A polysaccharide extract of Pleurotus citrinopileatus with the function of regulating flora and relieving constipation, as well as its preparation method and application

Abstract

The present invention discloses a polysaccharide extract of Pleurotus citrinopileatus with the function of regulating microflora and promoting bowel movements, as well as a preparation method and application thereof. The present invention provides the application of the Pleurotus citrinopileatus extract in the preparation of a medicament; the uses of the medicament are as follows: (a1) treating and / or preventing constipation; (a2) promoting intestinal peristalsis; (a3) ​​promoting defecation; (a4) increasing the water content of feces; (a5) improving the structure of intestinal microflora; (a6) regulating intestinal microflora. The preparation method of the Pleurotus citrinopileatus extract is as follows: (1) using Pleurotus citrinopileatus as a raw material to extract crude polysaccharide; (2) removing protein from the crude polysaccharide; (3) sequentially performing separation and purification by anion exchange column chromatography and molecular sieve chromatography; (4) obtaining the Pleurotus citrinopileatus extract. The present invention can be used to treat and / or prevent constipation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a polysaccharide extract of Pleurotus citrinopileatus with the function of regulating flora and relieving constipation, as well as a preparation method and application thereof. Background Art

[0002] Pleurotus citrinopileatus, also known as elm yellow mushroom, golden mushroom, and golden-topped pleurotus, belongs to the subdivision Basidiomycotina, class Hymenomycetes, order Agaricales, family Pleurotaceae, and genus Pleurotus. It is a valuable economic fungus. Its fruiting bodies are rich in crude protein, unsaturated fatty acids, 17 amino acids, vitamins, and various trace elements, meeting the human body's nutritional needs for essential amino acids, vitamins, and beneficial trace elements. It also has high medicinal value, with literature reporting its tonic and strengthening effects, promoting recovery, lowering cholesterol, and treating conditions such as kidney deficiency, impotence, and dysentery.

[0003] Constipation is typically caused by stool moving too slowly through the digestive tract or being unable to be effectively cleared from the rectum, resulting in dehydrated, hardened, and dry stool. Constipation is common in all populations, with an incidence rate of 2%-28%. The prevalence increases significantly with age, with 15%-20% of the elderly experiencing constipation symptoms. Treatment of constipation aims to relieve symptoms and restore normal intestinal motility and bowel function. Summary of the Invention

[0004] The present invention aims to provide a polysaccharide extract of Pleurotus citriodora with the function of regulating flora and relieving constipation, as well as a preparation method and application thereof.

[0005] The present invention provides the use of Pleurotus citrinopileatus extract in preparing medicines;

[0006] The use of the drug is at least one of the following (a1), (a2), (a3), (a4), (a5) and (a6):

[0007] (a1) Treatment and / or prevention of constipation;

[0008] (a2) Promote intestinal motility;

[0009] (a3) promote defecation;

[0010] (a4) Increase the water content of feces;

[0011] (a5) Improve intestinal flora structure;

[0012] (a6) Regulate intestinal flora.

[0013] The Pleurotus citrinopileatus extract can specifically be any of the Pleurotus citrinopileatus extracts described later.

[0014] The Pleurotus citrinopileatus extract can specifically be the Pleurotus citrinopileatus extract prepared by any of the subsequent methods.

[0015] The present invention provides a method for preparing a Pleurotus citrinopileatus extract, comprising the following steps in sequence:

[0016] (1) Extracting crude polysaccharides from Pleurotus citrinopileatus as raw material;

[0017] (2) Removal of proteins from crude polysaccharides;

[0018] (3) performing separation and purification by anion exchange column chromatography and separation and purification by molecular sieve chromatography in sequence;

[0019] (4) Obtaining the Pleurotus citrinopileatus extract.

[0020] The extraction adopts water extraction and alcohol precipitation method.

[0021] The "removal of protein from crude polysaccharide" adopts the Seveag method.

[0022] In the anion exchange column chromatography separation and purification, the filler of the anion exchange chromatography column is cellulose DE-52, and the mobile phase used for elution is NH4HCO3-NH3H2O ​​buffer solution;

[0023] In the molecular sieve chromatography separation and purification, the molecular sieve chromatography column is HiLoad 16 / 600 Superdex, and the mobile phase used for elution is water.

[0024] The present invention also provides a Pleurotus citrinopileatus extract, the preparation method of which comprises the following steps in sequence:

[0025] (1) Extracting crude polysaccharides from Pleurotus citrinopileatus as raw material;

[0026] (2) Removal of proteins from crude polysaccharides;

[0027] (3) performing separation and purification by anion exchange column chromatography and separation and purification by molecular sieve chromatography in sequence;

[0028] (4) Obtaining the Pleurotus citrinopileatus extract.

[0029] The extraction adopts water extraction and alcohol precipitation method.

[0030] The "removal of protein from crude polysaccharide" adopts the Seveag method.

[0031] In the anion exchange column chromatography separation and purification, the filler of the anion exchange chromatography column is cellulose DE-52, and the mobile phase used for elution is NH4HCO3-NH3H2O ​​buffer solution;

[0032] In the molecular sieve chromatography separation and purification, the molecular sieve chromatography column is HiLoad 16 / 600 Superdex, and the mobile phase used for elution is water.

[0033] “Using Pleurotus citrinopileatus as raw material to extract crude polysaccharides” specifically includes the following steps:

[0034] ① Crush the freeze-dried Pleurotus citrinopileatus fruiting body to obtain dry powder;

[0035] ② Take the dry powder obtained in step ①, add water, and let it stand at 2-6℃ for extraction;

[0036] ③After completing step ②, incubate at 85-95°C with shaking, and then collect the supernatant by centrifugation;

[0037] ④ After completing the remaining precipitate in step ③, add water, incubate at 85-95℃ with shaking, and then collect the supernatant by centrifugation;

[0038] ⑤ combining the supernatant collected in step ③ and the supernatant collected in step ④, and concentrating under reduced pressure to obtain a concentrated solution;

[0039] ⑥ Take the concentrated solution obtained in step ⑤, add anhydrous ethanol, and let it stand;

[0040] ⑦ After completing step ⑥, collect the precipitate by centrifugation to obtain the crude polysaccharide.

