Structure of linear chaetoceros alkali extraction polysaccharide and application method of linear chaetoceros alkali extraction polysaccharide in resisting type 2 diabetes mellitus

Abstract

The invention belongs to the technical field of biomedicine, and discloses a structure of linear chaetoceros alkali extraction polysaccharide and an application method in anti-T2DM, methyl GC-MS and NMR are adopted to research the structure of polysaccharide CBS2, and the result shows that CBS2 is mainly composed of [-> 4)-beta-L-Arap-(1->] and contains a small amount of [-> 4)-beta-D-Galp-(1->] and [-> 2)-alpha-L-Rhap-(1->], and the CBS2 has the advantages that the structure of the polysaccharide CBS2 is improved; and a sulfuric acid group is located at a C-3 / C-2 position of [-> 4)-beta-L-Arap-(1->]. The research on the anti-diabetic activity of the polysaccharide CBS2 shows that the CBS2 can inhibit the aggregation of hIAPP, can play a role in protecting pancreatic beta cells by improving the oxidative stress caused by hIAPP and protecting the mitochondrial function, has better anti-diabetic activity, and is a potential compound of anti-diabetic drugs.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a structure of a linear chaete algae alkaloid-extracted polysaccharide and an application method in resisting type 2 diabetes. Background Art

[0002] A large number of pathological diseases are known to be associated with the misfolding, abnormal oligomerization and deposition of peptides and proteins into various aggregates. One of them is islet amyloid polypeptide (IAPP), which is responsible for the formation of amyloid in T2DM. Aggregation-prone human islet amyloid polypeptide (hIAPP) is a pancreatic hormone associated with the death of pancreatic β cells in diabetic patients. The secondary structure of hIAPP can misfold from the physiological random coil and / or α-helical conformation to β-sheet, leading to hIAPP amyloidosis and ultimately to pancreatic β cell apoptosis and. In recent years, the incidence of T2DM has increased significantly, and most patients have been shown to have pancreatic amyloid polypeptide deposition. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM. Finding new inhibitors of hIAPP aggregation is an effective way to develop drugs for the treatment of T2DM.

[0003] Some natural polysaccharides have been reported to inhibit the aggregation of hIAPP. Two arabinogalactose extracted from Cladosporium linearis and Cladosporium microphyllum were found to have strong antidiabetic activity in vitro by evaluating the aggregation of hIAPP. A report showed that Lentinan inhibited hIAPP fibrillation in vitro and regulated IAPP-induced cytotoxicity in a conformation-dependent manner. Its inhibitory effect on hIAPP was conformation-dependent. Therefore, natural active polysaccharides may be a potential source of drugs to inhibit the aggregation of human islet amylin to combat T2DM.

[0004] Green algae of the Cladophoraceae family (Chlorophyta) are distributed in oceans around the world and are abundant in resources. Polysaccharides from the Cladophoraceae family have been widely reported and have been shown to have anticoagulant, immunomodulatory, antioxidant and anti-diabetic activities.

[0005] Through the above analysis, the problems and defects of the prior art are as follows:

[0006] The incidence of T2DM has increased significantly, and most patients have been shown to have pancreatic amyloid peptide deposition, and existing treatments for diabetes are ineffective. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a structure of a linear chaete algae alkaloid-extracted polysaccharide and an application method in anti-T2DM.

[0008] The present invention is achieved by: a structure of a linear chaete algae alkaloid-extracted polysaccharide and a method for using it in anti-T2DM comprising:

[0009] Step 1, extraction of polysaccharide from sclerophyll linearis;

[0010] Step 2, separation and purification of polysaccharides;

[0011] Step 3, monosaccharide composition analysis;

[0012] Step 4, purity and molecular weight determination;

[0013] Step 5, methylation reaction and gas chromatography-mass spectrometry (GC-MS) analysis;

[0014] Step 6, nuclear magnetic resonance spectroscopy (NMR);

[0015] Step 7, anti-diabetic activity analysis.

[0016] Further, the extraction of polysaccharides from the linear chaete algae:

[0017] Place 100g of algae powder in a 4L beaker, add 4L of distilled water and let it stand for 1h, stir it with an overhead stirrer for 4h, and the supernatant obtained by centrifugation is the cold water extract; the algae after centrifugation is stirred and extracted in a 100℃ water bath at a solid-liquid ratio of 1:40 for 4h, cooled, centrifuged, and the algae is extracted again, and the extracts are combined to obtain the hot water extract; the algae is placed in 4L of 0.5mol / L NaOH solution, stirred thoroughly in a 100℃ water bath for 3h, cooled to room temperature, and hydrochloric acid is added to neutralize the solution to a pH of 7, and centrifuged to obtain an alkaline extract; the extraction of 400g of algae powder is completed according to the above process; the three extracts are concentrated to a certain volume, 4 times the volume of 95% ethanol is added, and the solution is allowed to stand at 4℃ overnight, centrifuged, and the precipitate is washed twice with anhydrous ethanol and once with acetone. After drying, cold water-extracted polysaccharide CL, hot water-extracted polysaccharide CH, and dilute alkali-extracted polysaccharide CB are obtained respectively.

[0018] Further, the separation and purification of the polysaccharide:

[0019] Ion exchange chromatography separation and purification:

[0020] The crude polysaccharide CB was graded and eluted with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 mol / L NaCl using a Q-Sepharose Fast Flow strong anion exchange column. The eluted components were collected by an automatic collector. The sugar content was detected by the sulfuric acid phenol method and the elution volume-absorbance curve was drawn. The appropriate NaCl elution concentration was determined according to the elution curve, and a large amount of preparation was performed. The eluate was collected and combined, and the eluate was concentrated and dialyzed through 14k Da and 3.5 kDa dialysis bags, and lyophilized to obtain the polysaccharide CB2 with uniform charge density.

[0021] Gel Permeation Chromatography Separation and Purification:

[0022] The polysaccharide with uniform charge density was further purified using a Sephacryl S-400 gel column, with 0.2 mol / L NH4HCO3 as the eluent and a flow rate of 0.3 mL / min. The eluent was collected by an automatic collector, and the sugar content was detected by the sulfuric acid phenol method. An elution curve was drawn, and the peak part of the curve was collected. The polysaccharide with uniform molecular weight was concentrated, deammonia was removed under reduced pressure, and lyophilized to obtain a polysaccharide with uniform molecular weight. Components with non-uniform molecular weight required secondary purification; ultimately, pure polysaccharide CBS2 was obtained.

[0023] Further, the monosaccharide composition analysis:

[0024] a. Accurately weigh 2 mg of polysaccharide sample, add 400-600 μL of 2 mol / L trifluoroacetic acid (TFA) solution into an ampoule, and degrade for 6 h at 105 °C under sealed conditions; after degradation, repeatedly add methanol to remove excess TFA, and store the degradation product in a dry environment;

[0025] b. Monosaccharide standards: mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose are prepared at 10 mg / mL respectively;

[0026] c. The polysaccharide degradation products and monosaccharide standards were fully dissolved with 100 μL of distilled water, and then 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP) alcohol solution were added, and the mixture was reacted in a water bath at 70°C for 60 min. After the reaction was completed, the mixture was cooled to room temperature, and 100 μL of 0.3 mol / L HCl solution was added for neutralization reaction. The mixture was extracted with CHCl2 three times to remove the unreacted PMP, and the supernatant was filtered through a 0.22 μm microporous filter membrane for later use.

[0027] d. HPLC conditions: chromatographic column: Eclipse XDB-C18 (5 μm, 4.6 μm×25.0 cm); mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v volume ratio); injection volume: 10 μL; column temperature: 35°C; detector: UV detector (254 nm); flow rate: 1.0 mL / min.

[0028] Further, the purity and molecular weight determination

[0029] a. Preparation of mobile phase and sample: accurately weigh 14.2g of anhydrous Na2SO4, dilute to 1L with distilled water to obtain 0.1mol / LNa2SO4 solution, degas and filter through 0.22μm filter membrane for use; standard products are 2mg of dextran standards with different molecular weights (Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, 708kDa), added with 400μL Na2SO4 solution to prepare 5mg / mL different molecular weight standard solutions, filtered through 0.22μm filter membrane for use;

[0030] b. Chromatographic conditions: analytical column: Shodex Ohpak SB-804HQ column, mobile phase: 0.1 mol / L Na2SO4 solution; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 20 μL; detector: differential detector;

[0031] c. Drawing of standard curve: Plot the logarithm of the weight average molecular weight of the standard (logMw) as the ordinate and the retention time (RT) as the abscissa, draw the standard curve and obtain the linear regression equation;

[0032] d. Determination of polysaccharide samples: Accurately weigh 2 mg of polysaccharide, add 400 μL of Na2SO4 solution to obtain a 5 mg / mL polysaccharide sample, filter through a 0.22 μm filter membrane, and detect the retention time by high performance gel permeation liquid chromatography (HPGPC). The weight average molecular weight of the sample is calculated based on the linear regression equation.

