Sulfated arabino-oligosaccharides, methods for their preparation and uses thereof

Abstract

This invention discloses a sulfated arabinoglycoside, its preparation method, and its applications, belonging to the field of biomedical technology. The technical solution includes a sulfated arabinoglycoside derived from *Streptococcus linearis* polysaccharide; a degree of polymerization of 2-5; arabinose as the main monosaccharide, and containing galactose; the molar percentage of arabinose is not less than 80%, and the molar percentage of galactose is not more than 20%; the main chain is composed of (1→4)-β-L-pyranose arabinose groups, with the sulfate groups of the arabinose mainly substituted at the C-3 position. This invention is applied to inhibiting the abnormal aggregation of human pancreatic islet amyloid peptide (hIAPP), solving the problem of the lack of safe and effective intervention methods for the abnormal aggregation of human pancreatic islet amyloid peptide in existing technologies. It features stable raw material sources, high safety, a simple and mild preparation method suitable for large-scale preparation, and the ability to inhibit the abnormal aggregation of human pancreatic islet amyloid peptide and reduce pancreatic β-cell damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a sulfated arabinoligosaccharide, its preparation method, and its application. Background Technology

[0002] Type 2 diabetes is a metabolic disease characterized primarily by insufficient insulin secretion or insulin resistance, and its development is accompanied by gradual impairment of pancreatic β-cell function. Studies have shown that human islet amyloid polypeptide (hIAPP) is prone to abnormal folding and aggregation under pathological conditions, thereby inducing pancreatic β-cell damage, which is also a significant factor leading to pancreatic islet dysfunction. However, current interventions targeting islet damage associated with hIAPP abnormal aggregation are limited, and there is a lack of effective inhibitors that are safe, reliably sourced, and suitable for long-term use.

[0003] While small molecule compounds such as gallocatechin gallate (EGCG) and myricetin, as well as polypeptides like pramlineptide, have shown some inhibitory effects in in vitro experiments, their poor stability, insufficient biocompatibility, and potential toxic side effects limit their further applications. Seaweed sulfated polysaccharides and their degradation products have attracted widespread attention in recent years due to their good water solubility, biocompatibility, and diverse biological activities. However, current research mainly focuses on high-molecular-weight sulfated polysaccharides, which have large molecular weights, complex structures, and limited bioavailability, hindering precise regulation and functional applications.

[0004] Oligosaccharides, as small molecular fragments resulting from the degradation of polysaccharides, possess characteristics such as low molecular weight, good water solubility, and easy absorption, enabling them to cross the blood-brain barrier more efficiently and enter the bloodstream to exert their effects. For example, chromium mannuronic acid oligosaccharide complexes can exert anti-diabetic activity by improving insulin sensitivity in skeletal muscle cells and enhancing lipid metabolism. Furthermore, maltose oligosaccharides, lactulose oligosaccharides, and other oligosaccharides, due to their low-calorie and low-energy properties, can be used as functional food additives and have shown significant advantages in preventing obesity, hypertension, and diabetes. Sulfated arabinoligosaccharides, with their well-defined structures and low degree of polymerization, have potential advantages in inhibiting abnormal protein aggregation and protecting cell function.

[0005] However, sulfated arabinooligosaccharides with well-defined sources, clear structures, and suitability for large-scale preparation remain scarce. Therefore, developing a sulfated arabinooligosaccharide with safe sources, well-defined structures, controllable preparation processes, and the ability to inhibit abnormal hIAPP aggregation and protect pancreatic β-cell function, along with its preparation method, is of significant research importance and application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is the lack of safe and effective intervention methods for abnormal aggregation of human pancreatic islet amyloid peptides. This invention proposes a sulfated arabinoligosaccharide with stable raw material sources, high safety, a simple and mild preparation method suitable for large-scale preparation, and the ability to inhibit abnormal aggregation of human pancreatic islet amyloid peptides and reduce pancreatic β-cell damage. This sulfated arabinoligosaccharide can be used to prevent or alleviate diseases related to abnormal aggregation of hIAPP, along with its preparation method and applications.

[0007] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a sulfated arabinoligosaccharide derived from Streptomyces linearis polysaccharide; the degree of polymerization is 2-5; arabinose is the main monosaccharide, and galactose is also present; the molar percentage of arabinose is not less than 80%, and the molar percentage of galactose is not more than 20%; the main chain is composed of (1→4)-β-L-pyranose arabinose linked together, and the sulfate group of arabinose is mainly substituted at the C-3 position.

[0008] In some embodiments, the sulfate substitution of galactose occurs at the C-6 position.

[0009] In some embodiments, the structure of arabinobiose sulfate is Ara p (3SO4)-(1→4)-β-L-Ara p .

[0010] In some embodiments, the structure is as follows:

[0011] Wherein, R1 = H or HSO3 - R2 = H or HSO3 - .

[0012] Another aspect of the present invention provides a method for preparing sulfated arabinoglycosides according to any of the above technical solutions, comprising: extracting polysaccharides from Streptomyces linearis, enriching polysaccharide components with arabinose, and controllingly degrading them while maintaining the natural sulfate group substitution characteristics to obtain sulfated arabinoglycosides.

