A sulfated arabinogalactan derived from *Streptococcus linearis*, its preparation method and application

Abstract

This invention discloses an arabinogalactan sulfate derived from *Streptococcus filamentosa*, its preparation method, and its applications, belonging to the fields of natural product chemistry and marine biological resource development technology. The technical solution includes an arabinogalactan sulfate derived from *Streptococcus filamentosa*, with a monosaccharide composition comprising arabinose, galactose, and rhamnose, the molar percentage of arabinose being not less than 70%; the main chain backbone is modified with natural sulfate groups, with the sulfate groups replacing at least one hydroxyl site of the arabinose residues. This invention, when applied to anticoagulation, exhibits good safety and a significant anticoagulant effect.

Description

Technical Field

[0001] This invention belongs to the field of natural medicinal chemistry and marine biological resource development technology, and particularly relates to a sulfated arabinogalactan derived from linear sclerosis algae, its preparation method and application. Background Technology

[0002] Thrombosis is closely related to a variety of diseases, and its prevention and treatment mainly rely on traditional anticoagulants such as heparin and warfarin. However, existing anticoagulants generally have problems such as a narrow therapeutic window, high bleeding risk, or thrombocytopenia, which limit their long-term or preventative use. Natural sulfated polysaccharides, due to their structural diversity and high biocompatibility, are gradually becoming an important focus in the field of anticoagulant drug research.

[0003] Sulfated polysaccharides are a class of natural macromolecules with unique structural characteristics and biological activities, attracting widespread attention in areas such as anticoagulation, immune regulation, and antiviral activity. Currently, the most studied seaweed sulfated polysaccharides are mainly derived from brown and red algae. Brown algal sulfated polysaccharides are represented by alginate and sulfated fucoidan, while red algal sulfated polysaccharides are mainly sulfated galactan, with relatively fixed monosaccharide compositions and glycan backbone types. In contrast, green algae contain a class of sulfated arabinogalactan with arabinose as the main component unit, primarily distributed in algae such as *Streptococcus filamentosa*, *Cladosporium*, and *Pinus*. Their glycosidic bond linkages, branching characteristics, and sulfate group substitution positions differ from those of brown and red algal sulfated polysaccharides, and they generally exhibit high sulfation and high branching. *Streptococcus filamentosa*, a widely distributed green algal resource along my country's coast, possesses advantages such as large biomass, stable source, and high safety.

[0004] However, existing research lacks systematic reports on arabinogalactan sulfate derived from *Streptococcus linearis*, with arabinose as the main backbone and natural sulfate group modification characteristics. Its preparation methods and applications in the field of anticoagulation also require further development. Therefore, developing a novel arabinogalactan sulfate with a clearly defined source, novel structure, arabinose as the main backbone, and specific natural sulfation characteristics is of significant scientific and practical value for enriching the structural types of green algal sulfate polysaccharides and expanding the sources of novel anticoagulant active substances. Summary of the Invention

[0005] To address at least one shortcoming of the existing technology, this invention proposes a sulfated arabinogalactan derived from *Streptococcus linearis*, which has good safety and significant anticoagulant effects, as well as its preparation method and application.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: The present invention provides a sulfated arabinogalactan derived from *Streptococcus linearis*, the monosaccharide composition of which includes arabinose, galactose and rhamnose, with the molar percentage of arabinose not less than 70%; the main chain backbone is modified with natural sulfate groups, the sulfate groups replacing at least one hydroxyl site of the arabinose residues.

[0007] In some embodiments, the sulfate groups of sulfated arabinogalactan are mainly distributed at the C-2, C-4 and / or C-6 positions of arabinose.

[0008] In some embodiments, the sulfate groups in arabinogalactan sulfate are located at →4)-β-L-Ara p -(1→C-2 position, and →3)-β-L-Ara p -(1→C-4 bits.)

[0009] In some embodiments, the mass content of sulfate groups in arabinogalactan sulfate is 15-30%.

[0010] In some embodiments, the number average molecular weight of arabinogalactan sulfate is 30-40 kDa; arabinogalactan sulfate is a polysaccharide with a branched structure.

[0011] In some embodiments, the structure of arabinogalactan sulfate is as follows:

[0012] Where R = H or HSO3 R1=H,HSO3 Or a sidechain Gal p -(1→,Xyl p -(1→).

[0013] In another aspect, the present invention provides a method for preparing arabinogalactan sulfate according to any of the above technical solutions, which uses natural green algae polysaccharides as raw materials and prepares arabinogalactan sulfate through extraction, separation and purification steps.

[0014] In some embodiments, including: S1: Add ethanol to the linear fibrous algae powder, heat to defatting, centrifuge to collect the linear fibrous algae precipitate, and dry the precipitate; S2: Add distilled water to the dried precipitate, stir continuously at 100°C, and collect the supernatant by centrifugation; S3: The supernatant was concentrated using a rotary evaporator, and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; S4: Add ethanol to the concentrate for alcohol precipitation, let stand overnight, and then centrifuge to collect the precipitate; S5: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S6: The crude polysaccharide of *Streptococcus linearis* was separated and purified using a Q-Sepharose Fast Flow anion exchange column to obtain purified *Streptococcus linearis* polysaccharide. S7: The polysaccharide from *Streptococcus linearis* was further purified using a Sephacryl S-400 gel column to obtain polysaccharides with uniform molecular weight.

