CN112076211A - Glycosaminoglycan composition and preparation method and application thereof - Google Patents

Abstract

The invention provides a glycosaminoglycan composition and a preparation method and application thereof, the composition is prepared by extracting glycosaminoglycan from a ruminant heparin byproduct, consists of fast moving heparin and dermatan sulfate, has an anticoagulation and antithrombotic effect, has 60-100U/mg of anti-Xa activity depending on antithrombin III, 100-200U/mg of anticoagulation activity depending on heparin cofactor II, and 50-80U/mg of anticoagulation activity by a sheep plasma method. The composition can be developed into medicine or health product, and has clear nature. Moreover, the glycosaminoglycan serving as the raw material of organs of ruminants such as sheep and cattle or a byproduct produced by heparin is simple, convenient and easy to obtain, and has a remarkable economic effect.

Description

Glycosaminoglycan composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a glycosaminoglycan composition and a preparation method thereof.

Background

Glycosaminoglycans (GAGs), a natural unbranched long-chain polysaccharide polymer composed of repeating disaccharide units containing both hexuronic acid and hexosamine components, are widely distributed in the animal and human body in various organs. Glycosaminoglycans are acid mucopolysaccharides with negatively charged carboxyl or sulfate groups on the sugar chains. Glycosaminoglycans mainly include Heparin (HP), Heparan Sulfate (HS), Chondroitin Sulfate (CS) and Dermatan Sulfate (DS), which differ in the type of monosaccharide residues, the type of linkage between residues and the number and position of Sulfate groups. Glycosaminoglycans have very important physiological roles. In the bodies of higher animals and humans, dermatan sulfate is a main component of proteoglycan on the outer layer of blood vessel wall, and heparin is a natural anticoagulant substance widely present in various tissues and organs such as blood vessel wall, intestinal mucosa, lung, and the like.

The present invention relates generally to Fast Moving Heparin (FMH) and dermatan sulfate compositions.

Fast Moving Heparin (FMH), which is one of heparins derived from natural sources, is divided into fast Moving Heparin and Slow Moving Heparin (SMH) when separated by a barium salt-propylenediamine gel electrophoresis system. Among commercial Unfractional Heparin (UFH), fast-moving Heparin exists less and moves fast during electrophoresis [ p. bianchi et, j. Chomat, 196, 455 (1980) ], and mainly consists of low-to-medium molecular weight Heparin (Mw-7000 Da). Compared with unfractionated heparin and slow-moving heparin, the fast-moving heparin has low sulfation degree, high acetylation degree and low anticoagulation activity. Fast-moving heparins rely primarily on binding to antithrombin-III (AT-III) to produce some anti-Xa activity.

Dermatan sulfate, belonging to the chondroitin sulfate family, is mainly composed of the-IdoA-GalN 4S-repeating disaccharide unit, with an average molecular weight of 25,000 Da. Dermatan sulfate is occasionally sulfated at the 6-position of GalNAc and the 2-position of IdoA, a relatively more heterogeneous sugar structure. The special chemical structure of dermatan sulfate brings about a stronger antithrombotic effect. Dermatan sulfate is mainly dependent on the action of Heparin Cofactor II (HC II) and not on antithrombin-III.

Heparin is mainly used for preventing and treating venous thrombosis, but clinical use is often accompanied by obvious bleeding side effects, and the source is that heparin directly inhibits blood coagulation factors depending on antithrombin-III, so that the heparin is difficult to be applied to clinical antithrombotic treatment. As described above, dermatan sulfate is different from heparin, does not depend on antithrombin-III, but acts through heparin cofactor II, and has little bleeding side effect and strong antithrombotic effect. Due to different action principles, the fast moving heparin and the dermatan sulfate can be used for realizing synergistic effect on anticoagulation and anti-thrombosis. In vivo, both of them can also induce other important physiological actions, such as promoting the release of lipoprotein esterase, anti-inflammation, anti-angiogenesis, etc.

The structural formulas of the fast moving heparin and the dermatan sulfate are shown as follows,

on the other hand, clinical medical glycosaminoglycans including heparin and the like are mainly derived from pigs, but have the contradiction of insufficient supply, and have great uncontrollable risks due to single source.

