Sargassum fusiformis polysaccharide with enhanced sitagliptin hypoglycemic effect and preparation method and application thereof

Abstract

The present invention discloses a Sargassum polysaccharide having a synergistic hypoglycemic effect of sitagliptin, a preparation method and an application thereof, and belongs to the field of biomedical technology. The Sargassum polysaccharide SFPN4 of the present invention contains mannuronic acid and guluronic acid, and the molar ratio of the mannuronic acid to the guluronic acid is 0.695:0.305. The Sargassum polysaccharide SFPN4 prepared by the present invention has an α-glucosidase inhibitory effect and a hypoglycemic effect, and also has the effect of significantly enhancing the blood sugar control effect of sitagliptin, which is beneficial to reducing the dosage of sitagliptin and improving the drug efficacy and drug safety.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a Sargassum fusiformis polysaccharide having a synergistic sitagliptin blood sugar lowering effect, and a preparation method and application thereof. Background Art

[0002] With the rapid change of people's high-oil, high-fat and high-sugar eating habits, the incidence of diabetes mellitus (DM), especially type 2 diabetes mellitus (T2DM), has continued to rise, and the proportion of young and middle-aged people has increased significantly. About 6.7 million deaths worldwide are related to the deterioration of diabetes and its complications, and the corresponding medical expenditures are as high as 966 billion US dollars. Various signs show that diabetes has gradually developed into one of the diseases that seriously endangers human health and hinders economic development. Clinical data show that the drugs used to treat diabetes mainly include carbohydrate hydrolase inhibitors, insulin and its analogs, GLP-1 secretagogues and DPP-4 inhibitors, but some drugs may cause diarrhea, weight changes, organ damage and gastrointestinal reactions after long-term use, and combined use may also cause overlapping toxicity or multidrug resistance.

[0003] Sitagliptin (SIT) is a typical clinical drug for fasting and postprandial blood sugar management by selectively weakening DPP-4 activity. Compared with other drugs, SIT can reduce the incidence of hypoglycemia and fat in the treatment of diabetes, and can also reduce gastrointestinal side effects. However, clinically, SIT often needs to be used in combination with other oral drugs to achieve a good glucose control effect. At present, it has been reported that SIT combined with other commercially available drugs such as glycosidase inhibitors and metformin has better therapeutic effects, but it cannot avoid the adverse reactions caused by other drugs. Therefore, it is of great significance to develop food-type natural drug adjuvants, improve the therapeutic effect and application scope of SIT, and reduce or avoid multidrug resistance and overlapping toxicity caused by drug combinations. Summary of the invention

[0004] The purpose of the present invention is to provide a polysaccharide of Sargassum fusiformis with enhanced hypoglycemic effect of sitagliptin and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The Sargassum fusiformis polysaccharide SFPN4 prepared by the present invention has α-glucosidase inhibition and hypoglycemic effect, and also has the effect of significantly enhancing the blood sugar control effect of sitagliptin, which is beneficial to reduce the dosage of sitagliptin and improve the drug efficacy and drug safety.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Sargassum polysaccharide SFPN4 having a synergistic sitagliptin hypoglycemic effect, wherein the Sargassum polysaccharide SFPN4 contains mannuronic acid and guluronic acid, and the molar ratio of mannuronic acid to guluronic acid is 0.695:0.305;

[0007] The main chain of the Sargassum fusiformis polysaccharide SFPN4 is composed of →4)-β-ManA-(1→3,4)-β-ManA-(1→ and →3,4)-β-ManA-(1→4)-α-GulA-(1→; the side chains include α-GulA-(1→3,4)-β-ManA-(1→ and β-ManA-(1→3,4)-β-ManA-(1→;

[0008] The repeating unit structural formula of the Sargassum fusiformis polysaccharide SFPN4 is:

[0009]

[0010] The molecular weight of the Sargassum fusiformis polysaccharide SFPN4 is 89.83 kDa.

