Sri lankan sphaerotrichia multipolyssacharide with improved endothelial cell dysfunction and glycolipid metabolism disorder function

Abstract

The application discloses a Ceylonus sea wood ear polysaccharide with improved endothelial cell dysfunction and glycolipid metabolism disorder functions, and the polysaccharide is extracted from Ceylonus sea wood ear by using a composite enzyme. The application not only provides a theoretical basis for high-value utilization of the Ceylonus sea wood ear, but also provides a new idea for disease prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional polysaccharide extraction, in particular to a Ceylonus wood ear polysaccharide with the function of improving endothelial cell dysfunction and glycolipid metabolism disorder. BACKGROUND

[0002] Seaweeds are known as producers of various bioactive macromolecules (polyphenols, diterpenes, fibers, proteins, and especially polysaccharides), and polysaccharides, as an important component of seaweeds, have attracted strong research interest from scientists today due to their different structures and physicochemical properties and interesting functional characteristics. Due to the complexity of their chemical structure and the diversity of functional groups, seaweed polysaccharides have a variety of physical and chemical properties, thus also showing a wide range of biological activities, and can interact with many compounds, lipids, cellular proteins, and microbiota. Therefore, it is generally believed that seaweed polysaccharides have broad application prospects. For example, a recent study by Zhang et al. found that sulfated polysaccharides extracted from Undaria pinnatifida can regulate intestinal microbiota, thereby inhibiting weight gain and lipid metabolism, which may be a potential solution to obesity caused by high-fat diets (Zhang P, Jia J, Jiang P, et al. Polysaccharides from Edible Brown Seaweed Undaria Pinnatifida are Effective Against High-fat Diet-Induced Obesity in Mice Through the Modulation of Intestinal Microecology [J]. Food & Function, 2022, 13(5): 2581-2593). The functional properties of seaweed polysaccharides mainly depend on their complex polymer structure, such as glycosidic bonds, chain conformation, molecular weight, degree of sulfation, percentage of uronic acid, and monosaccharide composition. Therefore, it is necessary to understand the chemical properties of seaweed polysaccharides to further study the relationship between their structural characteristics and related biological activities. In addition, the difference in extraction methods is another factor that affects the biological activity of seaweed polysaccharides.

[0003] In order to extract polysaccharides from the cell walls of various algae, some techniques have been utilized to achieve an easy method for extracting polysaccharides. Traditional methods of extracting polysaccharides from algae mainly use hot water, acid, alkali, or a combination of these solvents as extraction media. The process involves placing pre-treated algae in different solvents at different temperatures for a certain period of time to obtain polysaccharides with minimal impurities. Although traditional methods can extract a certain amount of polysaccharides from algae, there are some limiting factors, such as long extraction time, low polysaccharide yield, the need for manual handling, thermal instability, and the threat of organic compounds to the environment, which limit the use of these methods.

[0004] Sarcodia ceylonensis and Sarcodia ceylanica. They mainly grow in the southeast coastal areas of China, and these sea areas provide suitable growth environment for them. Sarcodia contains rich seaweed polysaccharides and unsaturated fatty acids, and more than 20 kinds of nutrients unique to marine plants. These components endow Sarcodia with multiple pharmacological activities. Sarcodia is known as 'longevity vegetable' and'marine vegetable', and is deeply loved and respected by people. At present, the research on the active function of Sarcodia ceylonensis polysaccharide is still less, and further research and development of its value are needed. SUMMARY

[0005] The purpose of the present application is to provide a Sarcodia ceylonensis polysaccharide with the function of improving endothelial cell dysfunction and glycolipid metabolism disorder, which not only provides a theoretical basis for the high-value utilization of Sarcodia ceylonensis, but also provides a new idea for the prevention and treatment of diseases.

[0006] The technical scheme adopted by the present application to solve its technical problems is:

[0007] A Sarcodia ceylonensis polysaccharide with the function of improving endothelial cell dysfunction and glycolipid metabolism disorder is prepared by the following steps:

[0008] (1) Sarcodia ceylonensis is soaked with water, washed clean, then dried and crushed;

[0009] (2) Sarcodia ceylonensis powder is added to 95% ethanol at a solid-liquid ratio of 1g:4mL for heating to reflux for 3 hours for defatting and decolorizing treatment, and the ethanol is discarded;

[0010] (3) Step (2) is repeated twice, and then the defatted and decolorized Sarcodia ceylonensis powder is dried;

[0011] (4) The defatted Sarcodia ceylonensis powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1g:30-40mL, then a composite enzyme is added, the pH is adjusted, and then heated to 50-55 DEG C for enzymolysis reaction, after the enzymolysis reaction is completed, the enzyme is inactivated by heating, the supernatant is obtained by centrifugation, vacuum reduced pressure concentration, then anhydrous ethanol is added to precipitate at 4 DEG C overnight, the supernatant is discarded by centrifugation, and freeze-drying is carried out to obtain Sarcodia ceylonensis polysaccharide crude product;

[0012] (5) The Sarcodia ceylonensis polysaccharide crude product is dissolved in distilled water, and the Sevage method is used to remove protein;

[0013] (6) The deproteinized crude C. ceylonensis polysaccharide is dissolved in distilled water, and then loaded onto a DEAE-52 cellulose column, and then eluted with a 0.3 mol / L NaCl solution, and the eluate is collected, concentrated, dialyzed and freeze-dried to obtain the C. ceylonensis polysaccharide having the functions of improving endothelial cell dysfunction and glycolipid metabolism disorder.

[0014] In step (4), the amount of the complex enzyme is 1.5-2% of the weight of the defatted C. ceylonensis algal powder.

[0015] The amount of the complex enzyme is 1.6% of the weight of the defatted C. ceylonensis algal powder.