[0013] In some embodiments, including: S1: Add distilled water to defatted Streptomyces linearis powder, heat to extract Streptomyces linearis polysaccharides, and centrifuge to collect the supernatant; S2: The supernatant was concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag to obtain a concentrated solution. S3: Add ethanol to the concentrate for alcohol precipitation, let stand overnight, and then centrifuge to collect the precipitate; S4: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S5: The crude polysaccharide from *Streptococcus linearis* was separated and purified using an anion exchange column to obtain arabinogalactan sulfate. S6: Add 0.05 mol / L HCl to sulfated arabinose, heat in a water bath to degrade it, add saturated barium hydroxide solution to the degradation solution until no more precipitate is produced, centrifuge to collect the supernatant, dialyze the supernatant through a 100 Da dialysis bag, desalt it, and freeze-dry it to obtain an oligosaccharide mixture. After separation and purification of the oligosaccharide mixture by gel column, sulfated arabinooligosaccharide is obtained.

[0014] In some embodiments, in S1, the mass-to-volume ratio of defatted filamentous algae powder to distilled water is 1:5-10; in S3, the volume-to-volume ratio of concentrated liquid to ethanol is 1:4-8.

[0015] The present invention also provides a drug for inhibiting the abnormal aggregation of human pancreatic amyloid polypeptide hIAPP, comprising sulfated arabinoligosaccharides of any of the above-described technical solutions.

[0016] In some embodiments, the effective dose of sulfated arabinogalactosyl to inhibit the abnormal aggregation of hIAPP is 10-200 μg / mL.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a sulfated arabinoliposide, derived from the green algae *Streptococcus linearis*, which has a stable source, high safety, and is suitable for application in the food or pharmaceutical fields. The structural characteristics of the sulfated arabinoliposide are characterized by primary and secondary mass spectrometry analysis. The sulfated arabinoliposide exhibits low degree of polymerization, a well-defined structure, good water solubility and biocompatibility, which is beneficial for its biological activity. This invention provides a method for preparing sulfated arabinoligosaccharides. This method does not involve simple degradation of sulfated polysaccharides, but rather screening and enriching the polysaccharide components first, and then preparing oligosaccharide fragments with well-defined structures. While retaining the original sulfate substitution sites in the polysaccharides, the degree of polymerization of the oligosaccharides is clearly defined as 2-5 (molecular weight <1000), thereby obtaining a sulfated arabinoligosaccharide with a highly defined structure. Furthermore, this preparation method is simple, has mild conditions, and is suitable for large-scale preparation. This invention provides the application of sulfated arabinooligosaccharide in the preparation of a drug that inhibits the abnormal aggregation of human pancreatic islet amyloid peptide hIAPP. The sulfated arabinooligosaccharide of this invention can inhibit the abnormal aggregation of human pancreatic islet amyloid peptide, reduce pancreatic β-cell damage, and can be used to prevent or alleviate diseases related to abnormal aggregation of hIAPP. Attached Figure Description

[0018] Figure 1 Q Sepharose Fast Flow diagram of CP separation of Streptomyces linearis polysaccharide; Figure 2 Monosaccharide composition analysis of CP, a polysaccharide from Streptomyces linearis; Figure 3 The TLC chromatogram is shown for the degradation of linear sclerosis polysaccharide CP under 0.05 mol / L HCl conditions. Figure 4 The TLC chromatogram is shown for the polysaccharide CP of *Streptococcus linearis* under degradation conditions of 0.05 mol / L TFA. Figure 5 This is a chromatogram showing the separation and purification of polysaccharide CP degradation products using a Bio-Gel P4 column. Figure 6 This is the first-order mass spectrum of sulfated arabinolithosaccharide CPs1 from Streptomyces linearis; Figure 7 This is the first-order mass spectrum of sulfated arabinolithosaccharide CPs2 from *Streptococcus linearis*. Figure 8 This is the first-order mass spectrum of sulfated arabinolithosaccharide CPs3 from *Streptococcus linearis*. Figure 9 This is the first-order mass spectrum of sulfated arabinolithosaccharide CPs4 from the linear algae *Streptococcus linearis*. Figure 10 The image shows the secondary mass spectrum of sulfated arabinooligosaccharides (CPs) with a mass-to-charge ratio (m / z) of 229. In the image, a represents fragment ion information at position 229, b represents arabinooligosaccharide sulfated at position C-3, c represents arabinooligosaccharide sulfated at position C-2, and d represents arabinooligosaccharide sulfated at position C-4. Figure 11 The image shows the secondary mass spectrum of sulfated arabinooligosaccharide CPs with a mass-to-charge ratio (m / z) of 361. In the image, a represents fragment ion information at 361, b represents arabinobiose sulfated at C-3, and c represents arabinobiose sulfated at C-2. Figure 12 The image shows the second-order mass spectrum of sulfated arabinooligosaccharide CPs with a mass-to-charge ratio (m / z) of 493. In the image, a represents fragment ion information at 493, b represents non-reducing end sulfated arabinotrisaccharide, and c represents reducing end sulfated arabinotrisaccharide. Figure 13 The image shows a secondary mass spectrum of sulfated arabinooligosaccharide CPs with a mass-to-charge ratio (m / z) of 625. In the image, a represents fragment ion information at 625, b represents the non-reducing sulfated arabinotetrasaccharide, and c represents the reducing sulfated arabinotetrasaccharide. Figure 14 To detect the effect of CPs on hIAPP aggregation using the thioflavin T method; Figure 15To investigate the effect of CPs on hIAPP aggregation using transmission electron microscopy; Figure 16 The effect of CPs on the cell viability of pancreatic β cells; Figure 17 The effects of CPs on intracellular oxidative stress in pancreatic β cells; Figure 18 The effect of hIAPP on the survival rate of NIT-1 cells; Figure 19 The impact of hIAPP on MMP; Figure 20 This indicates the location of action of CPs within pancreatic β cells. Detailed Implementation

