Ganoderma lucidum polysaccharide extracted by three-phase method and application of ganoderma lucidum polysaccharide in intestinal inflammation treatment

Abstract

The invention discloses a method for extracting ganoderan through a three-phase method and application of the ganoderan in intestinal inflammation treatment, and the method comprises the following steps: drying, crushing and sieving ganoderma lucidum sporocarp, mixing the ganoderma lucidum sporocarp with hydrogen peroxide, ammonium sulfate and nonanoic acid-lauric acid (molar ratio is 2: 1-4: 1), extracting for 30-50 minutes at 70-90 DEG C under the microwave of 300-700 W, centrifuging, precipitating with ethanol, dissolving the precipitate in deionized water, and drying to obtain the ganoderan. The extraction yield under the optimal parameters reaches 12.05% + / -0.12%, and extra decoloration, deproteinization and impurity removal are not needed. The ganoderan can repair intestinal mucosa, reduce proinflammatory factors and improve intestinal flora and is used for treating intestinal inflammation such as ulcerative colitis, and the effective dose of the ganoderan is 100-200 mg / kg / d.

Description

Technical Field

[0001] The present invention relates to the technical field of biological extraction, and particularly to the extraction of ganoderma polysaccharide by a three-phase method and its application in the treatment of intestinal inflammation.

Background Art

[0002] Ganoderma lucidum belongs to the Basidiomycotina, Hymenomycetes, Ganodermatales, Ganodermataceae, Ganoderma in the fungal phylum, and is commonly known as single ganoderma, densely striated ganoderma, or old wood fungus, etc. As early as in "Shennong Ben Cao Jing", according to color and morphological characteristics, Ganoderma lucidum was divided into six types: red ganoderma, black ganoderma, blue ganoderma, white ganoderma, yellow ganoderma, and purple ganoderma, and it was regarded as a good medicine for regulating the human immune function and delaying aging. Archaeological discoveries show that in the early Neolithic period, the ancestors had already begun to collect and utilize Ganoderma lucidum. With the enhancement of people's awareness of health preservation and the revival of traditional Chinese medicine culture, the market demand for Ganoderma lucidum continues to grow. At present, Ganoderma lucidum is widely distributed in Europe, North America, and the Asia-Pacific region. It often grows in broad-leaved forests with scattered light, prefers an acidic environment, and also has certain requirements for humidity and light conditions. Ganoderma lucidum can be used both as a traditional Chinese medicine and processed into various foods. It is an extremely precious resource and has the largest global market share. Many effects of Ganoderma lucidum mainly stem from ganoderma polysaccharide. However, since most ganoderma polysaccharides exist in the cell wall and there are limitations in polysaccharide extraction technology, its individual pharmacological effects have not been deeply studied. If the extraction technology can be improved and its pharmacological effects can be deeply explored, the utilization value of Ganoderma lucidum will be greatly improved.

[0003] Ganoderma polysaccharide mainly exists in the cell wall of Ganoderma lucidum fruiting bodies. The cell wall of Ganoderma lucidum is a porous polymer complex located on the surface of the cytoplasmic membrane, composed of chitin microfibers, linear glucose polymers (glucans) and other compressive substances, as well as various cell wall proteins. The wall layer is thick and tough, so it is difficult to fully extract intracellular polysaccharides by traditional wall-breaking methods. Research has confirmed that ganoderma polysaccharide has the ability to resist oxidation and scavenge free radicals, which is of great significance in preventing diseases caused by oxidative stress and also has great application prospects in the fields of improving immunity, anti-cancer, and hypoglycemic. However, due to the limitations of polysaccharide extraction technology, the pharmacological effects of ganoderma polysaccharide have not been fully studied. Therefore, how to develop an efficient wall-breaking technology to extract ganoderma polysaccharide and what potential pharmacological effects ganoderma polysaccharide has are urgent to be studied and discovered.

[0004] The extraction rate of the traditional water extraction method (such as Comparative Example 1) is only 2.57%, and additional ethanol precipitation, activated carbon decolorization, and Sevag reagent protein removal steps are required (the steps are cumbersome); the extraction rate of the microwave-assisted method (Comparative Example 2) is 3.14%, and there are still problems such as low polysaccharide purity and activity damage. In the prior art, there is a lack of an efficient and synergistic integrated method for wall-breaking and impurity removal for the difficult dissolution of intracellular polysaccharides caused by the toughness of the Ganoderma lucidum cell wall.

[0005] To fully explore the potential value of Ganoderma lucidum polysaccharides, promote the development of the deep processing industry of Ganoderma lucidum, and develop relevant therapeutic strategies and functional foods, the present invention uses a microwave-assisted hydrogen peroxide-ammonium sulfate-medium-chain fatty acid (HP-SA-MCFA) three-phase extraction method to co-extract Ganoderma lucidum polysaccharides. The protective effects of the Ganoderma lucidum polysaccharides prepared by this method on memory and cognitive impairment, thinning of the intestinal mucosal layer, infiltration of inflammatory cells in the liver and intestine, mucosal gland damage, and intestinal flora disorder caused by inflammation in ICR mice are expected to provide technical support for the large-scale production and application of Ganoderma lucidum polysaccharides, and further enhance the comprehensive utilization value of Ganoderma lucidum resources.

[0006] Existing three-phase methods mostly rely on organic solvents or complex systems (such as the ternary system in Comparative Example 3), and there are problems such as high cost, high toxicity, or low extraction yield (7.31%). However, the present invention uses a low-toxic and low-cost medium-chain fatty acid (Medium-Chain Fatty Acids, MCFA) (selecting nonanoic acid and lauric acid) system, and the extraction yield is increased to 12.05%.

[0007] Therefore, there is an urgent need for an extraction method that combines efficient cell wall breaking, simultaneous impurity removal, and activity preservation to solve the problems of low extraction yield, complex purification, and impaired biological activity in the prior art.

Summary of the Invention

[0008] The object of the present invention is to solve the problems of low extraction yield of Ganoderma lucidum polysaccharides, cumbersome purification steps with residual impurities, insufficient retention of biological activity, and limited pharmacological effect research in the prior art, and provide a three-phase method for extracting Ganoderma lucidum polysaccharides and its application in the treatment of intestinal inflammation. Using Ganoderma lucidum fruiting bodies as raw materials, on the basis of drying and pulverizing pretreatment, with the help of microwave-assisted technology, using the HP-SA-MCFA system as a solvent, Ganoderma lucidum polysaccharides are effectively extracted, further improving its extraction yield (the extraction yield reaches 12.05% ± 0.12%) and biological activities such as alleviating inflammation, stress and cognitive impairment, and improving intestinal flora disorder.

[0009] To achieve the above object, the present invention proposes a three-phase method for extracting Ganoderma lucidum polysaccharides and its application in the treatment of intestinal inflammation. The Ganoderma lucidum polysaccharides are extracted according to the following steps:

[0010] (1) Dry the Ganoderma lucidum slices until the water content is below 8%, pulverize them into powder, and sieve them through different meshes to obtain Ganoderma lucidum powder;

[0011] (2) Take Ganoderma lucidum powder of different mesh sizes (60 - 80 mesh, 80 - 100 mesh, 100 - 150 mesh, <150 mesh) (preferably passing through 150 mesh); perform three-phase extraction, add 2 - 4 g of ammonium sulfate (preferably 3.5 g), at a hydrogen peroxide concentration of 1.2% - 2.7% (preferably 1.9%), add 10 - 20 mL (preferably 16 mL) of medium-chain fatty acid (MCFA, preferably nonanoic acid and lauric acid) extractant, under the condition of a microwave power of 400 - 600 W (watts), at a temperature of 70 - 90 °C (preferably 90 °C), and process for 30 - 50 min (preferably 49 min);

[0012] (3) Centrifuge the solution processed in step (2) (8000 rpm, 20 min), take 100 mL of the Ganoderma lucidum polysaccharide supernatant into a centrifuge tube, add 95% ethanol at a ratio of 1:4 (V / V), let it stand at 4 °C for 12 h, and then centrifuge at 4000 rpm for 10 min. Take the precipitate in the centrifuge tube, dissolve it in deionized water, after concentration, add 25 mL of Sevag reagent (chloroform - n-butanol, 4:1, V / V), mix well, oscillate for 20 - 30 min, then centrifuge at 4000 r / min for 5 min in a centrifuge to remove the precipitate, retain the supernatant, and repeat the above method about 3 times. Collect the supernatant, remove the organic solvent by rotary evaporation to obtain Ganoderma lucidum polysaccharide.

[0013] In the three-phase extraction system, nonanoic acid and lauric acid remove weakly polar components by destroying the lipid structure of the cell wall, ammonium sulfate (SA) precipitates proteins through salting-out, and microwave assistance promotes the generation of hydroxyl radicals. The three work together to achieve the integration of 'extraction - impurity removal', without the ethanol precipitation and activated carbon decolorization steps in the traditional process.