[0016] In step (4), the complex enzyme is composed of papain:cellulase = 2:1 by weight.

[0017] In step (4), the pH of the enzymatic reaction is controlled to be 5-5.5.

[0018] In step (4), the time of the enzymatic reaction is 120-140 min.

[0019] In step (6), the concentration of the deproteinized crude C. ceylonensis polysaccharide after being dissolved in distilled water is 5 mg / mL.

[0020] The C. ceylonensis polysaccharide is used for preparing a medicine for treating endothelial cell dysfunction.

[0021] The C. ceylonensis polysaccharide is used for preparing a medicine for treating glycolipid metabolism disorder.

[0022] The C. ceylonensis polysaccharide has the functions of improving endothelial cell dysfunction and glycolipid metabolism disorder, which provides a theoretical basis for high-value utilization of C. ceylonensis and provides a new idea for disease prevention and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 5 is the influence of different enzyme methods and auxiliary conditions on the extraction rate of C. ceylonensis SP (A) The influence of the amount of complex enzyme on the extraction rate; (B) The influence of the enzymolysis time on the extraction rate; (C) The influence of the enzymolysis temperature on the extraction rate; (D) The influence of the solid-liquid ratio on the extraction rate; (E) The influence of pH on the extraction rate. The results are represented as mean ± standard deviation (n = 3), and the values represented by different letters are significantly different (p < 0.05);

[0024] Figure 2 Figure 6 is the elution curve of the C. ceylonensis polysaccharide DEAE-52 ion exchange column chromatography;

[0025] Figure 3Figure 2: Effects of two SP components on LPS-induced inflammatory cytokines (TNF-α, INF-β) and NO secretion in HUVEC cells. (A) Effects of two SP components on NO content in HUVEC cells induced by LPS; (B) Effects of two SP components on TNF-α content in HUVEC cells induced by LPS; (C) Effects of two SP components on IFN-β content in HUVEC cells induced by LPS. Results are expressed as mean ± standard deviation (n = 3). Values ​​indicated by different letters are significantly different (p < 0.05).

[0026] Figure 4 The two SP components affect the content of antioxidant enzymes T-SOD, CAT, GPx, GST, and GR; the results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05);

[0027] Figure 5 Effects of SP on glucose consumption in IR-HepG2 cells; Results are expressed as mean ± SD (n = 3). Values ​​indicated by different letters are significantly different (p < 0.05);

[0028] Figure 6 This is a graph showing the inhibitory effect of Auricularia auricularia polysaccharide on α-glucosidase and pancreatic lipase;

[0029] Figure 7 The effect of SP-1 on HUVEC cell viability; the results are expressed as mean ± standard deviation (n = 4). The values ​​represented by different letters are significantly different (P < 0.05);

[0030] Figure 8 The effect of SP-1 on LPS-induced HUVEC cell hyperpermeability. (A) Cell migration and invasion were assessed using wound healing, transwell migration, and invasion assays. (B) Relative quantitative analysis of migration area was performed using ImageJ software. (C) and (D) Relative quantitative analysis of the number of migrating and invasive cells was performed using ImageJ software. Results are expressed as mean ± standard deviation (n = 3). Values ​​indicated by different letters are significantly different (P < 0.05).

[0031] Figure 9Effect of SP-1 on the parameters related to inflammation and anti-inflammatory ability of HUVEC cells. (A) and (B) Effect of SP-1 on the content and mRNA level of inflammatory factors of HUVEC cells; (C) and (D) Effect of SP-1 on the relative protein levels of p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-STAT1, STAT1, p-STAT3, STAT3, p-NF-κB, NF-κB, PECAM-1, ZO-1 and Occludin; the results are expressed as mean ± standard deviation (n = 3). The values represented by different letters are significantly different (P < 0.05);

[0032] Figure 10 Effect of SP-1 on the parameters related to oxidative damage and antioxidant ability of HUVEC cells. (A) and (B) Effect of SP-1 on the relative level of ROS caused by LPS; (C) and (D) Effect of SP-1 on the content and mRNA level of antioxidant enzymes Cu / ZnSOD, MnSOD, CAT, GPx, GST and GR; (E) and (F) Effect of SP-1 on the relative protein levels of N-Nrf2, T-Nrf2 and Keap1; the results are expressed as mean ± standard deviation (n = 3). The values represented by different letters are significantly different (P < 0.05);

[0033] Figure 11 Effect of SP-1 on the activity of HepG2 cells; the results are expressed as mean ± standard deviation (n = 4). The values represented by different letters are significantly different (P < 0.05);

[0034] Figure 12 Effect of SP-1 on the glucose metabolism disorder of IR-HepG2 cells induced by OA. (A) Effect of SP on the glucose consumption of HepG2 cells (B) Effect of SP-1 on the mRNA levels of PEPCK and G6pase; (C) and (D) Effect of EPS-2 on the 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 ± standard deviation (n = 3). The values represented by different letters are significantly different (P < 0.05);

[0035] Figure 13Effect of SP-1 on lipid metabolism disorder of OA-induced IR-HepG2 cells. (A) Oil red O staining (B) Effect of SP-1 on TG, TC, LDL and HDL content; (C) and (D) Effect of SP-1 on relative protein levels of p-AMPK, AMPK, p-ACC1, ACC1 and SREBP-1C; results are expressed as mean ± standard deviation (n = 3). Values denoted by different letters are significantly different (p < 0.05). DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further specifically described below through specific examples.

