Tin Lancea sarcodia polysaccharide with function of improving endothelial cell dysfunction and glycolipid metabolism disorder

Abstract

The invention discloses a sarcodia stannanensis polysaccharide with functions of improving endothelial cell dysfunction and glucose and lipid metabolism disorder. The polysaccharide is extracted from sarcodia stannanensis by utilizing a compound enzyme. The invention not only provides a theoretical basis for high-value utilization of the sarcodia stannanensis, but also provides a new idea for prevention and treatment of diseases.

Description

Technical Field

[0001] The invention relates to the technical field of functional polysaccharide extraction, in particular to a Ceylon auricularia auricula polysaccharide with the function of improving endothelial cell dysfunction and glucose and lipid metabolism disorder. Background Art

[0002] Seaweed is known as a producer of various bioactive macromolecules (polyphenols, diterpenes, fibers, proteins, and especially polysaccharides). As an important component of seaweed, polysaccharides have aroused strong research interest among scientists today due to their different structural and physicochemical properties and interesting functional characteristics. Due to their complex chemical structure and the diversity of functional groups, seaweed polysaccharides have a variety of physicochemical properties, thus 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 kelp can modulate intestinal microbiota, thereby inhibiting weight gain and lipid metabolism, which may become a potential solution to obesity caused by a high-fat diet (Zhang P, Jia J, Jiang P, et al. Polysaccharides from Edible Brown Seaweed Undaria Pinnatifidaare 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, uronic acid percentage, and monosaccharide composition. Therefore, it is necessary to understand the chemical properties of seaweed polysaccharides and further study the relationship between their structural characteristics and related biological activities. In addition, different extraction methods are another factor affecting the biological activity of seaweed polysaccharides.

[0003] In order to extract polysaccharides from the cell walls of various algae, several techniques have been utilized to achieve methods for easy extraction of polysaccharides. Traditional methods for extracting polysaccharides from algae mainly use hot water, acid, alkali, or a combination of these solvents as the extraction medium. The process involves placing the 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, manual handling, thermal instability issues, and environmental threats from organic compounds that limit the use of these methods.

[0004] Sarcodia ceylonensis, a member of the Rhodophyta, Gigarticales, and Sarcodiaceae, belongs to the Sarcodia genus. In China, this genus mainly includes two species: Sarcodia ceylonensis and Sarcodia ceylanica. They mainly grow in the southeastern coastal areas of my country, and these seas provide them with a suitable growth environment. Sarcodia ceylonensis is rich in seaweed polysaccharides and unsaturated fatty acids, as well as more than 20 nutrients unique to marine plants. These ingredients give Sarcodia ceylanica a variety of pharmacological activities. Sarcodia ceylonensis is known as the "longevity vegetable" and "marine vegetable", and is deeply loved and respected by people. At present, there is little research on the active functions of Sarcodia ceylonensis polysaccharides, and further research and development of its value is needed. Summary of the invention

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

[0006] The technical solution adopted by the present invention to solve its technical problem is: A Ceylon auricularia auricula polysaccharide having the function of improving endothelial cell dysfunction and glucose and lipid metabolism disorder is prepared by the following steps: (1) soaking the Ceylon fungus in water, rinsing it, drying it, and crushing it; (2) adding 95% ethanol to the Ceylon Auricularia auricula powder at a solid-liquid ratio of 1 g:4 mL, heating to reflux for 3 hours for degreasing and decolorization, and discarding the ethanol; (3) repeating step (2) twice, and then drying the defatted and decolorized Auricularia auricula powder; (4) mixing the defatted Auricularia auricularia powder obtained in step (3) with distilled water at a solid-liquid ratio of 1 g:30-40 mL, adding a composite enzyme, adjusting the pH and heating to 50-55° C. for enzymolysis, heating to inactivate the enzyme after the enzymolysis reaction, centrifuging to obtain the supernatant, concentrating under vacuum, adding anhydrous ethanol to precipitate at 4° C. overnight, centrifuging and discarding the supernatant, and freeze-drying to obtain a crude Auricularia auricularia polysaccharide; (5) The crude product of Auricularia auricularia polysaccharide was dissolved in distilled water and then deproteinized by Sevage method; (6) The deproteinized crude Auricularia auricularia polysaccharide was dissolved in distilled water and loaded onto a DEAE-52 cellulose column, and then eluted with a 0.3 mol / L NaCl solution. The eluate was collected, concentrated, dialyzed, and freeze-dried to obtain Auricularia auricularia polysaccharide with the function of improving endothelial cell dysfunction and lipid metabolism disorders.

[0007] In step (4), the dosage of the complex enzyme is 1.5-2% of the weight of the defatted Auricularia auricula powder.

[0008] The dosage of the complex enzyme is 1.6% of the weight of the defatted Ceylon sea fungus algae powder.

