Gymnadenia conopsea octasaccharide-decaose mixture as well as preparation method and application thereof

Abstract

The invention discloses a gymnadenia conopsea octasaccharide-decaose mixture as well as a preparation method and application thereof, and belongs to the technical field of medicines. The monosaccharide of the octasaccharide-decataccharide mixture of the gymnadenia conopsea comprises N-acetamino galactose, fucose, glucuronic acid or sugar alcohol thereof, galactose, N-acetylglucosamine, dehydrated talose and a sulfate group. The octasaccharide to decataccharide mixture of the gymnadenia conopsea disclosed by the invention can be used for remarkably prolonging the time of activating partial thromboplastin and prothrombin and effectively inhibiting endogenous and common coagulation pathways. The octasaccharide-decaose mixture of the gymnadenia conopsea can be used as a potential endogenous factor X enzyme inhibitor for preparing medicines for treating thrombotic cardiovascular diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to an eight-to-ten-saccharide mixture of Pterocarpus quadrangularis and a preparation method and application thereof. Background Art

[0002] Thromboembolism is one of the main causes of cardiovascular disease, and its treatment and prevention rely on the use of anticoagulants. Currently, commonly used anticoagulants in clinical practice include heparin, but its source and safety issues have attracted people's attention. Therefore, it is particularly important to study and develop new anticoagulants. Sea cucumber sulfated polysaccharides have attracted the attention of researchers due to their potent anticoagulant activity. Studies have shown that sea cucumber sulfated polysaccharides inhibit the activity of coagulation factors by activating antithrombin or heparin cofactor II in plasma, thereby exerting an anticoagulant effect. Different sulfation patterns and sulfate group contents have a significant effect on the anticoagulant activity of sea cucumber sulfated polysaccharides. In addition, oligosaccharide fragments of sea cucumber sulfated polysaccharides with lower molecular weight have stronger and safer anticoagulant activity. Colochirus quadrangularis is a sea cucumber species with abundant resources and low price, and the research on its polysaccharides has not been reported. Extracting sulfated polysaccharides from this type of sea cucumber and preparing oligosaccharide compounds may become an important way to develop new anticoagulant drugs. Previous studies have shown that sulfated polysaccharides extracted from other sea cucumber species have significant anticoagulant activity, such as fucosylated glycosaminoglycans (FG) extracted from Stichopus monotuberculatus and Apostichopus japonicus.

[0003] In summary, the sulfated polysaccharides of Pterochiura have the potential to become candidates for new anticoagulant drugs. They are novel in structure and rich in resources, and are important resources worthy of further research and development. Future research may focus on extraction, separation, structural identification, and activity evaluation in order to develop safer and more effective anticoagulant drugs. Summary of the invention

[0004] The present invention aims to provide a Pterochiron serrata octa- to deca-saccharide mixture and a preparation method and application thereof. The Pterochiron serrata octa- to deca-saccharide mixture can be used as a potential endogenous factor X enzyme inhibitor for preparing a drug for treating thrombotic cardiovascular diseases.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a mixture of octo-decaose of Pterochiron prismulata, wherein the monosaccharide composition of the mixture of octo-decaose of Pterochiron prismulata includes N-acetylgalactosamine, fucose, glucuronic acid or its sugar alcohol, galactose, N-acetylglucosamine, dehydrotalose and sulfate group; the structure of the mixture of octo-decaose of Pterochiron prismulata is as follows:

[0007]

[0008] In the formula, in the sugar groups a and b, R1=R3=-SO3Na, R2=H;

[0009] c, f, j sugar groups in which R1=R3=-SO3Na, R=H;

[0010] d In the sugar group, R1=R2=R=-SO3Na;

[0011] e. i In the sugar group, R1=R2=H, R3=-SO3Na;

[0012] In g and h sugar groups, R2 = -SO3Na, R1 = R3 = H;

[0013] In the k sugar group, R1=-SO3Na, R2=R3=H.