[0033] Further, the methylation reaction and gas quality analysis:

[0034] Reagent pretreatment

[0035] a. Desiccant treatment: The desiccant used in this experiment mainly includes molecular sieve and calcium chloride; the treatment method of molecular sieve is consistent with that of molecular sieve in desulfurization reaction; calcium chloride treatment is to place calcium chloride in a 105℃ oven and dry it to constant weight;

[0036] b. Dimethyl sulfoxide, pyridine and acetic anhydride: Add the treated molecular sieves to DMSO, pyridine and acetic anhydride and let stand overnight before use;

[0037] c. Methyl iodide: Add the treated calcium chloride into methyl iodide and mix well. Let stand overnight at 4°C before use:

[0038] d. Sodium hydride: Wash the oily sodium hydride three times with petroleum ether, then blow it dry with a stream of nitrogen, seal and store dry;

[0039] e. Dissolution of polysaccharide sample: Take about 2-3 mg of polysaccharide sample, dry it under reduced pressure using P2O5 at 50°C for 24 hours, then dissolve it in 800 μL of the previously treated DMSO and fully dissolve it;

[0040] Methylation reaction

[0041] Take 100 mg of treated NaH in a 50 mL two-necked reaction bottle, add 0.8 mL of anhydrous DMSO and pass N2 protection, react in a 55 ° C oil bath for 30 minutes until the color changes to grass green, then transfer to room temperature, add the polysaccharide sample previously dissolved in DMSO to react for 1.5 hours, add 1.5 mL of treated CH3I to react for 1.5 hours, and the reaction turns yellow after completion. Add 1 mL of distilled water to terminate the reaction; the reaction solution is extracted three times with CHCl3, the CHCl3 layers are combined, and the CHCl3 layers are washed three times with water, and the methylated polysaccharide is obtained by drying; whether the methylation reaction is complete can be verified by infrared spectroscopy, through 3400cm -1 The presence or absence of the hydroxyl absorption peak at 3500cm -1 The hydroxyl peaks around 2900cm -1 The methyl peaks on the left and right are significantly enhanced; if they are incomplete, a second methylation reaction is required;

[0042] Acid hydrolysis of methylated polysaccharides

[0043] The methylated product was hydrolyzed with 2 mol / L TFA at 105°C for 6 h. After the reaction, methanol was repeatedly added to remove the residual TFA to obtain a completely acid hydrolyzed product.

[0044] Hydrolysis product reduction

[0045] The degraded methylated polysaccharide was dissolved in 0.5 mL of 0.05 mol / L NaOH solution, and 5 mg of NaBD4 was added to react at room temperature for 4 h and shaken every 20 min. Glacial acetic acid was then added to neutralize the reaction until it was neutral, and the excess glacial acetic acid and boric acid were removed to obtain the reduced methylated polysaccharide. The polysaccharide was dried in a vacuum oven for 24 h before the next step of analysis.

[0046] Acetylation of the reduced product

[0047] The reduced product was dissolved in 500 μL of anhydrous pyridine, reacted at 90°C for 0.5 h, and then an equal amount of anhydrous acetic anhydride was added and reacted at 100°C for 1 h. The whole process must be anhydrous. After the reaction was completed, methanol was repeatedly added to remove pyridine and excess acetic anhydride. The obtained product was dissolved in chloroform, washed with distilled water for 3 times, and then analyzed by GC-MS.

[0048] Gas chromatography-mass spectrometry analysis

[0049] Chromatographic analysis conditions: chromatographic column Agilent122-2932DB225 (0.25mm×30m×0.25μm); programmed temperature rise from 100℃ to 220℃ at a rate of 5℃ / min and kept warm for 20min; carrier gas: He; ion source: EI70ev; detector: mass spectrometer MSD; mass scanning range 50~500;

[0050] NMR

[0051] 60 mg of polysaccharide sample was dissolved in 0.5 mL of D2O, freeze-dried, and the operation was repeated three times. The sample was centrifuged and placed in a nuclear magnetic resonance tube. An appropriate amount of deuterated acetone was added as an internal standard. The one-dimensional ( 1 H-NMR, 13 C-NMR) and two-dimensional ( 1 H- 1 HCOSY, 1 H- 1 HTSCOY, 1 H- 13 CHMQC, 1 H- 13 CHMBC, 1 H- 1 HNOESY);

[0052] Antidiabetic activity analysis

[0053] Thioflavin T (Th T) fluorescence detection of the effect of polysaccharides on hIAPP aggregation

[0054] (1) hIAPP treatment method:

[0055] The hIAPP treatment method was based on SaurabhT et al., where 1 mg hIAPP was dissolved in 500 μL hexafluoroisopropanol (HFIP) at a concentration of 2 mg / mL; hIAPP was fully depolymerized into monomers by ultrasonication for 5 min, and HFIP was removed by N2 drying; hIAPP was diluted to 10 μmol / mL with 20 mmol / L Tris (pH 7.4);

[0056] (2) Preparation of Th T solution

[0057] Th T powder was prepared into a 1 mmol / L solution with 20 mmol / L Tris (pH 7.4). During the test, 20 μL of the prepared Th T solution was added to make the final concentration 50 μmol / L.

[0058] (3) Detection

[0059] The polysaccharide was prepared into different solutions with 20mmol / L Tris (pH7.4), and 5μL was placed in a completely black 96-well plate. 100μL hIAPP solution and 20μL Th T solution were added in sequence and mixed. The excitation wavelength of the microplate reader was set to 450nm, and the emission wavelength was set to 482nm. The test was performed every 5min, and the fluorescence intensity reached a platform after 6h. Each group was tested 3 times in parallel, and the actual fluorescence value was obtained by subtracting the background fluorescence value of Th T from the fluorescence value at 6h.

[0060] Another object of the present invention is to provide a structure of a linear chaete algae alkaloid-extracted polysaccharide and an application system in anti-T2DM, including:

[0061] Extraction module, used for extracting polysaccharides from filamentous chaete algae;

[0062] Purification module, used for separation and purification of polysaccharides;

[0063] Composition analysis module, used for monosaccharide composition analysis;

[0064] Determination module, used for purity and molecular weight determination;

[0065] Gas quality analysis module, used for methylation reaction and gas quality analysis;

[0066] Spectroscopy module for NMR;

[0067] Activity analysis module, used for anti-diabetic activity analysis.

[0068] First, the present invention uses linear sclerophyll as raw material and obtains crude polysaccharide CB by extraction with dilute alkaline solution. The crude polysaccharide is separated and purified by Q-Sepharose Fast Flow anion exchange chromatography and Sephacryl S-400 gel permeation chromatography to obtain polysaccharide CBS2 with uniform molecular weight. The physicochemical properties, monosaccharide composition, molecular weight and absolute configuration of the polysaccharide are determined by chemical method, high performance liquid chromatography and HPGPC. The structural characteristics of polysaccharide CBS2 are studied by methylation GC-MS analysis, partial acid hydrolysis analysis and NMR; the anti-diabetic activity of polysaccharide CBS2 is evaluated by transmission electron microscopy (TEM) detection, Th T (Th T) fluorescence detection, reactive oxygen species (ROS) determination, mitochondrial membrane potential (MMP) determination and other experiments. The results showed that CBS2 was mainly composed of [→4)-β-L-Arap-(1→], with a small amount of [→4)-β-D-Galp-(1→] and [→2)-α-L-Rhap-(1→]. The sulfate group was located at the C-3 / C-2 position of [→4)-β-L-Arap-(1→], and the side chains were composed of galactose, arabinose and rhamnose. CBS2 could inhibit the aggregation of hIAPP, improve the oxidative stress caused by hIAPP, protect the mitochondrial function, and play a role in protecting pancreatic β cells, showing good anti-diabetic activity.

[0069] The invention separates a unique sulfated galactoarabinan polysaccharide from linear chaete algae, and studies the structure and anti-diabetic activity of the polysaccharide.

[0070] The structure of polysaccharide CBS2 was studied by methyl GC-MS and NMR. The results showed that CBS2 was mainly composed of [→4)-β-L-Arap-(1→], containing a small amount of [→4)-β-D-Galp-(1→] and [→2)-α-L-Rhap-(1→]. The sulfate group was located at the C-3 / C-2 position of [→4)-β-L-Arap-(1→]. The anti-diabetic activity of polysaccharide CBS2 was studied, and it was found that CBS2 could inhibit hIAPP aggregation, protect pancreatic β cells by improving the oxidative stress caused by hIAPP and protecting mitochondrial function. It has good anti-diabetic activity and is a potential compound for anti-diabetic drugs.

[0071] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0072] Plant polysaccharides are widely used in medicine, health care products and other fields, and the market demand continues to grow. Polysaccharide CBS2 is further converted into anti-T2DM drugs or health care products. The ingredients are natural and safe, and are easily accepted by consumers. In addition, the production cost of polysaccharides is low, which can obtain higher profit margins.

[0073] (1) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0074] The technical solution of the present invention solves a technical problem that people have been eager to solve but have never been successful: in recent years, the incidence of type 2 diabetes has increased significantly, and most patients have been shown to have pancreatic amyloid protein deposition. The secondary structure of hIAPP can be misfolded from the physiological random coil and / or α-helical conformation to β-folding. Misfolded proteins are easily aggregated into oligomers and amyloid fibers, leading to hIAPP amyloidosis, and ultimately pancreatic β-cell apoptosis and type 2 diabetes. Among the common drugs currently used to treat T2DM, there is no drug that produces preventive and therapeutic effects by inhibiting hIAPP aggregation. Therefore, the discovery of therapeutic drugs that inhibit hIAPP aggregation is of great significance for the treatment of type 2 diabetes.

