Application of sphingomonas exopolysaccharide in treatment of glycolipid metabolism disorder

Abstract

The invention discloses an application of sphingomonas extracellular polysaccharide in treatment of glucose and lipid metabolism disorder. The application range of the sphingomonas exopolysaccharide is expanded, the source is natural and safe, and a new thought is provided for prevention and treatment of diseases.

Description

Technical Field

[0001] The invention relates to the technical field of production of marine active substances, and in particular to application of Sphingomonas exopolysaccharide in treating glucose and lipid metabolism disorders. Background Art

[0002] The ocean is the region with the largest number of microorganisms on Earth. It is reported that more than 2 million species of eukaryotes and prokaryotes thrive in the ocean. Marine microorganisms have multiple functions: they can assess the health of marine ecology, maintain the balance of marine ecology, and are an important source of drugs and natural products. Many evidences show that bioactive components isolated from marine bacteria may have anti-inflammatory, hypoglycemic, hypolipidemic, immune and antioxidant activities. Although considerable progress has been made in the field of chemical synthesis and synthesis of bioactive compounds, the marine environment is still the richest and most diverse source of various bioactive substances. Marine bacteria can live in harsh marine environments such as deep sea and extreme cold, and have a variety of survival mechanisms to cope with environmental stress, thus producing bioactive substances with unique biological characteristics, which can be used in a variety of industrial and biotechnological applications. In nature, Sphingomonas are distributed in a variety of places, such as air, soil, deep sea and polar regions. Studies have shown that the reason why Sphingomonas can survive in such an environment is that in the face of environmental pressure, it can carry out efficient metabolic regulation, increase cell metabolism, and thus improve its own survival rate. Sphingomonasechinoides is a sphingomonas isolated from deep seawater. Therefore, Sphingomonas echinoides has great research value.

[0003] Cellular polymers contain many components such as polysaccharides, nucleic acids, proteins, lipids, etc. These substances are produced by microorganisms and secreted into their surrounding environment. Extracellular polymers can help bacteria adapt to the environment. Extracellular polymers have the function of assisting bacteria to survive on the surface of other substances and wrapping bacteria to resist changes in external dangerous environments. The physical properties of extracellular polymers are inseparable from their polysaccharide composition. The physical properties of extracellular polysaccharides depend not only on the structure of extracellular polysaccharides, but also on the form in which these residues are combined with each other. Therefore, bacterial extracellular polysaccharides have some functional characteristics of extracellular polymers, such as resisting changes in external dangerous environments and regulating the intracellular and extracellular environments. With the improvement of detection technology and people's analysis of sugar bond structure, carbohydrate research has become the preface of the life science neighborhood. Microbial extracellular polysaccharides (EPS) have their unique structure and genes, and can be regulated as expected during their production and modification. At the same time, they have excellent rheological properties and biodegradability, and have great application potential in many fields such as food and pharmaceuticals. Therefore, microbial EPS has great research significance.

[0004] At present, the technologies for preparing extracellular polysaccharides include: 1. Traditional fermentation method: At present, most methods for obtaining extracellular polysaccharide products use traditional fermentation methods, which may have the following shortcomings: (1) The content of extracellular polysaccharide products is too low; (2) The fermentation broth contains a variety of different substances and is a complex mixture; (3) It will produce an unpleasant odor that affects the subsequent use of extracellular polysaccharide products. In the face of such defects, the fermentation broth is usually concentrated, separated and purified multiple times.

[0005] 2. Optimizing culture conditions: The defects that may occur in traditional fermentation methods can be addressed by changing the culture conditions of bacteria to increase the production of bacterial exopolysaccharides. This is a widely used method to increase the production of exopolysaccharides and change the structural activity of exopolysaccharides. Specific methods include: (1) changing culture medium conditions such as comparison of multiple carbon sources and nitrogen sources; (2) changing culture conditions such as temperature, shaker speed, etc.