Taking heparin as an example, the current mainstream pharmacopoeias (european pharmacopoeia (EP), United States Pharmacopoeia (USP), chinese pharmacopoeia (ChP), Japanese Pharmacopoeia (JP), etc.) all specify or advocate that heparin and low molecular heparin are derived from porcine intestinal mucosa, which is obviously deficient. As in the current us market, heparin products (new drugs, imitation drugs, instruments, etc.) are all derived from porcine intestinal mucosa, however, 75% of the crude products for producing refined heparin are derived from china, single animals and regional sources, and there are serious uncontrollable risks (epidemics such as porcine reproductive and respiratory syndrome, war, trade dispute, etc., especially the recent large-scale outbreak african swine fever epidemic in china), thus seriously affecting the quality or supply of heparin products. In addition, the supply chain of the glycosaminoglycan products such as heparin is very complex, a large number of breeding households exist, more than 1500 intestinal mucosa of live pigs are needed for extracting 1 hundred million units of heparin, the supply and production chain relates to a series of links such as breeding households, slaughter houses, crude heparin production plants, crude heparin distributors, fine heparin production plants and fine heparin distributors, and all the links are easy to cause risks. Severe contamination events occurred with heparin from china in 2008, revealing that this single supply of porcine heparin is extremely fragile.

The ruminant such as the retrospective sheep, the cattle and the like, in particular to the domestic ruminant such as the sheep, the cattle and the like, including the cattle, the sheep, the goats and the like, the body or the organ of the ruminant also has rich heparin or fast moving heparin and dermatan sulfate, and the intestinal mucosa of the cattle, the intestinal mucosa of the sheep and the lung of the cattle are all practically applied to the extraction and production of the heparin. As is known, cattle and sheep are different from pigs in intensive large-scale breeding and concentrated slaughtering, and the difference in risk control is obvious compared with the scatter slaughtering and the like mainly produced in china by pigs.

In clinical application, the application of fast moving heparin and dermatan sulfate in specific clinical indications, especially in the prevention and treatment of antithrombotic and anticoagulant diseases, may have excellent curative effects.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a glycosaminoglycan composition and a preparation method and application thereof.

The purpose of the invention is realized by the following technical scheme:

a glycosaminoglycan composition is prepared by extracting glycosaminoglycan from a ruminant heparin byproduct, is composed of fast moving heparin and dermatan sulfate, has anticoagulation and antithrombotic effects, and has anticoagulation activity of 60-100U/mg depending on antithrombin III anti-Xa activity, 100-200U/mg depending on heparin cofactor II anticoagulation activity, and 50-80U/mg in a sheep plasma method anticoagulation activity. The fast moving heparin and dermatan sulfate fractions can be obtained by agarose gel electrophoresis analysis and nuclear magnetic hydrogen spectroscopy analysis.

Preferably, the fast moving heparin in the glycosaminoglycan composition, wherein the disaccharide unit is composed of hexuronic acid-hexosamine, the hexuronic acid being iduronic acid (IdoA-) or glucuronic acid (GlcA-), and the hexuronic acid being of hexuronic acid2-OMore than 40% of the positions are esterified with sulphate (IdoA 2S), hexosamine being glucosamine (GlcN) in the amino group6-O45% to 55% of the components in the positions are esterificated by sulfuric acid (GlcN 6S) atN-(GlcNS) or acetylated (GlcNAc) at a position wherein 15% to 25% of the component is acetylated. The above sugar unit characteristics of glycosaminoglycan compositions can be clearly classified by SAX-HPLC analysis of disaccharide content of glycosaminoglycan composition samples after heparinase hydrolysis.

The fast moving heparin described above, although also one of the heparins, differs from the pharmacopoeia-specified heparins such as USP sodium heparin in a significant characteristic in disaccharide composition. In SAX-HPLC analysis after heparinase hydrolysis, the main enzymatic disaccharide products in both fast-moving heparin and USP sodium heparin of the glycosaminoglycan composition are disaccharides IdoA-GlcNAc, IdoA-GlcNS, IdoA-GlcNAc6S, IdoA2S-GlcNAc, IdoA-GlcNS6S, IdoA2S-GlcNS and IdoA2S-GlcNS6S, which are the same in kind, but the composition percentages are very different, namely the percentage average values of the three disaccharides IdoA-GlcNAc, IdoA-Ac 6S and IdoA2S-GlcNS6S in fast-moving heparin and USP sodium heparin are respectively 18%vs1%、7% vs 3 percent and 34 percentvs63%。

Another ingredient of the glycosaminoglycan composition is dermatan sulfate, belonging to chondroitin sulfate family, and prepared from iduronic acid-6-A sulfate-galactosamine (-IdoA-GalN 4S-) repeating disaccharide unit with an average molecular weight of 25,000Da, which is occasionally of GalNAc6-Of bits and IdoA2-The sulfation is present at the site and is a relatively more heterogeneous sugar structure.