[0041] "Using Pleurotus citrinopileatus as raw material to extract crude polysaccharides" also includes the following steps: drying the precipitate obtained in step ⑦ to constant weight.

[0042] "Using Pleurotus citrinopileatus as raw material to extract crude polysaccharides" also includes the following steps: drying the precipitate obtained in step ⑦ to a constant weight, and then dissolving it in water to obtain a crude polysaccharide solution.

[0043] “Using Pleurotus citrinopileatus as raw material to extract crude polysaccharides” specifically includes the following steps:

[0044] ① Grinding the freeze-dried Pleurotus citrinopileatus fruiting body to obtain 60-100 mesh dry powder (specifically 80 mesh dry powder);

[0045] ② Take 10g of the dry powder obtained in step ①, add 400mL of deionized water, and let it stand at 4℃ for 10 hours;

[0046] ③After completing step ②, shake well, seal the tube, and then place it in a water bath shaker, incubate at 90°C and 100 rpm for 4 hours, then centrifuge at 7000 rpm for 30 minutes and collect the supernatant;

[0047] ④ Complete the remaining precipitate from step ③, add 200 mL of deionized water, shake well, seal the tube, and then place it in a water bath shaker, incubate at 90°C and 100 rpm for 4 h, then centrifuge at 7000 rpm for 30 min and collect the supernatant;

[0048] ⑤ The supernatant collected in step ③ and the supernatant collected in step ④ were combined and concentrated under reduced pressure (70°C, 0.1 MPa) to a density of 1.2 g / mL to obtain the concentrated solution;

[0049] ⑥ Take the concentrated solution obtained in step ⑤, add 4 times its volume of anhydrous ethanol, stir evenly, then cover with tin foil and let it stand for 12 hours;

[0050] ⑦ After completing step ⑥, centrifuge at 7000 r / min for 15 minutes, collect the precipitate, transfer the precipitate to a 60°C oven and dry it to constant weight, and grind it into powder, which is the crude polysaccharide of Pleurotus citrinopileatus.

[0051] “Using Pleurotus citrinopileatus as raw material to extract crude polysaccharides” also includes the following steps: dissolving 1g of Pleurotus citrinopileatus crude polysaccharides in 50mL of deionized water to obtain a crude polysaccharide solution.

[0052] Removing protein from crude polysaccharides specifically includes the following steps:

[0053] ① Take the crude polysaccharide solution, add Seveag reagent, vortex, and then centrifuge to collect the supernatant;

[0054] ② Take the supernatant obtained in the previous step, add Seveag reagent, vortex, and then centrifuge to collect the supernatant;

[0055] ③ Repeat step ② several times until no protein layer is observed, and collect the supernatant;

[0056] ④ Take the supernatant obtained in step ③, add anhydrous ethanol, let it stand, and then centrifuge to collect the precipitate.

[0057] The method for removing protein from crude polysaccharide further comprises the following step: drying the precipitate obtained in step ④ to a constant weight, which is the protein-removed Pleurotus citrinopileatus polysaccharide.

[0058] The method for removing protein from crude polysaccharide further comprises the following steps: drying the precipitate obtained in step ④ to a constant weight, and then dissolving the precipitate in water to obtain a polysaccharide solution of Pleurotus citrinopileatus.

[0059] Removing protein from crude polysaccharides specifically includes the following steps:

[0060] ① Take the crude polysaccharide solution, add one-third of its volume of Seveag reagent, vortex and shake for 5 minutes, then centrifuge at 4500 rpm for 15 minutes, and collect the supernatant;

[0061] ② Take the supernatant obtained in the previous step, add one-third of its volume of Seveag reagent, vortex for 5 minutes, then centrifuge at 4500 rpm for 15 minutes, and collect the supernatant;

[0062] ③ Repeat step ② several times until no protein layer is observed, and collect the supernatant;

[0063] ④ Take the supernatant obtained in step ③, add 4 times its volume of anhydrous ethanol, stir evenly, then cover with tin foil, let it stand for 12 hours, then centrifuge at 7000r / min for 15min, collect the precipitate, transfer the precipitate to a 60℃ oven and dry it to constant weight, and grind it into powder, which is the protein-removed Pleurotus citrinopileatus polysaccharide.

[0064] The method for removing protein from crude polysaccharide also includes the following steps: dissolving 1 g of protein-removed Pleurotus citriodora polysaccharide in 50 mL of deionized water to obtain a Pleurotus citriodora polysaccharide solution.

[0065] Seveag reagent: Prepared by mixing 4 parts by volume of chloroform and 1 part by volume of n-butanol.

[0066] The parameters of the anion exchange column chromatography are as follows: the filler is cellulose DE-52; the column size is 1×30 cm.

[0067] The polysaccharide solution of Pleurotus citrinopileatus was loaded, and eluted with NH4HCO3-NH3H2O ​​buffer solution as the mobile phase (the flow rate can be 2 ml / min), and the eluate corresponding to the elution peak with a retention volume of 13-84 ml was collected, which was called the D1 component.

[0068] NH4HCO3-NH3H2O ​​buffer solution: pH 9.4, 10mM.

[0069] The D1 component was transferred to a dialysis bag (molecular weight cut-off of 3 KDa), and then dialyzed in deionized water. The liquid phase in the dialysis bag was then collected for subsequent molecular sieve chromatography separation and purification.

[0070] The parameters of the molecular sieve chromatography are as follows: the molecular sieve chromatography column is HiLoad 16 / 600 Superdex 75 pg.

