Pleurotus eryngii polysaccharide, preparation method thereof and application of pleurotus eryngii polysaccharide in prevention and treatment of mastitis and / or intestinal flora disorder

Abstract

The invention belongs to the technical field of medical health food and medicines, and relates to pleurotus eryngii polysaccharide, a preparation method thereof and application of the pleurotus eryngii polysaccharide in prevention and treatment of mastitis and / or intestinal flora disorder. The pleurotus eryngii polysaccharide is neutral polysaccharide and is at least composed of galactose, glucose, mannose and fucose, residues of-> 2, 6)-alpha-D-Galp-(1->,-> 6)-alpha-D-Galp-(1-> form a main chain, branch sites are located at C-2 of-> 2, 6)-alpha-D-Galp-(1->, and residues of-> 6)-alpha-3-O-Me-D-Galp-(1->,-> 6)-3-OMe-alpha-D-Galp-(1-> exist; and the molecular weight at the peak position is 15800 to 15900 Da. The pleurotus eryngii polysaccharide provided by the invention has the pharmacological activity of protecting mastitis and regulating the abundance of intestinal flora.

Description

Technical Field

[0001] The invention belongs to the technical field of medical health food and medicine, and relates to a Pleurotus eryngii polysaccharide and a preparation method thereof and application thereof in preventing and treating mastitis and / or intestinal flora disorder. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cow mastitis is a local inflammation of the mammary tissue of dairy cows caused by a variety of pathogenic factors. It often manifests as redness, swelling, fever, pain and functional damage, and occurs frequently during the lactation period of dairy cows. The average incidence rate in farms around the world is as high as 25% to 70%. This not only affects milk production and quality, poses a threat to the healthy breeding of the dairy industry, but also seriously endangers food safety. The cause of cow mastitis is very complicated. Among them, LPS produced by Gram-negative bacteria such as pathogenic Escherichia coli stimulates mammary epithelial cells, which is one of the main causes of the disease. At present, antibiotic treatment is one of the commonly used methods for clinical prevention and treatment of cow mastitis. Although it has a certain preventive and therapeutic effect, excessive use of antibiotics can lead to drug resistance of related bacteria and cause drug residue problems in dairy products, which seriously threatens human health. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a Pleurotus eryngii polysaccharide and a preparation method thereof and its application in preventing and treating mastitis and / or intestinal flora disorder.

[0005] The present invention extracts Pleurotus eryngii polysaccharide by water extraction and alcohol precipitation method, and constructs a mouse mastitis model by microinjection of LPS into the milk pool. It is proved by experiments that Pleurotus eryngii polysaccharide has the pharmacological activity of protecting mastitis and regulating the abundance of intestinal flora. It is proved by blood routine, tissue homogenate index, staining section, immunofluorescence, Western blot and other experimental techniques that Pleurotus eryngii polysaccharide has the function of reducing the number and proportion of leukocytes and neutrophils, improving pathological damage such as inflammatory cell infiltration, interstitial congestion, edema, milk stasis, and alveolar integrity, reducing the expression of proinflammatory factors (TNF-α, IL-6, IL-1β), enhancing the activity of antioxidant enzymes (CAT, GSH, SOD, T-AOC), inhibiting cell apoptosis (Bcl-2 and Bax), and enhancing the blood-milk barrier (Occludin, Claudin-3, ZO-1); at the same time, it is clarified by 16S rRNA sequencing technology that it has the function of improving intestinal flora. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect, a Pleurotus eryngii polysaccharide is a neutral polysaccharide, which is composed of at least galactose, glucose, mannose and fucose, wherein the percentages of glucose, mannose, fucose and galactose are 37.700-37.800%, 1.100-1.150%, 0.710-0.720% and the remainder, respectively. In its chemical structure, the polysaccharide is composed of 2,6-α-D-Galp-(1→,

[0008] →6)-α-D-Galp-(1→ residues constitute the main chain, the branching site is located at C-2 of →2,6)-α-D-Galp-(1→, and there is

[0009] →6)-α-3-O-Me-D-Galp-(1→, →6)-3-OMe-α-D-Galp-(1→ residues; peak molecular weight is 15800~15900Da.

[0010] On the other hand, a method for preparing the above-mentioned Pleurotus eryngii polysaccharide comprises the following steps:

[0011] Using ethanol aqueous solution to extract Pleurotus eryngii under reduced pressure and reflux to remove fat-soluble substances, and obtaining Pleurotus eryngii residue;

[0012] The Pleurotus eryngii residue is boiled with water for extraction, and then insoluble components are removed to obtain a supernatant;

[0013] Adding ethanol or ethanol aqueous solution to the supernatant to adjust the ethanol volume fraction in the system to 77-83%, performing alcohol precipitation treatment, and removing protein from the collected precipitate to obtain crude polysaccharide;

[0014] First, the crude polysaccharide is subjected to column chromatography by using ion exchange column chromatography, wherein the component eluted by using distilled water is the distilled water component; and then the distilled water component is subjected to chromatography purification by using a gel filtration column.

[0015] The present invention uses Pleurotus eryngii as raw material and carefully prepares Pleurotus eryngii polysaccharide by water extraction and alcohol precipitation. Through animal experiments, it is confirmed for the first time that the polysaccharide has significant efficacy in treating mouse mastitis, demonstrating its outstanding value in the fields of health care and treatment.

[0016] In a third aspect, a use of the above-mentioned Pleurotus eryngii polysaccharide in preventing and treating mastitis and / or intestinal flora disorder in animals.

[0017] In a fourth aspect, a use of the above-mentioned Pleurotus eryngii polysaccharide in the preparation of a product for preventing and treating mastitis and / or intestinal flora disorder.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses Pleurotus eryngii as raw material, adopts water extraction and alcohol precipitation method to obtain Pleurotus eryngii polysaccharide, and proves for the first time through animal experiments that Pleurotus eryngii polysaccharide has the efficacy of preventing / treating mastitis and improving intestinal flora disorder, and has good health and therapeutic value. At the same time, the extraction and preparation method of the present invention is simple, easy to be converted into a modern industrial production process, and has great development potential, so it can achieve good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 This is the ion exchange column chromatography elution curve in the embodiment of the present invention.

