低抗凝肝素寡糖的制备、 结构表征及免疫活性评价

doi:10.7503/cjcu20150863

摘要/Abstract

摘要：

以猪肠黏膜来源低抗凝肝素为原料, 采用苄酯碱水解法制备了寡糖. 通过聚丙烯酰胺凝胶电泳(PAGE)、高效凝胶渗透色谱(HPGPC)-多角激光光散射法(MALLs)联用、核磁共振波谱(NMR)和液相色谱-质谱(LC-MS)联用技术对所制备寡糖的分子量分布及化学结构进行了表征. 以该寡糖对活化X因子(FXa)及人凝血酶(FⅡa)活性的抑制作用评价了其抗凝血活性, 并通过测定其对RAW264.7巨噬细胞吞噬能力及NO释放量的影响评价其免疫活性. 结果表明, 所制备寡糖的聚合度分布于2~22之间, 平均分子质量为5300, 无明显的抗凝血活性, 但具有显著的免疫增强活性.

关键词: 低抗凝肝素寡糖, 苄酯碱水解, 液相色谱-质谱联用, 免疫增强活性

Abstract:

Anticoagulant activities limit applications of heparin and enoxaparin sodium in immune-promoting activity. A novel and natural low anticoagulant heparin(LAH) was purified from crude porcine intestinal. In this work, oligosaccharide of LAH(BE-LAH) was prepared by benzyl ester alkaline hydrolysis. The molecular distribution and chemical structure of BE-LAH were determined based on combination technologies including polyacrylamide gel electrophoresis(PAGE), high performance gel permeation chromatography(HPGPC)-multi angel laser light scattering(MALLs), nuclear magnetic resonance(NMR) and liquid chromatography-mass spectrum(LC-MS). Meanwhile, the anticoagulant activities were evaluated by measuring the inhibitory activities on coagulation factor Xa(FXa) and thrombin(FⅡa), while the immunological activities were evaluated by its modulation effects on phagocytic ability and NO release of RAW 264.7 cells. The results demonstrated that BE-LAH was composed of fragments from dp2 to dp22 with an average molecular weight of 5300. Expectedly, BE-LAH showed no anticoagulant activity, but remarkable immune-promoting activity. A low anticoagulant heparin oligosaccharide was prepared and promised as an excellent immunoenhancer.

Key words: Low anticoagulant heparin oligosaccharide, Benzyl ester alkaline hydrolysis, Liquid chromatography-mass spectrum, Immune-promoting activity

中图分类号:

O629.1

TrendMD:

张晓, 单鑫迪, 赵小亮, 李国云, 王小江, 蔡超, 郝杰杰, 于广利. 低抗凝肝素寡糖的制备、结构表征及免疫活性评价. 高等学校化学学报, 2016, 37(7): 1335.

ZHANG Xiao, SHAN Xindi, ZHAO Xiaoliang, LI Guoyun, WANG Xiaojiang, CAI Chao, HAO Jiejie, YU Guangli. Preparation, Characterization and Immunological Activity Evaluation of Low Anticoagulant Heparin Oligosaccharides^†. Chem. J. Chinese Universities, 2016, 37(7): 1335.

图/表 7

Fig.1 Determination of molecular weight and its distribution of BE-LAH by HPGPC-MALLs

Fig.2 HILIC-FTMS total ion chromatogram(TIC) and major oligosaccharides quantified from BE-LAH (A) TIC of BE-LAH; (B) typical unsaturated oligosaccharide products quantification comparison of BE-LAH. Oligosaccharide compositions are given as △HexA, GlcN, HexA, Ac and SO3.

Table 1 Compositional analysis of BE-LAH, LAH and standard HP, HS, ES*

Sample	Composition(%)
Sample	Unsulfated disaccharides	Monosulfated disaccharides	Disulfated disaccharides	Trisulfated disaccharides	Total 3-O-sulfo group containing tetrasaccharides
HP-STD	1.69	5.17	10.98	75.96	6.20
ES-STD	2.07	9.02	11.81	71.28	5.82
LAH	4.30	28.92	17.07	49.13	0.57
BE-LAH	4.52	30.63	20.83	44.02
HS-STD	43.96	32.19	14.35	9.50

Fig.3 1H NMR(A), 13C NMR(B) of BE-LAH(a) and USP ES(b) A: glucosamine; M: mannosamine; G: glucuronic acid; I: iduronic acid; △U: 4-deoxy-α-L-threo-hex-4-enopyranosil uronic acid; peak a: ANS6S+ANS-C3; peak b: I2S-C3.

Fig.4 1H-13C HSQC spectrum of BE-LAH A: glucosamine; M: mannosamine; G: glucuronic acid; I: iduronic acid; △U: 4-deoxy-α-L-threo-hex-4-enopyranosil uronic acid; 1,6-an. A: 1,6-anhydro-glucosamine; 1,6-an. M: 1,6-anhydro-mannosamine; α/β red: terminal reducing residue with α/β configuration.

Table 2 Anti-Xa and anti-Ⅱa activities of samples

GAGs	Anti-Xa activity/(U·mg^-1)	Anti-Ⅱa activity/(U·mg^-1)	Anti-Xa/anti-Ⅱa(ratio)
HP	180	180	1.0
ES	160±5	49±4	3.3
HS	11±2	11±2	1.0
LAH	24±3	25±3	1.0
BE-LAH	9±1	7±1	1.3

Fig.5 Effects of different doses of GAGs on the phagocytosis index(A) and NO release(B) of the mouse macrophage cell line RAW 264.7Results are expressed as means ±S.D. * P<0.05, ** P<0.01, compared with blank control, as determined by unpaired student’s t-test.

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