中草药

蓝指海星新型糖胺聚糖分离纯化、结构解析与靶向内源性凝血途径的抗凝活性研究

PDF下载

符洁雯, 时响, 吕旭升, 侯小涛, 刘永宏, 袁清霞, 赵龙岩

中草药 | 海洋中药研究 2026,57(14): 5416-5427

收起

中草药 |海洋中药研究 2026 , 57 (14) : 5416 -5427

蓝指海星新型糖胺聚糖分离纯化、结构解析与靶向内源性凝血途径的抗凝活性研究

全屏

符洁雯, 时响, 吕旭升, 侯小涛, 刘永宏, 袁清霞, 赵龙岩

作者信息

通讯作者:

袁清霞

作者简介:

符洁雯: 符洁雯（1999—），女，硕士研究生，研究方向为海洋中药糖类新药研发。E-mail:weiwei1999777@163.com

Isolation, purification, structural characterization, and anticoagulant activity of a novel glycosaminoglycan from Linckia laevigata targeting intrinsic coagulation pathway

FU Jiewen, SHI Xiang, LYU Xusheng, HOU Xiaotao, LIU Yonghong, YUAN Qingxia, ZHAO Longyan

Affiliations

doi: 10.7501/j.issn.0253-2670.2026.14.005

文章导航

摘要

收起

目的从蓝指海星Linckia laevigata中挖掘新型糖胺聚糖，解析其精细结构，并评估其作为内源性凝血途径抑制剂的抗凝潜力。方法采用酶解-碱解协同提取蓝指海星总多糖，强阴离子交换色谱纯化获得糖胺聚糖；经脱酰-脱氨基解聚制备寡糖，采用“bottom up”策略联合高效凝胶渗透色谱（high-performance gel permeation chromatography，HPGPC）、1-苯基-3-甲基-5-吡唑酮衍生化高效液相色谱（1-phenyl-3-methyl-5-pyrazolone derivatization high-performance liquid chromatography，PMP-HPLC）、红外光谱（infrared spectroscopy，IR）及一维/二维核磁共振（one-dimensional/two-dimensional nuclear magnetic resonance，1D/2D NMR）进行结构表征；以活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、凝血酶时间（thrombin time，TT）、凝血酶原时间（prothrombin time，PT）评价体外抗凝活性，生色底物法测定内源性因子X酶（intrinsic factor Xase，iFXase）抑制活性。结果制备得到结构均一的硫酸化糖胺聚糖LLGAG（Mw≈38 000），其单糖组成为艾杜糖醛酸（iduronic acid，IdoA）∶乙酰氨基半乳糖（N-acetylgalactosamine，GalNAc）∶半乳糖（galactose，Gal）≈34.4∶59.7∶5.9。该聚糖核心由L-IdoA₂S₃S-α-1,3-D-GalNAc₄S₆S（2S3S4S6S）基序的二糖单元构成，兼含3S4S6S及2S3S6S等基序，为自然界中罕见的新型硫酸皮肤素样糖胺聚糖。体外实验中，LLGAG显著延长APTT与TT，活性与临床抗凝金标准依诺肝素钠相当；抑制iFXase的半数抑制浓度（median inhibition concentration，IC₅₀）为（17.75±1.12）ng/mL，较依诺肝素钠强约2.8倍。结论发现了1种结构新颖独特的蓝指海星来源糖胺聚糖，推断出其确切结构，发现其具有强效iFXase抑制活性，为开发海洋生物来源的肝素替代物提供理论依据与候选分子。