[0022] Figure 2 This is the gel filtration column chromatography elution curve of the sample PEP-E1 in the embodiment of the present invention.

[0023] Figure 3 It is the HPGPC chromatogram of PEP-E1 in the embodiment of the present invention; wherein A is PEPE1-F1 and B is PEPE1-F2.

[0024] Figure 4 It is the liquid chromatogram of the standard product and PEPE1-F1 in the embodiment of the present invention; wherein A is the standard sample and B is PEPE1-F1.

[0025] Figure 5 The effects of PEPE1-F1 and PEPE1-F2 Pleurotus eryngii polysaccharides in the examples of the present invention on inflammatory factors in an in vitro inflammatory model induced by LPS. (A) TNF-α content in mouse macrophages RAW264.7; (B) IL-6 content in mouse macrophages RAW264.7; (C) IL-1β content in mouse macrophages RAW264.7.

[0026] Figure 6 It is an apparent diagram of mouse mammary gland tissue in the embodiment of the present invention; wherein, A is a blank control group (NC), B is an LPS group (MC), C is a positive drug group (PC), D is a low-dose Pleurotus eryngii polysaccharide group (PEPL), E is a medium-dose Pleurotus eryngii polysaccharide group (PEPM), and F is a high-dose Pleurotus eryngii polysaccharide group (PEPH).

[0027] Figure 7Graph showing the effect of PEP on pathological changes of mouse mammary gland tissue in an embodiment of the present invention (200×); A is the NC group, B is the MC group, C is the PC group, D is the PEPL group, E is the PEPM group, F is the PEPH group, and G is the pathological score graph; red arrows represent neutrophil infiltration, yellow arrows represent milk stasis, blue arrows represent erythrocyte infiltration, and black arrows represent incomplete alveolar structure.

[0028] Figure 8 Graph showing the effect of PEP on blood routine, biochemistry, MPO activity and mammary gland index of mice in an embodiment of the present invention; A is the number of white blood cells, B is the number of monocytes, C is the ratio of the number of monocytes, D is the number of lymphocytes, E is the ratio of the number of lymphocytes, F is the ALT content in serum, G is the AST content in serum, H is the MPO activity, and I is the mammary gland organ index.

[0029] Fig. 9 Graph showing the effect of PEP on the expression of inflammatory cytokines in LPS-induced mouse mammary tissue in an embodiment of the present invention; wherein A, B, and C are the relative quantitative results of TNF-α, IL-6, and IL-1β mRNA levels in mammary tissue of each group, and D, E, and F are the expression levels of TNF-α, IL-6, and IL-1β protein levels in mammary tissue of each group, respectively.

[0030] Fig.10 Graph showing the effect of PEP on the NF-κB signaling pathway induced by LPS in mouse mammary gland tissue in an embodiment of the present invention; A is p65, B is p-65, C is p-65 / p65, D is IκB-α, E is p-IκB-α, F is p-IκB-α / IκB-α, and G is a protein blotting graph.

[0031] Fig.11 This is a graph showing the effect of PEP on the antioxidant system of LPS-induced mouse mastitis tissue in an embodiment of the present invention: wherein A, B, and C are the relative expression levels of CAT, GPX-1, and SOD-2 mRNA in each group, respectively; and D, E, F, G, and H are the relative expression levels of CAT, GSH, SOD, T-AOC, and MDA proteins in each group of mammary tissue, respectively.

[0032] Fig.12 Graph showing the effect of PEP on the Nrf2 / HO-1 signaling pathway in LPS-induced mouse mammary tissue in an embodiment of the present invention, A is a quantitative analysis bar graph, and B is a protein blot.

[0033] Fig.13Graph showing the effect of PEP on LPS-induced apoptosis of mouse mammary gland tissue in an embodiment of the present invention; wherein A, B, and C are the relative quantitative statistical results of Bcl-2, Bax, and Bcl-2 / Bax mRNA levels in each group, respectively; D, E, and F are the relative quantitative results of Bcl-2, Bax, and Bcl-2 / Bax protein levels in each group, respectively; G is the protein expression level of Bcl-2 and Bax molecules; and H is the statistical result of the number of TUNEL-positive cells.

[0034] Fig.14 Graph showing the effect of PEP on LPS-induced blood-milk barrier in mouse mammary tissue in an embodiment of the present invention; wherein, A, B, and C are the relative quantitative results of Claudin-3, Occludin, and ZO-1 mRNA expression levels; D, E, and F are the relative quantitative results of protein expression levels of Claudin-3, Occludin, and ZO-1 genes; G: Claudin-3, Occludin, and ZO-1 protein expression levels.

[0035] Fig.15 Graph showing the effect of PEP on the Alpha and Beta diversity of intestinal flora in an embodiment of the present invention; A is a rarefaction curve, B is principal coordinate analysis, C is PLS-DA analysis, D is a petal diagram of the number of OTUs in each group, and E, F, G, and H are alpha diversity analysis indicators Chao1, Observed species, Shannon, and Simpson, respectively.

[0036] Fig.16 It is a PEP analysis diagram of species composition at the phylum and level in an embodiment of the present invention; wherein A is the species composition at the phylum level, B is the species composition at the genus level, C is the abundance of the genus Firmicutes, D is the abundance of the genus Bacteroidetes, and E is the ratio of Firmicutes / Bacteroidetes.

[0037] Fig.17 It is a LEfSe analysis diagram of PEP on intestinal flora disorder in an embodiment of the present invention; wherein A is a distribution bar graph of LDA values ​​of significantly different species, and the LDA value is 3.0; and B is a species taxonomy branch diagram.

