Ganoderma lucidum polysaccharide efficient extraction method and function and application thereof

Abstract

The invention discloses an efficient ganoderma lucidum polysaccharide extraction method and functions and application thereof. According to the method, efficient and green extraction of the ganoderan is achieved by virtue of a free radical-DES synergistic effect. According to the method, hydrogen peroxide is heated to generate hydroxyl radicals to damage ganoderma lucidum cell walls, the release efficiency of intracellular polysaccharide is improved, parameters such as hydrogen peroxide concentration and treatment time are optimized, and damage to a polysaccharide structure due to excessive oxidation is avoided. On the basis, the released polysaccharide is dissolved and extracted by utilizing the dissolving capacity of a deep eutectic solvent (DES), and the dissolving and extracting effects of the DES on the polysaccharide are fully exerted by optimizing parameters such as the type of the DES, the water content of hydrated DES, the treatment temperature and time. The ganoderma lucidum polysaccharide with high yield and strong biological activity is finally obtained by taking the ganoderma lucidum polysaccharide extraction yield as a response value and by virtue of a BBD design method and an SEM scanning electron microscope.

Description

Technical Field

[0001] The present invention relates to the technical field of biological extraction, and particularly relates to a method for efficiently extracting ganoderma polysaccharide and its functions and applications.

Background Art

[0002] Ganoderma lucidum and its active ingredient ganoderma polysaccharide (GLP) have shown significant potential in the treatment of colitis due to their anti-inflammatory, antioxidant, and immunomodulatory properties. Existing studies have confirmed that ganoderma polysaccharide can reduce the expression of pro-inflammatory factors (TNF-α, IL-6, IL-1β) by inhibiting the NF-κB / MAPK signaling pathway, and improve intestinal barrier function by enhancing the expression of tight junction proteins (ZO-1, Occludin).

[0003] Current extraction techniques for ganoderma polysaccharide mainly include hot water extraction, dilute alkali extraction, biological enzyme method, and physical assistance method, but all have significant defects:

[0004] 1. Hot water extraction method: The dissolution rate of ganoderma polysaccharide is low, and high temperature easily destroys the active structure;

[0005] 2. Dilute alkali extraction method: Although it improves the extraction rate of acidic polysaccharides, high-concentration alkali solution causes glycosidic bond cleavage, and the subsequent purification cost is high;

[0006] 3. Biological enzyme method: The cost of enzymes is relatively high, and the activity of enzymes is easily affected by various factors, resulting in unstable extraction processes and difficulties in ensuring the consistency of product quality;

[0007] 4. Physical assistance method: including ultrasonic extraction method and microwave extraction method. Although it can improve the extraction efficiency to a certain extent, the equipment investment is large, the operation cost is high, and the technical requirements for operators are relatively high, which is not conducive to large-scale popularization and application.

[0008] With the continuous development of the concept of green chemistry, finding natural green solvents has become a key research focus. Deep eutectic solvents (DES), also known as natural deep eutectic solvents (NADES), are a type of novel green solvents. DES is formed by mixing hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), and its melting point is significantly lower than that of each component alone. In the study by Abbott et al. in 2003, it was reported that a deep eutectic solvent formed by mixing choline chloride and urea, and this mixture exhibited unique solvent properties. Compared with traditional organic solvents, DES has significant advantages such as lower toxicity and good biodegradability. In addition, it has broad application prospects in multiple fields such as extraction, dissolution, catalytic conversion, and nanomaterial preparation.

[0009] Hydrogen peroxide is an unstable compound that loses an electron when heated to form hydroxyl radicals, which can effectively break the tough and complex cell wall structure of Ganoderma lucidum, a basidiomycete. This wall-breaking effect makes the wall layer structure become loose, thereby increasing the release efficiency of polysaccharides. After hydrogen peroxide destroys the cell wall, DES can quickly penetrate into the cell interior, dissolve and extract polysaccharides. This synergistic effect not only improves the extraction yield of polysaccharides, but also reduces the dissolution of impurities and improves the purity of the extract. By combining the oxidative effect of hydrogen peroxide with the dissolution effect of DES in this way, the dissolution rate of polysaccharides is further increased. In the present invention, through the introduction of hydrogen peroxide pretreatment, the oxidative wall-breaking effect is used to enhance the cell wall permeability, which synergizes with the dissolution ability of DES, and the extraction yield is increased to 30.7%-35.6%, and the purity and biological activity of polysaccharides are significantly improved (see Examples 1-4).

[0010] In summary, although deep eutectic solvents (DES) show broad application prospects as a new type of green solvent in the field of biological extraction, currently, DES is mostly used alone for extraction. The present invention proposes a method for co-extracting Ganoderma lucidum polysaccharides with hydrogen peroxide-DES, which gives full play to the oxidative effect of hydrogen peroxide and the dissolution ability of DES, so as to achieve efficient extraction of Ganoderma lucidum polysaccharides. At the same time, the Ganoderma lucidum polysaccharides extracted by this method have unique biological activities and effectively enhance the body's antioxidant ability, relieve inflammation and other aspects of the ability.

Summary of the Invention

[0011] The object of the present invention is to solve the problems in the existing Ganoderma lucidum polysaccharide extraction technology, such as low extraction yield, damaged polysaccharide activity, many impurities, high cost and environmental pollution, and to propose a high-efficiency extraction method, function and application of Ganoderma lucidum polysaccharides, so as to achieve efficient, green and low-cost extraction of Ganoderma lucidum polysaccharides. This method uses the action of hydroxyl radicals generated by heating hydrogen peroxide to break the cell wall of Ganoderma lucidum, increasing the release efficiency of intracellular polysaccharides. At the same time, parameters such as hydrogen peroxide concentration and treatment time are optimized to avoid over-oxidation from damaging the polysaccharide structure. On this basis, the dissolved ability of deep eutectic solvent (DES) is used to dissolve and extract the released polysaccharides. By optimizing parameters such as the type of DES, the water content of hydrated DES, treatment temperature and time, the dissolution and extraction effects of DES on polysaccharides are fully exerted. Taking the extraction yield of Ganoderma lucidum polysaccharides as the response value, with the help of the BBD (Box-Behnken Design) method and SEM (Scanning Electron Microscope), Ganoderma lucidum polysaccharides with high yield and strong biological activities (such as reducing mouse colon permeability, protecting cells from oxidative damage, relieving inflammation, etc.) are finally obtained.

[0012] To achieve the above object, the present invention proposes a high-efficiency extraction method for Ganoderma lucidum polysaccharides, which includes the following steps:

[0013] (1) Ganoderma lucidum powder preparation: The Ganoderma lucidum slices are dried and then pulverized to obtain Ganoderma lucidum powder, which is divided and stored in a glass desiccator.

[0014] (2) Free radical pretreatment stage: The Ganoderma lucidum powder is mixed with hydrogen peroxide solution and treated at 80 - 100 °C for 60 - 200 min. The final concentration of hydrogen peroxide is 0.8 - 2.4% (mass fraction), and the liquid-solid ratio is 50 - 120 mL / g. Among them, the optimal parameters of this stage are determined by response surface optimization experiments (see Examples 1 - 4): the final concentration of hydrogen peroxide is 1.7%, the liquid-solid ratio is 110:1 mL / g, the treatment time is 145 min, and the temperature is 100 °C.

[0015] (3) Preparation of deep eutectic solvent (DES);

[0016] (4) Free radical - DES synergistic extraction stage: The DES is hydrated (water content 0 - 40%), and the hydrated DES is added to the mixture of Ganoderma lucidum powder and hydrogen peroxide solution, and extracted at 50 - 100 °C for 20 - 70 min. The DES liquid-solid ratio is 20 - 90 mL / g. Among them, the preferred DES is urea - choline chloride DES without water (molar ratio 2:1). The optimal parameters of this stage are: the DES liquid-solid ratio is 50 mL / g, the water content is 0%, the DES treatment temperature is 90 °C, and the DES treatment time is 20 min.

[0017] (5) Post-treatment stage: After centrifugation, high-purity Ganoderma lucidum polysaccharide is obtained by ethanol precipitation and protein removal by Sevag method.

[0018] Preferably, the preparation of the deep eutectic solvent (DES) is specifically as follows: Choline chloride is selected as the hydrogen bond acceptor, which is mixed with the hydrogen bond donor and magnetically stirred at a constant temperature of 75 - 85 °C for 2 hours until the solution is clear. The hydrogen bond donors include ethylene glycol, 1,4-butanediol, urea, triethylene glycol, and glycerol. The 1,4-butanediol is 1,4-butanediol. The molar ratios of each hydrogen bond donor to the hydrogen bond acceptor are respectively: ethylene glycol: choline chloride = 2:1; 1,4-butanediol: choline chloride = 1:2; triethylene glycol: choline chloride = 4:1; glycerol: choline chloride = 2:1; urea: choline chloride = 2:1.

