Preparation, Characterization and Immunological Activity Evaluation of Low Anticoagulant Heparin Oligosaccharides†

doi:10.7503/cjcu20150863

Abstract

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

CLC Number:

O629.1

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2016, 37(7): 1335.

Figures/Tables 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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