[0071] The liquid phase in the sample dialysis bag was eluted with deionized water as the mobile phase (the flow rate can be 1 ml / min), and the eluate corresponding to the elution peak with a retention volume of 37-45 ml was collected, which was called the SP1 component.

[0072] The SP1 component is freeze-dried to obtain the Pleurotus citrinopileatus extract.

[0073] The monosaccharide composition in any of the above-mentioned Pleurotus citrinopileatus extracts is mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose.

[0074] In any of the above-mentioned Pleurotus citrinopileatus extracts, the mass ratios of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose are: 11253:499:420:839439:14372:399:798.

[0075] In any of the above-mentioned Pleurotus citrinopileatus extracts, the contents of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose are 11253 mg / kg, 499 mg / kg, 420 mg / kg, 839439 mg / kg, 14372 mg / kg, 399 mg / kg and 798 mg / kg, respectively.

[0076] The contents of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose in any of the above Pleurotus citrinopileatus extracts are shown in Table 1, respectively.

[0077] The Mp (maximum peak molecular weight) of any of the above-mentioned Pleurotus citrinopileatus extracts is 7772833 g / mol.

[0078] The Mn (number average molecular weight) of any of the above-mentioned Pleurotus citrinopileatus extracts is 6173761 g / mol.

[0079] The Mw (weight average molecular weight) of any of the above Pleurotus citrinopileatus extracts is 7498604 g / mol.

[0080] The Mz (average molecular weight) of any of the above Pleurotus citrinopileatus extracts is 8522992 g / mol.

[0081] The monosaccharide linkage mode of any of the above-mentioned Pleurotus citrinopileatus extracts is shown in Table 3.

[0082] The present invention provides a method for preparing a Pleurotus citrinum extract and the Pleurotus citrinum extract prepared by the method. The Pleurotus citrinum extract is rich in polysaccharides, has the effects of improving intestinal flora structure and promoting defecation, and can be used to treat and / or prevent constipation. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is the elution curve of anion exchange chromatography separation and purification.

[0084] Figure 2 This is the elution curve of molecular sieve chromatography separation and purification.

[0085] Figure 3 These are the monosaccharide composition and content results of Pleurotus citrinopileatus extract.

[0086] Figure 4 The chromatogram is that of the standard.

[0087] Figure 5 This is the ink advancement rate result of animal experiment.

[0088] Figure 6 This is the total weight of feces from animal tests.

[0089] Figure 7 This is the result of the first red stool excretion time in animal experiments.

[0090] Figure 8 This is the result of the number of red stools in animal experiments.

[0091] Figure 9 This is the feces moisture content result from animal tests.

[0092] Figure 10 16S rRNA sequencing results of animal fecal fermentation products (abundance percentage of intestinal flora at the genus level).

[0093] Figure 11 It is the gas production result of animal feces fermentation.

[0094] Figure 12 This is a heat map of the bacterial composition and gas production of animal feces fermentation products.

[0095] Figure 13 These are the results of short-chain fatty acid determination in animal feces fermentation products.

[0096] Figure 14 Heat map of bacterial composition and short-chain fatty acids in animal feces fermentation products. DETAILED DESCRIPTION

[0097] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in this area or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Fructooligosaccharide: Shanghai Yuanye Biological Company, Article No. S11133. Unless otherwise specified, the quantitative tests in the following examples were repeated three times, and the results were averaged.

[0098] Intestinal motility and fecal data were statistically analyzed using DPS 7.05. P < 0.05 or P < 0.01 indicated statistical significance. Gas production and short-chain fatty acids (SCFAs) data were analyzed using nonparametric tests in GraphPad Prism 8 software. P < 0.05 or P < 0.01 indicated statistical significance. 16S rRNA sequencing results were analyzed using the recommended analysis methods of the Meiji Biotechnology One-Stop Research Service Platform.

[0099] The elm yellow mushroom used in the embodiment is elm yellow mushroom (Pleurotus citrinopileatus), also known as elm yellow mushroom (Pleurotus citrinopileatus Singer), which was collected on April 9, 2013 at the Forestry Microorganism Center of China Culture Collection Committee (also known as China Forestry Culture Collection Center, China Forestry Culture Collection Center, English abbreviation CFCC, referred to as Forestry Microorganism Center), with the deposit number CFCC89573. The strain is available to the public from CFCC since the date of collection.

[0100] Experimental animals: Kunming mice, male, 22-24 g (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., strain code 202).

[0101] Example 1. Preparation and identification of Pleurotus citrinopileatus extract

[0102] 1. Preparation of Pleurotus citrinopileatus Extract

[0103] 1. Extraction of crude polysaccharides by water extraction and alcohol precipitation

[0104] (1) The freeze-dried Pleurotus citrinopileatus fruiting bodies were placed in a high-speed universal crusher and crushed repeatedly for 4 times (20 seconds each time) to obtain a uniform dry powder (about 80 mesh).

[0105] (2) Weigh 10 g of the dry powder obtained in step (1), add 400 mL of deionized water, and let it stand at 4°C for 10 hours.

[0106] (3) After completing step (2), shake well, seal the tube, and then place it in a water bath shaker, incubate at 90°C and 100 rpm for 4 h, then centrifuge at 7000 rpm for 30 min and collect the supernatant.

[0107] (4) Complete the remaining precipitate from step (3), add 200 mL of deionized water, shake well, seal, and then place in a water bath shaker, incubate at 90°C and 100 rpm for 4 h, then centrifuge at 7000 rpm for 30 min, and collect the supernatant.

[0108] (5) The supernatant collected in step (3) and the supernatant collected in step (4) were combined and concentrated under reduced pressure (70° C., 0.1 MPa) to a density of 1.2 g / mL to obtain a concentrated solution.