[0019] 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.

[0020] This invention provides a sulfated arabinoliposaccharide derived from *Streptococcus linearis* polysaccharide; with a degree of polymerization of 2-5; primarily composed of arabinose monosaccharide and containing galactose; the molar percentage of arabinose is not less than 80%, and the molar percentage of galactose is not more than 20%; the main chain is composed of (1→4)-β-L-pyranose groups linked together, with the sulfate groups of the arabinose mainly substituted at the C-3 position. Further, the main chain linkage of the sulfated arabinoliposaccharide is (1→4)-β-L-Arap-(1→), where the sulfate groups are located at the C-3 position of either the non-reducing or reducing end of the arabinoliposaccharide.

[0021] The above-mentioned sulfated arabinoligosaccharide raw material is derived from the green algae *Streptococcus linearis*, which has a stable source, high safety, and is suitable for application in the food or pharmaceutical fields. By analyzing the first-order and second-order mass spectrometry of the sulfated arabinoligosaccharide of the present invention, its structural characteristics are characterized. The sulfated arabinoligosaccharide of the present invention has a low degree of polymerization, a well-defined structure, good water solubility and biocompatibility, which is conducive to exerting biological activity.

[0022] In some embodiments, the sulfate substitution of galactose occurs at the C-6 position. Further, in the above-described sulfated arabinoligosaccharides, the sulfate group of the arabinose unit is primarily substituted at the C-3 position, with a small amount substituted at the C-2 position; the sulfate substitution of the galactose unit occurs at the C-6 position.

[0023] In some embodiments, the structure of arabinobiose sulfate is Ara p (3SO4)-(1→4)-β-L-Arap .

[0024] In some embodiments, the structure is as follows:

[0025] Wherein, R1 = H or HSO3 - R2 = H or HSO3 - .

[0026] Another aspect of the present invention provides a method for preparing sulfated arabinoglycosides according to any of the above technical solutions, comprising: extracting polysaccharides from Streptomyces linearis, enriching polysaccharide components with arabinose, and controllingly degrading them while maintaining the natural sulfate group substitution characteristics to obtain sulfated arabinoglycosides.

[0027] This preparation method does not involve simple degradation of sulfated polysaccharides. Instead, it first screens and enriches the polysaccharide components, and then prepares oligosaccharide fragments with well-defined structures. While retaining the original sulfate substitution sites in the polysaccharides, it also clearly defines the degree of polymerization of the oligosaccharides as 2-5 (molecular weight <1000), thereby obtaining a sulfated arabinoligosaccharide with a highly defined structure. Furthermore, this preparation method is simple, mild, and suitable for large-scale preparation.

[0028] In some embodiments, including: S1: Add distilled water to defatted Streptomyces linearis powder, heat to extract Streptomyces linearis polysaccharides, and centrifuge to collect the supernatant; S2: The supernatant was concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag to obtain a concentrated solution. S3: Add ethanol to the concentrate for alcohol precipitation, let stand overnight, and then centrifuge to collect the precipitate; S4: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S5: The crude polysaccharide from *Streptococcus linearis* was separated and purified using an anion exchange column to obtain arabinogalactan sulfate. S6: Add 0.05 mol / L HCl to sulfated arabinose, heat in a water bath to degrade it, add saturated barium hydroxide solution to the degradation solution until no more precipitate is produced, centrifuge to collect the supernatant, dialyze the supernatant through a 100 Da dialysis bag, desalt it, and freeze-dry it to obtain an oligosaccharide mixture. After separation and purification of the oligosaccharide mixture by gel column, sulfated arabinooligosaccharide is obtained.

[0029] In some embodiments, in S1, the mass-to-volume ratio of defatted filamentous algae powder to distilled water is 1:5-10; in S3, the volume-to-volume ratio of concentrated liquid to ethanol is 1:4-8.

