Method for extracting tremella polysaccharide

Abstract

The invention discloses an extraction method of tremella polysaccharide, which comprises the following steps: (1) mixing tremella and a deep eutectic solvent according to a mass-volume ratio of 1g: (5-40) mL, swelling, and performing ultrasonic extraction for 40-180 minutes at the extraction temperature of 80-105 DEG C to obtain a tremella polysaccharide extracting solution; and (2) carrying out alcohol precipitation on the tremella polysaccharide extracting solution, and purifying to obtain the tremella polysaccharide, the eutectic solvent comprises water, a hydrogen bond donor and a hydrogen bond acceptor, the mass percentage of the water is 40-80%, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: (0.5-5). According to the extraction method provided by the invention, deproteinization is not needed, the extraction steps are simple, the extraction efficiency and the extraction purity are relatively high, and the extracted product has a remarkable effect on treating COPD.

Description

Technical Field

[0001] The invention relates to the field of purification, and in particular to a method for extracting tremella polysaccharide. Background Art

[0002] Tremella, also known as white fungus, has the effects of nourishing yin and moistening the lungs, nourishing the stomach and promoting fluid production. It is effective in treating lung heat cough, gastritis, facial spots, cancer tumors and other diseases. Most of the biological activities are related to Tremella polysaccharides, which is a traditional fungal food medicine in my country. Tremella polysaccharides have the effects of regulating immunity, lowering blood lipids, anti-radiation, anti-tumor, anti-aging, and anti-oxidation. At present, the extraction and purification process of Tremella polysaccharides is becoming more and more mature, but it has not yet been industrialized. The traditional water extraction efficiency and product purity are low, there are a series of problems, and the industrialization system needs to be improved.

[0003] Since Abbott et al. first proposed the concept of deep eutectic solvents (DESs) in 2003, the application of DESs in the extraction of bioactive ingredients has attracted much attention. DESs are a new type of green solvent with properties similar to ionic liquids, low price, easy to obtain, good biodegradability, stability, low toxicity or non-toxicity. They have been widely used in the extraction of polyphenols, flavonoids, and polysaccharides, but the application of deep eutectic solvents in the extraction of Tremella polysaccharides has not been reported.

[0004] Chronic obstructive pulmonary disease (COPD) is a disease characterized by recurrent respiratory symptoms and long-term airflow restriction. Clinically, it mainly manifests as dyspnea, cough and sputum production. In the later stages, emphysema and pulmonary fibrosis may occur. Long-term exposure to cigarette smoke (CSE) or exposure to environments containing toxic particles is one of the main causes of the disease. The mechanisms that cause COPD include oxidative stress, inflammatory response, imbalance of protease / antiprotease activity, and cell apoptosis. Although many achievements have been made in basic research on COPD, its exact pathogenic mechanism has not yet been fully elucidated, resulting in a single type of drug for the treatment of COPD and poor results. Summary of the invention

[0005] In order to overcome at least one technical problem existing in the above-mentioned prior art, one of the objects of the present invention is to provide a method for extracting Tremella polysaccharide.

[0006] A second object of the present invention is to provide a Tremella polysaccharide.

[0007] The third object of the present invention is to provide a method for extracting Tremella polysaccharide and the use of the extracted Tremella polysaccharide in the preparation of drugs for treating chronic obstructive pulmonary disease.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for extracting Tremella polysaccharide, comprising the following steps:

[0010] (1): After Tremella fuciformis and a low eutectic solvent are mixed and swollen in a mass volume ratio of 1 g: (5-40) mL, ultrasonic extraction is performed at an extraction temperature of 80-105° C. for 40-180 min to obtain a Tremella fuciformis polysaccharide extract;

[0011] (2): precipitating the Tremella polysaccharide extract with alcohol and purifying it to obtain the Tremella polysaccharide;

[0012] The low eutectic solvent comprises water, a hydrogen bond acceptor and a hydrogen bond donor, the mass percentage of water is 40-80%, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(0.5-5).