[0015] The present invention also provides a product for anticoagulation, comprising arabinogalactan sulfate from any of the above-described technical solutions.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a sulfated arabinogalactan derived from *Streptococcus linearis*, which differs from sulfated polysaccharides derived from brown and red algae in its monosaccharide composition, sugar chain backbone, and sulfation characteristics. It is also different from non-sulfated arabinogalactans derived from terrestrial plants, belonging to a novel type of natural sulfated polysaccharide. The sulfate groups in this sulfated arabinogalactan are naturally introduced, avoiding the structural inhomogeneity and safety risks that may arise from artificial sulfation modification. This sulfated arabinogalactan exhibits clear anticoagulant activity, exerting its effects by influencing endogenous and / or common coagulation pathways, and has the potential for further development as a novel anticoagulant active substance. This invention provides a method for preparing arabinogalactan sulfate derived from linear sclerosis algae. The raw materials used are stable and safe, the process is mild and highly controllable, suitable for large-scale preparation, and has good prospects for industrial application. Attached Figure Description

[0017] Figure 1 The Q Sepharose Fast Flow diagram shows the separation of arabinogalactan sulfate CHS2. Figure 2 Diagram showing the monosaccharide composition of arabinogalactan sulfate standard; Figure 3 This is a diagram showing the composition of the monosaccharide arabinogalactan CHS2. Figure 4 Infrared spectrum of arabinogalactan sulfate CHS2; Figure 5 For arabinogalactan CHS2 1 H spectrum; Figure 6 For arabinogalactan CHS2 13 C spectrum; Figure 7 For arabinogalactan CHS2 1 H- 1 H COSY spectrum; Figure 8 For arabinogalactan CHS2 1 H- 13 C HSQC spectrum; Figure 9 For arabinogalactan CHS2 1 H- 1 H NOESY spectrum; Figure 10 This is a schematic diagram of the structure of arabinogalactan CHS2. Figure 11 Image showing the in vivo APTT activity of arabinogalactan sulfate CHS2; Figure 12 Figure 1 shows the in vivo TT activity of arabinogalactan sulfate CHS2. Figure 13 Figure showing the in vitro thrombolytic activity of arabinogalactan CHS2; Figure 14 The effect of CHS2 on AT-III-mediated Xa factor inhibition; Figure 15 The effect of CHS2 on AT-III-mediated thrombin inhibition; Figure 16 The effect of CHS2 on HC-II-mediated thrombin inhibition. Detailed Implementation

[0018] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0019] Existing research indicates that arabinogalactan sulfate from different green algae sources exhibits different characteristics in its fine structure. Cladosporium (… Cladophora falklandica The arabinogalactan, with a molecular weight of 19 kDa, was derived from [source name missing]. The molar ratio of arabinose (Ara), xylose (Xyl), and galactose (Gal) was 1.0:0.2:0.4. Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis of some acid hydrolysis products showed that the polysaccharide backbone consisted of (1→4)-β-L-Ara [missing information]. p The composition, with the sulfate group located at (1→4)-β-L-Ara p At positions C-3 and C-2, the branching is (1→2)-β-D-Xyl p (1→5)-β-D-Gal f and (1→6)-β-D-Gal f Composition. Streptomyces ( Chaetomorpha anteninnaThe sulfated arabinogalactan contains 57.7% Ara, 38.5% Gal, and 3.8% rhamnose (Rha), with a sulfate group content of 11.9%, and is composed of (1→4)-β-L-Ara. p (1→3)-D-Gal p (1→4)-D-Gal p and (1→4)-α-L-Rha p The composition, with the sulfate group located at (1→4)-β-L-Ara p The C-2 position of the non-reducing terminal arabinose, the C-4 position of the non-reducing terminal galactose, and the C-3 position of the non-reducing terminal arabinose are all involved. The above research indicates that the structural characteristics of arabinogalactan sulfate from green algae are closely related to the algal species from which it is derived, and cannot be easily deduced from existing structural types.

[0020] The present invention provides a sulfated arabinogalactan derived from *Streptococcus linearis*, the monosaccharide composition of which includes arabinose, galactose and rhamnose, with the molar percentage of arabinose not less than 70%; the main chain backbone is modified with natural sulfate groups, the sulfate groups replacing at least one hydroxyl site of the arabinose residues.

