Low-molecular-weight flammulina velutipes polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and food, and discloses a low-molecular-weight flammulina velutipes polysaccharide as well as a preparation method and application thereof. The flammulina velutipes polysaccharide with low molecular weight is prepared by preparing flammulina velutipes crude polysaccharide through an ultrasonic-assisted hot water extraction method and purifying through ion-exchange chromatography and gel chromatography. Through detection, the flammulina velutipes polysaccharide with low molecular weight is neutral polysaccharide, has alpha-type pyranoside, and is glucan which takes-> 4)-alpha-D-Glcp-(1-> and-> 4, 6)-alpha-D-Glcp-(1-> as main chains and alpha-D-Glcp-(1-> as side chains. Pharmacological experiments show that the flammulina velutipes polysaccharide with low molecular weight has a very strong anti-inflammatory effect on inflammation stimulated by lipopolysaccharide (LPS), and can play the anti-inflammatory effect by down-regulating the levels of dimethyl phosphatidyl ethanolamine and histamine.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and food, and relates to polysaccharide substances, their extraction methods and their uses in the medical field. Specifically, it relates to a Flammulina velutipes polysaccharide, its preparation method and application. Background Art

[0002] As one of the most popular edible fungi in the world, Flammulina velutipes not only tastes delicious, but also has very rich nutritional value, being rich in carbohydrates, dietary fiber, protein, vitamins, etc. In recent years, polysaccharide, as one of the important bioactive components of Flammulina velutipes, has become a research hotspot in the fields of food and medicine. Many studies have shown that Flammulina velutipes polysaccharide (FVP) has functions such as antioxidant, anti-inflammatory, lipid-lowering, tumor inhibition, immune regulation, and memory improvement.

[0003] Currently, the reported Flammulina velutipes polysaccharides are mostly high-molecular-weight polysaccharides, up to hundreds of thousands, millions or even tens of millions of daltons. Little attention has been paid to low-molecular-weight Flammulina velutipes polysaccharides. The structure of polysaccharides is closely related to their activities. High-molecular-weight polysaccharides are not conducive to absorption and mostly play a role in health regulation through the mediation of intestinal flora. However, existing research mostly focuses on high-molecular-weight polysaccharides, and the relationship between the structure and function of low-molecular-weight polysaccharides is not yet clear, which limits their application potential. Summary of the Invention

[0004] In view of the above technical problems existing in the prior art, the present invention prepares crude Flammulina velutipes polysaccharide by ultrasonic-assisted hot water extraction method, and then purifies it by ion exchange chromatography and gel chromatography to obtain low-molecular-weight Flammulina velutipes polysaccharide. It is detected that the low-molecular-weight Flammulina velutipes polysaccharide is a neutral polysaccharide and there is α-type pyranoside. Pharmacological experiments show that the low-molecular-weight Flammulina velutipes polysaccharide has a strong anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated inflammation and can play an anti-inflammatory role by down-regulating the levels of dimethylphosphatidylethanolamine and histamine.

[0005] One object of the present invention is to provide a low-molecular-weight Flammulina velutipes polysaccharide, which is a neutral polysaccharide, has α-type pyranoside, is a glucan with →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ as the main chain and α-D-Glcp-(1→ as the side chain, and has a relative molecular mass of 6,181 Da.

[0006] Another object of the present invention is to provide a preparation method of the low-molecular-weight Flammulina velutipes polysaccharide.

[0007] Another object of the present invention is to provide the use of the low-molecular-weight Flammulina velutipes polysaccharide in anti-inflammatory drugs.

[0008] To achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a preparation method of low-molecular-weight Flammulina velutipes polysaccharide, and the preparation method includes the following steps:

[0010] S1: Preparation of Flammulina velutipes crude polysaccharide: The freeze-dried powder of Flammulina velutipes is defatted with ethanol, and the precipitate is obtained by centrifugation; deionized water is added to the precipitate, and ultrasonic extraction is carried out, and the supernatant is obtained by centrifugation; the supernatant is first rotary evaporated and then ethanol-precipitated, and the precipitate is obtained by centrifugation; deionized water is added to the precipitate for re-dissolution, Savage reagent is added for protein removal, stirred, and the supernatant is obtained by centrifugation; after rotary evaporation of the supernatant again, it is freeze-dried to obtain Flammulina velutipes crude polysaccharide;

[0011] S2: Separation and purification: The Flammulina velutipes crude polysaccharide is dissolved in deionized water, and the supernatant is obtained by centrifugation, passed through a DEAE Sepharose FF chromatography column, and eluted and separated with dH2O, repeated multiple times, the eluates are combined, concentrated, dialyzed, and freeze-dried; separated and purified multiple times with a Chromdex 75PG gel chromatography column to obtain low-molecular-weight Flammulina velutipes polysaccharide.