[0019] Preferably, the process is optimized by Box - Behnken Design. Based on the parameters of the above extraction method, a quadratic regression model is established with the polysaccharide yield as the response value: Y = 35.6 + 1.30833A - 0.166667B + 1.49167C + 0.8D - 0.775AB - 1.675AC + 2.325AD + 0.6BC + 0.925BD + 1.75CD - 1.34583A 2 - 2.23333B 2-2.97083C 2 -2.03333D 2 , where A is the solid-to-liquid ratio (g / mL), B is the hydrogen peroxide concentration (%), C is the hydrogen peroxide treatment time (min), and D is the DES addition amount (mL).

[0020] Preferably, under the optimal parameter conditions of the free radical pretreatment stage and the free radical-DES synergistic extraction stage, the extraction yield of the Ganoderma lucidum polysaccharide is 30.7%-35.6%; the free radical-DES synergistic extraction makes the cell wall show deep grooves, split layers and porous structures.

[0021] Preferably, the Ganoderma lucidum polysaccharide has biological activities such as anti-inflammatory, antioxidant and liver protection. Among them, the antioxidant effect can enhance the activities of antioxidant-related enzymes, reduce oxidative stress damage, and then play a synergistic role with biological activities such as anti-inflammatory and liver protection.

[0022] Preferably, the Ganoderma lucidum polysaccharide is used for preventing inflammatory bowel disease (IBD), plays a role by reducing the levels of TNF-α and IL-6, and at the same time enhances the activities of antioxidant-related enzymes and reduces oxidative stress damage to play a preventive role.

[0023] Preferably, the Ganoderma lucidum polysaccharide is used for preparing an antioxidant, can significantly improve the activity of serum GSH-Px (p<0.05), and reduce oxidative stress damage.

[0024] Preferably, the Ganoderma lucidum polysaccharide is used for liver protection, can significantly reduce the activity of serum ALT (p<0.05), and reduce hepatocyte damage.

[0025] Advantages of the present invention:

[0026] 1. The extraction efficiency and polysaccharide yield of the Ganoderma lucidum polysaccharide of the present invention are high. Hydrogen peroxide loses an electron when heated, and the formed hydroxyl radical, as a strong oxidant, can destroy the tough and thick structure of the Ganoderma lucidum cell wall, increase cell permeability, and promote the dissolution of polysaccharides. At the same time, hydrogen peroxide reduces subsequent purification steps by oxidizing and decomposing pigments and some impurities; combined with the unique screening of DES and DES water content, as well as the high polarity and hydrogen bond network characteristics of DES, the cell wall structure can be destroyed, and more Ganoderma lucidum polysaccharides can be promoted to dissolve. The synergistic effect of free radicals and DES realizes the efficient dissolution of polysaccharides while reducing the loss of the target. The extraction yield of the Ganoderma lucidum polysaccharide is 14.41 times higher than that of the Ganoderma lucidum polysaccharide extracted by traditional hot water, 9.75 times higher than that of the Ganoderma lucidum polysaccharide extracted by microwave-assisted extraction, 4.06 times higher than that of the Ganoderma lucidum polysaccharide extracted by hydrogen peroxide, and 4.98 times higher than that of the Ganoderma lucidum polysaccharide extracted by the enzymatic method.

[0027] 2. The polysaccharides of the present invention have better retained biological activity. In the free radical-DES co-extraction, the mild extraction environment (neutral pH) of DES is combined with the controllable oxidation of hydrogen peroxide, that is, the oxidation reaction rate of hydrogen peroxide in the DES system is controllable, avoiding over-oxidation to damage the main chain of polysaccharides, reducing the breakage of polysaccharide glycosidic bonds, and combining the design method of BBD and the broken wall morphology of Ganoderma lucidum cell wall by SEM scanning electron microscopy, fully ensuring the controllable crushing effect of Ganoderma lucidum cell wall, forming deep grooves, split layers and pore structures, which is not only conducive to the full dissolution of polysaccharides, but also conducive to the preparation of active polysaccharides with a narrow molecular weight distribution and the integrity of "active fragments" of polysaccharides not being damaged during the wall-breaking process. The extracted polysaccharides have the effects of enhancing the protection of cells from oxidative damage in mice in an inflammatory state, slowing down the intestinal permeability of mice, enhancing the activities of antioxidant enzymes SOD and GSH-px, reducing the levels of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and relieving inflammation; especially in enhancing the activity of SOD and reducing the level of IL-6, the effects are significantly better than those of polysaccharides extracted by hydrogen peroxide and hot water extraction (p<0.05).

[0028] 3. Green and environmentally friendly, with good safety. Compared with traditional alkali extraction and acid extraction, hydrogen peroxide decomposes into water and oxygen after the reaction, without residual toxicity, and is more in line with the requirements of green chemistry. At the same time, the DES used has the characteristics of non-toxicity and biodegradability, and the whole extraction process is operated in a neutral environment, reducing the use of harmful chemical reagents and reducing environmental pollution.

[0029] 4. Cost reduction. The costs of hydrogen peroxide and DES are relatively low, and the optimized extraction process reduces the reagent dosage and operation procedures, reducing the production cost and being conducive to large-scale industrial production.

[0030] The features and advantages of the present invention will be described in detail through examples in combination with the accompanying drawings.

Description of the Drawings

[0031] Figure 1 It is the standard curve graph of concentration-absorbance of different concentrations of glucose of the present invention;

[0032] Figure 2 It is the influence diagram of different DES on the extraction yield of Ganoderma lucidum polysaccharides of the present invention;

[0033] Figure 3 It is the influence diagram of the water content in hydrated DES on the extraction yield of Ganoderma lucidum polysaccharides of the present invention;

[0034] Figure 4 It is the influence diagram of different liquid-to-solid ratios on the extraction yield of Ganoderma lucidum polysaccharides of the present invention;

[0035] Figure 5 It is the influence diagram of different DES dosages on the extraction yield of Ganoderma lucidum polysaccharides of the present invention;

[0036] Figure 6 It is the graph of the effect of different hydrogen peroxide concentrations on the extraction yield of Ganoderma lucidum polysaccharide in the present invention;

[0037] Figure 7 It is the graph of the effect of different hydrogen peroxide treatment times on the extraction yield of Ganoderma lucidum polysaccharide in the present invention;

[0038] Figure 8 It is the graph of the effect of different DES treatment times on the extraction yield of Ganoderma lucidum polysaccharide in the present invention;

[0039] Figure 9 It is the graph of the effect of different DES treatment temperatures on the extraction yield of Ganoderma lucidum polysaccharide in the present invention;

[0040] Figure 10 It is the group of response surface graphs of the effect of free radical-DES co-extraction of Ganoderma lucidum polysaccharide on the polysaccharide yield in the present invention;

[0041] Figure 11 It is the group of SEM pictures of the surface characteristics of Ganoderma lucidum cell wall in the present invention;

[0042] Figure 12 It is the graph of the HE staining results of the colon of mice in each group in the present invention;

[0043] Figure 13 It is the graph of the HE staining results of the liver of mice in each group in the present invention;

[0044] Figure 14 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the TNF-α level in colon tissue;

[0045] Figure 15 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the IL-6 level in colon tissue;

[0046] Figure 16 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the LSP level in serum;

[0047] Figure 17 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the GSH-Px activity in serum;

[0048] Figure 18 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the SOD activity in serum;

[0049] Figure 19 It is the graph of the effect of three kinds of Ganoderma lucidum polysaccharides in the present invention on the ALT content in serum;

[0050] Figure 11 From left to right in it represent Ganoderma lucidum fruit body powder, hot water extraction, free radical-DES co-extraction respectively, and the magnification of the scanning electron microscope (SEM) image is ×1000;Figure 12 The magnification factors from left to right in it are 10 times, 40 times, and 60 times respectively; Figure 13 The magnification factor in it is 20 times.

Specific Embodiment

[0051] A method for efficiently extracting ganoderma polysaccharide, its functions and applications according to the present invention will be described in conjunction with the accompanying drawings.

[0052] Example 1

[0053] 1. Materials and Reagents

[0054] Ganoderma lucidum (provided by a company in Zhejiang), free of pests, diseases and rot. Ganoderma polysaccharide was purchased from a certain biotechnology company.

[0055] Phenol, H2O2, ethylene glycol, 1,4-butanediol, urea, triethylene glycol, glycerol, 95% ethanol, absolute ethanol, etc. are all of analytical grade; DSS (sodium dextran sulfate), 4% paraformaldehyde fixative, 2.5% glutaraldehyde, physiological saline; kits (GSH-px, SOD, glutamic pyruvic transaminase) were purchased from a certain research institute in Nanjing, and LPS, TNF Alpha and IL-6 kits were purchased from a certain company in Shanghai.

[0056] 2. Experimental Instruments

[0057] Small high-speed crusher (model WK-400A, a certain equipment company in Beijing), AL04 type electronic balance, SHA-B water bath shaker, DHG-9146A electrothermal constant temperature forced air drying oven, CT14R0 high-speed centrifuge, stirrer, UV1800PC ultraviolet-visible spectrophotometer, Itachi SU8010 type field emission scanning electron microscope (SEM), DS-5510DTH ultrasonic cleaner.