[0109] (6) Take the concentrated solution obtained in step (5), add 4 times its volume of anhydrous ethanol, stir evenly, then cover with tin foil and let it stand for 12 hours.

[0110] (7) After completing step (6), centrifuge at 7000 r / min for 15 min, collect the precipitate, transfer the precipitate to a 60°C oven and dry it to constant weight, and grind it into powder, which is the crude polysaccharide of Pleurotus citrinopileatus.

[0111] (8) Dissolve 1 g of crude polysaccharide from Pleurotus citrinopileatus in 50 mL of deionized water to obtain a crude polysaccharide solution.

[0112] 2. Removal of protein from crude polysaccharides of Pleurotus citrinopileatus (using Seveag method to remove protein)

[0113] Seveag reagent: Prepared by mixing 4 parts by volume of chloroform and 1 part by volume of n-butanol.

[0114] (1) Take the crude polysaccharide solution prepared in step 1, add one-third of its volume of Seveag reagent, vortex and shake for 5 minutes, then centrifuge at 4500 rpm for 15 minutes (free protein will produce a gel-like precipitate), and collect the supernatant.

[0115] (2) Take the supernatant obtained in the previous step, add one-third of its volume of Seveag reagent, vortex and shake for 5 minutes, then centrifuge at 4500 rpm for 15 minutes, and collect the supernatant.

[0116] (3) Repeat step (2) until no protein layer is observed, and collect the supernatant.

[0117] (4) Take the supernatant obtained in step (3), add 4 times the volume of anhydrous ethanol, stir evenly, then cover with tin foil, let it stand for 12 hours, then centrifuge at 7000r / min for 15 minutes, collect the precipitate, transfer the precipitate to a 60°C oven and dry it to constant weight, and grind it into powder, which is the protein-removed Pleurotus citrinopileatus polysaccharide.

[0118] (5) Dissolve 1 g of protein-removed Pleurotus citrinopileatus polysaccharide in 50 mL of deionized water to obtain a Pleurotus citrinopileatus polysaccharide solution.

[0119] 3. Anion exchange chromatography separation and purification

[0120] Anion exchange chromatography column: Cellulose DE-52 (Beijing Coolbo Technology Co., Ltd., Cat. No. CC3311) was used as the filler; column dimensions were 1 × 30 cm. NH₄HCO₃-NH₃H₂O buffer solution was used: pH 9.4, 10 mM.

[0121] The anion exchange chromatography column was equilibrated with NH4HCO3-NH3·H2O buffer solution, and then the Pleurotus citrinopileatus polysaccharide solution prepared in step 2 was loaded, followed by two-step elution, both at a flow rate of 2 ml / min.

[0122] The two-step elution process is as follows: after the sample is loaded, the first elution is carried out directly, and the mobile phase used is NH4HCO3-NH3H2O ​​buffer solution. The eluate after passing through the column is continuously collected with a collection tube (4 mL per tube), and 21 tubes are collected continuously; after the first elution is completed, the second elution is carried out directly, and the mobile phase used is NH4HCO3-NH3H2O ​​buffer solution containing 0.2M NaCl. The eluate is continuously collected with a collection tube (4 mL per tube), and 12 tubes are collected continuously.

[0123] The elution curve of anion exchange chromatography separation and purification is shown in Figure 1 .

[0124] The polysaccharide concentration of the solution in each collection tube was tested by sulfuric acid phenol method. The results are shown in Figure 1 .

[0125] Finally, the eluates from tubes 4 to 21 were combined (the retention volumes of the corresponding elution peaks were 13-84 ml) and called the D1 component.

[0126] 4. The D1 fraction obtained in step 3 was transferred to a dialysis bag (molecular weight cut-off of 3 KDa), and then dialyzed in deionized water for 4 hours, and the liquid phase in the dialysis bag was collected.

[0127] 5. Molecular sieve chromatography separation and purification

[0128] Molecular sieve chromatography column: HiLoad 16 / 600 Superdex 75 pg (GE Healthcare, catalog number 28-9893-33).

[0129] Load the liquid phase from step 4 into the dialysis bag (1 ml sample volume) and elute with deionized water at a flow rate of 1 ml / min. Continue collecting the eluate from the column using collection tubes (3 ml per tube) for a total of 41 tubes.

[0130] The elution curve of molecular sieve chromatography separation and purification is shown in Figure 2 .

[0131] The polysaccharide concentration of the solution in each collection tube was tested by sulfuric acid phenol method. The results are shown in Figure 2 .

[0132] Finally, the eluates from tubes 13 to 15 were combined (the retention volume of the corresponding elution peak was 37-45 ml), which was called the SP1 component.

[0133] 6. Take the SP1 component obtained in step 5, pre-freeze it at -80°C, and then freeze-dry it to obtain a dry powder, which is the Pleurotus citrinopileatus extract (expressed as PCP).

[0134] 2. Identification of Pleurotus citrinopileatus Extract

[0135] 1. Monosaccharide composition and content of Pleurotus citrinopileatus extract

[0136] The monosaccharide and uronic acid composition and mass ratio of PCP were determined by liquid chromatography using the method of polysaccharide alcohol acetate derivatives.

[0137] Reagents: trifluoroacetic acid, acetonitrile (chromatographic grade), phosphate buffer solution (pH = 6.8), monosaccharide and uronic acid standards (mannose, rhamnose, glucose, arabinose, fucose, xylose, galactose, galacturonic acid, glucuronic acid, etc.).

[0138] (1) Complete acid hydrolysis

[0139] Weigh an appropriate amount of PCP, add 1 mL of water, and then add 1 mL of 4 mol / L trifluoroacetic acid solution. Aerate with nitrogen and hydrolyze at 110°C for 120 min. Remove and cool to room temperature. Dry in a vacuum oven at 60°C for 2 h. Standard Preparation: First, prepare a 10 mg / mL standard solution and store at -20°C. Thaw when needed. Add 5 μL of each of the above standards to a sealable glass tube and mix thoroughly. Then, add 1 mL of 4 mol / L trifluoroacetic acid solution. Hydrolyze at 110°C for 120 min simultaneously with the sample. Blow dry with an air pump.