[0011] The present invention also provides a method for preparing the above-mentioned Sargassum fusiformis polysaccharide SFPN4, comprising the following steps:

[0012] (1) mixing Sargassum fusiformis with water and beating the obtained slurry, sterilizing the obtained slurry, inoculating a composite bacterial agent for fermentation, and obtaining a fermentation liquid;

[0013] (2) treating the fermented liquid in a water bath, centrifuging and obtaining the supernatant, and concentrating, precipitating with alcohol, removing ethanol, ultrafiltering and freeze-drying to obtain crude polysaccharide of Sargassum fusiformis;

[0014] (3) The crude polysaccharide of Sargassum fusiformis is prepared into a solution, and after ultrasonic and depigmentation treatment, eluted with 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 M NaCl solutions in sequence, using a DEAE Sepharose FF anion exchange column, collecting the sugar-containing solution eluted with the 0.4 M NaCl solution, dialyzing and concentrating to obtain a desalted solution; the desalted solution is freeze-dried to obtain the Sargassum fusiformis polysaccharide SFPN4.

[0015] Preferably, in step (1), the volume ratio of the Sargassum fusiformis to water is 1:10.

[0016] Preferably, in step (1), the composite bacterial agent is obtained by mixing thermophilic Streptococcus, plant lactobacillus and bulgaricus in a mass ratio of 1:1:1 and activating and culturing the mixture; the OD of the composite bacterial agent is 600 >0.5; the inoculation amount is 3%; the fermentation temperature is 37°C and the fermentation time is 36h.

[0017] Preferably, in step (2), the temperature of the water bath treatment is 90°C and the time is 2h; the concentration is 1 / 4 of the original volume; the alcohol precipitation method is: mixing the concentrated supernatant with 4 times the volume of anhydrous ethanol, and precipitating with alcohol at 4°C for 24h; the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is 10kDa; and the number of ultrafiltration is 6 times.

[0018] Preferably, in step (3), the concentration of the solution is 1 mg / mL; the power of the ultrasound is 300 W, and the time is 2 h; the depigmentation method is to use DA-201 macroporous resin to depigment until the color changes to light brown; and the elution flow rate is 1 mL / min.

[0019] The present invention also provides the use of the above-mentioned Sargassum polysaccharide SFPN4 or the Sargassum polysaccharide SFPN4 prepared by the above-mentioned preparation method in the preparation of a drug for treating diabetes.

[0020] The present invention also provides the use of the above-mentioned Sargassum polysaccharide SFPN4 or the Sargassum polysaccharide SFPN4 prepared by the above-mentioned preparation method in combination with sitagliptin in the preparation of a drug for treating diabetes.

[0021] The present invention also provides a medicine for treating diabetes, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the above-mentioned Sargassum fusiformis polysaccharide SFPN4 or the Sargassum fusiformis polysaccharide SFPN4 prepared by the above-mentioned preparation method.

[0022] The present invention discloses the following technical effects:

[0023] The present invention extracts polysaccharides from Sargassum fusiformis and systematically studies its physicochemical properties, structural characterization and activity determination. It is found that Sargassum fusiformis polysaccharide SFPN4 has an inhibitory effect on α-glucosidase, IC 50 The value was 469.03 μg / mL, which was lower than the IC of acarbose 50 value. The results of the starch loading capacity test showed that SFPN4 can effectively inhibit the increase in blood sugar caused by starch intake. Based on the diabetic mouse model, it was found that SFPN4 alone can significantly improve the abnormal glucose and lipid metabolism and insulin resistance of mice. Sitagliptin + SFPN4 treatment is better than sitagliptin and SFPN4 treatment alone in controlling the increase in blood sugar caused by starch, as well as the levels of fasting blood sugar, glycosylated hemoglobin, glucose tolerance, insulin resistance, total cholesterol, free fatty acids, etc. in diabetic animals, showing a significant synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a graph showing the molecular weight detection results of Sargassum fusiformis polysaccharide SFPN4;

[0026] Figure 2 This is a diagram showing the results of determining the monosaccharide composition of Sargassum fusiformis polysaccharide SFPN4;

[0027] Figure 3 Sargassum fusiformis polysaccharide SFPN4 1 H spectrum;

[0028] Figure 4 Sargassum fusiformis polysaccharide SFPN4 13 C spectrum;

[0029] Figure 5 COSY spectrum of Sargassum fusiformis polysaccharide SFPN4;

[0030] Figure 6 NOESY spectrum of Sargassum fusiformis polysaccharide SFPN4;

[0031] Figure 7 is the HSQC spectrum of Sargassum fusiformis polysaccharide SFPN4;

