A kind of fucosylated chondroitin sulfate fcs hm And its preparation method and application
A technology of chondroitin sulfate and fucosylation, which is applied in the field of biomedicine to achieve the effects of simple operation, high affinity, good anticoagulation and antithrombotic activity
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Embodiment 1
[0039] Example 1: Novel fucosylated chondroitin sulfate FCS hm preparation of
[0040] The novel fucosylated chondroitin sulfate FCS provided by the present invention hm , the FCS hm Derived from HolothuriaMexicana sea cucumber, its molecular weight is 50-200kDa, and the mass ratio of sulfate is 20-40%. The FCS hm The main chain is composed of chondroitin sulfate A (CSA) and chondroitin sulfate E (CSE), and its fine structure is analyzed by multi-dimensional nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry, and it is found that it is mainly composed of four structural fragments: two One is α-Fuc-2S4S and α-L-Fuc-4S linked to glucuronic acid (GlcA) through O-3, and the other two are α-L-Fuc-4S and α-L-Fuc-3S4S through O -6 is attached to acetylgalactosamine (GalNAc), and FCS hm The side chain contains 1-5 Fuc, the molar ratio of the structural fragment with 1 Fuc side chain is 40-80%, and the molar ratio of the structural fragment with 2-5...
Embodiment 2
[0047] Example 2: Novel fucosylated chondroitin sulfate FCS hm structural representation of
[0048] (1) Determination of absolute molecular weight and purity analysis: adopt high performance gel permeation chromatography (HPGPC)-multi-angle laser scattering method (MALLS) to measure the described FCS hm absolute molecular mass and purity.
[0049] The FCS that will be produced hm Sample with 0.1mol L -1 Na 2 SO 4 Dissolved to make 5mg·mL -1 aqueous solution. Chromatographic analysis conditions: the chromatographic column is used in series with Shodex OHpak SB-804HQ and Shodex OHpak SB-802.5 HQ, and the mobile phase is 0.1mol L -1 Na 2 SO 4 Aqueous solution, the injection volume is 100 μL, the column temperature is 35°C, and the flow rate is 0.6mL min -1 , the acquisition time is 45min, and the detector is a differential detector combined with a multi-angle laser scattering instrument. Experimental results such as figure 2 As shown, the differential and laser sign...
Embodiment 3
[0054] Example 3: Novel fucosylated chondroitin sulfate FCS hm Anticoagulation
[0055] (1) Determination of anticoagulant activity: The activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) were measured using a coagulation analyzer and a kit purchased from Nanjing Jiancheng Biotechnology Company. Such as Figure 10 As shown in A and 10B, compared with the negative control, FCShm The sample can significantly prolong the APTT and TT of plasma, but has no activity on PT, indicating that FCS exerts anticoagulant activity through the intrinsic coagulation pathway, and this effect is dose-dependent, and its activity is similar to that of low molecular weight heparin. In addition, CSA and CSE standard products have no anticoagulant activity, which proves that their fucose side chain is very important for their anticoagulant effect.
[0056] (2) Exploration of the anticoagulant mechanism: Experiments were carried out on 96-well plates using the...
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