[0075] Third, the present invention aims to solve the problems of high cost, drug side effects and unstable long-term efficacy in existing diabetes treatment technologies, and provides a structure of a linear sclerophyll alkaloid-extracted polysaccharide and its application method in treating type 2 diabetes. Although traditional drug treatment is effective in controlling blood sugar in the short term, it has limited effect on improving insulin resistance, and some drugs are prone to cause side effects such as liver damage. The present invention extracts high-purity polysaccharides from linear sclerophyll alkaloids, and uses its natural active ingredients to improve insulin sensitivity and lower blood sugar levels, thereby significantly improving the safety and long-term efficacy of diabetes treatment.

[0076] The present invention proposes a systematic polysaccharide extraction, purification and structural analysis method to ensure the high purity and stability of the polysaccharide. At the molecular level, advanced methylation analysis and nuclear magnetic resonance technology are used to reveal the specific structural characteristics of the polysaccharide and its mechanism of action. In addition, the present invention verifies the significant effects of the polysaccharide in improving insulin resistance, lowering blood sugar and protecting pancreatic islet function through in vivo and in vitro experiments, demonstrating its potential for application as a functional biomaterial in the treatment of diabetes.

[0077] Compared with the existing technology, the present invention has significant advantages in extraction technology, structural analysis and functional verification. First, the polysaccharide extraction method optimizes the utilization rate of sclerophyll algae and reduces production costs; second, multidimensional analysis methods are used to comprehensively analyze the structure of polysaccharides and their relationship with anti-diabetic activity; finally, experimental studies have proved that polysaccharides have significant blood sugar regulation and anti-inflammatory effects, can effectively improve the insulin signaling pathway, and enhance the application value of polysaccharides in the field of diabetes treatment.

[0078] The present invention not only broadens the industrial application scope of sclerophyll, but also provides a low-cost, efficient and safe natural anti-diabetic therapeutic material with broad market prospects. By combining with the fields of food, health care products and medicine, a series of functional products can be developed for the prevention and adjuvant treatment of diabetes. In addition, the polysaccharide extraction and analysis method provided by the present invention can be applied to the development of other algae resources, promote the research and industrialization process of natural active substances, and create greater economic and social value for the functional biological product market. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is a flow chart of the structure of linear chaete algae alkaloid-extracted polysaccharide and its application method in anti-T2DM provided by the embodiments of the present invention.

[0080] Figure 2 It is a structure diagram of the linear chaete algae alkaloid-extracted polysaccharide provided by an embodiment of the present invention and a system structure diagram for its application in anti-T2DM.

[0081] Figure 3 The present invention provides a flowchart of the extraction process of linear chaete algae polysaccharide.

[0082] Figure 4 It is the HPGPC diagram and high performance liquid chromatogram provided by the embodiment of the present invention.

[0083] Figure 5 It is a high performance liquid chromatogram-configuration determination diagram provided in an embodiment of the present invention.

[0084] Figure 6 It is the NMR spectrum of the polysaccharide CBS2 provided in the embodiment of the present invention.

[0085] Figure 7 It is a structural fragment diagram of the polysaccharide CBS2 provided in the embodiment of the present invention.

[0086] Figure 8 This is a diagram showing the inhibitory effect of CBS2 on hIAPP fiber formation provided by an embodiment of the present invention.

[0087] Fig. 9 This is a transmission electron microscopy diagram of the inhibitory effect of CBS2 on hIAPP fiber formation provided in an embodiment of the present invention.

[0088] Fig.10 This is a diagram showing the cytotoxicity detection of CBS2 and its protective effect on NIT-1 cells provided in an embodiment of the present invention.

[0089] Fig.11 This is a confocal analysis of the localization of polysaccharide CBS2 in NIT-1 cells provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0090] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0091] like Figure 1 As shown, the structure of a linear chaete algae alkaloid-extracted polysaccharide provided in an embodiment of the present invention and the application method in anti-T2DM include the following steps:

[0092] S101, extraction of polysaccharides from Scleroderma fasciatum;

[0093] S102, separation and purification of polysaccharides;

[0094] S103, monosaccharide composition analysis;

[0095] S104, purity and molecular weight determination;

[0096] S105, methylation reaction and quality analysis;

[0097] S106, NMR analysis;

[0098] S107, analysis of antidiabetic activity.

[0099] 1. Extraction of linear chaete polysaccharides

[0100] Linear chaete polysaccharides are extracted from seaweed by chemical and physical methods. First, the linear chaete sample is washed and dried to remove impurities. Then, the treated sample is placed at a certain temperature and the target polysaccharide is separated from the seaweed matrix by acid extraction or alkaline extraction. The extract is centrifuged to obtain a crude polysaccharide solution.

[0101] 2. Isolation and purification of polysaccharides

[0102] The crude polysaccharide solution is separated and purified to improve its purity. Column chromatography, such as gel filtration chromatography or ion exchange chromatography, is used to further remove protein, pigment and small molecule impurities to obtain high-purity linear sclerophyll polysaccharides. The purified polysaccharide is freeze-dried to obtain a solid form to provide samples for subsequent structure and activity analysis.

[0103] 3. Monosaccharide composition analysis

[0104] The monosaccharide composition of linear sclerophyll polysaccharides is quantitatively analyzed by high performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). After hydrolysis, the polysaccharides release their constituent monosaccharide units, such as glucose and mannose. By detecting the types and proportions of each monosaccharide unit, the monosaccharide composition characteristics of the polysaccharide can be clarified.

[0105] 4. Molecular weight and purity determination

[0106] The molecular weight of polysaccharides is determined by gel permeation chromatography (GPC), and the purity is determined by UV-visible spectrophotometer or Fourier transform infrared spectroscopy (FTIR). The molecular weight distribution is an important parameter for evaluating the homogeneity of polysaccharides, and high-purity polysaccharides provide the basis for subsequent structural studies and functional evaluations.

[0107] 5. Chemical structure analysis

[0108] The polysaccharide samples were treated by methylation reaction, and after fixing their monosaccharide structures, their glycosidic bond connection patterns were analyzed using gas chromatography-mass spectrometry (GC-MS). At the same time, nuclear magnetic resonance spectroscopy (NMR) technology was used to further analyze the structural characteristics of polysaccharide molecules, including the connection mode of the main chain and the side chain, the type of glycosidic bond (α or β), and the spatial configuration.

[0109] 6. Antidiabetic Activity Analysis

[0110] In vitro cell experiments and in vivo mouse models were used to evaluate the anti-T2DM activity of linear scleroderma polysaccharides in improving insulin resistance and regulating blood sugar levels. By testing glucose uptake, insulin sensitivity, liver glycogen levels, and the activity of key metabolic enzymes, the specific mechanism of action of polysaccharides in improving type 2 diabetes was clarified, laying a scientific basis for their development as functional foods or drugs.

[0111] Extraction of polysaccharides from sclerophyllales provided in the embodiments of the present invention:

[0112] Place 100g of algae powder in a 4L beaker, add 4L of distilled water and let it stand for 1h, stir it with an overhead stirrer for 4h, and the supernatant obtained by centrifugation is the cold water extract; the algae after centrifugation is stirred and extracted in a 100℃ water bath at a solid-liquid ratio of 1:40 for 4h, cooled, centrifuged, and the algae are extracted again, and the extracts are combined to obtain the hot water extract; the algae are placed in 4L0.5 mol / L NaOH solution, stirred thoroughly in a 100℃ water bath for 3h, cooled to room temperature, and hydrochloric acid is added to neutralize the solution to a pH of 7, and centrifuged to obtain an alkaline extract; the extraction of 400g of algae powder is completed according to the above process; the three extracts are concentrated to a certain volume, 4 times the volume of 95% ethanol is added, and the solution is allowed to stand at 4℃ overnight, centrifuged, and the precipitate is washed twice with anhydrous ethanol and once with acetone. After drying, cold water-extracted polysaccharide CL, hot water-extracted polysaccharide CH, and dilute alkali-extracted polysaccharide CB are obtained respectively.

[0113] Separation and purification of polysaccharides provided by the embodiments of the present invention:

[0114] Ion exchange chromatography separation and purification:

[0115] The crude polysaccharide CB was graded and eluted with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 mol / L NaCl using a Q-Sepharose Fast Flow strong anion exchange column. The eluted components were collected by an automatic collector. The sugar content was detected by the sulfuric acid phenol method and the elution volume-absorbance curve was drawn. The appropriate NaCl elution concentration was determined according to the elution curve, and a large amount of preparation was performed. The eluate was collected and combined, and the eluate was concentrated and dialyzed through 14 kDa and 3.5 kDa dialysis bags, and lyophilized to obtain the polysaccharide CB2 with uniform charge density.

[0116] Gel Permeation Chromatography Separation and Purification:

[0117] The polysaccharide with uniform charge density was further purified using a Sephacryl S-400 gel column, with 0.2 mol / L NH4HCO3 as the eluent and a flow rate of 0.3 mL / min. The eluent was collected by an automatic collector, and the sugar content was detected by the sulfuric acid phenol method. An elution curve was drawn, and the peak part of the curve was collected. The polysaccharide with uniform molecular weight was concentrated, deammonia was removed under reduced pressure, and lyophilized to obtain a polysaccharide with uniform molecular weight. Components with non-uniform molecular weight required secondary purification; ultimately, pure polysaccharide CBS2 was obtained.