[0038] Fig.18 : This is a screening analysis diagram of microorganisms with different intestinal flora markers in the embodiment of the present invention; wherein A is a random forest analysis at the genus level, B is a Venn diagram of LEfSe analysis, random forest analysis and abundance TOP20, and C is a Lactobacillus genus.

[0039] Figure 5 is the relative abundance of Lactobacillus in different groups, D is the relative abundance of COE1 in different groups, E is the relative abundance of Phocaeicola A 858004 in different groups, F is the relative abundance of Bacteroides_H in different groups, G is the relative abundance of Paraprevotella in different groups, and H is the relative abundance of Ligilactobacillus in different groups. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0041] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0042] The term "prevention and treatment" as used herein refers to prevention and / or treatment, and comprehensive measures for mastitis include not only direct treatment of these diseases to relieve existing symptoms, but also preventive treatment aimed at reducing the risk of disease occurrence or reducing the severity of symptoms. In addition, this concept also includes the prevention of disease recurrence, whether it is to prevent the disease from rebounding after treatment is completed, or to take measures to alleviate symptoms when the disease has already occurred.

[0043] In order to prevent and treat mastitis and intestinal flora disorder at the same time, the present invention proposes a Pleurotus eryngii polysaccharide and a preparation method thereof and application thereof in preventing and treating mastitis and / or intestinal flora disorder.

[0044] A typical embodiment of the present invention provides a Pleurotus eryngii polysaccharide, which is a neutral polysaccharide and is composed of at least galactose, glucose, mannose and fucose. The percentages of glucose, mannose, fucose and galactose are 37.700-37.800%, 1.100-1.150%, 0.710-0.720% and the remainder, respectively. In its chemical structure, the main chain is composed of →2,6)-α-D-Galp-(1→, →6)-α-D-Galp-(1→ residues, the branching site is located at C-2 of →2,6)-α-D-Galp-(1→), and there are →6)-α-3-O-Me-D-Galp-(1→, →6)-3-OMe-α-D-Galp-(1→ residues; the peak molecular weight is 15800-15900Da.

[0045] In some embodiments, the weight average molecular weight is 19150-19250 Da.

[0046] In some embodiments, the number average molecular weight is 13250-13300 Da.

[0047] Another embodiment of the present invention provides a method for preparing the above-mentioned Pleurotus eryngii polysaccharide, comprising the following steps:

[0048] Using ethanol aqueous solution to extract Pleurotus eryngii under reduced pressure and reflux to remove fat-soluble substances, and obtaining Pleurotus eryngii residue;

[0049] The Pleurotus eryngii residue is boiled with water for extraction, and then insoluble components are removed to obtain a supernatant;

[0050] Adding ethanol or ethanol aqueous solution to the supernatant to adjust the ethanol volume fraction in the system to 77-83%, performing alcohol precipitation treatment, and removing protein from the collected precipitate to obtain crude polysaccharide;

[0051] First, the crude polysaccharide is subjected to column chromatography by using ion exchange column chromatography, wherein the component eluted by using distilled water is the distilled water component; and then the distilled water component is subjected to chromatography purification by using a gel filtration column.

[0052] In some embodiments, in the vacuum reflux extraction, the mass volume ratio of Pleurotus eryngii to ethanol aqueous solution is 1:2-10 kg / L; preferably 1:4.5-5.5 kg / L. Specifically, the volume dispersion of ethanol aqueous solution is 92-96%. Specifically, the temperature of vacuum reflux is 70-90°C, preferably 83-87°C. Specifically, the vacuum reflux treatment is 2-4 times, each time for 1-4 hours.

[0053] In some embodiments, the cooking temperature is 90-100° C., preferably 95-100° C. Specifically, the cooking extraction time is 1-3 hours, preferably 1.8-2.2 hours, and the number of cooking extractions is 1-3 times, preferably 2 times.

[0054] In some embodiments, the standing time of the alcohol precipitation treatment is 10 to 40 hours, preferably 22 to 26 hours.

[0055] In some embodiments, the protein in the collected precipitate is removed by the Sevage method. Specifically, water is added to the precipitate at a mass volume ratio of 1:9-10 (g / mL) to obtain an aqueous solution, which is then mixed with a Sevage solution (the mass ratio of chloroform to n-butanol is 3.9-4.1:1) at a volume ratio of 3.5-4.5:1, and then centrifuged after shaking to obtain an upper polysaccharide solution, and this step is repeated 5-8 times (preferably 7 times) until the protein is completely removed.

[0056] In some embodiments, the ion exchange column chromatography is DEAE-Sepharose FF (DEAE Sepharose FF) ion exchange column chromatography. Specifically, the DEAE sepharose FF gel suspension is washed with about 3 volumes of ultrapure water until the filler has no alcohol taste, and distilled water of one-half to one times the volume of the sedimentation gel is added and stirred well for column loading. The pretreated DEAE sepharose FF gel suspension is stirred well and then slowly added to the XK chromatography column. After complete sedimentation, start the pump to flush the column with ultrapure water at a flow rate of 200 to 300 cm / h until the gel surface is stable, and complete the column loading; centrifuge the crude polysaccharide sample at 7000 to 9000 rpm for 5 to 15 minutes to remove the precipitate, filter with a 0.45 μm microporous filter membrane, and load the filtrate onto the equilibrated ion exchange chromatography column. The loading amount is 25 to 35% of the column volume. After all the sample liquid enters the chromatography column, it is sequentially filtered according to different salt concentrations (dH2O, 0.15 to 0.25 M NaCl, 0.45 to 0.55 M NaCl, 0.95 to 1.05 M The method comprises the following steps: 1.5 to 2.5 times column volume of each concentration of elution, an elution rate of 10 to 20 mL / min, and collecting the fractions using an automatic fraction collector. Each eluate gradient is collected in 90 to 110 tubes, and 9 to 11 mL is collected in each tube. The polysaccharide content in the eluate is tracked and detected by an anthrone-sulfuric acid method and an ELISA reader at 630 nm. The number of tubes is used as the abscissa and the absorbance is used as the ordinate to obtain a polysaccharide elution curve. The eluates of each tube at different elution peaks are collected respectively, and the eluate is repeatedly loaded on an ion exchange column for chromatography; the combined and collected eluate components are concentrated under reduced pressure, dialyzed with a dialysis bag at a cutoff value of 3.5 KDa, and vacuum freeze-dried to obtain components of each group; the dH2O elution component (named PEP-E1) is taken as a sample for gel filtration column chromatography.