[0014] Preferably, a and b sugar groups account for 30.8%-40.8% of the total side chain molar percentage, l, m, n, o, p, q and c, f, j sugar groups constitute the disaccharide side chains account for 20%-30% of the total side chain molar percentage, d sugar groups account for 7.4%-13.4% of the total side chain molar percentage, e and i sugar groups account for 7.1%-13.1% of the total side chain content, g and h sugar groups account for 15.7%-25.7% of the total side chain content, and k sugar groups account for 3.5%-5.5% of the total side chain content.

[0015] Preferably, the a and b sugar groups are 2,4-di-O-sulfated fucose side chains, l, m, n, o, p, q include one or more of sulfated galactose, sulfated acetylglucosamine or sulfated acetylglucosamine, c, f, j sugar groups are 2,4-di-O-sulfated fucose, the d sugar group is a 2,3,4-tri-O-sulfated fucose side chain, the e and i sugar groups are 4-O-sulfated fucose side chains, the g and h sugar groups are 3-O-sulfated fucose side chains, and the k sugar group is a 2-O-sulfated fucose side chain.

[0016] Preferably, it is obtained by depolymerization and separation of Pterochitrachytrae quadrangularis polysaccharide, and has a weight average molecular weight of 67.7-87.7 kDa calibrated with dextran, a uronic acid content of 10%-30%, and a sulfate content of 10%-30%.

[0017] Preferably, the Pterocarpus quadrangularis octa- to decasaccharide mixture has a molecular weight of 2.0-3.5 kDa and a sulfate ester content of 10%-45%.

[0018] The present invention also discloses a method for preparing the eight-to-ten sugar mixture of Pterocanthus quadrangularis, comprising the following steps:

[0019] S1. adding the homogenate or dry powder of Pterocarpus quadrangularis to papain for enzymatic hydrolysis, and adding the suspension after enzymatic hydrolysis to a sodium hydroxide solution with a final concentration of 0.1-1.0 M for alkaline hydrolysis;

[0020] S2, removing proteins at the isoelectric point, salting out and ethanol precipitation to obtain a crude polysaccharide extract of Pterochirum quadrangularis, purifying it by anion exchange column chromatography, collecting the eluted fractions with 1.0-2.0M sodium chloride aqueous solution, repeatedly precipitating with alcohol, concentrating and freeze-drying to obtain a purified polysaccharide of Pterochirum quadrangularis;

[0021] S3, adding 80% hydrazine hydrate or hydrazine monohydrate to the purified polysaccharide of Pterocarpus cylindricus obtained in step S3, carrying out hydrazine hydrolysis at 60-100° C. in the presence or absence of hydrazine sulfate, then adding 1-6M nitrous acid aqueous solution with a pH of 1-4, carrying out deamination depolymerization at room temperature or in an ice bath, adding sodium borohydride for reduction after the reaction, repeating alcohol precipitation, centrifuging, dissolving the obtained precipitate, dialyzing or ultrafiltration to remove salt, and freeze-drying to obtain the depolymerization product of Pterocarpus cylindricus polysaccharide;

[0022] S4. The depolymerization product of the Pteris quadrangularis polysaccharide obtained in step S3 is separated by a gel column chromatography column, eluted with a 0.1 or 0.2 M sodium chloride aqueous solution at a flow rate of 5 to 15 mL / h, and 2 to 8 mL / tube is received, and the phenol-sulfuric acid method is used for detection and an elution curve is drawn, and the fractions of each elution peak are combined according to the elution curve. The molecular weight of the eluted fractions is analyzed by high performance liquid chromatography for the combined samples, and the octa- to deca-saccharide components are combined, desalted, and freeze-dried to obtain a Pteris quadrangularis octa- to deca-saccharide mixture.

[0023] Preferably, the gel column is one of the Bio-Gel P series chromatographic columns P-6, P-10 or the Sephadex G series gel columns G-50, G-25.

[0024] The present invention also discloses an application of the Pterocarpus quadrangularis octa-decasaccharide mixture in preparing a medicine for treating and / or preventing thrombotic cardiovascular diseases.