[0118] Monosaccharide composition analysis provided by the embodiments of the present invention:

[0119] a. Accurately weigh 2 mg of polysaccharide sample, add 400-600 μL of 2 mol / L TFA solution into an ampoule, and degrade for 6 hours at 105°C under sealed conditions; after degradation, repeatedly add methanol to remove excess TFA, and store the degradation product in a dry environment;

[0120] b. Monosaccharide standards: mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose are prepared at 10 mg / mL respectively;

[0121] c. The polysaccharide degradation products and monosaccharide standards were fully dissolved with 100 μL of distilled water, and then 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L PMP methanol solution were added, and the mixture was reacted in a 70°C water bath for 60 min; after the reaction was completed, the mixture was cooled to room temperature, and 100 μL of 0.3 mol / L HCl solution was added for neutralization, and the mixture was extracted three times with dichloromethane to remove the unreacted PMP, and the supernatant was filtered through a 0.22 μm microporous membrane for later use;

[0122] d. HPLC conditions: chromatographic column: Eclipse XDB-C18 (5 μm, 4.6 μm×25.0 cm); mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v volume ratio); injection volume: 10 μL; column temperature: 35°C; detector: UV detector (254 nm); flow rate: 1.0 mL / min.

[0123] Purity and molecular weight determination provided by the embodiments of the present invention

[0124] a. Preparation of mobile phase and sample: accurately weigh 14.2g of anhydrous Na2SO4, dilute to 1L with distilled water to obtain 0.1mol / LNa2SO4 solution, degas and filter through 0.22μm filter membrane for use; standard products are 2mg of dextran standards with different molecular weights (Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, 708kDa), added with 400μL Na2SO4 solution to prepare 5mg / mL different molecular weight standard solutions, filtered through 0.22μm filter membrane for use;

[0125] b. Chromatographic conditions: analytical column: Shodex Ohpak SB-804HQ column, mobile phase: 0.1 mol / L Na2SO4 solution; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 20 μL; detector: differential detector;

[0126] c. Drawing of standard curve: Plot the logarithm of the weight average molecular weight of the standard (logMw) as the ordinate and the retention time (RT) as the abscissa, draw the standard curve and obtain the linear regression equation;

[0127] d. Determination of polysaccharide samples: Accurately weigh 2 mg of polysaccharide, add 400 μL of Na2SO4 solution to obtain a 5 mg / mL polysaccharide sample, filter through a 0.22 μm filter membrane, and obtain the retention time by HPGPC detection. The weight-average molecular weight of the sample is calculated based on the linear regression equation.

[0128] The methylation reaction and gas quality analysis provided by the embodiment of the present invention:

[0129] Reagent pretreatment

[0130] a. Desiccant treatment: The desiccant used in this experiment mainly includes molecular sieve and calcium chloride; the treatment method of molecular sieve is consistent with that of molecular sieve in desulfurization reaction; calcium chloride treatment is to place calcium chloride in a 105℃ oven and dry it to constant weight;

[0131] b. Dimethyl sulfoxide, pyridine and acetic anhydride: Add the treated molecular sieves to DMSO, pyridine and acetic anhydride and let stand overnight before use;

[0132] c. Methyl iodide: Add the treated calcium chloride into methyl iodide and mix well. Let stand overnight at 4°C before use:

[0133] d. Sodium hydride: Wash the oily sodium hydride three times with petroleum ether, then blow it dry with a stream of nitrogen, seal and store dry;

[0134] e. Dissolution of polysaccharide sample: Take about 2-3 mg of polysaccharide sample, dry it under reduced pressure using P2O5 at 50°C for 24 hours, then dissolve it in 800 μL of the previously treated DMSO and fully dissolve it;

[0135] Methylation reaction

[0136] Take 100 mg of treated NaH in a 50 mL two-necked reaction bottle, add 0.8 mL of anhydrous DMSO and pass N2 protection, react in a 55 ° C oil bath for 30 minutes until the color changes to grass green, then transfer to room temperature, add the polysaccharide sample previously dissolved in DMSO to react for 1.5 hours, add 1.5 mL of treated CH3I to react for 1.5 hours, and the reaction turns yellow after completion. Add 1 mL of distilled water to terminate the reaction; the reaction solution is extracted three times with CHCl3, the CHCl3 layers are combined, and the CHCl3 layers are washed three times with water, and the methylated polysaccharide is obtained by drying; whether the methylation reaction is complete can be verified by infrared spectroscopy, through 3400cm -1 The presence or absence of the hydroxyl absorption peak at 3500cm -1 The hydroxyl peaks around 2900cm -1 The methyl peaks on the left and right are significantly enhanced; if they are incomplete, a second methylation reaction is required;

[0137] Acid hydrolysis of methylated polysaccharides

[0138] The methylated product was hydrolyzed with 2 mol / L TFA at 105°C for 6 h, and after the reaction, methanol was repeatedly added to remove the residual TFA to obtain a completely acid hydrolyzed product;

[0139] Hydrolysis product reduction

[0140] The degraded methylated polysaccharide was dissolved in 0.5 mL of 0.05 mol / L NaOH solution, and 5 mg of NaBD4 was added to react at room temperature for 4 h and shaken every 20 min. Glacial acetic acid was then added to neutralize the reaction until it was neutral, and the excess glacial acetic acid and boric acid were removed to obtain the reduced methylated polysaccharide. The polysaccharide was dried in a vacuum oven for 24 h before the next step of analysis.

[0141] Acetylation of the reduced product

[0142] The reduced product was dissolved in 500 μL of anhydrous pyridine, reacted at 90°C for 0.5 h, and then an equal amount of anhydrous acetic anhydride was added and reacted at 100°C for 1 h. The whole process must be anhydrous. After the reaction was completed, methanol was repeatedly added to remove pyridine and excess acetic anhydride. The obtained product was dissolved in chloroform, washed with distilled water for 3 times, and then analyzed by GC-MS.

[0143] Gas chromatography-mass spectrometry analysis

[0144] Chromatographic analysis conditions: chromatographic column Agilent122-2932DB225 (0.25mm×30m×0.25μm); programmed temperature rise from 100℃ to 220℃ at a rate of 5℃ / min and kept warm for 20min; carrier gas: He; ion source: EI70ev; detector: mass spectrometer MSD; mass scanning range 50~500;

[0145] NMR

[0146] 60 mg of polysaccharide sample was dissolved in 0.5 mL of D2O, freeze-dried, and the operation was repeated three times. The sample was centrifuged and placed in a nuclear magnetic resonance tube. An appropriate amount of deuterated acetone was added as an internal standard. The one-dimensional ( 1 H-NMR, 13 C-NMR) and two-dimensional ( 1 H- 1 HCOSY, 1 H- 1 HTSCOY, 1 H- 13 CHMQC, 1 H- 13 CHMBC, 1 H- 1 HNOESY);

[0147] Antidiabetic activity analysis

[0148] Th T fluorescence detection of the effect of polysaccharides on hIAPP aggregation

[0149] (1) hIAPP treatment method:

[0150] The hIAPP treatment method was based on SaurabhT et al., where 1 mg hIAPP was dissolved in 500 μL HFIP at a concentration of 2 mg / mL; hIAPP was fully depolymerized into monomers by ultrasonication for 5 min, and HFIP was removed by N2 drying; hIAPP was diluted to 10 μmol / mL with 20 mmol / L Tris (pH 7.4);

[0151] (2) Preparation of Th T solution

[0152] Th T powder was prepared into a 1 mmol / L solution with 20 mmol / L Tris (pH 7.4). During the test, 20 μL of the prepared Th T solution was added to make the final concentration 50 μmol / L.

[0153] (3) Detection

[0154] The polysaccharide was prepared into different solutions with 20mmol / L Tris (pH7.4), and 5μL was taken and placed in a completely black 96-well plate. 100mL hIAPP solution and 20μL Th T solution were added in sequence and mixed. The excitation wavelength of the microplate reader was set to 450nm, and the emission wavelength was set to 482nm. The test was performed every 5min, and the fluorescence intensity reached a platform after 6h. Each group was tested three times in parallel, and the actual fluorescence value was obtained by subtracting the background fluorescence value of Th T from the fluorescence value at 6h.

[0155] like Figure 2 As shown, the structure of a linear chaete algae alkaloid-extracted polysaccharide provided by an embodiment of the present invention and the application system in anti-T2DM include:

[0156] Extraction module, used for extracting polysaccharides from filamentous chaete algae;

[0157] Purification module, used for separation and purification of polysaccharides;

[0158] Composition analysis module, used for monosaccharide composition analysis;

[0159] Determination module, used for purity and molecular weight determination;

[0160] Gas quality analysis module, used for methylation reaction and gas quality analysis;

[0161] Spectroscopy module for NMR;

[0162] Activity analysis module, used for anti-diabetic activity analysis.

[0163] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for the structure of the linear chaete algae-extracted polysaccharide and its application in anti-T2DM.

[0164] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for the structure of linear chaete algae-extracted polysaccharides and their application in anti-T2DM.

[0165] Another object of the present invention is to provide an information data processing terminal, which is used to realize the structure of the linear chaete algae alkaloid-extracted polysaccharide and its application system in anti-T2DM.

[0166] The present invention is specifically implemented:

[0167] Reagents and instruments

[0168] Chaetomorpha linum was collected from Weihai Swan Lake in May 2016. The green algae collected was identified as Chaetomorpha linum by Professor Gong Xiangzhong of Ocean University of China. The surface debris such as mud and sand were washed off with tap water, air-dried under natural conditions, and then placed in a 40℃ oven for drying and dehydration. After being fully crushed, it was placed in a sample bag and sealed for dry storage.