[0057] In some embodiments, the gel filtration column is a Chromdex 200PG gel filtration column. Specifically, the distilled water component is re-dissolved in water and added to the gel filtration column, the sample amount is 0.5-1.5% of the column volume, and the separation is eluted with ultrapure water at a flow rate of 1.5-2.0 mL / min. The eluate is collected by an automatic partial collector, 4.0-5.0 mL is collected in each tube, and 0.9-1.1 times the column volume is eluted. The eluate in the same elution peak area with higher peak height and better symmetry is collected, and the gel filtration column chromatography is repeated multiple times to enrich the purified polysaccharide. More specifically, the elution components in the 105-130min area are collected, the eluate components collected are combined, concentrated under reduced pressure, and vacuum freeze-dried to obtain the polysaccharide separated and purified by gel filtration column chromatography.

[0058] A third embodiment of the present invention provides a use of the above-mentioned Pleurotus eryngii polysaccharide in preventing and treating mastitis and / or intestinal flora disorder in animals.

[0059] In some embodiments, the animal is a non-human mammal; for example, mice, rats, rabbits, dogs, pigs, cows, sheep, etc.

[0060] A fourth embodiment of the present invention provides a use of the above-mentioned Pleurotus eryngii polysaccharide in the preparation of a product for preventing and treating mastitis and / or intestinal flora disorder.

[0061] In some embodiments, the product includes but is not limited to food, medicine, and feed, wherein the food is functional food and the feed is functional feed additive.

[0062] A fifth embodiment of the present invention provides a method for preventing and treating mastitis and improving intestinal flora disorder, comprising administering the above-mentioned Pleurotus eryngii polysaccharide to a subject.

[0063] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0064] Example 1: Preparation of Pleurotus eryngii polysaccharide

[0065] Extraction method of Pleurotus eryngii polysaccharide: weigh 500g of dry weight Pleurotus eryngii slices, cut them into pieces and place them in a distillation flask, add 2.5L 95% ethanol and heat to 80℃ for decompression reflux, 2.5h each time, reflux twice in total, aiming to remove pigments and fat-soluble small molecules. Then place the Pleurotus eryngii residue in a cool and ventilated place to dry to evaporate excess ethanol, then place it in a pot and add 10 times the volume of ultrapure water, heat and extract for 2h, extract twice in total, combine the filtrates of 3 times, and concentrate to a final concentration of 1g / mL. Add 90% ethanol to the filtrate, stirring while adding, so that the final concentration is 80%. After standing in a refrigerator at 4℃ for 24h, pour off the upper liquid and collect the precipitate. The alcohol precipitation part of 80% of the Pleurotus eryngii water extract is obtained, which is the Pleurotus eryngii crude polysaccharide PEP80, which is sealed and kept at -20℃ after freeze-drying for standby use.

[0066] Purification method of Pleurotus eryngii polysaccharide: Dissolve the freeze-dried Pleurotus eryngii crude polysaccharide PEP80 in ultrapure water to prepare a 0.1g / mL aqueous solution. Add Sevage reagent (chloroform: n-butanol = 4:1) at a ratio of 4:1, then shake on a shaker for 30 minutes, and repeat the operation 7 times to fully remove the protein component in the crude polysaccharide. Finally, remove the dissolved organic reagents by decompression using a rotary evaporator, and obtain the deproteinized Pleurotus eryngii polysaccharide after freeze-drying; then use DEAE-agarose gel FF (DEAE Sepharose FF) ion exchange column chromatography and Chromdex 200PG gel filtration column for chromatography purification.

[0067] The DEAE-agarose gel FF ion exchange column chromatography method includes: washing the DEAE sepharose FF gel suspension with about 3 times the volume of ultrapure water until the filler has no alcohol taste, adding distilled water of one-half to one times the volume of the sedimentation gel, stirring well for column loading. Stirring the pretreated DEAE sepharose FF gel suspension well, slowly add it to the XK chromatography column After complete sedimentation, start the pump at a flow rate of 250 cm / h to flush the column with ultrapure water until the gel surface is stable, and the column is loaded; the crude polysaccharide sample is centrifuged at 8000 rpm for 10 min to remove the precipitate, and filtered with a 0.45 μm microporous filter membrane. The filtrate is loaded onto the equilibrated ion exchange chromatography column, and the loading amount is 30% of the column volume. After all the sample liquid enters the chromatography column, it is sequentially filtered according to different salt concentrations (dH2O, 0.2M NaCl, 0.5M NaCl, 1.0M The eluent was eluted and separated with 2 times column volume for each concentration at an elution rate of 15 mL / min. The fractions were collected by an automatic fraction collector. 100 tubes were collected for each gradient eluent, and 10 ml was collected in each tube. The anthrone-sulfuric acid method was used and the polysaccharide content in the eluent was tracked and detected by an ELISA reader at 630 nm. The number of tubes was used as the horizontal coordinate and the absorbance was used as the vertical coordinate to obtain the polysaccharide elution curve. The eluents of each tube with different elution peaks were collected respectively, and the ion exchange column chromatography was repeated for multiple times. The combined and collected eluent components were concentrated under reduced pressure, dialyzed with a dialysis bag with a cutoff value of 3.5 KDa, and freeze-dried in a vacuum to obtain components of each group. The dH2O elution component (named PEP-E1) was taken as the sample for gel filtration column chromatography.