[0013] Preferably, the Tremella is prepared by removing the stalks of Tremella fruiting bodies, drying and then crushing them; further preferably, the Tremella is prepared by removing the stalks of Tremella fruiting bodies, drying and then crushing them and passing them through a 40-100 mesh sieve.

[0014] Preferably, the preparation method of the low eutectic solvent is: mixing a hydrogen bond donor, a hydrogen bond acceptor and water to obtain the solvent; further preferably, the preparation method of the low eutectic solvent is: mixing a hydrogen bond donor, a hydrogen bond acceptor and water to obtain the solvent at 90 to 95°C.

[0015] Preferably, the alcohol precipitation step in step (2) is: precipitating the Tremella polysaccharide extract with ethanol, and obtaining Tremella polysaccharide solid after centrifugation.

[0016] Preferably, the extraction temperature in step (1) is 80-100°C; further preferably, the extraction temperature in step (1) is 80-96°C; further preferably, the extraction temperature in step (1) is 90-96°C.

[0017] Preferably, in step (1), the swelling time is 0.5 to 2 hours; more preferably, the swelling time is 50 to 100 minutes; more preferably, the swelling time is 50 to 60 minutes.

[0018] Preferably, the hydrogen bond acceptor is selected from choline chloride.

[0019] Preferably, the hydrogen bond donor is selected from at least one of ethylene glycol, glycerol, 1,4-butanediol, urea, citric acid, DL-lactic acid, proline, malic acid, and propylene glycol; further preferably, the hydrogen bond donor is selected from citric acid or DL-lactic acid.

[0020] Preferably, in step (1), the mass volume ratio of Tremella fuciformis to the low eutectic solvent is 1 g: (10-31) mL.

[0021] Preferably, in step (1), the extraction time is 40 to 180 min; further preferably, in step (1), the extraction time is 75 to 145 min.

[0022] Preferably, in the low eutectic solvent, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(0.5-2).

[0023] Preferably, the mass percentage of water in the deep eutectic solvent is 55-70%.

[0024] Preferably, the low eutectic solvent is a mixture of choline chloride and citric acid in a molar ratio of 1: (0.5-1.5); further preferably, the low eutectic solvent is a mixture of choline chloride and citric acid in a molar ratio of 1: (0.9-1.2); more preferably, the low eutectic solvent is a mixture of choline chloride and citric acid in a molar ratio of 1:1.

[0025] Preferably, the purification step in step (2) is: eluting the solid after alcohol precipitation on a chromatography column using water and 0.1-0.3 mol / L sodium chloride solution in sequence, dialyzing the eluate and freeze-drying it to obtain purified Tremella polysaccharide.

[0026] Preferably, the chromatography column is a DEAE-52 column.

[0027] The second aspect of the present invention provides a Tremella polysaccharide, which is extracted by the Tremella polysaccharide extraction method provided by the first aspect of the present invention; the molecular weight of the Tremella polysaccharide is 13000-14000Da.

[0028] Preferably, the molecular weight of the Tremella fuciformis polysaccharide is 13100-13200 Da; further preferably, the molecular weight of the Tremella fuciformis polysaccharide is 13152 Da.

[0029] The third aspect of the present invention provides the use of Tremella polysaccharide extracted by the extraction method provided by the first aspect of the present invention in the preparation of a medicine for treating chronic obstructive pulmonary disease.

[0030] The beneficial effects of the present invention are as follows: the extraction method of the present invention uses a natural non-toxic low eutectic solvent for extraction, which can avoid the use of organic solvents, reduce potential human harm, and has good stability and safety. The extraction method provided by the present invention does not require deproteinization, has simple extraction steps, has high extraction efficiency and extraction purity, and the extracted product has significant efficacy in treating COPD, specifically, the protein content is reduced from 1.92% to 1.12%, the protein removal rate reaches 41.67%, and the extraction rate reaches 30.02%, which is significantly higher than the traditional water extraction method.