[0014] The high-frequency vibration generated by microwave assistance promotes the decomposition of hydrogen peroxide into hydroxyl radicals (·OH), which destroys the chitin microfibrils and glucan network of the Ganoderma lucidum cell wall. Medium-chain fatty acid (MCFA) dissolves the membrane lipids, reducing cell permeability. Ammonium sulfate precipitates proteins through salting-out effect. The three work together to increase the polysaccharide dissolution rate by 4.69 times (compared with the traditional hot water method), without additional decolorization steps.

[0015] Furthermore, the Ganoderma lucidum powder in step (1) is prepared as follows: After slicing the Ganoderma lucidum, place it in an oven, the drying temperature is 90 °C, the drying time is 12 hours, after drying, put it into a pulverizer to crush, and pass through a 150-mesh sieve to obtain Ganoderma lucidum powder.

[0016] Furthermore, the microwave conditions in step (2) are: power 400 - 600 W (preferably 600 W).

[0017] Furthermore, the volume dosage of the extractant in step (2) is 80 - 110 mL / g (preferably 90 mL / g) based on the mass of the Ganoderma lucidum powder in step (1).

[0018] Furthermore, in step (2), the chain fatty acids are a mixture of nonanoic acid and lauric acid in a molar ratio of 2:1 - 4:1. Through experimental screening (Table 4), the extraction yield (12.05%) of this combination is significantly higher than that of other MCFA combinations (such as octanoic acid - decanoic acid 10.318%, octanoic acid - lauric acid 6.094%), and the yield is the highest at a molar ratio of 3:1.

[0019] The nonanoic acid and lauric acid described in the present invention can be obtained through regular commercial purchase.

[0020] The Ganoderma lucidum polysaccharide of the present invention can effectively alleviate symptoms such as intestinal mucosal injury and atrophy, memory and cognitive impairment, hepatic and intestinal inflammatory cell infiltration, and intestinal flora disorder induced by dextran sulfate sodium (DSS) after intervention, and significantly reduce the levels of pro - inflammatory factors such as tumor necrosis factor - α (TNF - α) and interleukin - 6 (IL - 6) (p < 0.05); enhance the activity of antioxidant enzymes such as superoxide dismutase (SOD) (p < 0.05), reduce the level of endotoxin lipopolysaccharide (LPS) and the activity of alanine aminotransferase (ALT / GPT) (p < 0.05).

[0021] The present invention uses Ganoderma lucidum fruit bodies as raw materials. After drying and pulverizing, Ganoderma lucidum polysaccharide is extracted through a microwave - assisted three - phase extraction process. The technical key point of the present invention is that by using the microwave - assisted HP - SA - MCFA three - phase extraction method, under the optimal extraction parameters optimized by single - factor experiments and Box - Behnken test design, the polysaccharide in Ganoderma lucidum fruit bodies can be effectively dissolved and its biological activity can be maintained.

[0022] The application of the Ganoderma lucidum polysaccharide extracted by the method of the present invention in the preparation of drugs for treating intestinal inflammation is characterized in that the Ganoderma lucidum polysaccharide can:

[0023] (a) Repair the damage of the intestinal mucosal layer and inhibit inflammatory cell infiltration;

[0024] (b) Reduce the levels of pro - inflammatory factors tumor necrosis factor - α (TNF - α) and interleukin - 6 (IL - 6), and enhance the activity of antioxidant enzyme superoxide dismutase (SOD);

[0025] (c) Improve intestinal flora disorder, reduce the levels of serum endotoxin lipopolysaccharide (LPS) and liver injury marker alanine aminotransferase (ALT / GPT), have a dose - dependent anti - inflammatory effect, and have no obvious toxic reaction.

[0026] Furthermore, the intestinal inflammation includes ulcerative colitis and inflammatory bowel disease, and the drug is administered by gavage or orally, and the effective dose is 100 - 200 mg / kg / d.

[0027] Furthermore, the drug can also reduce dextran sulfate sodium (DSS)-induced hepatic and intestinal inflammatory cell infiltration, decrease the activity of serum alanine aminotransferase ALT / GPT, significantly improve DSS-induced memory impairment, and the effect is better than that of the positive control drug mesalazine (p<0.05).

[0028] Furthermore, the protein residue in the extract after the three-phase extraction is ≤0.1%.

[0029] Advantages of the present invention:

[0030] 1. High extraction efficiency and few impurities. The extraction yield of the traditional water extraction method (such as Comparative Example 1) is only 2.57%, and that of the microwave-assisted method (Comparative Example 2) is 3.14%. The extraction yield of the method of the present invention can reach 12.05%. Under the action of microwave, an electromagnetic wave that makes molecules vibrate, HP can generate a hydroxyl free radical, a strong oxidant. This free radical can destroy the tough and thick structure of the Ganoderma lucidum cell wall, increase cell permeability, and thus promote the release of polysaccharides. The advantage of the microwave-assisted HP-SA-MCFA three-phase extraction method is that it can maximize the efficiency of extraction and impurity removal in one step. By medium-chain fatty acids (MCFAs) (preferably nonanoic acid and lauric acid), the weakly polar and non-polar small molecule components in the Ganoderma lucidum fruiting body are removed, and by ammonium sulfate (SA), the proteins in the Ganoderma lucidum fruiting body are removed. The subsequent operations of removing weakly polar and non-polar small molecule components by ethanol precipitation, decolorization with activated carbon, and removing macromolecular proteins with Sevag reagent (chloroform-butanol, 4:1, V / V) are omitted. The traditional water extraction method is prone to dissolve impurities such as proteins and pigments, and additional steps (such as alcohol precipitation to remove small molecule impurities and Sevag method to remove proteins) are required for impurity removal and decolorization. However, the HP-SA-MCFA three-phase method can reduce the subsequent purification difficulty and achieve simple operation.

[0031] 2. Nonanoic acid and lauric acid are formulated in a molar ratio of 3:1, which has the advantages of low cost, low toxicity, easy preparation, etc. After extraction, it will not damage the activity of Ganoderma lucidum polysaccharides, enabling the extract to maintain good biological activity and other properties, and improving the purity of Ganoderma lucidum polysaccharides.

[0032] 3. The Ganoderma lucidum fruit body polysaccharide product extracted by microwave-assisted HP-SA-MCFA three-phase extraction method has no peculiar smell and good sensory properties. The extraction yield of Ganoderma lucidum polysaccharide is increased by 4.69 times compared with the traditional hot water method, 3.84 times compared with the microwave-assisted method, 1.65 times compared with the ternary system method, 1.95 times compared with the HP method, and 9.34 times compared with the MCFA system method. Compared with the negative control group, the increase in the DAI score of the mice in the Ganoderma lucidum polysaccharide intervention group was slow. Both the high-dose and low-dose groups of Ganoderma lucidum polysaccharide intervention could significantly reduce the pole-climbing time and maze time of the mice (p<0.05), and significantly increase the colon length of the mice (p<0.05); slow down the severe damage to the colonic mucosal layer and severe infiltration of inflammatory cells caused by DSS in the mice; effectively reduce liver cell damage and maintain the health of the liver tissue; significantly reduce the levels of TNF-α and IL-6 in the mice (p<0.05), significantly enhance the serum SOD activity (p<0.05), significantly reduce the serum LPS level (p<0.05), and significantly reduce the serum ALT / GPT activity (p<0.05).

[0033] 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

[0034] Figure 1 is the glucose standard curve graph of the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0035] Figure 2 is the graph group of the influence of different single factors on the extraction yield of Ganoderma lucidum polysaccharide by the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0036] Figure 3 is the response surface and contour graph of the influence of microwave-assisted HP-SA-MCFA three-phase extraction method on the extraction yield of Ganoderma lucidum polysaccharide by the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0037] Figure 4 is the graph of the influence of different doses of Ganoderma lucidum polysaccharide on the DAI score of mice by the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0038] Figure 5 is the graph group of the influence of different doses of Ganoderma lucidum polysaccharide on the pole-climbing test, maze test, and hanging test of mice by the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0039] Figure 6 is the graph of the influence of different doses of Ganoderma lucidum polysaccharide on the colon length of mice by the three-phase method for extracting Ganoderma lucidum polysaccharide of the present invention and its application in the treatment of intestinal inflammation;

[0040] Figure 7 This is a group of physical pictures showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the colon length of mice;

[0041] Figure 8 This is a HE staining section diagram showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the colon of mice;

[0042] Figure 9 This is a HE staining section (magnification: upper ×10, lower ×60) showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the liver of mice;

[0043] Figure 10 This is a group of diagrams showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the levels of TNF-α and IL-6 in the colon tissue of mice;

[0044] Figure 11 This is a diagram showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the SOD activity in the serum of mice;

[0045] Figure 12 This is a diagram showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the LPS content in the serum of mice;

[0046] Figure 13 This is a diagram showing the effects of different doses of Ganoderma lucidum polysaccharide extracted by the three-phase method of the present invention and its application in the treatment of intestinal inflammation on the ALT / GPT activity in the serum of mice.