[0068] The gel filtration column chromatography method includes: washing the gel filtration chromatography medium Chromdex 200PG gel suspension with about 3 times the volume of ultrapure water until the filler has no alcohol taste, adding distilled water of one-half to one times the volume of the sedimentation gel, stirring well for column loading. Stirring the pretreated gel filtration chromatography medium gel suspension well, slowly add it to the XK chromatography column After complete sedimentation, the column is rinsed with ultrapure water at a flow rate of 30 cm / h until the gel surface is stable, and the column is loaded; the PEP-E1 polysaccharide sample prepared by ion exchange column chromatography is dissolved with ddH2O, centrifuged at 8000 rpm for 10 minutes, the precipitate is removed, and filtered with a 0.45 μm microporous filter membrane, and the filtrate is loaded onto the equilibrated gel filtration chromatography, and the loading amount is 1% of the column volume. After all the sample liquid enters the chromatography column, it is eluted and separated with ultrapure water at a flow rate of 1.8 mL / min, and the eluate is collected by an automatic partial collector, 4.5 mL is collected in each tube, and 1 column volume is eluted, and the eluate with a differential detector is combined for online detection and collection, and the eluate with a higher peak height and better symmetry in the same elution peak area is collected, and the gel filtration column chromatography is repeatedly loaded multiple times to enrich the purified polysaccharide; specifically, the elution components in the 105-130 min area are collected; the combined and collected eluate components are concentrated under reduced pressure, and vacuum freeze-dried to obtain the polysaccharide separated and purified by gel filtration column chromatography.

[0069] The preliminary characterization results of Pleurotus eryngii polysaccharides are shown in Figure 1 , 2, 3, 4; The crude polysaccharide sample was chromatographed by ion exchange column, and gradient eluted in accordance with different salt concentrations of dH2O, 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl solutions in turn. The polysaccharide content of each tube was determined by anthrone-sulfuric acid method, and the elution curve was plotted as shown in Figure 1 As shown in the figure, the polysaccharide content of the dH2O elution fraction is significantly higher than that of the fractions eluted with 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl solutions. Based on the elution curve obtained by ion column chromatography and the molecular weight test results, the fraction sample PEP-E1 (dH2O elution fraction) was selected as the sample for gel filtration column chromatography. The elution curve obtained by gel filtration column chromatography is as follows: Figure 2 As shown, the elution components were collected at 105-130min and 150-160min, respectively, concentrated under reduced pressure, and freeze-dried in vacuum. After repeated gel filtration column chromatography, the isolated and purified polysaccharides were named PEPE1-F1 and PEPE1-F2. Figure 3 As shown in Figure 1, the HPGPC molecular weight test showed that the polysaccharide PEPE1-F1 and PEPE1-F2 components were both single symmetrical peaks, and their molecular weight results are shown in Table 1, indicating that they are homogeneous polysaccharides. PEPE1-F1 with a larger molecular weight was selected for subsequent monosaccharide composition and ratio analysis, and the results are shown in Table 1. Figure 4 They are the liquid chromatograms of the standard and PEPE1-F1, and the results show that PEPE1-F1 is composed of galactose (61.10%), glucose (37.78%), mannose (1.12%) and fucose (0.72%).

[0070] Table 1 Molecular weight results of polysaccharides PEPE1-F1 and PEPE1-F2

[0071]

[0072] Note: RT, Mp, Mw and Mn represent retention time, peak molecular weight, weight average molecular weight and number average molecular weight respectively.

[0073] Table 2 PEPE1-F1 monosaccharide composition and ratio

[0074]

[0075] The structure was identified by full methylation and the above-mentioned one-dimensional and two-dimensional NMR techniques. The preliminary structure was inferred to be

[0076] →2,6)-α-D-Galp-(1→, →6)-α-D-Galp-(1→) residues constitute the main chain, the branching site is located at C-2 of →2,6)-α-D-Galp-(1→, and there are a certain amount of →6)-α-3-O-Me-D-Galp-(1→, →6)-3-OMe-α-D-Galp-(1→ residues.

[0077] The prepared Pleurotus eryngii polysaccharides PEPE1-F1 and PEPE1-F2 were used in Example 2.

[0078] Example 2: PEPE1-F1 Pleurotus eryngii polysaccharide in treating LPS-induced in vitro inflammatory model

[0079] Model establishment: PEPE1-F1 and PEPE1-F2 were diluted in sterile DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and set up as blank control group, PEPE1-F1, PEPE1-F2 and LPS group, respectively. Mouse macrophage RAW264.7 in the logarithmic growth phase was taken and the cell density was adjusted to 6×10 5 / mL, inoculated in a 24-well plate, 500μL per well. After incubation for 12h, the culture medium was discarded, 500μL DMEM high-glucose culture medium was added to the blank control group and the model control group, and 500μL culture medium containing different Pleurotus eryngii polysaccharides was added to the drug-treated group. After culturing for 12h, 20μL supernatant was aspirated from each well, 20μL DMEM culture medium was added to the blank control group, and 20μL culture medium containing 0.025mg / mL LPS was added to the other groups to make the final concentration of LPS 1μg / mL. After adding LPS and culturing for 4h, 300μL supernatant was aspirated from each well into a 0.6mL EP tube for the determination of IL-6, TNF-α and IL-1β; the parallel plates were operated in the same way. The specific operations for the detection of each indicator were carried out according to the instructions of each kit.

[0080] The results of the inflammatory factor determination of mouse macrophage RAW264.7 showed that compared with the blank control group (NC), the content of TNF-α, IL-6, and IL-1β pro-inflammatory factors in the breast tissue of the model control group (MC) was extremely significantly increased (p<0.001). After PEPE1-F1 treatment, compared with the MC group, the content of TNF-α, IL-6, and IL-1β pro-inflammatory factors was extremely significantly decreased (p<0.001), but after PEPE1-F2 treatment, compared with the MC group, the content of TNF-α, IL-6, and IL-1β pro-inflammatory factors decreased, and the difference was not significant (p>0.05). This shows that only PEPE1-F1 treatment can inhibit the overexpression of TNF-α, IL-6, and IL-1β pro-inflammatory factors in vitro, thereby exerting an anti-inflammatory effect. Figure 5 shown.