[0058] 3. Experimental Methods

[0059] 3.1 Pretreatment of Ganoderma lucidum Powder

[0060] Place 100 g of Ganoderma lucidum slices in a drying oven for drying, set the oven temperature at 60 °C, and the drying time is 12 h. Crush the dried Ganoderma lucidum slices with a small high-speed crusher for 5 min to obtain unsieved Ganoderma lucidum powder, which is then sub-packed and stored in a glass desiccator for convenient subsequent use.

[0061] 3.2 Establishment of Quantitative Analysis Method for Ganoderma Polysaccharide

[0062] The phenol-sulfuric acid method was adopted. The method for preparing the standard curve is as follows. Weigh 100 mg of glucose dried to constant weight and dissolve it in deionized water, then dilute to a volume of 1 L in a volumetric flask to prepare a glucose standard solution (100 mg / L). Dissolve 5 g of phenol in a 100 mL volumetric flask and store it in the dark at 4 °C (brown bottle) to prepare 5% phenol. Add various reagents according to the amounts shown in Table 1 below.

[0063] Table 1 Dosages of various reagents in different groups

[0064]

[0065]

[0066] After vortex mixing, let it stand at room temperature for 10 min, then place it in a water bath at 30 °C for 20 min. Take an appropriate amount of the reaction solution and measure the absorbance at 490 nm. At the same time, use 0.5 mL of deionized water as a blank control.

[0067] Detection of polysaccharide content in the sample: Accurately measure a certain amount of Ganoderma lucidum polysaccharide solution (try to make the absorbance value fall between 0.2 and 0.8). Add reagents according to the amounts used in preparing the standard curve. Use a UV-visible spectrophotometer to measure the absorbance at 490 nm, and calculate the total sugar content according to the standard curve regression equation and calculate the extraction yield of Ganoderma lucidum polysaccharide in the sample according to the following formula.

[0068] Polysaccharide yield (%) = [c * V * F * 10-6 / M] * 100.

[0069] In the formula, c—the concentration (μg / mL) of the polysaccharide sample to be measured obtained from the standard curve ( Figure 1 ) ; V—the volume (mL) of the supernatant of the polysaccharide sample extract; F—the dilution factor; M—the mass (g) of Ganoderma lucidum fruit body powder.

[0070] Polysaccharide yield (%) = (mass of polysaccharide in the extract / mass of Ganoderma lucidum) × 100%.

[0071] 3.3 Selection of extractant

[0072] 3.3.1 Preparation of deep eutectic solvent (DES).

[0073] Choose choline chloride as the hydrogen bond acceptor, mix it with different hydrogen bond donors (ethylene glycol, 1,4-butanediol, urea, triethylene glycol and glycerol) at the specified molar ratios (as follows), and magnetically stir for 2 hours at a constant temperature of 75–85 °C until the solution becomes clear.

[0074] Ethylene glycol:choline chloride = 2:1;

[0075] 1,4-Butanediol:choline chloride = 1:2;

[0076] Triethylene glycol: choline chloride = 4:1;

[0077] Glycerol: choline chloride = 2:1;

[0078] Urea: choline chloride = 2:1.

[0079] 3.3.2 Screening of deep eutectic solvents (DES).

[0080] Five kinds of DES were prepared, namely ethylene glycol-choline chloride, butanediol-choline chloride, urea-choline chloride, triethylene glycol-choline chloride and glycerol-choline chloride. Weigh 1 g of Ganoderma lucidum powder (unsieved), add DES hydrated at 60% by volume according to the amount of 1 / 60 (g / mL), extract with a water bath shaker, extract for 60 min at 90 °C and a rotation speed of 125 rpm, then centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the yield.

[0081] 3.3.3 Screening of the water content in hydrated DES.

[0082] Weigh 1 g of Ganoderma lucidum powder (unsieved), and according to the ratio of 1 g:60 mL, add 60 mL of deionized water, 60 mL of hydrated DES with a volume concentration of 80% of deionized water (that is, a mixture of deep eutectic solvent and deionized water with a volume ratio of 20:80), 60 mL of hydrated DES with a volume concentration of 60% of deionized water (that is, a mixture of deep eutectic solvent and deionized water with a volume ratio of 40:60), 60 mL of hydrated DES with a volume concentration of 40% of deionized water (that is, a mixture of deep eutectic solvent and deionized water with a volume ratio of 60:40), 60 mL of hydrated DES with a volume concentration of 20% of deionized water (that is, a mixture of deep eutectic solvent and deionized water with a volume ratio of 80:20), 60 mL of DES (that is, without deionized water), the best DES obtained by the above DES screening, and carry out extraction under the same experimental conditions as the DES screening. Detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the yield.

[0083] 3.4 Process optimization of free radical-DES co-extraction of Ganoderma lucidum polysaccharide

[0084] 3.4.1 Liquid-to-material ratio

[0085] Weigh 1.0 g of Ganoderma lucidum powder (unsieved), and add hydrogen peroxide with a mass concentration of 30% and deionized water according to the liquid-to-solid ratios of 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, and 120:1 (mL / g) respectively, so that the final concentration of added hydrogen peroxide is 1.5% and the pH is 7.0. After extracting at 100 °C with a shaking speed of 125 rpm for 80 min in a water bath shaker, add 30 mL of hydrated DES (added according to the water content screening in hydrated DES), extract at 100 °C for 40 min, centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the yield.

[0086] 3.4.2 Dosage of hydrated DES

[0087] Weigh 1.0 g of Ganoderma lucidum powder (unsieved), add hydrogen peroxide with a mass concentration of 30% and deionized water according to the liquid-to-solid ratio of 1 g / 80 mL so that the final concentration of added hydrogen peroxide is 1.5% and the pH is 7.0. After extracting at 100 °C for 80 min, add 20, 30, 40, 50, 60, 70, 80, and 90 mL of hydrated DES. After extracting at 100 °C for 40 min, centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the yield.

[0088] 3.4.3 Concentration of hydrogen peroxide

[0089] Weigh Ganoderma lucidum powder (unsieved), add hydrogen peroxide with a mass concentration of 30% and deionized water according to the liquid-to-solid ratio of 1 / 80 (g / mL), so that the final concentrations of hydrogen peroxide are 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, and 2.4% respectively, with a pH of 7.0 and extract at 100 °C for 120 min. Then add 30 mL of hydrated DES (added according to the water content screening in hydrated DES). After extracting at 100 °C for 40 min, centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the yield.

[0090] 3.4.4 Treatment time of hydrogen peroxide

[0091] Weigh the Ganoderma lucidum powder (unsieved), add hydrogen peroxide with a mass concentration of 30% and deionized water according to the solid-liquid ratio of 1 g / 80 mL so that the final concentration of added hydrogen peroxide is 1.5%, pH 7.0. After extracting at 100 °C for 60, 80, 100, 120, 140, 160, 180, 200 min, add 20 mL of hydrated DES (added according to the water content screened in the hydrated DES). After extracting at 100 °C for 40 min, centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into a yield.

[0092] 3.4.5 DES treatment time

[0093] Weigh the Ganoderma lucidum powder (unsieved), add hydrogen peroxide with a mass concentration of 30% and deionized water according to the solid-liquid ratio of 1 g / 80 mL so that the final concentration of added hydrogen peroxide is 1.5%, pH 7.0. After extracting at 100 °C for 80 min, add 20 mL of hydrated DES (added according to the water content screened in the hydrated DES), and extract at 50 °C for 20, 30, 40, 50, 60, 70 min. Centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into a yield.

[0094] 3.4.6 Temperature during DES treatment

[0095] Weigh the Ganoderma lucidum powder (unsieved), add hydrogen peroxide with a mass concentration of 30% and deionized water according to the solid-liquid ratio of 1 g / 80 mL so that the final concentration of added hydrogen peroxide is 1.5%, pH 7.0. After extracting at 100 °C for 80 min, add 20 mL of hydrated DES (added according to the water content screened in the hydrated DES), and extract at 50, 60, 70, 80, 90, 100 °C for 40 min. Centrifuge at 8000 rpm for 15 min, and record the volume of the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into a yield.

[0096] 3.4.7 Response surface optimization of synergistic extraction of Ganoderma lucidum polysaccharide by free radicals - DES

[0097] According to the single-factor results, select the four factors that significantly affect the yield of Ganoderma lucidum polysaccharide, namely the solid-liquid ratio, hydrogen peroxide concentration, hydrogen peroxide addition time, and the amount of added hydrated DES, as independent variables, and the yield of Ganoderma lucidum polysaccharide as the response value. Design experiments using the Box-Behnken Design method to determine the optimal parameters.