[0031] The tremella polysaccharide extracted by the extraction method of the present invention can significantly reduce the level of the proinflammatory factor IL-6 in a COPD cell model formed by CSE-stimulated A549 cells, and significantly enhance the enzyme activities of SOD, catalase, and glutathione peroxidase, thereby achieving the efficacy of treating COPD by reducing the inflammation level of the model and alleviating the influence of oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a test chart of the extraction rate of Tremella polysaccharide extracted using different low-solubility reagents in Example 1.

[0033] Figure 2 This is a test chart of the extraction rate of Tremella polysaccharide extracted from the low eutectic solvent with different water contents in Example 2.

[0034] Figure 3 This is a test chart of the extraction rate of Tremella polysaccharide extracted using different ultrasonic times in Example 3.

[0035] Figure 4 This is a test chart of the extraction rate of Tremella polysaccharide extracted using different material-liquid ratios in Example 4.

[0036] Figure 5 This is a test chart showing the effect of TFP-1 extracted in Example 6 on the NO content in cells.

[0037] Figure 6 This is a test chart showing the effect of TFP-1 extracted in Example 6 on the IL-6 concentration in cells.

[0038] Figure 7 This is a test diagram of the effect of TFP-1 extracted in Example 6 on the activity of glutathione peroxidase in cells.

[0039] Figure 8 This is a test chart showing the effect of TFP-1 extracted in Example 6 on SOD enzyme activity in cells.

[0040] Fig. 9 This is a test chart showing the effect of cTFP extracted in Example 1 on the NO content in cells.

[0041] Fig.10 This is a test chart showing the effect of cTFP extracted in Example 1 on the IL-6 concentration in cells.

[0042] Fig.11 This is a test chart showing the effect of cTFP extracted in Example 1 on SOD enzyme activity in cells.

[0043] Fig.12 This is a test diagram of the effect of cTFP extracted in Example 1 on the activity of glutathione peroxidase in cells.

[0044] Fig.13 This is a standard curve diagram for determining the protein content of cTFP extracted in Example 1. DETAILED DESCRIPTION

[0045] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0046] The Tremella fruiting bodies used in the embodiments of the present invention are commercially available products. The extraction solvents used in the embodiments are all analytical grade. The water used is ultrapure water unless otherwise specified. The human non-small cell lung cancer cell line A549 was purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0047] Example 1

[0048] This example provides a method for extracting Tremella polysaccharide, which uses different deep eutectic solvents to extract Tremella polysaccharide, then measures the content of Tremella polysaccharide extracted by different deep eutectic solvents, and calculates the Tremella polysaccharide extraction rate. The specific steps are as follows:

[0049] (1) Preparation of low eutectic agents (DESs): Heat the hydrogen bond acceptors and hydrogen bond donors in Table 1 at 90-95°C and stir continuously until the liquid is evenly dissolved, then add water (the total mass percentage of the low eutectic agent is 100%, and the mass percentage of water is 40%) and mix evenly to obtain a low eutectic solvent for extraction experiments.

[0050] Table 1 Ratios of different eutectic reagents

[0051] Serial number Abbreviation Composition of DESs The amount of substance 1 DES-1 Choline chloride: ethylene glycol 1:2 2 DES-2 Choline chloride: glycerol 1:1 3 DES-3 Choline chloride: 1,4-butanediol 1:4 4 DES-4 Choline chloride: urea 1:2 5 DES-5 Choline chloride:propylene glycol:urea 1:1:1 6 DES-6 Choline chloride: DL-lactic acid 1:2 7 DES-7 Choline chloride: citric acid 1:1 8 DES-8 Choline chloride: Proline: Malate 1:1:1

[0052] (2) Preparation of Tremella powder: Select Tremella fruiting bodies that are free of mildew and insect pests, remove the stems and crush the sample, and pass it through a 40-mesh sieve to obtain Tremella powder.