[0032] Figure 8 is the HMBC spectrum of Sargassum fusiformis polysaccharide SFPN4;

[0033] Fig. 9 is the structural formula of Sargassum fusiformis polysaccharide SFPN4;

[0034] Fig.10 Figure 2 is the inhibitory effect of Sargassum fusiformis polysaccharide SFPN4 on α-glucosidase; A is the IC of acarbose and Sargassum fusiformis polysaccharide SFPN4 50 Value; B is the Lineweaver-Burk curve of α-glucosidase under different concentrations of SFPN4; C is the circular dichroism analysis result of α-glucosidase before and after the addition of SFPN4; D is the fluorescence spectrum analysis result of α-glucosidase at 25°C; E is the fluorescence spectrum analysis result of α-glucosidase at 37°C; F is the Stern-Volmer curve diagram;

[0035] Fig.11 The effect of Sargassum fusiformis polysaccharide SFPN4 supplementation on the starch loading capacity of sitagliptin;

[0036] Fig.12This is the effect of SFPN4 supplementation with Sargassum fusiformis polysaccharide on glucose metabolism in diabetic mice treated with sitagliptin; AD are the results of fasting blood glucose, glycosylated hemoglobin, glucose tolerance and insulin levels;

[0037] Fig.13 The effect of SFPN4 supplementation with Sargassum fusiformis polysaccharide on the blood lipid level of diabetic mice treated with sitagliptin; AE were the test results of TG, TC, HDL-C, LDL-C and FFA levels;

[0038] Fig.14 The effects of SFPN4 supplementation with Sargassum fusiformis polysaccharide on gene expression in the liver of diabetic mice treated with sitagliptin; AD are the results of relative expression of IRS-1, PI3K, Akt, and PPARγ;

[0039] Fig.15 This is the effect of Sargassum fusiformis polysaccharide SFPN4 supplementation on liver gene expression in diabetic mice treated with sitagliptin; AE are the expression detection results of AMPK-α, PPARα, CYP7A1, G-6-Pase and PEPCK, respectively. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1 Preparation of Sargassum fusiformis polysaccharide SFPN4

[0046] Wash the dried sample of Sargassum fusiformis with running water, wash off the salt and dust on the surface, and soak it in deionized water to allow it to absorb water until the leaves are full. After draining, add 10 times the volume of deionized water, beat into a paste, and then sterilize with high pressure steam at 121℃ for 20 minutes. The freeze-dried powder of thermophilic Streptococcus, plant lactobacillus, and bulgaricus (purchased from Shandong Pingao Pharmaceutical Co., Ltd.) was mixed in a mass ratio of 1:1:1, dissolved in physiological saline, and activated in a 37℃ water bath until OD 600 After the temperature is higher than 0.5, the mixture is inoculated into the cooled paste of Sargassum fusiformis at an inoculation amount of 3%. Finally, the mixture is fermented at 37°C for 36 hours under anaerobic conditions to obtain a fermentation liquid.

[0047] The fermentation liquid was adjusted to a neutral pH and placed in a 90°C water bath with a stirring paddle for 2 hours. The supernatant was centrifuged (10,000 rpm, 4°C, 20 min) and concentrated to 1 / 4 of the original volume by rotary evaporation. Four times the volume of anhydrous ethanol was added for alcohol precipitation. After alcohol precipitation at 4°C for 24 hours, the liquid was centrifuged again, the lower precipitate was removed, deionized water was added for redissolution, and the residual ethanol was removed by rotary evaporation. The resulting solution was ultrafiltered six times with an ultrafiltration membrane with a molecular weight cutoff of 10 KDa, and then freeze-dried to obtain crude polysaccharides from Sargassum fusiformis.

[0048] After the crude polysaccharide of Sargassum fusiformis was prepared into a 1 mg / mL solution, it was ultrasonically treated at a power of 300 W for 2 hours, and then depigmented by DA-201 macroporous resin until the color changed to light brown, and then eluted with a DEAE Sepharose FF anion exchange column. The elution process was: eluted with NaCl solutions of concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 M in sequence, with a flow rate of 1 mL / min. The total sugar content was measured by the phenol-sulfuric acid method, and finally the sugar-containing solution eluted with 0.4 M NaCl solution was selected for the next step of purification.

