Sargassum polysaccharide and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and in particular, to a Sargassum polysaccharide and a preparation method and application thereof. The Sargassum polysaccharide, as shown by the monosaccharide composition results, is mainly composed of fucose, guluronic acid and mannuronic acid; the molar ratio of the fucose, the guluronic acid and the mannuronic acid is 1.00:1.08:1.91. The Sargassum polysaccharide is an acidic polysaccharide that has the effects of lowering blood sugar and blood lipids at the same time, and can be used in the field of preparing drugs for treating hyperlipidemia and hyperglycemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to Sargassum polysaccharide and a preparation method and application thereof. Background Art

[0002] In recent years, the prevalence of hyperglycemia and hyperlipidemia has shown an increasing trend year by year, and the proportion of young and middle-aged patients is increasing, which may be related to a variety of factors, including bad eating habits, lack of exercise, obesity, smoking, alcoholism, etc. Continuous high blood sugar and high blood lipids often cause the body to have difficulty in utilizing basic nutrients, which in turn leads to systemic tissue lesions, such as kidney lesions, eye diseases, liver diseases, gastrointestinal diseases, nervous system lesions, etc.

[0003] Drugs for treating hyperlipidemia mainly include statins, fibrates, niacin, cholesterol absorption inhibitors and Chinese patent medicines. Drugs for treating hyperglycemia mainly include metformin, sulfonylureas, glinides, glycosidase inhibitors, DPP-4 inhibitors, etc. However, these drugs all have certain side effects (such as causing muscle pain, abnormal liver function, indigestion, skin flushing, gastrointestinal discomfort, abdominal pain and diarrhea, allergic reactions, etc.), and the application of some drugs is limited to certain groups of people. Therefore, it is of great significance to develop safer and more effective blood sugar and lipid-lowering drugs or functional foods.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a Sargassum polysaccharide and a preparation method and application thereof. The Sargassum polysaccharide has the effects of lowering blood lipids and blood sugar, and can be used to prepare drugs for treating and / or preventing hyperlipidemia and hyperglycemia.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a Sargassum polysaccharide. The monosaccharide composition results show that the Sargassum polysaccharide is mainly composed of fucose, guluronic acid and mannuronic acid;

[0008] The molar ratio of the fucose, the guluronic acid and the mannuronic acid is 1.00:1.08:1.91.

[0009] Some polysaccharides from natural sources have the characteristics of no toxic side effects and multi-target pharmacological effects, which have attracted the interest and attention of many scientific researchers. Based on the previous research accumulation of the inventor, Sargassum polysaccharides have good hypoglycemic and hypolipidemic effects and good biological safety.

[0010] The Sargassum polysaccharide is an acidic polysaccharide, which has the effects of lowering blood sugar and blood lipids at the same time, and can be used in the field of preparing drugs for treating hyperlipidemia and hyperglycemia.

[0011] Furthermore, the Sargassum polysaccharide was methylated and analyzed by GC-MS, and the presence of Fucp-(1→, →3)-Fucp-(1→, Gulp-(1→, →2,3)-Fucp-(1→, Manp-(1→, →4)-Manp-(1→ and →2,3)-Gulp-(1→ was detected.

[0012] On the basis of methylation, the main chain connection mode and the terminal group connection mode of the Sargassum polysaccharide were analyzed by one-dimensional and two-dimensional nuclear magnetic resonance spectra.

[0013] Furthermore, the main chain connection mode of the Sargassum polysaccharide is as follows:

[0014] →3)-α-Fucp-(1→3)-β-Fucp-1→3)-β-Fucp-(1→[4)-β-ManA-(1] 12 →[3)-α-GulA-(1) 2 →.

[0015] Furthermore, the terminal groups of the Sargassum polysaccharide include: β-ManA-(1→), α-Fucp-(1→) and α-GulA-(1→.

[0016] Furthermore, β-ManA-(1→) was connected to the main chain via O-2 of (→2,3)-β-Fucp-(1→).

[0017] Furthermore, α-Fucp-(1→) is connected to the main chain via (→2,3)-β-Fucp-(1→O-2).

[0018] Furthermore, α-GulA-(1→→2,3)-α-GulA-(1→O-2 is connected to the main chain.

[0019] On the basis of the main chain connection mode and the end group connection mode, the repeating unit structure of Sargassum polysaccharide can be obtained.

[0020] Furthermore, the structural formula of the repeating unit of the Sargassum polysaccharide is as follows:

[0021]

[0022] The purified Sargassum polysaccharide was subjected to preliminary structural characterization, and the molecular weight of the Sargassum polysaccharide was 24.11 kDa.

[0023] Another aspect of the present invention also relates to a method for preparing the Sargassum polysaccharide, comprising the following steps:

[0024] (a) subjecting the Sargassum soaked in an alkali solution to a high-voltage pulse electric field treatment, a first centrifugation and a concentration to obtain a concentrated solution;

[0025] (b) adding an organic solvent to the concentrated solution, performing a first stirring treatment and a second centrifugation to collect a lower layer of precipitate;

[0026] (c) adding an acid solution to the precipitate, performing a second stirring treatment and a third centrifugation to obtain a supernatant; adjusting the pH of the supernatant to neutral to obtain an extract; performing a first ultrafiltration and a first drying on the extract to obtain macromolecular substances and obtain a crude extract;

[0027] (d) subjecting the mixed solution containing the crude extract to ball milling, second ultrafiltration and second drying to obtain macromolecular substances to obtain crude Sargassum polysaccharides; and subjecting the solution containing the crude Sargassum polysaccharides to anion exchange chromatography and dialysis.

[0028] Furthermore, the alkali solution includes but is not limited to: NaOH solution.

[0029] Furthermore, the concentration of the alkali solution is 0.1 mol / L.

[0030] Furthermore, the soaking time is 4 hours.

[0031] Furthermore, the ratio of the Sargassum to the alkali solution is 1 g:12 mL.

[0032] Under certain conditions, soaking in alkaline solution can quickly destroy the cell structure and fully release the effective components in the cells, which is beneficial to the extraction of Sargassum polysaccharides and can greatly improve the efficiency of subsequent material-liquid separation. High-voltage pulsed electric field has the same effect and can also improve the extraction efficiency.

[0033] Furthermore, the number of pulses of the high-voltage pulse electric field treatment is 5 to 10, including but not limited to 5, 6, 7, 8, 9 or 10.

[0034] Furthermore, the electric field intensity of the high-voltage pulse electric field treatment is 15 to 20 kV / cm, including but not limited to 15 kV / cm, 16 kV / cm, 17 kV / cm, 18 kV / cm, 19 kV / cm or 20 kV / cm.

[0035] Furthermore, the high voltage pulse electric field treatment is repeated 1 to 3 times.

[0036] Furthermore, the speed of the first centrifugation is 5800-6000 rpm, including but not limited to 5800, 5850, 5900 rpm, 5950 rpm or 6000 rpm.