[0025] A pharmaceutical preparation comprises the eight to ten sugar mixture of Pteris quadrangularis.

[0026] A pharmaceutical preparation comprises the octa-decasaccharide mixture of Pteris quadrangularis as an active ingredient and a pharmaceutically acceptable carrier.

[0027] The pharmaceutical preparation includes the following dosage forms: freeze-dried powder for injection, aqueous solution for injection or oral solution.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] 1. The octa- to decasaccharide mixture of Pterospermum quadrangularis disclosed in the present invention is an oligosaccharide compound with a novel structural composition, containing 2-O, 3-O or 4-O monosulfate-substituted fucose side chains, with a relatively high content of 3-O-sulfate fucose side chains (>20%); containing novel structural disaccharide side chains, wherein the disaccharide side chains contain 2-O-sulfate galactose, 4-O-sulfate galactose, 2,3-di-O-sulfate galactose, 3,4-di-O-sulfate galactose, 4,6-di-O-sulfate acetylglucosamine, 4-O-sulfate acetylglucosamine and the like glycosyl groups, and are only connected to the O-3 position of 2,4-di-O-sulfate fucose; and do not contain 3,4-di-O-sulfate-substituted fucose side chains.

[0030] 2. The eight to ten sugar mixture of Pterocarpus quadrangularis disclosed in the present invention can significantly prolong the activated partial thromboplastin time (APTT) and prothrombin time (TT), and effectively inhibit the endogenous and common coagulation pathways. This pharmacological characteristic is different from other sea cucumber-derived oligosaccharides reported in the literature, indicating that it has unique advantages in anticoagulant effect.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The HPLC chromatogram of CqFG provided in Example 1;

[0033] Figure 2 The monosaccharide composition analysis chromatogram of CqFG provided in Example 1;

[0034] Figure 3 The CqFG provided in Example 1 1 H NMR detection spectrum;

[0035] Figure 4 The dCpFG elution curve detection diagram provided in Example 2;

[0036] Figure 5 The HPLC chromatogram of the eight to ten sugar mixture of Pterocarpus quadrangularis provided in Example 2;

[0037] Figure 6 The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H NMR detection spectrum;

[0038] Figure 7 The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H- 1 HCOSY detection spectrum;

[0039] Figure 8The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H- 1 HTOCSY detection map;

[0040] Fig. 9 The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H- 1 HROESY detection spectrum;

[0041] Fig.10 The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H- 13 CHSQC test spectrum;

[0042] Fig.11 The eight to ten sugar mixture of Pterospermum quadrangularis provided in Example 2 1 H- 13 CHMBC detection spectrum;

[0043] Fig.12 This is the result of the eight to ten sugar mixture of Pteris quadrangularis provided in Example 2 inhibiting the activity of endogenous factor X. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0045] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0046] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0047] Example 1 This example provides a Pterospermum quadrangularis polysaccharide, and the preparation method comprises the following steps:

[0048] S1. Crush 5.613 kg of Pterocaryum cylindraceum into a reactor, add 6.387 L of pure water, add 24 g of papain, and perform enzymolysis at 55°C for 4 h. Add the enzymolysis suspension into a sodium hydroxide solution with a final concentration of 0.5 M for alkaline hydrolysis and react for 2 h.