[0081] Therefore, the Pleurotus eryngii polysaccharide PEPE1-F1 prepared in Example 1 was used in Examples 3-4.

[0082] Example 3: Pleurotus eryngii polysaccharide in the treatment of LPS-induced mastitis in mice

[0083] Establishment of mouse mastitis model: Kunming female mice 6-8 days after giving birth were randomly divided into 6 groups, namely blank control group (NC, equal dose of normal saline), model control group (MC, equal dose of normal saline), positive control group (PC, 5 mg / KgDEX), Pleurotus eryngii polysaccharide low (PEPL, 400 mg / Kg), medium (PEPM, 800 mg / Kg), and high (PEPH, 1200 mg / Kg) dose groups, with 6 mice in each group; Pleurotus eryngii polysaccharide low, medium, and high treatment groups were gavaged with 400 mg / Kg, 800 mg / Kg, and 1200 mg / Kg polysaccharide solutions, respectively, at a concentration of 0.1 mL / 10 g BW, continued administration for 7 days, the positive control group was gavaged with 5mg / Kg of dexamethasone injection diluent, and the blank control group and model control group were gavaged with the same amount of normal saline as a control. After 7 days of administration, the offspring should be taken out and separated from the mother for weaning 3 hours before modeling. The Kunming mother mice were anesthetized and the fourth pair of mammary glands were disinfected with iodine tincture. The nipples of the mice were cut with sterile scissors and forceps to fully expose the milk ducts. 100μL 0.2mg / mL LPS solution was perfused into the milk pool with a sterile microinjector. After the modeling was completed, they were returned to the original cage for 24 hours. After the experiment, the Kunming mother mice were anesthetized and blood was collected by removing the eyeballs, and then killed, and mammary tissue and colon feces were collected.

[0084] Routine blood test and liver biochemical examination: Collect 3-5 drops of mouse blood in an anticoagulant tube (EDTA-K2), and use a veterinary fully automatic blood cell analyzer (BC-2800Vet) for routine blood test; centrifuge the blood at 3000 rpm for 15 minutes to obtain serum, and use a biochemical kit to detect the activity levels of ALT and AST in mouse serum to evaluate the liver function of mice.

[0085] ELISA examination: ELISA method was used to detect the protein expression of TNF-α, IL-6, and IL-1β in breast tissue to evaluate the anti-inflammatory ability of Pleurotus eryngii polysaccharide; biochemical kit was used to detect the protein expression of CAT, GSH, SOD, T-AOC and MDA in breast tissue to evaluate the antioxidant capacity of Pleurotus eryngii polysaccharide. The preparation method of 10% breast tissue homogenate includes: weighing 100 mg breast tissue, adding 900 μL sterile saline in advance into an EP tube, homogenizing at 50-60 Hz for 1-2 minutes, centrifuging at 3000 rpm for 20 minutes, collecting the supernatant and storing it in aliquots at -80 °C.

[0086] Real-time quantitative PCR (qRT-PCR) analysis: qPCR was used to detect the mRNA levels of inflammation (TNF-α, IL-6, IL-1β), oxidative stress (CAT, GPX-1, SOD-2), apoptosis (Bcl-2, Bax), and blood-milk barrier (Claudin-3, Occludin, and ZO-1). The specific method includes: taking an appropriate amount of breast tissue, extracting total RNA by the Ttizol method, and adjusting its concentration to be consistent, and synthesizing cDNA according to the instructions of the kit. AceQ qPCR SYBR Green Master Mix (Vazyme, Q111-02) was used for quantitative PCR (qPCR) and run on the BIO-RAD real-time PCR system. GAPDH was used as the control gene for normalization. The relative expression of the target gene was normalized to the relative expression of GAPDH by the ΔCt method.

[0087] Western blot analysis: Immunoblotting was used to detect the expression of key proteins in the NF-κB signaling pathway (p-65, p-p65, IκB-α, p-IκB-α) in breast tissue, the expression of proteins in the antioxidant Nrf2 / HO-1 signaling pathway (Nrf2, HO-1), the expression of apoptosis (Bax, Bcl-2) proteins, and the expression of blood-milk barrier (Claudin-3, Occludin and ZO-1) proteins. The specific method includes: taking an appropriate amount of breast tissue, lysing the breast with RIPA lysis buffer containing protease and phosphatase inhibitors for 30 minutes. Protein samples (40-50 μg) were separated by 10-12% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was incubated with the primary antibody (1:1000 dilution) at 4°C overnight, and then incubated with the secondary antibody (1:2000 dilution) at room temperature for 1 hour. Protein bands were imaged using an enhanced chemiluminescence kit (ECL, M0101) according to the manufacturer's instructions. Band intensities were quantified using Image J software.

[0088] like Figure 6 and Figure 7 As shown, the mammary glands of the mice in the blank control group (NC) were intact by naked eye observation, with no visible inflammatory damage such as redness, swelling, heat, or pain ( Figure 6 -A), under the microscope, the mammary gland alveolar structure is intact, there is no neutrophil infiltration in the alveoli, no congestion, edema, milk stasis, or alveolar necrosis ( Figure 7 -A), but the pathological results of the MC group showed that the mammary gland alveolar structure was severely damaged and unclear, and there was a large amount of inflammatory cell infiltration, red blood cell infiltration, interstitial congestion and edema in the alveoli ( Figure 7-B), indicating that the LPS milk pool perfusion method was successful in establishing a mouse mastitis model. After PEP treatment, inflammatory damage in mammary tissue was alleviated in a dose-dependent manner, with the medium-dose, high-dose polysaccharide groups and the positive drug group having good effects. Figure 8 As shown in the figure, compared with the MC group, the number and percentage of neutrophils and the total number of white blood cells were significantly reduced after treatment with low, medium and high doses of PEP and DEX (P<0.05 or P<0.001), the MPO activity was significantly inhibited, and the trend of mammary gland index reduction was significantly reversed (P<0.001). However, compared with the NC group, PEP treatment had no significant effect on ALT and AST in the liver of mice.

