WO2011054174A1

WO2011054174A1 - Depolymerized fucosylated glycosaminoglycan and preparation method thereof

Info

Publication number: WO2011054174A1
Application number: PCT/CN2010/001679
Authority: WO
Inventors: 赵金华; 吴明一; 康晖; 曾伟珍; 梁慧; 李姿; 徐世民; 冯汉林; 于琳
Original assignee: 深圳海王药业有限公司
Priority date: 2009-11-06
Filing date: 2010-10-25
Publication date: 2011-05-12
Also published as: CN101735336A; CN101735336B

Abstract

Disclosed is a method for preparing depolymerized fucosylated glycosaminoglycans which are obtained by depolymerizing fucosylated glycosaminoglycans through peroxide depolymerization catalyzed by the fourth period transition metal ions in water phase medium. The depolymerized fucosylated glycosaminoglycans obtained have no less than 80% of glycan molecular number taking GaINAc as reductive terminal, and weight average molecular weight of which is about 6000Da~ 20,000Da, and PDI of which is 1.0- 2.0.

Description

»^藻amine and its preparation method

The invention belongs to the technical field of medicine, and relates to a method for preparing a xyloganose (dFG) and a preparation method thereof, and a medicine group containing the same. Background technique

Fucosylated Glycosaminoglycan, Fucose-branched Glycosamino- glycan or fiicose-containing glycosaminoglycan (FG for short), or Fucosylated chondroitin sulfate (FCS), refers to a kind of skin animal A glycosaminoglycan derivative having a structure similar to that of chondroitin, but having a sucrose substitution in the body wall or viscera (J Biol Chem, 1988, 263 (34): 18176-83 and J Biol Chem, 1991, 266 (21) : 13530-6 X

Natural FG has ^ activity (Fan et al., Pharmacological Journal, 1980, 15: 267), which has potential therapeutic effects on certain blood pine diseases, but FG also has induced platelet aggregation activity (Jia- Zeng Li et al, Thronbosis and Haemostasis, 1988, 54(3): 435-9), and platelet-inducing activity severely affects the conversion of the potential therapeutic effects of FG to actual clinical applications. Recent studies have shown that i-quantity FG can retain a certain intensity of activity while eliminating or avoiding the platelet-inducing activity of prototypic polysaccharides (Fan et al., J. Biol. Chem., 1993, 9(2): 146- 151; Toshio Imanari et al, T3⁄4rombosis Research, 1997, 129: 27-31).

Low molecular weight FG can be derived from the fractionation of FG or from FG depolymerization. Hierarchical treatment [氐Ή FG method is simple (Fan et al., J. Biol. Chem., 1993, 9 (2): 146-151), but due to the fact that there are few ^ in the natural FG, the system is divided into two parts. ^flFG has a low yield and a serious waste of resources.

Enzymatic depolymerization and nitrous acid depolymerization methods commonly used for glycosaminoglycan depolymerization are not suitable for depolymerizing FG because: the presence of fucose bismuth makes known glycosaminoglycan endonucleases impossible. Hydrolyzed FG; ^ in the FG are all B-Tang, no free, so no ^fclLE Xiao poly.

The 3⁄40 ₂ method depolymerization FG has been found in ^gJi (Fan Hui-Zeng et al, WO 90/08784; Ken-ichiro Yoshida et al, Tetrahedron Lettere, 1992, 33 (34) 4959-62). Due to the existence of many different types of glycosidic bonds in FG, such as glucuronic acid (GlcUA) β(1→3) glycosidic bonds present in the main chain, acetylaminogalactose (GalNAc) β(1→4) sugars, and Tender α-fucose (Fuc) sugar truncation, etc., conventional 3⁄4 (3⁄4 depolymerization method has limited choice of these glycosidic bonds. Non-selective cleavage of the backbone glycosidic bond can form oligomeric groups with different '|3⁄4 ends The FG product, that is, the higher G?UA and GalNAc ends of the higher I:, thus affecting the homogeneity of the product; and the cleavage of the side-glycosidic bond can lead to the detachment of the side chain by the algalose.

^b, conventional H ₂ 0 ₂ method depolymerization? 0 required ^ cattle relatively intense, its ^ ί temperature requirements are higher, anti It should take a long time, and its key flaw is the controllability and repeatability.

Therefore, the development of a method for the production of FG with mild, reproducible and uniform product homogeneity is of great significance for the industrial production of FC. Summary of the invention

One object of the present invention is to overcome the deficiencies of the technology, and to provide uniform and controllable turbidity to prepare a highly reproducible oligomeric fucosylated glycosaminoglycan (Depolymerized fucose-containing glycosaminoglycan, called dFG). Or a method of oligomeric FG).

The method for preparing dFG according to the present invention comprises depolymerizing fucose strip glycan (FG) by catalytic depolymerization using a catalyst containing a fourth period ±> metal ion in a medium, comprising the following steps:

1) In the presence of a catalyst of i±3⁄4 metal ion in the fourth cycle, add i±| ^/ to the fucose-derived glycosaminoglycan from the target medium shield;

2) Suspend ^ J^, collect and purify the required amount of #3⁄4^藻糖-aminoglycan.