[0049] S2. After alkaline hydrolysis, add 6M hydrochloric acid solution, adjust pH to 2.8, let stand overnight, centrifuge at 4700rpm for 15min, take the supernatant, add 6M sodium hydroxide solution to neutralize, add anhydrous ethanol to a final concentration of 60%, precipitate with alcohol, centrifuge at 4500rpm for 15min, discard the supernatant, dissolve the precipitate in water, concentrate by low-pressure rotary evaporation under the conditions of vacuum degree of 10Pa, water bath temperature of 50℃, and rotation speed of 250rpm, pre-freeze at -40℃ for 120min, freeze-dry at 10℃ for 48h, add a certain volume of pure water to dissolve the freeze-dried product, and add Amberliter FPA98 (Cl after complete dissolution － ) anion resin, using gradient elution method to separate components. The gradient is as follows: pure water, 0.5M, 1.0M, 2.0M sodium chloride aqueous solution. The 2.0M sodium chloride elution fraction was collected, and after repeated alcohol precipitation to remove small molecules, it was pre-frozen at -40℃ for 120min, freeze-dried at 10℃ for 48h, and then eluted again with Amberliter FPA98 (Cl － ) anion exchange resin was used for separation, and the main components were collected. The cross-linked agarose gel (Sepharose CL-6B) filler was used for further separation and purification to obtain the main purified component, Pterocaryum square columnar purified polysaccharide, which was named CqFG.

[0050] The physicochemical properties of the purified polysaccharide CqFG from Pterochiura scutellaria provided in Example 1 were determined by the following experiments.

[0051] 1. Chemical composition: Determination of total sugar content: Phenol-sulfuric acid method was used, with glucose as the standard; Protein content was determined by Coomassie brilliant blue method, with bovine serum albumin as the standard; Uric acid content was determined by m-hydroxybiphenyl method, with galacturonic acid as the standard; Sulfate content was determined by barium chloride gelatin method, with K2SO4 as the standard; High performance liquid chromatography was used to determine the molecular weight of the purified polysaccharide CqFG from Pterocarya serrata. The standard was dextran of different molecular weights (9600, 21100, 47100 and 107000Da). The instrument was LC-2030C 3D HPLC, the chromatographic column was Shodex OHpak SB-804HQ (7μm, 8×300mm), the mobile phase was 0.1M sodium chloride aqueous solution, the flow rate was 0.5mL / min, the column temperature was 35℃, and the injection volume was 20μL. The results are shown in Table 1.

[0052] Table 1 Chemical composition of Pterocarpus quadrangularis polysaccharide CqFG

[0053]

[0054] Note: “-” means not detected.

[0055] As can be seen from Table 1, the molecular weight of CqFG measured by gel exclusion chromatography is 77.7KDa; the total sugar content of the purified polysaccharide component of Pterocaryum square column is 10.55±0.69%; the CqFG component does not contain protein; the uronic acid content of CqFG is 10.18±0.1%; the sulfuric acid-carboxylic acid molar ratio of CqFG measured by conductivity method is 2.06, and the sulfate ester content is calculated to be 33.7%; the sulfate ester content of CqFG measured by barium chloride-gelatin method is 33.9%, which is close to the result of conductivity measurement.

[0056] 2. The monosaccharide composition of the purified polysaccharide CqFG of Pterocarpus quadrangularis was determined by pre-column derivatization combined with high performance liquid chromatography. The monosaccharide standards were mannose (Man), acetylglucosamine (GlcNAc), acetylglucosamine (GalNAc), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal) and fucose (Fuc). HPLC detection conditions: Shimadzu LC-2030C 3D Plus high performance liquid chromatograph; detection column, Eclipse Plus C18 column (4.6mm×250mm, 5μm Venusil, USA); column temperature, 30℃; mobile phase, 0.1M PBS (pH6.7) and acetonitrile mixture (v:v=83:17); flow rate 1.0mL / min; detection wavelength, 245nm.

[0057] Polysaccharide NMR spectroscopy ( 1 H NMR analysis: 5 mg of polysaccharide sample was weighed, dissolved in 0.5 mL D2O, transferred to a NMR tube, and measured on an Advance III 600 MHz NMR spectrometer (Bruker, Germany). 1 H NMR spectrum. The results are as follows Figure 1-Figure 3 .

[0058] The extracted and purified polysaccharide CqFG from Pterochiura quadrangularis was detected as a single peak by high performance liquid chromatography (Shodex Ohpak SB-804HQ column). Figure 1 As shown, it indicates that the purity of the purified polysaccharide component of Pteris quadrangularis is relatively high after purification (purity>99.9%, area normalization method).