[0030] The above preparation method specifically includes: (1) Add distilled water to defatted Streptomyces linearis powder at a ratio of 1:10 (W / V), heat at 100°C for 4 hours to extract Streptomyces linearis polysaccharides, and collect the supernatant by centrifugation. (2) The supernatant in step (1) is concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; (3) Add 4 times the volume of 95% ethanol to the concentrate in step (2) for alcohol precipitation, let stand overnight at 4°C, and then collect the precipitate by centrifugation. (4) The precipitate in step (3) was decolorized and dehydrated using acetone and a large amount of ethanol, and dried at 55°C to obtain crude linear sclerosis polysaccharide. (5) The polysaccharide of Streptomyces linearis was separated and purified using a Q-Sepharose Fast Flow anion exchange column to obtain arabinogalactan sulfate; (6) Add 0.05 mol / L HCl to arabinose sulfate until the final concentration of arabinose sulfate is 10 mg / mL. Degrade it by heating in a water bath at 60°C for 5 h. Add saturated barium hydroxide solution to the degradation solution until no more precipitate is produced. Centrifuge and take the supernatant. Dialyze the supernatant through a 100 Da dialysis bag and desalt it. Then freeze-dry it to obtain an oligosaccharide mixture. The oligosaccharide mixture is further purified by Bio-Gel P4 gel column to obtain sulfated arabinooligosaccharide.

[0031] (7) Oligosaccharides were analyzed by electrospray mass spectrometry in negative ion mode. The sulfated arabinooligosaccharide sample was dissolved in acetonitrile-water = 1:1 (V / V) to achieve a concentration of 5-10 pmol / L. The injection volume was 5 µL.

[0032] Furthermore, in step (5), 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 4 mol / L NaCl solutions.

[0033] Furthermore, in step (6), the length of the Bio Gel P4 gel column is 100 cm and the diameter is 1.5 cm. The elution conditions are 0.2 mol / L NH4HCO3 solution and a flow rate of 0.3 mL / min.

[0034] Furthermore, in step (7), the mass spectrometry analysis uses N2 as the solvent for drying gas and spray gas with flow rates of 250 and 15 L / h, respectively. The mobile phase is acetonitrile-water = 1:1 (V / V), the capillary voltage is 3 kV, the cone voltage is 50 eV, and the evaporation temperatures of the ionic element and the solvent are 80 °C and 150 °C, respectively.

[0035] The present invention also provides a drug for inhibiting the abnormal aggregation of human pancreatic amyloid polypeptide hIAPP, comprising sulfated arabinoligosaccharides of any of the above-described technical solutions.

[0036] In some embodiments, the effective dose of sulfated arabinogalactosyl to inhibit the abnormal aggregation of hIAPP is 10-200 μg / mL.

[0037] It should be noted that the abnormal aggregation of hIAPP belongs to the protein conformational pathology mechanism, and its regulatory mechanism is not the same as the inflammatory or metabolic abnormality mechanism of diabetic nephropathy. The sulfated arabinoglycosides provided by this invention can inhibit the abnormal aggregation of human pancreatic islet amyloid peptides and reduce pancreatic β-cell damage. Specifically, sulfated arabinoglycosides can significantly inhibit hIAPP aggregation and effectively inhibit the formation of toxic fibers by changing the morphology of hIAPP aggregates; sulfated arabinoglycosides can alleviate pancreatic β-cell damage, intracellular oxidative stress and mitochondrial function caused by hIAPP aggregation; sulfated arabinoglycosides can enter cells to exert their effects and improve mitochondrial function and protect pancreatic β-cell function by localizing to the mitochondria, an organelle.

[0038] To provide a clearer and more detailed description of the sulfated arabinoligosaccharides, their preparation methods, and applications provided in the embodiments of the present invention, specific examples will be described below.

[0039] Example 1 Extraction, separation and purification of sulfated arabinoligosaccharides The extraction, separation, and purification of sulfated arabinoglycosides in this invention include 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 algae powder precipitate by centrifugation, and dry the precipitate at 55°C. (2) Add distilled water to the defatted algae powder in 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 linear filamentous algae polysaccharide sample was degraded with 0.05 mol / L HCl to a final concentration of 10 mg / mL. The sample was heated in a water bath at 60℃ for 5 h. The degradation solution was neutralized with NaOH, and the supernatant was collected by centrifugation. The supernatant was dialyzed and desalted using a 100 Da dialysis bag, and then freeze-dried to obtain an oligosaccharide mixture.

[0040] (8) The sulfated arabinooligosaccharides were separated and purified using a Q-Sepharose Fast Flow anion exchange column and a Bio-Gel P4 gel column to obtain sulfated arabinooligosaccharides CPs.

[0041] Example 2 Preparation and composition of linear spirulina polysaccharide (CP) 1. Ion exchange chromatography for the separation and purification of *Ulva prolifera* polysaccharides. 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 a polysaccharide with uniform charge density.

[0042] like Figure 1 As shown, the fraction eluted with 1.5 mol / L NaCl was collected, concentrated, lyophilized, and named CP.

[0043] 2. Determination of monosaccharide composition of CP by high performance liquid chromatography (1) Accurately weigh 2 mg of CP 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 CP degradation product and monosaccharide standard 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 composition of CP monosaccharides 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 As shown, the monosaccharide composition of CP consists of arabinose, galactose, and a small amount of rhamnose, with percentages of 83.62%, 13.77%, and 2.61%, respectively, indicating that CP is an arabinogalactan containing some rhamnose and galactose.