[0089] Table 3 Statistics of inflammation-related cells and percentages in routine blood examination

[0090]

[0091] Note: #(P<0.05), ##(P<0.01) and ###(P<0.001) indicate significant differences compared with the NC group, *(P<0.05), **(P<0.01) and ***(P<0.001) indicate significant differences compared with the MC group.

[0092] The results are as follows Fig. 9 As shown in the figure, the relative expression levels of TNF-α, IL-6, and IL-1β pro-inflammatory factors mRNA and protein in the MC group were significantly increased (P<0.001), but after PEP treatment, the relative expression levels of TNF-α, IL-6, and IL-1β in the PEP group were significantly decreased compared with the MC group (P<0.001). Fig.10 As shown in the results, PEP treatment could significantly reduce the expression of p-IκB-α / IκB-α and p-p65 / p65 proteins (P<0.01 or P<0.001), indicating that PEP can significantly inhibit the expression of TNF-α, IL-6, and IL-1β proinflammatory cytokines and relieve breast inflammation by inhibiting the phosphorylation of IκB-α and p65 proteins in the NF-κB signaling pathway.

[0093] In terms of oxidative stress, the results Fig.11 As shown in the results, PEP treatment can significantly enhance the relative expression of antioxidant genes (CAT, GPX-1 and SOD-2) mRNA levels and antioxidant enzymes (CAT, GSH, SOD, T-AOC and MDA) protein levels, and significantly inhibit the production of oxidative product MDA, indicating that PEP can participate in regulating the oxidative stress process and alleviate the inflammatory response of the mouse mammary gland. In terms of potential signaling pathways, the results are as follows Fig.12As shown in the results, PEP treatment can significantly enhance the expression of Nrf2 protein and HO-1 protein (P<0.01 or P<0.001), indicating that PEP can enhance the activity of antioxidant enzymes through the Nrf2 / HO-1 signaling pathway, thereby enhancing the body's antioxidant level and alleviating oxidative damage to breast cells.

[0094] In terms of apoptosis, the results Fig.13 As shown in the results, PEP treatment can significantly enhance the anti-apoptotic effect of Bcl-2, inhibit the expression of pro-apoptotic Bax mRNA and protein levels, and significantly increase the ratio of the relative expression of Bcl-2 to Bax proteins (P<0.001 or P<0.05), and significantly reduce the number of apoptosis-positive cells (P<0.001), indicating that PEP can alleviate inflammatory response by alleviating cell apoptosis damage.

[0095] In terms of blood-milk barrier, the results are as follows Fig.14 As shown in the results, PEP treatment can significantly enhance the expression of tight junction-related proteins (Claudin-3, Occludin and ZO-1) at the mRNA and protein levels, indicating that PEP can alleviate breast inflammatory damage by strengthening the blood-milk barrier and reducing intercellular permeability.

[0096] Example 4: Pleurotus eryngii polysaccharide improves LPS-induced intestinal flora disorder in mice.

[0097] Animal model: Same as Example 3.

[0098] Colon intestinal sample collection: The mouse colon contents collected in Example 3 were collected and stored in a -80°C refrigerator.

[0099] Detection of microbial diversity in colon intestinal contents: The genomic DNA of microorganisms in the collected colon contents was extracted using the CBCT method and stored at -20°C. The purity and concentration of the DNA were detected by 1% agarose gel electrophoresis, and an appropriate amount of sample was taken in a centrifuge tube and diluted to 1 ng / μl with sterile water for later use. The bacterial 16rRNA gene was PCR amplified in the V3-V4 region, and the amplification primers selected were 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'

[0100] -GGACTACNNGGGTATCTAAT-3'), detailed amplification parameters are shown in the instruction manual. The PCR product was then purified using 2% 1xTAE agarose gel electrophoresis, and the target band was recovered using the Universal DNA (TianGen, China) purification and recovery kit. The library was constructed using the Ultra DNA Library Prep Kit, and the constructed library was detected and quantified by Q-PCR using Agilent 5400; after the library was qualified, it was sequenced using the Illumina sequencing platform. The QIIME2 dada2 plug-in was used for quality control, trimming, denoising, and splicing, as well as removal of chimeras, removal of contaminated mitochondrial and chloroplast sequences, and classification unit (Operationaltaxonomic units, OUT) cluster analysis of each sample, and the Alpha diversity level of each sample was evaluated. At the same time, the distance matrix of each sample was calculated to measure the Beta diversity differences between different groups, and the marker difference microorganisms between different groups and samples were screened and identified.

[0101] Result analysis: In this example, LPS milk pool perfusion was used to induce mastitis in mice, resulting in intestinal flora disturbance. 16S rRNA sequencing was performed on the colon feces of mice to evaluate the regulatory effect on intestinal flora disturbance. The results are as follows: Fig.15 As shown, it was found that the rarefaction curves of each group tended to be flat, indicating that the sequencing depth of each sample group met the requirements ( Fig.15 -A). In the NC group, Chao1 (P<0.05) and Observed species (P<0.05) indexes were significantly higher than those in the MC group and other drug-treated groups, while Shannon (P=0.121) and Simpson (P=0.373) indexes were statistically different among the groups. Fig.15 -D showed that there were 239 common OUTs among the groups, and the numbers of OUTs unique to the NC, MC, PC, PEPM and PEPH groups were 704, 384, 378, 241 and 282, respectively, which was basically consistent with the results of Chao1 (P<0.05) and Observed species (P<0.05) indexes. At the same time, PCoA and PLS-DA analysis showed that LPS-induced mastitis in mice may have a certain inhibitory effect on the richness and diversity of intestinal flora, but there was no obvious improvement after DEX and different doses of PEP treatment.