[0047] Figure 7 Among them: from left to right are: blank control, negative control, positive control, high-dose Ganoderma lucidum polysaccharide, and low-dose Ganoderma lucidum polysaccharide.

Specific Embodiments

[0048] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0049] Example 1

[0050] 1 Materials and Reagents

[0051] Ganoderma lucidum (provided by a certain company in Zhejiang), free of pests, diseases, and rot;

[0052] Hydrogen peroxide, nonanoic acid, lauric acid, ammonium sulfate, phenol, concentrated sulfuric acid, glucose, dextran sulfate sodium, physiological saline, mesalazine, etc. are all of analytical grade;

[0053] The total superoxide dismutase (T-SOD) assay kit and alanine aminotransferase (ALT / GPT) assay kit were purchased from a research institute in Nanjing. The mouse lipopolysaccharide (LPS) enzyme-linked immunosorbent assay kit, mouse tumor necrosis factor-α (TNF-α) assay kit, and interleukin-6 (IL-6) assay kit were purchased from a company in Shanghai.

[0054] 2 Experimental instruments

[0055] Electronic balance (a scientific instrument company in Shanghai), microwave synthesis reactor XH-MC-1 (a technology development company in Beijing), bench-top high-speed centrifuge TG-16-WS (a company in Hunan), ultraviolet-visible spectrophotometer (a scientific instrument company in Shanghai), rotary evaporator RE-52 (an instrument factory in Shanghai), BIO-RAD iMark microplate reader (a medical product company), HH-S digital display constant temperature water bath (an instrument factory in Jintan).

[0056] 3 Experimental methods

[0057] 3.1 Pretreatment of Ganoderma lucidum powder

[0058] Place 100 g of Ganoderma lucidum slices in a hot air drying oven for drying. Set the oven temperature at 60 °C and the drying time at 12 hours. After drying the Ganoderma lucidum slices, crush them with a high-speed grinder and then sieve them through different mesh sizes to obtain Ganoderma lucidum powder with different particle sizes (>40 mesh, 40 - 60 mesh, 60 - 80 mesh, 80 - 100 mesh, 100 - 150 mesh, <150 mesh). Store them in a glass desiccator for subsequent use. 3.2 Establishment of quantitative analysis method for Ganoderma lucidum polysaccharide

[0059] Adopt the phenol-sulfuric acid method. Prepare the standard curve: Weigh 100 mg of glucose dried to constant weight and dissolve it in deionized water, then make up the volume to 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. Take 5 test tubes and add 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of glucose standard solution respectively, then add 0.5, 0.4, 0.3, 0.2, 0.1, and 0 mL of deionized water respectively. Then add 0.5 mL of 5% phenol and 2.5 mL of sulfuric acid to each tube. After vortex mixing, let it stand at room temperature for 10 min, then place it in a 30 °C water bath for 20 min. Take an appropriate amount of the reaction solution and measure the absorbance at 490 nm. The test tube with 0.5 mL of deionized water is used as the blank control.

[0060] Detection of polysaccharide content in samples: Accurately measure a certain amount of ganoderma polysaccharide solution (try to make the absorbance value fall between 0.2 and 0.8), add reagents according to the dosage for making the standard curve, use a visible spectrophotometer to measure the absorbance at 490 nm, calculate the total sugar content according to the standard curve regression equation, and calculate the extraction yield of ganoderma polysaccharide in the sample according to the following formula.

[0061] Polysaccharide yield (%) = [c * V * F * 10 -6 / M] * 100.

[0062] In the formula, c—the concentration of the polysaccharide sample to be measured obtained according to the standard equation (μg / mL); V—the volume of the supernatant of the polysaccharide sample extract (mL); F—dilution factor; M—the mass of ganoderma fruiting body powder (g).

[0063] 3.3 Screening of medium-chain fatty acid (MCFA) solvents

[0064] Accurately weigh 1.00 g of ganoderma sample and 3.00 g of ammonium sulfate, prepare MCFA solution according to Table 1, add MCFA according to 1:15 (g / mL), add hydrogen peroxide solution with a concentration of 2.1% according to 1:90 (g / mL), extract with a microwave synthesis reactor, extract for 30 min under the conditions of microwave power 600 W and 90 °C, then centrifuge for 20 min at 8000 r / min, record the volume of the supernatant, take 1 mL of the supernatant, and detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method and convert it into the yield.

[0065] Table 1 Composition and molar ratio of MCFA

[0066]

[0067] 3.4 Ganoderma polysaccharide extraction process

[0068] 3.4.1 Microwave-assisted HP-SA-MCFA three-phase extraction method for synergistically optimizing the extraction process of ganoderma polysaccharide

[0069] (1) Single-factor experiment

[0070] Effect of A raw material particle size on polysaccharide extraction yield. Weigh 1.00 g of ganoderma powder with different particle sizes (>40 mesh, 40 - 60 mesh, 60 - 80 mesh, 80 - 100 mesh, 100 - 150 mesh, and <150 mesh), add 3.00 g of ammonium sulfate, mix evenly with 2.4% hydrogen peroxide solution according to the solid-liquid ratio of 1:90 (g / mL), then add 15 mL of MCFA screened in method 3.3, mix well, extract at microwave power 600 W and temperature 90 °C for 40 min, centrifuge for 20 min (8000 rpm) after extraction, 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.

[0071] Effect of ammonium sulfate mass on the extraction yield of polysaccharide. On the basis of A, the mass of ammonium sulfate was changed to 1 - 5 g (1, 2, 3, 4, and 5 g), and other operations were the same as A.

[0072] Effect of hydrogen peroxide concentration on the extraction yield of polysaccharide. On the basis of A, the hydrogen peroxide concentration was changed to 1.5% - 2.7% (1.5, 1.8, 2.1, 2.4, and 2.7%), and other operations were the same as A.

[0073] Effect of MCFA dosage on the extraction yield of polysaccharide. On the basis of A, the addition amount of MCFA was changed to 5 - 25 mL (5, 10, 15, 20, and 25 mL), and other operations were the same as A.

[0074] Effect of extraction temperature on the extraction yield of polysaccharide. On the basis of A, the extraction temperature was changed to 50 - 90 °C (50, 60, 70, 80, and 90 °C), and other operations were the same as A.

[0075] Effect of extraction time on the extraction yield of polysaccharide. On the basis of A, the extraction time was changed to 10 - 50 min (10, 20, 30, 40, and 50 min), and other operations were the same as A.

[0076] (2) Response surface optimization

[0077] On the basis of single - factor experiments, taking the extraction yield of Ganoderma lucidum polysaccharide as the response value, according to the principle of central composite experiment, with time, hydrogen peroxide concentration, ammonium sulfate mass, and MCFA dosage as variables, a four - factor and three - level response surface analysis experiment was designed using Design - Expert 8.0.6 software for Box Benhnken Design response surface optimization.

[0078] 3.4 Dynamic tracking of proteins during the extraction process

[0079] According to the optimal process conditions optimized in 3.3, Ganoderma lucidum polysaccharide was extracted. After centrifugation at 8000rpm for 15min, 0.1mL of the supernatant was taken and the protein content was determined by Coomassie Brilliant Blue G-250 method. Then the supernatant was concentrated to one-fifth of the original volume by rotary evaporation, cooled to 4°C, 95% ethanol with a volume of 4 times the concentrate was slowly added and stirred evenly with a glass rod, placed in a 4°C refrigerator for alcohol precipitation for 12h, centrifuged (8000rpm, 5min), the precipitate in the centrifuge tube was taken, dissolved in deionized water, concentrated until the ethanol was completely removed, and 0.1mL was taken and the protein content was determined by Coomassie Brilliant Blue G-250 method. Sevag reagent was added to other solutions to remove protein, and after sufficient mixing and shaking, the solution was centrifuged in a centrifuge to remove the precipitate, retain the supernatant, and the above method was repeated about 2 times. The supernatant was collected, and after removing the organic solvent by rotary evaporation, 0.1mL was taken and the protein content was determined by Coomassie Brilliant Blue G-250 method.

[0080] Method for protein determination by Coomassie Brilliant Blue G-250: ① Preparation of standard curve. Take 6 clean 10mL stoppered test tubes and take samples according to Table 2. After plugging, invert the solution in each test tube vertically to mix, let stand for 5 minutes, use a 1cm optical diameter cuvette to compare the color at a wavelength of 595nm, use tube No. 1 to adjust to zero, and record the optical density OD of each tube. 595 nm, and make a standard curve. The standard equation is: A = 0.013C + 0.0211 (R 2 =0.9962).

[0081] Table 2 Standard curve preparation

[0082]

[0083] ②Determination of protein concentration in sample extract

[0084] Take two 10mL stoppered test tubes and take samples according to Table 3. Take 0.1mL of the extract (do three replicates) and put it into a stoppered graduated test tube. Add 5mL of Coomassie Brilliant Blue G-250 protein reagent and mix thoroughly. After leaving it for 5 minutes, use a 1cm optical path cuvette to compare the color at 595nm and record the optical density OD. 595 nm, and the protein content C (μg) in the sample extract to be tested is obtained through the standard curve. Test tube No. 1 of the standard curve is used as a blank.

