灰树花多糖的分离纯化、结构表征及精氨酸酶抑制活性

Isolation,purification,structural characterization and arginase inhibitory activity of polysaccharides from Grifola frondosa

采用碱提法,并以DEAE Sepharose Fast Flow离子柱层析和Sephacryl S-500 HR凝胶柱层析从灰树花(Grifola frondose)子实体中分离纯化得到水溶性多糖GFPN-2-A,通过高效尺寸排阻色谱-多角度激光散射-示差联用(high-performance size exclusion chromatography-multi-angle laser scattering-refractive index,HPSEC-MALLS-RI)、HPLC、傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FT-IR)、扫描电子显微镜、原子力显微镜、刚果红实验和比色法等对其进行结构表征及精氨酸酶抑制活性分析。结果表明,GFPN-2-A是分子质量为4.56×106 Da的β构型多糖,主要由葡萄糖以及少量的葡萄糖醛酸、甘露糖、核糖和半乳糖组成,物质的量比为54∶1∶0.51∶0.37∶0.68,其主要为碎屑状和片状,在水溶液中的构象呈现出圆柱和圆锥的块状特征,不含三螺旋结构。GFPN-2-A能够抑制精氨酸酶的活性,IC_(50)为(0.855±0.64)mg/mL。动力学分析表明,GFPN-2-A对精氨酸酶的抑制为竞争性可逆抑制,抑制常数K_(i)为0.284 mg/mL。该研究首次证明了灰树花多糖对精氨酸酶具有抑制作用,为灰树花多糖药物开发开辟新的药物靶点提供了理论依据。

The water-soluble polysaccharide of GFPN-2-A isolated by alkaline extraction and purified by DEAE Sepharose Fast Flow ion column chromatography and Sephacryl S-500 HR gel column chromatography from the fruiting bodies of Grifola frondosa.Its structural characterization and the arginase inhibitory activity analyzed by high-performance size exclusion chromatography-multi-angle laser light scattering-refractive index(HPSEC-MALLS-RI),HPLC,Fourier transform infrared spectroscopy,scanning electron microscopy,atomic force microscope,Congo red experiment and colorimetric method.The results showed that GFPN-2-A was aβ-configuration polysaccharide with the molecular weight 4.56×106 Da,composed of glucose and a small amount of glucuronic acid,mannose,ribose,and galactose in the molar ratio of 54:1:0.51:0.37:0.68,it mainly existed in the form of clasts and sheets,and its conformation in aqueous solution presented the characteristics of cylinder and cone,without triple helix structure.GFPN-2-A could inhibit the activity of arginase,the IC_(50) was(0.855±0.64)mg/mL.Kinetic analysis showed that the inhibition of arginase by GFPN-2-A was competitive and reversible,and the inhibition constant K_(i) was 0.284 mg/mL.This study proved for the first time that G.frondosa polysaccharides have an inhibitory effect on arginase and provided a theoretical basis for the development of new drug targets for G.frondosa polysaccharides.