[0006] At present, Western medicine is often used as the main drug for the treatment of glucose and lipid metabolism disorders in clinical practice. However, the treatment and medication regimen of Western medicine alone has a long way to go in terms of the therapeutic effect on patients, and the cost is high and the side effects are large. Therefore, it is very necessary to find a cheap, safe and effective method for prevention and treatment. At present, much research on Sphingomonas is concentrated in the field of environmental protection, but no research on its extracellular polysaccharides has been reported. Summary of the invention

[0007] The purpose of the present invention is to provide an application of Sphingomonas exopolysaccharide in treating glucose and lipid metabolism disorders, which is green and safe and provides a new idea for the prevention and treatment of diseases.

[0008] The technical solution adopted by the present invention to solve the technical problem is: Application of Sphingomonas exopolysaccharide in the preparation of drugs for treating glucose and lipid metabolism disorders. The present invention first discovered that Sphingomonas exopolysaccharide has the function of effectively alleviating glucose and lipid metabolism disorders, is natural in source, safe, and has little toxic and side effects, providing a new idea for the prevention and treatment of diseases.

[0009] The Sphingomonas is specifically Sphingomonas echinoidesh.

[0010] Glucose and lipid metabolism disorder is glucose and lipid metabolism disorder induced by oleic acid.

[0011] The Sphingomonas exopolysaccharide is prepared by the following steps: (1) inoculating Sphingomonas echinoides into 2216E liquid culture medium, culturing with shaking in a shaker at 20° C. for 5-10 hours, subjecting the fermentation liquid of Sphingomonas echinoides to ultrasonic treatment, and continuing the shaking culture for 30 hours after the ultrasonic treatment to obtain the fermentation liquid of Sphingomonas echinoides; (2) filtering the fermentation broth of Sphingomonas echinoides, centrifuging to obtain the fermentation broth supernatant, and concentrating by rotary evaporation to obtain a concentrate; (3) Sevage reagent and concentrate were mixed and shaken in a volume ratio of 1:4, and the precipitate was removed by centrifugation. This was repeated three times to obtain a deproteinized solution. (4) The deproteinized solution treated in step (3) is precipitated with anhydrous ethanol at 4° C. overnight, the precipitate is collected by centrifugation, and the precipitate is freeze-dried in vacuum to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide is dissolved in distilled water to form a crude exopolysaccharide solution, which is purified by a DEAE-Sepharose Fast Flow column, eluted with a 0.1 M NaCl solution, and the eluate is collected, concentrated, dialyzed, and freeze-dried to obtain purified exopolysaccharide; Or the crude extracellular polysaccharide is dissolved in distilled water to form a crude extracellular polysaccharide solution, which is purified on a DEAE-Sepharose Fast Flow column, and eluted with distilled water, a 0.1 M NaCl solution, and a 0.2 M NaCl solution, respectively, and the eluates are collected separately, and the three eluates are combined, concentrated, dialyzed, and freeze-dried to obtain purified extracellular polysaccharides.

[0012] The present invention improves the extraction method of sphingomonas exopolysaccharide, and ultrasound intervention at a specific time can significantly increase the yield of sphingomonas exopolysaccharide, the method is simple and efficient, and can extract the best exopolysaccharide component that effectively reduces the effect of sugar and lipid metabolism disorders. In step (5), 0.1M NaCl solution is used for elution to obtain an exopolysaccharide component, and distilled water, 0.1M NaCl solution, and 0.2M NaCl solution are used for elution to obtain three polysaccharide components, and mixed use has a more comprehensive effect.

[0013] In step (1), the ultrasonic treatment parameters are: ultrasonic power 30-90W, ultrasonic frequency 25 kHz, and ultrasonic time 20-40s.

[0014] In step (2), the concentrated solution is concentrated by rotary evaporation at 50° C. to obtain a concentrated solution of 1 / 5 of its original volume.

[0015] In step (3), the Sevage reagent is a mixture of chloroform: n-butanol in a volume ratio of 5:1.

[0016] In step (4), the volume ratio of the deproteinized liquid to anhydrous ethanol is 1:3-1:5.

[0017] In step (5), the flow rate of the NaCl solution is 1 mL / min.

