黄羽鸡肠多糖提取工艺、结构表征及其抗凝血活性

doi:10.3864/j.issn.0578-1752.2026.06.013

Abstract

Abstract:

【Objective】 This study aimed to establish an efficient extraction process for polysaccharides from yellow-feathered chicken intestine (YFCI-P), to analyze the structural characteristics of its key components, and to evaluate their in vitro anticoagulant and antithrombotic activities, thereby providing a theoretical basis for the high-value utilization of chicken intestinal by-products. 【Method】Single-factor experiments combined with Response Surface Methodology (RSM) were used to optimize the enzymatic hydrolysis extraction conditions of YFCI-P; The anticoagulant potency of the enzymatic hydrolysate was determined by the sheep plasma method. The polysaccharide components YFCI-P1 and YFCI-P2 were obtained by separation and purification through gel filtration column chromatography. Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) levels were assessed to evaluate the anticoagulant activity of the polysaccharides, alongside an assessment of in vitro thrombolytic activity. The molecular weight of YFCI-P1 was determined by High-Performance Gel Permeation Chromatography (GPC/HPLC). Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) techniques were applied to analyze its functional group composition and glycosidic linkage patterns. 【Result】The optimum enzymatic hydrolysis conditions were determined as follows: temperature 55 ℃, enzyme-to-substrate ratio 0.9%, time 3 h, sodium chloride concentration 3%, solid-to-liquid ratio 1:1 (g·mL^-¹), and pH 8.5. Under these conditions, the anticoagulant potency of the enzymatic hydrolysate reached 5.13 U·mL^-1. The predicted value from the response surface model showed good agreement with the experimental value. Two polysaccharide fractions, including YFCI-P1 and YFCI-P2, were obtained through separation and purification by gel filtration chromatography. In vitro anticoagulant activity studies demonstrated that at a concentration of 1 000 μg·mL^-1, YFCI-P significantly prolonged APTT to 485.7 s; YFCI-P1 significantly prolonged PT to 600 s; and YFCI-P2 significantly prolonged TT to 367 s, suggesting their anticoagulant effects via the intrinsic and extrinsic pathways, extrinsic and common pathways, and intrinsic and common pathways, respectively. Molecular characterization revealed that YFCI-P1 had a weight-average molecular weight (Mw) of 10.2 kDa and contained uronic acid, acetyl amino, and sulfate groups. Its backbone consisted of repeating disaccharide units composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) linked alternately by β-1, 4 and β-1, 3 glycosidic bonds, exhibiting the characteristic features of glycosaminoglycans. Furthermore, YFCI-P, YFCI-P1, and YFCI-P2 promoted tissue plasminogen activator (tPA) generation and demonstrated in vitro thrombolytic activity, with clot dissolution rates of 42.38%, 36.91%, and 19.74% at 1 000 μg·mL^-1, respectively. 【Conclusion】An efficient and stable extraction process for YFCI-P was successfully established, and the optimized parameter combination demonstrated promising potential for industrial application. YFCI-P1 was identified as a polysaccharide exhibiting structural features characteristic of glycosaminoglycans. Moreover, YFCI-P, YFCI-P1, and YFCI-P2 all exhibited significant in vitro anticoagulant and thrombolytic activities.

Key words: chicken intestines, polysaccharides, anticoagulant activity, structural characterization, glycosaminoglycan

ZHAO QingYao, WANG XiaoMing, XING Tong, LI LingYun, XU XingLian, ZHAO Xue. Extraction Optimization, Structural Characterization, and Anticoagulant Activity of Intestinal Polysaccharides from Yellow-Feathered Chickens[J].Scientia Agricultura Sinica, 2026, 59(6): 1317-1332.

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因素 Factor		水平 Level
因素 Factor		-1	0	1
酶解温度 Hydrolysis temperature A (℃)		50	55	60
酶底物比 Enzyme-to-substrate ratio B (%)		0.8	0.9	1.0
酶解时间 Hydrolysis time C (h)		2.5	3	3.5
RUN	A	B	C	Y (U·mL^-1)
1	0	0	0	5.20
2	-1	0	1	4.50
3	1	0	0	5.20
4	-1	-1	0	4.42
5	1	1	0	4.34
6	0	0	0	5.17
7	0	1	1	4.87
8	1	0	1	4.24
9	1	0	-1	4.26
10	0	0	0	5.19
11	0	-1	-1	4.71
12	-1	1	0	4.47
13	0	-1	1	4.65
14	-1	0	-1	4.43
15	0	1	-1	4.73
16	1	-1	0	4.10
17	0	0	0	5.18

方差来源 Source of variance	平方和 Sum of square	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value
模型 Model	2.3600	9	0.2620	1588.05	< 0.0001
A	0.0968	1	0.0968	586.67	< 0.0001
B	0.0351	1	0.0351	212.80	< 0.0001
C	0.0021	1	0.0021	12.80	0.0090
AB	0.0090	1	0.0090	54.70	0.0001
AC	0.0020	1	0.0020	12.27	0.0099
BC	0.0100	1	0.0100	60.61	0.0001
A²	1.6100	1	1.6100	9777.63	<0.0001
B²	0.2355	1	0.2355	1427.30	<0.0001
C²	0.1883	1	0.1883	1141.49	<0.0001
残差 Residual	0.0012	7	0.0002
失拟项 Lack of fit	0.0005	3	0.0002	0.9314	0.5034
误差项 Pure error	0.0007	4	0.0002
总和 Sum	2.3600	16

样品组 Sample group	浓度 Concentration (μg·mL^-1)	血凝块溶解率 Clot lysis rate (%)
生理盐水Saline solution	-	10.80±1.90
肝素钠Heparin sodium	8 U·mL^-1	10.10±2.17
YFCI-P	1000	42.38±2.30**
	500	31.40±1.96**
	50	11.04±1.47
YFCI-P1	1000	36.91±1.75**
	500	24.27±1.91**
	50	10.87±2.00
YFCI-P2	1000	19.74±1.67**
	500	11.06±2.49
	50	12.13±1.85

Extraction Optimization, Structural Characterization, and Anticoagulant Activity of Intestinal Polysaccharides from Yellow-Feathered Chickens

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