[0059] like Figure 2 As shown, the eight standard monosaccharide derivatives have good separation. Compared with the standards, it can be seen that CqFG is composed of glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal) and fucose (Fuc).

[0060] CqFG 1 H NMR spectrum Figure 3As shown. At 1.38ppm is the proton signal of the methyl group of Fuc, at 2.07ppm is the methyl proton signal of the acetyl group of GalNAc, and the ratio is about 1:0.6; 3.50-4.80ppm are the overlapping signals of the hydrogen of the sugar ring and the terminal hydrogen of GalNAc and GlcA; between 5.00-5.80ppm, there are the anomeric hydrogen signals of the Fuc and Gal side chains of various sulfate ester substitution types.

[0061] Example 2 This example provides a mixture of eight to ten sugars of Pteris quadrangularis

[0062] S1. Weigh 500 mg of CqFG prepared in Example 1, place in a 50 mL reaction bottle, add 155 mg of hydrazine sulfate and 15.5 mL of hydrazine hydrate in sequence, stir with a magnetic stirrer, and heat at 90 ° C for 30 h. After the reaction is completed, add 62.5 mL of anhydrous ethanol to a final concentration of 80% (v / v), centrifuge at 3900 rpm for 10 min, dissolve the resulting precipitate in 15.5 mL of water, precipitate with ethanol at a final concentration of 80% 4 times, centrifuge at 8000 rpm for 20 min, discard the supernatant, dissolve the precipitate in water, dialyze with a dialysis bag with a molecular weight cutoff of 1000 Da, concentrate the dialyzed retentate to 10 mL at 55 ° C, pre-freeze at -40 ° C for 120 min, and freeze-dry at -40 ° C for 48 h to obtain a deacetylated intermediate.

[0063] S2. Dissolve the deacetylated intermediate obtained in step S1 in 10 mL of water, add 20 mL of 5.5 M nitrous acid solution with a pH of 4, stir and react at room temperature for 10 min, add 1 M NaOH solution, adjust the pH to 7.0 to terminate the reaction, repeat the alcohol precipitation 4 times with 80% ethanol at a final concentration, centrifuge at 8000 rpm for 20 min, discard the supernatant, dissolve the precipitate in water, pre-freeze at -40°C for 120 min, and freeze-dry at -40°C for 48 h to obtain the FG depolymerization product dCqFG.

[0064] S3. Dissolve the FG depolymerization product dCqFG obtained in step S2 in 5 mL of water, adjust the solution pH to 9.0 with 1 M NaOH, add 10 mL of 0.25 M NaBH4 solution, heat and stir at 50°C for 2 h, cool to room temperature after the reaction is completed, adjust the pH to 3 with 0.5 M H2SO4 to remove excess NaBH4, and then adjust the pH to 7 with 0.5 M NaOH. Dialyze the solution with a dialysis bag with a molecular weight cutoff of 500-1000 Da, collect and concentrate the retentate, and freeze-dry at -40°C to obtain dCqFG.

[0065] S4, weigh 215 mg of dCqFG obtained in step S3, load it onto a balanced Bio-Gel P-10 gel column, collect it into an automatic fraction collector, the elution flow rate is 10 mL / h, 4 mL / tube is received, the elution curve is detected by phenol sulfuric acid method, and the flow fractions of each elution peak are combined according to the elution curve. The molecular weight of the eluted flow fractions is analyzed by high performance liquid chromatography for the combined sample, and the eight to ten sugar components are combined. The purified oligosaccharides are desalted with a Sephadex G-10 gel column, pre-frozen at -40°C for 120 min, and freeze-dried at -40°C for 24 h.

[0066] The physicochemical properties of the eight- to ten-saccharide mixture of Pterocarpus quadrangularis provided in Example 2 were measured by the following tests.

[0067] 1. Analyze using HPLC (SuperdexTM 30Increase 10 / 300GL column). Figure 4-Figure 5 .