[0098] 3.4.8 Processes for extracting Ganoderma lucidum polysaccharide by different methods

[0099] (1) Hot water extraction

[0100] Commercially available water-extracted Ganoderma lucidum polysaccharide was added to deionized water at a solid-liquid ratio of 1:50 g / mL, and extracted at 40 °C in a water bath for 90 min. After centrifugation at 8000 rpm for 20 min, the supernatant was rotary evaporated and concentrated to one-fifth of the original volume to obtain a concentrated solution. Four volumes of 95% ethanol were added to the concentrated solution, and after standing at 4 °C for 12 h, it was centrifuged at 1000 rpm for 5 min. The precipitate was taken and added with an appropriate amount of water, and rotary evaporated and concentrated at 65 °C until the ethanol was completely evaporated to obtain a concentrated solution. Sevag reagent (chloroform: n-butanol = 4:1) was added in a volume ratio of 1:5, shaken vigorously for 20 min, centrifuged at 3000 r / min for 10 min, and the protein layer was discarded. This was repeated 5 times. The aqueous layer solution was concentrated to 1 / 3 of the original volume to obtain deproteinized water-extracted Ganoderma lucidum polysaccharide.

[0101] (2) Hydrogen peroxide extraction

[0102] Ganoderma lucidum powder (unsieved) was weighed and added with hydrogen peroxide with a concentration of 1.50% (mass fraction) at a solid-liquid ratio of 1:110 g / mL, and extracted at an extraction temperature of 100 °C for 185 min. After centrifugation at 8000 rpm for 20 min, the subsequent steps were the same as those for hot water extraction to prepare deproteinized hydrogen peroxide-extracted Ganoderma lucidum polysaccharide.

[0103] 3.5 Observation of the microstructure of Ganoderma lucidum fruiting bodies and the residues after polysaccharide extraction by different methods using scanning electron microscopy (SEM)

[0104] 0.5 mg of Ganoderma lucidum fruiting body powder and the residue dried after polysaccharide extraction by the optimized method in 3.4 were taken respectively, coated on an MC1000 ion sputtering instrument (icn sputter), and observed using a German ZEISS GeminiSEM 300 scanning electron microscope (SEM).

[0105] 3.6 Functional evaluation method of Ganoderma lucidum polysaccharide extracted by free radical-DES synergistic extraction

[0106] 3.6.1 Animal experiment design

[0107] Male ICR mice, 6 - 8 weeks old, with a body weight of 28 - 32 g, were purchased from a certain limited liability company in Shanghai. The breeding temperature was 20℃ ± 2℃, and the relative humidity was 55 - 60%. During the adaptive feeding period after the mice were purchased, they had free access to food and water. One week later, the mice were randomly divided into 4 groups, with 8 mice in each group, including a normal control group (standard diet, normal saline), a model control group (modeling, normal saline), a high-dose sample group (including Ganoderma lucidum polysaccharides extracted by water extraction, Ganoderma lucidum polysaccharides extracted by hydrogen peroxide extraction, Ganoderma lucidum polysaccharides extracted by free radical-DES co-extraction, 300 mg / kg), and a low-dose sample group (including Ganoderma lucidum polysaccharides extracted by water extraction, Ganoderma lucidum polysaccharides extracted by hydrogen peroxide extraction, Ganoderma lucidum polysaccharides extracted by free radical-DES co-extraction, 150 mg / kg). All animal experiments complied with the specifications of the "Guide for the Care and Use of Laboratory Animals", and this study has passed the ethical review and approval.

[0108] One week after the adaptive feeding of the mice, the experimental period began. The normal control group continued to freely drink deionized water, and in the other groups, the deionized water was replaced with a 2.2% DSS (dextran sulfate sodium) solution on the 21st day. The experimental design is shown in Table 2 below.

[0109] Table 2 Animal experiment design

[0110]

[0111] 3.6.2 Determination of serum biochemical indexes

[0112] On the day when the experiment ended, blood was collected from the retro-orbital venous plexus of the mice. The blood was collected and allowed to stand overnight at 4℃, centrifuged (3000 rpm, 15 min), and the supernatant was collected and stored at -20℃ for later use after being aliquoted. After blood collection, the mice were dissected, and the liver and about 1 cm of the colon were directly immersed in 4% paraformaldehyde fixative, rinsed, and then immersed in 2.5% glutaraldehyde solution. The morphological changes of the liver and colon tissues were observed by HE staining.

[0113] The serum samples were thawed on ice, and the activities of lipopolysaccharide (LPS), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined according to the method described in the kit instructions.

[0114] 3.6.3 Determination of biochemical indexes in colon tissues

[0115] Mouse colon tissues were made into 10% homogenates using a handheld high-speed homogenizer, with normal saline as the homogenization medium. After further centrifugation (3000 rpm, 15 min), the precipitate was discarded and the supernatant was taken. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured according to the method described in the kit instructions.

[0116] 4. Experimental results

[0117] 4.1 Method for quantitative analysis of ganoderma polysaccharide

[0118] The concentration-absorbance standard curve of glucose at different concentrations was plotted, as shown in Figure 1 shown. Its linear regression equation was y = 7.635 + 0.0008, R 2 = 0.9989. This indicates that there is a good linear relationship between the concentration of glucose and the absorbance.

[0119] 4.2 Effect of extractant on the yield of ganoderma polysaccharide

[0120] 4.2.1 Effect of different DESs on the extraction yield of ganoderma polysaccharide

[0121] The five DESs were ethylene glycol-choline chloride, butanediol-choline chloride, urea-choline chloride, triethylene glycol-choline chloride, and glycerol-choline chloride. The extraction yields of ganoderma polysaccharide are shown in Figure 2 .

[0122] It can be seen from Figure 2 that different DESs have different effects on the extraction yield of ganoderma polysaccharide. Among them, the extraction yield of urea-choline chloride was the highest and was significantly higher than that of other DESs (p < 0.05); followed by 1,4-butanediol-choline chloride. Considering various factors such as the stability, safety, and cost of the solvent, urea-choline chloride was selected as the DES extractant for subsequent single-factor and response surface optimization to optimize the optimal process parameters for extracting ganoderma polysaccharide by free radical-DES.

[0123] 4.2.2 Screening of water content in hydrated DES

[0124] The extraction yields of ganoderma polysaccharide with five different water contents in hydrated DES, namely 80% water content, 60% water content, 40% water content, 20% water content, and 0% water content (i.e., without deionized water), are shown in Figure 3 .

[0125] It can be seen from Figure 3It can be seen that the water content in different hydrated DESs has different effects on the extraction yield of Ganoderma lucidum polysaccharides. Among them, the extraction yield is the highest when the water content in the hydrated DES is 0% (i.e., no deionized water is contained), and it is significantly higher than that of other hydrated DESs (p<0.05). When conducting single-factor and response surface optimizations subsequently, 0% water content (i.e., no deionized water is contained) in the hydrated DES is selected as the water content in the hydrated DES to obtain the optimal process parameters for extracting Ganoderma lucidum polysaccharides by the free radical-DES method.

[0126] 4.3 Single-factor experiment and response surface optimization for extracting Ganoderma lucidum polysaccharides by free radical-DES

[0127] 4.3.1 Liquid-to-material ratio

[0128] The liquid-to-material ratios are 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, and 120:1 (mL / g) respectively. The effects of different liquid-to-material ratios on the extraction yield of Ganoderma lucidum polysaccharides are as Figure 4 .

[0129] It can be Figure 4 seen that the effects of different liquid-to-material ratios on the extraction yield of Ganoderma lucidum polysaccharides are different. As the liquid-to-material ratio increases from 50:1 mL / g to 70:1 mL / g, the extraction yield of Ganoderma lucidum polysaccharides does not change significantly; when the liquid-to-material ratio continues to increase to 100:1 mL / g, the extraction yield of Ganoderma lucidum polysaccharides increases significantly (p<0.05) and reaches the peak. Subsequently, as the liquid-to-material ratio continues to increase, the extraction yield of Ganoderma lucidum polysaccharides shows a significant downward trend. Therefore, a liquid-to-material ratio of 100:1 mL / g is used as the central point in the subsequent optimization experiments.

[0130] 4.3.2 DES dosage

[0131] The dosages of DES are 20, 30, 40, 50, 60, 70, 80, and 90 mL respectively. The effects of different DES dosages on the extraction yield of Ganoderma lucidum polysaccharides are as Figure 5 .

[0132] It can be Figure 5 seen that the effects of different DES dosages on the extraction yield of Ganoderma lucidum polysaccharides are different. As the DES dosage increases from 20 mL to 40 mL, the extraction yield of Ganoderma lucidum polysaccharides shows a significant upward trend; when the DES dosage continues to increase to 90 mL, the extraction yield of Ganoderma lucidum polysaccharides shows a fluctuating trend of significant decrease, significant increase, and then significant decrease (p<0.05). The extraction yields are the highest when the DES dosages are 40 mL and 80 mL respectively. Considering various factors such as the safety and cost of DES, a DES dosage of 40 mL is used as the central point in the subsequent optimization experiments.

[0133] 4.3.3 Hydrogen peroxide concentration

[0134] The hydrogen peroxide concentrations were 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2% and 2.4% respectively. The effects of different hydrogen peroxide concentrations on the extraction yield of Ganoderma lucidum polysaccharide are as Figure 6 .