Preferably, the glycosaminoglycan composition has a significant prolongation of both APTT and TT and a reduction of fibrinogen content as a result of in vitro tests, which are based on human plasma studies. Specifically, after separating plasma from human blood, the influence of the glycosaminoglycan composition on blood coagulation conventions including APTT, TT, PT and the like is examined according to an automatic blood coagulator and a kit method. The glycosaminoglycan composition has obvious prolonging effects on APTT, TT and recalcification time, has a certain prolonging effect on PT, can reduce the content level of fibrinogen, and all the effects indicate that the glycosaminoglycan composition has a certain anticoagulation and antithrombotic effect.

Preferably, the preparation method of a glycosaminoglycan composition described above, comprises the steps of:

s1, selecting a byproduct glycosaminoglycan obtained after heparin is extracted from organs of ruminants as a raw material, wherein the ruminants are domestic sheep, cattle and goats.

S2, virus inactivation, dissolving the glycosaminoglycan as the heparin by-product, adding alkali for action, then adjusting pH, adding an oxidant for further virus inactivation and decoloration, and finally adding a filter aid for filtration to prepare a reaction solution.

S3, removing heavy metals, namely adding a metal ion chelating agent into the reaction solution obtained after virus inactivation in the S2, adjusting the pH, and finally precipitating a crude glycosaminoglycan composition by using an organic solvent.

S4, refining and purifying the glycosaminoglycan composition, redissolving the crude product in the S3, carrying out fractional precipitation by using an organic solvent, harvesting a precipitate, dehydrating, and finally drying to obtain the glycosaminoglycan composition.

Preferably, in step S1, the glycosaminoglycan as a by-product obtained after extraction of heparin from organs of ruminants such as sheep and cattle includes the glycosaminoglycan remaining after extraction of heparin, such as the part of the supernatant that is not precipitated during refining of heparin by fractional alcohol precipitation, and the part is precipitated with a higher concentration of organic solvent and is a glycosaminoglycan mixture.

Preferably, the raw material in step S1 may be crude heparin extracted from organs of ruminants such as sheep and cattle, and the processing steps are added because of the change of the raw material. The treatment step is to use a conventional heparin or mucopolysaccharide separation and purification mode, preferably comprises fractional precipitation of salt, fractional precipitation of an organic solvent and ion exchange resin purification; the salt preferably comprises sodium chloride, ammonium acetate, potassium acetate, more preferably sodium chloride; the organic solvent preferably comprises methanol, ethanol, isopropanol, acetone, more preferably ethanol; the ion exchange resin preferably comprises a strong anion exchange resin and a weak anion exchange resin, more preferably a strong anion exchange resin.

Preferably, the dissolving of the heparin by-product glycosaminoglycan in S2 refers to the redissolving of the heparin by-product glycosaminoglycan with the mass concentration of between 0 and 10 percent of saline water and the mass concentration of between 1 and 30 percent of saline water. The salt is preferably sodium chloride, the saline is a 5% sodium chloride solution, and the mass concentration of the heparin by-product glycosaminoglycan is preferably 10%.

Preferably, the alkali in the S2 is sodium hydroxide, the concentration is 0.01-5 mol/L, and more preferably 1 mol/L; the pH is adjusted to be between 7 and 11, preferably between 8 and 10; the oxidant is hydrogen peroxide or potassium permanganate.

Preferably, the filter aid in S2 is diatomaceous earth.

Preferably, the metal ion chelating agent in S3 is EDTA, and the final concentration of the metal ion chelating agent is preferably 0.1-50 mmol/L, the action temperature is preferably 10-50 ℃, and the action time is preferably 0.1-10 h.

Preferably, the final concentration of the metal ion chelating agent in S3 is 20 mmol/L EDTA, and the reaction is carried out for 4h at 30 ℃.

Preferably, the pH value of the S3 is adjusted to be between 4 and 6, so that the chelating agent is helpful for removing heavy metals.

Preferably, the organic solvent in S3 is acetone with a final concentration of 30% to 43%.

Preferably, the crude glycosaminoglycan composition in S4 is re-dissolved in saline, wherein the saline is 0-10% sodium chloride solution, and the concentration of the crude glycosaminoglycan is 1-30%.