[0068] Depend on Figure 4 It can be seen that the main peak of dCpFG obtained by depolymerization is octa-nonasaccharide.

[0069] like Figure 5 It can be seen that the octasaccharides to decasaccharides of Pteris quadrangularis are mainly composed of nonasaccharides, and also contain smaller amounts of octasaccharides and decasaccharides.

[0070] 2. Analysis of the structures of the eight to ten sugars of Pterochiola quadrangularis

[0071] 1 H- 1 The spin coupling system revealed by H COSY combines 1 HNMR, 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 1 HROESY, 1 H- 13 C HSQC, 1 H- 13 C HMBC, the results are as follows Figure 6-Figure 11 .

[0072] 1 H- 1 The spin coupling system revealed by H COSY combines 1 HNMR, 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 1 HROESY,1 H- 13 C HSQC, 1 H- 13 C HMBC, all C / H signals and related signals that can be attributed to the octasaccharides to decasaccharides of Pterocarpus serrata, and the results are shown in Table 2.

[0073] Table 2 Chemical shift assignment results of the NMR spectra of octa-deca-saccharides of Pterocarpus cylindricus

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Note: a in italics indicates sulfate ester linkage site; b in bold indicates glycosidic linkage site.

[0081] Depend on Figure 6-11 As shown in Table 2, the main chemical structures of the octa- to deca-saccharides of Pterocarpus quadrangularis are L-FucS-α1,3-D-GlcA-β1,3-{D-GalNAc 4S6S -β1,4-[L-FucS-α1,3]-D-GlcA-β1,3}2-D-anTal 4S6S -ol, where FucS represents fucose substituted with different sulfate groups. It has a main chain similar to CS-E in other sea cucumber species, while the FucS side chain is mainly connected to the C-3 position of GlcA in the main chain through an α1,3 glycosidic bond in the form of a monosaccharide side chain. The types of FucS monosaccharide side chains are richer than those of other sea cucumber species, mainly including Fuc 2S4S 、Fuc 3S 、Fuc 2S 、Fuc 4S The monosaccharide side chains of the following types account for 35.8%, 20.7%, 4.5% and 10.1% respectively. 1 H- 1 HROESY and 1 H- 13The relevant signals in the C HMBC spectrum show that the disaccharide side chain with novel structure is connected to the C-3 position of GlcA in the main chain through an α1,3 glycosidic bond. The disaccharide side chain contains 2-O-sulfated galactose, 4-O-sulfated galactose, 2,3-di-O-sulfated galactose, 3,4-di-O-sulfated galactose, 4,6-di-O-sulfated acetylglucosamine, 4-O-sulfated acetylglucosamine and other sugar groups, and is only connected to the O-3 position of 2,4-di-O-sulfated fucose. The fucose monosaccharide side chain without 3,4-di-O-sulfate substitution is obviously different from the polysaccharides from other species of sea cucumbers.

[0082] 3. Determination of the anticoagulant activity of eight to ten saccharides from Pteris quadrangularis

[0083] The effects of eight to ten sugars of Pterocarpus cylindricus on APTT, PT, and TT of standard human plasma were tested according to the instructions of the kit and literature (PNAS, 2015, 112 (27): 8284-8289). Specifically, 5 μL of eight to ten sugar samples of Pterocarpus cylindricus with a pipette were accurately taken and added to the test tube preheated at 37°C, and then 45 μL of coagulation quality control plasma was added, incubated at 37°C for 2 minutes, and 50 μL of APTT, PT, or TT reagent was added to the test tube after gentle shaking. When measuring APTT, after adding APTT reagent, incubate at 37°C for 3 minutes, and then add 50 μL of 0.02 mol / L calcium chloride solution preheated at 37°C, and time and record the coagulation time; when measuring PT or TT, time and record the coagulation time immediately after adding PT or TT reagent. Low molecular weight heparin (LMWH) solution and Tris-HCl buffer were used as positive control and blank control respectively according to the above operation. The results are shown in Table 3.

