罗汉果干果多糖的分离纯化、结构表征及其体外免疫调节活性

doi:10.7506/spkx1002-6630-20231103-025

食品科学 ›› 2025, Vol. 46 ›› Issue (4): 10-20.doi: 10.7506/spkx1002-6630-20231103-025

• 基础研究 • 上一篇

罗汉果干果多糖的分离纯化、结构表征及其体外免疫调节活性

张巧铃，蒋小华，张洁，卢凤来，韦玉璐，刘宏伟，宋静茹

（1.沈阳药科大学中药学院，辽宁沈阳 110016；2.广西植物功能物质与资源持续利用重点实验室，广西壮族自治区中国科学院广西植物研究所，广西桂林 541006）

发布日期:2025-02-07
基金资助:
广西科技重大专项（桂科AA23023035；桂科AA22096020）；广西科技基地和人才专项（桂科AA21196009）；广西植物功能物质与资源持续利用重点实验室主任基金项目（ZRJJ2023-11）；桂林市创新平台和人才计划项目（20210102-3）

Isolation, Purification and Structural Characterization of Polysaccharides from Dried Siraitia grosuenorii Fruits and Their Immunodulatory Activity in Vitro

ZHANG Qiaoling, JIANG Xiaohua, ZHANG Jie, LU Fenglai, WEI Yulu, LIU Hongwei, SONG Jingru

(1. School of Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; 2. Guangxi Key Laboratory of Plant Functional Phytochemicals and Sustainable Utilization, Guangxi Institute of Botany, Chinese Academy of Sciences, Guangxi Zhuang Autonomous Region, Guilin 541006, China)

Published:2025-02-07

摘要/Abstract

摘要： 探究罗汉果干果多糖（Siraitia grosuenorii polysaccharide，SGP）的结构及其免疫调节活性。利用水提醇沉的方法从罗汉果干果中提取粗多糖，进一步经Cellulose DE-52和Sephadex G-200柱分离纯化获得均一多糖组分SGP-C2和SGP-D1。采用PMP柱前衍生及配备有示差折光检测器的高效液相色谱系统测定其单糖组成和分子质量，并通过红外光谱与核磁共振对其结构进一步表征。采用中性红比色法检测SGP-C2和SGP-D1对RAW264.7细胞的吞噬活性，荧光探针DCFH-DA试剂检测其活性氧（reactive oxygen species，ROS）的释放，Griess法检测其NO生成以及利用酶联免疫吸附检测试剂盒检测其对肿瘤坏死因子-α（tumor necrosis factor，TNF）-α与白细胞介素-6（interleukin-6，IL-6）分泌水平的影响。SGP-C2和SGP-D1是由葡萄糖醛酸、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖（galactose，Gal）、阿拉伯糖组成的杂多糖，其单糖物质的量之比分别为0.78∶1.84∶26.57∶1.31∶0.49∶0.06和1.37∶2.00∶20.79∶0.61∶0.12∶0.25，分子质量分别为5.0×105 Da和7.6×105 Da。核磁共振谱图显示SGP-C2主链结构为→4)-α-D-GalpA-(1→和→4)-α-D-GalpAMe-(1→，而SGP-D1主链结构为→4)-α-D-GalpA-(1→。同时，6.25～25 µg/mL质量浓度范围的SGP-C2和SGP-D1具有促进巨噬细胞吞噬、NO生成、TNF-α和IL-6分泌的作用，SGP-D1还具有促进ROS释放的活性。结果表明，罗汉果多糖SGP-C2和SGP-D1具有体外免疫调控活性，为罗汉果多糖资源的开发利用提供了科学依据和技术路线。

关键词: 罗汉果；多糖；纯化；结构表征；免疫调节

Abstract: We undertook this study to evaluate the structure and immunomodulatory activity of polysaccharides from dried Siraitia grosuenorii fruits. The crude polysaccharides were obtained by water extraction followed by alcohol precipitation and purified and separated into two homogeneous fractions: SGP-C2 and SGP-D1 through sequential Cellulose DE-52 and Sephadex G-200 column chromatography. The monosaccharide compositions and molecular masses of SGP-C2 and SGP-D1 were analyzed by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by high performance liquid chromatography (HPLC) equipped with a refractive index detector (RID). Their structures were characterized by Fourier transform infrared spectrometer (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. Their immunomodulatory activity was evaluated by the neutral red colorimetric assay, their activity to induce the release of reactive oxygen species (ROS) from RAW264.7 cells was tested using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as the fluorescent probe, and their activity to induce NO production and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in RAW264.7 cells was examined using a Griess kit and an enzyme-linked immunosorbent assay (ELISA) kit, respectively. SGP-C2 and SGP-D1 were heteropolysaccharides composed of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose with mole ratios of 0.78:1.84:26.57:1.31:0.49:0.06 and 1.37:2.00:20.79:0.61:0.12:0.25, respectively. Their relative molecular masses were 5.0 × 105 and 7.6 × 105 Da, respectively. NMR analysis showed that the backbone chain structure of SGP-C2 was →4)-α-D-GalpA-(1→ and →4)-α-D-GalpAMe-(1→, while that of SGP-D1 was →4)-α-D-GalpA-(1→. In the concentration range of 6.25–25 µg/mL, both polysaccharides promoted macrophage phagocytosis, NO production, and the secretion of TNF-α and IL-6. Besides, SGP-D1 significantly promoted the release of ROS. These results showed that SGP-C2 and SGP-D1 had immune-enhancing effects in vitro. This study provides the scientific basis for the development and application of polysaccharides from S. grosuenorii.

Key words: Siraitia grosuenorii; polysaccharide; purification; structural characterization; immunomodulatory

中图分类号:

TS201.2

张巧铃，蒋小华，张洁，卢凤来，韦玉璐，刘宏伟，宋静茹. 罗汉果干果多糖的分离纯化、结构表征及其体外免疫调节活性[J]. 食品科学, 2025, 46(4): 10-20.

ZHANG Qiaoling, JIANG Xiaohua, ZHANG Jie, LU Fenglai, WEI Yulu, LIU Hongwei, SONG Jingru. Isolation, Purification and Structural Characterization of Polysaccharides from Dried Siraitia grosuenorii Fruits and Their Immunodulatory Activity in Vitro[J]. FOOD SCIENCE, 2025, 46(4): 10-20.

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罗汉果干果多糖的分离纯化、结构表征及其体外免疫调节活性

Isolation, Purification and Structural Characterization of Polysaccharides from Dried Siraitia grosuenorii Fruits and Their Immunodulatory Activity in Vitro

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