[0012] In a further technical solution, the ethanol defatting includes: adding an ethanol aqueous solution with a volume fraction of 85% to the freeze-dried powder of Flammulina velutipes at a material-liquid ratio of 1:15, and soaking at room temperature for 8-24 h.

[0013] In a further technical solution, the ultrasonic extraction includes: adding deionized water to the precipitate at a material-liquid ratio of 1:30, carrying out ultrasonic extraction at a ultrasonic power of 620 W, an ultrasonic extraction time of 50 min, and an ultrasonic extraction temperature of 45 °C.

[0014] In a further technical solution, the vacuum degree of the rotary evaporation is 60-70, the temperature is 55 °C, and the rotary evaporation is carried out until the original volume is reduced to 1 / 4.

[0015] In a further technical solution, the ethanol precipitation includes: adding three times the volume of an ethanol aqueous solution with a volume fraction of 95% to the rotary-evaporated solution, and keeping warm at 4 °C for 8-24 h.

[0016] In a further technical solution, the rotation speed of the centrifugation is 5000 rpm, and the centrifugation time is 12-15 min; when re-dissolving, deionized water is added at a material-liquid ratio of 1:30 for re-dissolution.

[0017] In a further technical solution, the Savage reagent is prepared by mixing n-butanol and chloroform at a volume ratio of 1:4, and added to the solution at a material-agent ratio of 1:0.2; the stirring time is 30 min.

[0018] The present invention also provides a low - molecular - weight Flammulina velutipes polysaccharide, which is prepared by the above - mentioned preparation method of the low - molecular - weight Flammulina velutipes polysaccharide. The low - molecular - weight Flammulina velutipes polysaccharide is a neutral polysaccharide, with α - type pyranoside glycosides, and is a glucan with →4)-α - D - Glcp-(1→ and →4,6)-α - D - Glcp-(1→ as the main chain and α - D - Glcp-(1→ as the side chain, and its relative molecular mass is 6,181 Da.

[0019] The present invention also provides the use of the low - molecular - weight Flammulina velutipes polysaccharide in anti - inflammatory drugs, which exerts an anti - inflammatory effect by down - regulating the levels of dimethylphosphatidylethanolamine and histamine.

[0020] The present invention also provides a pharmaceutical composition, which comprises the above - mentioned low - molecular - weight Flammulina velutipes polysaccharide.

[0021] The present invention also provides a functional product, which comprises the above - mentioned low - molecular - weight Flammulina velutipes polysaccharide.

[0022] The present invention has the following beneficial effects:

[0023] The present invention prepares a low - molecular - weight Flammulina velutipes polysaccharide through ultrasonic - assisted hot - water extraction method and after separation and purification. The low - molecular - weight Flammulina velutipes polysaccharide has a strong anti - inflammatory effect on LPS - stimulated inflammation, can improve the stimulation of LPS on macrophages, and exerts an anti - inflammatory effect by down - regulating the levels of dimethylphosphatidylethanolamine and histamine. The low - molecular - weight polysaccharide is non - toxic to Caco - 2 cells. The transmembrane transport experiment shows that the low - molecular - weight Flammulina velutipes polysaccharide is a polysaccharide with medium absorption, and there is an energy - consuming active transport. The transport mechanism experiment shows that the low - molecular - weight Flammulina velutipes polysaccharide is not a substrate of P - gp efflux transporter, and its transport is not related to the endocytosis pathways mediated by clathrin and caveolin. The low - molecular - weight Flammulina velutipes polysaccharide is absorbed by Caco - 2 cells through the endocytosis pathway mediated by macropinocytosis. The drug with the low - molecular - weight Flammulina velutipes polysaccharide as the active ingredient can be absorbed by intestinal cells and then exert an anti - inflammatory effect on cells. Description of the Drawings

[0024] Figure 1 It is the polysaccharide elution curve of the Flammulina velutipes crude polysaccharide by ion - exchange chromatography in Example 1 of the present invention;

[0025] Figure 2 It is the HPLC chromatogram of the purified fraction of the Flammulina velutipes crude polysaccharide by gel in Example 1 of the present invention;

[0026] Figure 3 It is the HPGPC chromatogram of the low - molecular - weight Flammulina velutipes polysaccharide in Example 1 of the present invention;

