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A low anticoagulant heparin, its oligosaccharides, its preparation method and its application in the preparation of anti-Alzheimer's disease medicine

A low anticoagulant and oligosaccharide technology, applied in the field of medicine, can solve the problems of waste of biological resources, low anticoagulant heparin by-products, etc., and achieve low cost, abundant raw material sources, and good anti-Alzheimer's effect

Active Publication Date: 2017-12-05
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] my country is the main producer and exporter of crude heparin in the world. In the refining process of crude heparin, there are a large number of by-products of low anticoagulant heparin. Since no new use has been found, most of them are discarded, resulting in waste of biological resources.

Method used

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  • A low anticoagulant heparin, its oligosaccharides, its preparation method and its application in the preparation of anti-Alzheimer's disease medicine
  • A low anticoagulant heparin, its oligosaccharides, its preparation method and its application in the preparation of anti-Alzheimer's disease medicine
  • A low anticoagulant heparin, its oligosaccharides, its preparation method and its application in the preparation of anti-Alzheimer's disease medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of low anticoagulant heparin

[0035] The preparation method of low anticoagulant heparin of the present invention specifically comprises the following steps:

[0036] 1. Chondroitinase ABC enzyme removes chondroitin impurities: dissolve pig intestinal mucosa crude heparin (10g) purchased from a heparin production plant in 3L enzymolysis buffer (0.05mol L -1 Ammonium acetate, 2mmol·L -1 Calcium chloride), 50 Units of chondroitinase ABC were added thereto, reacted on a shaking table at 35° C. for 6 hours, and inactivated in a boiling water bath for 10 minutes.

[0037] 2. Hydrochloric acid precipitation to remove nucleic acid impurities: centrifuge the enzymatic solution and take the supernatant. Add 1 mol·L dropwise to the supernatant with stirring -1 hydrochloric acid until the pH of the enzymolysis solution is 3.0, centrifuge, take the supernatant and use 1mol·L -1 neutralized to pH 7.0 with NaOH.

[0038] 3. Ethanol precipitation: After co...

Embodiment 2

[0040] Example 2: Structural characterization of the low anticoagulant heparin

[0041](1) Absolute molecular weight determination and purity analysis: The absolute molecular mass of the low anticoagulant heparin was determined and the purity was analyzed by high performance gel permeation chromatography (HPGPC)-octadecanine laser light scattering method (MALLs).

[0042] Chromatographic conditions: Column: Shodex OHPak SB804HQ column connected in series with Shodex OHPak SB802.5HQ column; mobile phase: 0.1mol L -1 Na 2 SO 4 Aqueous solution; The molecular weight of low anticoagulant heparin is detected and determined in series by a differential detector and an octagonal laser light scattering instrument in series; the sample is in the middle of a single symmetrical peak in the HPGPC spectrogram (such as figure 1 Shown), the sample purity is very high, and its absolute molecular mass is measured as 10.24kDa.

[0043] (2) Analysis of disaccharide composition: make low antico...

Embodiment 3

[0049] Example 3: Anticoagulant activity of low anticoagulant heparin

[0050] Follow the steps indicated in the kit to determine the inhibitory effect of the sample on the activity of FXa and FIIa, the initial concentration of the sample is 0.001, 0.01, 0.1, 1, 10 and 100 μg·mL -1 . Add 20 μL antithrombin III (ATIII) and 20 μL sample solution to each well of the 96-well plate, incubate at 37°C for 1 min, then add 40 μL FXa or FIIa, incubate at 37°C for 1 min, add 100 μL FXa or FIIa substrate, 37 Incubate at ℃ for 4min, finally add 100μL of stop solution, detect the absorbance at 405nm wavelength, and calculate the inhibition rate: the result ( Figure 4 ) showed that LAH had weak anticoagulant activity, which was much lower than that of HP and ES standards, and slightly higher than that of HS standards.

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Abstract

The invention relates to a low anticoagulant heparin, its oligosaccharide, its preparation method and its application in the preparation of anti-Alzheimer's disease medicine. The present invention removes chondroitin sulfate and hyaluronic acid through chondroitinase ABC treatment of the crude heparin from porcine small intestine mucosa, removes nucleic acid by hydrochloric acid precipitation, ethanol precipitation and anion exchange resin purification to finally obtain the high-purity low anticoagulant heparin, and then Further degrade with heparanase or β-elimination method to obtain oligosaccharides of different molecular weights, the molecular weight of the low anticoagulant heparin obtained in the present invention is between 10-20 kDa, and the molecular weight of oligosaccharides is between 1-7 kDa, Each disaccharide unit contains an average of 1.5‑2.5 sulfate groups. The low anticoagulant heparin and its oligosaccharides prepared by the invention can significantly inhibit the activity of BACE1 through experiments, and can be used to prepare anti-Alzheimer's medicine.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a low-anticoagulant heparin, its oligosaccharides, a preparation method thereof, and an application in preparation of anti-Alzheimer's medicine. Background technique [0002] Heparin (Heparin, HP) has a variety of biological functions. Currently, it is mainly used as an anticoagulant to prevent thrombosis. In addition, it also has various functions such as adjusting blood lipid concentration, enhancing immunity, anti-inflammation, and anti-allergy. However, due to side effects such as hemorrhage, thrombocytopenia, hyperkalemia and osteoporosis in the clinical application of heparin, its clinical application in other active aspects is limited. Therefore, research on low anticoagulant heparin (LAH) products has become a hot spot. Compared with heparin, low anticoagulant heparin has lower anticoagulant activity, does not bind to platelet factor 4 (PF4) antibody (platel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/10A61K31/727A61P25/28
Inventor 于广利张晓李国云蔡超李芹英
Owner OCEAN UNIV OF CHINA