[0140] (2) PMP derivatization

[0141] To the sample obtained after hydrolysis and drying, add 0.5 ml each of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent and 0.3 mol / L NaOH solution dissolved in anhydrous methanol. Mix thoroughly and react in a water bath at 70°C for 60 min. Cool to room temperature, add 0.5 ml of 0.3 mol / L HCl, and mix thoroughly. Add 0.5 ml of chloroform, shake thoroughly, and extract. Centrifuge (5000 rpm, 5 min) to remove the chloroform layer. Extract three times. Filter the aqueous layer (minimum 0.4 ml) through a 0.22 μm filter membrane before loading onto the analyzer.

[0142] (3) Instrument conditions

[0143] Chromatographic column: SHISEIDO C18 column (4.6×250 mm, 5 μm);

[0144] Mobile phase: 0.1 mol / L pH 6.8 phosphate buffer (PB): acetonitrile = 82:18 (v / v);

[0145] Flow rate: 1.0 mL / min;

[0146] Column temperature: 25°C;

[0147] Injection volume: 10 μL

[0148] Wavelength: 245nm.

[0149] Instrument: Agilent 1200 high performance liquid chromatograph.

[0150] The results are shown in Table 1 and Figure 3 The chromatogram of the standard is shown in Figure 4 .

[0151] Table 1 Monosaccharide composition and content results

[0152] Monosaccharide composition Content (mg / kg) Mannose 11253.33 Rhamnose 499.2 Glucuronic acid 420.27 glucose 839438.93 Galactose 14372.27 arabinose 398.93 Fucose 797.87

[0153] 2. Molecular weight of Pleurotus citriodora extract

[0154] The molecular weight of PCP was determined by gel chromatography.

[0155] Gel chromatography: Wyatt, ELEOS System;

[0156] Chromatographic column: Shodex OHpak series SB-806 series 803;

[0157] Mobile phase: 0.02 g / 100 ml NaN3 aqueous solution;

[0158] Detector: Waters 515 pump laser detector (LS) differential refractive index detector (DRI).

[0159] The results are shown in Table 2.

[0160] Table 2 PCP molecular weight test results

[0161]

[0162]

[0163] 3. Analysis of monosaccharide linkage patterns of Pleurotus citrinopileatus extract

[0164] PCP was submitted to Qingdao Kechuang Quality Inspection Co., Ltd. for polysaccharide methylation analysis to determine the polysaccharide linkage characteristics and content. The results of the analysis of monosaccharide linkage patterns within PCP are shown in Table 3.

[0165] Table 3 Analysis results of monosaccharide connection mode within PCP

[0166] Connection method Relative molar ratio (%) T-Manp 1.076 T-Glcp 16.527 3-Glcp 0.182 2-Glcp 0.749 6-Manp 0.678 4-Glcp 64.379 6-Galp 2.652 3,4-Glcp 2.095 4,6-Glcp 10.345 2,5-Araf 0.623 3,4,6-Glcp 0.694

[0167] Example 2: Application of Pleurotus citrinopileatus extract

[0168] 1. Group processing

[0169] The experimental animals were randomly divided into 5 groups, with 10 animals in each group.

[0170] The five groups were: negative control group, FO positive control group, model group, PCP low-dose group, and PCP high-dose group.

[0171] From the 1st to the 7th day of the experiment, each group of experimental animals was fed in an environment with a temperature of 22°C and a humidity of 50% to 60% for one week. From the 8th to the 21st day of the experiment, the low-dose PCP group was gavaged with PCP solution once a day (the single dose of PCP was 1.0 g / kg body weight). From the 8th to the 21st day of the experiment, the high-dose PCP group was gavaged with PCP solution once a day (the single dose of PCP was 2.0 g / kg body weight). From the 8th to the 21st day of the experiment, the FO positive control group was gavaged with oligofructose solution once a day (the single dose of oligofructose was 1.0 g / kg body weight). From the 8th to the 21st day of the experiment, the negative control group was gavaged with distilled water once a day. From the 8th to the 21st day of the experiment, the model group was gavaged with distilled water once a day.

[0172] The PCP solution was obtained by dissolving the PCP prepared in step 1 of Example 1 in distilled water.

[0173] The oligofructose solution is obtained by dissolving oligofructose in distilled water.

[0174] The oral gavage volume for a single experimental animal was 0.5-1 ml.

[0175] During the experiment, the experimental animals were fed a normal diet, and their morphology and respiratory characteristics were observed every day.

[0176] During the experiment, the hair, skin and respiratory morphology of each group of experimental animals were normal. The results showed that PCP had no significant effect on the growth and development of the experimental animals.

[0177] 2. Intestinal motility experiment

[0178] After completing step 1, 5 experimental animals were randomly selected from each group to perform an intestinal motility experiment.

[0179] The intestinal motility experiment was carried out in the following steps:

[0180] 1. On the 22nd day of the experiment (counted consecutively with the number of days in step 1), all experimental animals were fasted but not watered for 16 hours.

[0181] 2. After completing step 1, each group of experimental animals shall undergo the following operations:

[0182] The experimental animals in the FO positive control group, model group, PCP low-dose group and PCP high-dose group were all gavaged with sucralfate suspension once (the dose of sucralfate was 4g / kg body weight). The experimental animals in the negative control group were gavaged with distilled water.

[0183] Sucralfate suspension was prepared by suspending sucralfate in distilled water. The oral volume for each test animal was 1 ml.