[0018] The beneficial effects of the present invention are: 1. The present invention improves the method for extracting extracellular polysaccharides of Sphingomonas, which is simple and efficient and can extract the best extracellular polysaccharide components that effectively alleviate the disorder of glucose and lipid metabolism.

[0019] 2. It expands the application scope of Sphingomonas extracellular polysaccharides, which are natural and safe, and provide new ideas for the prevention and treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effects of different ultrasound-assisted conditions on the EPS extraction of Sphingomonas (a) The effect of different ultrasound powers on the EPS extraction; (b) The effect of different ultrasound times on the EPS extraction; (c) The effect of different alcohol precipitation ratios on the EPS extraction; (d) The effect of different ultrasound stages on the EPS extraction. The results are expressed as mean ± SD (n = 3), and the values ​​represented by different letters are significantly different (p < 0.05).

[0021] Figure 2 These are SEM micrographs of Sphingomonas exopolysaccharides. (a) Exopolysaccharide morphology before ultrasound, ×1000; (b) Exopolysaccharide morphology after ultrasound, magnification ×1000.

[0022] Figure 3 This is the elution curve of EPS after DEAE-Sepharose FF ion exchange chromatography.

[0023] Figure 4 The results are expressed as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05).

[0024] Figure 5 Effects of crude polysaccharides and three EPS fractions on OA-induced glucose consumption in HepG2 cells. The results are expressed as mean ± SD (n = 3). The values ​​indicated by different letters are significantly different (p < 0.05).

[0025] Figure 6The results are shown in Table 1. The effects of three EPS fractions on the inhibition rate of α-glucosidase and pancreatic lipase. (a) The effects of three EPS fractions on the inhibition rate of α-glucosidase; (b) The effects of three EPS fractions on the inhibition rate of pancreatic lipase. The results are expressed as mean ± SD (n = 3).

[0026] Figure 7 The results are expressed as mean ± SD (n = 4). The values ​​with different letters are significantly different (p < 0.05).

[0027] Figure 8 The effect of EPS-2 on OA-induced glucose metabolism disorder in IR-HepG2 cells. (a) Effect of EPS-2 on glucose consumption rate (b) Effect of EPS-2 on mRNA levels of PEPCK and G6pase; (c) and (d) Effect of EPS-2 on relative protein levels of p-IRS-1, IRS-1, p-PI3K, PI3K, p-AKT, AKT, p-GSK-3β, GSK-3β, p-FoxO1, FoxO1, PEPCK, and G6Pase; The results are expressed as mean ± SD (n = 3). The values ​​indicated by different letters are significantly different (p < 0.05).

[0028] Fig. 9 The effect of EPS-2 on lipid metabolism disorder in IR-HepG2 cells induced by OA. (a) Oil Red O staining (b) Effect of EPS-2 on TG, TC, LDL and HDL content; (c) and (d) Effect of EPS-2 on relative protein levels of p-AMPK, AMPK, p-ACC1, ACC1 and SREBP-1C; Results are expressed as mean ± SD (n = 3). Values ​​indicated by different letters are significantly different (p < 0.05). DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below through specific embodiments.

[0030] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0031] The Sphingomonas echinoides strain was purchased from the China Marine Microbial Culture Collection Center, and the strain was Sphingomonas echinoides isolated from deep sea water.

[0032] Example 1: Application of Sphingomonas exopolysaccharide in the preparation of drugs for treating glucose and lipid metabolism disorders.