[0049] The sugar-containing solution was dialyzed for 48 hours, repeatedly concentrated by rotary evaporation until the salinity of the internal solution was 0 as detected by a salinity meter, and then freeze-dried to obtain the Sargassum fusiformis polysaccharide SFPN4.

[0050] Example 2 Structural Characterization of Sargassum fusiformis Polysaccharide SFPN4

[0051] 1. Determination of molecular weight

[0052] The Sargassum fusiformis SFPN4 prepared in Example 1 was dissolved in a 0.05% sodium azide solution (m / v), centrifuged (10000 rpm, 10 min), and the supernatant was taken and passed through a 0.22 μm membrane before loading. 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 results are shown in FIG. Figure 1 As shown, the molecular weight of SFPN4 is 89.83 kDa.

[0053] 2. Determination of monosaccharide composition

[0054] Prepare SFPN4 into a 10 mg / mL sample solution, take 1 mL into an ampoule, add trifluoroacetic acid, burn and seal it in an oven, and hydrolyze it at 110°C for 8 hours; 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 100 μL NaOH (0.6M) and 100 μL 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°C for 100 minutes. After cooling to room temperature, add 100 μL hydrochloric acid (0.3M), make up the volume to 2 mL with water, add 2 mL chloroform, centrifuge repeatedly (2000rpm, 3min), remove the chloroform phase for extraction, until the organic phase is clear and transparent, indicating that PMP has been removed, and then take the aqueous phase through the membrane for sampling and analysis. At the same time, prepare 10 mg / mL of guluronic acid, mannuronic acid, mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose standards and mixed standards, treat them according to the above derivatization method, filter them through a 0.22 μm filter membrane and then operate on the machine. The results are as follows Figure 2 As shown, the ion chromatogram of the monosaccharide composition of SFPN4 showed that SFPN4 was mainly composed of mannuronic acid and guluronic acid, and the molar ratio of the two was 0.695:0.305.

[0055] 3. Analysis of glycosidic bond connection mode

[0056] SFPN4 was subjected to uronic acid reduction, methylation, complete acid hydrolysis and acetylation and then monitored and analyzed by gas chromatography-mass spectrometry. The results are shown in Table 1. SFPN4 methylation was analyzed by GC-MS, and five glycosidic bonds were detected, mainly composed of T-Gulp (8.9%), T-Man (13.5%), 1,4-Gulp (16.2%), 1,4-Man (31.2%) and 1,3,4-Man (2.0%).

[0057] Table 1 Gas chromatography-mass spectrometry results of SFPN4 methylation product acid hydrolyzate derivatization of sugar alcohol acetate

[0058]

[0059] 4. Analysis of the fine structure of SFPN4

[0060] SFPN4 was dissolved in 0.5 mL D2O and analyzed using a 600 MHz Bruker NMR spectrometer. 1 H and 13 The glycosidic bond composition of SFPN4 was analyzed by C spectrum and two-dimensional NMR COSY, NOESY, HSQC and HMBC spectra. Figure 3-Figure 8 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 The anomeric proton signals of the H spectrum were mainly concentrated in δ3.2-4.0 ppm, and a total of five proton peaks were identified, namely δ4.96, 4.95, 4.63, 4.61, and 4.56. 13 The anomeric carbon signals of the C spectrum are concentrated between δ93 and 105, and a total of five anomeric carbons were identified, which are δ101.33, 100.86, 99.92, 99.91, and 99.41. In addition, 172-176 ppm is the uronic acid signal peak. Through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ99.92 and the corresponding anomeric hydrogen signal is δ4.57. Through HH-COSY, the signals of H1-2 are 4.57 / 3.95; the signals of H2-3 are 3.95 / 3.69; the signals of H3-4 are 3.69 / 3.81; the signals of H4-5 are 3.81 / 3.68; it can be inferred that H1, H2, H3, H4, H5 are δ4.57, 3.95, 3.69, 3.81, 3.68 respectively, and the corresponding C1-C5 are 99.92, 69.8775.55, 77.76, 71.34; the chemical shift of C6 is δ174.99. Therefore, this signal should be attributed to the glycosidic bond →4)-β-ManA-(1→. All glycosidic bond signals are assigned according to similar rules, as shown in Table 2.