[0021] The sulfated arabinogalactan of this invention is derived from the green algae *Streptococcus linearis*. Its monosaccharide composition is predominantly neutral sugars, such as arabinose, galactose, and rhamnose, and it contains no amino sugars or uronic acid units. It differs from sulfated polysaccharides derived from brown and red algae, as well as from non-sulfated arabinogalactans derived from terrestrial plants, in its monosaccharide composition, sugar chain backbone, and sulfation characteristics. It is a novel natural sulfated polysaccharide. The sulfate groups in this sulfated arabinogalactan are naturally introduced, avoiding the structural inhomogeneity and safety risks that may arise from artificial sulfation modification. This sulfated arabinogalactan exhibits clear anticoagulant activity, exerting its effects by influencing endogenous and / or common coagulation pathways, and has the potential for further development as a novel anticoagulant substance.

[0022] In some embodiments, the percentages of arabinose, galactose, and xylose in arabinogalactan sulfate are 83.62%, 13.77%, and 2.61%, respectively.

[0023] The above-mentioned sulfated arabinogalactan is in the form of β-(1→4)-L-Ara p and β-(1→3)-L-Ara p It forms the backbone. Furthermore, arabinose is composed of →2,4)-β-L-Ara p -(1→,→3,4)-β-L-Ara p -(1→and→4)-β-L-Ara p -(1→ is composed of; galactose is composed of→4)-β-D-Gal p -(1→and→6)-β-D-Galp -(1→ is composed of; xylose is mainly located at the terminal group of this polysaccharide, i.e., Xyl p -(1→).

[0024] In some embodiments, the sulfate groups of sulfated arabinogalactan are primarily located at the C-2, C-4, and / or C-6 positions of arabinose. In some embodiments, the sulfate groups in sulfated arabinogalactan are located at →4)-β-L-Ara p -(1→C-2 position, and →3)-β-L-Ara p -(1→ at the C-4 position. The sulfate groups of the above-mentioned arabinogalactan sulfate are mainly distributed at the C-2, C-4 and / or C-6 positions of arabinose, and there is no 3-O-sulfation structure. Furthermore, the sulfate groups in arabinogalactan sulfate are located at →4)-β-L-Ara p -(1→ at C-2 and C-3 positions, the side chain is composed of galactose and xylose.

[0025] In some embodiments, the sulfate group content in arabinogalactan is 15-30% by mass. Further, the sulfate group content is 28.27%.

[0026] In some embodiments, the number-average molecular weight of arabinogalactan sulfate is 30-40 kDa; arabinogalactan sulfate is a polysaccharide with a branched structure. Further, the molecular weight of arabinogalactan sulfate is 38.0 kDa, the molar percentage of arabinose is 83.62%, and it contains natural sulfate groups modified in the sugar chain.

[0027] In some embodiments, the structure of arabinogalactan sulfate is as follows:

[0028] Where R = H or HSO3 R1=H,HSO3 Or a sidechain Gal p -(1→,Xyl p -(1→).

[0029] In another aspect, this invention provides a method for preparing arabinogalactan sulfate according to any of the above-mentioned technical solutions, using natural green algae polysaccharides as raw materials, and preparing arabinogalactan sulfate through extraction, separation, and purification steps. Further, arabinogalactan sulfate is prepared from linear scabies algal powder through hot water extraction, alcohol precipitation, and column chromatography.

[0030] In some embodiments, including: S1: Add ethanol to the linear fibrous algae powder, heat to defatting, centrifuge to collect the linear fibrous algae precipitate, and dry the precipitate; S2: Add distilled water to the dried precipitate, stir continuously at 100°C, and collect the supernatant by centrifugation; S3: The supernatant was concentrated using a rotary evaporator, and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; S4: Add ethanol to the concentrate for alcohol precipitation, let stand overnight, and then centrifuge to collect the precipitate; S5: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S6: The crude polysaccharide of *Streptococcus linearis* was separated and purified using a Q-Sepharose Fast Flow anion exchange column to obtain purified *Streptococcus linearis* polysaccharide. S7: The polysaccharide from *Streptococcus linearis* was further purified using a Sephacryl S-400 gel column to obtain polysaccharides with uniform molecular weight.

[0031] The above method for preparing arabinogalactan sulfate does not include the artificial sulfation reaction step, and specifically includes the following steps: (1) Add 95% ethanol to the linear fibrous algae powder at a ratio of 1:5 (W / V), heat at 80°C for 4 hours to degrease, collect the linear fibrous algae precipitate by centrifugation, and dry the precipitate at 55°C. (2) Add distilled water to the dried linear briar algae precipitate in step (1) at a ratio of 1:10 (W / V), stir continuously at 100°C for 4 hours, and collect the supernatant by centrifugation. (3) The supernatant in step (2) is concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; (4) Add 95% ethanol to the concentrate from step (3) at a ratio of 1:4 (V / V) for alcohol precipitation. After standing overnight at 4°C, collect the precipitate by centrifugation. (5) The precipitate in step (4) was decolorized and dehydrated using acetone and a large amount of ethanol, and dried at 55°C to obtain crude linear fibrous polysaccharide. (6) The polysaccharide of *Streptococcus linearis* was separated and purified using a Q-Sepharose Fast Flow anion exchange column to obtain the purified polysaccharide of *Streptococcus linearis*. (7) The polysaccharide of *Streptococcus linearis* was further purified using a Sephacryl S-400 gel column to obtain a polysaccharide with uniform molecular weight.