Preferably, the organic solvent for fractional precipitation in the step S4 is ethanol with a final concentration of 40-60%, and the temperature during precipitation is 4-35 ℃ and the time is 1-48 h.

Preferably, the method for preparing a glycosaminoglycan composition comprises the steps of separately purifying a fast-moving heparin component and a dermatan sulfate component of a ruminant animal, and mixing the separately extracted fast-moving heparin component and dermatan sulfate component to prepare the glycosaminoglycan composition. The preparation method used for the respective purification is that the conventional separation and purification mode of heparin or mucopolysaccharide is used, and preferably comprises the steps of fractional precipitation of salt, fractional precipitation of organic solvent and purification of ion exchange resin; the salt preferably comprises sodium chloride, ammonium acetate, potassium acetate, more preferably sodium chloride; the organic solvent preferably comprises methanol, ethanol, isopropanol, acetone, more preferably ethanol; the ion exchange resin preferably comprises a strong anion exchange resin and a weak anion exchange resin, more preferably a strong anion exchange resin.

A pharmaceutical composition comprising a glycosaminoglycan composition of any of the above and a pharmaceutically acceptable carrier. The pharmaceutical composition is prepared into sterile injection or capsules or tablets by using water for injection.

Use of the glycosaminoglycan composition of any of the above in the preparation of a medicament or health product or halal product for the prevention and treatment of thromboembolic disorders, vasculopathy disorders and vascular disorders at risk of thrombosis.

The invention relates to drying in each preparation step, which comprises natural drying, vacuum drying or freeze drying and other drying modes. Preferably vacuum drying or freeze drying; in the drying process, stirring, grinding, powdering and the like can be preferably carried out so as to improve the drying effect.

The invention has the beneficial effects that: provides a novel glycosaminoglycan composition which is prepared from ruminants such as sheep, cattle and the like, has a certain anticoagulation antithrombotic effect, low bleeding risk, and also has the effects of preventing and repairing angiopathy, can be developed into medicines or health-care products, and has trueness. The glycosaminoglycan which is the by-product produced by organs of ruminants such as sheep and cattle or heparin and is used as the raw material is simple, convenient and easy to obtain, and has obvious economic effect.

Drawings

FIG. 1: SAX-HPLC chromatogram of disaccharide after enzymolysis of glycosaminoglycan composition sample by heparinase.

FIG. 2: schematic representation of agarose gel electrophoresis analysis of glycosaminoglycan compositions.

FIG. 3: of samples of glycosaminoglycan compositions¹Schematic representation of H-NMR.

Detailed Description

The technical scheme of the invention is specifically described in the following by combining with the embodiment, and the invention discloses a novel glycosaminoglycan composition and a preparation method and application thereof.

The first embodiment is as follows: preparation of glycosaminoglycan composition 1

The side product glycosaminoglycan of heparin of sheep intestinal mucosa is obtained in the process of purifying and preparing heparin of sheep intestinal mucosa. The method specifically comprises the following steps of preparing crude sheep heparin sodium (manufacturer: Shandong Shen combined biological science and technology Co., Ltd., batch No. 20150326, obtained by extracting sheep intestinal mucosa), purifying heparin by a process well known in the industry, namely dissolving the crude sheep heparin sodium in water, performing salt hydrolysis and alkaline hydrolysis, adding protease for hydrolysis, adjusting pH of hydrolysate to weak alkali, adsorbing and eluting by anion exchange resin, performing alcohol precipitation to prepare sheep heparin, adding ethanol into supernatant obtained after separation of sheep heparin as a sheep heparin byproduct glycosaminoglycan, supplementing ethanol to a final mass concentration of 70%, recovering the precipitate, and drying.

800g of glycosaminoglycan, which is a heparin by-product of the intestinal mucosa of sheep, was weighed and dissolved in 8000mL of 5% saline with stirring, and then 400g of sodium hydroxide was added thereto to react at room temperature for 0.5 hour. Adjusting the pH value to 9.0 by using dilute hydrochloric acid, adding 1.3L of hydrogen peroxide, and stirring for reacting for 6 hours. 800g of diatomaceous earth was added thereto, and the mixture was stirred for 5min and then filtered. 40g of ETDA was added to the filtrate, and the mixture was reacted at room temperature for 6 hours. The pH was controlled at 5.5, cold acetone was added to a final concentration of 42%, stirred for 15 min and allowed to stand overnight. The supernatant was carefully drained, the pellet redissolved in 5000mL of 5% sodium chloride solution, adjusted to pH 6.1, and precipitated overnight at room temperature with ethanol to a final concentration of 46%. The precipitate was dehydrated with ethanol and dried under vacuum to obtain 430g of glycosaminoglycan composition as a product with a loss on drying of 5.3%.