[0033] The Sphingomonas exopolysaccharide is prepared by the following steps: (1) Sphingomonas echinoides stored in -80℃ glycerol tubes was inoculated into 2216E solid culture medium (Qingdao Haibo Biotechnology Co., Ltd.) for activation twice. A single colony was picked and inoculated into 100 mL 2216E liquid culture medium (Qingdao Haibo Biotechnology Co., Ltd.), and cultured in a 20℃ shaker at 120 rpm for 12 h as a seed fermentation liquid. The seed fermentation liquid was inoculated into 500 mL 2216E liquid culture medium at a ratio of 2% (v / v), and cultured in a 20℃ shaker at 160 rpm. During the 10 h of culture, the Sphingomonas echinoides fermentation liquid was ultrasonically treated. The ultrasonic probe was disinfected with alcohol and cleaned with sterile water, and inserted 1 cm below the liquid level. The sample was kept in an ice bath during the treatment process. The ultrasonic frequency was 25 kHz, the ultrasonic power was 60 W, and the ultrasonic time was 40 s. After ultrasonic treatment, the culture was continued to shake for 30 hours to obtain the Sphingomonas echinoides fermentation liquid; (2) The fermentation broth of Sphingomonas echinoides was filtered and centrifuged at 5000 rpm for 15 min to obtain the fermentation broth supernatant, which was concentrated to 1 / 5 of the original volume by rotary evaporation at 50°C to obtain a concentrate; (3) Sevage reagent (chloroform: n-butanol = 5:1 volume ratio) and concentrate were mixed and shaken in a volume ratio of 1:4, and centrifuged at 10,000 rpm and 4 °C for 20 min to remove the precipitate. This was repeated three times to obtain a deproteinized solution. (4) The deproteinized solution treated in step (3) was precipitated with anhydrous ethanol, the volume ratio of the deproteinized solution to anhydrous ethanol was 1:4, the solution was kept at 4°C overnight, and the precipitate was collected by centrifugation at 8000 rpm for 15 min. The precipitate was vacuum freeze-dried to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide was dissolved in distilled water to form a crude exopolysaccharide solution with a concentration of 0.5 g / mL, and then purified on a DEAE-Sepharose Fast Flow column. First, the chromatography column (2.6×50 cm) was cleaned with distilled water. After the ion column was loaded, it was fully eluted with distilled water to fully balance the anion column. Elution was performed with a 0.1 M NaCl solution at a flow rate of 1 mL / min. The eluate was collected, concentrated, dialyzed (300 Da), and freeze-dried to obtain purified exopolysaccharide.

[0034] Single factor experiment based on Example 1: Experiment 1: The ultrasonic power was selected as 0, 30, 60 (Example 1), 90, 120, and 150 W; Experiment 2: The ultrasound time was selected as 0, 20, 40 (Example 1), 60, 80, 100, and 120 s; Experiment 3: The volume ratio of deproteinized liquid to anhydrous ethanol was 1:1, 1:2, 1:3, 1:4 (Example 1), 1:5, 1:6; Experiment 4: The Sphingomonasechinoides fermentation broth was subjected to ultrasonic treatment at 0 h, 5 h, 10 h (Example 1), 15 h, 20 h, and 25 h of cultivation; Experiment 5: The eluent was replaced with distilled water and NaCl solution (0.1 (Example 1), 0.2, 0.3, 0.4, 0.5 M) for elution. 5 mL of eluent was collected from each tube, and the polysaccharide content was detected by the phenol-sulfuric acid method. The elution curve was drawn with the absorbance value as the ordinate and the number of tubes as the abscissa.

[0035] Example 2: Application of Sphingomonas exopolysaccharide in the preparation of drugs for treating glucose and lipid metabolism disorders.

[0036] The difference from Example 1 is that: (5) the crude exopolysaccharide is dissolved in distilled water to form a crude exopolysaccharide solution with a concentration of 0.5 g / mL, and then purified on a DEAE-Sepharose Fast Flow column. First, the chromatography column (2.6×50 cm) is cleaned with distilled water, and after the ion column is loaded, it is fully eluted with distilled water to fully balance the anion column. Distilled water, a 0.1 M NaCl solution, and a 0.2 M NaCl solution are used for elution, and the eluates are collected respectively. The three eluates are combined, concentrated, dialyzed, and freeze-dried to obtain purified exopolysaccharide.