[0061] Table 2 Attribution of hydrogen and carbon signals

[0062]

[0063] Main chain analysis: In the HMBC spectrum, the anomeric hydrogen of the glycosidic bond →4)-β-ManA-(1→ has a correlation signal peak with its own C4; in addition, the anomeric carbon has a correlation signal peak with its own H4; indicating the existence of a connection mode of →4)-β-ManA-(1→4)-β-ManA-(1→. In addition, the anomeric hydrogen of →4)-β-ManA-(1→ has a correlation peak with the C4 of the glycosidic bond →3,4)-β-ManA-(1→, indicating the existence of a glycosidic bond →4)-β-ManA-(1→3,4)-β-ManA-(1→; the anomeric hydrogen of →3,4)-β-ManA-(1→ has a correlation peak with the C4 of the glycosidic bond →4)-α-GulA-(1→, indicating the existence of a glycosidic bond →3,4)-β-ManA-(1→4)-α-GulA-(1→.

[0064] Branch chain analysis: H1 of α-GulA-(1→) and H3 of →3,4)-β-ManA-(1→) have correlation peaks, indicating the existence of glycosidic bond α-GulA-(1→3,4)-β-ManA-(1→. H1 of β-ManA-(1→) and H3 of →3,4)-β-ManA-(1→) have correlation peaks, indicating the existence of glycosidic bond β-ManA-(1→3,4)-β-ManA-(1→.

[0065] Based on this analysis, the structure of SFPN4 is Fig. 9 shown.

[0066] Example 3 Activity verification of Sargassum fusiformis polysaccharide SFPN4

[0067] 1. Inhibitory effect of SFPN4 on α-glucosidase

[0068] Take 40 μL of SFPN4 samples with different concentrations, add 30 μL of α-glucosidase solution (0.2 U / mL), incubate at 37 ° C for 10 min, then add 30 μL of p-NPG solution (5 mM) for 15 min, then add 100 μL of Na2CO3 (0.2 M) to terminate the reaction, and finally measure its absorbance at a wavelength of 405 nm. PBS was the blank group and acarbose was the positive control group. The inhibition rate of α-glucosidase and IC 50 value.

[0069] The results are as follows Fig.10 As shown in A, the IC of SFPN4 50 The value was 469.03 μg / mL, which was lower than the IC of acarbose 50 value (657 μg / mL), which indicated that SFPN4 had potential hypoglycemic activity.

[0070] 2. Inhibitory mechanism of SFPN4 on α-glucosidase

[0071] From the Lineweaver-Burk curve ( Fig.10 B) It can be seen that when the concentration of α-glucosidase remains unchanged, when different concentrations of SFPN4 (0, 200, 800 μg / mL) are added, the fitting curves of the reciprocal of the reaction rate (1 / V) and the reciprocal of the substrate concentration (1 / pNPG) show a linear relationship and intersect at the X-axis, and as the inhibitor concentration increases, the Michaelis constant Kmax remains unchanged and the maximum reaction rate Vmax decreases, indicating that SFPN4 can form an inhibitor-enzyme-substrate complex with α-glucosidase and the substrate, so that the substrate cannot be decomposed into products, and the inhibition type belongs to non-competitive inhibition.

[0072] Circular dichroism spectroscopy can be used to explore the effect of SFPN4 on the secondary structure of α-glucosidase, such as Fig.10 As shown in C and Table 3, after the addition of SFPN4 (2 mg / mL), the proportion of α-helix of α-glucosidase increased significantly, and the proportion of β-folding decreased significantly, which indicated that SFPN4 interacted with α-glucosidase to change the secondary structure of the enzyme, thereby inhibiting the enzyme activity.

[0073] Table 3 Changes in the secondary structure of α-glucosidase

[0074]

[0075] Different concentrations of SFPN4 were mixed and incubated with α-glucosidase at 25°C or 37°C, and then the fluorescence spectrum of α-glucosidase was detected. Fig.10 As shown in DF (in D and E, 0, 5, 10, 15, 20, 25, 30, 35, and 40 μg / mL represent the fluorescence values ​​of SFPN4 after incubation with α-glucosidase at this concentration, and SFPN4 represents the fluorescence value of the pure SFPN4 solution). It can be seen that the fluorescence intensity of the pure SFPN4 solution (40 μg / mL) is very weak and can be ignored. The emission spectrum of α-glucosidase is concentrated around 339 nm. As the concentration of added SFPN4 increases, the fluorescence intensity of α-glucosidase gradually decreases, indicating that SFPN4 can interact with α-glucosidase and cause the endogenous fluorescence quenching of α-glucosidase. In addition, the λem of α-glucosidase does not change significantly, indicating that polysaccharides do not change the internal microenvironment of α-glucosidase. At different temperatures, the Stern-Volmer correction curves of SFPN4 on the fluorescence quenching of α-glucosidase showed a good linear relationship, and with the increase of temperature, the effective quenching constant Ka increased, indicating that the fluorescence quenching of α-glucosidase by SFPN4 was dynamic quenching.