Example two: preparation of glycosaminoglycan composition 2

Collecting glycosaminoglycan as a byproduct in the purification of the bovine intestinal mucosa heparin, wherein the glycosaminoglycan as the byproduct is obtained in the refining and purification of crude bovine intestinal mucosa heparin (the crude bovine intestinal mucosa heparin is extracted from small intestinal mucosa of slaughtered beef cattle in northeast China), is clear liquid rich in glycosaminoglycan in the heparin process, and is prepared by obtaining precipitate with ethanol with the final concentration of 70% and drying.

100g of glycosaminoglycan, which is a heparin by-product of bovine intestinal mucosa, was weighed and dissolved in 1000mL of 2% saline with stirring, and then 40g of sodium hydroxide was added thereto to react at 25 ℃ for 2 hours. The pH value is adjusted to 7.0 by dilute hydrochloric acid, 1g of potassium permanganate is added, and the mixture is stirred and reacts for 30 min. 100g of diatomaceous earth was added thereto, and the mixture was stirred for 5min and then filtered through a filter paper. 2.2g of ETDA was added to the filtrate, and the mixture was reacted at room temperature for 4 hours. Controlling pH at 5.0, adding cold acetone to final concentration of 40%, stirring for 5min, and standing overnight. The supernatant was carefully drained off, the precipitate was redissolved with 1000mL of 5% sodium chloride solution, adjusted to pH6.2, and precipitated at room temperature for 20h with methanol to a final concentration of 63%. The precipitate was dehydrated with ethanol and dried under vacuum to obtain 47.8g of glycosaminoglycan composition as a product having a loss on drying of 3.7%.

Example three: preparation of glycosaminoglycan composition 3

30g of glycosaminoglycan, which is a by-product of bovine pulmonary heparin, was dissolved in 500mL of 3% saline with stirring, and then 3g of sodium hydroxide was added thereto to react at 35 ℃ for 2 hours. Adjusting the pH value to 7.8 by using dilute hydrochloric acid, adding 10 mL of hydrogen peroxide, stirring, standing and decoloring for 5 hours. 30g of diatomaceous earth was added, stirred for 5min and filtered through a filter paper. 1.3g of ETDA was added to the filtrate, and the mixture was reacted at room temperature for 3.5 hours. Adjusting pH to 5.5, adding cold acetone to a final concentration of 41%, stirring thoroughly, centrifuging, and collecting precipitate. The precipitate was dissolved in 300mL of 5% sodium chloride solution, adjusted to pH5.8, added with ethanol to a final concentration of 48%, and allowed to stand overnight at room temperature. Centrifuging to remove supernatant, dehydrating the precipitate with 95% ethanol, and vacuum drying to obtain 14.2g glycosaminoglycan composition with 7.1% loss on drying.

Example four: preparation of glycosaminoglycan composition 4

5g of the glycosaminoglycan compositions prepared in the first to third examples are respectively taken, mixed, pulverized and homogenized to obtain the mixed glycosaminoglycan composition.

Example five: preparation of glycosaminoglycan composition 5

20g of the heparin byproduct glycosaminoglycan of the sheep intestinal mucosa in the first embodiment is weighed, dissolved by stirring with 1000mL of 1% saline water, filtered by 0.45 mu m, and loaded onto a treated DEAE-FF anion exchange column with the volume of 1000 mL. After loading, the column was equilibrated with 1% sodium chloride solution for 6 column volumes, and then eluted with a linear gradient of 0% to 100% 15% sodium chloride solution for 10 column volumes, one collected per 500 mL. Supplementing sodium chloride to 15% of each collected eluent, adding 1250ml of methanol, stirring for 5 minutes, standing, finally centrifuging, collecting precipitate, dehydrating the precipitate with methanol, and drying. The components are detected by electrophoresis, and are combined to prepare 4.3g of pure fast moving heparin and 5.6g of pure dermatan sulfate. Accurately weighing 4.0g of fast-moving heparin and 0.5g of dermatan sulfate, dissolving in 50mL of purified water, adjusting the pH value to 6.5, and freeze-drying to obtain 4.5g of glycosaminoglycan composition.