[0135] As Figure 6 can be seen, different hydrogen peroxide concentrations have different effects on the extraction yield of Ganoderma lucidum polysaccharide. As the hydrogen peroxide concentration increases from 0.8% to 1.6%, the extraction yield of Ganoderma lucidum polysaccharide continuously increases significantly. When the hydrogen peroxide concentration is 1.6%, the extraction yield reaches the peak. Subsequently, as the hydrogen peroxide concentration continues to increase, the extraction yield of Ganoderma lucidum polysaccharide shows a significant downward trend. Therefore, a hydrogen peroxide concentration of 1.6% is used as the central point in the subsequent optimization experiments.

[0136] 4.3.4 Hydrogen peroxide treatment time

[0137] The hydrogen peroxide treatment times were 60, 80, 100, 120, 140, 160, 180 and 200 min respectively. The effects of different treatment times on the extraction yield of Ganoderma lucidum polysaccharide are as Figure 7 .

[0138] As Figure 7 can be seen, different hydrogen peroxide treatment times have different effects on the extraction yield of Ganoderma lucidum polysaccharide. As the hydrogen peroxide treatment time increases from 60 min to 140 min, the extraction yield of Ganoderma lucidum polysaccharide continuously increases significantly. When the hydrogen peroxide treatment time is 140 min, the extraction yield reaches the peak. Subsequently, as the hydrogen peroxide treatment time continues to increase, the extraction yield of Ganoderma lucidum polysaccharide shows a significant downward trend. Therefore, a hydrogen peroxide treatment time of 140 min is used as the central point in the subsequent optimization experiments.

[0139] 4.3.5 DES treatment time

[0140] The DES treatment times were 20, 30, 40, 50, 60 and 70 min respectively. The effects of different treatment times on the extraction yield of Ganoderma lucidum polysaccharide are as Figure 8 .

[0141] As Figure 8 can be seen, different DES treatment times have different effects on the extraction yield of Ganoderma lucidum polysaccharide. As the DES treatment time increases from 20 min to 50 min, the extraction yield of Ganoderma lucidum polysaccharide does not change significantly. Subsequently, as the DES treatment time continues to increase, the extraction yield of Ganoderma lucidum polysaccharide shows a significant downward trend. Therefore, this factor will not be optimized in the subsequent experiments, and a DES treatment time of 20 min is selected.

[0142] 4.3.6 Temperature during DES treatment

[0143] The DES treatment temperatures were 50, 60, 70, 80, 90, and 100 °C respectively. The effects of different DES treatment temperatures on the extraction yield of Ganoderma lucidum polysaccharide are as follows Figure 9 .

[0144] As Figure 9 can be seen, different DES treatment temperatures have different effects on the extraction yield of Ganoderma lucidum polysaccharide. As the DES treatment temperature gradually increases, the extraction yield of Ganoderma lucidum polysaccharide also gradually increases. When the treatment temperature is 90 °C, the extraction yield of Ganoderma lucidum polysaccharide reaches the peak. When the temperature continues to rise to 100 °C, the extraction yield of Ganoderma lucidum polysaccharide shows a downward trend but there is no difference compared with that at 90 °C. Therefore, this factor will not be optimized in the subsequent experiments, and the DES treatment temperature is selected as 90 °C.

[0145] 4.3.7 Response surface optimization

[0146] 4.3.7.1 Model fitting and testing

[0147] Based on the single-factor experiments, using soluble polysaccharide as the screening index, according to the principle of central composite experiment, a three-factor and three-level response surface analysis experiment was designed using Design-Expert.V8.0.6 software. The factor levels of the response surface experiment are shown in Table 3, and the experimental design and results are shown in Table 4.

[0148] Table 3 Factor levels of the experimental design for the co-extraction of Ganoderma lucidum polysaccharide by free radicals-DES

[0149]

[0150] Table 4 Experimental design and experimental results for the co-extraction of Ganoderma lucidum polysaccharide by free radicals-DES

[0151]

[0152]

[0153] Performing multiple regression fitting on the experimental data in Table 4, a regression equation with the polysaccharide yield (Y) as the response value was obtained

[0154] Y = 35.6 + 1.30833A - 0.166667B + 1.49167C + 0.8D - 0.775AB - 1.675AC + 2.325AD + 0.6BC + 0.925BD + 1.75CD - 1.34583A 2 - 2.23333B 2 - 2.97083C 2 - 2.03333D 2

[0155] Performing variance analysis on the regression equation, the results are shown in Table 5.

[0156] Table 5 Regression model analysis of synergistic extraction of Ganoderma lucidum polysaccharide by free radical - DES

[0157]

[0158] From Table 5, the p - value was used as a tool to check the significance of each coefficient. The p - value of the model was less than 0.0001, indicating that the model was significant (p < 0.01). R 2 was 0.9000, indicating that the predicted value and the simulation test value were well - fitted within the range of experimental parameters; the adjusted R 2 was 0.8000, indicating that the equation could explain 80.00% of the response value change, and the fitting degree was good. The C.V value was 3.88%, indicating a relatively high credibility of the model. The p - value of the lack - of - fit term was 0.0750 and was not significant, indicating that the residuals were caused by random errors and were not significant relative to the pure error (p > 0.05). Therefore, the regression equation could be used to predict the experimental results.

[0159] From the results of variance analysis, among the four factors, the linear terms A, C, and D had extremely significant effects on the response value; among the quadratic terms, A 2 , B 2 , C 2 , D 2 had extremely significant effects on the response value curve effect; the interaction terms AC, AD, and CD had extremely significant effects on the response surface effect, while the interaction terms AB, BC, and BD had no significant effects on the response surface effect, indicating that there were obvious synergistic effects between the liquid - to - material ratio and the hydrogen peroxide treatment time, the liquid - to - material ratio and the DES addition amount, and the hydrogen peroxide treatment time and the DES addition amount, while there was no synergistic effect between the liquid - to - material ratio and the hydrogen peroxide concentration, the hydrogen peroxide concentration and the hydrogen peroxide treatment time, and the hydrogen peroxide concentration and the DES addition amount; from the magnitude of the F - value, it could be inferred that the primary and secondary order of the four factors affecting the extraction rate was: C > A > D > B, that is, the hydrogen peroxide treatment time > the liquid - to - material ratio > the DES addition amount > the hydrogen peroxide concentration.

[0160] Based on the regression equation, the response surface diagrams of the interaction factors were made as shown in Figure 10 .

[0161] A large slope of the response surface diagram indicates a large effect of the factor on the response value. Dense and elliptical contour lines indicate a large interaction between two factors, while a gentle slope and circular contour lines are the opposite. Figure 10 Among them, the contour lines of the three - group interaction effects of the four factors tended to be elliptical, indicating that there were interactions between any two in the three groups.

[0162] 4.3.7.2 Model verification

[0163] The optimal conditions optimized by the model are as follows: hydrogen peroxide concentration 1.681%, hydrogen peroxide treatment time 143.943 min, liquid-to-solid ratio 109.912 mL / g, and DES addition amount 49.802 mL. Under these conditions, the extraction yield of ganoderma polysaccharide can reach 36.528%. Considering the actual conditions in the laboratory, the optimal adjustment is: hydrogen peroxide concentration 1.7%, time 145 min, liquid-to-solid ratio 110 mL / g, and DES addition amount 50 mL. To further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out according to the optimal adjusted extraction conditions. The yield of ganoderma polysaccharide can reach 35.6% ± 0.37%, which is consistent with the predicted value, indicating that the response surface analysis method is effective, fits well with the actual situation, and verifies the reliability of the regression equation.

[0164] 4.3.8 Observation of the surface characteristics of ganoderma cell wall by scanning electron microscopy (SEM)

[0165] SEM images of the raw material of ganoderma fruiting body and the filter residue after obtaining ganoderma polysaccharide by the synergistic action of hot water and free radical-DES under the optimal extraction conditions are shown in Figure 11 . As Figure 11 can be seen, the cell wall and mycelium of the ganoderma fruiting body are basically intact, with few cracks and holes. The surface of the sample treated with hot water shows a flocculent structure and has slight cracks. For the sample treated with hydrogen peroxide, rod-like shapes appear on the cell wall surface and have a clear porous structure. For the sample treated with the synergistic action of free radical-DES, the surface has a large degree of rupture, deep grooves, split layers and hole structures. It can be seen that different treatment methods have a great impact on the microscopic structure of the ganoderma fruiting body cell wall, and it is speculated that they also have a great impact on the polysaccharide structure inside the cell wall. The extraction yield, molecular structure and biological activity of polysaccharides strongly depend on the adopted cell wall breaking treatment method, suggesting that there are also differences in the molecular structure and biological activity of the polysaccharides obtained by several methods.