[0032] Furthermore, in step (6), the length of the Q-Sepharose Fast Flow anion exchange column is 40 cm and the diameter is 3.5 cm. The elution conditions are 0 mol / L, 0.5 mol / L, 1 mol / L and 2 mol / L NaCl solutions.

[0033] Furthermore, in step (7), the length of the Sephacryl S-400 gel column is 100 cm and the diameter is 2.5 cm. The elution conditions are 0.2 mol / L NH4HCO3 solution and a flow rate of 0.3 mL / min.

[0034] This invention also provides a product for anticoagulation, comprising arabinogalactan sulfate from any of the above-described technical solutions. This product can be a pharmaceutical, health food, or functional food.

[0035] The present invention also provides the application of arabinogalactan sulfate from any of the above-mentioned technical solutions in the preparation of anticoagulant drugs.

[0036] Furthermore, the effective dose of arabinogalactan sulfate to exert its anticoagulant activity is 5-30 mg / kg; the effective dose of arabinogalactan sulfate to exert its thrombolytic activity is 5-15 mg / kg.

[0037] The aforementioned arabinogalactan sulfate can effectively prolong activated partial thromboplastin time (APTT) and thrombin time (TT) and reduce fibrinogen (FIB) levels within a certain concentration range, exerting anticoagulant activity by acting on intrinsic and / or common coagulation pathways. Furthermore, the aforementioned arabinogalactan sulfate can inhibit coagulation factors IIa and Xa to a certain extent, dependent on antithrombin III (AT-III) and heparin cofactor II (HC-II). Furthermore, the aforementioned arabinogalactan sulfate can increase the dissolution rate of blood clots, exhibiting significant thrombolytic activity.

[0038] It should be noted that the sulfated arabinogalactan provided by the present invention regulates the relevant factors in the coagulation cascade reaction through the negative charge distribution formed by the sulfate groups at multiple sites. It belongs to a non-heparin anticoagulation mechanism and has the characteristics of mild effect and high safety.

[0039] To more clearly and in detail introduce the arabinogalactan sulfate derived from *Streptococcus linearis*, its preparation method, and its applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0040] Example 1 Extraction, separation and purification of arabinogalactan sulfate from Streptomyces linearis The extraction, separation, and purification of arabinogalactan sulfate in this invention includes the following steps: (1) Add 95% ethanol to 500g of linear brittle algae powder at a ratio of 1:5 (W / V), heat at 80°C for 4h for degreasing, collect the linear brittle algae precipitate by centrifugation, and dry the precipitate at 55°C. (2) Add distilled water to the linear sclerosis precipitate from step (1) at a ratio of 1:10 (W / V), stir continuously for 4 hours at room temperature, and collect the supernatant by centrifugation. (3) The supernatant in step (2) is concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag; (4) Add 95% ethanol to the desalted concentrate from step (3) at a ratio of 1:4 (V / V) for alcohol precipitation. After standing overnight at 4°C, collect the precipitate by centrifugation. (5) The precipitate in step (4) was decolorized and dehydrated using acetone and anhydrous ethanol, and dried at 55°C to obtain crude linear sclerosis polysaccharide. (6) The polysaccharide of linear sclerosis was separated and purified using a Q-Sepharose Fast Flow anion exchange column and a Sephacryl S-400 gel column to obtain arabinogalactan CHS2.

[0041] Example 2 Basic structural features of arabinogalactan CHS2 1. Ion exchange chromatography for the separation and purification of polysaccharides from Streptomyces linearis. The linear spirulina polysaccharide prepared in Example 1 was fractionated and eluted using a Q-Sepharose Fast Flow strong anion exchange column with NaCl at concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 4.0 mol / L. The eluent fractions were collected using an automated collector, and the sugar content was determined using the sulfuric acid-phenol method to plot the elution volume-absorbance curve. Based on the elution curves, a suitable NaCl elution concentration was determined, and large-scale preparations were performed. The eluents were collected and combined, concentrated, dialyzed through a 3.5 kDa dialysis bag, and lyophilized to obtain polysaccharides with uniform charge density.

[0042] like Figure 1 As shown, the fraction eluted with 1.0 mol / L NaCl was collected, concentrated, lyophilized, and named CHS2.

[0043] 2. Determination of monosaccharide composition of CHS2 by high performance liquid chromatography (1) Accurately weigh 2 mg of CHS2 prepared in Example 1, add 400 μL of 2 mol / L trifluoroacetic acid and degrade it under sealed conditions at 105 °C for 6 h. After degradation, remove excess trifluoroacetic acid.

[0044] (2) Dissolve the CHS2 degradation products and monosaccharide standards in 100 μL of distilled water, add 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L PMP methanol solution, and incubate in a water bath at 70 °C for 60 min. After cooling, add HCl solution for neutralization reaction, extract three times with dichloromethane, and filter through a 0.22 μm filter membrane for later use.