[0085] Table 3 Determination of protein concentration of test solution

[0086]

[0087]

[0088] ③Result calculation

[0089]

[0090] In the formula: C is the protein content (μg) obtained from the standard curve.

[0091] 3.5 Protective effect of Ganoderma lucidum polysaccharide on ulcerative colitis

[0092] 3.5.1 Establishment of colitis mouse model

[0093] After 50 ICR mice were adaptively fed for one week under the environmental conditions of room temperature 22±2°C and relative humidity 60%±5%, they were randomly divided into 5 groups: blank control group (normal mice); high and low dose groups of Ganoderma lucidum polysaccharide; positive control group was mesalazine group; negative control group was model group.

[0094] Days 1-18: On the basis of the mice's diet. Equal amounts of normal saline were intragastrically administered to the blank group and the negative control group; 100 mg / kg / d of polysaccharide was intragastrically administered to the low dose group; 200 mg / kg / d of polysaccharide was intragastrically administered to the high dose group; 30 mg / kg / d of mesalazine was intragastrically administered to the positive control group of mesalazine.

[0095] Days 19-25: Except for the blank group and the negative group, a 3% DSS solution was added to the drinking water of the other 3 groups of mice to induce acute colitis in mice. The water bottles were cleaned every day, and distilled water and the modeling agent were replaced. The specific model construction method is shown in Table 4.

[0096] Table 4 Construction of mouse colitis model

[0097]

[0098] 3.5.2 DAI score

[0099] During the DSS modeling period, the body weight of the mice was recorded, the fecal status was observed, and the diarrhea and bleeding conditions of the mice were checked before daily intragastric administration. The DAI score was used to quantify the degree of inflammation. The DAI scoring criteria are shown in Table 5.

[0100] Table 5 DAI scoring criteria for mice

[0101]

[0102] Note: *Normal: formed stools; Loose: pasty, semi-formed stools and not adhering to the anus; Watery stools: watery stools adhering to the anus.

[0103] 3.5.3 Mouse behavioral detection

[0104] 3.5.3.1 Pole climbing test

[0105] Make a simple climbing pole by winding a wooden pole with a diameter of 1 cm and a length of 60 cm with two layers of gauze (for anti-slip), and fix a round wooden plug at the top of the pole. Place the mouse on the wooden plug. When the mouse starts to crawl downward naturally with its head facing down, record the time it takes for the mouse to crawl naturally from the top of the pole to the bottom platform (with both front paws touching the ground). Conduct 3 experiments on each animal, with an interval of at least 10 minutes each time, and take the average value for statistical analysis.

[0106] 3.5.3.2 Hanging test

[0107] For the hanging test, fix a 5-mm steel wire horizontally. Let the mouse grasp the steel wire with its two front paws and observe whether its two hind paws can grasp the steel wire. Gently lift the mouse's tail to invert the mouse, and release the mouse's tail after its front limbs just grasp the stainless steel rod. If the mouse grasps the steel wire with its two hind paws, record 3 points; if it grasps the steel wire with one hind paw, record 2 points; if the mouse's two hind paws cannot grasp the steel wire, record 1 point. Conduct 3 experiments on each animal, with an interval of at least 10 minutes each time. Finally, record the total score of each mouse and take the average value for statistical analysis.

[0108] 3.5.3.3 Maze test

[0109] Adaptation stage: First, let the mouse freely explore the maze for a period of time, generally 5 - 10 minutes, to make it familiar with the maze environment.

[0110] Testing stage: Place the mouse at a starting point of the maze and record the time it takes for the mouse to reach the end point. Conduct 3 experiments on each animal, with an interval of at least 10 minutes each time. Finally, record the time-consuming situation of each mouse and take the average value for statistical analysis.

[0111] 3.5.4 Sample collection

[0112] 3.5.4.1 Collection of mouse blood specimens

[0113] After the gavage intervention, all mice are fasted but allowed to drink water for 12 hours. Then, anesthetize the mice with ether, quickly collect blood from the orbital venous plexus, let it stand at room temperature for 2 hours for natural separation of serum, and centrifuge at 2000 rpm for 15 minutes to collect the upper-layer serum and store it at -80°C.

[0114] 3.5.4.2 Collection of mouse colon tissue

[0115] Dissect the mouse and measure the length between the ileocecum and the proximal rectum. Take about 1 cm of the middle segment of the colon tissue and fix it in 4% paraformaldehyde fixative. Then, take out the colon tissue to make pathological sections. Stain the tissue sections using the hematoxylin-eosin (H&E) staining method, seal the sections with neutral gum and let them dry. After that, place the prepared pathological sections of the colon tissue under a microscope for observation.

[0116] 3.5.4.3 Mouse liver tissue collection

[0117] After dissecting the mouse and removing the liver, rinse the surface with pre-cooled physiological saline, then blot dry, weigh, quickly freeze in liquid nitrogen, and then transfer to an -80°C refrigerator for storage for subsequent experimental detection.

[0118] 3.5.4.4 Preparation of tissue homogenate

[0119] Weigh and cut the liver and colon into pieces respectively, transfer them to 1.5 mL centrifuge tubes, add 9 times the weight of pre-cooled physiological saline, and grind up and down in an ice-water bath using a handheld high-speed homogenizer, 10 seconds each time, with an interval of about 30 s, repeat several times until there is no obvious precipitate in the centrifuge tube. Centrifuge the centrifuge tube at high speed, and the supernatant is 10% tissue homogenate, which is stored at 4°C for later use.

[0120] 3.4.5 Determination of indicators related to redox status

[0121] Collect mouse serum and measure the activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and the level of lipopolysaccharide (LPS) in the serum according to the instructions of the kit.

[0122] 3.5.6 Determination of the levels of inflammatory factors in colon tissue

[0123] Accurately weigh the mouse colon tissue, grind it into a homogenate, and measure the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the tissue homogenate according to the instructions of the kit.

[0124] 3.6 Data processing

[0125] Experimental data are expressed as Means±SD. One-way analysis of variance (ANOVA) in IBM SPSS Statistics 25 software is used for homogeneity of variance test and LSD multiple comparison analysis for significant difference analysis. p<0.05 indicates significant difference.

[0126] 4 Experimental results

[0127] 4.1 Quantitative analysis method of ganoderma polysaccharide

[0128] Draw a standard curve of the concentration (abscissa, mg / mL) - absorbance (ordinate) of ganoderma polysaccharide at different concentrations, see Figure 1As shown, its linear regression equation is y = 4.4873x - 0.0039, R 2 = 0.9935. This indicates that there is a good linear relationship between the concentration of Ganoderma lucidum polysaccharide and absorbance.

[0129] 4.2 Effects of types and ratios of MCFA on the extraction yield of Ganoderma lucidum polysaccharide

[0130] MCFAs with different types and ratios have different effects on the extraction of Ganoderma lucidum polysaccharide. The screening results are shown in Table 6. It is found that the MCFAs prepared by mixing nonanoic acid and lauric acid in a ratio of 3:1 have the highest yield for extracting Ganoderma lucidum polysaccharide, and the MCFAs prepared by mixing octanoic acid and decanoic acid in a ratio of 2:1 have the second-best effect, with significant differences in the extraction yields compared to the other six extractants. Considering various factors such as stability, safety, cost, and environmental friendliness, when extracting Ganoderma lucidum polysaccharide subsequently, the MCFAs prepared by mixing nonanoic acid and lauric acid in a ratio of 3:1 are selected for single-factor and response surface optimization to obtain the optimal technological parameters for Ganoderma lucidum polysaccharide extraction.

[0131] Table 6 Experimental results of extracting Ganoderma lucidum polysaccharide with MCFA solutions of different compositions

[0132]

[0133]

[0134] 4.3 Single-factor experiments and response surface optimization of microwave-assisted HP-SA-MCFA three-phase extraction method for extracting Ganoderma lucidum polysaccharide

[0135] The effects of Ganoderma lucidum powder particle size, ammonium sulfate mass, hydrogen peroxide concentration, MCFA addition amount, extraction time, and extraction temperature on the extraction yield of Ganoderma lucidum polysaccharide are shown in Figure 2 .

[0136] As Figure 2 can be seen, as the extraction time increases from 10 min to 40 min, the extraction yield of Ganoderma lucidum polysaccharide shows a continuously significant upward trend. When the time increases to more than 40 min, the extraction yield of Ganoderma lucidum polysaccharide shows a significant downward trend. When the extraction time is 40 min, the extraction yield of Ganoderma lucidum polysaccharide is significantly higher than that at other times. Therefore, 40 min is used as the central point in the subsequent optimization experiments.