[0076] 3. Starch loading experiment

[0077] The SFP grade Kunming mice used in the present invention were purchased from the Guangdong Medical Animal Experiment Center. Animal feed (SFP grade) was provided by the Guangdong Medical Experiment Animal Center and stored at 4°C. The temperature of the feeding room was maintained at 25±1°C, the relative humidity was maintained at 55±5%, and the lighting was maintained for 12 hours per day by controlling the lighting. During the experiment, the animals were not restricted in diet and drinking water. After one week of adaptive feeding, the mice were grouped and processed according to Table 4.

[0078] Table 4 Starch loading experiment grouping and treatment method

[0079]

[0080] In the formal experiment, all animals were fasted for 12 hours and then gavaged according to the experimental groups, treatment methods and doses designed in Table 4. The blood glucose levels of each mouse at 0, 30, 60 and 120 minutes were recorded using an Omron blood glucose meter HGM-111. Fig.11 As shown, the blood sugar level in the control group increased rapidly after 30 minutes. In contrast, the blood sugar level in the other experimental groups increased slowly. The blood sugar level in the sitagliptin + SFPN4 group increased the slowest and had the lowest blood sugar level. The results showed that sitagliptin-SFPN4 was significantly superior to single sitagliptin intervention and single SFPN4 intervention in inhibiting the increase in blood sugar level caused by starch intake, and the combined use of SFPN4 and sitagliptin had a synergistic effect.

[0081] 4. Effect of SFPN4 supplementation on the hypoglycemic effect of sitagliptin

[0082] 4.1 Construction and grouping of diabetic mouse models

[0083] The present invention relates to Kunming mice (SPF grade, male), weighing 20±2g. The breeding environment is 25±1°C, relative humidity 55±5%, 12 hours of light per day, and the mice can freely eat and drink. The mouse feed is divided into normal maintenance feed and high-sugar and high-fat feed.

[0084] After all experimental animals were fed with normal maintenance feed for one week, they were randomly divided into a blank group and a high sugar and high fat (HSFD) group. The NC group (n = 10) continued to be fed with normal maintenance feed, and the HSFD group was fed with high sugar and high fat feed for 8 weeks. Then, all experimental animals were fasted for 12 hours, and mice in the HSFD group were intraperitoneally injected with 1.5 mL of STZ (prepared with low-temperature sterile pH = 4.4, 0.1 M citrate buffer solution) at a dose of 25 mg / kg, and the NC group was injected with the same volume of normal saline. The injection was continued for 3 days, and the feeding situation remained unchanged during the period. The fasting blood glucose of mice in the HSFD group was measured using a blood glucose meter, and mice with blood glucose higher than 11.1 mM were selected to enter the formal experiment.

[0085] The selected HSFD group mice were regrouped into 4 groups: model group, sitagliptin group, SFPN4 group, sitagliptin+SFPN4 group. All mice were treated according to Table 5.

[0086] Table 5 Experimental groups and treatment methods

[0087]

[0088]

[0089] The fasting blood glucose of all mice was recorded weekly. At the end of the experiment, all mice were fasted for 12 hours and then anesthetized with ether. Whole blood was collected and centrifuged (1000g, 4°C) for 10 minutes to collect serum; after sacrifice, the liver was collected and placed in liquid nitrogen for molecular biological analysis.

[0090] 4.2 Data Analysis

[0091] 4.2.1 During the administration period, body weight was measured once a week and blood was taken from the tail tip of the mice to measure fasting blood glucose using a blood glucose meter. An oral glucose tolerance test was performed one week before the end of the experiment: after fasting for 12 hours, 2 g / kg body weight glucose was given by gavage, and blood glucose levels were measured and recorded at 0, 30, 60, 90, 120, and 180 min, and the area under the blood glucose curve was calculated. The test kit was used to detect the patient's glycated hemoglobin and insulin levels, and the insulin resistance index was calculated according to the above method.