[0037] In the present application, unless specified, the raw materials and equipment used can be purchased from the market or commonly used in the art. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0038] Cellulase 10000 U / mg, purchased from Beijing Solayebio Technology Co., Ltd.; papain 10000 U / mg, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0039] Example 1:

[0040] A Ceylonus sea wood ear polysaccharide with the function of improving endothelial cell dysfunction and glycolipid metabolism disorder is prepared by the following steps:

[0041] (1) Ceylonus sea wood ear (dry product) is soaked with water, rinsed clean, and then dried in a 55℃ air-drying oven, and pulverized;

[0042] (2) The Ceylonus sea wood ear powder is added to 95% ethanol at a solid-liquid ratio of 1g:4mL, heated to reflux for 3 hours for defatting and decolorizing treatment, and the ethanol is discarded;

[0043] (3) Step (2) is repeated twice, and then the defatted and decolorized Ceylonus sea wood ear powder is dried;

[0044] (4) The defatted Ceylonus sea wood ear powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1g:30mL, then a composite enzyme is added, the composite enzyme is composed of papain:cellulase at a weight ratio of 2:1, the amount of the composite enzyme is 1.5% of the weight of the defatted Ceylonus sea wood ear powder, after adjusting the pH to 5, the enzyme is heated to 50℃ for enzymolysis reaction for 140min, after the enzymolysis reaction is completed, the enzyme is inactivated by heating, the supernatant is obtained by centrifugation, vacuum reduced pressure concentration, then anhydrous ethanol is added to precipitate at 4℃ overnight, the supernatant is discarded by centrifugation, and freeze-drying is performed to obtain a crude Ceylonus sea wood ear polysaccharide;

[0045] (5) The crude Ceylonus sea wood ear polysaccharide is dissolved in distilled water to form a solution with a concentration of 5mg / mL, and the Sevage method is used for deproteinization;

[0046] (6) The deproteinized crude C. zeylanica polysaccharide is dissolved in distilled water, and then loaded onto a DEAE-52 cellulose column. The eluate is collected, concentrated, dialyzed, and freeze-dried to obtain C. zeylanica polysaccharide with improved endothelial cell dysfunction and glycolipid metabolism disorder function.

[0047] Example 2:

[0048] The difference between this example and Example 1 is that:

[0049] (4) The defatted C. zeylanica algal powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1 g: 35 mL, and then a complex enzyme is added. The complex enzyme is composed of papain: cellulase = 2: 1 by weight, and the amount of the complex enzyme is 2% of the weight of the defatted C. zeylanica algal powder. After adjusting the pH to 5.5, the enzyme hydrolysis reaction is carried out at 55°C for 120 min. After the enzyme hydrolysis reaction is completed, the enzyme is inactivated by heating. The supernatant is obtained by centrifugation, and then concentrated under vacuum and reduced pressure. Anhydrous ethanol is added and precipitated at 4°C overnight. The supernatant is discarded by centrifugation, and then freeze-dried to obtain the crude C. zeylanica polysaccharide. The rest is the same as in Example 1.

[0050] Example 3:

[0051] The difference between this example and Example 1 is that:

[0052] (4) The defatted C. zeylanica algal powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1 g: 40 mL, and then a complex enzyme is added. The complex enzyme is composed of papain: cellulase = 2: 1 by weight, and the amount of the complex enzyme is 1.6% of the weight of the defatted C. zeylanica algal powder. After adjusting the pH to 5.2, the enzyme hydrolysis reaction is carried out at 50.4°C for 120 min. After the enzyme hydrolysis reaction is completed, the enzyme is inactivated by heating. The supernatant is obtained by centrifugation, and then concentrated under vacuum and reduced pressure. Anhydrous ethanol is added and precipitated at 4°C overnight. The supernatant is discarded by centrifugation, and then freeze-dried to obtain the crude C. zeylanica polysaccharide. The rest is the same as in Example 1.

[0053] Experimental part: Three groups of repeated tests were set in this study, and the test results were expressed as mean ± standard deviation (SD). SPSS23.0 was used to analyze all the results by one-way analysis of variance (ANOVA) to determine whether there was a significant difference between the test groups. It is considered statistically significant when p<0.05.

[0054] 1.1 Pretreatment of C. zeylanica

[0055] Sri Lankan Gracilaria dura (dry) was soaked in water and repeatedly washed to remove impurities such as sand, and then the washed Sri Lankan Gracilaria dura was placed in a 55°C air-drying oven for drying. The dried Sri Lankan Gracilaria dura was crushed by a pulverizer, and then the Sri Lankan Gracilaria dura powder was added to 95% ethanol at a solid-liquid ratio of 1:4 (w / v) for 3h of reflux defatting and decolorizing treatment, and the defatting and decolorizing treatment was repeated twice. Then the ethanol was discarded, and the defatted Sri Lankan Gracilaria dura powder was dried at 50°C and stored in a dry and dark place.

[0056] 1.2 Single-factor condition setting for extraction of crude polysaccharides from Sri Lankan Gracilaria dura

[0057] Single-factor experiments were performed with respect to the composite enzyme addition amount (0.5%, 1%, 1.5%, 2%, 2.5%), enzyme hydrolysis time (60, 80, 100, 120 and 140 min), enzyme hydrolysis temperature (45, 50, 55, 60 and 65°C), solid-liquid ratio (1:20, 1:30, 1:40, 1:50 and 1:60 g:mL) and pH (3, 4, 5, 6 and 7) to analyze the effect of each factor on the extraction rate of polysaccharides from Sri Lankan Gracilaria dura by composite enzyme-assisted extraction.