[0037] Under different ultrasound-assisted conditions, the amount of Sphingomonas exopolysaccharide extracted changes as follows Figure 1 As shown. Figure 1 a It can be seen that when the ultrasonic power increases from 0 W to 30 W, the EPS (extracellular polysaccharide) extraction amount increases significantly. When it increases from 30 W to 60 W, the EPS extraction amount increases significantly and the increase is greater than 0-30 W. When the ultrasonic power is 60-120 W, the EPS extraction amount begins to drop sharply. When the ultrasonic power is 120-150 W, the EPS extraction amount tends to be stable. Figure 1 As shown in Figure b, when the ultrasonic time increased from 0 s to 40 s, the EPS extraction amount increased significantly, while when the ultrasonic time was 40-80 s, the EPS extraction amount decreased significantly. When the ultrasonic time was 80-120 s, the EPS extraction amount tended to be stable. Figure 1c It can be seen that when the alcohol precipitation ratio (volume ratio of deproteinized liquid to anhydrous ethanol) increases from 1:1 to 1:3, the EPS extraction amount increases significantly. When the alcohol precipitation ratio is between 1:3 and 1:6, the EPS extraction amount is basically at the same level. Figure 1 It can be seen that when the ultrasonic stage was 0-10 h, the EPS extraction amount increased significantly, when the ultrasonic stage was 10-15 h, the EPS extraction amount decreased significantly, and when the ultrasonic stage was 15-25 h, the EPS extraction amount tended to be stable.

[0038] The conditions of Example 1 were used as the control, and no ultrasound-assisted extraction was performed. The microstructure of the EPS of Sphingomonas echinoides without ultrasound-assisted extraction was observed by SEM to be granular, and the EPS particles treated with ultrasound were finer and more uniform ( Figure 2 ).

[0039] The conditions of Example 1 were used to investigate the elution effects of different eluents: distilled water, NaCl solution (0.1, 0.2, 0.3, 0.4, 0.5 M) for elution. The polysaccharide content was detected by the phenol-sulfuric acid method. Since the polysaccharide content was positively correlated with the absorbance value, the absorbance value was used to represent the polysaccharide content. The elution curve was drawn with the absorbance value as the ordinate and the number of tubes as the abscissa. Figure 3 ), the crude polysaccharide EPS was separated by anion column to obtain three components, named EPS-1 (eluted with distilled water), EPS-2 (eluted with 0.1 M NaCl solution) and EPS-3 (eluted with 0.2 M NaCl solution).

[0040] Effects of EPS fractions on the proliferation activity of HepG2 cells Before the biological activity evaluation, the effects of crude polysaccharide and three EPS fractions on the proliferation activity of HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in the concentration range of 100-300 μg / mL were first determined. Figure 4 As shown. It was found that 200 μg / mL was the concentration of each component that had the best effect on the proliferation activity of HepG2 cells (p < 0.05). Therefore, 200 μg / mL of each component was selected as the concentration for preliminary activity screening.

[0041] The present invention utilizes OA to construct a glucose and lipid metabolism disorder model and studies the effect and related mechanism of Sphingomonas exopolysaccharide on regulating glucose and lipid metabolism disorder.

[0042] Control group: untreated cells; OA group: treated with OA (1 mmol / L) for 24 h; OA+EPS group: HepG2 cells were treated with OA (1 mmol / L) for 24 h and then treated with EPS-2 (200 μg / mL) for 24 h.

[0043] Effects of EPS fractions on glucose consumption rate of HepG2 cells induced by OA The effects of crude polysaccharides and three EPS (200 μg / L) on the glucose consumption rate of HepG2 cells induced by OA were detected to preliminarily screen the optimal EPS components for improving the glucose consumption rate of HepG2 cells induced by OA. Figure 5 As shown, crude polysaccharides and three EPS fractions significantly reversed the decrease in glucose consumption rate induced by OA (p < 0.05), and the glucose consumption rate after EPS-2 treatment was significantly higher than that after EPS-1 and EPS-3 treatments (p < 0.05).

