摘要
目的:提取纯化香附粗多糖,制备纯化香附多糖(PPX),测定PPX单糖组成和相对分子量并研究其免疫调节作用。方法:采用单因素试验和响应面试验优化香附粗多糖的提取工艺,采用醇沉法制备香附粗多糖,进一步通过除蛋白、透析和SephadexG-50凝胶柱层析制备PPX。采用高效液相色谱法(HPLC)探究PPX的单糖组成,采用高效凝胶渗透色谱法(HPGPC)测定PPX的相对分子质量。采用噻唑蓝(MTT)法、中性红实验、一氧化氮(NO)试剂盒和酶联免疫吸附分析(ELISA)检测小鼠巨噬细胞RAW264.7的增殖、吞噬及其释放NO、分泌白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的能力,评价PPX的免疫活性。结果:香附粗多糖最佳提取工艺为料液比1 g∶25 mL,100℃提取90 min,提取3次,香附粗多糖的质量分数为(17.57±0.29)%。PPX主要由甘露糖(Man)、鼠李糖(Rha)、D-葡萄糖醛酸(D-GlcA)、D-半乳糖醛酸(D-GalA)、D-葡萄糖(D-Glc)、D-半乳糖(D-Gal)、D-木糖(D-Xyl)、L-阿拉伯糖(L-Ara)组成,各单糖的质量分数分别为Man3.38%、Rha4.62%、D-GlcA1.88%、D-GalA7.54%、D-Glc54.24%、D-Gal10.29%、D-Xyl8.06%、L-Ara9.99%;相对分子量为8912.5 Da。体外免疫活性实验表明,在质量浓度12.5~200.0μg/mL范围内,PPX能够促进巨噬细胞RAW264.7的增殖、吞噬及其释放NO、分泌TNF-α和IL-6。结论:香附粗多糖的提取工艺可靠,PPX具有较好的免疫调节作用,本研究可为香附多糖的开发和应用提供基础。
Objective:To extract and purify crude polysaccharides of Xiangfu(Cyperi Rhizoma),process and purify polysaccharides from Xiangfu(PPX),determine the monosaccharide composition and relative molecular mass of PPX,and to study its immunomodulative effect.Methods:The extraction technology of crude polysaccharides from Xiangfu was optimized by single factor test and response surface experiment.The crude polysaccharides from Xiangfu were prepared by alcohol precipitation method,and PPX was further processed by protein removal,dialysis and Sephadex G-50 Gel column chromatography.The monosaccharide composition of PPX was investigated by high performance liquid chromatography(HPLC),and the relative molecular mass of PPX was determined by high performance gel permeation chromatography(HPGPC).The proliferation,phagocytosis of mouse macrophage RAW264.7 cells and its ability to release nitric oxide(NO),to secrete interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected by thiazolyl blue(MTT)assay,neutral red assay,NO detection kit and enzyme-linked immunosorbent assay(ELISA)to evaluate the immune activity of PPX.Results:The optimum extraction technology of crude polysaccharides from Xiangfu was refluxing three times with water at 100℃and a solid-liquid ratio of 1 g∶25 mL,90 minutes each time,and the mass fraction of crude polysaccharides from Xiangfu was(17.57±0.29)%.PPX was mainly composed of annose(Man),rhamnose(Rha),D-glucuronic acid(D-GlcA),Dgalacturonic acid(D-GalA),D-glucose(D-Glc),D-galactose(D-Gal),D-xylose(D-Xyl),and L-arabinose(L-Ara).The mass fraction of each monosaccharide were Man 3.38%,Rha 4.62%,D-GlcA 1.88%,D-GalA 7.54%,DGlc 54.24%,D-Gal 10.29%,D-Xyl 8.06%,L-Ara 9.99%.The relative molecular mass was 8912.5 Da.In vitro immune activity experiments showed that PPX could promote the proliferation,phagocytosis of macrophage RAW264.7 cells and NO release,secretion of TNF-αand IL-6 within the mass concentration range of 12.5~200.0μg/mL.Conclusions:The extraction technology of crude polysaccharides from Xiangfu is reliable,and PPX has good immunomodulatory effect.This study can provide a basis for the development and application of polysaccharides from Xiangfu.
作者
邓碧琦
郑真
陈雪梅
朱立俏
盛华刚
DENG Biqi;ZHENG Zhen;CHEN Xuemei;ZHU Liqiao;SHENG Huagang(School of Pharmacy,Shandong Uinversity of Trational Chinese Medicine,Jinan 250355,China)
出处
《山东中医药大学学报》
2025年第4期503-512,共10页
Journal of Shandong University of Traditional Chinese Medicine
基金
山东省自然科学基金面上项目(编号:ZR2022MH173)。
关键词
香附多糖
提取纯化
单糖组成
相对分子量
免疫活性
巨噬细胞RAW264.7
polysaccharides from Xiangfu(Cyperi Rhizoma)
extraction and purification
monosaccharide composition
relative molecular mass
immune activity
macrophage RAW264.7 cells