[0037] Furthermore, the first centrifugation time is 15 to 20 min, including but not limited to 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0038] Furthermore, the solid content of the concentrate is 10wt% to 20wt%, including but not limited to 10wt%, 12wt%, 14wt%, 16wt%, 18wt% or 20wt%.

[0039] Furthermore, the organic solvent includes but is not limited to: anhydrous ethanol.

[0040] Furthermore, the volume ratio of the concentrate to the organic solvent is 1:3-5, including but not limited to 1:5, 1:4 or 1:5.

[0041] Furthermore, the temperature of the first stirring treatment is 2-6°C, including but not limited to 2°C, 3°C, 4°C, 5°C or 6°C.

[0042] Furthermore, the first stirring treatment time is 10 to 14 hours, including but not limited to 10 hours, 11 hours, 12 hours, 13 hours or 14 hours.

[0043] Further, the speed of the second centrifugation is 5800-6200 rpm, including but not limited to 5800 rpm, 5850 rpm, 5900 rpm, 5950 rpm, 6000 rpm, 6050 rpm, 6100 rpm, 6150 rpm or 6200 rpm.

[0044] Furthermore, the temperature of the second centrifugation is 22-28°C, including but not limited to 22°C, 24°C, 26°C or 28°C.

[0045] Furthermore, the second centrifugation time is 8 to 12 minutes, including but not limited to 8 minutes, 9 minutes, 10 minutes, 11 minutes or 12 minutes.

[0046] Furthermore, the volume ratio of the lower precipitate to the acid solution is 1:2-4, including but not limited to 1:2, 1:3 or 1:4.

[0047] Furthermore, the acid solution includes but is not limited to: hydrochloric acid solution.

[0048] Furthermore, the concentration of the acid solution is 0.5 to 2 mol / L, including but not limited to 0.5 mol / L, 1 mol / L, 1.5 mol / L or 2 mol / L.

[0049] Furthermore, the temperature of the second stirring treatment is 45-55°C, including but not limited to 45°C, 47°C, 49°C, 51°C, 53°C or 55°C.

[0050] Furthermore, the second stirring treatment time is 2 to 4 hours, including but not limited to 2 hours, 3 hours or 4 hours.

[0051] Furthermore, the condition of the third centrifugation is 5800-6200 rpm, including but not limited to 5800 rpm, 5850 rpm, 5900 rpm, 5950 rpm, 6000 rpm, 6050 rpm, 6100 rpm, 6150 rpm or 6200 rpm.

[0052] Further, the pH of the supernatant is adjusted to 7.0-7.5, including but not limited to 7.0, 7.2, 7.4 or 7.5.

[0053] Furthermore, the molecular cut-off of the first ultrafiltration is 3 kDa. The molecular cut-off of the first ultrafiltration is designed according to the molecular weight of Sargassum polysaccharide.

[0054] Furthermore, the first ultrafiltration is repeated 4 to 6 times.

[0055] Furthermore, the ball milling time is 44 to 52 hours, including but not limited to any one of 44 hours, 46 hours, 48 ​​hours, 50 hours or 52 hours or a range between any two of them.

[0056] Furthermore, the molecular cut-off of the second ultrafiltration is 3 kDa. The molecular cut-off of the second ultrafiltration is designed according to the molecular weight of Sargassum polysaccharide.

[0057] Furthermore, the second ultrafiltration is repeated 4 to 6 times.

[0058] Furthermore, the anion exchange chromatography column of the anion exchange chromatography includes but is not limited to: DEAE-cellulose, with an inner diameter of 6.0 cm and a height of 28 cm.

[0059] Further, the anion exchange chromatography comprises:

[0060] The first eluent, the second eluent, the third eluent, the fourth eluent, the fifth eluent, the sixth eluent and the seventh eluent are used for elution in sequence, and the sugar-containing solution eluted by the third eluent is collected for dialysis.

[0061] Furthermore, the first eluent includes 0 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the second eluent includes 0.2 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the third eluent includes 0.4 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the fourth eluent includes 0.6 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the fifth eluent includes 0.8 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the sixth eluent includes 1 mol / L NaCl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3; the seventh eluent includes 2 mol Nacl solution and 0.01 mol / L NaOH solution in a volume ratio of 1:3.

[0062] Anion exchange chromatography is carried out according to certain steps and can accurately collect and obtain Sargassum polysaccharides.

[0063] Furthermore, the eluent flow rate of the anion exchange chromatography is 0.8 to 1.2 mL / min, including but not limited to 0.8, 0.9, 1.0, 1.1 or 1.2.

[0064] Furthermore, the molecular weight cut-off of the dialysis is 500Da.

[0065] Furthermore, the dialysis time is 24 hours.

[0066] Another aspect of the present invention also relates to the use of the Sargassum polysaccharide in the preparation of drugs for lowering blood lipids and / or blood sugar.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) The Sargassum polysaccharide provided by the present invention has high purity and clear structural characteristics, and has the effects of lowering blood sugar and blood lipids, with small toxic and side effects, and can be used in the field of preparing drugs for treating hyperlipidemia and hyperglycemia.

[0069] (2) The present invention found that the single administration of Sargassum polysaccharide (SCP-B) can significantly reduce the fat content in Caenorhabditis elegans by establishing a high-fat nematode model and Pdhs::dhs-3::GFP Caenorhabditis elegans. The single administration of SCP-B can significantly reduce the glycogen content in Caenorhabditis elegans by establishing a high-fat nematode model, and it is also confirmed that the administration of SCP-B has no side effects on Caenorhabditis elegans. By measuring the inhibitory effect of SCP-B on Escherichia coli OP50, it is confirmed that the presence of SCP-B and SCP-B at different concentrations do not affect the amount of food for nematodes and reduce food intake, thereby reducing the fat content in the nematodes. By establishing a high-fat nematode model, it is found that the single administration of SCP-B has little effect on the movement ability of nematodes. Through the nematode tropism experiment, it is found that the presence of 1.0 mg / mL of SCP-B has basically no effect on the food preference of nematodes, which excludes the possibility that SCP-B reduces the food preference of nematodes and affects the feeding of nematodes, thereby reducing the fat content in nematodes.

[0070] (3) The present invention has confirmed the potential of Sargassum polysaccharide SCP-B to prevent blood sugar from rising due to eating through starch loading experiments; the db / db diabetic mouse experiment further confirmed the experimental results of the nematode model, and SCP-B intervention can significantly reduce the blood sugar and blood lipid levels of diabetic mice, reduce insulin resistance, and increase the level of glucagon-like peptide (GLP-1). In addition, it can also repair liver and kidney damage complicated by high blood sugar and high blood lipids.