In the step 1), the fucose ^#amine polyglycoside G) of the reference source refers to an acid mucopolysaccharide containing the fucoid 4 extracted and extracted from the animal of the sea cucumber family. Its structural characteristics are: GlcUA (gluconic acid) and GaJNAc (2-M2-J^N-B-semitylate) exist in near-equal molar ratio (1:1±0.3) And GlcUA and GaJNAc are linked to each other by β1→3 and β1→4 sugar, respectively, and the fucose (Fuc) or its sulfate is linked to the main chain in a side chain, in terms of molar ratio, Fuc The GalNAc ratio can range from about 0.5 to 2.5. The FG backbone structural unit can be expressed as equation (I):

In the formula (I): -OR is a group (-OH), a sulfate group (-OS (V), or a chelate-fucosyl group represented by the formula (Π):

In the formula (Π): -OR is a hydroxyl group (-OH), and an acid ester group (-OS0 ₃ - ) is as defined above.

The proportion of side chain fucose bismuth from FG from different sea cucumber species/variety and its linkage to the main chain position may be different; the same sea cucumber varieties but different tissue sources or differences in extraction methods may also lead to the proportion of monosaccharide composition of FG There are differences in the form of side chain and the degree of polysaccharide formation, but these differences do not involve the FG structural characteristics and the changes of the sugar bond type, and do not affect the effective application of the method of the present invention. Therefore, in the invention, the FC may be derived from sea cucumbers of different varieties, including but not limited to sea cucumber, green sea cucumber, sea cucumber, black sea cucumber, black sea cucumber, and sea cucumber.

In step 1), the solution is carried out in a medium, and the metal ion is used as a catalyst to catalyze the solution to generate ^^ FG.

The compound can generate free radicals in the ^Ji system and pass through the free radical chain FG glycosidic linkages to form the dFG product. These peroxides include, but are not limited to, peroxyacetic acid, hydrogen peroxide, 3-chloro-perbenzoic acid, hydroperoxyolefin, sodium persulfate, benzoyl peroxide, and salts or esters thereof, preferably paste hydrogen .

The mass fraction of the FG in the system is about 0.05% "15%, and the mass fraction in the system is about 0.5% to about 30%. During the depolymerization of FG, the peroxide can react. In the former one-time ^ plus T ^ ^ i system, ^ ^ 1⁄2 can also be gradually added to the system by ^ or intermittent method. The present invention ^ ^ will continue to add the reaction system according to the rate Medium.

The metal ion as a catalyst is a fourth periodic transition metal ion, including Cu+, Cu ²⁺ , Fe ²⁺ , Fe ³⁺ , Cr ³⁺ , Cr ₂ 0 ₇ ² -, Mn ²⁺ , Zn ²⁺ , Ni ^2+, etc., these metal ions can be used alone or in combination with each other. Among them, the catalysts are Cu+, Cu ²⁺ Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , and the most ^ Cu ²⁺ . Since metal ions are not chemical reagents that exist independently, the actual organic salts of these metal ions are. In the reaction system, the concentration of the metal ion may range from about 1 nmol/L to 0.1 mol/L, and the range of ^ ranges from 10 nmol/L to 10 mmol/L.

The conventional process parameters of the depolymerization process are: a temperature range of 10 ° C to 75 ° C; a reaction time of 20 minutes to 8 hours; the reaction can be carried out under normal pressure or pressure; the reaction can be selected from nitrogen, inert gas Under protection, it can also be carried out under atmospheric pressure with the atmosphere ring*N.

At the end of the reaction, a chelate can be added to the system to inhibit it by chelation with the metal ion catalyst.

Then it is terminated by technical means such as ^ P, and agent. ^^ refers to a physical shield capable of forming a metal ion, including but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 3-propylenediaminetetraacetic acid (PDTA), Triacetic acid ammonia (NTA) or a salt thereof. The process of the invention is preferably disodium edetate or a hydrate thereof. The reaction product precipitation method is a method of adding a chelating agent to a system of a polysaccharide material by adding a salt (for example, potassium acetate), and the hydrazine solvent includes decyl alcohol, ethanol, acetone, and the like. Low carbitol/ketone, of which ethanol and acetone are preferred.

The reaction product dFG can be purified by methods known in the art, for example by dialysis or ultrafiltration. ^"Salt, further purification by means of analysis or DEAE ion exchange, etc., the obtained dFG product can also be prepared by cation exchange to prepare a single salt form, such as sodium salt, potassium salt or calcium salt, etc. In the middle, the amount of dFG can be 4%. The molecular weight of the dialysis membrane should be cut off, and the molecular weight cut off should be 3000 Da. The dialysis time should be determined by specific treatment conditions, usually not less than 6 hours. The salt formation process of dFG product ύ ^ The dynamic ion exchange salt method, wherein the strong acid cation exchange resin can be selected for use. The pretreatment of the tree column, the sample loading and the sample can be carried out according to the usual method. The sample mass of the sample of the ύί4 in the method of the invention ^ about 2 ° / ^ 5%.

The method of the invention can significantly improve the reaction conditions of the ruthenium peroxide depolymerization FG, that is, when the same peroxide or the original FG starting material is used to prepare the same or near-order amount of dFG, at temperature and the like In the approximate case, the method can increase the reaction rate and shorten the reaction time course relative to the direct i3⁄4 compound depolymerization method (ie, the method of peroxide depolymerization in the absence of a metal ion catalyst); similarly, in controlling the reaction time Under the conditions, the method of the invention can significantly lower the temperature required for the reaction, so that the desired JS can be completed at room temperature.