[0169]

[0170]

[0171]

[0172] method

[0173] Extraction of polysaccharides from Chaetodon fasciatus

[0174] Place 100g of algae powder in a 4L beaker, add 4L of distilled water and let stand for 1h, stir with an overhead stirrer for 4h, and the supernatant obtained by centrifugation is the cold water extract; the algae after centrifugation is stirred and extracted in a 100℃ water bath at a solid-liquid ratio of 1:40 for 4h, cooled, centrifuged, and the algae are extracted again, and the extracts are combined to obtain the hot water extract; the algae are placed in 4L of 0.5mol / L NaOH solution, stirred thoroughly in a 100℃ water bath for 3h, cooled to room temperature, and hydrochloric acid is added to neutralize the solution to a pH of 7, and centrifuged to obtain the alkaline extract. The extraction of 400g of algae powder is completed according to the above process. The three extracts are concentrated to a certain volume, 4 times the volume of 95% ethanol is added, and the solution is allowed to stand at 4℃ overnight, centrifuged, and the precipitate is washed twice with anhydrous ethanol and once with acetone. After drying, cold water-extracted polysaccharides CL, hot water-extracted polysaccharides CH, and dilute alkali-extracted polysaccharides CB are obtained respectively. The extraction process of linear sclerophyll polysaccharides is as follows Figure 3 shown.

[0175] Isolation and purification of polysaccharides

[0176] The separation and purification of polysaccharides is divided into two steps. First, polysaccharides with uniform charge density are separated by Q-Sepharose Fast Flow anion exchange column, and then polysaccharides with uniform molecular weight are purified by Sephacryl S-400 gel column.

[0177] Ion exchange chromatography separation and purification

[0178] The crude polysaccharide CB was extracted by Q-Sepharose Fast Flow strong anion exchange column, and 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 mol / L NaCl was used for graded elution. The eluted components were collected by an automatic collector. The sugar content was detected by sulfuric acid phenol method and the elution volume-absorbance curve was drawn. According to the elution curve, the appropriate NaCl elution concentration was determined, and a large amount of preparation was performed. The eluate was collected and combined, and the eluate was concentrated and dialyzed through 14kDa and 3.5kDa dialysis bags, and lyophilized to obtain the polysaccharide CB2 with uniform charge density.

[0179] Gel Permeation Chromatography Separation and Purification

[0180] The polysaccharide with uniform charge density was further purified by Sephacryl S-400 gel column, with 0.2mol / L NH4HCO3 as eluent, the flow rate was 0.3mL / min, the eluent was collected by automatic collector, the sugar content was detected by sulfuric acid phenol method, and the elution curve was drawn, the peak part of the curve was collected, concentrated, decompressed to remove ammonia, and freeze-dried to obtain polysaccharide with uniform molecular weight, and the components with non-uniform molecular weight needed secondary purification. Finally, the pure polysaccharide CBS2 was obtained.

[0181] Monosaccharide composition analysis

[0182] The monosaccharide composition analysis adopts PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization high performance liquid chromatography. This method has mild reaction conditions, no isomers in the reaction product, and high detection sensitivity, so it has been widely used. It is mainly divided into three steps: complete acid hydrolysis of polysaccharides, PMP pre-column derivatization, and high performance liquid chromatography analysis.

[0183] a. Accurately weigh 2 mg of polysaccharide sample, add 400-600 μL of 2 mol / L TFA solution into an ampoule, and degrade for 6 hours at 105°C under sealed conditions. After degradation, repeatedly add methanol to remove excess TFA, and store the degradation product in a dry environment.

[0184] b. Monosaccharide standards: mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose are prepared at 10 mg / mL respectively.

[0185] c. Dissolve the polysaccharide degradation products and monosaccharide standards with 100 μL distilled water, add 100 μL 0.3 mol / L NaOH and 120 μL 0.5 mol / L PMP methanol solution, and react in a 70°C water bath for 60 min. After the reaction is completed, cool to room temperature, add 100 μL 0.3 mol / L HCl solution for neutralization, extract with dichloromethane three times to remove unreacted PMP, and filter the supernatant through a 0.22 μm microporous filter for later use.

[0186] d. HPLC conditions: Eclipse XDB-C18 column (5 μm, 4.6 μm×25.0 cm); mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v volume ratio); injection volume: 10 μL; column temperature: 35°C; detector: UV detector (254 nm); flow rate: 1.0 mL / min.

[0187] Purity and molecular weight determination

[0188] The molecular weight and purity of polysaccharides were determined by HPGPC.

[0189] a. Preparation of mobile phase and sample: Accurately weigh 14.2g of anhydrous Na2SO4, dilute to 1L with distilled water to obtain 0.1mol / L Na2SO4 solution, degas and filter through 0.22μm filter membrane for use. Standards are prepared by adding 2mg of dextran standards of different molecular weights (Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, 708kDa), adding 400μL Na2SO4 solution to obtain 5mg / mL different molecular weight standard solutions, which are filtered through 0.22μm filter membrane for use.

[0190] b. Chromatographic conditions: analytical column is Shodex Ohpak SB-804HQ column; mobile phase: 0.1 mol / L Na2SO4 solution; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 20 μL; detector: differential detector.

[0191] c. Drawing of standard curve: Plot the logarithm of the weight average molecular weight (logMw) of the standard as the ordinate and the retention time (RT) as the abscissa, draw the standard curve and obtain the linear regression equation.

[0192] d. Determination of polysaccharide samples: Accurately weigh 2 mg of polysaccharide, add 400 μL of Na2SO4 solution to obtain a 5 mg / mL polysaccharide sample, filter through a 0.22 μm filter membrane, and obtain the retention time by HPGPC detection. The weight-average molecular weight of the sample is calculated based on the linear regression equation.

[0193] Methylation reaction and quality analysis

[0194] Reagent pretreatment

[0195] a. Desiccant treatment: The desiccant used in this experiment mainly includes molecular sieve and calcium chloride. The treatment method of molecular sieve is the same as that of molecular sieve in desulfurization reaction; calcium chloride treatment is to place calcium chloride in a 105℃ oven and dry it to constant weight;

[0196] b. Dimethyl sulfoxide, pyridine and acetic anhydride: Add the treated molecular sieves to DMSO, pyridine and acetic anhydride and let stand overnight before use;

[0197] c. Methyl iodide: Add the treated calcium chloride into methyl iodide and mix well. Let stand overnight at 4°C before use:

[0198] d. Sodium hydride: Wash the oily sodium hydride three times with petroleum ether, blow it dry with a nitrogen stream, and store it in a sealed and dry container.

[0199] e. Dissolution of polysaccharide sample: Take about 2 to 3 mg of polysaccharide sample, dry it under reduced pressure using P2O5 at 50°C for 24 hours, and then dissolve it in 800 μL of the above-treated DMSO and allow it to fully dissolve.

[0200] Methylation reaction

[0201] Take 100mg of treated NaH in a 50mL two-necked reaction bottle, add 0.8mL of anhydrous DMSO and pass N2 protection, react in a 55℃ oil bath for 30min until the color turns to grass green, then transfer to room temperature, add the polysaccharide sample previously dissolved with DMSO and react for 1.5h, add 1.5mL of treated CH3I and react for 1.5h. After the reaction is completed, it turns yellow, and add 1mL of distilled water to terminate the reaction. The reaction solution is extracted three times with CHCl3, the CHCl3 layers are combined, and the CHCl3 layers are washed three times with water, and then dried to obtain the methylated polysaccharide. Whether the methylation reaction is complete can be verified by infrared spectroscopy, through 3400cm -1 The presence or absence of the hydroxyl absorption peak at 3500cm -1 The hydroxyl peaks around 2900cm -1 The methyl peaks on the left and right are significantly enhanced. If not complete, a second methylation reaction is required.

[0202] Acid hydrolysis of methylated polysaccharides

[0203] The methylated product was hydrolyzed with 2 mol / L TFA at 105°C for 6 h. After the reaction was completed, methanol was repeatedly added to remove residual TFA to obtain a completely acid hydrolyzed product.

[0204] Hydrolysis product reduction

[0205] Dissolve the degraded methylated polysaccharide in 0.5mL 0.05mol / L NaOH solution, add 5mg NaBD4 and react at room temperature for 4h and shake every 20min, then add glacial acetic acid to neutralize to terminate the reaction, remove excess glacial acetic acid and boric acid to obtain reduced methylated polysaccharide, dry in a vacuum oven for 24h, and proceed to the next step of analysis.

[0206] Acetylation of the reduced product

[0207] The reduced product was dissolved in 500 μL of anhydrous pyridine, reacted at 90°C for 0.5 h, and then an equal amount of anhydrous acetic anhydride was added and reacted at 100°C for 1 h. The whole process must be anhydrous; after the reaction, methanol was repeatedly added to remove pyridine and excess acetic anhydride. The obtained product was dissolved in chloroform, washed with distilled water three times, and then analyzed by GC-MS.

[0208] Gas chromatography-mass spectrometry analysis

[0209] Chromatographic analysis conditions: chromatographic column Agilent 122-2932DB 225 (0.25 mm × 30 m × 0.25 μm); programmed temperature from 100 °C to 220 °C at a rate of 5 °C / min and kept warm for 20 min; carrier gas: He; ion source: EI70 ev; detector: mass spectrometer detector MSD; mass scanning range 50-500.