[0053] (3) Weigh Tremella powder and low eutectic solvent at a solid-liquid ratio of 1 g:10 mL (solid-liquid ratio refers to the mass-volume ratio of Tremella powder to low eutectic solvent), extract at an extraction temperature of 95°C for 40 min, and obtain a Tremella polysaccharide crude extract. Add anhydrous ethanol at a volume ratio of 1:4 to the crude Tremella polysaccharide extract, centrifuge and discard the supernatant, add ultrapure water for reconstitution, and obtain a Tremella polysaccharide extract, which is recorded as cTFP.

[0054] (4) The phenol-sulfuric acid method was used to determine the extraction rate of Tremella polysaccharide. The specific method is as follows: take 150 μL of Tremella polysaccharide extract in a 2 mL centrifuge tube, add 75 μL of 6% phenol solution, mix on an oscillator, and insert into ice. Add 375 μL of concentrated sulfuric acid, mix on an oscillator, and insert into ice to cool for 3 minutes. After cooling, boil in a boiling water bath for 10 minutes, insert into ice to cool for 3 minutes, and start the measurement when the liquid temperature returns to room temperature. Pipette 200 μL of the sample solution into a 96-well plate and measure at 490 nm on an enzyme reader. The extraction rate of Tremella polysaccharide is obtained based on the standard curve. The specific test results are as follows. Figure 1 As shown. In order to screen the low eutectic reagent with the highest extraction rate of Tremella polysaccharide, the extraction rates of Tremella polysaccharide of 8 DESs were tested. Taking water extraction as the control, the extraction rates of DES-6 and DES-7 were significantly higher than those of water extraction (p<0.0001, p<0.0001), and the extraction rate of DES-7 (9.61%) was higher than that of DES-6 (6.92%), so DES-7 was selected for subsequent experiments. After testing, the number average molecular weight of the Tremella polysaccharide extracted in this example was 13152Da.

[0055] Example 2

[0056] This example provides a method for extracting Tremella polysaccharide, which uses low eutectic solvents with different water contents to extract Tremella polysaccharide, then measures the content of the extracted Tremella polysaccharide, and calculates the Tremella polysaccharide extraction rate. The specific steps are as follows:

[0057] (1) Take Tremella powder and swell it in DES-7 for 60 min. The extraction time is fixed at 40 min and the solid-liquid ratio is 1 g:10 mL. The effect of water content in DES-7 (40%, 50%, 60%, 70%, 80%) on the extraction effect is investigated.

[0058] (2) Extraction was performed at an extraction temperature of 95°C to obtain a crude extract of Tremella polysaccharide. Anhydrous ethanol was added at a volume ratio of 1:4 between the crude extract of Tremella polysaccharide and anhydrous ethanol, the supernatant was discarded after centrifugation, and ultrapure water was added for re-dissolution. The Tremella polysaccharide content under each single factor condition (i.e., different water contents) was determined, and the Tremella polysaccharide extraction rate was calculated. The specific test results are as follows: Figure 2 shown.

[0059] Depend on Figure 2It can be seen that when the water content of DES-7 is in the range of 40-60%, the extraction rate of Tremella polysaccharide increases with the increase of water content. This may be because the water content can affect the viscosity, surface tension and dissolution and dispersion ability of DES-7, thereby affecting the dissolution rate of Tremella polysaccharide. Compared with the water content of 40 and 50% in DES-7, the extraction rate of Tremella polysaccharide is the highest when the water content of DES-7 is 60%, reaching 13.95%. When the water content of DES-7 exceeds 60%, the extraction rate of Tremella polysaccharide decreases with the increase of water content. This is because too high water content will reduce the interaction between DES-7 components, affect the formation of hydrogen bonds, and destroy the structure of low eutectic reagents, thereby reducing the extraction rate of Tremella polysaccharide.

[0060] Example 3

[0061] This example provides a method for extracting Tremella polysaccharide, which uses different ultrasonic times to extract Tremella polysaccharide, then measures the content of the extracted Tremella polysaccharide, and calculates the Tremella polysaccharide extraction rate. The specific steps are as follows:

[0062] (1) Take Tremella fuciformis powder and swell it in DES-7 for 60 min. The water content is fixed at 60% and the solid-liquid ratio is fixed at 1 g:10 mL. The effect of ultrasonic time (40 min, 75 min, 110 min, 145 min, 180 min) on the extraction effect is investigated.