[0058] 1.3 Extraction process of polysaccharides from Sri Lankan Gracilaria dura

[0059] The defatted Sri Lankan Gracilaria dura powder was mixed with distilled water. Then, the composite enzyme (papain:cellulase = 2:1) was added according to the substrate mass, and the pH of the solution was adjusted with hydrochloric acid or NaOH. Then, the solution was placed in a constant-temperature water bath shaker for constant-temperature oscillation extraction, and then heated in a 90°C water bath for 10 min to inactivate the enzyme. Then, the mixture was centrifuged at 8000 rpm for 15 min and the supernatant was collected, and the supernatant was concentrated to about 1 / 4 of the volume under vacuum at 50°C, and then anhydrous ethanol was added at a ratio of (1:3, v / v) to precipitate overnight at 4°C. Then, the supernatant was discarded by centrifugation at 4500 rpm for 15 min, and the precipitate was freeze-dried to obtain the crude polysaccharide sample from Sri Lankan Gracilaria dura. The freeze-dried polysaccharide sample was stored in a desiccator until use, and the polysaccharide extraction rate was determined by the phenol-sulfuric acid method.

[0060] 1.4 Results of single-factor experiment for extraction of crude polysaccharides from Sri Lankan Gracilaria dura

[0061] The effects of different enzyme-assisted methods on the extraction rate of polysaccharides from Sri Lankan Gracilaria dura are shown in FIG. 1A. As shown in FIG. 1A, when the composite enzyme addition amount was 0.5%-1.5%, the polysaccharide extraction rate from Sri Lankan Gracilaria dura was significantly improved; when the enzyme amount was 1.5%-2%, the polysaccharide extraction rate began to decrease; and when the enzyme amount was 2%-2.5%, the polysaccharide extraction rate tended to be stable. Figure 1 Figure 1 ​BIt can be seen that the extraction rate of crude polysaccharide from C. ceylonensis increased significantly in the 60-120 min of enzymatic hydrolysis, and then tended to be stable in the following time. Figure 1 CAs shown in Fig. 2C, when the enzymatic hydrolysis temperature was lower than 50℃, the extraction rate of polysaccharide from C. ceylonensis gradually increased with the increase of temperature; however, once the temperature exceeded 50℃, the extraction rate of polysaccharide from C. ceylonensis began to decrease. From the above results, it can be seen that the optimal enzymatic hydrolysis temperature for the extraction of polysaccharide from C. ceylonensis was 50℃. Figure 1 DAs shown in Fig. 2D, when the solid-liquid ratio was less than 1:40 g / mL, the extraction rate of polysaccharide from C. ceylonensis gradually increased, and when the solid-liquid ratio was greater than 1:40 g / mL, the extraction began to decrease rapidly. From the above results, it can be seen that the optimal solid-liquid ratio for the extraction of polysaccharide from C. ceylonensis was 1:40 g / mL. Figure 1 EAs shown in Fig. 2E, the extraction rate of crude polysaccharide from C. ceylonensis first increased with the increase of pH, and then decreased significantly with the increase of pH.

[0062] 1.5Box-Behnken response surface optimization of polysaccharide extraction process

[0063] On the basis of single-factor experiments, Box-Behnken experimental design was carried out with enzyme addition amount (A), enzymatic hydrolysis temperature (B), and pH (C) as experimental factors and the extraction rate of crude polysaccharide from C. ceylonensis as response value. The process of compound enzyme-assisted extraction of crude polysaccharide from C. ceylonensis was optimized by response surface analysis. The factor level table of response surface analysis is shown in Table 1.

[0064] Table 1 Factor level table of response surface analysis

[0065]

[0066] The experimental conditions and the extraction rate of polysaccharide are shown in Table 2. The results showed that the extraction rate of polysaccharide was between 14.01-21.07%. The regression coefficient value was calculated to obtain a second-order polynomial equation: Y = 20.70 + 0.62A + 0.65B + 0.78C + 0.53AB + 0.29AC + 0.21BC - 1.16A 2 -4.90B 2 -2.20C 2 . According to the F value, the influence of A, B, and C on the extraction rate of crude polysaccharide from C. ceylonensis was in the order of C (pH) > B (enzymatic hydrolysis temperature ℃) > A (enzyme addition amount %).

[0067] Table 2 Response surface experimental design scheme and results

[0068]

[0069] 1.6Optimization of the optimal conditions and verification of the results

[0070] From the above experiment, it can be concluded that the optimum conditions for the composite enzyme-assisted extraction of Ceylonese Sarcodia polysaccharides are: the composite enzyme addition amount is set to 1.66%, the enzymolysis temperature is controlled at 50.43°C, and the pH value is adjusted to 5.2. Under these precise conditions, the predicted extraction rate of Ceylonese Sarcodia polysaccharides can reach 20.90%. In order to ensure the operability of the experimental process, the process parameters are slightly adjusted: the composite enzyme addition amount is adjusted to 1.6%, the enzymolysis temperature is set to 50.4°C, and the pH value is kept at 5.2. The test results show that under these modified conditions, the polysaccharide extraction rate is 20.86%, which is similar to the result obtained from the predicted value, proving the accuracy of the model.

[0071] 1.7 The Ceylonese Sarcodia polysaccharides extracted by the enzyme-assisted method are crude polysaccharides, which also contain impurities such as proteins and pigments. The structure and activity of polysaccharides will be affected by these impurities. In this experiment, Sevag method was used to remove proteins, and then DEAE-52 anion exchange column was used to separate and purify Ceylonese Sarcodia polysaccharides. At the same time, the best active component that can reduce endothelial dysfunction and glycolipid metabolism disorder was screened through in vitro experiments, and its physicochemical analysis and structural characterization were carried out.

[0072] The crude Ceylonese Sarcodia polysaccharide was dissolved in distilled water to form a solution with a concentration of 5 mg / mL, then mixed with Sevage reagent [V(trichloromethane):V(n-butanol)=4:1] at a ratio of 1:4, and fully oscillated to achieve the purpose of removing proteins. Then centrifugal treatment was carried out, the supernatant was dialyzed for 48 h, and then the extracted solution after dialysis was alcohol precipitated overnight. After centrifugation, the precipitate was freeze-dried to obtain the Ceylonese Sarcodia deproteinized sample.