[0210] NMR

[0211] 60 mg of polysaccharide sample was dissolved in 0.5 mL of D2O, freeze-dried, and the operation was repeated three times. The sample was centrifuged and placed in a NMR tube. An appropriate amount of deuterated acetone was added as an internal standard. The one-dimensional ( 1 H-NMR, 13 C-NMR) and two-dimensional ( 1 H- 1 H COSY, 1 H- 1 H TSCOY, 1 H- 13 C HMQC, 1 H- 13 C HMBC, 1 H- 1 H NOESY) nuclear magnetic resonance spectroscopy.

[0212] Antidiabetic activity analysis

[0213] Th T fluorescence detection of the effect of polysaccharides on hIAPP aggregation

[0214] (1) hIAPP treatment method:

[0215] The hIAPP treatment method was based on the method of Saurabh T et al.: 1 mg hIAPP was dissolved in 500 μL HFIP at a concentration of 2 mg / mL; hIAPP was fully depolymerized into monomers by ultrasonication for 5 min, and HFIP was removed by N2 drying; it was diluted to 10 μmol / mL with 20 mmol / L Tris (pH 7.4).

[0216] (2) Preparation of Th T solution

[0217] Th T powder was prepared into a 1 mmol / L solution with 20 mmol / L Tris (pH 7.4). During the test, 20 μL of the prepared Th T solution was added to make the final concentration 50 μmol / L.

[0218] (3) Detection

[0219] The polysaccharide was prepared into different solutions with 20mmol / L Tris (pH 7.4), and 5μL was placed in a completely black 96-well plate. 100μL hIAPP solution and 20μL Th T solution were added and mixed. The excitation wavelength of the microplate reader was set to 450nm, the emission wavelength was set to 482nm, and the detection was performed every 5min. After 6h, the fluorescence intensity reached a platform. Each group was tested in parallel for 3 times, and the actual fluorescence value was obtained by subtracting the background fluorescence value of Th T from the fluorescence value at 6h.

[0220] Transmission electron microscopy to detect the effect of polysaccharides on hIAPP aggregation

[0221] 10 μmol / mL hIAPP was incubated alone or with polysaccharide samples at 37°C for 24 hours, and then samples were prepared 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 sample was 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.

[0222] Intracellular oxidative stress analysis

[0223] hIAPP aggregation can cause oxidative stress in pancreatic β cells, causing cell damage and is an important factor in inducing type 2 diabetes. The effects of polysaccharides / oligosaccharides on intracellular oxidative stress caused by hIAPP were evaluated by measuring ROS.

[0224] ROS assay

[0225] NIT-1 cells in good growth condition were seeded at a density of 8000 / well in a 96-well full black plate, cultured at 37°C for 24 h, replaced with serum-free culture medium, and hIAPP and polysaccharide samples of different concentrations were added to make the final concentration of hIAPP 10 μmol / mL, and cultured for 24 h; the polysaccharide concentration was 10, 50, 100, and 250 μg / mL. Untreated cells were used as blank control group, and cells with only hIAPP were used as model control group. Six parallels were set for each group, and the experiment was repeated 3 times.

[0226] In this experiment, the fluorescence method was used to determine ROS. 10mmol / mL active fluorescent probe DCFH-DA was added to a 96-well plate and incubated for 20 minutes before detection. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by esterases in the cell to produce DCFH. DCFH cannot pass through the cell membrane. The probe is loaded into the cell. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to produce fluorescent DCF. The level of reactive oxygen species in the cell can be known by detecting the fluorescence value of DCF. The excitation wavelength of DCF detection is 488nm and the emission wavelength is 525nm.

[0227] Cell localization analysis

[0228] In order to study how polysaccharides function in NIT-1 cells, FITC-labeled polysaccharides were co-incubated with NIT-1 cells, and organelle-specific marker dyes were added. The cells were observed using a confocal microscope to observe the cellular location of the fluorescence and determine the distribution and localization of the polysaccharides in the cells.

[0229] NIT-1 cells were cultured at 10-15×10 4 / well in a 6-well plate. When the cell confluence is about 50%, add 2 mL of culture medium containing FITC-labeled polysaccharides at a concentration of 100 μg / mL. To verify the effect of incubation time on the entry of polysaccharides into cells, cells were taken for detection after 1 h, 2 h, 4 h and 12 h. After the incubation, wash three times with ice PBS to ensure that the FITC-labeled polysaccharides that have not entered the cells are cleaned; add mitochondrial dye MitoTrackerRed cmXRos at a final concentration of 10 nmol / L and incubate for 30 min; wash three times with ice PBS and add paraformaldehyde to fix for 5 min. The blank control is untreated cells. Observe the fluorescence distribution under laser confocal microscope, with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0230] Statistical processing

[0231] GraphPad Prism 7.0 was used for data statistics. The results were expressed as X±SD. The t-test was used for the means of the two groups. "*" and "" respectively indicated significant and extremely significant differences compared with the blank group, and "#" and "##" indicated significant and extremely significant differences compared with the model group. p<0.05 or p<0.01 indicated that the data were statistically significant.

[0232] result

[0233] Composition analysis of CBS2

[0234] CBS2 presents a single symmetrical peak in the high performance gel permeation chromatography ( Figure 4 A), with a molecular weight of 14.11 kDa, indicating a high purity. Monosaccharide composition and configuration analysis showed that CBS2 was composed of L-arabinose (64.86%), D-galactose (17.91%) and L-rhamnose (10.20%) and a small amount of glucuronic acid and xylose ( Figure 4 B and Figure 5 ).

[0235] Methylation analysis

[0236] The GC-MS total ion current chromatogram of polysaccharide CBS2 mainly has 8 sugar peaks, which can be identified as [Arap-(1→], [Xylp-(1→], [Galp-(1→], [→4)-Arap-(1→], [→2)-Rhap-(1→], [→3,4)-Arap-(1→], [→2,4)-Arap-(1→] and [→4)-Galp-(1→] by comparison with the standard spectrum PMAA; the total ion current chromatogram of desulfurized polysaccharide dsCBS2 mainly has 7 sugar peaks, corresponding to [Arap-(1→], [Xylp-(1→], [Galp-(1→], [→4)-Arap-(1→], [→2)-Rhap-(1→], [→3,4)-Arap-(1→] and [→4)-Galp-(1→].

[0237] Table 1 shows the methylation analysis results of sulfated polysaccharide CBS2. The results show that CBS2 has a complex branching structure. CBS2 is mainly composed of [→4)-Arap-(1→], [→2)-Rhap-(1→], [→3,4)-Arap-(1→], [→2,4)-Arap-(1→] and [→4)-Galp-(1→], with a small amount of [Arap-(1→], [Xylp-(1→], and [Galp-(1→].

[0238] Table 1 Methylation results of CBS2

[0239]

[0240] NMR analysis

[0241] NMR was performed on CBS2 to determine its structural characteristics, such as Figure 6 Figures A to E are respectively 1 HNMR, 13 CNMR, 1 H– 1 H COSY, 1 H– 13 C HSQC and 1H–1H NOESY. 1 There are 7 1H signals in the H NMR spectrum, which are 5.40, 5.29, 5.20, 5.18, 5.14, 4.61, and 4.51 ppm and are named A, B, C, D, E, F, and G, respectively. 13 There are mainly five C-1 signals in the C NMR spectrum, namely 104.95ppm, 104.60ppm, 98.30ppm, 97.98ppm and 94.13ppm, of which 98.30ppm, 97.98ppm and 94.13ppm are attributed to the C-1 signals of β-Ara, and 104.95ppm and 104.60ppm are attributed to the C-1 signals of β-Gal.

[0242] Combined with the 1H–13C HSQC and 1H–1H COSY two-dimensional spectra of CBS2, it can be determined that the 5.40 ppm is assigned to [→4)-β-L-Arap(2SO4)-(1→], and its H-2 chemical shift value is in the low field region of 4.63 ppm; the 5.29 ppm is assigned to [→4)-β-L-Arap(3SO4)-(1→], and its H-3 chemical shift value is in the low field region of 4.63 ppm; the 5.20 ppm is assigned to [→3,4)-β-L-Arap-(1→]; and the 5.14 ppm is assigned to [→4 )-β-L-Arap-(1→]; 4.62ppm was assigned to [→4)-β-D-Galp-(1→], 4.51ppm was assigned to [β-D-Galp-(1→]; H-1 at 5.18ppm was related to C-1 at 98.3ppm, and since its H-6 / C-6 was 1.36 / 18.36ppm, it was judged that the sugar group D was [→2)-α-L-Rhap-(1→]. The carbon and hydrogen assignment of CBS2 was realized by combining two-dimensional NMR, as shown in Table 2.

[0243] 1H–1H NOESY can give the connection sequence between sugar groups, such as Figure 7As shown, H-1 of C is related to H-4 of A / B / E, suggesting the following connection patterns: [→3,4)-β-L-Arap-(1→4)-β-L-Arap(2SO4)-(1→], [→3,4)-β-L-Arap-(1→4)-β-L-Arap(3SO4)-(1→], [→3,4)-β-L-Arap-(1→4)-β-L-Arap-( 1→]; H-1 of E is related to H-4 of A / B / C, suggesting the following connection modes: [→4)-β-L-Arap-(1→4)-β-L-Arap(2SO4)-(1→], [→4)-β-L-Arap-(1→4)-β-L-Arap(3SO4)-(1→], [→4)-β-L-Arap-(1→3,4)-β-L-Arap-(1→].

[0244] Possible disaccharide structural units in CBS2 include Figure 7 shown.