During the repeated preparation of dFG, the batch-to-batch variation of the products obtained by the method of the present invention was significantly lower than that of the direct oxide depolymerization method under the same or similar ^ cattle. For the preparation of dFG of different target molecular weights, the method of the present invention can be conveniently achieved by changing the conditions, and for the direct peroxide depolymerization method, the uncertainty of changing the post-cow reaction product deviation from the expected result is more serious (the obtained dFG) The difference between the amount and the target molecular weight can reach 5% or more).

The method of the invention significantly improves the repeatability and controllability of the preparation of dFG, which may be related to the activation energy of the catalyst and the steady rate of the shape, and may also shorten the temperature and the time course to shorten the isothermal bovine and its cattle. Controllability is related to qualitative improvement.

For a relatively pure, homogeneous FG feedstock, the product obtained by the process of the invention has a high degree of repeatability under the same reaction conditions; however, for the FG "crude polysaccharide" containing more impurity components, the depolymerization reaction The stability and controllability of the present invention are still quite different. The present inventors have now found that the presence of an organic salt having a certain ionic strength in the reaction system can improve the stability of the solution.

To this end, the present invention provides a method for improving and improving the controllability of FG solution, that is, adding a certain concentration of ^^/ or organic to a metal ion-catalyzed system of lyophilized polysaccharides. salt. The i^bM^/ or organic salt includes a salt formed of a metal element (such as an alkali metal, a metal element, etc.) with a halogen, an organic acid, or the like, a salt of an organic or inorganic acid and an organic base, and a combination thereof. Salt, which evades sodium chloride, chlorinated clock, sodium acetate, sodium acetate trihydrate, acetic acid clock. In the method of the present invention, the salt ion concentration of the valence or organic salt used for improving the depolymerization and the controllability of the reaction is from about 0.1 mmol/L to about 1.0 mol/L. The mechanism by which salt ions improve the J^H cattle cannot be fully elucidated. For the method of the present invention, it may be related to the conformation of the stable reactant FG.

The FG of the present invention is a fucose t-aminoglycan derived from the echinoderms of the echinoderms, which is characterized by the presence of GlcUA and GalNAc (^^-ester) in a molar ratio of about 1:1 ± 0.3 and different molar ratios. Fucose Acid ester). Differences in sea cucumber varieties and their tissue sources or differences in extraction methods may lead to differences in the ratio of monosaccharides, the forms of existence, and the degree of sulfate in the polysaccharides of FG, but these differences do not involve the essential changes in the type of FG structural polyglycoside bonds. Therefore, the practical application of the depolymerization method of the present invention is not affected. The source animal of the FG of the present invention may be selected from, but not limited to, sea cucumber, green sea cucumber, sea cucumber, black sea cucumber, black sea cucumber, and ginseng. Obviously, it will be understood by those skilled in the art that for other varieties of sea cucumbers produced in the world, the fucosylated glycosaminoglycans of the above-mentioned structural features can be depolymerized by the method of the present invention to obtain the desired The oligomeric FG product, therefore, is not limited by the particular sea cucumber variety.

The reduced end of the dFG product obtained in the present invention can be detected by NMR. NMR detection can also be conveniently used to confirm the Fuc/GaJNAc composition ratio in the product.

Comparing the direct i3⁄4 compound depolymerization with the catalytic compound depolymerization product, the present inventors have found that the Mh depolymerization method has an unexpected sugar ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ It is less than 80% or more than 95%) and less than GlcUA, which is different from the direct depolymerization product in the high GfUA end of the GfUA, indicating that the dFG obtained by the catalytic peroxide depolymerization method is better. End uniformity, thus better quality uniformity and controllability

There is no significant change in the composition ratio of Fuc GalNAc; in the direct peroxygen do not depolymerization process, the ratio may have about 5% enthalpy, which proves the choice of the sugar of the method of the present invention.

Therefore, another object of the present invention is to provide an oligomeric FG (dFG) prepared by the method of the present invention, wherein the dFG has a terminal mainly of GalNAc and is subjected to nuclear magnetic resonance (NMR) technology. It can be confirmed that the composition ratio thereof is not less than 80%, and accordingly, in the dFG, the number of glycans in which GlcUA is used as the reduction 'f sister' is less than 20%. In the dFG of the present invention, the number of polysaccharides having GalNAc as 4 sisters is not less than 90%.

The amount of dFG product can be measured by high-efficiency sword-cross chromatography. Reserved FG jk¾ from active learning in ½-induced platelet aggregation activity Free considerations, weight average ^^ meter, the present invention is selected from ^ _£ ^ ^^ 0 in an amount ranging from about 6,000 ~ 20,000Da, V escape molecular weight ranging from about 10,000 to 15,000 Da.

The multi-dimensional index (PDI, ratio of weight average molecular weight to number average molecular weight, Mw/Mn) of the dFG of the present invention is between 1.0 and 2.0; for the oligomeric FG of bismuth, the PDI is 1.2. Between 1.6.

The dFG of the present invention may be a salt of a pharmaceutically acceptable alkali metal, a metal or the like, such as a sodium salt, a potassium salt and a calcium salt; and the like, and the oligomeric FG of the present invention may also be a pharmaceutical thereof. An acceptable ester of an alkaline organic group.