[0071] (4) The preparation method of Sargassum polysaccharide provided by the present invention overcomes the defects of the traditional method such as strong water absorption of algae, difficulty in separating the material and liquid, and low extraction efficiency. The method is simple and easy to operate without relying on complicated processes and equipment. The prepared Sargassum polysaccharide has the effects of lowering blood sugar and lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. The drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0073] Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 The nuclear magnetic resonance spectrum of Sargassum polysaccharide SCP-B provided by the present invention;

[0074] Figure 7 The inhibitory effect of SCP-B provided by the present invention on Escherichia coli OP50;

[0075] Figure 8 The effect of SCP-B provided by the present invention on the motor ability of a wild-type Caenorhabditis elegans high-fat model;

[0076] Fig. 9 and Fig.10 The effect of SCP-B provided by the present invention on the fat content of wild-type Caenorhabditis elegans high-fat model;

[0077] Fig.11 and Fig.12 The effect of SCP-B provided by the present invention on the lipid droplet size of Pdhs-3::dhs-3::GFP Caenorhabditis elegans;

[0078] Fig.13 and Fig.14 The effect of SCP-B provided by the present invention on the glycogen content of the wild-type Caenorhabditis elegans high-fat model;

[0079] Fig.15 The effect of SCP-B provided by the present invention on the tropism of wild-type Caenorhabditis elegans high-fat model;

[0080] Fig.16 The effect of SCP-B provided by the present invention on the feeding rate of wild-type Caenorhabditis elegans high-fat model;

[0081] Fig.17 The effect of SCP-B provided by the present invention on the defecation cycle of a wild-type Caenorhabditis elegans high-fat model;

[0082] Fig.18 The effect of the high-fat model of SCP-B wild-type Caenorhabditis elegans provided by the present invention on the number of offspring;

[0083] Fig.19 The effect of SCP-B provided by the present invention on starch loading capacity;

[0084] Fig. 20 The effects of SCP-B provided by the present invention on fasting blood glucose, fasting insulin, insulin resistance index and GLP-1 in diabetic mice;

[0085] Fig.21 The effect of SCP-B provided by the present invention on blood lipid indexes of diabetic mice;

[0086] Fig. 22 The effects of SCP-B provided by the present invention on liver function indicators of diabetic mice;

[0087] Fig.23 The invention discloses the effects of SCP-B on renal function indexes of diabetic mice. DETAILED DESCRIPTION

[0088] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific implementation methods. However, those skilled in the art will understand that the embodiments described below are only part of the embodiments of the present invention, rather than all of the embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.

[0089] Example 1 Preparation of Sargassum polysaccharide SCP-B

[0090] The method for preparing Sargassum polysaccharide provided in this embodiment comprises the following steps:

[0091] (1) taking fresh Sargassum, washing it, draining it, drying it, grinding it into powder and sieving it;

[0092] (2) After the Sargassum powder was added to a 0.1 mol / L NaOH solution (W / V=1:12) and soaked for 4 hours, it was treated with a high-voltage pulse electric field with 8 pulses, an electric field strength of 18 kV / cm, and 2 extractions. The supernatant was centrifuged and concentrated to a solid content of 15%, and 4 times the volume of anhydrous ethanol was added;

[0093] (3) Stirring at 4°C for 12 hours, centrifuging at 6000 rpm, 25°C for 10 minutes, collecting the lower precipitate; adding 3 times the volume of 1 mol / L hydrochloric acid solution, stirring at 50°C for 3 hours, centrifuging to obtain the supernatant, and adjusting the pH to neutral;

[0094] (4) ultrafiltration the extract 5 times using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa and then drying to obtain macromolecular substances to obtain a crude extract;

[0095] (5) The dried crude extract was prepared into a 1% solution with pure water, added into a planetary ball mill, and treated for 48 hours, and then ultrafiltered 5 times with a 3KDa ultrafiltration membrane and dried to obtain macromolecular substances to obtain Sargassum crude polysaccharide;

[0096] (6) The crude polysaccharide of Sargassum was prepared into a 10 mg / mL solution with ultrapure water, and the supernatant was obtained by centrifugation;

[0097] Loading on DEAE-cellulose (6.0 x 28 cm) anion exchange column chromatography, eluting with the first eluent, the second eluent, the third eluent, the fourth eluent, the fifth eluent, the sixth eluent and the seventh eluent in sequence, the flow rate is 1.0 mL / min, and collecting the sugar-containing solution eluted by the third eluent;

[0098] (7) The product was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 500 Da, concentrated and freeze-dried to obtain Sargassum polysaccharide (SCP-B).

[0099] Example 2 Structural Characterization of Sargassum Polysaccharide SCP-B

[0100] (1) Molecular weight determination of Sargassum polysaccharide SCP-B

[0101] SCP-B was dissolved in 0.05% sodium azide solution (m / v), and the supernatant was taken after centrifugation at a speed of 10000rpm for 10min, and the sample was loaded after passing through a 0.22μm membrane. The molecular weight of the polysaccharide was analyzed by size exclusion gel chromatography combined with an 18-angle laser light scattering instrument and a differential detector, and the data was processed with ASTRA6.1 software. The analysis showed that the molecular weight of SCP-B was 24.11kDa.

[0102] (2) Determination of monosaccharide composition of Sargassum polysaccharide SCP-B

[0103] Prepare SCP-B into a 10mg / mL sample solution, take 1mL and add trifluoroacetic acid to the ampoule, burn and seal it, put it in an oven at 110℃ for hydrolysis for 8h; take it out and transfer it to a small rotating bottle after cooling to room temperature, add methanol to dissolve, and evaporate it under reduced pressure. Repeat the operation 5-6 times to remove trifluoroacetic acid. Sample derivatization: After the sample is evaporated to dryness, add 100μL NaOH (0.6M) and 100μL PMP methanol solution in sequence. After the sample is fully dissolved, transfer it to a sealed glass tube and derivatize it in a hot water bath at 70℃ for 100min. After cooling to room temperature, add 100μL hydrochloric acid (0.3M), make up the volume to 2mL with water, add 2mL chloroform, centrifuge it repeatedly at a speed of 2000rpm for 3min, remove the chloroform phase, extract it until the organic phase is clear and transparent to prove that PMP has been removed, and take the aqueous phase through the membrane for sampling and analysis. At the same time, guluronic acid, mannuronic acid, mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose standards and mixed standards were prepared at 10 mg / mL and derivatized as described above. After passing through a 0.22 μm filter membrane, the samples were put into the machine for operation. The monosaccharide composition results showed that SCP-B was mainly composed of fucose, guluronic acid and mannuronic acid, with a molar ratio of 1.00:1.08:1.91.

[0104] (3) Analysis of glycosidic bond connection mode of Sargassum polysaccharide SCP-B

[0105] The product was subjected to uronic acid reduction, methylation, complete acid hydrolysis and acetylation, and then monitored and analyzed by gas chromatography-mass spectrometry. As shown in the results in Table 1, SCP-B was detected to contain Fucp-(1→, →3)-Fucp-(1→, Gulp-(1→, →2,3)-Fucp-(1→, Manp-(1→, →4)-Manp-(1→ and →2,3)-Gulp-(1→. Combined with the monosaccharide composition and uronic acid analysis, Gulp-(1→, Manp-(1→, →4)-Manp-(1→ and →2,3)-Gulp-(1→ represent GulA-(1→, ManA-(1→, →4)-ManA-(1→ and →2,3)-GulA-(1→, respectively.