[0084] Table 3. Effects of eight to ten sugars of Pterocarpus quadrangularis on APTT, TT and PT

[0085] Sample APTT (μg / ml) TT (μg / ml) PT (μg / ml) LMWH 3.28±0.12 1.09±0.10 >128 QUR 2.24±0.07 1.81±0.04 40.73±0.83 dC 6.52±0.19 92.80±3.09 >128 dp8-10 8.95±0.12 99.35±1.94 >128

[0086] As shown in Table 3, LMWH, CqFG, dCqFG and eight to ten saccharides of Pterochironium cylindraceum all have strong anticoagulant activity. Among them, the concentration required for eight to ten saccharides of Pterochironium cylindraceum to double the APTT time of human standard plasma is 8.95 μg / mL, which is close to LMWH. Eight to ten saccharides of Pterochironium cylindraceum can significantly prolong the TT time, and the drug concentration required to double the TT time of human standard plasma is 99.35 μg / mL, indicating that it can significantly inhibit the common coagulation pathway, which is significantly different from the pharmacological action characteristics of sea cucumber polysaccharides studied in the past. Eight to ten saccharides of Pterochironium cylindraceum have no effect on the PT time, indicating that the sample has no effect on the exogenous coagulation pathway.

[0087] Detection of the inhibition of endogenous factor X enzyme activity by eight to ten saccharides from Pteris quadrangularis:

[0088] The activity of the eight to ten sugars of Pterospermum quadrangularis to inhibit endogenous factor X enzymes was determined by reference (PNAS, 2015, 112 (27): 8284-8289) and using the FVIII:C kit. Take a 96-well plate, add 30μL of the eight to ten sugar sample solutions of Pterospermum quadrangularis with a series of gradient concentrations, 30μL of FVIII solution (2IU / mL), and 30μL of R2 solution in sequence, and incubate at 37.0℃ for 2min; take 30μL of R1 solution and add it to the reaction solution, and incubate at 37.0℃ for 1min; take 30μL of R3 solution preheated at 37.0℃ and add it to the reaction solution, shake and mix, and read the absorbance at 405nm continuously. LMWH and Tris-HCl buffer were used as positive control and blank control, respectively. The results are as follows Fig.12 .

[0089] Depend on Fig.12 It can be seen that LMWH, CqFG, dCqFG and Pterocarpus quadrangularis octa-deca-saccharides all have strong inhibitory activity against factor X. Among them, the IC50 value of Pterocarpus quadrangularis octa-deca-saccharides for inhibiting endogenous factor X is 193.4 ng / mL, which is very active.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A mixture of eight to ten sugars of Pteris quadrangularis, characterized in that The monosaccharide composition of the octa- to deca-saccharide mixture of Pterospermum quadrangularis includes N-acetylgalactosamine, fucose, glucuronic acid or its sugar alcohol, galactose, N-acetylglucosamine, dehydrated talose and sulfate group; the structure of the octa- to deca-saccharide mixture of Pterospermum quadrangularis is as follows: In the formula, in the sugar groups a and b, R1=R3=-SO3Na, R2=H; c, f, j sugar groups in which R1=R3=-SO3Na, R=H; d In the sugar group, R1=R2=R=-SO3Na; e. i In the sugar group, R1=R2=H, R3=-SO3Na; In g and h sugar groups, R2 = -SO3Na, R1 = R3 = H; In the k sugar group, R1=-SO3Na, R2=R3=H.

2. The eight-to-ten sugar mixture of Pterocanthus quadrangularis according to claim 1, characterized in that: a and b sugar groups account for 30.8%-40.8% of the total side chain molar percentage, l, m, n, o, p, q and c, f, j sugar groups constitute disaccharide side chains account for 20%-30% of the total side chain molar percentage, d sugar groups account for 7.4%-13.4% of the total side chain molar percentage, e and i sugar groups account for 7.1%-13.1% of the total side chain content, g and h sugar groups account for 15.7%-25.7% of the total side chain content, and k sugar groups account for 3.5%-5.5% of the total side chain content.