[0137] The extraction yield of Ganoderma lucidum polysaccharide shows a continuously significant upward trend with the increase of MCFA addition amount. When the MCFA addition amount increases to 15 mL, the extraction yield of Ganoderma lucidum polysaccharide is the highest. Continuing to increase the MCFA addition amount, the extraction yield of Ganoderma lucidum polysaccharide shows a significant downward trend. Therefore, 15 mL is used as the central point for the MCFA addition amount in the subsequent optimization experiments.

[0138] With the increase in the mass of ammonium sulfate, the extraction yield of Ganoderma lucidum polysaccharide showed a significant upward trend. When the mass of ammonium sulfate was 3.0 g, the extraction yield of Ganoderma lucidum polysaccharide was the highest. Continuing to increase the mass of ammonium sulfate, the extraction yield of Ganoderma lucidum polysaccharide showed a significant downward trend. Therefore, 3.0 g of ammonium sulfate mass was used as the central point in the subsequent optimization experiments.

[0139] The extraction yield of Ganoderma lucidum polysaccharide increased significantly with the increase in hydrogen peroxide concentration. When the hydrogen peroxide concentration increased to 2.1% and 2.4%, the extraction yield of Ganoderma lucidum polysaccharide reached the peak, and there was no difference in the effect of the two concentrations on the extraction yield of Ganoderma lucidum polysaccharide. When the hydrogen peroxide concentration continued to increase to 2.7%, the extraction yield of Ganoderma lucidum polysaccharide showed a significant downward trend. Therefore, 2.1% was selected as the central point for the subsequent optimization experiments.

[0140] With the continuous increase in temperature, the extraction yield of Ganoderma lucidum polysaccharide showed a continuous significant increase trend. When the temperature reached 90 °C, the extraction yield of Ganoderma lucidum polysaccharide was the highest. There was no inflection point in the effect of temperature on the extraction yield of Ganoderma lucidum polysaccharide. Therefore, 90 °C was selected for the temperature in the subsequent optimization experiments.

[0141] With the continuous decrease in particle size, the extraction yield of Ganoderma lucidum polysaccharide showed a continuous significant increase trend. When the particle size reached 80 - 100 mesh, a significant decrease in the extraction yield of Ganoderma lucidum polysaccharide occurred. When the particle size continued to decrease, the extraction yield of Ganoderma lucidum polysaccharide showed a continuous significant increase trend again. When the particle size was <150 mesh, the extraction yield of Ganoderma lucidum polysaccharide reached the peak. There was no inflection point in the effect of particle size on the extraction yield of Ganoderma lucidum polysaccharide. Therefore, a particle size of less than 150 mesh was selected for the subsequent optimization experiments.

[0142] Based on the analysis of the results of single-factor experiments, according to the principle of central composite experimental design, with the extraction yield of Ganoderma lucidum polysaccharide as the response value, Design-Expert 8.0.6 software was used for response surface analysis. The factor levels of the response surface optimization experiments are shown in Table 7, and the experimental design and results are shown in Table 8.

[0143] Table 7 Factor levels of the experimental design for extracting Ganoderma lucidum polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method

[0144]

[0145] Table 8 Experimental design and experimental results for extracting Ganoderma lucidum polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method

[0146]

[0147] Perform multiple regression fitting on the test data in Table 8 to obtain a regression equation with the polysaccharide yield (Y) as the response value: Y = 13.07 + 1.66A + 0.6958B + 0.7816C - 1.30D - 0.1139AB - 0.0456AC - 1.17AD - 1.20BC + 0.7605BD + 0.0412CD - 1.82A 2 - 2.63B 2 - 1.88C 2 - 3.33D 2

[0148] Perform an analysis of variance on the regression equation, and the results are shown in Table 9.

[0149] Table 9 Regression model analysis of microwave-assisted HP-SA-MCFA three-phase extraction for Ganoderma lucidum polysaccharide

[0150]

[0151]

[0152] From Table 9, the p-value is used as a tool to check the significance of each coefficient. The p-value of the model is less than 0.0001, indicating that the model is significant (p < 0.01). R 2 is 0.9546, indicating that within the range of experimental parameters, the predicted values fit well with the simulation test values; the adjusted R 2 is 0.9092, indicating that the equation can explain 90.02% of the response value changes and has a good fitting degree. The C.V value is 8.82%, indicating a relatively high credibility of the model. The p-value of the lack-of-fit term is 0.3161 and is not significant, indicating that the residuals are caused by random errors and are not significant relative to the pure error (p > 0.05). Therefore, this regression equation can be used to predict the experimental results.

[0153] Based on the experimental results, a response surface plot is made, that is, a three-dimensional space plot composed of the response value Y and the corresponding factors A, B, C, D, which can intuitively reflect the influence of each factor on the response value Y. According to the response surface optimization plot, the interaction between various factors during the dissolution process of Ganoderma lucidum polysaccharide can be seen, so as to determine the appropriate process conditions.

[0154] The quadratic multiple regression model of Ganoderma lucidum polysaccharide yield (Y) with time (A), hydrogen peroxide concentration (B), ammonium sulfate mass (C), and MCFA addition amount (D) is:

[0155] Y = 13.07 + 1.66A + 0.6958B + 0.7816C - 1.30D - 0.1139AB - 0.0456AC - 1.17AD - 1.20BC + 0.7605BD + 0.0412CD - 1.82A2 - 2.63B2 - 1.88C2 - 3.33D2

[0156] From Table 9, the order of the influence of each factor on the extraction rate of Ganoderma lucidum polysaccharide is extraction time > MCFA addition amount > ammonium sulfate mass > hydrogen peroxide concentration. The statistical data in Table 9 show that the interaction terms AD and BC have a significant effect on the response value curve effect, and there is an obvious interaction between extraction time and MCFA addition amount, and between hydrogen peroxide concentration and ammonium sulfate mass; the interaction terms AB, AC, BD, and CD have no significant effect on the response value surface effect, indicating that there is no obvious synergistic effect between time and hydrogen peroxide concentration, extraction time and ammonium sulfate mass, hydrogen peroxide concentration and MCFA addition amount, and ammonium sulfate mass and MCFA addition amount.

[0157] Based on the regression equation, the response surfaces of the interaction factors are as Figure 3 .

[0158] A large slope of the response surface graph indicates a large influence 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 3 Among them, the contour lines of the two interaction effects of AD and BC approach an ellipse, indicating that there is an interaction between them.

[0159] The optimal conditions optimized by the model are: time 48.87 min, hydrogen peroxide concentration 1.863%, ammonium sulfate mass 3.475 g, MCFA addition amount 15.983 mL. Under these conditions, the polysaccharide extraction rate can reach 11.968%. Considering the actual experimental conditions, the optimal adjustment is time 49 min, hydrogen peroxide concentration 1.9%, ammonium sulfate mass 3.5 g, and MCFA addition amount 16 mL.

[0160] In order to further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out according to the optimized extraction conditions. The yield of Ganoderma lucidum polysaccharide can reach 12.05% ± 0.12%, 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.

[0161] 4.4 Dynamic changes of polysaccharide and protein during separation and extraction

[0162] Extract Ganoderma lucidum polysaccharide under the optimal technological conditions optimized according to 3.3. For the extraction supernatant after centrifugation at 8000 rpm for 15 min, rotary evaporate and concentrate the supernatant to one-fifth of the original volume. After cooling to 4 °C, slowly add 95% ethanol with a volume four times that of the concentrated solution and stir evenly with a glass rod. Place it in a 4 °C refrigerator for alcohol precipitation for 12 h, then centrifuge (8000 rpm, 5 min). Take the precipitate in the centrifuge tube, dissolve it in deionized water, and concentrate it until the ethanol is completely removed to obtain the alcohol-precipitated concentrated solution. Add Sevag reagent to remove proteins, mix and shake well, then centrifuge in a centrifuge to remove the precipitate and retain the supernatant. Repeat the above method about 2 times. Collect the supernatant, remove the organic solvent by rotary evaporation to obtain the protein-depleted concentrated solution. The yields of polysaccharides (detected by the phenol-sulfuric acid method in 3.1) and proteins (determined by the Coomassie brilliant blue G-250 method in 3.4) in the extraction supernatant, alcohol-precipitated concentrated solution, and protein-depleted concentrated solution are shown in Table 10.

[0163] Table 10 Yields of polysaccharides and proteins during the separation and extraction process

[0164] Polysaccharide yield (%) Protein yield (%) Extracted supernatant 12.05 0.084 Alcohol precipitation concentrate 10.43 0.046 Protein-depleted concentrate 8.14 0.027

[0165] As can be seen from Table 10, when extracting Ganoderma lucidum polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method, the yield of polysaccharides will show an obvious downward trend during the separation and purification process. However, the yield of proteins is less than 0.1%. Although the yield of proteins will decrease during the separation and purification process, the overall yield is low and the degree of change is not large. This shows that the microwave-assisted HP-SA-MCFA three-phase extraction method can more thoroughly remove proteins in the extract during extraction, without further subsequent separation and purification, which can not only effectively avoid the loss of polysaccharides, but also simplify the separation and extraction process.