关键词

蓝指海星 / 多糖 / 糖胺聚糖 / 结构表征 / 抗凝血活性 / 内源性因子X酶

Abstract

收起

Objective To identify novel glycosaminoglycans from Linckia laevigata, elucidate their fine structures, and evaluate their anticoagulant potential as inhibitors of the intrinsic coagulation pathway. Methods Total polysaccharides were extracted from the starfish L. laevigata using a combination of enzymatic and alkaline hydrolysis, and then purified by strong anion-exchange chromatography. Oligosaccharides were prepared through deacetylation-deamination depolymerization. A “bottom-up” strategy integrating high-performance gel permeation chromatography (HPGPC), 1-phenyl-3-methyl-5-pyrazolone derivatization high-performance liquid chromatography (PMP-HPLC), infrared spectroscopy (IR), and one-dimensional/two-dimensional nuclear magnetic resonance (1D/2D NMR) was employed for structural characterization. In vitro anticoagulant activity was assessed by activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) assays, while inhibition of intrinsic factor Xase (iFXase) was measured via a chromogenic substrate assay. Results A homogeneous sulfated glycosaminoglycan, designated LLGAG (molecular weight, M_w≈38 000), was obtained with a monosaccharide composition of iduronic acid (IdoA)∶N-acetylgalactosamine (GalNAc)∶galactose (Gal) ≈34.4∶59.7∶5.9. The polymer core comprised disaccharide units featuring the L-IdoA_2S3S-α-1,3-D-GalNAc_4S6S (2S3S4S6S) motif, together with 3S4S6S and 2S3S6S motifs, representing a rare and novel dermatan sulfate-like glycosaminoglycan. In vitro, LLGAG significantly prolonged APTT and TT, showing activity comparable to that of the clinical gold-standard enoxaparin sodium. Its half-maximal inhibitory concentration (IC₅₀) for iFXase inhibition was (17.75 ± 1.12) ng/mL, approximately 2.8-fold more potent than that of enoxaparin sodium. Conclusion This study identified a structurally unique glycosaminoglycan from L. laevigata, elucidated its precise structure, and demonstrated its potent iFXase inhibitory activity, thus providing both a theoretical foundation and a candidate molecule for the development of marine-derived heparin alternatives.

Key words

Linckia laevigata (Linnaeus) / polysaccharide / glycosaminoglycan / structural characterization / anticoagulant activity / intrinsic factor Xase

引用本文

符洁雯, 时响, 吕旭升, 侯小涛, 刘永宏, 袁清霞, 赵龙岩. 蓝指海星新型糖胺聚糖分离纯化、结构解析与靶向内源性凝血途径的抗凝活性研究. 中草药, 2026 , 57 (14) : 5416 -5427 . DOI: 10.7501/j.issn.0253-2670.2026.14.005

FU Jiewen, SHI Xiang, LYU Xusheng, HOU Xiaotao, LIU Yonghong, YUAN Qingxia, ZHAO Longyan. Isolation, purification, structural characterization, and anticoagulant activity of a novel glycosaminoglycan from Linckia laevigata targeting intrinsic coagulation pathway[J]. Chinese Traditional and Herbal Drugs, 2026 , 57 (14) : 5416 -5427 . DOI: 10.7501/j.issn.0253-2670.2026.14.005

基金

收起

国家自然科学基金资助项目 (82404503); 国家自然科学基金资助项目 (32160220); 国家自然科学基金资助项目 (82373788); 广西杰出青年科学基金 (2020GXNSFFA297005); 中药壮瑶药创新药物教育部工程研究中心 (ZYZYY2025034)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