[0063] (2) Extraction was performed at an extraction temperature of 95°C to obtain a crude extract of Tremella polysaccharide. Anhydrous ethanol was added at a volume ratio of 1:4 between the crude extract of Tremella polysaccharide and anhydrous ethanol, the supernatant was discarded after centrifugation, and ultrapure water was added for re-dissolution. The contents of the extracted Tremella polysaccharide were determined respectively, and the extraction rate of Tremella polysaccharide was calculated. The specific test results are as follows: Figure 3 shown.

[0064] Depend on Figure 3 It can be seen that when the extraction time is within the range of 40-110min, the extraction rate of Tremella polysaccharide increases with the extension of extraction time; when the extraction time is 110min, the extraction rate of Tremella polysaccharide is relatively high, reaching 24.55%. When the extraction time is 145min or 180min, the extraction rate of Tremella polysaccharide begins to decrease with the extension of extraction time. This is because the extraction time is too long, which leads to the degradation and oxidation of Tremella polysaccharide, resulting in a decrease in the extraction rate of Tremella polysaccharide.

[0065] Example 4

[0066] This example provides a method for extracting Tremella polysaccharide, which uses different material-liquid ratios to extract Tremella polysaccharide, then determines the content of the extracted Tremella polysaccharide, and calculates the Tremella polysaccharide extraction rate. The specific steps are as follows:

[0067] (1) Take Tremella powder and swell it in DES-7 for 60 minutes. Fix the DES water content at 60% and the extraction time at 110 minutes to explore the effect of the solid-liquid ratio (1g:5mL, 1g:10mL, 1g:20mL, 1g:30mL, 1g:40mL) on the extraction effect.

[0068] (2) Extraction was performed at an extraction temperature of 95°C to obtain a crude extract of Tremella polysaccharide. Anhydrous ethanol was added at a volume ratio of 1:4 between the crude extract of Tremella polysaccharide and anhydrous ethanol, the supernatant was discarded after centrifugation, and ultrapure water was added for re-dissolution. The contents of Tremella polysaccharide extracted under different material-liquid ratios were determined, and the extraction rate of Tremella polysaccharide was calculated. The specific test results are as follows: Figure 4 shown.

[0069] Depend on Figure 4 It can be seen that within the range of 1g:5mL-1g:10mL of the solid-liquid ratio, as the relative amount of DES-7 reagent increases, the extraction rate of Tremella polysaccharide increases. Among them, when the solid-liquid ratio is 1g:10mL, the extraction rate of Tremella polysaccharide reaches 28.48%. When the amount of DES-7 reagent input is too high (that is, after the solid-liquid ratio is greater than 1g:30mL), the extraction rate of Tremella polysaccharide gradually decreases. This is because as the solid-liquid ratio increases, the mass proportion of Tremella powder decreases. When the polysaccharide dissolution tends to be saturated, the extraction rate decreases as the volume of the solvent increases.

[0070] Embodiment 5 to Embodiment 18

[0071] The extraction method of Tremella polysaccharide in Examples 5 to 18 is: using DES-7 reagent with a water content of 56.72 to 70% for extraction, the extraction time is 75 to 145 min, the solid-liquid ratio during extraction (i.e., the mass volume ratio of Tremella to the low eutectic solvent) is 1 g: 20 to 40 mL, and the specific extraction conditions are shown in Table 2 below. According to the specific extraction conditions recorded in Table 2 below and with reference to the extraction method of Tremella polysaccharide recorded in Example 4, the extraction is carried out, and the actual extraction rate of Tremella polysaccharide is calculated after extraction, and the calculation result of the actual extraction rate is recorded in Table 2 below.