[0073] In order to further purify the Ceylonese Sarcodia deproteinized sample, we used ultrapure water and 0.1, 0.3 and 0.5 M NaCl solutions to elute the 5 mg / mL Ceylonese Sarcodia deproteinized polysaccharide sample loaded into the DEAE-52 cellulose column. During the elution process, every 5 mL was collected, and the absorbance of the eluate was detected at 490 nm wavelength by the phenol-sulfuric acid method. Then the elution curve was drawn with the number of tubes as the horizontal coordinate and the absorbance as the vertical coordinate. According to the curve, the peak high component was collected, then concentrated, dialyzed, and finally freeze-dried for standby.

[0074] Figure 2 As shown in the figure. The Ceylonese Sarcodia deproteinized sample was separated by DEAE-52 anion column, and two obvious peaks were obtained at pure water and 0.3 mol / L NaCl, named SP-0 and SP-1, and freeze-dried for standby.

[0075] 1.7 Effect of SP components on the secretion levels of inflammatory factors (TNF-a, IFN-β) and NO in HUVEC cells The polysaccharide components were screened according to the contents of NO, TNF-a and IFN-β1. The cells were divided into three groups: untreated cells (Control), LPS-treated cells and cells treated with SP followed by LPS (LPS+SP-0, LPS+SP-1). After 1 h of treatment with SP-0, SP-1, LPS was added, and then the cells were incubated in a 37 °C, 5% CO2 incubator for 24 h. The supernatant was collected, and the contents of NO, TNF-a and IFN-β1 were detected according to the ELISA kit instructions, respectively.

[0076] The optimal SP component for inhibiting the inflammatory response of LPS-induced HUVEC cells was preliminarily screened by detecting the effects of two SPs (150 μg / mL) on the secretion levels of inflammatory factors (TNF-a, IFN-β) and NO in LPS-induced HUVEC cells. The effects of two SP isolated components on the secretion levels of inflammatory factors (TNF-a, INF-β) and NO in LPS-induced HUVEC cells are shown in Figure 3 SP-1 treatment significantly reduced the increase of TNF-a, INF-β and NO caused by LPS (p<0.05), and the secretion levels of TNF-a, INF-β and NO after SP-1 treatment were significantly lower than those after SP-0 treatment (p<0.05).

[0077] 1.8 Effect of SP components on the antioxidant enzymes in HUVEC cells

[0078] The cells were divided into three groups: untreated cells (Control), LPS-treated cells and cells treated with SP followed by LPS (LPS+SP-0, LPS+SP-1). After 1 h of treatment with SP-1, the HUVEC cells were treated with LPS (1.0 μg / mL) for 24 h. Then, the cells were collected and lysed on ice with a cell disrupter, and the protein concentration was determined according to the BCA method. The antioxidant enzyme activity levels were measured according to the manufacturer's instructions.

[0079] The optimal SP component for antioxidant effect was preliminarily screened by detecting the effects of two SPs (150 μg / L) on the antioxidant enzyme activity in LPS-induced HUVEC cells. The effects of two SP isolated components on the antioxidant enzyme activity in LPS-induced HUVEC cells are shown in Figure 4 SP-1 treatment significantly increased the activities of T-SOD, CAT, GPx, GST and GR in HUVEC cells (p<0.05), and the activities of T-SOD, CAT, GPx, GST and GR after SP-1 treatment were significantly higher than those after SP-0 treatment (p<0.05).

[0080] 1.9 Effect of SP on glucose consumption in IR-HepG2 cells

[0081] IR-HepG2 cell model was established using oleic acid (OA) induction for subsequent experiments. Cells were divided into three groups: untreated cells (Control), OA-treated group and OA-treated cells followed by SP treatment (OA+SP-0, OA+SP-1). When the cell density increased to 10 5 cells were seeded in 96-well plates. After cell attachment, 100 μm OA was added and incubated for 24 h. Then, SP-0 and SP-1 were added and incubated for another 24 h. After that, glucose concentration in the culture medium was determined using a glucose assay kit. Cell glucose consumption = blank group (no cells, only medium) glucose concentration - each experimental group glucose concentration.

[0082] The best hypoglycemic SP component was screened by detecting the effect of two SPs (200 μg / mL) on glucose consumption in IR-HepG2 cells. The effect of two SP separated components on glucose consumption in IR-HepG2 cells is shown in Figure 5 SP treatment significantly reversed the decrease in glucose consumption rate in IR-HepG2 cells caused by OA (p < 0.05), and the glucose consumption rate after SP-1 treatment was significantly higher than that after SP-0 treatment (p < 0.05).

[0083] 1.10 Effect of SP components on pancreatic lipase and α-glucosidase inhibition rate

[0084] The pancreatic lipase inhibitory activity was evaluated using p-nitrophenyl butyrate (p-NPB) as a substrate. A 50 μL aliquot of each sample solution at different concentrations (0.25, 0.5, 1.0, 1.5, and 2.0 mg / mL) was mixed with 50 μL of pancreatic lipase solution (50 mg / mL) that had been prepared in Tris buffer (0.1 mol / L, pH 8.2). The reaction was initiated by adding 50 μL of p-NPB (final concentration 5 mM) prepared in phosphate buffer (pH 7.3). The control reaction (without sample) used a similar procedure, which reflected 100% enzyme activity. After incubation at ambient temperature for 1 h, the absorbance of the reaction mixture was measured at 405 nm using a microplate reader. As a positive control, orlistat was determined using the same procedure. A suitable control without enzyme was added for each sample to remove the background absorbance. The inhibition of pancreatic lipase was estimated using the following formula:

[0085]

[0086] Where A reflects the control absorbance (reaction vial containing live pancrelipase and buffer); B reflects the control blank absorbance (buffer only, no pancrelipase); C indicates sample absorbance (sample and pancrelipase), D reflects sample blank absorbance (no pancrelipase, sample and buffer only).