The dFG of the present invention has an exact activity and thus has a clear pharmaceutical potential. dFG has a good water solubility and is therefore easy to prepare into a solution preparation or a lyophilized product thereof. As a polysaccharide ingredient, its mouth! The use of the material is limited, due to the preparation of the gastrointestinal preparation dosage form, the preparation of the preparation can be in accordance with the field Well-known technical methods are carried out.

A further object of the present invention is to provide a pharmaceutical composition comprising the dFG of the present invention and a pharmaceutically acceptable excipient. The dFG of the present invention has a certain intensity of activity, and thus can be used for the prevention and treatment of different degrees of i=f diseases, such as JI^ creative cardiovascular disease, cerebrovascular disease, pulmonary veins, peripheral veins ώ ^, deep veins, peripheral arteries JW congruent. Accordingly, the present invention can provide use in the preparation of a medicament for the treatment of cardiovascular diseases. DRAWINGS

Figure 1 shows the HPGPC spectrum of FG and its degradation product dFG;

2 is a ¹ H NMR spectrum showing a Fuc/GalNA molar ratio (integral area ratio of a thiol signal) in FG and dFG;

Figure 3A is a ^- ¹ !! COSY spectrum of dFG-2;

Figure 3B is a cross-section of the COSY spectrum at 5.22 ppm of Figure 3A showing the NMR signals of the α-Η of the reducing ends GalNAc and GlcUA.

^^ Reality

The following examples are intended to further illustrate the invention and are not to be construed as limiting the scope of the invention.

Example 1 Metal ion catalyzed t ^ depolymerization of FG

1.1 Materials:

FG: Fucoid-derived rock algae 4 Tang ^^ Tang glycosaminoglycan, according to the text! ^ Method (J Biol Chem, 1991, 266 (21):

13530 · 6) Extraction preparation. Purity 98% (HPGPC, area normalization), ^"mount (Mw), 69800.

Reagents for H ₂ 0 ₂ , CH ₃ COONa-3H ₂ 0, NaCl, NaOH, CuCl ₂ , FeCl ₂ , ZnCl _{2 ,} etc.: All are commercially available analytical reagents.

1.2 method:

Dissolve 5.0g of each of the four sea cucumbers FG in 180ml water in a round bottle, keep warm in a 45 °C water bath and continue to evenly cake, add 10ml pure water or 20mmol/L concentration of chlorine (Cu ²⁺ ) solution, chlorine Ferrous (Fe ²⁺ ) solution: ^Zinc chloride (Zn ²⁺ ) dissolved, 10% 3⁄4 (3⁄4) was added at a rate of 15 ml/h within 2 hours. The pH was controlled with 1N NaOH solution during the process. The value ranged from 7.2 to 7.8, and the above half-reaction was carried out for 2 to 8 hours. At the 30 min, lh, 2 h, 6 h, and 8 h after the start, 15 mg of EDTA disodium salt was added from each ^ I»i 5 ml, and mixed. Ice 7j ^ P, add 3, 95% ethanol to determine the polysaccharide, 5ml 60% ethanol washed twice, hot air blow dry ethanol, 10ml pure water to volume, high performance cross-chromatography (HPGPC) method to determine the molecular weight of the product.

1 result:

Depolymerization of hydrogen peroxide to sea cucumber FG in the presence or absence of a fourth periodic transition metal ion catalyst is shown in Table 1 and Figure 1. Table 1: FG depolymerization product measurement

Depolymerization product molecular weight at different time points (Da)

,

Omin 30min 60min 120 min 360 min 480 min

3⁄4 (3⁄4+ pure water 69,800 60,500 58,700 54,100 48,030 41,010

HiOz + Cu ² " 69,800 33,906 21,952 14,996 12,450 8,805

H ₂ (3⁄4+Fe ²⁺ 69,800 343)0 26,700 19,854 16,906 12,400

H ₂ (3⁄4+Zn ²⁺ 69,800 32,805 27,903 18,031 15,762 11,080) The results of Table 1 show that the metal ions Cu ²⁺ , Fe ²⁺ , Zn ²⁺ have a significant catalytic effect on the F ^ ^ ^ 3⁄41⁄2 « " The polymerization rate is significantly improved. By comparing the catalytic efficiencies of the three catalysts, it can be seen that the catalytic efficiency of the metal ion Cu ²⁺ is high, and the preparation of dFG is realized in the time of the bean. NMR Spectroscopic Detection of dFG Products Obtained by Depolymerization and Metal Ion Catalytic Peroxide Depolymerization

2.1 Materials:

FG: Fucose-derived fucose ^ ^-glycan, prepared by the method "J Biol Chem, 1991, 266 (21): 13530-6). Purity 98% (HPGPC, area normalization) Method), quantity (Mw), 69800.

H2O2, CH ₃ COONa-3H ₂ 0, NaCl, NaOH, Cu (CH3COO 3⁄40, etc. Reagents used: All are commercially available analytical reagents.