[0009] In step (4), the complex enzyme is composed of papain:cellulase in a weight ratio of 2:1.

[0010] In step (4), the pH of the enzymatic hydrolysis reaction is controlled to be 5-5.5.

[0011] In step (4), the enzymatic hydrolysis reaction time is 120-140 min.

[0012] In step (6), the concentration of the deproteinized Auricularia auricularia polysaccharide crude product after dissolving in distilled water is 5 mg / mL.

[0013] The application of the Ceylon Auricularia auricularia polysaccharide in the preparation of a medicine for treating endothelial cell dysfunction.

[0014] The invention discloses an application of the Ceylon Auricularia auricularia polysaccharide in preparing a medicine for treating glucose and lipid metabolism disorders.

[0015] The beneficial effect of the present invention is that through the specific extraction method of the present invention, Ceylon sea fungus polysaccharide with a new function of improving endothelial cell dysfunction and glucose and lipid metabolism disorder is obtained, which provides a theoretical basis for the high-value utilization of Ceylon sea fungus and provides a new idea for the prevention and treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effects of different enzyme-assisted conditions on the extraction rate of Auricularia auricula SP (A) The effect of the amount of compound enzyme added on the extraction rate; (B) The effect of enzymatic hydrolysis time on the extraction rate; (C) The effect of enzymatic hydrolysis temperature on the extraction rate; (D) The effect of the solid-liquid ratio on the extraction rate; (E) The effect of pH on the extraction rate. The results are expressed as mean ± standard deviation (n = 3), and the values ​​represented by different letters are significantly different (p < 0.05); Figure 2 This is the elution curve of Ceylon Auricularia auricula polysaccharide on DEAE-52 ion exchange column chromatography; Figure 3 The two SP components have an effect on the secretion levels of inflammatory factors (TNF-α, INF-β) and NO in HUVEC cells induced by LPS. (A) The effect of the two SP components on the NO content of HUVEC cells induced by LPS; (B) The effect of the two SP components on the TNF-α content of HUVEC cells induced by LPS; (C) The effect of the two SP components on the IFN-β content of HUVEC cells induced by LPS; The results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05); 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); Figure 5 The effect of SP on glucose consumption rate in IR-HepG2 cells; the results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05); Figure 6 This is a graph showing the inhibitory effect of Auricularia auricularia polysaccharide on α-glucosidase and pancreatic lipase; 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); Figure 8 The effect of SP-1 on LPS-induced HUVEC cell hyperpermeability. (A) Cell migration and invasion were detected by 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 migration and invasion cell numbers was performed using ImageJ software, and the results were expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters were significantly different (P < 0.05); Fig. 9 The effects of SP-1 on parameters related to inflammation and anti-inflammatory ability of HUVEC cells. (A) and (B) The effects of SP-1 on the content and mRNA levels of inflammatory factors in HUVEC cells; (C) and (D) The effects 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); Fig.10 The effects of SP-1 on parameters related to oxidative damage and antioxidant capacity of HUVEC cells. (A) and (B) The effects of SP-1 on the relative level of ROS induced by LPS; (C) and (D) The effects of SP-1 on the content and mRNA levels of antioxidant enzymes Cu / ZnSOD, MnSOD, CAT, GPx, GST, and GR; (E) and (F) The effects 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); Fig.11 The 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); Fig.12 The effect of SP-1 on OA-induced glucose metabolism disorder in IR-HepG2 cells. (A) Effect of SP on glucose consumption in HepG2 cells (B) Effect of SP-1 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 ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (P < 0.05); Fig.13 The effect of SP-1 on lipid metabolism disorder in IR-HepG2 cells induced by OA. (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 ​​represented by different letters are significantly different (p < 0.05). DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below through specific embodiments.

[0018] 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.