[0184] 3. 30 minutes after completing the gavage in step 2, each group of experimental animals underwent the following operations (all single gavage):

[0185] FO positive control group: oral administration of fructooligosaccharide carmine solution (the dosage of fructooligosaccharide was 1.0 g / kg body weight);

[0186] PCP low-dose group: PCP carmine solution was administered orally (the PCP dosage was 1.0 g / kg body weight);

[0187] PCP high-dose group: PCP carmine solution was administered orally (the PCP dosage was 2.0 g / kg body weight);

[0188] Negative control group: carmine solution was administered orally;

[0189] Model group: Carmine solution was administered orally.

[0190] The PCP carmine solution is obtained by dissolving the PCP prepared in step 1 of Example 1 in the carmine solution.

[0191] The fructooligosaccharide carmine solution is obtained by dissolving fructooligosaccharide in carmine solution.

[0192] Carmine solution: Sigma, product number 41325.

[0193] The gavage volume for a single experimental animal was 0.5-1 ml.

[0194] 4. 25 minutes after completing the oral gavage in step 3, sacrifice the animal by cervical dislocation. Open the abdominal cavity and excise the intestinal tract from the pylorus to the cecum. Without pulling, lay the intestinal tract flat and straighten it. Measure the "total small intestine length" and "ink propagation length" and calculate the ink propagation rate. The "ink propagation length" is the distance from the pylorus to the carmine front.

[0195]

[0196] The results are shown in Table 4 and Figure 5 (Compared with the model group, # represents P < 0.05, ## represents P < 0.01). After the mouse constipation model was established with sucralfate, the ink propulsion rate of the mice in the model group was significantly lower than that in the negative control group, and the difference was statistically significant, indicating that the small intestinal peristalsis was inhibited by sucralfate and the mouse constipation model was successfully established. The ink propulsion rates of mice in the FO positive control group, PCP low-dose group, and PCP high-dose group were all higher than those in the model group, and the difference was statistically significant. The ink propulsion rate of mice in the PCP high-dose group was higher than that in the FO positive control group. The results showed that PCP can promote intestinal peristalsis in constipated mice, and the promoting effect of high-dose PCP is the most obvious.

[0197] Table 4 Ink advancement rate (%)

[0198]

[0199]

[0200] 3. Fecal Experiment

[0201] After completing step 1, 5 mice were randomly selected from each group for fecal experiment.

[0202] The feces experiment was carried out in the following steps:

[0203] 1. Same as step 1 in step 2.

[0204] 2. Same as step 2.

[0205] 3. Same as step 3 in step 2.

[0206] Eat normally and observe continuously.

[0207] The time of the first appearance of red feces after drug administration was recorded for each animal.

[0208] Record the number of red fecal pellets of each animal within 5 hours (starting from the completion of gavage in step 3).

[0209] Weigh the total weight of red feces (W1, in g) of each animal within 5 h (starting from the completion of gavage in step 3), measure the weight after drying (W2, in g), and calculate the water content of feces.

[0210]

[0211] The results of the total weight of feces of each mouse at 5 h are shown in Tables 5 and Figure 6(Compared with the model group, # represents P < 0.05, ## represents P < 0.01). After the mouse constipation model was established with sucralfate, the total fecal weight of the model group mice was significantly lower than that of the negative control group, and the difference was statistically significant, indicating that the mouse constipation model was successfully established. The total fecal weight of the mice in the FO positive control group, PCP low-dose group, and PCP high-dose group was higher than that in the model group, and the difference was statistically significant. The results showed that PCP can increase the amount of stool in constipated mice and promote defecation in constipated mice.

[0212] Table 5 Total weight of mouse feces (g)

[0213] Model Group FO positive control group Negative control group PCP low-dose group PCP high-dose group 0.07±0.01 <![CDATA[0.19±0.06 ## ]]> <![CDATA[0.12±0.03 # ]]> <![CDATA[0.24±0.02 ## ]]> <![CDATA[0.19±0.03 ## ]]>

[0214] The time of first appearance of red stool is shown in Table 6 and Figure 7 (Compared with the model group, # represents P < 0.05, ## represents P < 0.01). After the constipation model of mice was established with sucralfate, the time to first defecation of the mice in the model group was significantly longer than that in the negative control group, and the difference was statistically significant, indicating that the constipation model of mice was successfully established. Compared with the model group, the time to first defecation of the mice in the FO positive control group, the low-dose PCP group, and the high-dose PCP group was significantly reduced, and the difference was statistically significant. The results show that PCP can shorten the defecation time of constipated mice.

[0215] Table 6 Time of first red stool excretion in mice (min)

[0216] Model Group FO positive control group Negative control group PCP low-dose group PCP high-dose group 207.7±7.64 <![CDATA[92.8±28.30 ## ]]> <![CDATA[117.0±30.05 ## ]]> <![CDATA[112.5±24.75 ## ]]> <![CDATA[141.3±15.15 ## ]]>

[0217] The number of red feces pellets per mouse at 5 h is shown in Table 7 and Figure 8 (Compared with the model group, # represents P < 0.05, ## represents P < 0.01). After the constipation model of mice was established with sucralfate, the number of red fecal pellets in the model group was significantly lower than that in the negative control group, and the difference was statistically significant, indicating that the constipation model of mice was successfully established. Compared with the model group, the number of red fecal pellets in the FO positive control group, the PCP low-dose group, and the PCP high-dose group were significantly increased, and the difference was statistically significant. The results show that PCP can increase the amount of stool in constipated mice and promote defecation.