[0047] Example 3 Preparation and primary analysis of sulfated arabinoligosaccharides (CPs) 1. Optimization of degradation conditions for sulfated arabinoligosaccharides Trifluoroacetic acid (TFA) or hydrochloric acid (HCl) of different concentrations were used to hydrolyze the polysaccharide of Streptomyces linearis. The effects of different reaction temperatures and reaction times on the polysaccharide degradation products during the hydrolysis process were studied by thin-layer chromatography. Figure 3 and 4 The TLC results show the degradation of polysaccharide CP in 0.05 mol / L HCl and 0.05 mol / L TFA at 60℃ and 80℃, respectively. The degree of degradation increased with time. Compared to degradation at 80℃, the degradation at 60℃ was less severe. At 80℃, the oligosaccharide content gradually increased with time, indicating a greater degree of degradation. Compared to the HCl degradation results, the TFA degradation trend was weaker. Therefore, the mild degradation conditions of 0.05 mol / L HCl at 60℃ were selected for partial acid degradation of the polysaccharide for 5 hours. After the degradation reaction, the polysaccharide was neutralized with sodium hydroxide, dialyzed using a 100 Da dialysis bag, concentrated, and the resulting supernatant was concentrated and then lyophilized.

[0048] 2. Preparation of sulfated arabinoligosaccharides (CPs) Weigh an appropriate amount of linear spirulina polysaccharide, add 0.05 mol / L HCl to a final concentration of 10 mg / mL, and degrade in a 60℃ water bath for 5 h. After the reaction is complete, add sodium hydroxide to neutralize, centrifuge, and collect the supernatant. Dialyze the supernatant through a 100 Da dialysis bag, desalt it, and then freeze-dry to obtain an oligosaccharide mixture. Further purification of the oligosaccharide mixture using a Bio-Gel P4 gel column yielded arabinogalactosyl sulfate, named CPs. The sugar content of the eluent from the Bio-Gel P4 column was determined using the phenol-sulfuric acid method, and elution curves were plotted. Figure 5As shown, six oligosaccharide components were separated and purified. The peaks were collected, and the same oligosaccharide fractions were collected repeatedly after multiple loadings. The fractions were then repeatedly removed with water to remove ammonia, concentrated, and lyophilized.

[0049] 3. Determination of the degree of polymerization of sulfated arabinoligosaccharides (CPs) Oligosaccharides were analyzed using electrospray mass spectrometry in negative ion mode. 2 mg of oligosaccharide was dissolved in acetonitrile-water at a ratio of 1:1 (v / v) to achieve a concentration in the range of 5-10 pmol / L, with an injection volume of 5 µL. During mass spectrometry analysis, the flow rates of the drying gas and spray gas using N2 as solvent were 250 L / h and 15 L / h, respectively; the mobile phase was acetonitrile-water at a ratio of 1:1 (v / v), injected via a syringe at a flow rate of 10 µL / min under pump power; the capillary voltage was 3 kV, the cone voltage was 50 eV, and the evaporation temperatures of the ionogen and solvent were 80 °C and 150 °C, respectively.

[0050] The results are as follows Figure 6 , 7 Figures 8 and 9 show the primary mass spectra of the arabinose sulfate fraction, where R, A, G, and S represent rhamnose, arabinose, galactose, and sulfate groups, respectively.

[0051] Figure 6 The main single-charge ion peaks are m / z 229, m / z 259, m / z 243 and m / z 163. Since the monosaccharide composition of CP is mainly arabinose, galactose and rhamnose, m / z 229 is monosulfated arabinose (AS), m / z 259 is monosulfated galactose (GS), m / z 243 is monosulfated rhamnose (R), and m / z 163 is unsulfated rhamnose (RS). Figure 7 The presence of single-charged ion peaks at m / z 259 and m / z 361 suggests that m / z 361 represents sulfated arabinobiose A2S. Figure 8 The main ion peaks are m / z 361, m / z 441, m / z 493 and m / z 573, where m / z 441 is arabinobiose disulfate A2S2, m / z 493 is arabinotriose sulfate A3S and m / z 573 is arabinotriose disulfate A3S2. Figure 9 Among them, there are many oligosaccharide fragments with high degree of polymerization, which are all sulfated or non-sulfated arabinose and galactose, and some of them have double charges, such as double-charged ion peaks m / z 220, m / z 286 and m / z 352.

[0052] Example 4 Secondary mass spectrometry analysis of sulfated arabinoligosaccharides (CPs) Electrospray collision-induced fragmentation secondary mass spectrometry (ESI-CID-MS / MS) in negative ion mode was used to resolve oligosaccharide structures. Secondary mass spectrometry, building upon primary mass spectrometry, performs specific fragmentation on ions with specific mass-to-charge ratios, yielding rich structural information. By assigning these fragments, the structure of oligosaccharides can be determined. In the secondary mass spectrometry of oligosaccharides, fragmentation can occur not only at glycosidic bonds but also within the sugar ring, and may also occur through the loss of small molecules.