[0019] Cellulase 10000 U / mg was purchased from Beijing Solebow Technology Co., Ltd.; papain 10000 U / mg was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] Embodiment 1: A Ceylon auricularia auricula polysaccharide having the function of improving endothelial cell dysfunction and glucose and lipid metabolism disorder is prepared by the following steps: (1) soaking the Ceylon sea fungus (dried product) in water, rinsing it, drying it in a forced air drying oven at 55° C., and crushing it; (2) adding 95% ethanol to the Ceylon Auricularia auricula powder at a solid-liquid ratio of 1 g:4 mL, heating to reflux for 3 hours for degreasing and decolorization, and discarding the ethanol; (3) repeating step (2) twice, and then drying the defatted and decolorized Auricularia auricula powder; (4) mixing the defatted Auricularia auricula powder obtained in step (3) with distilled water at a solid-liquid ratio of 1 g:30 mL, and then adding a composite enzyme, wherein the composite enzyme is composed of papain and cellulase in a weight ratio of 2:1, and the amount of the composite enzyme is 1.5% of the weight of the defatted Auricularia auricula powder; adjusting the pH to 5 and heating to 50° C. for enzymolysis reaction for 140 min; heating to inactivate the enzyme after the enzymolysis reaction is completed, centrifuging to obtain the supernatant, concentrating under vacuum, adding anhydrous ethanol to precipitate at 4° C. overnight, centrifuging and discarding the supernatant, and freeze-drying to obtain a crude Auricularia auricula polysaccharide; (5) The crude product of Auricularia auricularia polysaccharide was dissolved in distilled water to form a solution with a concentration of 5 mg / mL, and then deproteinized by the Sevage method; (6) The deproteinized crude Auricularia auricularia polysaccharide was dissolved in distilled water and loaded onto a DEAE-52 cellulose column, and then eluted with a 0.3 mol / L NaCl solution. The eluate was collected, concentrated, dialyzed, and freeze-dried to obtain Auricularia auricularia polysaccharide with the function of improving endothelial cell dysfunction and lipid metabolism disorders.

[0021] Embodiment 2: The difference between this embodiment and embodiment 1 is that: (4) The defatted Ceylon auricularia auricularia powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1 g:35 mL, and then a composite enzyme is added, wherein the composite enzyme is composed of papain:cellulase in a weight ratio of 2:1, and the amount of the composite enzyme is 2% of the weight of the defatted Ceylon auricularia auricularia powder. The pH is adjusted to 5.5 and then heated to 55° C. for enzymolysis reaction for 120 min. After the enzymolysis reaction is completed, the enzyme is heated to inactivate the enzyme, and the supernatant is centrifuged and concentrated under vacuum. Anhydrous ethanol is added to precipitate at 4° C. overnight, the supernatant is discarded by centrifugation, and the crude Ceylon auricularia auricularia polysaccharide is obtained after freeze-drying; the rest is the same as in Example 1.

[0022] Embodiment 3: The difference between this embodiment and embodiment 1 is that: (4) The defatted Ceylon auricularia auricularia powder obtained in step (3) is mixed with distilled water at a solid-liquid ratio of 1 g:40 mL, and then a composite enzyme is added, wherein the composite enzyme is composed of papain:cellulase in a weight ratio of 2:1, and the amount of the composite enzyme is 1.6% of the weight of the defatted Ceylon auricularia auricularia powder. The pH is adjusted to 5.2 and then heated to 50.4° C. for enzymolysis reaction for 120 min. After the enzymolysis reaction is completed, the enzyme is heated to inactivate the enzyme, and the supernatant is centrifuged and concentrated under vacuum. Anhydrous ethanol is added to precipitate at 4° C. overnight, the supernatant is discarded by centrifugation, and the crude Ceylon auricularia auricularia polysaccharide is obtained after freeze-drying; the rest is the same as in Example 1.

[0023] Experimental part: This study set up three groups of repeated experiments, and the experimental results were expressed as mean ± standard deviation (SD). SPSS23.0 was used to perform one-way analysis of variance (ANOVA) on all results to determine whether there were significant differences between the experimental groups. P < 0.05 was considered statistically significant.

[0024] 1.1 Pretreatment of Ceylon Auricularia auricula The Ceylon sea fungus (dried product) is soaked in water and repeatedly rinsed to remove impurities such as gravel, and then the washed Ceylon sea fungus is placed in a 55°C forced air drying oven for drying for use. The dried Ceylon sea fungus is crushed with a grinder, and then the Ceylon sea fungus powder is added with 95% ethanol at a material-liquid ratio of 1:4 (w / v) for 3h reflux degreasing and decolorization, and the degreasing and decolorization operation is repeated twice. Then the ethanol is discarded, and the defatted Ceylon sea fungus algae powder is dried at a low temperature of 50°C and stored in a dry and dark place at room temperature.

[0025] 1.2 Single factor condition setting for the extraction of Ceylon Auricularia auricularia polysaccharide Single factor experiments were carried out with the addition amount of complex enzyme (0.5%, 1%, 1.5%, 2%, 2.5%), enzymatic hydrolysis time (60, 80, 100, 120 and 140 min), enzymatic 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 effects of each factor on the extraction rate of Auricularia auricularia polysaccharide assisted by complex enzyme.