[0106] Table 1 SCP-B partially methylated alditol acetate (PMAAs) methylation gas analysis results

[0107]

[0108] (4) Detailed structural analysis of Sargassum polysaccharide SCP-B

[0109] SCP-B (50 mg) was dissolved in 0.5 mL D 2 O and transferred to a 5 mm NMR tube. The samples were analyzed using a nuclear magnetic resonance spectrometer (600M AVANCE II, Bruker Technology Co., Ltd, Bergisch Gladbach, Germany) according to the one-dimensional NMR spectrum. 1 H and 13 The glycosidic bond composition of SCP-B was analyzed by C spectrum and two-dimensional NMR spectrum COSY, NOESY, HSQC and HMBC spectra. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 The types of monosaccharide residues and sugar ring configurations in polysaccharides must first be determined based on 1 H spectrum, 13 The signals of the anomeric proton and anomeric carbon were identified in the C spectrum and HSQC spectrum. 1 H spectrum, are δ5.15, 4.96, 4.97, 4.85, 4.79, 4.62, and 4.57 respectively. 13 The anomeric carbon signals of the C spectrum are concentrated between δ93 and 105, and a total of seven anomeric carbons were identified, namely δ100.81, 100.09, 99.99, 99.93, 99.85, 93.59, and 93.51. Figure 3), in residue A, the signals of H1-2, H2-3, H3-4 and H4-5 correspond to δ4.62 / 3.95ppm, δ3.95 / 3.69ppm, δ3.69 / 3.81ppm and δ3.81 / 3.68ppm, respectively. It can be inferred that the signals of H1, H2, H3, H4 and H5 are δ4.62, 3.95, 3.69, 3.81, 3.68ppm, respectively. Therefore, the corresponding C1-C5 signals are δ99.93, 69.87, 75.55, 77.76, 71.34ppm, respectively. The carbon chemical shift at about 175ppm indicates the presence of hexonic acid of the carboxylic acid group. Combined with the low-field shift of the C4 carbon signal, therefore, the signal should be attributed to the glycosidic bond as →4)-β-ManA-(1→. All glycosidic bond signals are assigned according to similar rules, as shown in Table 2.

[0110] Table 2 Attribution of hydrogen and carbon signals

[0111]

[0112] Main chain analysis: By analyzing the cross peaks in HMBC and NOESY spectra ( Figure 5 and Figure 6 ), the connection pattern of glycosidic bonds in SCP-B can be determined. The correlation peaks of residue F at δ4.58 / 77.95ppm (FH1 / FC3) and δ3.95 / 99.85ppm (FH3 / FC1) appeared, indicating the existence of the connection type of →2,3)-β-Fucp-(1→2,3)-β-Fucp-(1→

[0113] The glycosidic bond →3)-α-Fucp-(1→'s H1 and β-Fucp-(1→'s H3 have related signal peaks, which indicates the existence of the glycosidic bond →3)-α-Fucp-(1→2,3)-β-Fucp-(1→. The anomeric hydrogen of →2,3)-β-Fucp-(1→ has a related signal peak with its own C1; in addition, the anomeric hydrogen has a related signal peak with its own C3; indicating the existence of the connection mode of →2,3)-β-Fucp-(1→2,3)-β-Fucp-(1→. The anomeric carbon of the glycosidic bond →2,3)-β-Fucp-(1→ →4)-β-ManA-(1→ has a related signal peak, indicating the existence of →2,3)-β-Fucp-(1→ 4)-β-ManA-(1→. The anomeric carbon of the glycosidic bond→4)-β-ManA-(1→ has a correlated signal peak with H3 of the glycosidic bond2,3)-α-GulA-(1→, indicating the existence of→4)-β-ManA-(1→2,3)-α-GulA-(1→. The glycosidic bond→4)-β-ManA-(1→ has a correlated signal peak with its own H2, indicating the existence of→4)-β-ManA-(1→4)-β-ManA-(1→. The glycosidic bond→2,3)-α-GulA-(1→ has a correlated signal peak with its own H3, indicating the existence of→2,3)-α-GulA-(1→2,3)-α-GulA-(1→.

[0114] Branch chain analysis: The H1 of the glycosidic bond α-Fucp-(1→) and the H2 of 2,3)-β-Fucp-(1→ have related signal peaks, indicating the existence of the glycosidic bond α-Fucp-(1→2,3)-β-Fucp-(1→. The H1 of the glycosidic bond β-ManA-(1→) and the H2 of 2,3)-β-Fucp-(1→ have related signal peaks, indicating the existence of the glycosidic bond β-ManA-(1→2,3)-β-Fucp-(1→. The H1 of the glycosidic bond α-GulA-(1→) and the H2 of 2,3)-α-GulA-(1→ have related signals, indicating the existence of the glycosidic bond α-GulA-(1→2,3)-α-GulA-(1→.

[0115] Example 3 Inhibitory Effect of Sargassum Polysaccharide SCP-B on Escherichia coli OP50

[0116] Take 10μL of SCP-B mixed with OP50 samples of different concentrations, add 20mL LB culture medium, and culture in a shaker at 37℃ and 200rmp. At 0h, 2h, 4h, 6h, 8h, 9h, and 10h, draw 1mL of liquid from each centrifuge tube into the centrifuge tube, draw 1mL of culture medium of different concentrations at different time periods and store it in a -80℃ refrigerator, and finally measure its absorbance at a wavelength of 600nm. E. coli OP50 is the control group, and each concentration of SCP-B sample is measured three times in parallel. Graphpad Prism 10 is used to statistically analyze and draw the growth curve of E. coli OP50 in different concentrations of SCP-B, and compare whether there is a significant difference between the control group and the experimental group.

[0117] The results are as follows Figure 7 As shown, the growth of E. coli OP50 in the three groups of SCP-B mixed with E. coli OP50 samples at 0.1, 1.0, and 10.0 mg / mL was not significantly different from that in the control group (p>0.05), indicating that SCP-B has no inhibitory effect on E. coli OP50. Because inhibiting the growth of E. coli OP50 can reduce the amount of food obtained by nematodes and reduce food intake, the experiment shows that the presence of SCP-B and SCP-B at different concentrations do not affect the amount of food obtained by nematodes and reduce food intake, thereby reducing the fat content in the nematodes.

[0118] Example 4 Effect of Sargassum polysaccharide SCP-B on the motor ability of a high-fat wild-type Caenorhabditis elegans model

[0119] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). Cooked egg yolks and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, the culture medium was protected from light. After nematode synchronization, the nematodes were transferred to the culture medium according to Table 3 for 48 hours before the experiment.