[0045] (3) The monosaccharide composition of CHS2 was determined by HPLC. The chromatographic conditions were as follows: Eclipse XDB-C18 (5μm, 4.6μm×25.0cm) column; mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v); injection volume: 10μL; column temperature: 35℃; UV detector (254nm); flow rate: 1.0mL / min.

[0046] The results are as follows Figure 2 , 3 As shown, the monosaccharide composition of CHS2 consists of arabinose, galactose, and a small amount of xylose, with percentages of 83.62%, 13.77%, and 2.61%, respectively, indicating that CHS2 is an arabinogalactan containing some galactose and xylose.

[0047] 3. The infrared spectrum of CHS2 was determined using the KBr pellet method. The polysaccharide sample CHS2 was dried under reduced pressure with P2O5 at 50℃ for 48 hours. Approximately 1 mg of the sample was ground with an appropriate amount of dried potassium bromide powder, pressed into a transparent thin film, and then subjected to infrared spectroscopy. The infrared spectrometer parameters were as follows: Nicolet Nexus 470 infrared spectrometer; background scans: 32; scan range: 400-4000 cm⁻¹ -1 Resolution: 4.0cm -1 Detector: DTGS.

[0048] The results are as follows Figure 4 As shown, in the FT-IR spectrum of CHS2, at 3420 cm⁻¹ -1 The characteristic absorption band at 1617 cm⁻¹ is attributed to the stretching vibration of -OH. -1 The absorption signal at 1332 cm⁻¹ is attributed to the bending vibration of the -OH group. -1 The peak at 1000 cm⁻¹ corresponds to the absorption peak of the CH bond angle change vibration. -1 The peak at 1252 cm⁻¹ originates from the stretching vibration of CO. Several peaks corresponding to sulfate groups were also observed. -1 865cm -1 and 826cm -1 The stretching vibrations at this point are due to S=O. Infrared spectroscopy analysis indicates that CHS2 is a highly sulfated arabinogalactan.

[0049] 4. Analysis of the methylation reaction and the connection mode of CHS2 by gas chromatography-mass spectrometry The modified Hakomori method was used to methylate CHS2. The methylated product was completely acid-hydrolyzed, reduced, and acetylated before being analyzed by GC-MS. The linkage mode of the sugar groups could be determined by comparing the MS spectrum with that of the standard.

[0050] Based on the peak areas of the gas phase spectrum, the molar ratios between different linkage methods were calculated. The comparative analysis results of the methylation of sulfated polysaccharide CHS2 and its desulfurized sample dsCHS2 are shown in Table 1: Ara in CHS2 p -(1→、→4)-Ara p -(1→、→3,4)-Ara p -(1→、→2,4)-Ara p -(1→exists in a molar ratio of 1.00:4.97:5.77:3.51,→4)-Gal p -(1→、→6)-Gal p -(1→ exists in a molar ratio of 1.00:1.28, and also contains a small amount of Xyl p -(1→。Compared to CHS2, the desulfurization component contains →4)-Ara p -(1→content increased from 25.60% to 49.17%,→3,4)-Ara p -(1→、→2,4)-Ara p The content of -(1→ decreases, therefore it is inferred that the sulfate group is located at →4)-Ara p -(1→C-3 and C-2 bits, where 17.07% →4)-Ara p -(1→) is replaced by a sulfate group at the C-3 position.

[0051] Table 1. Methylation analysis results of polysaccharides CHS2 and dsCHS2

[0052] 5. Polysaccharide nuclear magnetic resonance spectroscopy analysis Polysaccharide samples were dissolved in D2O, lyophilized, centrifuged, and placed in NMR tubes. One-dimensional NMR spectroscopy was performed using an Agilent DDZ 500 MHz NMR spectrometer. 1 H-NMR, 13 C-NMR (nuclear magnetic resonance) spectroscopy. Deuterated acetone was added as an internal standard. 1 The chemical shift of H was determined to be 2.225 ppm. 13 The chemical shift of C was determined to be 31.07 ppm, and nuclear magnetic resonance spectroscopy analysis was performed.

[0053] CHS2 1H NMR such as Figure 5 As shown, the six H⁻¹ signals at 5.40, 5.25, 5.18, 5.14, 4.61, and 4.57 ppm are labeled A, B, C, D, E, and F, respectively; the signals at 3.58–4.73 ppm are attributed to the hydrogen signals of H₂–H₆ on the sugar ring. (CHS₂) 13 CNMR such as Figure 6 As shown, in the C spectrum, 98.5 ppm is assigned to the C-1 signal of β-configured arabinopyranose, 104.2 ppm is assigned to the C-1 signal of β-configured galactopyranose, and 61.1-82.6 ppm is assigned to the C signals of C2-C6.