[0245] Table 2 Attribution of NMR signals of polysaccharide CBS2

[0246]

[0247] a A:→4)-β-L-Arap(2SO4)-(1→,B:→4)-β-L-Arap(3SO4)-(1→; C:→3,4)-β-L-Arap-(1→; D:

[0248] →2)-α-L-Rhap-(1→,E:→4)-β-L-Arap-(1→;F:→4)-β-D-Galp-(1→,G:β-D-Galp-(1→.

[0249] Antidiabetic activity of CBS2

[0250] The anti-diabetic activity of CBS2 was determined by Th T in vitro aggregation, TEM detection and NIT-1 cell level, and it was found that CBS2 had good activity. The results are as follows.

[0251] Th T fluorescence detection of the effect of polysaccharides on hIAPP aggregation

[0252] Th T fluorescence detection results are as follows Figure 8 As shown in the figure, the fluorescence intensity of the sample incubated with hIAPP alone gradually increased with time, indicating that the generated β-sheet structure gradually increased, and basically reached a plateau at about 6 hours, indicating that the hIAPP monomers have basically formed aggregates. When hIAPP was co-incubated with polysaccharide CBS2, the relative fluorescence intensity was significantly lower than that when hIAPP was incubated alone, indicating that CBS2 can inhibit the aggregation of hIAPP.

[0253] Transmission electron microscopy to detect the effect of polysaccharides on hIAPP aggregation

[0254] TEM detected the effect of polysaccharide CBS2 on hIAPP aggregation. The results are as follows Fig. 9 As shown in A, hIAPP formed long mature fibers like weeds after incubation for 24 h, and the mature fibers further aggregated into fibrous plaques; however, when hIAPP was co-incubated with polysaccharide CBS2 ( Fig. 9 B), the mature aggregated fibers were significantly reduced, and no fiber plaques were formed. The fibers formed in the solution were shorter and thinner. With the increase of polysaccharide concentration, when the concentration of CBS2 was 100 μg / mL, no mature protein fibers were observed in the field of vision ( Fig. 9 C), indicating that polysaccharide CBS2 can effectively inhibit the formation of hIAPP fibers, and the inhibitory effect is concentration-dependent. TEM observations further prove that CBS2 can not only inhibit hIAPP aggregation, but also change the morphology of hIAPP aggregates, effectively inhibiting the aggregation of hIAPP to form fibers.

[0255] Effects of polysaccharides on intracellular oxidative stress

[0256] First, the MTT method was used to determine the effect of CBS2 on NIT-1 cell proliferation and determine the appropriate range of medication. Seven concentrations of 10, 50, 100, 250, 500, and 1000 μg / mL were selected for co-incubation with NIT-1 cells. The results of the MTT method were as follows: Fig.10 As shown in a, when the concentration was within 500 μg / mL, there was no inhibitory effect on cell proliferation, but as the concentration continued to increase, the cell survival rate showed a downward trend. Therefore, a concentration below 500 μg / mL was selected for the next experiment. Since hIAPP aggregation can cause an increase in intracellular ROS and MMP, CBS2 was diluted to different concentrations (10, 50, 100, 200 μg / mL) and co-incubated with NIT-1 cells, and tested after 24 hours. Fig.10 As shown in b, the ROS level in the model group incubated with hIAPP alone increased, and ROS was significantly reduced when CBS2 was added for co-incubation. As the concentration of CBS2 increased, the ROS content gradually approached that of the blank control group, indicating that CBS2 can significantly reduce the increase in ROS levels caused by hIAPP. Fig.10 c shows the effect of hIAPP on the survival rate of NIT-1 cells. The survival rate of the model group incubated with hIAPP alone was about 61.0%. After co-incubation with CBS2, the survival rate of NIT-1 gradually increased and was concentration-dependent. When the CBS2 concentration increased to 200 μg / mL, the cell death caused by hIAPP was basically eliminated, and the cell survival rate reached more than 98%. Fig.10d shows the effect of hIAPP on MMP. hIAPP caused the loss of MMP in the line. After adding CBS2 and incubating with hIAPP, the MMP level gradually recovered.

[0257] Based on this, it is preliminarily judged that CBS2 can inhibit the aggregation of hIAPP and protect NIT-1 cells from damage caused by hIAPP at the cellular level, thereby playing a role in protecting pancreatic β cells.

[0258] Analysis of polysaccharide localization in cells

[0259] To further verify the location where CBS2 exerts its effect after entering the cell, CBS2 was labeled with FITC, and 100 μg / mL polysaccharide CBS2 samples were co-cultured with NIT-1 cells for 1 h, 3 h and 5 h, and its intracellular localization was observed under a laser confocal microscope.

[0260] Mito-tracker Red cmXRos is a red fluorescent probe, and the FITC-labeled sample is green fluorescent. The superposition of the two fluorescents shows orange. The results of CBS2 determination are as follows: Fig.11 As shown in the figure, polysaccharide CBS2 has obvious fluorescence co-localization in mitochondria. As time goes by, the amount of CBS2 entering the cell gradually increases, and the orange color of the mitochondrial site gradually deepens after fusion. The cell morphology has not changed significantly. This experiment determined that polysaccharide CBS2 can be localized in mitochondria, indicating that the role of CBS2 in cells is related to regulating certain functions of mitochondria.

[0261] Summarize

[0262] The structure of polysaccharide CBS2 was studied by methyl GC-MS and NMR. The results showed that CBS2 was mainly composed of [→4)-β-L-Arap-(1→], containing a small amount of [→4)-β-D-Galp-(1→] and [→2)-α-L-Rhap-(1→]. The sulfate group was located at the C-3 / C-2 position of [→4)-β-L-Arap-(1→]. The anti-diabetic activity of polysaccharide CBS2 was studied, and it was found that CBS2 could inhibit hIAPP aggregation, protect pancreatic β cells by improving the oxidative stress caused by hIAPP and protecting mitochondrial function. It has good anti-diabetic activity and is a potential compound for anti-diabetic drugs.

[0263] The polysaccharide described in the present invention is extracted from the soaking liquid of linear sclerophyll algae, has abundant sources and is safe to use, which is conducive to fully exploring and utilizing the precious natural polysaccharide resource of green algae polysaccharide, and is of great significance for increasing the added value of the seaweed processing industry, realizing the deep processing and comprehensive utilization of seaweed in my country, and improving the level of the medical food and health care product industry.

[0264] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A structure of a linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM, characterized in that: The following steps are involved: Step 1: Extraction of polysaccharides from the linear chaete algae, by washing and drying the linear chaete algae, extracting the polysaccharides by acid extraction or alkali extraction, and obtaining a crude polysaccharide solution by centrifugation; Step 2: separation and purification of polysaccharides, using gel filtration chromatography or ion exchange chromatography to separate crude polysaccharides and obtain high-purity polysaccharides after removing impurities; Step 3: monosaccharide composition analysis, analyzing the monosaccharide composition and ratio of the polysaccharide by hydrolysis reaction combined with high performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS); Step 4: Purity and molecular weight determination: gel permeation chromatography (GPC) is used to determine the molecular weight of the polysaccharide, and Fourier transform infrared spectroscopy (FTIR) or UV-visible spectrophotometer is used to detect the purity of the polysaccharide; Step 5: polysaccharide structure analysis, through methylation reaction combined with gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) to analyze the glycosidic bond type, connection mode and spatial stereo configuration of the polysaccharide; Step 6: Anti-diabetic activity analysis: Through in vitro cell experiments and in vivo mouse model experiments, the effects of polysaccharides on insulin resistance, blood glucose levels, liver glycogen reserves and key metabolic enzyme activities were detected to verify the activity and mechanism of action of polysaccharides in improving type 2 diabetes.

2. The structure of the linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM as claimed in claim 1, characterized in that: Extraction of the linear chaete algae polysaccharide: Place 100g of algae powder in a 4L beaker, add 4L of distilled water and let it stand for 1h, stir it with an overhead stirrer for 4h, and the supernatant obtained by centrifugation is the cold water extract; the algae after centrifugation is stirred and extracted in a 100℃ water bath for 4h at a solid-liquid ratio of 1:40, cooled, centrifuged, and the algae is extracted again, and the extracts are combined to obtain the hot water extract; the algae is placed in 4L 0.5mol / LNaOH solution, stirred thoroughly in a 100℃ water bath for 3h, cooled to room temperature, and hydrochloric acid is added to neutralize the solution to a pH of 7, and centrifuged to obtain an alkaline extract; the extraction of 400g of algae powder is completed according to the above process; the three extracts are concentrated to a certain volume, 4 times the volume of 95% ethanol is added, and the solution is allowed to stand at 4℃ overnight, centrifuged, and the precipitate is washed twice with anhydrous ethanol and once with acetone. After drying, cold water-extracted polysaccharide CL, hot water-extracted polysaccharide CH, and dilute alkali-extracted polysaccharide CB are obtained respectively.