[0053] The mass-to-charge ratio (m / z) at 229 is monosulfated arabinose, and its ESI-CID-MS... 2 The diagram is as follows Figure 10 As shown, m / z 97 is caused by the removal of a sulfate group. The high abundance of m / z 97 indicates a large amount of sulfated arabinose at position 3. The m / z 139 and m / z 168 ions are respectively... 0,2 X and 0,2 The formation of A-fractionation indicates that the sulfate group may be located at the C-3, C-4, and C-2 positions of arabinose. Based on the abundance of ions at m / z 97, m / z 139, and m / z 168, it is inferred that AS mainly consists of C-3 sulfated arabinose, with a relatively low content of C-2 sulfated arabinose.

[0054] At mass-to-charge ratio (m / z) 361, there is monosulfated arabinobiose, such as Figure 11 As shown, fragment ions m / z 211 and m / z 229 are generated by the breaking of the arabinose rings and are assigned to B1, C1 or Z1, Y1, respectively. m / z 301 and m / z 271 are generated by the breaking of the rings within the rings. 0,2 A2 and 2,4 The A2 ion fragment did not show the ion peak at m / z 169, therefore the reduction end is 1,4 connected. 0,2 The formation of fragment A ions requires a free hydroxyl group at the C-3 position to assist in the breaking of the C2-C3 chemical bond. Therefore, it is determined that the sulfate group is located at the non-reducing end of the C-3 position. Considering the generation of m / z 139 ion fragments, the sulfate group may also be located at the reducing end of the C-2 position. 0,2 X1 fracture occurs. Therefore, the structure of m / z 361 is inferred to be β-L-Ara. p (3SO4)-(1→4)-β-L-Ara p or β-L-Ara p -(1→4)-β-L-Ara p (2SO4).

[0055] The mass-to-charge ratio (m / z) at 493 is a secondary mass spectrum of arabinotriose sulfate. Figure 12As shown, m / z 361, m / z 343, and m / z 211 are interring fracture ions B1, B2, and Cl; m / z 433, m / z 403, and m / z 301 are ions generated by intraring fracture. 0, 2 A3 0,2 X3 and 0,2 A2, proving that the sulfate group is located at the C-3 position of either the non-reducing or reducing end, suggests that the structure of sulfated arabinotrisaccharide is β-L-Ara. p (3SO4)-(1→4)-β-L-Ara p -(1→4)-β-L-Ara p or β-L-Ara p -(1→4)-β-L-Ara p -(1→4)-β-L-Ara p (3SO4).

[0056] The mass-to-charge ratio (m / z) at 625 is the secondary mass spectrum of arabinotetraose sulfate. Figure 13 As shown, m / z 493, m / z 475, m / z 343, and m / z 211 are interring fracture ions C3, B3, B2, and B1; m / z 565, m / z 535, and m / z 433 are generated by intraring fracture. 0,2 A4 0,2 X4 0,2 A3; Proving that the sulfate group is located at the C-3 position of either the non-reducing or reducing end, we deduce that the structure of sulfated arabinotetrasaccharide is β-L-Ara. p (3SO4)-(1→4)-β-L-Ara p -(1→4)-β-L-Ara p -(1→4)-β-L-Ara p or β-L-Ara p -(1→4)-β-L-Ara p -(1→4)-β-L-Ara p -(1→4)-β-L-Ara p (3SO4).

[0057] Example 5 Impact of CPs on hIAPP aggregation The effects of CPs on hIAPP aggregation were detected by thioflavone T (Th T) fluorescence assay and transmission electron microscopy (TEM).

[0058] 1. Effect of thioflavone T fluorescence detection on the aggregation of polysaccharides on hIAPP hIAPP was dissolved in hexafluoroisopropanol to a concentration of 2 mg / mL, sonicated for 5 min to fully depolymerize into monomers, dried with N2 to remove HFIP, and diluted to 10 μmol / mL with 20 mmol / L Tris (pH 7.4). ThT powder was also prepared into a 1 mmol / L solution with 20 mmol / L Tris (pH 7.4). CPs were prepared into different solutions with 20 mmol / L Tris (pH 7.4), and 5 μL of each solution was placed in a completely black 96-well plate. 100 μL of hIAPP solution and 20 μL of ThT solution were added sequentially and mixed thoroughly. The excitation wavelength of the microplate reader was set to 450 nm and the emission wavelength to 482 nm. Detection was performed every 5 min, and the fluorescence intensity plateaued after 6 h.

[0059] Th T fluorescence detection results are as follows Figure 14 As shown, the fluorescence intensity of the sample incubated with hIAPP alone gradually increased with time, indicating that the number of β-sheet structures gradually increased. It reached a plateau around 6 hours, indicating that the hIAPP monomers had essentially formed aggregates. When hIAPP was co-incubated with oligosaccharides CPs, the relative fluorescence intensity was significantly lower than that of hIAPP incubated alone, indicating that CPs could inhibit the aggregation of hIAPP.