2.2 Method:

Preparation of dFG: 5.0 g of each of the two sea cucumbers FG was dissolved in a 180 ml 7j solution in a round tile, respectively, to 70. C and 35. C water bath insulation and continuous uniform stirring, then add 10ml pure water or 60mmol / L concentration of copper acetate (Cu ² ) solution, then add 10% 3⁄4 at a rate of 10ml / h within 2 hours (3⁄4, the reaction process The pH range was controlled from 1N NaOH solution to 7.2 ~ 7.8. After the continuous simmering for 4 hours, add 500 mg EDTA disodium salt to the solution and mix well, ice τ ^ Ρ, add 3 95% ethanol, centrifuged;; fixed, 100ml 60% ethanol wash twice, centrifuged to obtain H.; dissolved in 150ml water, 001x7 type cationic resin, 4 sodium salt, then Interception ^ "3500Da dialysis membrane dialysis ό hour, the product was intercepted, lyophilized, respectively, to obtain 3.23g (dFG-l, direct 3⁄4 (3⁄4 depolymerization product) and 3.52 g (dFG-2, Cu ²⁺ catalyzed 3⁄4 ( 3⁄4 depolymerization product) depolymerized sample.

^^ ί: HPGPC ^i ^ ¹ amount and distribution; Conductivity detection - OSC ACOa molar ratio.

NMR^i:

Instrument, AVANCEAV400 superconducting nuclear magnetic resonance [Yi (Swiss Bruker 400MHz);

Solvent, I3⁄4099.9Atom%D (Norell);

Internal standard, trimethylsilyl-propionic acid (TSP-D4); temperature, 45 °C.

2.3 Results The test results are shown in Table 2 below. The NMR spectrum is shown in Figure 2.

Table 2: FG, (^0-1 and ^0-2, ^^^^^^^^

NMR detection

Only mouth

Mw n GalNAc(CH ₃ ) Ga Ac/

PDI -OS(3⁄47-COO- (Da) (Da) /Fuc(CH ₃ ) GlcUA*

FG 68,740 63,468 1.08 3.46 1: 1.02 - dFG-1 18,960 11,760 1.61 3.32 1: 0.93 1:0.45 dFG-2 14,930 10,660 1.40 3.45 1:1.00 1:0.08

Mw: weight average ^ ^ quantity; Mn: number average; PDI: quantity multi-index (PDI=Mw/Mn)

* ι^ 3⁄4 sugar ratio by 1 ^ 矣 poly, catalyzed i ± 匕 ^ ^ ^ (70 ° C / 4h, 35 ° C / 4 h) and the molecular weight of the two reaction products (about 1.9kDa and about 1.5 kDa) It can be seen that in the presence of Cu ²⁺ ions, (3⁄4 depolymerization FG reaction temperature is higher, the efficiency is higher.

From the physicochemical and spectral analysis results of the two products, it can be seen that the PDI values of dFG-1 and dFG-2 are higher than those of FG, which is related to the 4 free polyhime of mucopolysaccharide, and the practice of dFG-2. Right lower.

From -OSC ACOa ratio ^f, the value of dFG-1 is 4% to FG 4 mesh, while dFG-2 is unchanged.

According to the NMR methyl signal product, the GalNAcFuc molar ratio (the fucose sulfhydryl peak signal is about 1.3 ppm, and the 33⁄4^ 1/2 tang 曱 base peak is about 2.1 ppm), dFG-1 is more FG. About 9%, while dFG-2 did not change. In the presence of Cu ²⁺ ions, Η ₂ (3⁄4 depolymerized FG has no effect on the fucoids. Obviously, according to the direct ±^t^ solution, the Fuc ratio of dFG-1 is changed. It can be judged that the direct depolymerization can lead to the formation of "alcohol".

From Fig. 3A, the molar ratio of GalNAc and GlcUA at the depolymerization product end can be judged, wherein the non-reducing end and the bridgehead hydrogen of GalNAc and GlcUA in the polysaccharide main chain are both β-configuration, and the NMR signal appears to be about 4.4 to 4.6 ppm. Meanwhile, the 'ends of GaJNAc and GlcUA each have about half of the α-configuration bridgehead hydrogen. ^{In the} H NMR spectrum, the α-configuration bridgehead hydrogen of GalNAc and GlcUA is about 5.2 ~ 5.3 ppm, which is difficult to distinguish between them, but the H2#^J chemistry associated with them is about 4.2 ~ 4.5 ppm. And 3.6 ~ 3.9 ppm, so it can be seen in the "PuD" to distinguish (the prototype FG due to ^" large amount, the number of terminal glycosyl groups is small and it is difficult to obtain a sufficiently strong distinguishing signal).

It can be seen from Fig. 3B that the molar ratios of GalNAc to GlcUA at the 'ends of dGAG-1 and dGAG-2 are about 1:0.45 and about 1:0.08, respectively, which shows that the Cu ²⁺ catalyzed 3⁄4 polymerization method For FG The GalNAc β (1→4) sugar has the choice i of ^;

The research of the present invention shows that the metal ion catalysis can significantly improve the selection of the sugar bit bond by the it^t^ depolymerization method. In spite of the fact that there is a 3⁄4iiCu ²⁺ ion catalyzed ^ ^ t in the process of depolymerization, a certain choice of f is shown, ^ "the exact mechanism is unknown. According to the mechanism of the solution, the selection of the catalytic solution of the present invention should be matched with the catalyst. The effects of activation energy of free radicals of different types of sugars ^ ^ are related.