[0087] The 40 μL of sample solution with different concentrations (0.25, 0.5, 1.0, 1.5 and 2.0 mg / mL) was added into 96-well plate containing 30 μL of α-glucosidase solution (0.2 U / mL), and then incubated at 37 °C for 10 min. Subsequently, 30 μL of p-NPB was mixed with the reaction mixture, and incubated at 37 °C for 0.5 h, followed by adding 0.2 mol / L sodium carbonate solution (50 μL) to stop the reaction. The mixed solution was incubated at 25 °C for 5 min, and its absorbance at 405 nm was measured, with acarbose as a positive control group. The α-glucosidase inhibition rate was determined as follows:

[0088]

[0089] Where A1 is the absorbance of the mixture reaction solution, A2 is the absorbance of the sample reaction solution without enzyme system, and A0 is the absorbance of the sample reaction solution without sample.

[0090] By detecting the effects of two SP separation components (0.25-2.0 mg / mL) on the levels of α-glucosidase and pancrelipase inhibition, the active polysaccharide with the greatest impact on glycolipid metabolism was preliminarily screened. As shown in Figure 6 A. It was found that both SP-0 and SP-1 could inhibit the activity of α-glucosidase in a dose-dependent manner, but the inhibition rate of α-glucosidase tended to be flat after the dose increased to 1 mg / mL, and the effect of SP-1 was stronger than that of SP-0 at the same concentration level. As shown in Figure 6 B. It was found that SP-1 had no obvious effect on the pancrelipase inhibition than SP-0 at 0.25-0.5 mg / mL; when the concentrations of the two polysaccharides increased from 1.0 mg / mL to 2.0 mg / mL, the inhibition rates of pancrelipase of the two SP components tended to be flat; and it was found that the inhibition effect of SP-1 on pancrelipase was better than that of SP-0 at each concentration.

[0091] 1.11 Two polysaccharides, SP-0 and SP-1, were isolated from the Ceylon sea fungus (Auricularia auricularia). Initial in vitro screening identified SP-1 as the optimal component for alleviating endothelial cell dysfunction and glucose and lipid metabolism disorders. Physicochemical analysis and structural characterization of SP-1 revealed a total sugar content of 80.89%, a protein content of 1.3%, a uronic acid content of 2.41%, and a sulfate content of 12.52%. Ultraviolet absorption spectroscopy also revealed minimal protein and nucleic acid content. SP-1 has a molecular weight of 18.453 kDa, and its monosaccharide composition is 8.18%, 84.16%, 2.26%, and 5.40% for galactose, glucose, xylose, and mannose, respectively.

[0092] 2.1 Ceylon Auricularia polysaccharide alleviates lipopolysaccharide-induced endothelial cell dysfunction. Currently, there are no reports on the effects of Ceylon Auricularia polysaccharide on endothelial dysfunction. Therefore, this study used lipopolysaccharide (LPS) to establish an endothelial injury cell model to investigate the effects of Ceylon Auricularia polysaccharide on endothelial cell dysfunction and its related mechanisms.

[0093] Control group: untreated cells; LPS group: HUVEC cells were treated with LPS (1.0 μg / mL) for 24 h; LPS+SP-1 group: HUVEC cells were treated with SP-1 (200 μg / mL) for 1 h and then with LPS (1.0 μg / mL) for 24 h.

[0094] Effect of SP-1 on HUVEC cell viability

[0095] Cell viability was determined by CCK-8 assay. Figure 7 Compared with the control group, LPS treatment led to a significant decrease in HUVEC cell viability (p<0.05); compared with the LPS group, the LPS+SP-1 group significantly increased HUVEC cell viability (p<0.05), indicating that SP-1 helps reverse the negative effect of LPS on HUVEC cell viability.

[0096] Effect of 2.3SP-1 on LPS-induced HUVEC cell hyperpermeability

[0097] Cell scratch assay: HUVEC cells from each group in the logarithmic growth phase were seeded in a six-well plate. When cells reached 90% confluence, a vertical line was made along the midpoint using a 200 μL pipette tip. The old culture medium was discarded, and the cells were washed twice with PBS. The cells were cultured for an additional 24 hours and images were observed using an inverted fluorescence microscope. Quantitative analysis was performed using ImageJ software, and images were observed using an inverted fluorescence microscope.

[0098] Cell migration and invasion experiments: Methods see Jin B R, Kim H J, Kim E Y, et al. 6'-Sialyllactose Ameliorates In Vivo and In Vitro Benign Prostatic Hyperplasia by Regulating the E2F1 / pRb-AR Pathway [J]. Nutrients, 2019, 11(9): 2203.

[0099] Figure 8 The effect of SP-1 on LPS-induced HUVEC cell hyperpermeability is shown. As shown in Figure 8 A-D. In cell scratch, migration and invasion experiments, LPS treatment resulted in a significant decrease in the total number of migrated and invaded cells compared to the control group; LPS+SP-1 group increased the total number of migrated and invaded cells compared to the LPS alone treatment group. The results showed that SP-1 can alleviate LPS-induced HUVEC cell hyperpermeability.

[0100] 2.4 Effect of SP-1 on HUVEC cell inflammation and anti-inflammatory ability related parameters

[0101] The effect of SP-1 on LPS-induced HUVEC cell hyperpermeability is shown. As shown in

[0102] As shown in Figure 9 A. Compared with the control group, the LPS alone treatment group significantly increased the content of IL-1β, IL-6, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells (p<0.05); compared with the LPS alone treatment group, the LPS+SP-1 group reduced the content of IL-1β, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells (p<0.05), but the content of IL-6 had no significant change (p>0.05).