[0026] 1.3 Extraction process of Auricularia auricula polysaccharide Defatted Ceylon sea fungus algae powder was mixed with distilled water. Then, a composite enzyme (papain: cellulase = 2:1) was added according to the mass of the substrate, and the pH of the solution was adjusted with hydrochloric acid or NaOH. The solution was then placed in a constant temperature water bath oscillator for constant temperature oscillation extraction, and then heated in a 90°C water bath for 10 minutes to inactivate the enzyme. The mixture was then centrifuged at 8000 rpm for 15 minutes and the supernatant was collected. The supernatant was concentrated to about 1 / 4 volume under vacuum conditions at 50°C, and then anhydrous ethanol was added at a ratio of (1:3, v / v) to precipitate overnight at 4°C. After that, the supernatant was discarded by centrifugation at 4500 rpm for 15 minutes, and the precipitate was freeze-dried to obtain a Ceylon sea fungus crude polysaccharide sample. The freeze-dried polysaccharide sample was stored in a desiccator until use, and the polysaccharide extraction rate was determined using the phenol-sulfuric acid method.

[0027] 1.4 Results of single factor test on the extraction of crude polysaccharides from Auricularia auricularia Effects of different enzyme-assisted extraction methods on the extraction rate of Auricularia auricularia polysaccharides Figure 1As shown in 1A, when the amount of compound enzyme added is 0.5%-1.5%, the extraction rate of polysaccharides from Ceylon sea fungus is significantly improved; when the amount of enzyme is 1.5%-2%, the extraction rate of polysaccharides begins to decrease; when the amount of enzyme is 2%-2.5%, the extraction rate of polysaccharides tends to be stable. Figure 1 B shows that during the 60-120 min of enzymatic hydrolysis, the extraction rate of Ceylon Auricularia auricula crude polysaccharides increased significantly, and then the extraction rate of polysaccharides tended to be stable. Figure 1 As shown in Figure C, when the enzymatic hydrolysis temperature is lower than 50°C, the extraction rate of Auricularia auriculariae polysaccharides gradually increases with the increase of temperature; however, once the temperature exceeds 50°C, the extraction rate of Auricularia auriculariae polysaccharides begins to decrease. Figure 1 D It can be seen that when the solid-liquid ratio is less than 1:40 g / mL, the extraction rate of Auricularia auricularia polysaccharide gradually increases, and when the solid-liquid ratio is greater than 1:40 g / mL, the extraction rate begins to decline rapidly. Figure 1 E It can be seen that the extraction of crude polysaccharides from Auricularia auricularia Ceylon first increases with the increase of pH, and then decreases significantly with the increase of pH.

[0028] 1.5 Box-Behnken response surface optimization of polysaccharide extraction process On the basis of the single factor experiment, the enzyme addition amount (A), enzymolysis temperature (B), and pH (C) were used as experimental factors, and the extraction rate of Ceylon sea fungus crude polysaccharide was used as the response value. A three-factor three-level Box-Behnken experimental design was carried out, and the process of composite enzyme-assisted sea fungus crude polysaccharide was optimized by response surface analysis. The response surface analysis factor level table is shown in Table 1.

[0029] Table 1. Response surface analysis factor level table

[0030] The experimental conditions and polysaccharide extraction rates are shown in Table 2. The results show that the polysaccharide extraction rate is between 14.01% and 21.07%. The regression coefficient values ​​were 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 The influence of factors A, B and C on the yield of Ceylon Auricularia auricularia crude polysaccharide was judged according to the F value. The order of influence from large to small is: C (pH) > B (enzyme hydrolysis temperature ℃) > A (enzyme addition %).

[0031] Table 2 Response surface experimental design and results

[0032] 1.6 Optimization of optimal conditions and verification of results From the above experiments, it can be concluded that the optimal conditions for the complex enzyme-assisted extraction of Ceylon sea fungus polysaccharides are: the amount of complex enzyme added is set to 1.66%, the enzymatic hydrolysis temperature is controlled at 50.43℃, and the pH value is adjusted to 5.2. Under these precise conditions, the predicted extraction rate of Ceylon sea fungus polysaccharides can reach 20.90%. For the operability of the experimental process, the process parameters were fine-tuned: the amount of complex enzyme added was adjusted to 1.6%, the enzymatic hydrolysis temperature was set to 50.4℃, and the pH value was maintained at 5.2. The experimental results showed that under these corrected conditions, the polysaccharide extraction rate was 20.86%, and the results obtained were similar to those obtained from the predicted values, proving the accuracy of the model.

[0033] 1.7 Purification of crude polysaccharides The polysaccharides extracted by enzyme-assisted extraction 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, the Sevag method was used to remove proteins, and then the polysaccharides from Ceylon sea fungus were separated and purified by DEAE-52 anion exchange column. At the same time, the best active components that reduce endothelial cell dysfunction and lipid metabolism disorders were screened out through in vitro experiments, and physical and chemical analysis and structural characterization were performed on them.