[0092] The fasting blood glucose of mice before, during and after the experiment was Fig.12 As shown in A. Compared with the blank group, the blood glucose level of mice in the model group was significantly increased (P<0.05), and the blood glucose level remained high in the following 8 weeks. Compared with the model group, the fasting blood glucose levels of mice in the sitagliptin group, SFPN4 group, and sitagliptin + SFPN4 group showed a gradual downward trend (P<0.05). At the 8th week of intervention, compared with the model group, the fasting blood glucose levels of mice in the sitagliptin group, SFPN4 group, and sitagliptin + SFPN4 group decreased by 38.88%, 40.74%, and 60.15%, respectively. The sitagliptin + SFPN4 group had a better effect on reducing the FBG level of diabetic mice than the single sitagliptin group and the single SFPN4 group.

[0093] Glycated hemoglobin level is an important marker of diabetes and reflects the severity of diabetic mice. Fig.12 As shown in Figure B, compared with the blank group, the glycated hemoglobin level in the model group was significantly increased, indicating that the diabetic mice in the model group were severely damaged. After intervention with sitagliptin, SFPN4, and sitagliptin + SFPN4, the glycated hemoglobin level of mice decreased significantly.

[0094] Oral glucose tolerance test was used to evaluate the glucose tolerance ability. Fig.12 C represents the area under the blood glucose curve (AUC) in the experiment. It can be seen that sitagliptin treatment alone, SFPN4 treatment alone and combined sitagliptin + SFPN4 administration can significantly restore the glucose tolerance of high-fat diet and STZ injection.

[0095] Homeostatic indices of insulin resistance Fig.12 D shows the insulin resistance of diabetic mice. The insulin resistance index level in the model group was significantly higher than that in the blank group. After 8 weeks of intervention, the insulin resistance index levels in the sitagliptin group, SFPN4 group, and sitagliptin + SFPN4 group were significantly lower than those in the model group.

[0096] The above experimental results show that the combined use of sitagliptin and SFPN4 has a better hypoglycemic effect than the use of sitagliptin alone.

[0097] 4.2.2 Serum parameters were detected by fully automatic biochemical analyzer, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and free fatty acids (FFA).

[0098] The results are as follows Fig.13 As shown in the figure, compared with the blank group, the serum TC, TG, HDL-C, LDL-C and FFA levels of the model group mice were significantly increased (P<0.05), indicating that diabetic mice had severe dyslipidemia; compared with the model group, sitagliptin, SFPN4 and sitagliptin + SFPN4 treatment can reduce the levels of TG, TC, LDL-C and FFA (P<0.05). In terms of reducing TC and FFA, the combined treatment of sitagliptin + SFPN4 was more effective than sitagliptin alone and SFPN4 alone (P<0.05). Sitagliptin + SFPN4 has a better effect on improving hyperlipidemia in diabetic mice.

[0099] 4.2.3 Gene expression analysis was performed on the livers collected from each group of mice using RT-qPCR technology to study the synergistic mechanism of sitagliptin + SFPN4 at the mRNA level. The primer sequences used for RT-qPCR are shown in Table 6. After normalization to β-actin, the mRNA expression of the mRNA was analyzed by 2 -ΔΔCt The expression level was calculated by the formula.

[0100] Table 6 RT-qPCR primer sequences

[0101]

[0102] The results are as follows Fig.14 and Fig.15As shown in the results, compared with the blank group, the model group had significantly decreased IRS-1, PI3K, Akt and PPARγ mRNA expressions in the liver of mice, and significantly increased G-6-Pase, PEPCK and CYP7A1 mRNA expressions (P<0.05). Compared with the model group, sitagliptin + SFPN4 administration effectively upregulated the relative expression of IRS-1, PI3K, Akt, AMPK-α, PPARγ and CYP7A1 (P<0.05), and significantly downregulated the relative expression of G-6-Pase and PEPCK (P<0.05). Among them, the expression levels of IRS-1, PI3K, Akt, AMPKα, PPARγ and CYP7A1 were significantly higher than those in the sitagliptin group and SFPN4 group (P<0.05), and the expression level of PEPCK was significantly lower than that in the sitagliptin group and SFPN4 group (P<0.05), indicating that sitagliptin + SFPN4 treatment is more conducive to the regulation of glucose and lipid metabolism-related pathways than single sitagliptin treatment and single SFPN4 treatment.