[0054] According to the two-dimensional map 1 H- 1 H COSY ( Figure 7 )and 1 H- 13 C HSQC ( Figure 8 The assignment of hydrogens A, B, C, E, and F in the sugar ring was achieved. Based on the analysis of methylation data, the A glycosyl group was assigned as →4)-β-L-Ara. p (2SO4)-(1→; β-glycosyl group is assigned to →4)-β-L-Ara p (3SO4)-(1→; C glycosyl group is assigned to →3,4)-β-L-Ara p -(1→;D-glycosyl group is classified as →4)-β-L-Ara p -(1→;E-glycosyl group is classified as→4)-β-d-Gal p -(1→;glycosyl F is classified as →6)-β-d-Gal p -(1→。Combined) 1 H- 1 H NOESY ( Figure 9 Two-dimensional spectroscopy can infer the linkage between sugar groups in CHS2, such as... Figure 10 As shown, arabinose is expressed as →4)-β-L-Ara p -(1→ is composed of alternating links, with sulfate substituents at C-3 and C-2 positions.

[0055] Example 3 Analysis of the anticoagulant activity of arabinogalactan sulfate 1. In vitro anticoagulant activity The anticoagulant activity of polysaccharides was evaluated by in vitro measurement of APTT, PT, TT, and FIB; low molecular weight heparin was used as a positive control and physiological saline was used as a blank control.

[0056] Sheep plasma was centrifuged at 3000 r / min for 15 min, and the supernatant plasma was collected for later use. Polysaccharide samples were dissolved in physiological saline to prepare appropriate concentrations of 0, 5, 10, 25, 50, 100, and 200 μg / mL for later use.

[0057] Polysaccharide samples were mixed with plasma at a 1:9 ratio. 100 μL of the plasma to be tested was incubated at 37°C for 60 s. APTT, TT, and PT reagents were preheated at 37°C, and 100 μL of each were added to the plasma. Incubation was continued at 37°C for 120 s. For the APTT test, 100 μL of preheated 0.25 mol / L CaCl2 solution was added, and the coagulation time was recorded. For the PT and TT tests, the coagulation time was recorded directly. For FIB content determination, the reconstituted plasma was diluted with buffer. 200 μL of the plasma was preheated at 37°C for 180 s, and 100 μL of thrombin solution was added. The coagulation time was recorded for calibration. Polysaccharide-containing plasma was prepared and diluted 1:10 with buffer. 200 μL of the plasma to be tested was preheated at 37°C for 180 s, and 100 μL of thrombin solution was added. The plasma coagulation time was recorded, and the FIB content was calculated according to the standard curve equation.

[0058] Table 2 shows that the prolongation effect of polysaccharide CHS2 on APTT gradually increases with increasing concentration. When the CHS2 concentration is 100 μg / mL, the APTT value is prolonged to 197.1 s, and the clotting time is close to saturation (200 s), indicating that CHS2 can effectively prolong APTT. TT measurement results show that polysaccharide CHS2 has a weak prolongation effect at the same concentration as heparin. When the sample concentration increases to 200 μg / mL, polysaccharide CHS2 prolongs TT to 45.1 s. PT measurement results show that polysaccharide did not show an effective prolongation effect on PT. FIB measurement results show that the FIB content gradually decreases with increasing polysaccharide CHS2 concentration, indicating that it has certain fibrinolytic activity, but its activity is weaker than heparin. To achieve the same effect as heparin, the effective concentration of the polysaccharide sample needs to be increased.

[0059] Based on the experimental results, it was preliminarily determined that polysaccharide CHS2 can act on intrinsic and / or common coagulation pathways, while reducing the content of FIB, and has no effect on extrinsic coagulation pathways, indicating that the anticoagulant mechanism of polysaccharide CHS2 is different from that of heparin.

[0060] Table 2. Anticoagulation results of CHS2

[0061] Note: Results are expressed as mean ± standard deviation. Compared with the control group, *P<0.05, **P<0.01.

[0062] 2. In vivo anticoagulant activity In vivo anticoagulant activity was mainly determined by measuring the levels of APTT, TT, and PT in the plasma of treated rats.

[0063] (1) Animal grouping: 36 SD rats were randomly divided into 6 groups, with 6 rats in each group. The groups were: blank group (physiological saline), heparin group (0.5 mg / kg), 5 mg / kg polysaccharide group, 7.5 mg / kg polysaccharide group, 15 mg / kg polysaccharide group and 30 mg / kg polysaccharide group; free access to water and food was maintained before the experiment.

[0064] (2) Experimental procedure: Fasting was performed for 12 hours before the experiment, but water was allowed. SD rats were anesthetized with 30% urethane and then injected with the corresponding group drug via the lingual vein; the dosage was 0.1 mL / 100 g; after administration, the rats were placed flat for 0.5 h and blood was collected from the abdominal aorta. The collected blood was centrifuged at 3000 r / min for 10 min and the supernatant plasma was taken to measure the APTT, TT and PT values.

[0065] To further investigate whether the polysaccharide CHS2 can exert anticoagulant activity in vivo, the polysaccharide solution was first injected into the lingual vein of rats. Blood was collected 30 minutes later, and APTT, TT, and PT were analyzed. The experimental results are as follows: Figure 11 and 12 As shown in the figure. Using heparin as a positive control, APTT and TT gradually prolonged with increasing drug concentration. When the sample concentration was 15 mg / kg, APTT and TT showed highly significant differences compared with the blank control group (P < 0.001). Polysaccharide CHS2 did not prolong PT. This is consistent with the in vitro results, indicating that the anticoagulant activity of polysaccharide CHS2 increases with increasing concentration, inhibiting intrinsic and common coagulation pathways, but not extrinsic coagulation pathways.