[0120] Table 3 Grouping of sports ability experiment

[0121]

[0122] In the formal experiment, synchronized L4 nematodes were picked up with a biological pick and placed in NG medium (without OP50) with 5 μL M9 buffer. The nematodes were allowed to adapt for 15 seconds. The number of bending movements during the movement in 30 seconds was counted using a hand counter. The left and right swings of the nematodes were observed under a microscope and recorded once. 15 parallels were made for each group, and each group of data was recorded separately. New NGM medium was used for each group. If the nematodes lost their ability to move or died in the new NGM medium, they needed to be picked up again for the experiment. The results are shown in the figure below. Figure 8As shown in the figure, according to the comparison between the control group and the model group, the mobility of the model group was significantly reduced. Compared with the experimental group and the model group, SCP-B had little effect on the mobility of nematodes.

[0123] Example 5: Lipid-lowering effect of Sargassum polysaccharide SCP-B in a wild-type Caenorhabditis elegans high-fat model

[0124] Wild-type Caenorhabditis elegans (hermaphrodites) were used. Cooked egg yolk and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0125] Prepare high-fat model food, mix cooked egg yolk and 10× E. coli OP50, operate 10× E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10× E. coli OP50 (V / V=1:5). Then vortex for 2-3 minutes, shake up and down for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration for 5-10 minutes to break them up until there are no large yolk particles visible to the naked eye. After nematode synchronization, transfer wild-type Caenorhabditis elegans eggs using a pipette to the wild-type Caenorhabditis elegans cultured for 56 hours according to Table 4 for experiments.

[0126] Table 4 Grouping of nematode Oil Red O staining experiment

[0127]

[0128] The specific experimental process is as follows: (1) After culturing wild-type Caenorhabditis elegans for 56 hours, prepare a sufficient number of 1.5 mL centrifuge tubes in advance, and use a pipette to draw 1000 μL of M9 buffer to rinse the NGM medium containing nematodes. Tilt the plate and rinse the liquid from top to bottom 4-5 times until the nematodes are almost completely rinsed, and transfer the turbid liquid to a labeled 1.5 mL centrifuge tube. Centrifugation conditions: 0.5 min, 3.5×10 3 rcf, remove the supernatant and collect the precipitate; (2) Take the precipitate obtained in step (1), use a pipette to absorb 500 μL of MRWB fixative, and add it to a 1.5 mL centrifuge tube. Shake on a shaker with tin foil in the dark for 1 h, 200 rpm, 20°C. Centrifugation conditions: 0.5 min, 3.5×10 3 rcf, remove the supernatant and collect the precipitate; (3) take the precipitate obtained in step (2), use a pipette to draw 500 μL of 1× PBS and add it to a centrifuge tube, centrifuge conditions: 0.5 min, 3.5×10 3rcf, remove the supernatant after centrifugation, collect the precipitate, and wash it twice; (4) take the precipitate obtained in step (3), use a pipette to draw 500 μL of 60% isopropanol into a centrifuge tube, incubate for 15 minutes, incubate once at room temperature, centrifuge conditions: 0.5 min, 3.5×10 3 rcf, after centrifugation, remove the supernatant and collect the precipitate; (5) Take the precipitate obtained in step (4), use a pipette to absorb 500 μL of saturated ORO dye, add it to a centrifuge tube, and shake it in a shaker in tin foil in the dark for 8 hours, 200 rpm, 20°C. After centrifugation, remove the supernatant and collect the precipitate; (6) Take the precipitate obtained in step (5), use a pipette to absorb 500 μL of 0.01% TritonX-100 solution, add it to a centrifuge tube, centrifuge conditions: 0.5 min, 3.5×10 3 rcf, remove the supernatant after centrifugation and collect the precipitate, and wash it repeatedly twice. (7) Take the precipitate obtained in step (6), keep 100μL of the supernatant, use a 200μL pipette to suck out the nematodes at the bottom of the centrifuge tube and drop them into a sterile place of uncontaminated NGM culture medium; (8) Take the wild-type Caenorhabditis elegans in NGM culture medium in step (7). Select two groups, each with 8 wild-type Caenorhabditis elegans of similar size at the L4 stage, place them on the 3% agarose pad on the slide, and align the head and tail of each nematode. Use a color camera to take pictures.

[0129] The body fat of nematodes was stained with Oil Red O staining, and pictures were taken with a color camera and processed using Image J software. The Oil Red O signals from the pharyngeal pump site to the tail region of 16 C. elegans in each group were counted.

[0130] The data obtained by Oil Red O staining are as follows: Fig. 9 and Fig.10 As shown. Compared with the control group, the fat content of the nematodes in the model group increased significantly (P<0.05), verifying that the high-fat nematode model was successfully established. The fat content of the experimental groups of 0.1, 1.0, and 10.0 mg / mL SCP-B was significantly reduced compared with the model group (P<0.05). The experiment showed that different concentrations of SCP-B reduced the fat content of wild-type Caenorhabditis elegans to different degrees. The 1.0 mg / mL SCP-B experimental group was used for further experiments.

[0131] Example 6 Effect of Sargassum polysaccharide SCP-B on lipid droplet size of Pdhs-3::dhs-3::GFP Caenorhabditis elegans

[0132] The nematodes used were Pdhs-3::dhs-3::GFP Caenorhabditis elegans (hermaphrodites). Cooked egg yolk and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0133] Prepare high-fat model food, mix cooked egg yolk and 10× E. coli OP50, operate 10× E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10× E. coli OP50 (V / V=1:5). Then vortex for 2-3 minutes, shake up and down for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration for 5-10 minutes to break them up until there are no large yolk particles visible to the naked eye. After nematode synchronization, transfer Pdhs-3::dhs-3::GFP Caenorhabditis elegans eggs to the nematode culture according to Table 5 for 48 hours, and then take pictures of specific areas near the nematode intestine under fluorescence.

[0134] Table 5 Grouping of experiments for determining lipid droplet size of Pdhs-3::dhs-3::GFP Caenorhabditis elegans

[0135]

[0136] The nematodes were anesthetized by dropping 2 μL of 25 mM levamisole hydrochloride solution on a 2% agarose pad on a glass slide. The size of lipid droplets expressing green fluorescent protein (GFP) in C. elegans was quantitatively analyzed using Image J software (Wayne Rasband, NIH, USA). Fig.11 and Fig.12 As shown in the figure, compared with the control group, the size of lipid droplets in the intestinal area of ​​the model group was significantly increased (P<0.05), and compared with the model group, the size of lipid droplets in the 1.0 mg / mL SCP-B experimental group was significantly reduced (P<0.05). The experiment proved that the model group was successfully established, and the 1.0 mg / mL SCP-B experimental group had a fat content-reducing effect in the high-fat model of nematodes.