[0218] Table 7 Number of red stools in mice (granules)

[0219] Model Group FO positive control group Negative control group PCP low-dose group PCP high-dose group 4.3±1.53 <![CDATA[15.0±4.97 ## ]]> <![CDATA[7.7±3.06 ## ]]> <![CDATA[15.0±0.00 ## ]]> <![CDATA[14.5±3.70 ## ]]>

[0220] The fecal water content is shown in Table 8 and Figure 9(Compared with the model group, # represents P < 0.05, and ## represents P < 0.01.) After establishing a constipation model in mice with sucralfate, the fecal water content of the model group was significantly lower than that of the negative control group, with a statistically significant difference, indicating that the constipation model was successfully established. The fecal water content of mice in the FO positive control group, the low-dose PCP group, and the high-dose PCP group was higher than that of the model group, with statistically significant differences. These results indicate that PCP can increase fecal water content.

[0221] Table 8 Water content of mouse feces (%)

[0222] Model Group FO positive control group Negative control group PCP low-dose group PCP high-dose group 36.2±3.50 <![CDATA[48.8±3.75 # ]]> <![CDATA[47.4±3.42 # ]]> <![CDATA[51.4±1.48 ## ]]> <![CDATA[50.9±1.53 ## ]]>

[0223] Example 3: Effect of Pleurotus citrinopileatus Extract on Intestinal Flora

[0224] 1. Group processing

[0225] The experimental animals were randomly divided into 4 groups, with 10 animals in each group.

[0226] The four groups were: negative control group, FO positive control group, model group, and PCP group.

[0227] From day 1 to day 7 of the experiment, each group of experimental animals was fed an adaptive diet at a temperature of 22°C and a humidity of 50% to 60% for one week. From day 8 to day 21 of the experiment, the PCP group was gavaged with PCP solution once daily (the single dose of PCP was 2.0 g / kg body weight). From day 8 to day 21 of the experiment, the FO positive control group was gavaged with oligofructose solution once daily (the single dose of oligofructose was 1.0 g / kg body weight). From day 8 to day 21 of the experiment, the negative control group was gavaged with distilled water once daily. From day 8 to day 21 of the experiment, the model group was gavaged with distilled water once daily.

[0228] The PCP solution was obtained by dissolving the PCP prepared in step 1 of Example 1 in distilled water.

[0229] The oligofructose solution is obtained by dissolving oligofructose in distilled water.

[0230] The oral gavage volume for a single experimental animal was 0.5-1 ml.

[0231] 2. Further processing

[0232] After completing step 1, each group of experimental animals will follow the following steps:

[0233] 1. On the 22nd day of the experiment (counted consecutively with the number of days in step 1), all experimental animals were fasted but not watered for 16 hours.

[0234] 2. After completing step 1, each group of experimental animals shall undergo the following operations:

[0235] The experimental animals in the FO positive control group, model group, and PCP group were all gavaged with sucralfate suspension once (the dosage of sucralfate was 4 g / kg body weight); the experimental animals in the negative control group were gavaged with distilled water.

[0236] Sucralfate suspension was prepared by suspending sucralfate in distilled water. The oral volume for each test animal was 1 ml.

[0237] 3. After completing step 2, collect feces from each experimental animal within 5 hours (starting from the completion of gavage in step 2).

[0238] 3. Testing

[0239] PCP represents the PCP group. FOS represents the FO positive control group. MOD represents the model group. CON represents the negative control group.

[0240] 1. In vitro fermentation

[0241] Feces from each group of mice were collected and suspended in PBS buffer to a concentration of 100 g wet weight / L to prepare a fecal suspension. The fecal suspension was filtered through a sterile metal sieve with a pore size of 0.125 mm, and the filtrate was collected. 500 μL of the filtrate was inoculated into a culture flask containing 5 ml of YCFA medium (Qingdao Haibo Biotechnology, HB9212). The flask was then sealed and incubated at 37°C for 24 h.

[0242] 2. Gas production detection

[0243] After completing step 1, use a gas meter to measure the content of methane (CH4), ammonia (NH3), hydrogen sulfide (H2S), hydrogen (H2) and carbon dioxide (CO2) in the culture bottle and record them.

[0244] 3. Short-chain fatty acid determination and 16S rRNA sequencing

[0245] After completing step 1, centrifuge at 9000 rpm for 3 minutes, and collect the supernatant and precipitate. The supernatant was analyzed for short-chain fatty acid (SCFA) metabolism using gas chromatography. Total DNA was extracted from the precipitate and then analyzed by 16S rRNA sequencing.

[0246] 4. Results

[0247] (1) 16S rRNA sequencing results

[0248] See the results Figure 10. The relative abundance of Parabacteroides in the model group was lower than that in the negative control group (P<0.05), and the relative abundance of Lachnospiraceae in the model group was lower than that in the negative control group (P<0.05), indicating that modeling affects the relative abundance of Parabacteroides and Lachnospiraceae. Under the premise of successful model establishment, compared with the model group, the relative abundance of Parabacteroides in the PCP group increased (P<0.05), and the relative abundance of Lachnospiraceae increased (P<0.05). The results show that Pleurotus citriodora polysaccharides can improve the structure of intestinal flora and alleviate dysbacteriosis.

[0249] (2) Gas production results

[0250] The results are shown in Table 9 and Figure 11 (Compared with the model group, # represents P < 0.05, ## represents P < 0.01). There were no significant differences in the gas production between the negative control group and the model group (P > 0.05), and the differences were not statistically significant, indicating that modeling had little effect on gas production. Compared with the model group, H2S production in the PCP group and the FO positive control group decreased by 99.72% (P = 0.001) and 98.23% (P = 0.001), respectively, with statistically significant differences. NH3 production decreased by 90.43% (P = 0.0015) and 88.69% (P = 0.0016), respectively, with statistically significant differences. CH4 production decreased by 76.03% (P = 0.0001) and 35.51% (P = 0.0292), respectively, with statistically significant differences. There were no significant differences in H2 and CO2 production among the groups (P > 0.05).