[0103] As shown in Figure 9 B. Compared with the control group, the LPS alone treatment group significantly increased the mRNA levels of IL-1β, IL-6, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 (p<0.05); compared with the LPS alone treatment group, the LPS+SP-1 group significantly reduced the mRNA levels of IL-1β, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells (p<0.05), but there was no significant difference in IL-6 (p>0.05).

[0104] AsFigure 9 C and 9D. Compared with the control group, LPS alone increased the expression of p-ERK, p-JNK, p-p38, p-STAT1, p-STAT3 and p-NF-κB proteins, and decreased the expression of PECAM-1, ZO-1 and Occludin proteins (p<0.05); compared with the LPS alone group, LPS+SP-1 group decreased the expression of p-ERK, p-JNK, p-p38, p-STAT1, p-STAT3 and p-NF-κB proteins, and increased the expression of PECAM-1, ZO-1 and Occludin proteins (p<0.05). There was no significant difference in the expression of ERK, JNK, p-p38, p38, p-STAT1, STAT-1, STAT-3 and NF-κB proteins among the groups (p>0.05).

[0105] 2.5 Effect of SP-1 on oxidative damage and antioxidant capacity-related parameters of HUVEC cells

[0106] DCFH-DA staining was used to determine the content of ROS. The effect of SP-1 on oxidative damage and antioxidant capacity-related parameters of HUVEC cells is shown in Figure 10 A and B. Compared with the control group, LPS alone significantly increased the relative ROS level of HUVEC cells (p<0.05); compared with LPS alone, LPS+SP-1 group decreased the relative ROS level in HUVEC cells (p<0.05). Figure 10

[0107] As shown in Figure 10 C. Compared with the control group, LPS treatment led to decreased activities of Cu / Zn-SOD, CAT, GPx, GST and GR (p<0.05); compared with LPS alone, LPS+SP-1 group significantly increased the activities of Cu / Zn-SOD, CAT, GPx, GST and GR in HUVEC cells (p<0.05). There was no significant difference in MnSOD activity among the groups (p>0.05).

[0108] RNA was detected by qRT-PCR. As shown in Figure 10 D. Compared with the control group, LPS treatment significantly decreased the mRNA levels of Cu / Zn-SOD, CAT, GST and GR (p<0.05); compared with LPS alone, LPS+SP-1 group significantly increased the mRNA levels of Cu / Zn-SOD, CAT, GST and GR in HUVEC cells (p<0.05). There was no significant difference in the mRNA levels of MnSOD and GPx among the groups (p>0.05).

[0109] ​Western-blotting detection of proteins, such as Figure 10 As shown in E and F, compared with the control group, LPS treatment significantly reduced N-Nrf2 protein expression (p < 0.05) and increased Keap1 protein expression (p < 0.05). Compared with LPS treatment alone, the LPS + SP-1 group significantly increased N-Nrf2 protein expression, while significantly decreased Keap1 protein expression. No significant differences in T-Nrf2 protein levels were observed among the groups (p > 0.05).

[0110] 2.6 In summary, SP-1 has an alleviating effect on LPS-induced endothelial dysfunction in HUVEC cells. First, SP-1 effectively reversed the negative effects of LPS on HUVEC cell viability. Second, SP-1 significantly reduced LPS-induced HUVEC cell hyperpermeability and decreased the content and mRNA levels of inflammatory factors. This effect may be closely related to the MAPK (p-ERK, p-JNK, p-p38) signaling pathway. In addition, SP-1 reduced the content of ROS and increased the activity and mRNA expression levels of antioxidant enzymes, which may be attributed to the activation of the Nrf2 / Keap1 signaling pathway. This study provides a theoretical basis for SP-1 as a potential endothelial dysfunction improver for the prevention and treatment of cardiovascular diseases.

[0111] 3. Ceylon Auricularia auricularia polysaccharide alleviates oleic acid-induced glucose and lipid metabolism disorders in IR-HepG2 cells

[0112] The IR-HepG2 cell model was established by inducing oleic acid (OA) for subsequent experiments. The cells were divided into three groups: untreated cells (Control), OA-treated group (100 μM OA, incubated for 24 h), and cells treated with SP-1 after OA treatment (OA+SP-1). 5 When the cells were at a concentration of 1 μg / mL, the cells were seeded in a 96-well plate. After the cells attached, 100 μM OA was added and incubated for 24 hours. SP-1 (200 μg / mL) was then added and incubated for another 24 hours.

[0113] 3.1 Cell viability

[0114] Cell viability was determined by CCK-8 assay. Figure 11 Compared with the control group, the viability of HepG2 cells was significantly reduced after OA treatment alone (p<0.05). However, compared with the OA treatment alone group, the OA+SP-1 group significantly increased the viability of HepG2 cells (p<0.05), indicating that SP-1 can effectively reverse the negative effects of OA on HepG2 cell viability.

[0115] Effects of SP-1 on glucose metabolism disorders in IR-HepG2 cells

[0116] Figure 12 The effects of SP-1 on OA-induced IR-HepG2 cell glucose metabolism disorder related parameters were presented. As shown in Figure 12 A, compared with the control group, the glucose consumption rate after OA treatment was significantly reduced (p<0.05); while compared with the OA treatment group, the glucose consumption rate in the OA+SP-1 group was significantly increased (p<0.05).

[0117] qRT-PCR was used to detect RNA. As shown in Figure 12 B, compared with the control group, the PEPCK and G6Pase mRNA levels after OA treatment were significantly increased (p<0.05); however, compared with the OA alone treatment group, the PEPCK and G6Pase mRNA levels in the OA+SP-1 group were significantly reduced (p<0.05).