[0034] The crude product of Ceylon sea fungus polysaccharide was dissolved in distilled water to form a solution with a concentration of 5 mg / mL, and then mixed with Sevage reagent [V (chloroform): V (n-butanol) = 4:1] in a ratio of 1:4, and fully shaken to achieve the purpose of deproteinization. Then centrifugation was performed, the supernatant was dialyzed for 48 hours, and the dialyzed extract was precipitated with alcohol overnight, and the precipitate was centrifuged and freeze-dried to obtain the Ceylon sea fungus deproteinized sample.

[0035] In order to further purify the deproteinized sample of Ceylon sea fungus. We used ultrapure water and 0.1, 0.3 and 0.5M NaCl solutions to elute the 5mg / mL Ceylon sea fungus deproteinized polysaccharide sample loaded on the DEAE-52 cellulose column. During the elution process, one tube was collected for every 5mL, and the absorbance of the eluent was detected by the phenol-sulfuric acid method at a wavelength of 490nm. Subsequently, the elution curve was drawn with the number of tubes as the horizontal coordinate and the absorbance as the vertical coordinate. According to this curve, the peak height components were collected, then concentrated, dialyzed, and finally freeze-dried for standby use.

[0036] Figure 2 The deproteinized sample of Auricularia auricula was separated by DEAE-52 anion column, and two obvious peaks were obtained at the positions of pure water and 0.3 mol / L NaCl, named SP-0 and SP-1, which were freeze-dried and used for later use.

[0037] 1.7 Effect of SP components on the secretion levels of inflammatory factors (TNF-α, IFN-β) and NO in HUVEC cells Polysaccharide components were screened based on the content of NO, TNF-α and IFN-β1. The cells were divided into three groups: untreated cells (Control), LPS-treated group and cell group with LPS added after SP treatment (LPS+SP-0, LPS+SP-1). SP-0 and SP-1 were added for 1 hour, LPS was added, and then placed in a 37°C, 5% CO2 incubator for 24 hours, the supernatant was collected, and the NO, TNF-α and IFN-β1 contents were detected according to the instructions of the ELISA kit.

[0038] By detecting the effects of two SPs (150 μg / mL) on the secretion levels of inflammatory factors (TNF-α, IFN-β) and NO in HUVEC cells induced by LPS, the optimal SP component for inhibiting the inflammatory response of HUVEC cells induced by LPS was preliminarily screened. The effects of two SP separation components on the secretion levels of inflammatory factors (TNF-α, IFN-β) and NO in HUVEC cells induced by LPS are shown in Figure 2. Figure 3 SP-1 treatment significantly reduced the increase of TNF-α, INF-β and NO induced by LPS (p<0.05), and the secretion levels of TNF-α, INF-β and NO after SP-1 treatment were significantly lower than those after SP-0 treatment (p<0.05).

[0039] Effects of 1.8SP components on antioxidant enzymes in HUVEC cells The cells were divided into three groups: untreated cells (Control), LPS-treated group, and cell groups treated with SP followed by LPS (LPS+SP-0, LPS+SP-1). HUVEC cells were treated with LPS (1.0 μg / mL) for 24 h after being treated with SP-1 for 1 h. Then, the cells were collected and lysed on ice using a cell disruptor, and the protein concentration was determined according to the BCA method. Antioxidant enzyme activity levels were measured according to the manufacturer's instructions.

[0040] By detecting the effects of two SPs (150 μg / L) on the antioxidant enzyme activities in HUVEC cells induced by LPS, the SP fraction with the best antioxidant effect was preliminarily screened. 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).

[0041] Effect of 1.9SP on glucose consumption in IR-HepG2 cells IR-HepG2 cell model was established by induction with oleic acid (OA) for subsequent experiments. The cells were divided into three groups: untreated cells (Control), OA-treated group, and cells treated with SP after OA treatment (OA+SP-0, OA+SP-1). 5 When the concentration of cells was 100 μM / mL, the cells were inoculated in a 96-well plate. After the cells adhered to the wall, 100 μM OA was added and incubated for 24 h. SP-0 and SP-1 were then added and incubated for another 24 h. After that, the glucose concentration in the culture medium was determined using a glucose determination kit. Cellular glucose consumption = glucose concentration of the blank group (no cells, only culture medium) - glucose concentration of each experimental group.

[0042] The best hypoglycemic SP fraction was screened by detecting the effects of two SPs (200 μg / mL) on glucose consumption in IR-HepG2 cells. Figure 5 SP treatment significantly reversed the decrease in glucose consumption rate of IR-HepG2 cells induced 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).