[0060] 2. Transmission electron microscopy analysis of the effect of CPs on hIAPP aggregation After incubating 10 μmol / mL hIAPP alone or separately with CPs samples at 37 °C for 24 h, samples were collected for electron microscopy. 10 μL of sample was spotted on a copper grid and dried at room temperature. 10 μL of 1.5% phosphotungstic acid (pH 7.4) was added to each copper grid. After the samples dried, the morphology of protein aggregates was observed under a transmission electron microscope (JEOL 1200), and the differences in the fibers formed between the groups were compared.

[0061] The results are as follows Figure 15 As shown, hIAPP alone, after 24 hours of incubation, forms relatively long, mature fibers resembling weeds, which further aggregate into fiber plaques. However, when hIAPP is co-incubated with oligosaccharides (CPs), the number of mature aggregated fibers is significantly reduced, no fiber plaques are formed, and the fibers formed in the solution are all shorter and finer. With increasing oligosaccharide concentration, no mature protein fibers can be observed in the field of view at a CPs concentration of 100 μg / mL, indicating that CPs effectively inhibit hIAPP fiber formation. TEM observations further demonstrate that CPs not only inhibit hIAPP aggregation but also alter the morphology of hIAPP aggregates, effectively suppressing hIAPP fiber formation.

[0062] Example 6 Research on the reduction of islet cell damage induced by CPs by hIAPP In a mouse pancreatic islet tumor (NIT-1) cell model, the protective effects of CPs on NIT-1 cells were examined, including the intracellular localization of CPs, their influence on intracellular oxidative stress response, and their effects on mitochondrial function.

[0063] 1. Assay of pancreatic β-cell viability Cell viability was assessed using the MTT assay. Healthy NIT-1 cells were seeded at a density of 8000 cells / well in 96-well plates and cultured at 37°C for 24 h. Serum-free medium was then added, and the cells were cultured for another 24 h. hIAPP and different concentrations of polysaccharide samples were added to achieve a final hIAPP concentration of 10 μmol / mL. After cell culture, 20 μL of 0.5 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 h. The medium was then aspirated, and 150 μL of DMSO was added to each well. The cells were incubated at 37°C for 30 min to allow the crystals to dissolve completely. The OD value was then measured at 570 nm. Cells without hIAPP and polysaccharides served as the blank control group, while cells with only hIAPP served as the model control group. Each group was divided into six replicates, and the experiment was repeated three times. Cell viability was calculated using the following formula: Cell viability (%) = (Average OD value of drug-treated wells / Average OD value of control group) × 100% The results are as follows Figure 16 As shown, CPs did not inhibit cell proliferation at concentrations below 500 μg / mL, but cell viability decreased with increasing concentration. Therefore, a concentration below 500 μg / mL was chosen for the next stage of the experiment.

[0064] 2. Intracellular oxidative stress analysis NIT-1 cells in good growth condition were seeded at a density of 8000 cells / well in 96-well all-black plates and cultured at 37°C for 24 h. The medium was then replaced with serum-free medium, and hIAPP and different concentrations of CPs were added to achieve a final hIAPP concentration of 10 μmol / mL, followed by 24 h of culture. CPs concentrations of 10, 50, 100, and 250 μg / mL were used. Untreated cells served as a blank control group, and cells treated with only hIAPP served as a model control group. Each group was divided into six replicates, and the experiment was repeated three times. ROS were measured using a fluorescence method. 10 mmol / mL of the active fluorescent probe DCFH-DA was added to the 96-well plate and incubated for 20 min before detection. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. After entering the cell, it is hydrolyzed by intracellular esterases to generate DCFH. The fluorescence value of DCFH indicates the intracellular reactive oxygen species level. The excitation wavelength was 488 nm, and the emission wavelength was 525 nm.

[0065] The results are as follows Figure 17 As shown, the ROS level increased in the model group incubated with hIAPP alone, while the ROS level decreased significantly in the group with added CPs. As the concentration increased, the ROS level gradually approached that of the blank control group, indicating that CPs can significantly reduce the increase in ROS level caused by hIAPP.

[0066] 3. Mitochondrial membrane potential measurement Changes in mitochondrial membrane potential were detected using the fluorescent probe JC-1. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form polymers that emit strong red fluorescence. In damaged mitochondria, due to a decrease or loss of membrane potential, JC-1 exists only as monomers in the cytoplasm, producing green fluorescence. Cells were cultured for 24 h in 96-well all-black cells with hIAPP and different concentrations of CPs (10, 50, 100, and 250 μg / mL). Untreated cells served as a blank control group, and cells treated with only hIAPP served as a model control group. Each group was divided into six replicates, and the experiment was repeated three times. Cells were stained with JC-1 dye, and detection was performed 30 min later. Changes in mitochondrial membrane potential could be detected by comparing the fluorescence ratio of JC-1 aggregates to monomers or by the change from red to green fluorescence.

[0067] Figure 18 The study showed the effect of hIAPP on the survival rate of NIT-1 cells. The cell survival rate of the model group incubated with hIAPP alone was about 61.0%. After co-incubation with CPS, the cell survival rate gradually increased in a concentration-dependent manner. When the CPS concentration increased to 200 μg / mL, the cell death induced by hIAPP was basically eliminated, and the cell survival rate reached more than 98%. Figure 19 The study showed the effect of hIAPP on MMP. After incubation with CPS and hIAPP, MMP gradually recovered, and the protective effect became more obvious with increasing concentration.