[0103] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Sargassum polysaccharide SFPN4 having a synergistic hypoglycemic effect of sitagliptin, characterized in that: The Sargassum fusiformis polysaccharide SFPN4 contains mannuronic acid and guluronic acid, and the molar ratio of mannuronic acid to guluronic acid is 0.695:0.305; The main chain of the Sargassum fusiformis polysaccharide SFPN4 is composed of →4)-β-ManA-(1→3,4)-β-ManA-(1→ and →3,4)-β-ManA-(1→4)-α-GulA-(1→; the branched chain includes α-GulA-(1→3,4)-β-ManA-(1→ and β-ManA-(1→3,4)-β-ManA-(1→; The repeating unit structural formula of the Sargassum fusiformis polysaccharide SFPN4 is: The molecular weight of the Sargassum fusiformis polysaccharide SFPN4 is 89.83 kDa.

2. A method for preparing the Sargassum fusiformis polysaccharide SFPN4 according to claim 1, characterized in that: The steps include: (1) mixing Sargassum fusiformis with water and beating the obtained slurry, sterilizing the obtained slurry, inoculating a composite bacterial agent for fermentation, and obtaining a fermentation liquid; (2) treating the fermented liquid in a water bath, centrifuging and obtaining the supernatant, and concentrating, precipitating with alcohol, removing ethanol, ultrafiltering and freeze-drying to obtain crude polysaccharide of Sargassum fusiformis; (3) preparing the crude polysaccharide of Sargassum fusiformis into a solution, subjecting it to ultrasonic and depigmentation treatment, and then eluting it with 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 M NaCl solutions as eluents, using a DEAE Sepharose FF anion exchange column for elution, collecting the sugar-containing solution eluted with the 0.4 M NaCl solution, dialyzing and concentrating it to obtain a desalted solution; The desalted solution is freeze-dried to obtain the Sargassum fusiformis polysaccharide SFPN4.

3. The preparation method according to claim 2, characterized in that: In step (1), the volume ratio of the Sargassum fusiformis to water is 1:

10.

4. The preparation method according to claim 2, characterized in that: In step (1), the composite bacterial agent is obtained by mixing thermophilic Streptococcus, plant lactobacillus and bulgaricus in a mass ratio of 1:1:1 and activating and culturing; the OD600 of the composite bacterial agent is greater than 0.5; the inoculation amount is 3%; the fermentation temperature is 37° C. and the time is 36 hours.

5. The preparation method according to claim 2, characterized in that: In step (2), the temperature of the water bath treatment is 90° C. and the time is 2 h; the concentration is 1 / 4 of the original volume; the alcohol precipitation method is: mixing the concentrated supernatant with 4 times the volume of anhydrous ethanol, and precipitating with alcohol at 4° C. for 24 h; the molecular weight cutoff of the ultrafiltration membrane used for the ultrafiltration is 10 kDa; and the number of ultrafiltrations is 6 times.

6. The preparation method according to claim 2, characterized in that: In step (3), the concentration of the solution is 1 mg / mL; the power of the ultrasound is 300 W and the time is 2 h; the depigmentation method is to use DA-201 macroporous resin to depigment until the color changes to light brown; the elution flow rate is 1 mL / min.

7. Use of the Sargassum fusiformis polysaccharide SFPN4 according to claim 1 or the Sargassum fusiformis polysaccharide SFPN4 prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a drug for treating diabetes.

8. Use of the Sargassum fusiformis polysaccharide SFPN4 according to claim 1 or the Sargassum fusiformis polysaccharide SFPN4 prepared by the preparation method according to any one of claims 2 to 6 in combination with sitagliptin in the preparation of a drug for treating diabetes.

9. A drug for treating diabetes, characterized in that: The invention comprises active ingredients and pharmaceutically acceptable excipients; the active ingredients comprise the Sargassum fusiformis polysaccharide SFPN4 described in claim 1 or the Sargassum fusiformis polysaccharide SFPN4 prepared by the preparation method described in any one of claims 2 to 6.

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