[0066] Example 4 Thrombolytic activity analysis of arabinogalactan sulfate Before the experiment, SD rats were fasted for 12 hours, but water was allowed. They were anesthetized with 30% urethane. After the rats were placed flat for half an hour, blood was collected from the abdominal aorta. The blood was allowed to coagulate naturally at room temperature. The supernatant serum was removed, and the rats were washed three times with physiological saline. After removing excess liquid from the surface of the blood clot, the clot was cut into pieces of approximately 0.3g each. The exact weight of each piece was measured and placed into appropriately sized EP tubes, and the corresponding weight was recorded as Wt. Blood clot grouping: The cut blood clots were randomly divided into 5 groups, with 6 blood clots in each group. The negative control group was added with 1 mL of normal saline, the positive control group with 1 mL of 100 U / mL urokinase, and the polysaccharide groups with 1 mL of 5 mg / mL, 7.5 mg / mL, and 15 mg / mL, respectively. The clots were incubated in a 37℃ constant temperature shaker for 24 h. After incubation, undissolved blood clots were removed, rinsed three times with normal saline, and weighed (Ws). The blood clot dissolution rate was calculated using the following formula: (Wt - Ws) / Wt × 100%.

[0067] Results of the blood clot dissolution experiment of polysaccharide CHS2 are as follows Figure 13 As shown, the positive control group was co-incubated with 100 U / mL urokinase. The blood clot dissolution rate in the saline group was 19.35%, and when the CHS2 concentration was 5 mg / mL, the dissolution rate was 13.63%, which was significantly different from the saline group (p < 0.01). With the increase of polysaccharide concentration, the blood clot dissolution rate increased. When the concentration was 15 mg / mL, the dissolution rate increased to 62.43%, which was highly significant compared with both the blank group and the positive control group (p < 0.001). This indicates that the polysaccharide has significant thrombolytic activity, and its antithrombotic activity in vivo can be further explored to investigate its potential as an antithrombotic drug.

[0068] Example 5 Analysis of the anticoagulant mechanism of arabinogalactan sulfate The study on the anticoagulation mechanism of polysaccharides mainly involves measuring the effects of polysaccharides on coagulation factors IIa and Xa under the regulation of heparin cofactor II (HC-II) and antithrombin III (AT-III).

[0069] Heparin was used as a positive control, and physiological saline was used as a blank control.

[0070] (1) Preparation of Tris-HCl buffer: Accurately pipette 1 mL of 1 mol / L Tris-HCl and dissolve it in physiological saline in a 50 mL volumetric flask to obtain a 20 mmol / L Tris-HCl solution, and store it at 4℃ for later use.

[0071] (2) Accurately weigh 0.2g of PEG8000 and dissolve it in 200mL of prepared Tris-HCl buffer. Mix well and store at 4℃ for later use.

[0072] (3) Preparation of reagents: S-2238 and S-2765 were dissolved in 10 mL of Tris-HCl buffer solution to obtain 1 mmol / L S-2238 and 1 mmol / L S-2765, which were stored at 4℃ for later use. Coagulation factor IIa and coagulation factor Xa were dissolved in 1 mL of Tris-HCl buffer solution respectively and stored at 4℃ for later use.

[0073] Determination of the effects of polysaccharide on coagulation factors IIa and Xa in the absence of AT-III and HC-II: 12 μL of polysaccharide solution, 20 μL of 2 nmol / mL coagulation factor Xa or 2 nmol / mL coagulation factor IIa, and 90 μL of Tris-HCl PEG8000 solution were added sequentially to a 96-well plate. The plate was incubated at 37℃ for 120 s, and then 60 μL of chromogenic substrate S-2238 or S-2765 was added. The absorbance value at 405 nm was measured and recorded every 1 min for 60 min. The absorbance value was plotted as a function of time, and the rate of change in absorbance was used to determine the inhibitory effect of the drug on coagulation factors.

[0074] The effects of polysaccharides on coagulation factors IIa and Xa in the presence of AT-III and HC-II were determined as follows: 12 μL of polysaccharide solution, 20 μL of 2 nmol / mL coagulation factor Xa or 2 nmol / mL coagulation factor IIa, 12 μL of 10 nmol / L AT-III or 2 nmol / L coagulation factor IIa, and 78 μL of Tris-HCl PEG 8000 solution were added sequentially to a 96-well plate. The plate was incubated at 37°C for 120 s, and then 60 μL of chromogenic substrate S-2238 or S-2765 was added. The absorbance value at 405 nm was recorded every 1 min for 60 min. The absorbance value was plotted as a function of time, and the rate of change in absorbance was used to determine the inhibitory effect of the drug on coagulation factors.