[0068] 4. Cell localization analysis FITC-labeled CPs were co-incubated with NIT-1 cells, and organelle-specific labeling dyes were added. The location of fluorescence within the cells was observed using a confocal microscope to determine its distribution and localization. NIT-1 cells were incubated at 10⁻¹⁵ × 10⁶ cells / cells. 4Cells were seeded at a density of 1 / 2 well in 6-well plates. When the cell confluence reached approximately 50%, 2 mL of medium containing FITC-labeled CPs (100 μg / mL) was added. Cells were analyzed at 1 h, 2 h, 4 h, and 12 h. After incubation, cells were washed three times with ice-cold PBS to ensure that any FITC-labeled polysaccharides that had not entered the cells were removed. MitoTracker Red CMXRos (mitochondrial dye) was added to a final concentration of 10 nmol / L, and the cells were incubated for 30 min. Cells were then washed three times with ice-cold PBS and fixed with paraformaldehyde for 5 min. The blank control consisted of untreated cells. The excitation wavelength was 488 nm, and the emission wavelength was 520 nm.

[0069] Test results as follows Figure 20 As shown, the mitochondrial dye Mito-tracker Red CMXRos is a red fluorescent probe, while the FITC-labeled sample exhibits green fluorescence. The superposition of these two fluorescences results in an orange color. Oligosaccharide CPs show significant fluorescent co-localization in mitochondria. With prolonged exposure, the green fluorescence displayed by FITC gradually intensifies, indicating an increase in sample entering the cell; the orange color in the mitochondrial region also gradually deepens after fusion. No significant changes were observed in cell morphology. This experiment confirms that oligosaccharide CPs can be localized in mitochondria, and their intracellular function is related to improving certain mitochondrial functions.

[0070] In summary, sulfated arabinoligosaccharides have a mitigating effect on pancreatic β-cell damage caused by hIAPP aggregation.

Claims

1. A sulfated arabinoligosaccharide, characterized in that, It is derived from polysaccharides of *Streptococcus linearis*; the degree of polymerization is 2-5; arabinose is the main monosaccharide, and it also contains galactose; the molar percentage of arabinose is not less than 80%, and the molar percentage of galactose is not more than 20%; the main chain is composed of (1→4)-β-L-pyranose linked together, and the sulfate group of the arabinose is mainly substituted at the C-3 position.

2. The sulfated arabinooligosaccharide according to claim 1, characterized in that, The sulfate substitution of the galactose occurs at the C-6 position.

3. The sulfated arabinooligosaccharide according to claim 1, characterized in that, The structure of arabinobiose sulfate is Ara p (3SO4)-(1→4)-β-L-Ara p .

4. The sulfated arabinoligosaccharide according to claim 1, characterized in that, The structure is as follows: Wherein, R1 = H or HSO3 - R2 = H or HSO3 - .

5. The method for preparing sulfated arabinooligosaccharides according to any one of claims 1-4, characterized in that, In order, they include: The sulfated arabinolithosaccharide was obtained by extracting polysaccharides from *Streptococcus linearis*, enriching polysaccharide components with arabinose, and controlling its degradation while maintaining the natural sulfate group substitution characteristics.

6. The preparation method according to claim 5, characterized in that, include: S1: Add distilled water to defatted Streptomyces linearis powder, heat to extract Streptomyces linearis polysaccharides, and centrifuge to collect the supernatant; S2: The supernatant is concentrated using a rotary evaporator and then desalted using a 3500Da dialysis bag to obtain a concentrated solution; S3: Add ethanol to the concentrated solution for alcohol precipitation, let stand overnight, and then collect the precipitate by centrifugation; S4: The precipitate was decolorized and dehydrated using acetone and ethanol, and then dried to obtain crude linear fibrous polysaccharide. S5: The crude polysaccharide from *Streptococcus linearis* was separated and purified using an anion exchange column to obtain arabinogalactan sulfate. S6: Add 0.05 mol / L HCl to the sulfated arabinose, heat in a water bath to degrade it, add saturated barium hydroxide solution to the degradation solution until no more precipitate is produced, centrifuge to collect the supernatant, dialyze the supernatant through a 100 Da dialysis bag, desalt it, freeze dry it to obtain an oligosaccharide mixture, and then purify the oligosaccharide mixture by gel column separation to obtain the sulfated arabinooligosaccharide.

7. The preparation method according to claim 6, characterized in that, In S1, the mass-to-volume ratio of defatted filamentous algae powder to distilled water is 1:5-10; in S3, the volume-to-volume ratio of concentrated solution to ethanol is 1:4-8.

8. A drug, characterized in that, The drug is used to inhibit the abnormal aggregation of human pancreatic islet amyloid polypeptide hIAPP, and comprises the sulfated arabinoligosaccharide according to any one of claims 1-4.

9. The medicament according to claim 8, characterized in that, The effective dose of the sulfated arabinooligosaccharide for inhibiting the abnormal aggregation of hIAPP is 10-200 μg / mL.

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