[0166] 4.5 Intervention ability of Ganoderma lucidum polysaccharide on ulcerative colitis (UC)

[0167] 4.5.1 Effects of Ganoderma lucidum polysaccharide intervention on the weight change rate and disease activity index score of UC mice

[0168] UC usually shows clinical symptoms such as diarrhea, rectal bleeding, abdominal pain, and weight loss. Since DSS can induce experimental animals such as mice to present symptoms and pathological characteristics similar to those of human UC, it is widely used in the study of the pathogenesis and potential treatment methods of UC. During the DSS modeling period, the mice in the blank control group and the positive control group were in good mental state and had shiny hair, and the DAI score was always maintained within the range of (0, 1). The mice in the other experimental groups began to drink a DSS solution containing 3% from the 14th day of the experiment, showing symptoms of listlessness, loose stools, and the DAI score showed an upward trend since the 1st day of DSS modeling. As Figure 4As shown in the figure, after 5 days of modeling, the negative group mice had visible blood in the stool, the feces were unformed or loose, and the DAI score was significantly increased compared with the blank group (p<0.05). Compared with the negative control group, the DAI score of mice in the different doses of Ganoderma lucidum polysaccharide intervention groups increased slowly. In summary, different doses of Ganoderma lucidum polysaccharide intervention have a certain effect on the development of colitis in mice, and can slow down the weight loss of mice caused by UC and the increase in DAI score, and the high dose is better than the low dose.

[0169] 4.5.2 Effects of Ganoderma lucidum polysaccharide intervention on memory and cognitive flexibility in UC mice

[0170] The effects of colitis on memory and cognitive flexibility in mice were mainly reflected in cognitive impairment and reduced hippocampal neurogenesis. In addition, leaky gut can lead to dysregulation of gene networks related to immune activation, oxidative stress, and myelination in the brain, which may affect cognitive function. Figure 5 As shown in the results, compared with the blank control group, the ability levels of mice in the negative control group in climbing poles, mazes, and hanging tests were significantly reduced (p<0.05). Compared with the negative control group, both the high-dose and low-dose groups of Ganoderma lucidum polysaccharide intervention can significantly reduce the climbing pole time and maze time of mice (p<0.05), and the effect in the climbing pole experiment is better than that of the positive control group (p<0.05). Both high- and low-dose Ganoderma lucidum polysaccharides and positive drugs can reduce the hanging time of mice compared with the negative control group, although there is no significant difference (p>0.05). This shows that Ganoderma lucidum polysaccharides have a good ability to repair the memory ability and cognitive flexibility of mice, and have a certain repair effect on the strength of mouse hanging.

[0171] 4.5.3 Effect of Ganoderma lucidum polysaccharide intervention on colon length in UC mice

[0172] The inflammatory response of UC causes damage to the colonic mucosa, which in turn forms ulcers and causes colon atrophy in mice. Therefore, the severity of UC can be assessed based on the length of the mouse colon. Figure 6 and Figure 7 As shown in the figure, the colons of mice in the blank control group and the positive control group were uniform and smooth, without edema and congestion, and the intestinal contents were full and granular; and the colon length of mice in the positive control group was significantly longer than that in the negative control group (p<0.05). After the action of DSS, the colon tissue of mice in the negative control group was damaged, the colon length was significantly shortened, the intestinal contents were yellow-green watery, and the colon tissue was fragile and easy to break. Compared with the negative control group, the colon length of mice in the high-dose and low-dose groups of Ganoderma lucidum polysaccharide was significantly increased (p<0.05), and the intestinal contents were observed to be viscous and slightly pasty, indicating that Ganoderma lucidum polysaccharide can protect intestinal tissue and alleviate colon atrophy caused by inflammatory response.

[0173] 4.5.4 Effect of Ganoderma lucidum polysaccharide intervention on pathological morphology of colitis mice

[0174] Hyperemia, necrosis, and edema of colonic tissue are typical features of UC. The DSS-induced UC mouse model exhibits severe pathological symptoms such as mucosal ulceration, destruction of crypt structure, and absence of goblet cells. As Figure 8 shown, the surface of the colonic mucosa layer of mice in the blank control group and the positive control group is smooth, the intestinal gland epithelial tissue is arranged neatly and tightly, and the crypts are covered with a large number of goblet cells. The colonic mucosa layer of mice in the negative control group is severely damaged, the crypt structure is destroyed, goblet cells are absent, and severe inflammatory cell infiltration appears. Compared with the negative control group, the colonic injury of mice in the Ganoderma lucidum polysaccharide intervention group is lighter, the crypt structure is more complete, and the number of goblet cells is more than that in the negative control group. Among them, there is mild inflammatory cell infiltration in the low-dose group of mice, and no inflammatory cell infiltration is seen in the high-dose group of mice. This shows that Ganoderma lucidum polysaccharide has a good protective effect on the mouse colon, and its intervention can significantly slow down the colonic tissue damage of colitis mice and improve the degree of colonic inflammation.

[0175] Liver changes are one of the common complications caused by colitis. As Figure 9 shown, the hepatic tissue structure of mice in the blank control group is normal, the hepatocytes are arranged neatly, the cell nuclei are clear, and there is no obvious inflammatory cell infiltration or tissue damage. Compared with the blank control group, the arrangement of hepatocytes in the negative control group is more disordered, the cell gaps are enlarged, and inflammatory cell infiltration appears, indicating that the UC inflammatory response induced by DSS has triggered pathological changes in liver cells. Compared with the negative control group, the arrangement of hepatocytes under the intervention of different doses of Ganoderma lucidum polysaccharide only shows slight disorder and mild inflammatory response; the size and shape of the cell nuclei have changed, but the overall structure is still relatively complete. The results show that Ganoderma lucidum polysaccharide has a significant intervention effect on the liver complications caused by mouse colitis, can effectively reduce liver cell damage, and maintain the health of liver tissue.

[0176] 4.5.5 Effect of Ganoderma lucidum polysaccharide intervention on inflammatory factors in colonic tissue of UC mice

[0177] TNF-α is one of the important indicators reflecting intestinal inflammation. TNF-α is mainly synthesized by T lymphocytes and further promotes the expression of IL-6 through the NF-KB signaling pathway. IL-6 plays a key role in acute, chronic inflammation, and autoimmunity, and the increase in its level reflects the degree of the body's inflammatory response. As Figure 10As shown, compared with the blank control group, the levels of pro-inflammatory factors TNF-α and IL-6 in the mice of the negative control group increased significantly (p < 0.05). Compared with the negative control group, the levels of TNF-α and IL-6 in the high-dose and low-dose groups of mice intervened with Ganoderma lucidum polysaccharide were significantly lower than those in the negative control group (p < 0.05), and the levels of pro-inflammatory factors were in the order of low-dose group < high-dose group < negative control group. Among them, the level of IL-6 in the high- and low-dose groups of Ganoderma lucidum polysaccharide in mice was significantly lower than that in the positive control group (p < 0.05), and there was no difference in the IL-6 level between the positive control group and the negative control group (p > 0.05). It is suggested that interventions with different doses of Ganoderma lucidum polysaccharide can effectively inhibit the increase in the levels of pro-inflammatory factors TNF-α and IL-6 caused by DSS-induced inflammation, and reduce the colonic inflammatory cascade reaction.

[0178] 4.5.6 Effect of Ganoderma lucidum polysaccharide intervention on the oxidative stress level in UC mice

[0179] Abnormal oxidative stress in UC can lead to overactivation of intestinal immune cells, exacerbate intestinal inflammatory responses and intestinal flora imbalance, thus accelerating the development of the disease. As an important antioxidant enzyme, SOD reduces the damage of oxidative stress to cells by scavenging superoxide anion radicals in cells. The SOD activity reflects the oxidative stress level in colonic tissue cells. As Figure 11 shown, compared with the blank control group, the SOD activity in the serum of mice in the negative control group decreased significantly (p < 0.05), indicating that the DSS-induced UC inflammatory response triggered cellular oxidative stress, resulting in the consumption of SOD. Compared with the negative control group, interventions with different doses of Ganoderma lucidum polysaccharide could significantly enhance the SOD activity in the serum (p < 0.05). There was no difference in the effect of the high-dose Ganoderma lucidum polysaccharide group on the SOD activity in the serum compared with the positive control group, while the ability of the low-dose Ganoderma lucidum polysaccharide group to enhance the SOD activity in the serum was significantly better than that of the positive control group and the high-dose group (p < 0.05). It shows that both high- and low-dose Ganoderma lucidum polysaccharide can significantly enhance the SOD activity in the serum of mice, thereby enhancing the antioxidant capacity of the body, alleviating the inflammatory state in vivo, and reducing the over-immune response induced by DSS and the oxidative stress it causes; there is no dose-dependent relationship between Ganoderma lucidum polysaccharide and the SOD activity in the serum of mice.

