刺五加果多糖ASPF的结构表征及其体外抗肺癌活性研究

doi:10.11924/j.issn.1000-6850.casb2021-0779

摘要/Abstract

摘要：

从刺五加果实体中纯化得到一种均一分子量多糖ASPF,对其结构表征及体外抗肺癌活性进行研究。将提取得到的粗多糖通过DEAE-52柱层析分离得到3个组分,进行体外抗肺癌活性筛选后,将ASPB-3通过Sephadex G-200凝胶柱色谱得到ASPF,选择GPC、IR、HPLC方法对该多糖进行结构解析,并采用CCK-8法研究其对肺癌细胞体外存活率的影响。测得ASPF的糖醛酸含量为22.9%,是一种酸性果胶类结构多糖,其相对分子质量M_W为9.7462×10⁴,IR扫描结果显示其具有吡喃环结构,单糖测定结果显示其由鼠李糖、半乳糖醛酸、葡萄糖、木糖、半乳糖、阿拉伯糖6种单糖组成,对A549细胞存在抑制效果,其机制可能与作用于凋亡基因Bcl-2/Bax有关。ASPF对人肺癌细胞具有一定体外抑制作用。

关键词: 刺五加果实体, 多糖, 纯化, 结构表征, 抗肺癌

Abstract:

Homogenous molecular weight polysaccharide ASPF was purified from the fructification of Acanthopanax senticosus, and its structure characterization and in vitro anti-lung cancer activity were explored. The extracted crude polysaccharides were separated by DEAE-52 column chromatography to obtain 3 components, after screening for in vitro anti-lung cancer activity, ASPB-3 was obtained by Sephadex G-200 gel column chromatography to get ASPF. GPC, IR and HPLC methods were selected to analyze the structure of the polysaccharide, and the CCK-8 method was used to study its effect on the survival rate of lung cancer cells in vitro. The content of uronic acid in ASPF was 22.9%. It was an acidic pectin-like structure polysaccharide. Its relative molecular mass M_W was 9.7462×10⁴. IR scanning results showed that it had a pyran ring structure, which was composed of rhamnose and galacturonic acid. The results of monosaccharide determination showed that it was composed of six monosaccharides: rhamnose, galacturonic acid, glucose, xylose, galactose, and arabinose. There was an inhibitory effect on A549 cells, and its mechanism might be related to the effect on the apoptosis gene Bcl-2/Bax. In summary, ASPF has a certain in vitro inhibitory effect on human lung cancer cells.

Key words: Acanthopanax senticosus fructification, polysaccharide, purification, structural characterization, anti-lung cancer

中图分类号:

R284.2

崔雪娇, 佟潇禹, 张彦龙, 曾伟民. 刺五加果多糖ASPF的结构表征及其体外抗肺癌活性研究[J]. 中国农学通报, 2022, 38(22): 157-164.

CUI Xuejiao, TONG Xiaoyu, ZHANG Yanlong, ZENG Weimin. Polysaccharide ASPF from Acanthopanax senticosus Fructification: Structural Characterization and Its Anti-lung Cancer Activity in Vitro[J]. Chinese Agricultural Science Bulletin, 2022, 38(22): 157-164.

图/表 14

基因名称		序列(5'→3')
Bcl-2	上游	GTGGAGGAGCTCTTCAGGGA
Bcl-2	下游	CCGGTTCAGGTACTCAGTCATC
Bax	上游	AGGATGATTGCCGCCGT
Bax	下游	GGCCTTGAGCACCAGTTTG
β-actin	上游	TAGTTGCGTTACACCCTTTCTTG
β-actin	下游	TCACCTTCACCGTTCCAGTTT

表1. 引物序列

图1. DEAE-52纤维素离子柱洗脱曲线

图2. DEAE-52柱层析各组分对A549细胞增殖活性的影响

图3. Sephadex G-200凝胶柱洗脱曲线

图4. ASPF HPGPC色谱图

图5. ASPF的IR扫描图

图6. 单糖标准品液相色谱图

图7. ASPF单糖组成液相色谱图

单糖	标准品保留时间/min	样品保留时间/min
Rha	26.707	26.755
Gal UA	35.985	36.011
Glc	42.225	42.253
Xyl	48.826	48.851
Gal	51.373	51.413
Ara	53.829	53.866

表2. 标准品、样品中单糖的保留时间

图8. A549细胞凋亡形态变化的倒置显微镜图片(200×) A为对照组,B、C、D、E分别为100、200、400、800 μg/mL ASPF处理48 h,F为阳性对照组（5 μg/mL姜黄素处理48 h）

图9. 不同浓度ASPF对A549细胞存活率的影响表示与空白组相比差异显著(P<0.05),**表示与空白组相比差异极显著(P<0.01),下同

图10. ASPF对A549细胞Caspase酶活性的影响

图11. RNA电泳检测图

图12. ASPF对Bcl-2/Bax的mRNA表达量影响

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