Example six: anticoagulant antithrombotic Activity assay of glycosaminoglycan compositions

The anti-Xa activity assay was performed according to EP9.0 heparin sodium, the whole sheep plasma method activity assay was performed according to methods well known in the heparin art (equivalent to the activity assay of USP32 heparin sodium), and the heparin cofactor II (HC II) anticoagulation activity was determined by the chromogenic substrate method.

The activity of each sample from example one to example five is shown in table 1.

Table 1: comparison of biological Activity of glycosaminoglycan composition samples

Name (R)	Anticoagulation by whole sheep plasma method (U/mg)	anti-Xa (U/mg)	Heparin cofactor II anticoagulation (U/mg)
				Glycosaminoglycan composition sample 1	61.7	86.2	143.2
Glycosaminoglycan composition sample 2	56.2	78.6	137.1
				Glycosaminoglycan composition sample 3	74.5	92.3	178.4
Glycosaminoglycan composition sample 4	67.2	89.1	170.4
				Glycosaminoglycan composition sample 5	62.4	85.2	151.5

The results show that: five groups of glycosaminoglycan composition samples all have certain anticoagulation and anti-thrombus activity, and the activity is similar.

Example seven: heparinase enzymatic disaccharide analysis of glycosaminoglycan compositions

The SAX-HPLC method after heparinase hydrolysis was examined with reference to the appendix "1, 6-anhydride derivative of enoxaparin sodium" of USP39, but without reduction of the disaccharide sodium borohydride. Analysis of disaccharides in the glycosaminoglycan composition, which are present in the rapidly moving heparin fraction that can be enzymatically hydrolyzed by heparinase, is shown in FIG. 1, and indicates that: the main enzymatic hydrolysis disaccharide products comprise IdoA-GlcNAc (18.4%), IdoA-GlcNS (10.8%), IdoA-GlcNAc6S (6.9%), IdoA2S-GlcNAc (1.4%), IdoA-GlcNS6S (8.9%), IdoA2S-GlcNS (8.7%), IdoA2S-GlcNS6S (33.6%) and the like; of hexosamine (glucosamine, GlcN)N-The site is acylated or acetylated by sulfuric acid, wherein acetylation accounts for-20%, namely the acetyl-containing component accounts for-20%; of hexosamine6-O50% of the components are esterificated by sulfuric acid, calculated as an integral; hexuronic acid (iduronic acid, IdoA after enzymolysis),2-Omore than 40% of the positions are esterified by sulfuric acid; of the hexosamines, glucosamine, which is generally considered to be associated with anticoagulant activity3-OThe sulfate ester group accounts for about 1.5 percent and is obviously less than the average level of 2.4 percent of USP heparin, but the sulfate ester group also has certain anticoagulant activity。

Example eight: agarose gel electrophoresis analysis of glycosaminoglycan compositions

According to the method described in the literature [ P. Bianchini et al, J. Chomat, 196, 455 (1980) ], 0.5% agarose gel was electrophoretically separated with barium acetate buffer solution and diaminopropane, respectively, and CTAB was fixed, dried, stained and decolored after electrophoresis was finished, and the obtained gel was scanned to record the pattern. Standard controls include chondroitin sulfate, heparin (or slow moving heparin, SMH), Fast Moving Heparin (FMH), and Dermatan Sulfate (DS). The electrophoresis results are shown in FIG. 2, wherein lane 1 in FIG. 2, the fast moving heparin standard; lane 2, fast moving heparin standard + dermatan sulfate standard (concentrated); lane 3, fast moving heparin standard + dermatan sulfate standard (diluted); lane 4, dermatan sulfate standard; lane 5, glycosaminoglycan composition sample 1; lane 6, glycosaminoglycan composition sample 2. Glycosaminoglycan compositions are composed of fast moving heparin and dermatan sulfate, with little to no slow moving heparin and dermatan sulfate.