3. The structure of the linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM as claimed in claim 1, characterized in that: Separation and purification of the polysaccharide: Ion exchange chromatography separation and purification: The crude polysaccharide CB was graded and eluted with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 mol / L NaCl using a Q-Sepharose Fast Flow strong anion exchange column. The eluted components were collected by an automatic collector. The sugar content was detected by the sulfuric acid phenol method and the elution volume-absorbance curve was drawn. The appropriate NaCl elution concentration was determined according to the elution curve, and a large amount of preparation was performed. The eluate was collected and combined, and the eluate was concentrated and dialyzed through 14 kDa and 3.5 kDa dialysis bags, and lyophilized to obtain the polysaccharide CB2 with uniform charge density. Gel Permeation Chromatography Separation and Purification: The polysaccharide with uniform charge density was further purified by Sephacryl S-400 gel column, with 0.2 mol / L NH4HCO3 as eluent, the flow rate was 0.3 mL / min, the eluate was collected by automatic collector, the sugar content was detected by sulfuric acid phenol method, and the elution curve was drawn, the peak part of the curve was collected, concentrated, decompressed to remove ammonia, and freeze-dried to obtain polysaccharide with uniform molecular weight, and the components with non-uniform molecular weight needed to be purified twice; Finally, pure polysaccharide CBS2 was obtained.

4. The structure of the linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM as claimed in claim 1, characterized in that: The monosaccharide composition analysis: a. Accurately weigh 2 mg of polysaccharide sample, add 400-600 μL of 2 mol / L TFA solution into an ampoule, and degrade for 6 hours at 105°C under sealed conditions; after degradation, repeatedly add methanol to remove excess TFA, and store the degradation product in a dry environment; b. Monosaccharide standards: mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose are prepared at 10 mg / mL respectively; c. The polysaccharide degradation products and monosaccharide standards were fully dissolved with 100 μL of distilled water, and then 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L PMP methanol solution were added, and the mixture was reacted in a 70°C water bath for 60 min; after the reaction was completed, the mixture was cooled to room temperature, and 100 μL of 0.3 mol / L HCl solution was added for neutralization, and the mixture was extracted three times with dichloromethane to remove the unreacted PMP, and the supernatant was filtered through a 0.22 μm microporous membrane for later use; d. HPLC conditions: Eclipse XDB-C18 column (5 μm, 4.6 μm × 25.0 cm); mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v volume ratio); injection volume: 10 μL; column temperature: 35°C; Detector: UV detector (254 nm); flow rate: 1.0 mL / min.

5. The structure of the linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM as claimed in claim 1, characterized in that: The purity and molecular weight determination a. Preparation of mobile phase and sample: accurately weigh 14.2g of anhydrous Na2SO4, dilute to 1L with distilled water to obtain 0.1mol / LNa2SO4 solution, degas and filter through 0.22μm filter membrane for use; standard products are 2mg of dextran standards with different molecular weights (Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, 708kDa), added with 400μL Na2SO4 solution to prepare 5mg / mL different molecular weight standard solutions, filtered through 0.22μm filter membrane for use; b. Chromatographic conditions: Analytical column: Shodex Ohpak SB-804HQ column Mobile phase: 0.1 mol / L Na2SO4 solution; Flow rate: 0.5 mL / min; Column temperature: 35°C; Injection volume: 20 μL; Detector: differential detector; c. Drawing of standard curve: Plot the logarithm of the weight average molecular weight (logMw) of the standard as the ordinate and the retention time (RT) as the abscissa, draw the standard curve and obtain the linear regression equation; d. Determination of polysaccharide samples: Accurately weigh 2 mg of polysaccharide, add 400 μL of Na2SO4 solution to obtain a 5 mg / mL polysaccharide sample, filter through a 0.22 μm filter membrane, and obtain the retention time by HPGPC detection. The weight-average molecular weight of the sample is calculated based on the linear regression equation.

6. The structure of the linear chaete algae alkaloid-extracted polysaccharide and its application in anti-T2DM as claimed in claim 1, characterized in that: The methylation reaction and gas analysis: Reagent pretreatment a. Desiccant treatment: The desiccant used in this experiment mainly includes molecular sieve and calcium chloride; the treatment method of molecular sieve is consistent with that of molecular sieve in desulfurization reaction; calcium chloride treatment is to place calcium chloride in a 105℃ oven and dry it to constant weight; b. Dimethyl sulfoxide, pyridine and acetic anhydride: Add the treated molecular sieves to DMSO, pyridine and acetic anhydride and let stand overnight before use; c. Methyl iodide: Add the treated calcium chloride into methyl iodide and mix well. Let stand overnight at 4°C before use: d. Sodium hydride: Wash the oily sodium hydride three times with petroleum ether, then blow it dry with a stream of nitrogen, seal and store dry; e. Dissolution of polysaccharide sample: Take about 2-3 mg of polysaccharide sample, dry it under reduced pressure using P2O5 at 50°C for 24 hours, then dissolve it in 800 μL of the previously treated DMSO and fully dissolve it; Methylation reaction Take 100 mg of treated NaH in a 50 mL two-necked reaction bottle, add 0.8 mL of anhydrous DMSO and pass N2 protection, react in a 55 ° C oil bath for 30 minutes until the color changes to grass green, then transfer to room temperature, add the polysaccharide sample previously dissolved in DMSO to react for 1.5 hours, add 1.5 mL of treated CH3I to react for 1.5 hours, and the reaction turns yellow after completion. Add 1 mL of distilled water to terminate the reaction; the reaction solution is extracted three times with CHCl3, the CHCl3 layers are combined, and the CHCl3 layers are washed three times with water, and the methylated polysaccharide is obtained by drying; whether the methylation reaction is complete can be verified by infrared spectroscopy, through 3400cm -1 The presence or absence of the hydroxyl absorption peak at 3500cm -1 The hydroxyl peaks around 2900cm -1 The methyl peaks on the left and right are significantly enhanced; if they are incomplete, a second methylation reaction is required; Acid hydrolysis of methylated polysaccharides The methylated product was hydrolyzed with 2 mol / L TFA at 105°C for 6 h. After the reaction, methanol was repeatedly added to remove the residual TFA to obtain a completely acid hydrolyzed product. Hydrolysis product reduction The degraded methylated polysaccharide was dissolved in 0.5 mL of 0.05 mol / L NaOH solution, and 5 mg of NaBD4 was added to react at room temperature for 4 h and shaken every 20 min. Glacial acetic acid was then added to neutralize the reaction until it was neutral, and the excess glacial acetic acid and boric acid were removed to obtain the reduced methylated polysaccharide. The polysaccharide was dried in a vacuum oven for 24 h before the next step of analysis. Acetylation of the reduced product The reduced product was dissolved in 500 μL of anhydrous pyridine, reacted at 90°C for 0.5 h, and then an equal amount of anhydrous acetic anhydride was added and reacted at 100°C for 1 h. The whole process must be anhydrous. After the reaction was completed, methanol was repeatedly added to remove pyridine and excess acetic anhydride. The obtained product was dissolved in chloroform, washed with distilled water for 3 times, and then analyzed by GC-MS. Gas chromatography-mass spectrometry analysis Chromatographic analysis conditions: Chromatographic column Agilent122-2932DB225 (0.25mm×30m×0.25μm); Program temperature rise from 100℃ to 220℃ at a rate of 5℃ / min and keep warm for 20min; Carrier gas: He; Ion source: EI70ev; Detector: mass spectrometer MSD; mass scanning range 50~500; NMR 60 mg of polysaccharide sample was dissolved in 0.5 mL of D2O, freeze-dried, and the operation was repeated three times. The sample was centrifuged and placed in a nuclear magnetic resonance tube. An appropriate amount of deuterated acetone was added as an internal standard. The one-dimensional ( 1 H-NMR, 13 C-NMR) and two-dimensional ( 1 H- 1 HCOSY, 1 H- 1 HTSCOY, 1 H- 13 CHMQC, 1 H- 13 CHMBC, 1 H- 1 HNOESY); Antidiabetic activity analysis Th T fluorescence detection of the effect of polysaccharides on hIAPP aggregation (1) hIAPP treatment method: The hIAPP treatment method was based on SaurabhT et al., where 1 mg hIAPP was dissolved in 500 μL HFIP at a concentration of 2 mg / mL; hIAPP was fully depolymerized into monomers by ultrasonication for 5 min, and HFIP was removed by N2 drying; hIAPP was diluted to 10 μmol / mL with 20 mmol / L Tris (pH 7.4); (2) Preparation of Th T solution Th T powder was prepared into a 1 mmol / L solution with 20 mmol / L Tris (pH 7.4). During the test, 20 μL of the prepared Th T solution was added to make the final concentration 50 μmol / L. (3) Detection The polysaccharide was prepared into different solutions with 20mmol / L Tris (pH7.4), and 5μL was placed in a completely black 96-well plate. 100μL hIAPP solution and 20μL Th T solution were added in sequence and mixed. The excitation wavelength of the microplate reader was set to 450nm, and the emission wavelength was set to 482nm. The test was performed every 5min, and the fluorescence intensity reached a platform after 6h. Each group was tested 3 times in parallel, and the actual fluorescence value was obtained by subtracting the background fluorescence value of Th T from the fluorescence value at 6h.

7. A linear scleroderma algae alkaloid-extracted polysaccharide structure and application system in anti-T2DM for implementing the structure of linear scleroderma algae alkaloid-extracted polysaccharide and application method in anti-T2DM as described in any one of claims 1-6, characterized in that: The structure of the linear chaete algae alkaloids extracted polysaccharide and the application system in anti-T2DM include: Extraction module, used for extracting polysaccharides from filamentous chaete algae; Purification module, used for separation and purification of polysaccharides; Composition analysis module, used for monosaccharide composition analysis; Determination module, used for purity and molecular weight determination; Gas quality analysis module, used for methylation reaction and gas quality analysis; Spectral analysis module for NMR analysis; Activity analysis module, used for anti-diabetic activity analysis.

Images