The results of the present invention are 1⁄2i3⁄4. For the metal ion-catalyzed peroxide depolymerization method of FG, the number of glycan molecules with GalNAc as the reducing end in the dFG product is usually more than 80%, and in this embodiment, GalNAc is used as ' The ratio of the 13⁄4 end is 95%. Example 3 Comparison of repeatability and controllability of dFG prepared by direct ± depolymerization and metal ion catalysis ijA depolymerization

3.1 Materials:

Same as 2.1

3.2 Method:

Direct hydrogen process depolymerization F & five parts of sea cucumber FG each 300g dissolved in 9L water, 60 ° C water bath insulation and continuous uniform half, then in a 2 hour at a rate of 0.6L / h drop 15% (3⁄4, in the process Use 1 N NaOH solution to control the pH range from 7.2 to 7.8. After 6.5 hours of continuous treatment, add 3.0 g of EDTA disodium salt to the solution and mix well. Add ice to 3 ^, 95%. Ethanol'; fixed polysaccharide, centrifuged; J, washed twice with 0.6L of 60% ethanol, then dissolved in 10L of water with a cut-off of 3000Da modified cellulose 3⁄4 filter, 6 hours, cut off the product, freeze-dried, calculate the product Rate and detect the amount of product and its distribution.

Cu ²⁺ catalyzed dehydrogenation of FG by hydrogen peroxide: 300 g of each of the five sea cucumbers FG was dissolved in 9 L of water, 45. C water bath insulation and continuous uniformity of 4 and a half, then add 0.6L pure water or 20mmol / L concentration of copper acetate (Cu ²⁺ ) solution, and then add 15% H ₂ 0 at a rate of 0.6L / h within 2 hours ₂ , ^^ process with 1 N NaOH solution to control the pH range of 7.2 ~ 7.8. After 4 hours and 4 hours in a row, 3.0 g of EDTA disodium salt was added to the liquid and mixed, ice 7j Hp, 3 ^^P, 95% ethanol; 3⁄4 定定 polysaccharide, centrifuged to precipitate, It was washed twice with 0.6 L of 60% ethanol, then dissolved in 10 L of water to filter the modified cellulose ball with a molecular weight of 3000 Da for 6 hours, the product was withdrawn, freeze-dried, the yield of the product was calculated, and the amount and distribution of the product were measured.

3.3 Results

Result 3. Table 3: Direct hydrogen peroxide depolymerization FG and (^ ²⁺ catalytic hydrogen depolymerization FG products

Solution!^ Method Product Mw (Da) Mn (Da) PDI

1 17,360 10200 1.70

2 19,320 12350 1.56 Direct 3 18,690 13,100 1.43

Fooling together 4 16,780 11,560 1.45

5 16,820 9,980 1.69

RSD (%) 6.47 11.78 8.16

1 10,500 8,040 1.31

2 11,020 8,760 1.26

Cu ²⁺ catalysis

3 10,780 7,900 1.36

Turn

Na Ju 4 10,660 7,890 1.35

5 11340 8,030 1.41

RSD (%) 3.03 4.46 4.20

Mw: weight average, Mn: number average ^ ^ quantity, PDI: quantity more ^ L index (PDI = Mw / Mn)

RSD: relative standard deviation

The results in Table 3 show that the direct D3G product has a large difference between the batches, and the RSD values of the weight average molecular weight and the number average molecular weight are both greater than 5%; and the product obtained by catalytic hydrogen depolymerization The difference between batches is small, and the RSD values of both the weight average and the number average are less than 5%. Moreover, the RSD of the three detection values of the products Mw, Mn, and PDI obtained by directly dehydrating the hydrogen is significantly larger than that obtained by the method of the present invention, indicating that the catalytic hydrogen peroxide depolymerization method of the present invention is more stable. Sex and controllability. Example 4 ^3 metal ion catalyzed 3⁄4 compound method to dissolve jade ginseng crude polysaccharide

4.1 Materials:

Jade ginseng: Commercial varieties

Reagents: same as 2.1

4.2 Method:

Preparation of jade ginseng fucoidan sylvestris (refer to grammar preparation): Take dried jade ginseng 1000g, pulverize, ^ bath, add 10L soak. A solid hydrogenation clock was added to a concentration of 1 N, and the temperature was kept at 60 ° C for 100 min. After cooling, όN hydrochloric acid was adjusted to pH 8.5, 50 g of trypsin was added, and the mixture was incubated at 50 ° C for 3 hours. After that, centrifuge to remove the sputum. The supernatant was taken, and the pH was adjusted to 2.5 with hydrochloric acid, and the acidic protein was removed by centrifugation. Take the supernatant, neutralize with 2N NaOH solution, centrifuge ^ redundancy. Take the supernatant and add 95% ethanol to make the concentration reach 60%. C overnight. Centrifuge to obtain a precipitate, add 10 times the weight of water, and remove it by centrifugation. The supernatant was taken and potassium acetate was added to dissolve the mixture to a final concentration of 2.0 mol/L, and 95% ethanol was added until the final ^^ was 30%, and the precipitate was allowed to stand. The precipitate obtained by centrifugation was washed successively with 95% ethanol and absolute ethanol, and vacuum-dried under reduced pressure to obtain 8.1 g of 4:3⁄4 ginseng polysaccharide.