[0251] Table 9 Gas production (ppm)

[0252] Negative control group PCP group FO positive control group Model Group <![CDATA[CH4]]> 45±22.23 <![CDATA[35±4.51 ## ]]> <![CDATA[85±2.89 # ]]> 130±5.77 <![CDATA[NH3]]> 96±25.77 <![CDATA[6±1.15 ## ]]> <![CDATA[9±0.58 ## ]]> 60±20.82 <![CDATA[H2]]> 5339±1813.23 1223±467.66 241±321.71 1893±866.38 <![CDATA[H2S]]> 2000±526.54 <![CDATA[41±10.02 ## ]]> <![CDATA[5±1.53 ## ]]> 1519±279.75 <![CDATA[CO2]]> 1.1±0.15 1.2±0.15 0.9±0.15 1.1±0.06 Total 7340.1±1253.71 1265.2±400.89 246.9±509.68 3413.1±1780.73

[0253] Heat map see Figure 12The heat map shows that the production of CH4 is mainly related to Psychrobacter, Sporosarcina and Atopostipes; while Glutamicibacter, Staphylococcus, Sporosarcina, Psychrobacter, Jeotgalicoccus and Clostridium_sensu_stricto_1 ) and Atopostipes mainly metabolize to produce NH3; the production of H2 and H2S is related to the metabolism of Glutamicibacter, Staphylococcus, Streptococcus and Enterorhabdus, indicating that Pleurotus citrino polysaccharides can reduce gas production by inhibiting the metabolism of intestinal flora such as Glutamicibacter, Staphylococcus and Streptococcus.

[0254] (3) Analysis of short-chain fatty acids (SCFAs) determination results

[0255] The results are shown in Table 10 and Figure 13 (Compared with the model group, # represents P < 0.05, and ## represents P < 0.01.) Compared with the negative control group, the production of acetic acid, propionic acid, and butyric acid in the model group decreased by 82.33% (P < 0.0001), 85.95% (P = 0.0117), and 80.59% (P = 0.0359), respectively. These differences were statistically significant. There was no significant difference in the production of valeric acid, isovaleric acid, and isobutyric acid in the model group (P > 0.05), indicating that establishing a constipation model reduces the production of acetic acid, propionic acid, and butyric acid, but has no significant effect on the production of valeric acid, isovaleric acid, and isobutyric acid. On the premise that the model was successfully established, compared with the model group, the acetic acid production in the PCP group and the FO positive control group increased by 80.03% (P<0.0001) and 67.10% (P=0.0222), respectively, and the differences were statistically significant. There was no significant difference in the production of propionic acid, butyric acid, valeric acid, isovaleric acid and isobutyric acid among the groups (P>0.05).

[0256] Table 10 Short-chain fatty acid content (mmol / L)

[0257] Negative control group PCP group FO positive control group Model Group Acetic acid <![CDATA[6.8704±1.22 ## ]]> <![CDATA[5.8312±0.23 ## ]]> <![CDATA[2.543±0.66 # ]]> 1.3642±0.31 Propionic acid <![CDATA[2.0633±0.77 # ]]> 0.4688±0.19 0.0981±0.08 0.2552±0.08 Isobutyric acid 0.0562±0.03 0.0127±0.00 0.007±0.00 0±0.00 Butyric acid <![CDATA[0.9978±0.32 # ]]> 0.1275±0.07 0.1914±0.10 0.2148±0.08 Isovalerate 0.2283±0.11 0.0353±0.01 0.017±0.01 0.036±0.01 Valeric acid 0.0342±0.01 0.0108±0.00 0.0158±0.00 0.0087±0.02 Total 10.2502±2.42 6.4863±0.44 2.8723±0.76 1.8789±0.45

[0258] Heat map see Figure 14 . Figure 14In Chinese: Ace represents acetic acid; Val represents valeric acid; Isob represents isobutyric acid; But represents butyric acid; Pro represents propionic acid; Isov represents isovaleric acid. The heat map shows that Clostridia and Rikenella genera primarily metabolize to produce acetic acid, propionic acid, butyric acid, and isobutyric acid; Odoribacter primarily metabolizes to produce propionic acid, butyric acid, and isobutyric acid; Desulfovibrio primarily metabolizes to produce acetic acid and isobutyric acid; Parabacteroides metabolizes to produce acetic acid and propionic acid; and Alistipes metabolizes to produce acetic acid and butyric acid. Acetate production is also associated with Ruminococcaceae and Oscillospiraceae, while propionic acid production is also associated with Gordonibacter. These results suggest that Pleurotus citrinoides polysaccharides may increase SCFA production by promoting the metabolism of related intestinal microbiota.

[0259] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application of Pleurotus citrinopileatus extract as a single active ingredient in the preparation of medicines; The use of the medicine is to treat and / or prevent constipation; The monosaccharide composition in the Pleurotus citrinopileatus extract is mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose.

2. A method for preparing a Pleurotus citrinopileatus extract, comprising the following steps: (1) Extracting crude polysaccharides from Pleurotus citrinopileatus as raw material; (2) Removal of proteins from crude polysaccharides; (3) performing separation and purification by anion exchange column chromatography and separation and purification by molecular sieve chromatography in sequence; (4) obtaining an extract of Pleurotus citrinopileatus; The monosaccharide composition in the Pleurotus citrinopileatus extract is mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose and fucose.

3. The Pleurotus citrinopileatus extract according to claim 2, wherein: The extraction adopts water extraction and alcohol precipitation method.

4. The Pleurotus citrinopileatus extract according to claim 2, wherein: The "removal of protein from crude polysaccharides" adopts the Seveag method.

5. The Pleurotus citrinopileatus extract according to claim 2, wherein: In the anion exchange column chromatography separation and purification, the filler of the anion exchange chromatography column is cellulose DE-52, and the mobile phase used for elution is NH4HCO3-NH3H2O ​​buffer solution; In the molecular sieve chromatography separation and purification, the molecular sieve chromatography column is HiLoad 16 / 600S uperdex, and the mobile phase used for elution is water.

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