[0137] Example 7 Effect of Sargassum polysaccharide SCP-B on glycogen content in a high-fat wild-type Caenorhabditis elegans model

[0138] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). Cooked egg yolk, Escherichia coli OP50, and elemental iodine were stored at 4°C. The incubator temperature was maintained at 20±1°C, and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0139] Prepare high-fat model food, mix cooked egg yolk and 10× E. coli OP50, operate 10× E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10× OP50 (V / V=1:5). Then vortex for 2-3 minutes, shake up and down for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration for 5-10 minutes to break them up until there are no large egg yolk particles visible to the naked eye. Iodine stored at 4°C needs to be placed at room temperature of 25°C 30 minutes in advance to restore the iodine to room temperature.

[0140] After synchronization of the nematodes, the wild-type Caenorhabditis elegans eggs were transferred using a pipette to the wild-type Caenorhabditis elegans cultured according to Table 6 for 56 h before the experiment.

[0141] Table 6 Grouping of experiments for determination of glycogen content in nematodes

[0142]

[0143] Glycogen staining was used. 500 μL of M9 buffer was dropped into the NGM medium for culturing nematodes. The medium was gently shaken on the table, and then the M9 buffer with nematodes was pipetted into a 1.5 mL centrifuge tube. The tube was allowed to settle naturally for 2-3 minutes, and the supernatant was removed to collect the precipitate. The nematodes were pipetted onto a glass slide with a 3% agarose pad and the slide was inverted. The nematodes were stained with iodine vapor for 2 minutes. After 4 minutes, 16 nematodes in each group were photographed with a color camera with a 10x objective lens and adjusted with ZEN blue software.

[0144] The Glycogen staining signal of each group of images was obtained by processing the images in Image J and then statistically analyzed by Graph pad Prism 10 software, such as Fig.13 and Fig.14 As shown, the control group was compared with the model group, and the Glycogen staining signal of the model group was significantly increased (P<0.05). The 1mg / mL SCP-B experimental group was compared with the model group, and the Glycogen staining signal of the experimental group was significantly reduced (P<0.05). The experiment shows that 1mg / mL SCP-B helps to reduce the glycogen content in wild-type Caenorhabditis elegans.

[0145] Example 8 Experiment on the tropism of Sargassum polysaccharide SCP-B to wild-type Caenorhabditis elegans high-fat model

[0146] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). The wild-type Caenorhabditis elegans (hermaphrodites) were cultured in an uncontaminated NGM medium with OP50 at 20°C for 48 hours to obtain wild-type Caenorhabditis elegans in the L4 period. A dividing line was made through the central origin of the 60mm uncontaminated NGM medium, and the central origin was used as the nematode drop area. The control group and the experimental group drop areas were respectively located on the same straight line passing through the central origin on both sides of the dividing line and at the same distance from the central origin. After the nematodes were synchronized, the wild-type Caenorhabditis elegans eggs were cultured for 48 hours and then the experiment was performed according to Table 7.

[0147] Table 7 Grouping of experiments for determination of nematode tropism

[0148]

[0149] After incubation at 20 ± 1 °C in the dark for 6 h, the number of nematodes in each area was recorded and counted. Statistical analysis using Graphpad Prism 10 software, such as Fig.15 As shown, there was no significant difference in the percentage of nematodes between the control group and the 1.0 mg / mL SCP-B experimental group, which proved that the presence of 1.0 mg / mL SCP-B had basically no effect on the food preference of nematodes, and ruled out that SCP-B reduced the food preference of nematodes and affected the nematodes' feeding, causing the fat content in the nematodes to decrease.

[0150] Example 9 Effect of Sargassum polysaccharide SCP-B on the feeding rate of wild-type Caenorhabditis elegans high-fat model

[0151] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). Cooked egg yolk and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0152] Prepare high-fat model food, mix cooked egg yolk and 10× E. coli OP50, operate 10× E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10× E. coli OP50 (V / V=1:5). Then vortex for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration is performed for 5-10 minutes to break them up until there are no large yolk particles visible to the naked eye. After nematode synchronization, wild-type Caenorhabditis elegans eggs are transferred using a pipette to the experiment after culturing wild-type Caenorhabditis elegans for 56 hours according to Table 8.

[0153] Table 8 Grouping of experiments for determining nematode feeding rate

[0154]

[0155] The feeding rate of nematodes can be reflected by observing the beating of the pharyngeal pump of nematodes. Each group has 15 parallels. Each parallel is observed for 15 seconds in the NGM medium of nematodes. Each beating of the pharyngeal pump of nematodes is recorded once. Fig.16 As shown in the figure, through experimental records and statistical analysis, it was found that compared with the model group, the model group significantly reduced the feeding rate of nematodes (P<0.05), but compared with the model group, there was no significant difference in the pharyngeal pump beating rate between the 1 mg / mL SCP-B experimental group and the model group (P>0.05), indicating that SCP-B would not further affect the feeding rate of nematodes and reduce the body fat content of nematodes.

[0156] Example 10 Effect of Sargassum polysaccharide SCP-B on the defecation cycle of wild-type Caenorhabditis elegans high-fat model

[0157] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). Cooked egg yolk and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0158] Prepare high-fat model food, mix cooked egg yolk and 10×OP50, operate 10×E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10×E. coli OP50 (V / V=1:5). Then vortex for 2-3 minutes, shake up and down for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration for 5-10 minutes to break them up until there are no large yolk particles visible to the naked eye. After nematode synchronization, wild-type Caenorhabditis elegans eggs were transferred to the experiment according to Table 9 after culturing wild-type Caenorhabditis elegans for 56 hours.

[0159] Table 9 Grouping of experiments for determining the defecation movement cycle of nematodes

[0160]

[0161]

[0162] There are many ultrarhythmic processes in C. elegans. Defecation is a well-defined and tractable model of controlled rhythmic behavior. Defecation depends on the defecation motor program (DMP). Underlying the rhythmic execution of this motor program is an oscillator encoded in the intestinal cells of the animal. The defecation cycle refers to the period from one pBoc to the next.

[0163] The excretion rate of nematodes was reflected by observing the posterior body wall muscle contraction (PBoc) and excretion (Exp) cycles of nematodes. The experiment recorded the time from the start of excretion (Exp) to the posterior body wall muscle contraction (PBoc) and the time from the posterior body wall muscle contraction (PBoc) to excretion (Exp) in each parallel. At room temperature of 25°C, 8 parallels were observed in each group, and each parallel cycled for two cycles. Fig.17 As shown, compared with the model group and the control group, there was no significant difference in the DMP cycle period from the last excretion (Exp) to the posterior body wall muscle contraction (PBoc) and the DMP cycle period from the posterior body wall muscle contraction (PBoc) to excretion (Exp) of the nematodes in the model group (P>0.05). Compared with the model group, there was no significant difference in the DMP cycle period from the last excretion (Exp) to the posterior body wall muscle contraction (PBoc) and the DMP cycle period from the posterior body wall muscle contraction (PBoc) to excretion (Exp) of the nematodes in the 1 mg / mL SCP-B experimental group (P>0.05). The experiment concluded that the high-fat nematode model and the 1.0 mg / mL SCP-B experimental group had no obvious effect on the defecation cycle of nematodes.