[0102] The TOP10 at the phylum level and TOP20 at the genus level of the intestinal flora community were analyzed, and the results were as follows: Fig.16 As shown. In the phylum level difference analysis, it was found that PEP supplementation could reduce the abundance of Firmicutes, increase the abundance of Bacteroidetes, and reduce the ratio of Firmicutes / Bacteroidetes (F / B), reversing the intestinal flora disorder caused by LPS. At the genus level, the linear discriminant analysis effect size (LEfSe) was performed to analyze the differences in community composition between different groups, and the LDA value was set to 3.0. The results are shown in Fig.17As shown in Figure 2, there are 25 species that pass this threshold. The dominant bacterial community (TOP20 important bacterial genera) was screened by random forest analysis, and the abundance TOP20, LEfSe and TOP20 bacterial genera in random forest were comprehensively analyzed by Venn diagram. The results are shown in Figure 2. Fig.18 As shown in the figure, it was found that there were 3 common bacterial genera among the three, and the different bacterial genera were Lactobacillus, COE1, and Phocaeicola A 858004), there were 9 genera in total in both groups. The abundance of different genera in each group was analyzed statistically. Compared with the NC group, the Lactobacillus genus in the MC group increased significantly (P<0.01), but after treatment with DEX, low, medium and high doses of PEP, the PEPM group and PEPH group were significantly higher than the MC group. Compared with the NC group, the COE1 genus in the MC group decreased significantly (P<0.05), but the trend was not reversed after treatment with drugs in each group. There was no statistical difference in the Paecilomyces genus between the NC and MC groups, but after treatment with DEX and different doses of PEP, it showed a gradual increase trend, among which the PEPH group was significantly higher than the MC group (P<0.05). There was no statistical difference in the Bacteroides genus among the NC, MC, PC and PEPM groups, but the PEPH group was significantly higher than other groups (P<0.05). Compared with the NC group, the Prevotella genus decreased in the MC group, and there was a reversal trend after treatment with drugs in each group, but there was no statistical difference. Therefore, Lactobacillus and COE1 can be used as potential markers of LPS-induced mastitis, while Paecilomyces, Bacteroides, and Prevotella can be used as potential markers of PEP to regulate intestinal flora and alleviate LPS-induced mastitis. The above results show that pre-administration of different doses of PEP has a certain improvement effect on the intestinal flora disorder of LPS-induced mastitis in mice.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Pleurotus eryngii polysaccharide, characterized in that: The Pleurotus eryngii polysaccharide is a neutral polysaccharide, which is composed of at least galactose, glucose, mannose and fucose. The percentages of glucose, mannose, fucose and galactose are 37.700-37.800%, 1.100-1.150%, 0.710-0.720% and the remainder, respectively. In its chemical structure, the polysaccharide is composed of 2,6-α-D-Galp-(1→, →6)-α-D-Galp-(1→ residues constitute the main chain, the branching site is located at C-2 of →2,6)-α-D-Galp-(1→, and there is →6)-α-3-O-Me-D-Galp-(1→, →6)-3-OMe-α-D-Galp-(1→ residues; peak molecular weight is 15800~15900Da.

2. The Pleurotus eryngii polysaccharide according to claim 1, characterized in that: The weight average molecular weight is 19150~19250Da; Or, the number average molecular weight is 13250 to 13300 Da.

3. A method for preparing Pleurotus eryngii polysaccharide according to claim 1, characterized in that: The steps include: Using ethanol aqueous solution to extract Pleurotus eryngii under reduced pressure and reflux to remove fat-soluble substances, and obtaining Pleurotus eryngii residue; The Pleurotus eryngii residue is boiled with water for extraction, and then insoluble components are removed to obtain a supernatant; Adding ethanol or ethanol aqueous solution to the supernatant to adjust the ethanol volume fraction in the system to 77-83%, performing alcohol precipitation treatment, and removing protein from the collected precipitate to obtain crude polysaccharide; First, the crude polysaccharide is subjected to column chromatography by using ion exchange column chromatography, wherein the component eluted by using distilled water is the distilled water component; and then the distilled water component is subjected to chromatography purification by using a gel filtration column.

4. The method for preparing Pleurotus eryngii polysaccharide according to claim 3, characterized in that: In the vacuum reflux extraction, the mass volume ratio of Pleurotus eryngii to ethanol aqueous solution is 1:2-10, kg / L; preferably 1:4.5-5.5, kg / L; preferably, the volume dispersion of ethanol aqueous solution is 92-96%; preferably, the vacuum reflux temperature is 70-90°C, preferably 83-87°C; preferably, the vacuum reflux treatment is 2-4 times, each time for 1-4 hours.

5. The method for preparing Pleurotus eryngii polysaccharide according to claim 3, characterized in that: The cooking temperature is 90-100°C, preferably 95-100°C; preferably, the cooking extraction time is 1-3h, preferably 1.8-2.2h, and the number of cooking extractions is 1-3 times, preferably 2 times; Alternatively, the standing time of the alcohol precipitation treatment is 10 to 40 hours, preferably 22 to 26 hours.

6. The method for preparing Pleurotus eryngii polysaccharide according to claim 3, characterized in that: The protein in the collected precipitate was removed by Sevage method; Or, the ion exchange column chromatography is DEAE-Sepharose FF ion exchange column chromatography; Or, the gel filtration column is a Chromdex 200PG gel filtration column.

7. Use of the Pleurotus eryngii polysaccharide according to claim 1 or 2 in preventing and treating mastitis and / or intestinal flora disorder in animals.

8. The use according to claim 7, characterized in that: The animal is a non-human mammal.

9. Use of the Pleurotus eryngii polysaccharide according to claim 1 or 2 in the preparation of products for preventing and treating mastitis and / or intestinal flora disorders.

10. The use according to claim 9, characterized in that: The product is food, medicine or feed.

Images