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采用分级沉淀法研究铁皮石斛多糖的理化性质及其体外初步抗氧化和抗肝癌细胞HepG2活性

Physicochemical properties of polysaccharides from Dendrobium officinale by fractional precipitation and their preliminary antioxidant and anti-HepG2 cells activities in vitro.

作者信息

Xing Shangping, Zhang Xiaofeng, Ke Hannu, Lin Ji, Huang Yuechun, Wei Gang

机构信息

School of Pharmaceutical Science, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China.

The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510006, China.

出版信息

Chem Cent J. 2018 Sep 25;12(1):100. doi: 10.1186/s13065-018-0468-4.

Abstract

BACKGROUND

Dendrobium officinale as a precious traditional Chinese herb is widely used in medicines and health supplements. Thus the extraction, purification and biological activities of polysaccharides from the stem of Dendrobium officinale have significant meaning on theory and application value.

METHODS

The crude Dendrobium officinale polysaccharide (DOP) was obtained by hot water extraction- ethanol precipitation method, and four new polysaccharide fractions (DOP-40, DOP-50, DOP-60, and DOP-70) were further obtained from the crude DOP by fractional precipitation with ethanol method, then four fractions were further purified by Toyopearl-H65F gel resin. The molecular weight and monosaccharide composition of four purified fractions were determined by high performance anion exchange chromatography and high performance liquid chromatography. The antioxidant activities of them were evaluated by the reducing power assay, and the superoxide anion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and hydroxyl free radicals scavenging assays, respectively. Finally, the anticancer activities of them were investigated via the MTT assay and the western blot analysis using HepG2 cells.

RESULTS

Among these four purified fractions were mainly composed of D-mannose and D-glucose with different molar ratios, and their average molecular weights were 999, 657, 243 and 50.3 kDa, respectively. What's more, DOP-70 always exhibited the strongest antioxidant and anticancer activities, while DOP-40 and DOP-60 showed very close antioxidant and anticancer activities which were better than that of DOP-50. The western blotting analysis also showed that DOP-40, DOP-60, and DOP-70 induced apoptosis in HepG2 human liver cancer cells through the Bcl-2 and Bax-dependent pathway.

CONCLUSIONS

Fractional precipitation with ethanol could successfully apply to extract four new polysaccharide fractions from Dendrobium officinale stems, and the polysaccharide fractions possessed efficient antioxidant and anticancer activities, especially DOP-70.

摘要

背景

铁皮石斛作为一种珍贵的传统中药材,被广泛应用于医药和保健品领域。因此,从铁皮石斛茎中提取、纯化多糖及其生物活性研究具有重要的理论意义和应用价值。

方法

采用热水提取 - 乙醇沉淀法获得铁皮石斛粗多糖(DOP),再通过乙醇分级沉淀法从粗多糖中进一步得到4个新的多糖级分(DOP - 40、DOP - 50、DOP - 60和DOP - 70),然后用Toyopearl - H65F凝胶树脂对这4个级分进行进一步纯化。采用高效阴离子交换色谱和高效液相色谱法测定4个纯化级分的分子量和单糖组成。分别通过还原力测定、超氧阴离子、2,2 - 二苯基 - 1 - 苦基肼(DPPH)和羟基自由基清除试验评估它们的抗氧化活性。最后,通过MTT试验和使用HepG2细胞的蛋白质印迹分析研究它们的抗癌活性。

结果

这4个纯化级分主要由不同摩尔比的D - 甘露糖和D - 葡萄糖组成,它们的平均分子量分别为999、657、243和50.3 kDa。此外,DOP - 70始终表现出最强的抗氧化和抗癌活性,而DOP - 40和DOP - 60表现出非常接近的抗氧化和抗癌活性,且优于DOP - 50。蛋白质印迹分析还表明,DOP - 40、DOP - 60和DOP - 70通过Bcl - 2和Bax依赖性途径诱导HepG2人肝癌细胞凋亡。

结论

乙醇分级沉淀法可成功用于从铁皮石斛茎中提取4个新的多糖级分,这些多糖级分具有高效的抗氧化和抗癌活性,尤其是DOP - 70。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d47b/6768022/d11dab945c86/13065_2018_468_Fig1_HTML.jpg

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