[0164] Example 11 Effect of Sargassum polysaccharide SCP-B on the number of offspring in a high-fat model of wild-type Caenorhabditis elegans

[0165] The nematodes used were wild-type Caenorhabditis elegans (hermaphrodites). Cooked egg yolk and Escherichia coli OP50 were stored at 4°C. The incubator temperature was maintained at 20±1°C and the relative humidity was maintained at 70±5%. During the experiment, all culture media were protected from light in order to unify the variables.

[0166] Prepare high-fat model food, mix cooked egg yolk and 10× E. coli OP50, operate 10× E. coli OP50 and cooked egg yolk in a clean bench, and use a weighing spoon to scoop out the egg yolk. Mix cooked egg yolk and 10× E. coli OP50 (V / V=1:5). Then vortex for 2-3 minutes, shake up and down for 2-3 minutes, or if the egg yolk particles are too large, ultrasonic vibration for 5-10 minutes to break them up until there are no large yolk particles visible to the naked eye. After nematode synchronization, transfer wild-type Caenorhabditis elegans eggs using a pipette to the wild-type Caenorhabditis elegans cultured according to Table 10 for 48 hours before conducting experiments.

[0167] Table 10 Experimental groups for determining the number of nematode offspring

[0168]

[0169] After culturing the synchronized nematodes for 48 hours, 36 hermaphroditic L4 nematodes of similar size and normal movement ability were selected, with 4 nematodes in each NGM culture medium, and 3 parallel groups were made, and cultured at a constant temperature of 20°C. The original nematodes were transferred to new NGM every 24 hours to continue laying eggs. Each group of nematodes laid eggs for 72 hours, and the 4 mother nematodes in each group were transferred twice. After culturing at 20°C for 36-48 hours, the number of nematodes was recorded using a hand calculator, and the data was recorded three times for each parallel. After all the eggs developed to the L4 stage, the number of offspring was calculated and the number of nematode offspring after 72 hours was determined. Fig.18 As shown in the experimental results, there was no significant difference in the number of offspring in the model group compared with the control group, but the number of offspring in the 1.0 mg / mL SCP-B experimental group was reduced to a certain extent compared with the model group.

[0170] Example 12 Effects of Sargassum polysaccharide SCP-B on lowering blood sugar and blood lipids

[0171] (1) Starch loading experiment

[0172] The SFP grade Kunming mice used were purchased from the Guangdong Medical Animal Experiment Center, with an age of 5-6 weeks. Animal feed (SFP grade) was provided by the Guangdong Medical Animal Experiment Center and stored at 4°C. The temperature of the breeding room was maintained at 25±1°C, the relative humidity was maintained at 55±5%, and the lighting was controlled to maintain 12h lighting and 12h darkness every day. During the experiment, the animals were not restricted in diet and drinking water.

[0173] Table 11 Starch loading experiment grouping

[0174]

[0175] In the formal experiment, all animals were fasted for 12 hours and then gavaged according to the experimental groups, intervention methods and doses designed in Table 3. The blood glucose levels of each mouse at 0, 30, 60, 120 and 150 minutes were recorded using an Omron blood glucose meter HGM-111. Fig.19 As shown, according to the comparison between the control group and the starch group, the blood sugar level increased rapidly in 30 minutes, and the other experimental groups and the starch group all had lower blood sugar levels. The test of starch loading capacity showed that within 30-120 minutes, the SCP-B200 group and the SCP-B400 group had a significant effect in inhibiting the increase in blood sugar caused by starch intake.

[0176] (2) Study on the regulatory effect of SCP-B on glucose and lipid metabolism in mice

[0177] This experiment involved SPF db / db diabetic mice and SPF wild-type db / m mice. The temperature of the breeding room was maintained at 25±1℃, the relative humidity was maintained at 55±5%, and the lighting was controlled to maintain 12 hours of lighting and 12 hours of darkness every day. During the experiment, the animals were not restricted in food and water intake.

[0178] All animals were randomly divided into groups after 7 days of adaptive feeding, with 5 mice per cage and 2 cages per group. 10 SPF db / m mice were registered as the normal control (NC) group, and 1 mL of saline was used for intervention during the experiment. 40 db / db mice were randomly divided into 4 groups, with 10 mice in each group, and were registered as the model (DC) group, metformin (MET) group, SCP-B200 group, and SCP-B400 group, respectively. The experimental grouping is shown in Table 12.

[0179] Table 12 Grouping of SCP-B hypoglycemic effect experiment

[0180]

[0181] After 56 days, all animals were fasted for 12 hours, blood was collected by pulling out the eyeballs, and the animals were killed by dislocating the neck and then dissected. The colon contents of each group of experimental animals were collected by saline flushing and placed in cryopreservation tubes, frozen in liquid nitrogen, and then stored in a -80℃ refrigerator for high-throughput sequencing.

[0182] Effects of SCP-B on fasting blood glucose, fasting insulin, insulin resistance index and GLP-1 in diabetic mice:

[0183] Fasting blood glucose (FBG) of mice Fig. 20 As shown. Compared with the control group, the blood glucose level of mice in the model group was significantly increased (P<0.05). Compared with the model group, the blood glucose levels of mice in the metformin group, SCP-B200 group, and SCP-B400 group were significantly reduced (P<0.05). Compared with the metformin group, the blood glucose level of mice in the SCP-B400 group was significantly reduced (P<0.05), and the blood glucose lowering effect of the SCP-B400 group was better than that of the metformin group.

[0184] The insulin level of mice changes with the dynamic changes of blood glucose. The fasting insulin (FINS) of mice is Fig. 20 As shown in the figure, compared with the control group, the insulin level of the model mice was significantly increased (P<0.05). Compared with the model group, the fasting insulin levels of mice in the metformin group, SCP-B200 group, and SCP-B400 group were significantly reduced (P<0.05), and the fasting insulin level of mice in the SCP-B400 group was significantly lower than that in the metformin group and SCP-B200 group (P<0.05).

[0185] Homeostasis insulin resistance index (HOMA-IRI) Fig. 20 As shown, it shows the insulin resistance of diabetic mice. The HOMA-IRI level in the model group was significantly higher than that in the control group. After 8 weeks of intervention, the HOMA-IRI level in each experimental group was significantly lower than that in the model group (P<0.05), and the HOMA-IRI level in the SCP-B400 group was significantly lower than that in the metformin group and the SCP-B200 group (P<0.05).

[0186] Glucagon-like peptide-1 (GLP-1) is a key incretin-secreting peptide hormone secreted by intestinal L cells. GLP-1 and its receptor have become one of the most important targets for the development of diabetes and obesity drugs in recent years. Fig. 20 As shown, the results showed that the GLP-1 level of mice in the model group was significantly lower than that in the control group (P<0.05), and the GLP-1 level of mice in each experimental group was significantly restored (P<0.05). The GLP-1 level of mice in the SCP-B400 group was significantly increased compared with the SCP-B200 group.