[0166] 4.4 Functional evaluation of ganoderma polysaccharide extracted by the synergistic action of free radical-DES

[0167] 4.4.1 Observation of the morphology of colon and liver tissues

[0168] The HE staining results of the colon of mice in each group are as Figure 12As shown, the colonic structure of the mice in the K (normal control) group was intact, and the glands and epithelial cells were arranged neatly. Dextran sulfate sodium could damage the tight junctions and basement membranes of epithelial cells, leading to increased intestinal permeability, promoting the infiltration of intestinal flora and antigens, activating the immune system, and inducing inflammatory responses. Affected by dextran sulfate sodium, the colonic structure of the mice in the N (model control) group was significantly damaged, the number of goblet cells and crypts decreased significantly, and there were obvious inflammatory cell infiltrations and ulcers in many places. Compared with the mice in the N group, the colonic damage of the mice in the P, Lg, Ld, Yg, Yd, Wg, and Wd groups was less severe, the decrease in the number of goblet cells and crypts was not obvious, and the degree of inflammatory cell infiltration was lighter. Moreover, the intestinal health status of the mice in the high-dose groups of Ganoderma lucidum polysaccharides extracted by the three methods was better than that of the low-dose groups of the same polysaccharide. Among them, the colonic recovery effect of the mice in the Yg (i.e., the high-dose Ganoderma lucidum polysaccharide extracted by the free radical-DES co-extraction method) group was obvious, and the intestinal protection effect was better, indicating that the Ganoderma lucidum polysaccharide extracted by this method had a protective and reparative effect on the inflammation of mice, and there might be a certain dose-dependence.

[0169] The results of HE staining of the livers of the mice were as Figure 13 shown (the picture was magnified 20 times). As shown in the figure, the liver structure of the mice in the K group was intact, the structure of the hepatic lobules was clear, and the hepatic cords and hepatic sinusoids were arranged neatly. Intestinal leakage caused by dextran sulfate sodium could lead to the entry of intestinal bacteria or toxins into the portal vein, affecting the liver, resulting in oxidative stress, inflammatory cell infiltration, and fatty degeneration. Affected by dextran sulfate sodium, there were obvious inflammatory cell infiltrations in many places in the portal area of the central vein of the liver tissue of the mice in the N (model control) group, and vacuoles were present in some hepatocytes, indicating pathological changes in their livers. Compared with the mice in the N group, the liver damage and inflammatory infiltration of the mice in the P, Lg, Ld, Yg, Yd, Wg, and Wd groups were less severe, and the effects of the Yg and Wg (water-extracted Ganoderma lucidum polysaccharide) groups were more obvious. This indicated that Ganoderma lucidum polysaccharide had a certain protective effect on the liver inflammation of mice.

[0170] 4.4.2 Effects of three Ganoderma lucidum polysaccharides on the levels of TNF-α and IL-6 in colonic tissues

[0171] Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are representative pro-inflammatory cytokines, both of which are cytokines with a wide range of biological functions. Among them, TNF-α is the core mediator of the inflammatory response, which can activate other inflammatory factors (such as IL-1β, IL-6) and chemokines, promote the infiltration of inflammatory cells, and is an important indicator for judging the body's immune function. TNF-α can cause apoptosis of intestinal epithelial cells, increase intestinal mucosal permeability, damage the barrier function, and exacerbate the inflammatory response. Interleukin-6 (IL-6) is produced by a variety of cells such as macrophages, T cells, B cells, and fibroblasts. It is a polypeptide substance mainly expressed in the inflammatory response (released by inflammatory cells). It is the main mediator of the acute-phase response, which can induce the production of C-reactive protein (CRP) and fibrinogen, and promote the inflammatory response. The effects of Ganoderma lucidum polysaccharides prepared by different extraction methods on the TNF-α level in colon tissue are shown in Figure 14 .

[0172] By Figure 14 , the TNF-α level in the colon tissue of mice in group N was significantly increased compared with that in group K (p<0.05), indicating that dextran sulfate sodium increased the inflammatory level of mice. Compared with the mice in group N, the TNF-α level in the colon tissue of mice in other sample groups was significantly decreased except for the Wd group (p<0.05). Among them, there was no significant difference in the TNF-α level in the colon tissue of mice in the Lg and Wg groups, but there were significant differences compared with the P group, Ld group, Yg group, and Yd group (p<0.05). The TNF-α level in the colon tissue of mice in the Ld group was the lowest, followed by the Yd group and Yg group, and the effects of these three groups were better than those of the P group (p<0.05). It shows that Ganoderma lucidum polysaccharides extracted by free radical-DES and hydrogen peroxide can significantly inhibit the increase in the level of inflammatory factors in the colon tissue of mice, reduce the damage of dextran sulfate sodium to the intestinal mucosal barrier, protect the barrier function, and the effect is better than that of water-extracted Ganoderma lucidum polysaccharides.

[0173] By Figure 15 It can be seen that the IL-6 level in the colon tissue of mice in group N was significantly increased compared with that in group K (p<0.05), indicating that dextran sulfate sodium increased the inflammatory level of mice. Compared with the mice in group N, there was no difference in the IL-6 content in the colon tissue of mice in the P group and Ld group (p>0.05), indicating that the positive drug and low-dose Ganoderma lucidum polysaccharides extracted by hydrogen peroxide could not inhibit the increase in the IL-6 level in the colon tissue of mice; while the IL-6 content in the colon tissue of mice in the Lg, Yg, Yd, Wg, and Wd groups was significantly decreased compared with that in group N (p<0.05), indicating that Ganoderma lucidum polysaccharides extracted by these methods could effectively reduce the increase in IL-6 caused by dextran sulfate sodium at a certain dosage.

[0174] Comprehensively Figure 14 and Figure 15As a result, both high and low doses of Ganoderma lucidum polysaccharides extracted by free radical-DES could significantly reduce the levels of TNF-α and IL-6 in the colon tissues of mice, thereby inhibiting the attack of immune cells on their own tissues, alleviating inflammation, and relieving the inflammatory symptoms of colitis.

[0175] 4.4.3 Effects of Three Kinds of Ganoderma lucidum Polysaccharides on Serum Biochemical Indexes

[0176] The level of lipopolysaccharide (LPS) in mice is an index reflecting intestinal permeability. Dextran sulfate sodium can damage the integrity of the intestinal mucosa by inhibiting the DNA synthesis of epithelial cells and interfering with cell proliferation, resulting in the migration of Gram-negative bacteria and their metabolites LPS in the intestinal tract to the lamina propria; at the same time, the expression of tight junction proteins (such as ZO-1) in the colon tissues of mice is down-regulated, the intestinal permeability is significantly increased, and LPS enters the systemic circulation through the portal vein system, causing the serum LPS level to rise accordingly. As Figure 16 , the serum LPS level of the N group of mice was significantly increased compared with that of the K group (p < 0.05). Compared with the N group of mice, both high and low doses of Ganoderma lucidum polysaccharides extracted by the three methods could reduce the serum LPS level of mice, but except for the Yg group, there was no significant difference in the serum LPS level between the other 5 groups (Lg, Ld, Yd, Wg, and Wd) and the N group of mice (p > 0.05). The high-dose Ganoderma lucidum polysaccharide extracted by free radical-DES (Yg group) could not only effectively reduce the serum LPS level of mice, but also the reduction effect was better than that of the K group (p < 0.05), indicating that the Ganoderma lucidum polysaccharide extracted by this method could effectively slow down the increase in colon permeability of mice. Since after serum LPS binds to Toll-like receptor 4 (TLR4), it can trigger the activation of the NF-κB signaling pathway, promote the release of pro-inflammatory factors such as TNF-α and IL-6, and exacerbate the systemic inflammatory response. The effect of the high-dose Ganoderma lucidum polysaccharide extracted by free radical-DES on the serum LPS level of mice was consistent with the results in 4.4.2.

[0177] Oxidative damage and oxidative stress levels are one of the important markers for evaluating inflammation. Glutathione peroxidase (GSH-Px), as an important antioxidant enzyme in the body, can catalyze glutathione (GSH) to reduce hydrogen peroxide (H2O2) to water, and at the same time reduce lipid peroxides to the corresponding alcohols, thereby scavenging reactive oxygen species in cells, protecting cells from oxidative stress, and inhibiting inflammation and oxidative stress-induced regulated cell death. As Figure 17, the GSH-Px activity in the serum of N group of mice was significantly lower than that in the K group (p<0.05). There was no significant difference in the effect of low-dose Ganoderma lucidum polysaccharides (Ld, Yd, and Wd) extracted by three methods on the GSH-Px activity in the serum of mice compared with that in the N group of mice (p>0.05); while there were significant differences in the effects of high-dose Ganoderma lucidum polysaccharides (Lg, Yg, and Wg) extracted by these three methods and the positive control (P group) on the GSH-Px activity in the serum of mice compared with that in the N group of mice (p<0.05). It indicated that all three Ganoderma lucidum polysaccharides could reduce the damage of oxidative stress to cells and tissues, resist oxidative damage, and the effect had a concentration-dependent relationship.