Cu ²⁺ catalyzed hydrogenation of FG: 5.0 g of crude polysaccharides of five parts of jade ginseng dissolved in a circle; 190 ml of water in a bottle, 35. C water bath ί 显 and ^ uniform halving, then add 50mmol / L concentration of copper acetate (Cu ²⁺ ) solution 10ml, then add 10% H ₂ 0 ₂ at a rate of 10ml / h, 1 N during the reaction The NaOH solution controls the pH range from 7.2 to 7.8. After the above ^ 2 cattle for 2 ^ 3 hours, add 500mg EDTA disodium salt to the liquid and mix, ice 7j p, add 3 of 95% ethanol ϋ polysaccharide, centrifuge to determine, with 100ml 60% ethanol Wash twice, 100ml 7J dissolved, add acetic acid clock to dissolve the final concentration of 2.0mol / L, stand overnight, collect by centrifugation; determine, 100ml water soluble, and then dialyzed for 8 hours with a molecular weight cut off of 3500Da dialysis membrane, the product is freeze-dried Calculate the yield of the product and measure the molecular weight and distribution of the product.

Cu ²⁺ catalyzed by ionic cesium hydrogen depolymerization F & five parts of crude polysaccharides of jasper ginseng each 5.0g dissolved in round watts containing 12.21g of sodium acetate trihydrate and 6.02g of chlorine! 3⁄4 of 190ml of water, 35 . C water bath insulation and uniform cake, then add 50ml / L concentration of copper acetate (Cu ²⁺ ) solution 10ml, and then drop 10% at 10ml / h rate (3⁄4, process with 1N NaOH solution to control pH The value range is 7.2 ~ 7.8. After the above ^ ί cattle for 43⁄4! half ^ ^ 4 hours, add 500mg EDTA disodium salt to the liquid and mix well, ice τ ^ Ρ, add 3 ^ Μ?, 95% Ethanol ^ ^ polysaccharide, centrifuged to obtain iKJ, 100ml 60% ethanol washed twice, 100ml water soluble, add potassium acetate ^ ^ ^ dissolved to a final concentration of 2.0mol / L, left overnight, centrifuged to collect, 100ml The solution was lyophilized for 8 hours with a 3500 Da dialysis membrane, and the cut-off product was freeze-dried to calculate the product yield and the amount and distribution of the product.

4.3 Results

Result 4.

Table 4: Dehydrogenation of crude polysaccharides from Jade

Solution 3⁄4 ^ method product 4^ under Mn (Da) PDI

1, 13,320 9,560 1.39

2 11,090 8270 1.34

Cu ²⁺ catalysis 3 9,790 7,120 1.38

Hydrogen

Depolymerization 4 12,800 9,430 1.36

5 10,750 8,040 1.34

RSD (%) 12.70 12.01 0.84 1, 11,690 8,660 1.35

1.0 M ion 2 11,720 8,450 1.39

Under strength

Cu ²⁺ catalysis ³ 12,030 9,030 1.33

10,940 8,140 1.34

5 11,830 3⁄4530 1.38

RSD (%) 3.60 3.79 1.91

Mw: weight average ^ "quantity, Mn: number average ^ ^ quantity, PDI: ^ ^ quantity more ^ t index (PDI = Mw / Mn)

RSD: relative standard deviation

The results in Table 4 show that for crude polysaccharides, due to the relatively high impurity content, metal ion-catalyzed polymerization is used at this time, and the obtained product can still be subjected to large batch-to-batch variation, which is mainly manifested in average molecular weight. There is a significant difference (RSD > 10%), while the dispersion is different.

Adding a certain intensity of non-organic or organic ions to the reaction medium can significantly improve the reproducibility of the depolymerization reaction, although the addition of these ions may be a certain degree of enthalpy. ^ or the mechanism by which organic ions improve the repeatability of the reaction is not fully understood and may be thought to be related to maintaining a stable conformation of the polysaccharide.

The related research of the present invention shows that in the ±| 匕 depolymerization reaction of the non-pure FG of different species of sea cucumber, the suitable cation strength is about 0.1 mol/L to 1.0 mol/L for the ii LW or organic ion. Example 5 ^ k activity of low saccharose ^ ^ teratamine (dFG)

5.1 material

The series of jade ginseng dFG: Mw are 25,380, 18,050, 14,350, 11,450, 8,800 respectively. Prepared according to Cu ²⁺ catalyzed depolymerization method in Example 4. (dimerization time is different);

Reagents: rabbit anemia small ^ pulp, Guangzhou Rui Te Biotechnology Co., Ltd.;

Activated partial thromboplastin time (ΑΡΤΓ) assay kit (ellagic acid): Shanghai Sun Biotechnology Co., Ltd.

Instrument: BICO Double Letter it r3⁄4 instrument, Minivolt (Italy).

5.2 method

Accurately miscellaneous ^ 1100mg of sheep, dissolved and dissolved to 100ml, then thin needle times. The time of each sample was measured according to the method of the kit, and the clotting time of the blank plasma was subtracted, which is the time ΔΑΡΤΤ (Sec) of the sample to prolong the blood coagulation.