[0187] All the results of this experiment showed that the SCP-B400 group had a better hypoglycemic effect than the metformin group.

[0188] Effects of Sargassum polysaccharide SCP-B on blood lipid indexes in diabetic mice

[0189] Serum index detection uses a fully automatic biochemical analyzer to detect serum indicators, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and free fatty acids (FFA).

[0190] according to Fig.21 As shown in the results, compared with the control group, the levels of serum TG, TC, LDL-C, and FFA in the model group mice were significantly increased (P<0.05), indicating that diabetic mice had severe dyslipidemia; compared with the model group, all experimental groups could reduce the levels of serum TG, TC, LDL-C, and FFA in mice (P<0.05). In terms of reducing the levels of serum TG, TC, LDL-C, and FFA in mice, the treatment effect of the SCP-B400 group was more significant than that of the metformin group and the SCP-B200 group (P<0.05).

[0191] The metformin group, SCP-B200 group, and SCP-B400 group all improved the hyperlipidemia of diabetic mice, but the SCP-B400 group had a better lipid-lowering effect.

[0192] Effects of Sargassum polysaccharide SCP-B on liver damage and inflammatory response indicators in diabetic mice

[0193] Serum index detection uses a fully automatic biochemical analyzer to detect aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acid (TBA) levels. Fig. 22 As shown in the results, compared with the control group, the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acid (TBA) in the model group mice were significantly increased on average (P<0.05); compared with the model group, the aspartate aminotransferase (AST) in the metformin group was significantly increased (P<0.05), and the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acid (TBA) in the SCP-B200 group and SCP-B400 group mice were significantly decreased (P<0.05); compared with the SCP-B200 group, the levels of serum aspartate aminotransferase (AST) and total bile acid (TBA) in the SCP-B400 group mice were significantly decreased. The experiment showed that the SCP-B400 group had the best therapeutic effect on liver damage and inflammatory response in diabetic mice.

[0194] Effects of Sargassum polysaccharide SCP-B on renal function parameters in diabetic mice

[0195] Serum index detection uses automatic biochemical analyzer to detect urea (BUN), creatinine (CREA) and uric acid (UA) levels. Fig.23 As shown, compared with the control group, the levels of urea (BUN), creatinine (CREA) and uric acid (UA) in the serum of mice in the model group were significantly increased (P<0.05); compared with the model group, the levels of urea (BUN), uric acid (UA) and creatinine (CREA) in the serum of mice in each experimental group were significantly decreased (P<0.05); compared with the SCP-B200 group, the level of urea (BUN) in the serum of mice in the SCP-B400 group and the metformin group was significantly decreased (P<0.05); compared with the SCP-B200 group, the level of uric acid (UA) in the serum of mice in the SCP-B400 group was significantly decreased. The experiment proved that the SCP-B400 group had the best therapeutic effect on renal damage in diabetic mice.

[0196] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for preparing Sargassum polysaccharide, characterized in that: The following steps are involved: (a) subjecting the Sargassum soaked in an alkali solution to a high-voltage pulse electric field treatment, a first centrifugation and a concentration to obtain a concentrated solution; (b) subjecting the mixed system containing the concentrated solution and the organic solvent to a first stirring treatment and a second centrifugation to collect a lower layer of sediment; (c) subjecting the mixed system containing the lower precipitate and the acid solution to a second stirring treatment and a third centrifugation to obtain a supernatant; and adjusting the pH of the supernatant to neutral to obtain an extract; The extract is subjected to a first ultrafiltration and a first drying to obtain macromolecular substances and obtain a crude extract; (d) subjecting the mixed solution containing the crude extract to ball milling, second ultrafiltration and second drying to obtain macromolecular substances to obtain crude Sargassum polysaccharide; subjecting the solution containing the Sargassum crude polysaccharide to anion exchange chromatography and dialysis; The anion exchange chromatography comprises: eluting with a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence, collecting the sugar-containing solution eluted by the third eluent for dialysis; The first eluent includes a 0mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the second eluent includes a 0.2mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the third eluent includes a 0.4mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the fourth eluent includes a 0.6mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the fifth eluent includes a 0.8mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the sixth eluent includes a 1mol / L NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:3; the seventh eluent includes a 2moL NaCl solution and a 0.01mol / L NaOH solution in a volume ratio of 1:

3.

2. The method for preparing Sargassum polysaccharide according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The number of pulses in the high-voltage pulse electric field treatment is 5 to 10; (2) The electric field strength of the high voltage pulse electric field treatment is 15-20 kV / cm; (3) The molecular cut-off of the first ultrafiltration is 3 kDa; (4) The molecular retention capacity of the second ultrafiltration is 3 kDa.

3. The method for preparing Sargassum polysaccharide according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The temperature of the first stirring treatment is 2-6°C; (2) The first stirring treatment time is 10 to 14 hours; (3) The temperature of the second stirring treatment is 45-55°C; (4) The second stirring treatment time is 2 to 4 hours.

4. A Sargassum polysaccharide prepared by the method for preparing Sargassum polysaccharide according to any one of claims 1 to 3, characterized in that: The Sargassum polysaccharide is composed of fucose, guluronic acid and mannuronic acid; The molar ratio of the fucose, the guluronic acid and the mannuronic acid is 1.00:1.08:1.

91.

5. The Sargassum polysaccharide according to claim 4, characterized in that The main chain connection mode of the Sargassum polysaccharide is as follows: →3)- α -Fucp-(1→3)- β- Fucp-1→3) - β- Fucp-(1→[4)- β -ManA-(1] 12 →[3)- α -GulA-(1]2→; and / or, the terminal group of the Sargassum polysaccharide comprises: β -ManA-(1→), α -Fuc p -(1→) and α -GulA-(1→.

6. The Sargassum polysaccharide according to claim 5, characterized in that Includes at least one of the following technical features: (1) β -ManA-(1→) through→2,3)- β -Fuc p -(1→'s O-2 is connected to the main chain; (2) α -Fuc p -(1→) through →2,3)- β -Fuc p -(1→O-2 is connected to the main chain; (3) α -GulA-(1→through→2,3)- α -GulA-(1→O-2 is connected to the main chain.

7. The Sargassum polysaccharide according to claim 6, characterized in that The structural formula of the repeating unit of the Sargassum polysaccharide is as follows: 。 8. The Sargassum polysaccharide according to any one of claims 4 to 7, characterized in that The molecular weight of the Sargassum polysaccharide is 24.11 kDa.

9. Use of the Sargassum polysaccharide according to any one of claims 4 to 8 in the preparation of a medicament for lowering blood lipids and / or blood sugar and / or repairing liver and kidney damage complicated by hyperglycemia and hyperlipidemia.

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