[0178] Superoxide dismutase (SOD) is an important antioxidant enzyme. During the inflammatory response, immune cells will release a large amount of superoxide anion free radicals, exacerbating inflammatory damage. SOD can reduce the inflammatory response, decrease the release of inflammatory factors, and relieve inflammatory symptoms. In addition, SOD can catalyze the conversion of superoxide anion free radicals (O2 - ) into oxygen and hydrogen peroxide, reduce the oxidative damage of free radicals to cells, delay aging and reduce the risk of diseases. SOD can also maintain the integrity of cell membranes, prevent lipid peroxidation damage; at the same time, protect mitochondrial function and reduce apoptosis induced by oxidative stress. It can be Figure 18 seen that the SOD activity in the serum of N group of mice was significantly lower than that in the K group (p<0.05). There was no significant difference in the effect of low-dose Ganoderma lucidum polysaccharides (Ld and Yd) on the SOD activity in the serum of mice compared with that in the N group of mice (p>0.05); while there were significant differences in the effects of high-dose Ganoderma lucidum polysaccharides (Lg and Yg) extracted by these two methods and the positive control (P group) on the SOD activity in the serum of mice compared with that in the N group of mice (p<0.05). It indicated that the effect of Ganoderma lucidum polysaccharides extracted by these two methods on the SOD activity in the serum of mice had a concentration-dependent relationship. Compared with the N group of mice, water-extracted Ganoderma lucidum polysaccharides (Wg and Wd) could significantly increase the SOD activity in the serum of mice (p<0.05), but there was no difference between the two doses, indicating that there was no concentration-dependent relationship in the effect on the SOD activity in the serum of mice. Among the Ganoderma lucidum polysaccharides extracted by three methods, the high-dose Ganoderma lucidum polysaccharide (Yg group) extracted by free radical-DES had the best effect, which could not only effectively reduce the LPS level in the serum of mice, but also the decreasing effect was better than that in the K group (p<0.05). LPS induces the generation of reactive oxygen species (ROS), reduces the activities of antioxidant enzymes (such as SOD, GSH-Px) in the liver and serum, resulting in oxidative damage. Figure 17 - 18 In line with Figure 16 the results, it indicated that Ganoderma lucidum polysaccharides (especially the high-dose Ganoderma lucidum polysaccharide extracted by free radical-DES) could effectively scavenge free radicals, reduce oxidative damage and inflammatory response.

[0179] Alanine aminotransferase (ALT) is mainly present in liver cells. An increase in ALT is mainly directly related to liver cell damage. Inflammatory factors reach the liver through the blood circulation, which may cause liver cell damage and increase ALT. From Figure 19 it can be seen that the serum ALT activity of the mice in group N was significantly increased compared with that in group K (p<0.05), indicating that the livers of the mice in the model control group were damaged to a certain extent. Compared with the mice in group N, the positive control (group P) and the high- and low-dose Ganoderma lucidum polysaccharides extracted by the three methods could significantly reduce the serum ALT content of the mice (p<0.05), indicating that the Ganoderma lucidum polysaccharides extracted by the three methods could effectively reduce the degree of liver cell damage, reduce the release of ALT in liver cells into the blood, thereby reducing the ALT content in the blood and maintaining the integrity and normal function of liver cells. Intestinal inflammation causes lipopolysaccharide (LPS) to enter the blood and activate the liver immune response, which can indirectly aggravate liver cell damage and increase ALT; excessive production of reactive oxygen species (ROS) can damage the mitochondrial membrane potential of liver cells, resulting in the release of ALT. The effect of Ganoderma lucidum polysaccharide (especially the high-dose Ganoderma lucidum polysaccharide extracted by free radical-DES) on the ALT content was consistent with the effects of LSP, GSH-Px and SOD, indicating that the Ganoderma lucidum polysaccharide extracted by free radical-DES could effectively slow down the intestinal permeability of mice, protect cells from oxidative damage, relieve inflammation, and then protect the normal physiological function of cells.

[0180] Example 2: Optimal extraction process

[0181] Based on the optimal extraction process parameters optimized by the free radical-DES co-extraction of Ganoderma lucidum polysaccharide in Example 1, the Ganoderma lucidum fruiting bodies dried at 50°C for 12 h were pulverized in a pulverizer to obtain Ganoderma lucidum powder (unsifted). Weigh 1.0 g of Ganoderma lucidum powder and place it in a conical flask, add hydrogen peroxide with a concentration of 1.7% (the solid-liquid ratio is 1 g:110 mL, that is, take 6.23 mL of hydrogen peroxide and add 103.8 mL of deionized water to make 110 mL of 1.7% hydrogen peroxide solution), mix well, and extract in a water bath shaker at 100°C for 145 min. Then add 50 mL of DES extractant with a water content of 0% and a molar ratio of urea:choline chloride = 2:1, extract in a water bath shaker at 90°C for 20 min, centrifuge at 8000 rpm for 15 min, and 0.356 g of Ganoderma lucidum polysaccharide is contained in the supernatant. The supernatant was rotary evaporated and concentrated to one-fifth of the original volume to obtain a concentrated solution. Add 4 times the volume of 95% ethanol to the concentrated solution, let it stand at 4°C for 12 h, centrifuge at 1000 rpm for 5 min, take the precipitate, add an appropriate amount of water, and rotary evaporate and concentrate at 65°C until the ethanol is completely evaporated to obtain a concentrated solution. Add Sevag reagent (chloroform:n-butanol = 4:1) in a volume ratio of 1:5, shake vigorously for 20 min, centrifuge at 3000 r / min for 10 min, discard the protein layer, and repeat 5 times. The aqueous layer solution was concentrated to 1 / 3 of the original volume to obtain 0.178 g of deproteinized Ganoderma lucidum polysaccharide co-extracted by free radical-DES.

[0182] Using the detection method in 3.2 of Example 1, before alcohol precipitation for protein removal, the average yield of Ganoderma lucidum polysaccharide in the extracted supernatant was 35.60%; using the detection method in 3.6 of Example 1, after alcohol precipitation for protein removal, Ganoderma lucidum polysaccharide caused less damage to the colon tissue and liver tissue of mice induced by dextran sulfate sodium, the reduction of goblet cells and crypt numbers was not obvious, and the degree of inflammatory cell infiltration was light; and it could significantly reduce the levels of TNF-α and IL-6 in the colon tissue of mice, enhance the activities of serum GSH-Px and SOD, and reduce the levels of serum ALT and LPS, with significant differences compared with the negative control group (p < 0.05).

[0183] Comparative Example 1

[0184] 1.0 g of Ganoderma lucidum fruit body powder was added with 110 mL of deionized water according to the solid-liquid ratio of 1 g:110 mL, extracted in a water bath at 100 °C for 145 min, then centrifuged (8000 rpm, 20 min), and the supernatant was taken to detect the polysaccharide concentration. The polysaccharide detection method was the same as that in Example 1, and the results are shown in Table 6.

[0185] Table 6 Yield indexes of Ganoderma lucidum fruit body polysaccharide extracted by different methods

[0186]

[0187] As can be seen from Table 6, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the polysaccharide extraction yield. The synergistic extraction of free radical-DES can increase the extraction yield of Ganoderma lucidum polysaccharide by 14.41 times compared with the Ganoderma lucidum polysaccharide extracted by traditional hot water extraction.

[0188] Comparative Example 2

[0189] 1.0 g of Ganoderma lucidum fruit body powder was taken and added with 135 mL of deionized water, and extracted in a microwave chemical reactor at a power of 600 W and a temperature of 70 °C for 105 min. Centrifuged at 8000 rpm for 20 min, and the supernatant was taken to detect the polysaccharide concentration. The polysaccharide detection method was the same as that in Example 1, and the results are shown in Table 7.

[0190] Table 7 Yield indexes of Ganoderma lucidum fruit body polysaccharide extracted by different methods

[0191]

[0192] As can be seen from Table 7, the extraction process of Ganoderma lucidum polysaccharide has a relatively large influence on the polysaccharide extraction yield. The synergistic extraction of free radical-DES can significantly increase the extraction yield of Ganoderma lucidum polysaccharide, which is 9.75 times higher than that of Ganoderma lucidum polysaccharide extracted by microwave-assisted extraction.

[0193] Comparative Example 3

[0194] Take 1.0 g of Ganoderma lucidum fruit body powder, add 103.8 mL of deionized water, then add 6.23 mL of 30% hydrogen peroxide and mix well. Use a water bath shaker to extract at 100 °C for 145 min. Centrifuge at 8000 rpm for 20 min to obtain the Ganoderma lucidum polysaccharide solution. The polysaccharide detection method is the same as in Example 1, and the results are shown in Table 8.

[0195] Table 8 Yield indexes of Ganoderma lucidum fruit body polysaccharides extracted by different methods

[0196]

[0197] As can be seen from Table 8, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the extraction yield of polysaccharide. The synergistic extraction of free radical-DES can significantly improve the extraction yield of Ganoderma lucidum polysaccharide, which is 4.06 times higher than that of Ganoderma lucidum polysaccharide extracted by hydrogen peroxide.

[0198] Comparative Example 4

[0199] Take 1.0 g of Ganoderma lucidum fruit body powder, add 0.015 g of cellulase (activity 10,000 U / g, purchased from a certain biochemical technology company in Shanghai), add 110 mL of deionized water and mix well. Adjust the pH to 5.6 with hydrochloric acid. Use a water bath shaker to extract at 55 °C for 145 min. Centrifuge at 8000 rpm for 20 min to obtain the Ganoderma lucidum polysaccharide solution. The polysaccharide detection method is the same as in Example 1, and the results are shown in Table 9.