[0043] 1.10 Effects of SP components on the inhibition rate of pancreatic lipase and α-glucosidase Pancreatic lipase inhibitory activity was assessed using p-nitrophenylbutyrate (p-NPB) as a substrate. 50 μL aliquots of each sample solution of different concentrations (0.25, 0.5, 1.0, 1.5 and 2.0 mg / mL) were mixed with 50 μL pancreatic lipase solutions (50 mg / mL) prepared in Tris buffer (0.1 mol / L, pH 8.2). The reaction was initiated by adding 50 μL p-NPB (final concentration 5 mM) prepared in phosphate buffer (pH 7.3). A similar procedure was used for the control reaction (without sample), which reflected 100% enzyme activity. After incubation for 1 h at ambient temperature, 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. Suitable controls without enzymes were added to each sample to remove background absorbance. The inhibitory effect of pancreatic lipase was estimated using the following formula: Where A reflects the control absorbance (reaction bottle containing live pancreatic lipase and buffer); B reflects the control blank absorbance (buffer only, no pancreatic lipase); C represents the sample absorbance (sample and pancreatic lipase), and D reflects the sample blank absorbance (no pancreatic lipase, only sample and buffer).

[0044] 40 μL of sample solutions of different concentrations (0.25, 0.5, 1.0, 1.5 and 2.0 mg / mL) were added to a 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, incubated at 37°C for 0.5 h, and then 0.2 mol / L sodium carbonate solution (50 μL) was added 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 the positive control group. The α-glucosidase inhibition rate was determined as follows: Where A1 is the absorbance of the mixture reaction solution, A2 is the absorbance of the sample reaction solution without the enzyme system, and A0 is the absorbance of the sample reaction solution without the sample.

[0045] By testing the effects of two SP fractions (0.25-2.0 mg / mL) on α-glucosidase levels and pancreatic lipase inhibition rates, we preliminarily screened the active polysaccharides that have the greatest impact on glucose and lipid metabolism. Figure 6 A. It was found that both SP-0 and SP-1 inhibited the activity of α-glucosidase in a dose-dependent manner, but the inhibition rate of α-glucosidase tended to be flat after the dose was increased to 1 mg / mL, and the effect of SP-1 was stronger than that of SP-0 at the same concentration level. Figure 6 B. It was found that at 0.25-0.5 mg / mL, the inhibitory effect of SP-1 on pancreatic lipase was not as obvious as that of SP-0; when the concentration of the two polysaccharides increased from 1.0 mg / mL to 2.0 mg / mL, the inhibition rate of pancreatic lipase of the two SP components tended to be flat; at the same time, it was found that the inhibitory effect of SP-1 on pancreatic lipase at all concentrations was better than that of SP-0.

[0046] 1.11 Two polysaccharides with high content, SP-0 and SP-1, were isolated from Ceylon sea fungus. Through in vitro experiments, it was initially screened that the best component for alleviating endothelial cell dysfunction and lipid metabolism disorders was SP-1. Physical and chemical analysis and structural characterization of SP-1 showed that the total sugar content of SP-1 was 80.89%, the protein content was 1.3%, the uronic acid content was 2.41%, and the sulfate content was 12.52%. The ultraviolet absorption spectrum also showed that there were very few protein nucleic acid substances. The molecular weight of SP-1 was 18.453KDa, and the monosaccharide composition was galactose, glucose, xylose and mannose, with the percentage of each monosaccharide composition being 8.18%, 84.16%, 2.26% and 5.40%.

[0047] 2.1 Ceylon Auricularia polysaccharide alleviates lipopolysaccharide-induced endothelial cell dysfunction At present, there are no reports on the effects of Ceylon Auricularia polysaccharide on endothelial dysfunction. Therefore, this study used lipopolysaccharide (LPS) to construct an endothelial injury cell model to study the role and related mechanisms of Ceylon Auricularia polysaccharide in regulating endothelial cell dysfunction.

[0048] Control group: untreated cells; LPS group: 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.

[0049] 2.2 Effect of SP-1 on HUVEC cell viability The cell viability was determined by CCK-8 method. Figure 7 Compared with the control group, LPS treatment caused a significant decrease in the viability of HUVEC cells (p<0.05); compared with the LPS group, the LPS+SP-1 group significantly increased the viability of HUVEC cells (p<0.05), indicating that SP-1 helps reverse the negative effect of LPS on the viability of HUVEC cells.

[0050] 2.3 Effect of SP-1 on LPS-induced HUVEC cell hyperpermeability Cell scratch test: HUVEC cells from each group in the logarithmic growth phase were inoculated in a six-well plate. When the cells were confluent to 90%, a vertical line was drawn along the midpoint with a 200 μL pipette tip, the old culture medium was discarded, and the cells were washed twice with PBS and cultured for 24 hours. The images were observed using an inverted fluorescence microscope. ImageJ software was used for quantitative analysis, and images were observed using an inverted fluorescence microscope.

[0051] Cell migration and invasion assay: For the method, see Jin BR, Kim HJ, Kim EY, et al. 6′-Sialyllactose Ameliorates In Vivo and In Vitro Benign Prostatic Hyperplasiaby Regulating the E2F1 / pRb–AR Pathway[J]. Nutrients, 2019, 11(9): 2203.