5.3 Results

The actual 3^ fruit is shown in Table 5. Table 5: Effect of different molecular weight dFG derived from Jade foot sea cucumber on plasma sputum time in rabbits

dFG amount

25 80 18,050 14^50 11,450 8,800 (Mw )

AAPTT(Sec) 95.1 76.0 64.2 40.9 15.3 The results in Table 5 show that dFG derived from jade ginseng can prolong the pulping time, indicating that it can inhibit endogenous 3⁄41. Example 6 lyophilized product of low > ^ alginate glycosaminoglycan (dFG)

6.1 Materials

Sea cucumber dFG: Mw 14,350. Prepared by the Cu ²⁺ -catalyzed depolymerization method in Example 3.

20mmol/L pH7.0 phosphate buffer (PBS)

6.2 prescription

6.3 preparation process

The prescribed amount of sea cucumber dFG 20mmol/L pH 7.0 PBS was weighed to the full amount and the solution was completed. Add 0.6% medicinal activated carbon, half 20 min; Buchner funnel and 3.0 μπι microporous filter ship carbon. Measuring intermediate ^ f:. After passing the test, use 0.22μιη micro-lUi membrane±consult; fill in the control vial, 0.5ml per bottle, monitor the filling volume during the filling process, half-press plug, freeze dry slag, ^^ freeze-drying curve Freeze-dried, tamped, out of the box, rolled, and qualified.

Freeze-drying process: Place the sample into the box and lower the partition to >0. C, hold 3h; cold trap drops to -50. C, start pumping vacuum to 300nbar. Start sublimation: lh heat up to -30 at a constant rate. C, ^ 2h; 2h at a constant rate to -20. C,

8h, vacuum ^ 200 ~ 300 bar; then dry: 2h to -5. C, ^ 2h, vacuum

150 ~ 200Mbar; 0.5h to 10. C, 2h, vacuum ^ 80 ~ lOOnbar; 0.5h to 40. C,

4h, vacuum is drawn to the lowest.

Claims

^Change the book

1. A method for producing oxalate amines, ^ in the presence of a catalyst in the N-shi shield, urging ill sulphate miscible saponin, wherein:

Is a kind of echinoaraminin or organism derived from echinoderms, and its structural feature is that the FG contains a glucosyl group or a ketone of a molar ratio ranging from 0.7 to 1.3, and contains a rock. Alginose or ^ m vine, algae ^ ^ ester and B

The molar ratio of the acid ester ranges from 0.5 to 2.5;

The catalyst is a metal ion forming agent from the elemental cycle of the element; the algae ^3⁄4|amine » refers to a fucose amine edge having a weight average ^ * of 6000 ~ 20000Da

2. The method according to claim 1, wherein the ^^ is a catalyst of a metal ion of four cycles from the element; ^ Cu ⁺ , Οι ,

Formed by Ni; salt, salt, or a combination thereof.

3. The method of claim 2, wherein the metal ion is

4. The method of claim 2, wherein in the reaction system, the concentration of the catalyst is 0.001 mmol/L to 100 mmol Lo

5, the right spear J ^ seeking 1 method, which is selected from i3⁄4 acetic acid, hydrogen, 3-chloro-: ^ formic acid, ! Tl ^ ene, sodium ^^, itl t ^ 曱 St its salt or ester.

6. The method of claim 5, wherein the hydrogen is obtained.

7. The method according to the invention, wherein in the system, the mass fraction of the FG is 0.05% to 15%, the mass fraction of 1±^ is 0.5% to 30%, and the catalyst is 1⁄2 ^ 0.001 mmolL ~ 100 mmol / L. The range of δ ^ - 3⁄4> is 10 ° C - 75 ⁰ C.

The method according to claim 1, wherein the reaction system further comprises 1 mmol L to 1.0 mol/L.

ϋ^^/ Machine ^^ from chlorine, chlorinated clock, sodium acetate, potassium acetate and their water ^, and combinations thereof.

9,

a salt and/or an ester of the compound or a pharmaceutically acceptable salt thereof, wherein the glucan>R^amine contains a grape with a molar ratio of 1 _: 1 ±0.3 ^ and B LS ^ ' t or ^ ^ ester,

Among the glycosaminoglycans, GalNAc is used as i^'tt ^! The number of 3⁄43⁄4^ is not less than 80%, and the number of multiple entries is ^; 1.0 - 2.0.

10, find the right ^^ tree alginate amine 9 ^! Or a pharmaceutically salt thereof and / or esters, wherein the weight average amine fucose strip ^ ¹ in an amount of 10,000 Da -15,000 Da, multiple index entries 1.2-1.6, the number of glycan molecules having GalNAc as a reducing end is not less than 90%.

The hypoglycosylated glycosaminoglycan or a pharmaceutically acceptable salt and/or ester thereof according to claim 40, wherein the acceptable salts are metal and /^i metal salts.

14

Correction page (Article 91)

12. The sucrose 1⁄2 amine» or a pharmaceutically acceptable salt and/or ester thereof according to claim 11, wherein the pharmaceutically acceptable salts are sodium salts, potassium salts and salts.

13, a drug group ^ ^, in, ^ effective dose of the right ^ ^ seeking 9 described alginose ^ I amine! ^ or its pharmaceutically acceptable salts and / or esters, in order to learn from the auxiliary materials.

14. Use of a sucrose glycosaminoglycan or a pharmaceutically acceptable salt and/or ester thereof according to the invention for the preparation of a medicament for the treatment and/or prevention of blood sputum disease.

15

Correction page (Article 91)