[0044] Effects of EPS fractions on the inhibition rate of α-glucosidase and pancreatic lipase By testing the effects of three EPS fractions (0.25-2.0 mg / mL) on the inhibition rates of α-glucosidase and pancreatic lipase, the active polysaccharides with the greatest impact on glucose and lipid metabolism were preliminarily screened. Figure 6 a, EPS-1, EPS-2 and EPS-3 were found to inhibit the activity of α-glucosidase in a dose-dependent manner, but the inhibition rate of α-glucosidase did not change much after the dose was increased to 1.5 mg / mL, and the effect of EPS-2 was stronger than that of EPS-1 and EPS-3 at the same concentration level. Figure 6 b. It was found that except for EPS-1, which had no improvement in the inhibitory effect on pancreatic lipase at 0.25-0.5 mg / mL, the inhibitory effects of the other groups were all improved. When the concentration of the three polysaccharides increased from 1.5 mg / mL to 2.0 mg / mL, unlike the effect of EPS-1 on the inhibition rate of pancreatic lipase, the inhibition rates of the other two EPS separation components on pancreatic lipase began to decrease. At the same time, it was found that the inhibitory effect of EPS-2 at each concentration on pancreatic lipase was better than that of the other two components.

[0045] Three extracellular polysaccharides, EPS-1, EPS-2 and EPS-3, were isolated from the fermentation broth of Sphingomonas. After activity screening, the best component for alleviating lipid metabolism disorders was EPS-2. After testing, the basic physical and chemical properties of EPS-2: the total sugar content of EPS-2 accounted for as high as 74.3%, the protein content was 1.3%, the uronic acid content was low at 1.2%, and the ash content was 23.2%, indicating that EPS-2 had a high purity.

[0046] Effects of EPS-2 on HepG2 cell viability: Figure 7As shown in the data, compared with the control group, OA treatment alone significantly reduced the viability of HepG2 cells (p < 0.05); compared with the OA treatment group alone, the viability of HepG2 cells in the OA+EPS-2 group was significantly increased (p < 0.05), indicating that EPS-2 helps to reverse the negative effect of OA on the viability of HepG2 cells.

[0047] Effects of EPS-2 on glucose metabolism disorder induced by OA in HepG2 cells EPS-2 alleviates the glucose and lipid metabolism disorder in HepG2 cells induced by light oleic acid Effects of EPS-2 on parameters related to glucose metabolism disorder induced by OA in IR-HepG2 cells Figure 8 As shown. Figure 8 As shown in a, compared with the control group, OA treatment significantly reduced the glucose consumption rate (p < 0.05); compared with the OA alone treatment group, the OA+EPS-2 group significantly reversed the decrease in glucose consumption rate (p < 0.05).

[0048] like Figure 8 As shown in b, compared with the control group, the mRNA levels of PEPCK and G6Pase in OA-treated HepG2 cells were significantly increased (p < 0.05); compared with the OA-treated group, the mRNA levels of PEPCK and G6Pase in HepG2 cells in the OA+EPS-2 group were significantly decreased (p < 0.05).

[0049] like Figure 8 As shown in c, d, compared with the control group, OA treatment significantly reduced the protein levels of p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxO1, and significantly increased the protein levels of PEPCK and G6Pase (p < 0.05); compared with the OA alone treatment group, the protein levels of p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxO1 in the OA+EPS-2 group were significantly increased, while the protein levels of PEPCK and G6Pase were significantly decreased (p < 0.05). There was no significant difference in the protein levels of IRS-1, PI3K, AKT, GSK-3β and FoxO1 among the groups (p > 0.05).

[0050] Effects of EPS-2 on lipid metabolism disorder induced by OA in HepG2 cells Effects of EPS-2 on parameters related to lipid metabolism disorders in HepG2 cells induced by OA Fig. 9 As shown. Fig. 9 As shown in a, compared with the control group, the lipid secretion of HepG2 cells increased after OA treatment alone; compared with the OA alone treatment group, the lipid secretion of HepG2 cells in the OA+EPS-2 group decreased.

[0051] like Fig. 9 As shown in (b), compared with the control group, OA treatment significantly increased the contents of TG, TC and LDL, and significantly decreased the content of HDL (p < 0.05); compared with the OA alone treatment group, the contents of TG, TC and LDL in HepG2 cells in the OA+EPS-2 group were significantly decreased, and the content of HDL was significantly increased (p < 0.05).