[0118] Western-blotting was used to detect protein, as shown in Figure 12 C-D. Compared with the control group, OA treatment resulted in significantly reduced p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxOl protein expression, while PEPCK and G6Pase protein expression was increased (p<0.05); compared with the OA alone treatment group, the p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxOl protein expression in the OA+SP-1 group was significantly increased, while the PEPCK and G6Pase protein expression was significantly decreased (p<0.05). There was no significant difference in IRS-1, PI3K, AKT, GSK-3β and FoxOl protein expression between groups (p>0.05).

[0119] 3.3 Effects of SP-1 on lipid metabolism disorder in IR-HepG2 cells

[0120] Figure 13 The effects of SP-1 on OA-induced HepG2 cell lipid metabolism disorder related parameters were presented. As shown in Figure 13 A, it was visually shown that compared with the control group, the lipid accumulation in HepG2 cells increased after OA treatment; while compared with the OA alone treatment group, the lipid in HepG2 cells in the OA+SP-1 group was reduced.

[0121] The levels of TC, TG, LDL and HDL were detected using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). As shown in Figure 13OA treatment caused TG, TC and LDL levels to increase and HDL levels to decrease (p<0.05) compared with the control group; these changes were reversed in the OA+SP-1 group compared with the OA alone group, with TG, TC and LDL levels significantly reduced and HDL levels significantly increased (p<0.05).

[0122] Western-blotting was used to detect proteins such as Figure 13 OA treatment reduced p-AMPK and p-ACC1 protein expression (p<0.05) and increased SREBP-1C protein expression (p<0.05) compared with the control group; p-AMPK and p-ACC1 protein expression was significantly increased and SREBP-1C protein expression was significantly reduced in the OA+SP-1 group compared with the OA alone group (p<0.05). No significant differences in AMPK and ACC1 protein expression were observed between the groups (p>0.05).

[0123] 3.4 Summary

[0124] In summary, SP-1 significantly improved the glycolipid metabolic disorder of OA-induced IR-HepG2 cells. First, SP-1 effectively reversed the adverse effects of OA on HepG2 cell viability. Second, SP-1 significantly increased the α-glucosidase inhibition rate in OA-induced IR-HepG2 cells, which may be related to the activation of the IRS / PI3K / Akt / GSK-3β / FoxO1 signaling pathway, which in turn down-regulates the mRNA and protein levels of PEPCK and G6Pase. In addition, SP-1 may also improve the changes in TG, TC, LDL and HDL levels induced by OA by activating the AMPK / ACC1 / SREBP-1C signaling pathway. The results of this study provide theoretical support for SP-1 as a potential glycolipid metabolic disorder improver.

[0125] The above-described embodiments are only a preferred scheme of the present application and do not limit the present application in any form. Other variants and modifications can be made without exceeding the technical scheme recited in the claims.

Claims

1. A Ceylon fungus polysaccharide having the function of improving endothelial cell dysfunction and glycolipid metabolism disorder, characterized in that: Prepared by the following steps: (1) Soak the Ceylon fungus in water, rinse it clean, then dry it and grind it; (2) Add 95% ethanol to the Ceylon fungus powder at a solid-liquid ratio of 1 g:4 mL and heat to reflux for 3 hours for degreasing and decolorization, and discard the ethanol; (3) repeating step (2) twice, and then drying the defatted and decolorized Ceylon fungus powder; (4) The defatted Ceylon fungus algae powder obtained in step (3) is mixed with distilled water at a material-liquid ratio of 1 g:30-40 mL, and then a complex enzyme is added. The pH is adjusted and the mixture is heated to 50-55° C. for enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, the enzyme is inactivated by heating, and the supernatant is obtained by centrifugation. After vacuum concentration, anhydrous ethanol is added to precipitate the mixture at 4° C. overnight, the supernatant is discarded by centrifugation, and the crude Ceylon fungus polysaccharide is obtained after freeze-drying. (5) The crude polysaccharide of Auricularia auricularia Ceylon was dissolved in distilled water and deproteinized by Sevage method; (6) The deproteinized crude polysaccharide from Auricularia auriculariae was dissolved in distilled water and loaded onto a DEAE-52 cellulose column. The column was then eluted with a 0.3 mol / L NaCl solution. The eluate was collected, concentrated, dialyzed, and freeze-dried to obtain Auricularia auriculariae polysaccharide with the function of improving endothelial cell dysfunction and glycolipid metabolism disorders.

2. The Ceylon Auricularia auricularia polysaccharide according to claim 1, characterized in that In step (4), the dosage of the complex enzyme is 1.5-2% of the weight of the defatted Ceylon sea fungus algae powder.

3. The Ceylon Auricularia auricularia polysaccharide according to claim 2, characterized in that The dosage of the complex enzyme is 1.6% of the weight of the defatted Ceylon sea fungus algae powder.

4. The Ceylon Auricularia auricularia polysaccharide according to claim 1, characterized in that In step (4), the complex enzyme consists of papain:cellulase in a weight ratio of 2:

1.

5. The Ceylon Auricularia auricularia polysaccharide according to claim 1, characterized in that In step (4), the pH of the enzymatic hydrolysis reaction is controlled to be 5-5.

5.

6. The Ceylon Auricularia auricularia polysaccharide according to claim 1, characterized in that In step (4), the enzymatic hydrolysis reaction time is 120-140 min.

7. The Ceylon Auricularia auricularia polysaccharide according to claim 1, characterized in that In step (6), the deproteinized Auricularia auricularia polysaccharide crude product is dissolved in distilled water to a concentration of 5 mg / mL.

8. Use of the Auricularia Ceylon polysaccharide according to claim 1 in the preparation of a medicament for treating endothelial cell dysfunction.

9. Use of the Auricularia auriculariae polysaccharide according to claim 1 in the preparation of a medicament for treating glycolipid metabolism disorders.

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