[0075] The inhibitory effect of polysaccharide-dependent AT-III on coagulation factor Xa, such as Figure 14 As shown, in the absence of AT-III in the reaction system, the activity of coagulation factor Xa did not change significantly with variations in heparin and polysaccharide concentrations, indicating that CHS2 has no direct inhibitory effect on coagulation factor Xa. After adding AT-III to the reaction system, the activity of coagulation factor Xa decreased significantly with increasing CHS2 concentration. The inhibitory effect of CHS2 on coagulation factor Xa activity showed a similar trend to that of heparin, exhibiting a dose-dependent effect, but weaker than that of heparin. At a heparin concentration of 10 μg / mL, the activity of coagulation factor Xa was essentially below 5%, while this effect was achieved at a CHS2 concentration of 100 μg / mL.

[0076] The inhibitory effect of polysaccharide-dependent AT-III on coagulation factor IIa, such as Figure 15As shown, in the absence of AT-III in the reaction system, the activity of coagulation factor IIa did not change significantly with variations in heparin and polysaccharide concentrations, indicating that CHS2 has no direct inhibitory effect on coagulation factor IIa. After adding AT-III to the reaction system, the activity of coagulation factor IIa decreased significantly with increasing CHS2 concentration. The inhibitory effect of CHS2 on coagulation factor IIa activity showed a similar trend to that of heparin, exhibiting a dose-dependent effect, but weaker than that of heparin. At a heparin concentration of 1 μg / mL, the activity of coagulation factor IIa was essentially below 5%, while this effect was achieved at a CHS2 concentration of 1000 μg / mL.

[0077] The inhibitory effect of polysaccharide-dependent HC-II on coagulation factor IIa, such as Figure 16 As shown, in the absence of HC-II in the reaction system, the activity of coagulation factor IIa did not change significantly with changes in heparin and polysaccharide concentrations, indicating that CHS2 has no direct inhibitory effect on coagulation factor IIa. After adding HC-II to the reaction system, the activity of coagulation factor IIa decreased significantly with increasing CHS2 concentration. The inhibitory effect of CHS2 on coagulation factor IIa activity showed a similar trend to heparin, exhibiting a dose-dependent effect, but its effect was weaker. At 1 μg / mL, heparin reduced the activity of coagulation factor IIa to approximately 40%, while CHS2 achieved the same effect at a concentration of 1000 μg / mL. This indicates that CHS2 has a weak inhibitory effect on coagulation factor IIa mediated by HC-II, but CHS2 does not inhibit coagulation factor Xa mediated by HC-II.

Claims

1. An arabinose sulfate derived from linear kelp, characterized in that, The monosaccharide composition includes arabinose, galactose and rhamnose, wherein the molar percentage of arabinose is not less than 70%; the main chain backbone is modified with natural sulfate groups, wherein the sulfate groups are substituted at at least one hydroxyl site of the arabinose residues.

2. The arabinogalactan sulfate according to claim 1, characterized in that, The sulfate groups of the sulfated arabinogalactan are mainly distributed at the C-2, C-4 and / or C-6 positions of arabinose and galactose.

3. The arabinogalactan sulfate according to claim 2, characterized in that, The sulfate groups in the arabinogalactan sulfate are located at →4)-β-L-Ara p -(1→C-2 position, and →3)-β-L-Ara p -(1→C-4 bits.) 4. The arabinogalactan sulfate according to claim 1, characterized in that, The sulfated arabinose contains 15-30% by mass of sulfate groups.

5. The arabinogalactan sulfate according to claim 1, characterized in that, The number-average molecular weight of the arabinogalactan sulfate is 30-40 kDa; the arabinogalactan sulfate is a polysaccharide with a branched structure.

6. The arabinogalactan sulfate according to claim 1, characterized in that, The structure of the sulfated arabinogalactan is as follows: Where R = H or HSO3 R1=H,HSO3 Or a sidechain Gal p -(1→,Xyl p -(1→).

7. The method for preparing arabinogalactan sulfate according to any one of claims 1-6, characterized in that, The sulfated arabinogalactan was prepared by using natural green algae polysaccharides as raw materials through extraction, separation, and purification steps.

8. The preparation method according to claim 7, characterized in that, include: S1: Add ethanol to the linear fibrous algae powder, heat to defatting, centrifuge to collect the linear fibrous algae precipitate, and dry the precipitate; S2: Add distilled water to the dried precipitate, stir continuously at 100°C, and collect the supernatant by centrifugation; S3: The supernatant is concentrated using a rotary evaporator, and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; S4: Add ethanol to the concentrated solution for alcohol precipitation, let stand overnight, and then centrifuge to collect the precipitate; S5: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S6: The crude Streptomyces linearis polysaccharide was separated and purified using a Q-Sepharose Fast Flow anion exchange column to obtain purified Streptomyces linearis polysaccharide. S7: The polysaccharide from *Streptococcus linearis* was further purified using a Sephacryl S-400 gel column to obtain a polysaccharide with uniform molecular weight.

9. A product for anticoagulation, characterized in that, Includes the arabinogalactan sulfate according to any one of claims 1-6.

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