Example nine: nuclear magnetic hydrogen spectroscopy analysis of glycosaminoglycan compositions

From nuclear magnetic hydrogen spectroscopy analyses of samples from the examples, the apparatus was zeroed using a 400MHz NMR spectrometer from the university of Suzhou analytical testing center, sodium 3-trimethylsilylpropionate-d 4 (TSP). The results of the analysis are shown in FIG. 3, showing: the main peak positions of the nuclear magnetic hydrogen spectrum of the glycosaminoglycan composition are 2.05 ppm, 2.08 ppm, 3.27 ppm, 3.38 ppm, 3.54 ppm, 3.80 ppm, 4.05 ppm, 4.20 ppm, 4.34 ppm, 4.68 ppm, 4.73 ppm, 5.22 ppm and 5.42 ppm; wherein 2.04 ppm, 3.27 ppm, 4.34 ppm, 5.22 ppm and 5.42 ppm are the main characteristic hydrogen peaks of fast moving heparin; and 2.08 ppm, 3.38 ppm, 3.54 ppm, 3.80 ppm, 4.05 ppm, 4.20 ppm, 4.68 ppm, and 4.73 ppm are the main characteristic hydrogen peaks of dermatan sulfate, well indicating that the glycosaminoglycan composition is a mixture of fast-moving heparin and dermatan sulfate.

Example ten: in vitro anticoagulation and antithrombotic testing of glycosaminoglycan compositions

The experimental method comprises the following steps: 3mL of 5 human peripheral venous blood is taken each time, 3.8% sodium citrate anticoagulant is used for anticoagulation at a ratio of 1:9, and the Platelet Poor Plasma (PPP) is separated after 3000 r/min centrifugation. According to the kit method, the detection is carried out on a machine (full-automatic hemagglutination instrument, Stago Compact). The experimental groups were as follows: a sample group of a sample of a glycosaminoglycan composition for intestinal mucosa of sheep (described in example one), a sample group of a glycosaminoglycan composition for intestinal mucosa of cattle (described in example two), a sample group of a glycosaminoglycan composition for lung of cattle (described in example three) and a sample group of a glycosaminoglycan composition (described in example five), all groups had a final concentration of 2mg/L, and the experiment was carried out with physiological saline as a blank.

Results and analysis:

1) APTT, PT and TT

The results of the experiment are shown in table 2:

table 2: effect on APTT, PT and TT in vitro

Group of	APTT	PT	TT
				Glycosaminoglycan composition sample 1	134.1±16.7 s	12.9±0.5 s	70.3±13.2 s
Glycosaminoglycan composition sample 2	127±7.9 s	13.5±0.7 s	71.9±9.6 s
				Glycosaminoglycan composition sample 3	144.3± 20.4 s	12.9±0.5 s	67.3±14.2 s
Glycosaminoglycan composition sample 4	114.1±18.4 s	12.4±0.6 s	65.3±11.5 s
				Glycosaminoglycan composition sample 5	134.6± 20.4 s	13.1±0.9 s	71.3±12.6 s
Blank control	38.1±6.4 s	11.6±0.6 s	16.6±3.7 s

As can be seen from Table 2, 5 samples of glycosaminoglycan compositions showed significant prolongation of APTT and TT, and also some prolongation of PT in vitro, and the samples of glycosaminoglycan compositions showed similar effects on APTT, TT and PT, with no significant difference therebetween.

APTT, PT and TT are clinically important coagulation indexes, the numerical values are prolonged, and the glycosaminoglycan composition has a certain anticoagulation and anti-thrombus effect.

2) Fibrinogen and recalcification time:

the results of the experiment are shown in table 3:

table 3: external effects on fibrinogen and recalcification time

Group of	Fibrinogen	Calcium recovery time
			Glycosaminoglycan composition sample 1	1.98±0.44 g/L	31.00±0.00 s*
Glycosaminoglycan composition sample 2	2.11±0.36 g/L	31.00±0.00 s*
			Glycosaminoglycan composition sample 3	2.09±0.41 g/L	31.00±0.00 s*
Glycosaminoglycan composition sample 4	2.07±0.53 g/L	31.00±0.00 s*
			Glycosaminoglycan groupCompound sample 5	2.22±0.27 g/L	31.00±0.00 s*
Blank control	2.57±0.25 g/L	9.95±0.40 s

*: all exceed the detection range

As can be seen from Table 3, five samples of glycosaminoglycan compositions all reduced fibrinogen content in vitro, while the recalcification time was greatly prolonged, and exceeded the detection range.

Fibrinogen participates in thrombus formation, fibrinogen levels are also associated with blood viscosity, and glycosaminoglycan compositions help to interfere with thrombus formation, reduce blood viscosity, and improve blood flow by reducing fibrinogen levels. The recalcification time is greatly prolonged, which indicates that the increase of the anticoagulant substances in the plasma of the test group influences the coagulation of the blood, namely, the glycosaminoglycan composition has the effects of anticoagulation and thrombus resistance.