3. The eight to ten sugar mixture of Pterospermum quadrangularis according to claim 1 or 2, characterized in that: The a and b sugar groups are 2,4-di-O-sulfated fucose side chains, l, m, n, o, p, q include one or more of sulfated galactose, sulfated acetylglucosamine or sulfated acetylglucosamine, c, f, j sugar groups are 2,4-di-O-sulfated fucose, d sugar groups are 2,3,4-tri-O-sulfated fucose side chains, e and i sugar groups are 4-O-sulfated fucose side chains, g and h sugar groups are 3-O-sulfated fucose side chains, and k sugar groups are 2-O-sulfated fucose side chains.

4. The eight-to-ten sugar mixture of Pteris quadrangularis according to claim 1, characterized in that: The polysaccharide is obtained by depolymerization and separation of Pterochiura quadrangularis polysaccharide, and has a weight average molecular weight of 67.7-87.7 kDa as calibrated with dextran, a uronic acid content of 10%-30%, and a sulfate content of 10%-30%.

5. The eight-to-ten sugar mixture of Pteris quadrangularis according to claim 1, characterized in that: The octa-deca-saccharide mixture of Pterocarpus quadrangularis has a molecular weight of 2.0-3.5 kDa and a sulfate ester group content of 10%-45%.

6. A method for preparing the eight-to-ten sugar mixture of Pterospermum quadrangularis as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: S1. adding the homogenate or dry powder of Pterocarpus quadrangularis to papain for enzymatic hydrolysis, and adding the suspension after enzymatic hydrolysis to a sodium hydroxide solution with a final concentration of 0.1-1.0 M for alkaline hydrolysis; S2, removing proteins at the isoelectric point, salting out and ethanol precipitation to obtain a crude polysaccharide extract of Pterochirum quadrangularis, purifying it by anion exchange column chromatography, collecting the eluted fractions with 1.0-2.0M sodium chloride aqueous solution, repeatedly precipitating with alcohol, concentrating and freeze-drying to obtain a purified polysaccharide of Pterochirum quadrangularis; S3, adding 80% hydrazine hydrate or hydrazine monohydrate to the purified polysaccharide of Pterocarpus cylindricus obtained in step S3, carrying out hydrazine hydrolysis at 60-100° C. in the presence or absence of hydrazine sulfate, then adding 1-6M nitrous acid aqueous solution with a pH of 1-4, carrying out deamination depolymerization at room temperature or in an ice bath, adding sodium borohydride for reduction after the reaction, repeating alcohol precipitation, centrifuging, dissolving the obtained precipitate, dialyzing or ultrafiltration to remove salt, and freeze-drying to obtain the depolymerization product of Pterocarpus cylindricus polysaccharide; S4. The depolymerization product of the Pteris quadrangularis polysaccharide obtained in step S3 is separated by a gel column chromatography column, eluted with a 0.1 or 0.2 M sodium chloride aqueous solution, with an elution rate of 5-15 mL / h, and 2-8 mL / tube is received, and the phenol-sulfuric acid method is used for detection and an elution curve is drawn, and the fractions of each elution peak are combined according to the elution curve, and the molecular weight of the eluted fractions is analyzed by high performance liquid chromatography for the combined samples, and the octa- to deca-saccharide components are combined, desalted, and freeze-dried to obtain a Pteris quadrangularis octa- to deca-saccharide mixture.

7. Use of the octasaccharide to decasaccharide mixture of Pteris quadrangularis as claimed in any one of claims 1 to 5 in the preparation of a medicament for treating and / or preventing thrombotic cardiovascular diseases.

8. A pharmaceutical preparation comprising one or more octa- to decasaccharide mixtures of Pteris quadrangularis as claimed in any one of claims 1 to 5.

9. A pharmaceutical preparation according to claim 8, characterized in that The invention comprises the mixture of eight to ten sugars of Pteris quadrangularis as active ingredients and a pharmaceutically acceptable carrier.

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