山豆根多糖的制备、单糖组成及体外抗肿瘤作用  

Preparation,monosaccharide composition and in vitro antitumor effect of polysaccharide from Sophora tonkinensis Radix et Rhizoma

作　　者：王淑娜 杨丽雅 余登香 傅月朦 邓中平[1] WANG Shu-na;YANG Li-ya;YU Deng-xiang;FU Yue-meng;DENG Zhong-ping(Innovation Research Institute of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学创新中药研究院,上海201203

出　　处：《中国新药杂志》2023年第11期1155-1162,共8页Chinese Journal of New Drugs

摘　　要：目的:制备山豆根多糖(polysaccharide of Sophorae Tonkinensis Radix et Rhizoma, SRP)并分析其单糖组成,探索SRP的体外抗肿瘤作用。方法:水提醇沉法制备SRP,紫外-可见分光光度法测定SRP中多糖、黄酮、皂苷、酚类和蛋白质含量,高效液相色谱法测定生物碱含量,返滴定法测定有机酸含量。通过高效液相色谱仪,色谱柱采用Waters X Bridge C_(18)柱(250 mm×4.6 mm, 5μm),以乙腈:磷酸盐缓冲液(0.05 mol·L^(-1),pH=6.8)=18∶82为流动相,等梯度洗脱,流速为0.8 mL·min^(-1),柱温为30℃,检测波长为245 nm,建立SRP的单糖组成分析方法,测定SRP中各单糖含量并计算摩尔比。采用MTT法检测SRP抑制肿瘤细胞增殖的活性。结果:水提醇沉法制得SRP,棕黄色粉末,得率为3.95%,其中不含生物碱成分,多糖、黄酮、皂苷、有机酸和酚类含量分别为66.08%,7.17%,3.45%,2.54%和4.33%,蛋白质含量可忽略不计。SRP由甘露糖、鼠李糖、葡糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖组成,各单糖的摩尔比为1.25∶0.31∶0.28∶0.50∶40.47∶1.00∶10.46,SRP的质量分数为64.97%。SRP对Hepa1-6,HepG2,HT29和4T1细胞均具有显著的增殖抑制作用且呈浓度依赖性,IC_(50)值分别为770,1 910,1 870和1 880μg·mL^(-1)。结论:本研究制备的SRP中多糖含量大于60%,已知成分含量大于80%,其中不含生物碱。1-苯基-3-甲基-5-吡唑啉酮(1-phenyl-3-methyl-5-pyrazolo, PMP)柱前衍生化-HPLC法精密度、重复性和准确性良好,可用于分析SRP的单糖组成。SRP是一类以葡萄糖和阿拉伯糖为主要成分的杂多糖。SRP具有明显的细胞毒作用,提示SRP可通过抑制肿瘤细胞增殖发挥抗肿瘤作用。本研究为推进SRP成分的基础研究和促进SRP的抗肿瘤作用研究提供了参考。Objective:To extract and separate polysaccharide from Sophora tonkinensis Radix et Rhizoma(SRP),determine the monosaccharide composition and content of SRP,and investigate the antitumor effects of SRP in vitro.Methods:SRP was prepared by water extraction and alcohol precipitation.The contents of polysaccharides,flavonoids,saponins,total phenols and proteins in SRP were determined by UV-Vis spectrophotometry,the contents of alkaloids were determined by high performance liquid chromatography(HPLC),and the content of organic acid was measured by back titration method.The monosaccharide analysis method of SRP was established by HPLC.A Waters X Bridge C_(18) column(250 mm×4.6 mm,5μm)was used with acetonitrile:phosphate buffer(0.05 mol·L^(-1),pH=6.8)at the ration of 18:82 as mobile phase,the flow rate was 0.8 mL·min^(-1),the column temperature was 30℃,and the detection wavelength was 245 nm.The contents of monosaccharides in SRP were determined and the mole ratio was calculated.MTT assay was used to detect the antitumor effects of SRP in vitro.Results:SRP,which is brown powder,was obtained by water extraction and alcohol precipitation with a yield of 3.95%.The contents of polysaccharides,flavonoids,saponins,organic acids and total phenols in SRP were 66.08%,7.17%,3.45%,2.54%and 4.33%,respectively.No alkaloids and proteins were detected.SRP was composed of mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose and arabinose.The mole ratios were 1.25:0.31:0.28:0.50:40.47:1.00:10.46,and the percentage of polysaccharide was 64.97%.SRP had inhibitory effect on Hepal-6,HepG2,HT29 and 4T1 cells in a concentration-dependent manner with IC_(50) of 770,1910,1870 and 1880μg·mL^(-1),respectively.Conclusion:The SRP prepared in this study contained more than 60%polysaccharide and more than 80%known components,among which no alkaloids were detected.HPLC with pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolo has good precision,reproducibility and accuracy,and is suitable for the monosaccharide composion a

关 键 词：山豆根多糖 单糖组成 Hepa1-6细胞 HEPG2细胞 HT29细胞 4T1细胞 抗肿瘤 

分 类 号：R969.1[医药卫生—药理学]