Mackman N, Bergmeier W, Stouffer G A, et al. Therapeutic strategies for thrombosis: New targets and approaches [J]. Nat Rev Drug Discov, 2020, 19(5): 333-352.
Douaisi M, Paskaleva E E, Fu L, et al. Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin [J]. Proc Natl Acad Sci USA, 2024, 121(14): e2315586121.
Xu Y M, Masuko S, Takieddin M, et al. Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins [J]. Science, 2011, 334(6055): 498-501.
高玥, 许倩楠, 蔡明刚, 等. 海洋来源药食同源品开发利用研究进展[J]. 中草药, 2021, 52(17): 5455-5464.
李泽宇, 郝二伟, 李卉, 等. 抗肿瘤作用的海洋中药[J]. 中草药, 2022, 53(14): 4527-4544.
刘梓栋, 符洁雯, 时响, 等. 海洋棘皮动物多糖新药挖掘进展: 分离制备、结构解析及药理活性[J]. 药学学报, 2025, 60(7): 2106-2121.
Li H, Yuan Q X, Lv K L, et al. Low-molecular-weight fucosylated glycosaminoglycan and its oligosaccharides from sea cucumber as novel anticoagulants: A review [J]. Carbohydr Polym, 2021, 251: 117034.
Zhao L Y, Wu M Y, Xiao C, et al. Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan [J]. Proc Natl Acad Sci U S A, 2015, 112(27): 8284-8289.
徐思嘉, 肖宁, 曾晓起. 中国海域海盘车科(棘皮动物门, 海星纲)种类记述[J]. 海洋科学, 2018, 42(10): 53-63.
Williams S T, Heyworth S M, Kano Y, et al. The blue advantage: A novel blue carotenoprotein pigment in the tropical seastar Linckia Laevigata is an antioxidant defence against extreme environmental stress [J]. Mar Biol, 2025, 172(2): 31.
王云达, 李高燕, 郭庆梅, 等. 市售海星类药材的鉴别研究[J]. 中药材, 2024, 47(4): 863-868.
Ustyuzhanina N E, Bilan M I, Dmitrenok A S, et al. Oversulfated dermatan sulfate and heparinoid in the starfish Lysastrosoma Anthosticta: Structures and anticoagulant activity [J]. Carbohydr Polym, 2021, 261: 117867.
Shi X, Shi M H, Fu J W, et al. Structure–activity relationship of a potent anti-FXase and antithrombotic chondroitin sulfate–dermatan sulfate hybrid bearing 2, 3-O-sulfated motifs from Anthenoides Laevigatus [J]. Carbohydr Polym, 2026, 375: 124752.
李军, 蒋碧蓉, 王海妹, 等. 海星多糖的分离纯化及鉴定[J]. 食品工业科技, 2010, 31(02): 122–124.
Mackman N. Triggers, targets and treatments for thrombosis [J]. Nature, 2008, 451(7181): 914-918.
Yuan Q X, Li H, Wang Q, et al. Deaminative-cleaved S. monotuberculatus fucosylated glycosaminoglycan: Structural elucidation and anticoagulant activity [J]. Carbohydr Polym, 2022, 298: 120072.
Dodgson K S, Price R G. A note on the determination of the ester sulphate content of sulphated polysaccharides [J]. Biochem J, 1962, 84(1): 106-110.
Yuan Q X, Zhao L Y, Cha Q Q, et al. Structural characterization and immunostimulatory activity of a homogeneous polysaccharide from Sinonovacula constricta [J]. J Agric Food Chem, 2015, 63(36): 7986-7994.
Yao Y, Tang H, Ma H Q, et al. Chondroitin sulfate/dermatan sulfate hybrid chains from swim bladder: Isolation, structural analysis, and anticoagulant activity [J]. Mar Drugs, 2024, 22(1): 9.
Zhao L Y, Lai S S, Huang R, et al. Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage [J]. Carbohydr Polym, 2013, 98(2): 1514-1523.
Yuan Q X, Liang R Y, Lv K L, et al. Structural characterization of a Chlorella heteropolysaccharide by analyzing its depolymerized product and finding an inducer of human dendritic cell maturation [J]. Carbohydr Polym, 2024, 333: 122000.
Tang H, Huang J W, Yuan Q X, et al. A regular Chlorella mannogalactan and its sulfated derivative as a promising anticoagulant: Structural characterization and anticoagulant activity [J]. Carbohydr Polym, 2023, 314: 120956.
Zhang Y Y, Fonslow B R, Shan B, et al. Protein analysis by shotgun/bottom-up proteomics [J]. Chem Rev, 2013, 113(4): 2343-2394.
赵宁, 韩著, 简颖琳, 等. 中药多糖结构表征及质量评价研究进展[J]. 中草药, 2024, 55(21): 7491-7506.
Mulloy B, Forster M J, Jones C, et al. The effect of variation of substitution on the solution conformation of heparin: A spectroscopic and molecular modelling study [J]. Carbohydr Res, 1994, 255: 1-26.
Sudo M, Sato K, Chaidedgumjorn A, et al. 1H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate [J]. Anal Biochem, 2001, 297(1): 42-51.
Pan Y, Sun H F, Gu X, et al. Oligosaccharide-assisted resolution of holothurian fucosylated chondroitin sulfate for fine structure and P-selectin inhibition [J]. Carbohydr Polym, 2025, 351: 123145.
Yuan Q X, Liu Z D, Yao Y, et al. A novel glycosaminoglycan from the brittlestar Trichaster Palmiferus: Structure and low-bleed anticoagulant–antithrombotic action [J]. Carbohydr Polym, 2026, 379: 125036.
Bilan M I, Anisimova N Y, Tokatly A I, et al. Glycosaminoglycans from the starfish Lethasterias Fusca: Structures and influence on hematopoiesis [J]. Mar Drugs, 2023, 21(4): 205.
Lin L S, Zhao L Y, Gao N, et al. From multi-target anticoagulants to DOACs, and intrinsic coagulation factor inhibitors [J]. Blood Rev, 2020, 39: 100615.
Wu M Y, Wen D D, Gao N, et al. Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase [J]. Eur J Med Chem, 2015, 92: 257-269.
Xiao C, Zhao L Y, Gao N, et al. Nonasaccharide inhibits intrinsic factor xase complex by binding to factor IXa and disrupting factor IXa–factor VIIIa interactions [J]. Thromb Haemost, 2019, 119(5): 705-715.

2026年第57卷第14期

PDF下载

引用本文

BibTeX

文章信息

doi: 10.7501/j.issn.0253-2670.2026.14.005

接收时间：2026-03-14
首发时间：2026-09-10

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2026-03-14

基金

作者信息

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/zcy/CN/10.7501/j.issn.0253-2670.2026.14.005

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT