125
[16]	Friesen J B, Pauli G F. J Liq Chromatogr R T, 2005, 28(17): 2777
[17]	Wang J, Gu D, Wang M. et al. J Chromatogr B, 2017, 1053: 16
[18]	Liu Y, J. Brent F, Edyta M, et al. J Chromatogr A, 2017, 1504: 46
[19]	Ito Y. J Chromatogr A, 2005, 1065(2): 145
[20]	Ding Q. [MS Dissertation]. Changsha: Zhongnan University, 2014
[20]	丁琼. [硕士学位论文]. 长沙: 中南大学, 2014
[21]	Hosny A. Phytochem, 1994, 36(1): 159
[22]	Dong J Y, Yue L, Shao W W, et al. China Journal of Chinese Materia Medica, 2012, 37(6): 814
[22]	董建勇, 岳磊, 邵薇薇, 等. 中国中药杂志, 2012, 37(6): 814
[23]	Wu P L, Lin F W, Wu T S, et al. Chem Pharm Bull, 2004, 52(3): 345
[24]	Miao J, Zhang J, Deng S M, et al. Chinese Herbal Medicines, 2012, 43(3): 432
[24]	苗静, 张洁, 邓世明, 等. 中草药, 2012, 43(3): 432