[0072] Table 2 Extraction conditions and extraction rate of Tremella polysaccharide

[0073]

[0074]

[0075] It can be seen from Table 2 that when the water content in the DES-7 reagent is 56.72-70%, the extraction time is 75 min, and the solid-liquid ratio is 1 g: (20-30.63) mL, the extraction rate of Tremella polysaccharide exceeds 29%.

[0076] Embodiment 19

[0077] This example provides a method for purifying Tremella polysaccharide, which comprises the following steps:

[0078] Purification: Install a 2.6 × 50cm chromatography column, fill it with DEAE-52 filler, and balance it with ultrapure water for 24h. After the balance is completed, load the sample with a 10mg / mL Tremella polysaccharide solution (the Tremella crude polysaccharide sample extracted in Examples 1 to 18), and use distilled water, 0.1mol / L, 0.2mol / L, and 0.3mol / L NaCl solutions as eluents in sequence. Set the parameters of the constant flow pump, and adjust the automatic collector collection speed to 1mL / min, 12min / tube. After the elution is completed, 150μL sample solution is drawn by the tube, and the phenol-sulfuric acid method measures the absorbance of each tube and draws the absorbance curve, collects the eluent corresponding to each absorption peak, and is concentrated by a rotary evaporator, and freeze-dried after dialysis to obtain purified Tremella polysaccharide TFP-1, and the molecular weight of purified Tremella polysaccharide TFP-1 is 13152Da.

[0079] Therefore, the molecular weight of the Tremella polysaccharides extracted in Examples 1 to 18 of the present invention is 13152 Da.

[0080] Comparative Example 1

[0081] This example provides a method for extracting Tremella polysaccharide by a traditional water extraction method, and the specific steps are as follows:

[0082] (1) Preparation of Tremella powder: Select Tremella fruiting bodies that are free of mildew and insect pests, remove the stems and crush the sample, and pass it through a 40-mesh sieve to obtain Tremella powder.

[0083] (2) Mix Tremella powder and ultrapure water at a solid-liquid ratio of 1:10, extract for 40 min at an extraction temperature of 95°C to obtain a crude Tremella polysaccharide extract. Add anhydrous ethanol in a certain proportion, centrifuge and discard the supernatant, add ultrapure water to re-dissolve, obtain a Tremella polysaccharide extract, determine the Tremella polysaccharide content, and calculate the Tremella polysaccharide extraction rate. The specific test results are as follows: Figure 1 As shown, Figure 1 The test result corresponding to water is the extraction rate of Tremella polysaccharide extracted by water extraction method in this example, and the extraction rate is 3.28%.

[0084] Performance Testing:

[0085] Add approximately 1 × 10 5A549 cells were cultured overnight, and 20% cigarette smoke extract (CSE) was added to the A549 cells to model the COPD model cells. The COPD cell model was stimulated with different concentrations of TFP-1 purified in Example 19 (10 μg / mL, 50 μg / mL, 100 μg / mL), and a blank control group (denoted as con) was set up. After 24 hours of action, the culture supernatant was aspirated to test the NO content and IL-6 concentration; lysis solution was added to lyse the cells, and the SOD enzyme activity and glutathione enzyme activity in the lysate were measured. In order to evaluate the effects of different concentrations of drug treatment on the COPD cell model, this study used the Griess reagent method to determine the NO content. The test results are as follows: Figure 5 As shown. Figure 5 It can be seen that TFP-1 treatment at low concentration (10μg / mL) had no significant effect on the NO content produced by A549 cells. When the concentration increased to 50μg / mL and 100μg / mL, the NO content decreased significantly in a dose-dependent manner (p<0.05, p<0.0001). To explore whether different drugs can achieve the effect of treating COPD through the secretion of cytokines in the process of acting on the A549 cell model of COPD, this study used the Elisa method to determine IL-6. The specific test results are as follows: Figure 6 As shown. Figure 6 The results showed that after treatment with 10μg / mL, 50μg / mL and 100μg / mL TFP-1, the IL-6 content of A549 cells was significantly reduced in a dose-dependent manner (p<0.01, p<0.0001, p<0.0001). Nrf2 expression is increased in COPD patients. The body reverses the effect of Nrf2 overexpression and promotes ferroptosis by enhancing the levels of glutathione peroxidase and SOD and inhibiting the release of proinflammatory cytokines. Figure 7 It can be seen that after treatment with TFP-1, the glutathione peroxidase activity of the COPD cell model A549 cells increased in a dose-dependent manner within the concentration range of 10μg / mL, 50μg / mL and 100μg / mL (p>0.05, p<0.0001, p<0.0001). SOD is a scavenger of superoxide anion free radicals in the normal energy metabolism of the human body. It is a metal-active enzyme that can scavenge oxygen free radicals in the body and prevent oxidative lysis of the cell nucleus. COPD patients have chronic airway inflammation, continuous destruction of the alveolar capillary bed, and oxygen exchange disorders, which lead to ischemia and hypoxia of organ tissues, increased free radical production, and reduced SOD production and activity. The ability to scavenge oxygen free radicals is significantly reduced, and excessive free radicals lead to damage to tissue cells. Figure 8It can be seen that after treatment with TFP-1, the SOD enzyme activity of A549 cells increased significantly in a dose-dependent manner within the concentration range of 10μg / mL, 50μg / mL and 100μg / mL (p>0.05, p<0.05, p<0.01).