[0052] Figure 8 The effect of SP-1 on LPS-induced HUVEC cell hyperpermeability is shown. Figure 8AD. In the cell scratch, migration and invasion assays, LPS treatment resulted in a significant decrease in the total number of migrating and invasive cells compared with the control group; the LPS+SP-1 group increased the total number of migrating and invasive cells compared with the LPS treatment group alone. The results showed that SP-1 could alleviate the LPS-induced hyperpermeability of HUVEC cells.

[0053] 2.4 Effects of SP-1 on parameters related to inflammation and anti-inflammatory ability of HUVEC cells The enzyme-linked immunosorbent assay (ELISA) was used to determine the effects of LPS on the levels of inflammatory factors in HUVEC cells.

[0054] like Fig. 9 As shown in A. Compared with the control group, the LPS treatment group alone significantly increased the levels 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 treatment group alone, the LPS+SP-1 group reduced the levels of IL-1β, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells (p<0.05), while the IL-6 content did not change significantly (p>0.05).

[0055] like Fig. 9 As shown in B. Compared with the control group, the LPS treatment group alone 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 treatment group alone, 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).

[0056] like Fig. 9C and 9D. Compared with the control group, the LPS treatment group 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 treatment group, the 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).

[0057] Effects of 2.5SP-1 on oxidative damage and antioxidant capacity-related parameters of HUVEC cells The DCFH-DA staining method was used to determine the ROS content. The effects of SP-1 on parameters related to oxidative damage and antioxidant capacity of HUVEC cells were as follows: Fig.10 As shown. Fig.10 As shown in A and B. Compared with the control group, LPS treatment alone significantly increased the relative ROS level of HUVEC cells (p<0.05); compared with LPS treatment alone, LPS+SP-1 group reduced the relative ROS level in HUVEC cells (p<0.05).

[0058] like Fig.10 C. Compared with the control group, LPS treatment led to decreased activities of copper-zinc superoxide dismutase (Cu / Zn-SOD), CAT, GPx, GST and GR (p<0.05); compared with LPS treatment alone, LPS+SP-1 group significantly increased the activities of Cu / Zn-SOD, CAT, GPx, GST and GR in HUVEC cells (p<0.05). No significant difference was found in MnSOD activity among the groups (p>0.05).

[0059] qRT-PCR detection of RNA. Fig.10 D. Compared with the control group, LPS treatment significantly reduced the mRNA levels of Cu / Zn-SOD, CAT, GST and GR (p<0.05); compared with LPS treatment alone, LPS+SP-1 group significantly increased the mRNA levels of Cu / Zn-SOD, CAT, GST and GR in HUVEC cells (p<0.05). The mRNA levels of MnSOD and GPx were significantly different among the groups (p>0.05).

[0060] Western-blotting detection of proteins, such as Fig.10 As shown in E and F. Compared with the control group, LPS treatment significantly reduced the expression of N-Nrf2 protein (p<0.05) and increased the expression of Keap1 protein (p<0.05); compared with LPS treatment alone, LPS+SP-1 group significantly increased the expression of N-Nrf2 protein, while the expression of Keap1 protein was significantly reduced. There was no significant difference in the level of T-Nrf2 protein among the groups (p>0.05).

[0061] 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. Secondly, SP-1 significantly reduced LPS-induced HUVEC cell hyperpermeability, reduced the content and mRNA levels of inflammatory factors, and 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, increased the activity of antioxidant enzymes and mRNA expression levels, 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.

[0062] 3. Auricularia auricula polysaccharide alleviates the glucose and lipid metabolism disorders of IR-HepG2 cells induced by oleic acid

[0063] IR-HepG2 cell model was established by oleic acid (OA) induction 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 100 μg / mL, the cells were seeded in a 96-well plate, and after the cells adhered, 100 μM OA was added and incubated for 24 h. SP-1 (200 μg / mL) was then added and incubated for another 24 h.

[0063] 3.1 Cell viability The cell viability was determined by CCK-8 method. Fig.11 As shown. Compared with the control group, the viability of HepG2 cells after OA treatment alone was significantly reduced (p<0.05). However, compared with the OA treatment group alone, 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 the viability of HepG2 cells.

[0064] 3.2 Effect of SP-1 on glucose metabolism disorders in IR-HepG2 cells Fig.12The effects of SP-1 on parameters related to glucose metabolism disorders induced by OA in IR-HepG2 cells are presented. Fig.12 As shown in A, compared with the control group, the glucose consumption rate after OA treatment was significantly reduced (p<0.05); and compared with the OA treatment group, the glucose consumption rate of the OA+SP-1 group was significantly increased (p<0.05).