All the above data reveal that glycosaminoglycan compositions have good anticoagulant and antithrombotic effects in vitro.

There are, of course, many other specific embodiments of the invention and these are not to be considered as limiting. All technical solutions formed by using equivalent substitutions or equivalent transformations fall within the scope of the claimed invention.

Claims (10)

1. The glycosaminoglycan composition is prepared by extracting glycosaminoglycan from a ruminant heparin byproduct, consists of fast moving heparin and dermatan sulfate, has an anticoagulation and antithrombotic effect, and has an anti-Xa activity dependent on antithrombin III of 60-100U/mg, an anticoagulation activity dependent on heparin cofactor II of 100-200U/mg, and an anticoagulation activity of 50-80U/mg by a sheep plasma method.

2. The glycosaminoglycan composition of claim 1, wherein the fast moving heparin in the glycosaminoglycan composition is comprised of hexuronic-hexuronic acid unitsAmine, the hexuronic acid is iduronic acid or glucuronic acid, and the hexuronic acid is2-OMore than 40% of the positions are esterified with sulphate, hexosamine being glucosamine in the amino group6-O45-55% of the components are esterified by sulfuric acid esterN-In situ sulfation or acetylation, wherein 15% to 25% of the components are acetylated; dermatan sulfate in said glycosaminoglycan composition is comprised of iduronic acid-6-Sulfate-galactosamine repeating disaccharide unit, average molecular weight is 25,000 Da.

3. A glycosaminoglycan composition according to claim 1 or 2, characterized in that it has a significant prolongation of both APTT and TT and a reduction of fibrinogen content as a result of in vitro tests on human plasma.

4. The method for preparing a glycosaminoglycan composition according to claim 1, comprising the steps of:

s1, selecting glycosaminoglycan as a by-product obtained after heparin is extracted from organs of ruminants, wherein the ruminants comprise cows, sheep or goats;

s2, virus inactivation, namely dissolving glycosaminoglycan which is a heparin by-product, adding alkali for action, then adjusting pH, adding an oxidant for further virus inactivation and decoloration, and finally adding a filter aid for filtration to prepare a reaction solution;

s3, removing heavy metals, namely adding a metal ion chelating agent into the reaction solution obtained after virus inactivation in the S2, adjusting the pH, and finally precipitating a crude glycosaminoglycan composition by using an organic solvent;

s4, refining and purifying the glycosaminoglycan composition, redissolving the crude product in the S3, carrying out fractional precipitation by using an organic solvent, harvesting a precipitate, dehydrating, and finally drying to obtain the glycosaminoglycan composition.

5. The method of claim 4, wherein the dissolving of the heparin by-product glycosaminoglycan in S2 is performed by re-dissolving the heparin by-product glycosaminoglycan with a mass concentration of 1% to 30% by mass in saline water with a mass concentration of 0% to 10%; the alkali action refers to the treatment by adopting sodium hydroxide with the concentration of 0.01-5 mol/L; the pH adjustment refers to adjusting the pH to 7-11; the oxidant comprises hydrogen peroxide or potassium permanganate; the filter aid is diatomite.

6. The method for preparing a glycosaminoglycan composition according to claim 4, wherein the metal ion chelating agent in the S3 means EDTA at a concentration of 0.1 to 50 mmol/L; the pH value is adjusted to be between 4 and 6; the organic solvent precipitation refers to the precipitation of crude glycosaminoglycan composition with acetone at a final concentration of 30% -43%.

7. The method of claim 4, wherein the step of S4 comprises dissolving crude glycosaminoglycan composition in saline, the saline being a 0% to 10% sodium chloride solution, and the crude glycosaminoglycan having a concentration of 1% to 30%; the organic solvent fractional precipitation refers to precipitation by using ethanol or methanol or acetone with the final concentration of 40-60%.

8. The method for preparing a glycosaminoglycan composition according to claim 1, comprising the steps of separately purifying the fast-moving heparin component and the dermatan sulfate component of ruminants, and mixing the separately extracted fast-moving heparin component and dermatan sulfate component to prepare the glycosaminoglycan composition.

9. A pharmaceutical composition comprising the glycosaminoglycan composition of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

10. Use of the glycosaminoglycan composition of any of claims 1 to 3 for the preparation of a medicament or a nutraceutical or halal product for the prevention and treatment of thrombo-embolic diseases, vasculopathy diseases and vascular diseases at risk of thrombosis.

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