[0052] like Fig. 9 As shown in c and d, compared with the control group, OA treatment significantly reduced the protein levels of p-AMPK and p-ACC1 (p < 0.05), and significantly increased the protein level of SREBP-1C (p < 0.05); compared with the OA alone treatment group, the protein levels of p-AMPK and p-ACC1 in the OA+EPS-2 group were significantly increased, and the protein level of SREBP-1C was significantly decreased (p < 0.05). There was no significant difference in the protein levels of AMPK and ACC1 among the groups (p > 0.05).

[0053] In summary, EPS-2 alleviated OA-induced glucose and lipid metabolism disorders in IR-HepG2 cells. First, in HepG2 cells, EPS-2 reversed the negative effects of OA on HepG2 cell viability. Secondly, EPS-2 increased the inhibition rate of α-glucosidase in OA-induced IR-HepG2 cells, which may be related to the activation of the IRS-1 / PI3K / Akt / GSK-3β / FoxO1 signaling pathway to downregulate the mRNA and protein levels of PEPCK and G6Pase. Furthermore, EPS-2 improved the changes in TG, TC, LDL and HDL levels induced by OA, which may be related to the activation of the AMPK / ACC1 / SREBP-1C signaling pathway. The present invention provides a theoretical basis for EPS-2 as an effective ingredient for potentially improving glucose and lipid metabolism disorders.

[0054] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. Application of Sphingomonas exopolysaccharide in the preparation of drugs for treating glucose and lipid metabolism disorders.

2. The use according to claim 1, characterized in that: The Sphingomonas is specifically Sphingomonas echinoidesh.

3. The use according to claim 1, characterized in that: Glucose and lipid metabolism disorder is glucose and lipid metabolism disorder induced by oleic acid.

4. The use according to claim 1, characterized in that: The Sphingomonas exopolysaccharide is prepared by the following steps: (1) inoculating Sphingomonas echinoides into 2216E liquid culture medium, shaking and culturing in a shaker at 20° C. for 5-10 hours, ultrasonically treating the fermentation liquid of Sphingomonas echinoides, and continuing to shake and culture for 30 hours after the ultrasonic treatment to obtain a fermentation liquid of Sphingomonas echinoides; (2) filtering the fermentation broth of Sphingomonas echinoides, centrifuging to obtain the fermentation broth supernatant, and concentrating by rotary evaporation to obtain a concentrate; (3) Sevage reagent and concentrate were mixed and shaken in a volume ratio of 1:4, and the precipitate was removed by centrifugation. This was repeated three times to obtain a deproteinized solution. (4) The deproteinized solution treated in step (3) is precipitated with anhydrous ethanol at 4° C. overnight, the precipitate is collected by centrifugation, and the precipitate is freeze-dried in vacuum to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide is dissolved in distilled water to form a crude exopolysaccharide solution, which is purified by a DEAE-Sepharose FastFlow column, eluted with a 0.1 M NaCl solution, and the eluate is collected, concentrated, dialyzed, and freeze-dried to obtain purified exopolysaccharide; Or the crude extracellular polysaccharide is dissolved in distilled water to form a crude extracellular polysaccharide solution, which is purified on a DEAE-Sepharose FastFlow column, and eluted with distilled water, a 0.1 M NaCl solution, and a 0.2 M NaCl solution, respectively, and the eluates are collected separately, and the three eluates are combined, concentrated, dialyzed, and freeze-dried to obtain purified extracellular polysaccharides.

5. The use according to claim 1, characterized in that: In step (1), the ultrasonic treatment parameters are: ultrasonic power 30-90W, ultrasonic frequency 25kHz, and ultrasonic time 20-40s.

6. The use according to claim 1, characterized in that: In step (2), the concentrated solution is concentrated by rotary evaporation at 50° C. to obtain a concentrated solution of 1 / 5 of its original volume.

7. The use according to claim 1, characterized in that: In step (3), the Sevage reagent is a mixture of chloroform: n-butanol in a volume ratio of 5:

1.

8. The use according to claim 1, characterized in that: In step (4), the volume ratio of the deproteinized liquid to anhydrous ethanol is 1:3-1:

5.

9. The use according to claim 1, characterized in that: In step (5), the flow rate of the NaCl solution is 1 mL / min.

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