[0086] Add approximately 1 × 10 5 A549 cells were cultured overnight and 20% CSE was added for modeling, which was recorded as the modeling group; dexamethasone stimulated the modeling group cells as a positive control; a blank control group (recorded as the control group) was set up. After 24 hours of action, the culture supernatant was aspirated to measure the NO content and IL-6 concentration; lysis solution was added to lyse the cells, and the SOD enzyme activity and glutathione enzyme activity in the lysate were measured. The test showed that compared with the control group, the NO content in the modeling group was significantly increased (p<0.0001), indicating that the modeling was successful. And the dexamethasone solution with a concentration of 1μg / mL and 10μg / mL can significantly reduce the NO content produced by the COPD cell model (p<0.001, p<0.0001). Compared with the control group, the IL-6 content in the modeling group was significantly increased (p<0.0001), indicating that 20% CSE led to an increase in the proinflammatory factor IL-6 in the COPD cell model, and the modeling was successful. Dexamethasone solution at a concentration of 1μg / mL and 10μg / mL can significantly reduce the IL-6 content produced by the COPD cell model (p<0.0001, p<0.0001). Compared with the control group, the SOD enzyme activity in the modeling group was significantly reduced (p<0.05), indicating that 20% CSE led to a decrease in the SOD enzyme activity in the COPD cell model, and the modeling was successful. And the dexamethasone solution at a concentration of 1μg / mL can significantly reverse the effect of 20% CSE on the SOD enzyme activity of the COPD A549 cell model, which is manifested by a significant increase in the measured SOD enzyme activity (p<0.01). Compared with the control group, the glutathione peroxidase activity in the modeling group was significantly reduced (p<0.0001), indicating that 20% CSE led to a decrease in the glutathione enzyme activity in the COPD cell model, and the modeling was successful. Moreover, a dexamethasone solution with a concentration of 1 μg / mL was able to significantly reverse the effect of 20% CSE on the glutathione peroxidase activity in the COPD A549 cell model, as evidenced by a significant increase in the measured glutathione peroxidase activity (p<0.0001).