[0180] LPS is an endotoxin that is usually associated with intestinal inflammation. High LPS levels may lead to impaired intestinal barrier function, thereby triggering or exacerbating colitis. As Figure 12As shown, compared with the blank control group, the serum LPS level of the mice in the negative control group was significantly increased (p < 0.05); the serum LPS level of the mice in the positive control group was slightly higher than that of the blank control group but there was no statistically significant difference (p > 0.05). There was no difference in the serum LPS content of the mice in the high- and low-dose Ganoderma lucidum polysaccharide groups compared with the blank control group and the positive control group (p > 0.05), but it was significantly lower than that of the negative control group (p < 0.05); there was no difference between the high- and low-dose Ganoderma lucidum polysaccharide groups, and the effect of Ganoderma lucidum polysaccharide on the serum LPS level of mice did not show a dose-dependent relationship.

[0181] ALT / GPT is a marker of liver cell injury, and the increase in its activity reflects the injury of liver cells. In intestinal inflammation, the liver may be indirectly affected by intestinal inflammation, resulting in the release of alanine aminotransferase into the serum. As Figure 13 , compared with the blank control group, the serum ALT / GPT activity of the mice in the negative control group was significantly increased (p < 0.05), indicating that the UC inflammatory response induced by DSS caused liver cell injury, resulting in an increase in the serum ALT / GPT activity. Interventions with different doses of Ganoderma lucidum polysaccharide could restore the decrease in the serum ALT / GPT activity of mice induced by DSS, and the serum ALT / GPT activity of the mice in the high- and low-dose Ganoderma lucidum polysaccharide groups was significantly different from that of the negative control group (p < 0.05), and there was no statistical difference compared with the positive control group (p > 0.05); there was no difference between the high- and low-dose Ganoderma lucidum polysaccharide groups (p > 0.05). It is suggested that the hepatic tissue cell structure of the DSS-treated mice changed, the degree of liver cell injury increased, and the activity of alanine aminotransferase in the serum increased. Different doses of Ganoderma lucidum polysaccharide had protective effects on extraintestinal symptoms such as the liver caused by colitis, and the effect did not show a dose-dependent relationship.

[0182] Optimal extraction process of Example 2

[0183] Based on the optimal extraction process parameters optimized by the microwave-assisted HP-SA-MCFA three-phase extraction method in Example 1, the dried Ganoderma lucidum fruiting bodies were placed in a grinder and pulverized, and then passed through a 150-mesh sieve to obtain Ganoderma lucidum fruiting body powder. Then, the Ganoderma lucidum polysaccharide was extracted using the optimal extraction process parameters of the microwave-assisted HP-SA-MCFA three-phase extraction method.

[0184] Put 1.0 g of Ganoderma lucidum fruit body powder (same as in Example 1) into a three-necked flask, add 84.3 mL of deionized water and 5.7 mL of 30% hydrogen peroxide according to the material-liquid ratio of 1 g:90 mL, then add 3.5 g of ammonium sulfate and 3.532 g of lauric acid (the density of lauric acid is 0.88 g / mL, and 3.532 g is equivalent to 4 mL), and finally add 12 mL of nonanoic acid. After mixing, extract at a temperature of 90 °C and a microwave power of 600 W for 49 min. Centrifuge at a speed of 8000 rpm for 20 min. The middle layer solution is detected by the detection method in 3.2 of Example 1, and the average yield of Ganoderma lucidum polysaccharide is 12.05%.

[0185] Using the detection method in 3.4 of Example 1, compared with the negative control group, the increase in the DAI score of the mice in the Ganoderma lucidum polysaccharide intervention group was slow. Both the high-dose and low-dose groups of Ganoderma lucidum polysaccharide intervention could significantly reduce the pole-climbing time and maze time of the mice (p < 0.05), and significantly increase the colon length of the mice (p < 0.05); slow down the serious damage to the colonic mucosa layer and severe infiltration of inflammatory cells caused by DSS; effectively reduce liver cell damage and maintain the health of the liver tissue; significantly reduce the levels of TNF-α and IL-6 in the mice (p < 0.05), significantly enhance the serum SOD activity (p < 0.05), significantly reduce the serum LPS level (p < 0.05), and significantly reduce the serum ALT / GPT activity (p < 0.05).

[0186] Comparative Example 1

[0187] Take the washed Ganoderma lucidum fruit body and place it in an oven, dry it at 50 °C until constant weight, and crush it through a 150-mesh sieve. Take 1.0 g of Ganoderma lucidum fruit body powder, add 90 mL of deionized water, and extract Ganoderma lucidum polysaccharide under the condition of a hot water bath with an extraction temperature of 90 °C, an extraction time of 49 min, and a liquid-material ratio of 90:1. After centrifuging at a speed of 8000 rpm for 20 min, the supernatant is detected by the detection method in 3.2 of Example 1, and the extraction yield of Ganoderma lucidum polysaccharide is 2.57%.

[0188] In Example 2, the extraction yield of Ganoderma lucidum polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method reached 12.05%, and the extraction yield of Ganoderma lucidum polysaccharide was significantly improved, which was 4.69 times higher than that of the traditional hot water method.

[0189] Comparative Example 2

[0190] Extract Ganoderma lucidum polysaccharide by microwave-assisted hot water extraction. Take 1.0 g of Ganoderma lucidum fruit body powder (passed through a 150-mesh sieve), add 90 mL of deionized water, use a microwave chemical reactor, extract at a microwave power of 600 W and 90 °C for 49 min, then centrifuge at a speed of 8000 rpm for 20 min. The supernatant is detected by the detection method in 3.2 of Example 1, and the extraction yield of Ganoderma lucidum polysaccharide is 3.14%.

[0191] In Example 2, the extraction yield of ganoderma polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method reached 12.05%, and the extraction yield of ganoderma polysaccharide was significantly improved, which was 3.84 times higher than that of the microwave-assisted method.

[0192] Comparative Example 3

[0193] Choline chloride, lactic acid and guaiacol were mixed in a molar ratio of 1:1:1, and magnetic stirring was carried out at 300 rpm and 80 °C until a homogeneous and transparent ternary system was obtained. Subsequently, 1.0 g of ganoderma powder and 90 mL of the prepared ternary system (containing 50 wt% H2O) were placed in a constant temperature water bath and treated at 90 °C for 49 min. After centrifugation at 8000 rpm for 20 min, the supernatant was detected by the method of 3.2 in Example 1, and the extraction yield of ganoderma polysaccharide was 7.31%.

[0194] In Example 2, the extraction yield of ganoderma polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method reached 12.05%, and the extraction yield of ganoderma polysaccharide was significantly improved, which was 1.65 times higher than that of the ternary system method.

[0195] Comparative Example 4

[0196] 1.0 g of ganoderma fruiting body powder (screened through 150 mesh) was taken, 84.3 mL of deionized water was added, and then 5.7 mL of 30% hydrogen peroxide (HP) was added and mixed evenly. The extraction was carried out using a water bath shaker at 90 °C for 49 min. After centrifugation at 8000 rpm for 20 min, the supernatant was detected by the method of 3.2 in Example 1, and the extraction yield of ganoderma polysaccharide was 6.17%.

[0197] In Example 2, the extraction yield of ganoderma polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method reached 12.05%, and the extraction yield of ganoderma polysaccharide was significantly improved, which was 1.95 times higher than that of the HP method.

[0198] Comparative Example 5

[0199] Lauric acid and nonanoic acid were mixed in a molar ratio of 1:3, and magnetic stirring was carried out at 300 rpm and 80 °C until a homogeneous and transparent MCFA system was obtained. Subsequently, 1.0 g of ganoderma powder and 106 mL of the MCFA system (containing 35 wt% CH3CH2OH and 50 wt% H2O) were placed in a constant temperature water bath and treated at 90 °C for 49 min. After centrifugation at 8000 rpm for 20 min, the supernatant was detected by the method of 3.2 in Example 1, and the extraction yield of ganoderma polysaccharide was 1.29%.

[0200] In Example 2, the extraction yield of Ganoderma lucidum polysaccharide by microwave-assisted HP-SA-MCFA three-phase extraction method reached 12.05%, and the extraction yield of Ganoderma lucidum polysaccharide increased significantly, which was 9.34 times higher than that of the MCFA system method.

[0201] Example 3

[0202] (1) Place the dried Ganoderma lucidum in a grinder and crush it, then pass through a 80-100 mesh sieve to obtain Ganoderma lucidum powder.

[0203] (2) Take 1 g of the Ganoderma lucidum powder prepared in step (1), add 84.3 mL of deionized water and 5.7 mL of 30% hydrogen peroxide according to the solid-liquid ratio of 1 g:90 mL, then add 3.0 g of ammonium sulfate and 3.532 g of lauric acid, and finally add 12 mL of nonanoic acid. After mixing, microwave treat at a temperature of 80 °C and a microwave power of 400 W for 49 min. Centrifuge at a speed of 8000 rpm for 20 min and take the middle layer solution.