[0200] Table 9 Yield indexes of Ganoderma lucidum fruit body polysaccharides extracted by different methods

[0201]

[0202] As can be seen from Table 9, the extraction process of Ganoderma lucidum polysaccharide has a great influence on the extraction yield of polysaccharide. The synergistic extraction of free radical-DES can significantly improve the extraction yield of Ganoderma lucidum polysaccharide, which is 4.98 times higher than that of Ganoderma lucidum polysaccharide extracted by the enzymatic method.

[0203] Example 3

[0204] Accurately weigh 1.0 g of Ganoderma lucidum fruit body powder, add hydrogen peroxide with a concentration of 1.4% (the material-liquid ratio is 1 g:90 mL, that is, take 4.2 mL of hydrogen peroxide and add 85.8 mL of deionized water to make 90 mL of 1.4% hydrogen peroxide solution), mix well, and extract in a water bath shaker at 100 °C for 145 min. Then add 50 mL of DES extractant with a water content of 0% and a molar ratio of urea:choline chloride = 2:1, extract in a water bath shaker at 100 °C for 30 min, and centrifuge at 8000 rpm for 15 min. The supernatant contains 0.313 g of Ganoderma lucidum polysaccharide. The average extraction yield of Ganoderma lucidum polysaccharide is 31.3%.

[0205] Example 4

[0206] Accurately weigh 1.0 g of Ganoderma lucidum fruit body powder, add hydrogen peroxide with a concentration of 1.8% (the material-liquid ratio is 1 g: 110 mL, that is, take 6.6 mL of hydrogen peroxide and add 103.4 mL of deionized water to make 110 mL of 1.8% hydrogen peroxide solution). After mixing evenly, extract in a water bath shaker at 90 °C for 120 min. Then add 30 mL of DES extractant with a water content of 0% and a molar ratio of urea: choline chloride = 2: 1. After extracting in a water bath shaker at 90 °C for 40 min, centrifuge at 8000 rpm for 15 min. The supernatant contains 0.307 g of Ganoderma lucidum polysaccharide. The average yield of Ganoderma lucidum polysaccharide is 30.7%.

[0207] Example 5

[0208] Accurately weigh 1.0 g of Ganoderma lucidum fruit body powder, add hydrogen peroxide with a concentration of 1.7% (the material-liquid ratio is 1 g: 90 mL, that is, take 5.1 mL of hydrogen peroxide and add 84.9 mL of deionized water to make 90 mL of 1.7% hydrogen peroxide solution). After mixing evenly, extract in a water bath shaker at 100 °C for 160 min. Then add 30 mL of DES extractant with a water content of 0% and a molar ratio of urea: choline chloride = 2: 1. After extracting in a water bath shaker at 90 °C for 20 min, centrifuge at 8000 rpm for 15 min. The supernatant contains 0.327 g of Ganoderma lucidum polysaccharide. The average yield of Ganoderma lucidum polysaccharide is 32.7%.

[0209] Example 6

[0210] Accurately weigh 1.0 g of Ganoderma lucidum fruit body powder, add hydrogen peroxide with a concentration of 1.8% (the material-liquid ratio is 1 g: 100 mL, that is, take 6 mL of hydrogen peroxide and add 94 mL of deionized water to make 100 mL of 1.8% hydrogen peroxide solution). After mixing evenly, extract in a water bath shaker at 100 °C for 145 min. Then add 40 mL of DES extractant with a water content of 0% and a molar ratio of urea: choline chloride = 2: 1. After extracting in a water bath shaker at 100 °C for 30 min, centrifuge at 8000 rpm for 15 min. The supernatant contains 0.322 g of Ganoderma lucidum polysaccharide. The average yield of Ganoderma lucidum polysaccharide is 32.2%.

[0211] The above embodiments are illustrative of the present invention, not restrictive of the present invention. Any simple transformation of the present invention belongs to the protection scope of the present invention.

Claims

1. A method for efficiently extracting ganoderma polysaccharide, characterized in that: It includes the following steps: (1) Ganoderma lucidum powder preparation: Dry Ganoderma lucidum slices and then crush them to obtain Ganoderma lucidum powder, which is packed and stored in a glass desiccator; (2) Free radical pretreatment stage: Mix Ganoderma lucidum powder with hydrogen peroxide solution and treat it at 80 - 100 °C for 60 - 200 min. The final concentration of hydrogen peroxide is 0.8 - 2.4% (mass fraction), and the liquid-to-solid ratio is 50 - 120 mL / g. Among them, the optimal parameters for this stage are: the final concentration of hydrogen peroxide is 1.7%, the liquid-to-solid ratio is 110:1 mL / g, the treatment time is 145 min, and the temperature is 100 °C; (3) Preparation of deep eutectic solvent (DES); (4) Free radical-DES synergistic extraction stage: Hydrate DES (water content 0 - 40%), add hydrated DES to the mixture of Ganoderma lucidum powder and hydrogen peroxide solution, and extract at 50 - 100 °C for 20 - 70 min. The DES liquid-to-solid ratio is 20 - 90 mL / g. Among them, the preferred DES is urea-choline chloride DES without water (molar ratio 2:1). The optimal parameters for this stage are: DES liquid-to-solid ratio 50 mL / g, water content 0%, DES treatment temperature 90 °C, and DES treatment time 20 min; (5) Post-treatment stage: After centrifugation, high-purity Ganoderma lucidum polysaccharide is obtained through ethanol precipitation and protein removal by the Sevag method.

2. The high-efficiency extraction method of ganoderma polysaccharide according to claim 1, characterized in that: The preparation of the deep eutectic solvent (DES) is specifically as follows: Select choline chloride as the hydrogen bond acceptor, mix it with the hydrogen bond donor, and magnetically stir at a constant temperature of 75–85 °C for 2 hours until the solution is clear; the hydrogen bond donors include ethylene glycol, 1,4-butanediol, urea, triethylene glycol, and glycerol. The 1,4-butanediol is 1,4-butanediol. The molar ratios of each hydrogen bond donor to the hydrogen bond acceptor are respectively: Ethylene glycol: Choline chloride = 2:1; 1,4-Butanediol: Choline chloride = 1:2; Triethylene glycol: Choline chloride = 4:1; Glycerol: Choline chloride = 2:1; Urea: Choline chloride = 2:

1.

3. The highly efficient extraction method of ganoderma lucidum polysaccharide according to claim 1, characterized in that: The process was optimized by Box-Behnken Design. Based on the parameters of the extraction method in Claim 1, a quadratic regression model was established with the polysaccharide yield as the response value: Y = 35.6 + 1.30833A - 0.166667B + 1.49167C + 0.8D - 0.775AB - 1.675AC + 2.325AD + 0.6BC + 0.925BD + 1.75CD - 1.34583 A 2 - 2.23333B 2 - 2.97083C 2 - 2.03333D 2 , where A is the solid-liquid ratio (g / mL), B is the hydrogen peroxide concentration (%), C is the hydrogen peroxide treatment time (min), and D is the DES addition amount (mL).

4. A method for efficiently extracting ganoderma polysaccharide according to any one of claims 1 to 3, characterized in that: Under the optimal parameter conditions of the free radical pretreatment stage and the free radical-DES synergistic extraction stage in claim 1, the extraction yield of the Ganoderma lucidum polysaccharide is 30.7% - 35.6%; the free radical-DES synergistic extraction makes the cell wall show deep grooves, split layers, and porous structures.

5. The Ganoderma lucidum polysaccharide extracted by the method for efficiently extracting Ganoderma lucidum polysaccharide according to any one of claims 1 to 3, characterized in that: The Ganoderma lucidum polysaccharide has biological activities such as anti-inflammatory, antioxidant, and liver protection. Among them, the antioxidant effect can enhance the activity of antioxidant-related enzymes, reduce oxidative stress damage, and then synergistically play a role with anti-inflammatory and liver protection biological activities.

6. Use of Ganoderma lucidum polysaccharide extracted by the method for highly efficient extraction of Ganoderma lucidum polysaccharide according to any one of claims 1 to 3, characterized in that: The Ganoderma lucidum polysaccharide is used for the prevention of inflammatory bowel disease (IBD), and it plays a role by reducing the levels of TNF-α and IL-6, and at the same time enhancing the activity of antioxidant-related enzymes and reducing oxidative stress damage to play a preventive role.

7. Use of Ganoderma lucidum polysaccharide extracted by the method for highly efficient extraction of Ganoderma lucidum polysaccharide according to any one of claims 1 to 3, characterized in that: The Ganoderma lucidum polysaccharide is used for the preparation of an antioxidant, which can significantly increase the activity of serum GSH-Px (p < 0.05) and reduce oxidative stress damage.

8. Use of Ganoderma lucidum polysaccharide extracted by the method for efficiently extracting Ganoderma lucidum polysaccharide according to any one of claims 1 to 3, characterized in that: The Ganoderma lucidum polysaccharide is used for liver protection, which can significantly reduce the activity of serum ALT (p < 0.05) and reduce liver cell damage.

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