[0087] Add approximately 1 × 10 5A549 cells were cultured overnight and 20% CSE was added for modeling, which was recorded as the modeling group; 10μg / mL, 100μg / mL, and 500μg / mL cTFP were added to the modeling group to stimulate A549 cells, and a blank control group (recorded as con) was set up. After 24 hours of action, the culture supernatant was aspirated to measure the NO content and IL-6 concentration, and the lysis solution was added to lyse the cells, and the SOD enzyme activity and glutathione enzyme activity in the lysate were measured. The specific test results are as follows Figures 9 to 12 As shown. Fig. 9 It can be seen that cTFP (10 μg / mL) promotes the release of NO at low concentrations, which may be due to its combined effect with 20% CSE to aggravate inflammation. As the concentration increases, the NO content produced by the cTFP treatment group decreases, but there is still no significant difference with the modeling group. Fig.10 It can be seen that the cTFP treatment group at a low concentration (10 μg / mL) significantly reduced the IL-6 content produced by A549 cells (p<0.0001). When the concentration increased to 100 μg / mL, there was no significant reduction effect. When the concentration continued to increase to 500 μg / mL, the inhibitory effect on IL-6 increased (p<0.0001), which was not dose-dependent. Fig.11 It can be seen that the cTFP treatment group at three concentrations (10μg / mL, 100μg / mL, 500μg / mL) can significantly increase the SOD enzyme activity of A549 cells (p<0.001, p<0.001, p<0.001). Fig.12 It can be seen that the cTFP treatment group at 10μg / mL, 100μg / mL, and 500μg / mL had no significant effect on increasing the glutathione peroxidase activity of A549 cells (p>0.05).

[0088] The total protein content in the Tremella fruiting body was measured by the Kjeldahl method to be 1.92%. The protein content in the Tremella polysaccharide sample extracted by DES-7 in Example 1 was tested by the BCA method to be 1.12%. The specific test results are as follows: Fig.13 As shown, it can be seen that the protein content of the extracted Tremella polysaccharide can be reduced from 1.92% to 1.12% by the extraction method of the present invention, and the protein removal rate reaches 41.67%.

[0089] In summary, the Tremella polysaccharides extracted by the present invention have a certain effect in treating COPD. Compared with the crudely extracted Tremella polysaccharides, the purified TFP-1 has a more significant overall therapeutic effect on the COPD cell model.

[0090] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for extracting Tremella polysaccharide, characterized in that: The following steps are involved: (1): After Tremella fuciformis and a low eutectic solvent are mixed and swollen in a mass volume ratio of 1 g: (5-40) mL, ultrasonic extraction is performed at an extraction temperature of 80-105° C. for 40-180 min to obtain a Tremella fuciformis polysaccharide extract; (2): precipitating the Tremella polysaccharide extract with alcohol and purifying it to obtain the Tremella polysaccharide; The low eutectic solvent comprises water, a hydrogen bond acceptor and a hydrogen bond donor, the mass percentage of water is 40-80%, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(0.5-5).

2. The method for extracting Tremella polysaccharide according to claim 1, wherein: The hydrogen bond acceptor is selected from choline chloride; the hydrogen bond donor is selected from at least one of ethylene glycol, glycerol, 1,4-butanediol, urea, citric acid, DL-lactic acid, proline, malic acid, and propylene glycol.

3. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: In the step (1), the mass volume ratio of Tremella fuciformis to the low eutectic solvent is 1 g: (10-31) mL.

4. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: In the step (1), the ultrasonic extraction time is 80 to 120 minutes.

5. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: In the low eutectic solvent, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(0.5-2).

6. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: The mass percentage of water in the deep eutectic solvent is 55-70%.

7. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: The deep eutectic solvent has a molar ratio of 1: A mixture of choline chloride (0.5-1.5) and citric acid.

8. The method for extracting Tremella polysaccharide according to claim 1, characterized in that: The purification step in step (2) is as follows: the solid after alcohol precipitation is eluted on a chromatography column using water and a 0.1-0.3 mol / L sodium chloride solution in sequence, and the eluate is dialyzed and freeze-dried to obtain purified Tremella polysaccharide.

9. A Tremella polysaccharide, characterized in that: The tremella polysaccharide is extracted by the tremella polysaccharide extraction method according to any one of claims 1 to 8; the number average molecular weight of the tremella polysaccharide is 13000 to 14000 Da.

10. Use of the Tremella polysaccharide extracted by the method for extracting Tremella polysaccharide according to any one of claims 1 to 8 in preparing a medicine for treating chronic obstructive pulmonary disease.

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