[0065] qRT-PCR detection of RNA. Fig.12 As shown in B. Compared with the control group, the mRNA levels of PEPCK and G6Pase were significantly increased after OA treatment (p<0.05); however, compared with the OA alone treatment group, the mRNA levels of PEPCK and G6Pase in HepG2 cells in the OA+SP-1 group were significantly decreased (p<0.05).

[0066] Western-blotting detection of proteins, such as Fig.12 CD. Compared with the control group, OA treatment led to a significant decrease in the expression of p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxO1 proteins, while an increase in the expression of PEPCK and G6Pase proteins (p<0.05); compared with the OA alone treatment group, the OA+SP-1 group had a significant increase in the expression of p-IRS-1, p-PI3K, p-AKT, p-GSK-3β and p-FoxO1 proteins, while a significant decrease in the expression of PEPCK and G6Pase proteins (p<0.05). There was no significant difference in the expression of IRS-1, PI3K, AKT, GSK-3β and FoxO1 proteins among the groups (p>0.05).

[0067] Effect of SP-1 on lipid metabolism disorders in IR-HepG2 cells Fig.13 The effects of SP-1 on parameters related to lipid metabolism disorders in HepG2 cells induced by OA were demonstrated. Fig.13 A visually shows that compared with the control group, the lipid accumulation of HepG2 cells increased after OA treatment; while compared with the OA alone treatment group, the lipid accumulation of HepG2 cells in the OA+SP-1 group decreased.

[0068] The levels of TC, TG, LDL, and HDL were measured using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Fig.13 As shown in B. Compared with the control group, OA treatment led to an increase in TG, TC and LDL levels, and a decrease in HDL content (p<0.05); compared with the OA alone treatment group, these changes were reversed in the OA+SP-1 group, with a significant decrease in TG, TC and LDL levels, and a significant increase in HDL content (p<0.05).

[0069] Western-blotting detection of proteins, such as Fig.13 CD. Compared with the control group, OA treatment reduced the expression of p-AMPK and p-ACC1 proteins (p<0.05), and increased the expression of SREBP-1C protein (p<0.05); compared with the OA alone treatment group, the expression of p-AMPK and p-ACC1 proteins in the OA+SP-1 group was significantly increased, and the expression of SREBP-1C protein was significantly reduced (p<0.05). No significant differences were found in the expression of AMPK and ACC1 proteins among the groups (p>0.05).

[0070] 3.4 Summary In summary, SP-1 has a significant improvement effect on OA-induced glucose and lipid metabolism disorders in IR-HepG2 cells. First, in HepG2 cells, SP-1 effectively reversed the adverse effects of OA on HepG2 cell viability. Second, SP-1 significantly increased the inhibition rate of α-glucosidase in OA-induced IR-HepG2 cells, which may be related to the activation of the IRS / PI3K / Akt / GSK-3β / FoxO1 signaling pathway and the downregulation of 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 improver of glucose and lipid metabolism disorders.

[0071] 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. A Ceylon Auricularia auricula polysaccharide having the function of improving endothelial cell dysfunction and glucose and lipid metabolism disorders, characterized in that: Prepared by the following steps: (1) soaking the Ceylon fungus in water, rinsing it, drying it, and crushing it; (2) adding 95% ethanol to the Ceylon Auricularia auricula powder at a solid-liquid ratio of 1 g:4 mL, heating to reflux for 3 hours for degreasing and decolorization, and discarding the ethanol; (3) repeating step (2) twice, and then drying the defatted and decolorized Auricularia auricula powder; (4) mixing the defatted Auricularia auricularia powder obtained in step (3) with distilled water at a solid-liquid ratio of 1 g:30-40 mL, adding a composite enzyme, adjusting the pH and heating to 50-55° C. for enzymolysis, heating to inactivate the enzyme after the enzymolysis reaction, centrifuging to obtain the supernatant, concentrating under vacuum, adding anhydrous ethanol to precipitate at 4° C. overnight, centrifuging and discarding the supernatant, and freeze-drying to obtain a crude Auricularia auricularia polysaccharide; (5) The crude product of Auricularia auricularia polysaccharide was dissolved in distilled water and then deproteinized by Sevage method; (6) The deproteinized crude Auricularia auricularia polysaccharide was dissolved in distilled water and loaded onto a DEAE-52 cellulose column, and then eluted with a 0.3 mol / L NaCl solution. The eluate was collected, concentrated, dialyzed, and freeze-dried to obtain Auricularia auricularia polysaccharide with the function of improving endothelial cell dysfunction and lipid metabolism disorders.

2. The Ceylon Auricularia auricula 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 Auricularia auricula powder.

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

1.

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

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

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

9. Use of the Auricularia auricula polysaccharide according to claim 1 in the preparation of a medicament for treating glucose and lipid metabolism disorders.

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