[0204] (3) Take 1.0 mL of the middle layer solution, adopt the detection method 3.2 in Example 1, calculate the extraction yield of Ganoderma lucidum polysaccharide according to Example 1, and set 3 parallels. The average value of the polysaccharide extraction yield is 10.06%.

[0205] Example 4

[0206] (1) Place the dried Ganoderma lucidum in a grinder and crush it, then pass through a 100-150 mesh sieve to obtain Ganoderma lucidum powder.

[0207] (2) Take 1 g of the Ganoderma lucidum powder prepared in step (1), add 76 mL of deionized water and 4 mL of 30% hydrogen peroxide according to the solid-liquid ratio of 1 g:80 mL, then add 2 g of ammonium sulfate and 3.532 g (equivalent to 4 mL) of lauric acid, and finally add 12 mL of nonanoic acid. After mixing, microwave treat at a temperature of 90 °C and a microwave power of 500 W for 50 min. Centrifuge at a speed of 8000 rpm for 20 min and take the middle layer solution.

[0208] (3) Take 1.0 mL of the middle layer solution, adopt the detection method 3.2 in Example 1, calculate the extraction yield of Ganoderma lucidum polysaccharide according to Example 1, and set 3 parallels. The average value of the polysaccharide extraction yield is 9.26%.

[0209] Example 5

[0210] (1) Place the dried Ganoderma lucidum in a grinder and crush it, then pass through a 60-80 mesh sieve to obtain Ganoderma lucidum powder.

[0211] (2) Take 1 g of Ganoderma lucidum powder prepared in step (1), add 91 mL of deionized water and 9 mL of 30% hydrogen peroxide according to the material-liquid ratio of 1 g:100 mL, then add 3.5 g of ammonium sulfate and 3.532 g (equivalent to 4 mL) of lauric acid, and finally add 12 mL of nonanoic acid. After mixing, perform microwave treatment at a temperature of 70 °C and a microwave power of 600 W for 40 min. Centrifuge at a speed of 8000 rpm for 20 min and take the middle layer solution.

[0212] (3) Take 1.0 mL of the middle layer solution, use the detection method in 3.2 of Example 1, convert it to the extraction rate of Ganoderma lucidum polysaccharide according to Example 1, and set 3 parallels. The average value of the polysaccharide extraction rate is 9.67%.

[0213] Example 6

[0214] (1) Place the dried Ganoderma lucidum in a pulverizer and crush it, then pass through a 150-mesh sieve to obtain Ganoderma lucidum powder.

[0215] (2) Take 1 g of Ganoderma lucidum powder prepared in step (1), add 84.3 mL of deionized water and 5.7 mL of 30% hydrogen peroxide according to the material-liquid ratio of 1 g:90 mL, then add 4 g of ammonium sulfate and 4.415 g (equivalent to 5 mL) of lauric acid, and finally add 15 mL of nonanoic acid. After mixing, perform microwave treatment at a temperature of 90 °C and a microwave power of 400 W for 30 min. Centrifuge at a speed of 8000 rpm for 20 min and take the middle layer solution.

[0216] (3) Take 1.0 mL of the middle layer solution, use the detection method in 3.2 of Example 1, convert it to the extraction rate of Ganoderma lucidum polysaccharide according to Example 1, and set 3 parallels. The average value of the polysaccharide extraction rate is 10.42%.

[0217] Example 7

[0218] (1) Place the dried Ganoderma lucidum in a pulverizer and crush it, then pass through a 150-mesh sieve to obtain Ganoderma lucidum powder.

[0219] (2) Take 1 g of Ganoderma lucidum powder prepared in step (1), add 84.3 mL of deionized water and 5.7 mL of 30% hydrogen peroxide according to the material-liquid ratio of 1 g:90 mL, then add 3.5 g of ammonium sulfate and 2.208 g (equivalent to 2.5 mL) of lauric acid, and finally add 7.5 mL of nonanoic acid. After mixing, perform microwave treatment at a temperature of 90 °C and a microwave power of 600 W for 49 min. Centrifuge at a speed of 8000 rpm for 20 min and take the middle layer solution.

[0220] (3) Take 1.0 mL of the middle layer solution, use the detection method in 3.2 of Example 1, convert it to the extraction rate of Ganoderma lucidum polysaccharide according to Example 1, and set 3 parallels. The average value of the polysaccharide extraction rate is 10.85%.

[0221] The above embodiments are illustrative of the present invention and not restrictive thereof. Any solution obtained by simply transforming the present invention falls within the protection scope of the present invention.

Claims

1. A method for extracting ganoderma polysaccharide by a three-phase method, characterized in that: It includes the following steps: (1) Pretreatment: Dry the Ganoderma lucidum fruiting body until the water content ≤ 8%, crush it and sieve it through a 40 - 150 mesh sieve to obtain Ganoderma lucidum powder; (2) Microwave-assisted hydrogen peroxide-ammonium sulfate-medium chain fatty acid (HP-SA-MCFA) three-phase extraction method: According to the solid-liquid ratio of 1 g:80 - 110 mL, mix the Ganoderma lucidum powder with hydrogen peroxide solution, ammonium sulfate and medium chain fatty acid (MCFA), and extract at 70 to 90 °C for 30 - 50 min under the condition of microwave power of 400 - 600 W. During the microwave-assisted three-phase extraction process, microwave assistance promotes the decomposition of hydrogen peroxide into hydroxyl radicals (·OH), destroys the chitin microfibrils and glucan network of the Ganoderma lucidum cell wall. Medium chain fatty acid (MCFA) dissolves cell membrane lipids and reduces cell permeability. Ammonium sulfate precipitates proteins through salting-out effect. The three work together to achieve "extraction - impurity removal" integration, eliminating the need for separate ethanol precipitation to remove weakly polar components and activated carbon decolorization steps in traditional processes, and also reducing the operation complexity of using Sevag reagent to remove proteins later; the MCFA is a mixture of nonanoic acid and lauric acid in a molar ratio of 2:1 - 4:1, preferably the molar ratio of nonanoic acid to lauric acid is 3:1; (3) Separation and purification: Centrifuge the extract and take the supernatant, add 1 - 5 volumes of 95% ethanol to precipitate polysaccharides, dissolve the precipitate in deionized water, and obtain Ganoderma lucidum polysaccharides after rotary evaporation and concentration.

2. The method according to claim 1, wherein: The Ganoderma lucidum powder in step (1) is prepared as follows: Cut Ganoderma lucidum into slices, place them in an oven, the drying temperature is 90 °C, the drying time is 12 hours, dry and then crush, and sieve through a 150 mesh sieve to obtain Ganoderma lucidum powder.

3. The method according to claim 1, characterized in that: In step (2), the concentration of hydrogen peroxide is 1.2% - 2.7%, preferably the concentration of hydrogen peroxide is 1.9%, the dosage of ammonium sulfate is 2 - 4 g / 1 g of Ganoderma lucidum powder, and the dosage of MCFA is 10 - 20 mL / 1 g of Ganoderma lucidum powder.

4. The method according to claim 3, characterized in that: The molar ratio of nonanoic acid to lauric acid is 3:1, the concentration of hydrogen peroxide is 1.9%, the mass of ammonium sulfate is 3.5 g, the dosage of MCFA is 16 mL, the extraction time is 49 min, the temperature is 90 °C, and the microwave power is 600 W.

5. Ganoderma lucidum polysaccharide extracted by the method according to claim 4, characterized in that: The yield of the Ganoderma lucidum polysaccharides can reach 12.05% ± 0.12% (determined by the phenol-sulfuric acid method).

6. Use of the ganoderma polysaccharide extracted by the method according to any one of claims 1-5 in the preparation of a drug for treating intestinal inflammation, characterized in that, The Ganoderma lucidum polysaccharides can: (a) Repair the damage of the intestinal mucosal layer and inhibit the infiltration of inflammatory cells; (b) Reduce the levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and enhance the activity of antioxidant enzyme superoxide dismutase (SOD); (c) Improve the disorder of the intestinal flora, reduce the levels of serum endotoxin lipopolysaccharide (LPS) and liver injury marker alanine aminotransferase (ALT / GPT), have a dose-dependent anti-inflammatory effect, and have no obvious toxic reaction.

7. The application according to claim 6, wherein The intestinal inflammation includes ulcerative colitis and inflammatory bowel disease. The drug is administered by gavage or orally, and the effective dose is 100 - 200 mg / kg / d.

8. The application according to claim 6, characterized in that, The drug can also reduce the infiltration of hepatic and intestinal inflammatory cells induced by dextran sulfate sodium (DSS), reduce the activity of serum alanine aminotransferase ALT / GPT, significantly improve the memory impairment induced by DSS, and the effect is better than that of the positive control drug mesalazine (p < 0.05).

9. The method according to any one of claims 1-8, characterized in that: The protein residue in the extract after the three-phase extraction is ≤ 0.1%.

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