[0027] Figure 4It is the HPLC chromatogram of the monosaccharide composition of the mixed standard product and low-molecular-weight Flammulina velutipes polysaccharide in Example 1 of the present invention;

[0028] Figure 5 It is the infrared spectrum of low-molecular-weight Flammulina velutipes polysaccharide in Example 1 of the present invention;

[0029] Figure 6 It is the total ion current chromatogram of low-molecular-weight Flammulina velutipes polysaccharide in Example 1 of the present invention;

[0030] Figure 7 It is the nuclear magnetic resonance spectrum of low-molecular-weight Flammulina velutipes polysaccharide in Example 1 of the present invention (where, Figure 7 A is the 1H NMR curve, Figure 7 B is the 13C NMR curve, Figure 7 C is the COSY curve, Figure 7 D is the HSQC curve, Figure 7 E is the HSQC-TOCSY curve, Figure 7 F is the HMBC curve, Figure 7 G is the NOESY curve);

[0031] Figure 8 It is the ultraviolet spectrum of low-molecular-weight Flammulina velutipes polysaccharide and FFVP in Example 2 of the present invention;

[0032] Figure 9 It is the FITC standard curve in Example 2 of the present invention;

[0033] Figure 10 It is the bar graph of the effect of different concentrations of FFVP on the viability of Caco-2 cells in Example 2 of the present invention;

[0034] Figure 11 It is the microscopic photograph of Caco-2 cells at different times in Example 2 of the present invention (where, Figure 11 A) to Figure 11 I) correspond to the 0th, 2nd, 4th, 6th, 9th, 12th, 15th, 18th, and 21st days);

[0035] Figure 12 It is the curve of the transepithelial electrical resistance value of the Caco-2 cell monolayer in Example 2 of the present invention;

[0036] Figure 13 It is the alkaline phosphatase activity on the apical and basolateral sides of the Caco-2 cell monolayer in Example 2 of the present invention;

[0037] Figure 14 It is the fluorescein sodium standard curve in Example 2 of the present invention;

[0038] Figure 15Permeation rates of sodium fluorescein through Transwell polyester membranes and Caco-2 cell monolayers at different time points in Example 2 of the present invention;

[0039] Figure 16 Fluorescence intensity standard curve of FFVP in Example 2 of the present invention;

[0040] Figure 17 Apparent permeability of different concentrations of FFVP from the AP side to the BL side at different times in Example 2 of the present invention;

[0041] Figure 18 Apparent permeability of different concentrations of F-F from the BL side to the AP side at different times in Example 2 of the present invention;

[0042] Figure 19 Effect of four transport inhibitors on the apparent permeability of FFVP in Example 2 of the present invention;

[0043] Figure 20 Effect of different concentrations of low molecular weight Flammulina velutipes polysaccharide on the viability of RAW264.7 cells in Example 3 of the present invention;

[0044] Figure 21 Effect of different concentrations of LPS on the viability of RAW264.7 cells in Example 3 of the present invention;

[0045] Figure 22 Effect of different concentrations of LPS on the production of NO by RAW264.7 cells in Example 3 of the present invention;

[0046] Figure 23 Effect of different concentrations of low molecular weight Flammulina velutipes polysaccharide on the production of NO, IL-1β, IL-6 and TNF-α induced by LPS in RAW264.7 cells in Example 3 of the present invention (wherein, Figure 23 A corresponds to NO, Figure 23 B corresponds to IL-1β, Figure 23 C corresponds to IL-6, Figure 23 D corresponds to TNF-α);

[0047] Figure 24 PCA score plots of the control group, model group and drug administration group in Example 3 of the present invention;

[0048] Figure 25 A to Figure 25 D are respectively the OPLS-DA score plots of the model group and the control group, the 200-time permutation test plots of the model group and the control group, the OPLS-DA score plots of the drug administration group and the model group, and the 200-time permutation test plots of the drug administration group and the model group in Example 3 of the present invention;

[0049] Figure 26This is the volcano plot of differential metabolites between groups in Example 3 of the present invention (where, Figure 26 A corresponds to the control group and the model group, Figure 26 B corresponds to the administration group and the model group);

[0050] Figure 27 This is the correlation heat map of differential metabolites and inflammatory mediators in Example 3 of the present invention;

[0051] Figure 28 This is the metabolic pathway analysis in Example 3 of the present invention. Detailed implementation mode

[0052] The following combines specific examples and drawings to more detailedly illustrate the technical solution of the present invention. However, it should be noted that the following examples are only some relatively superior implementation modes of the technical solution of the present invention, rather than a limitation to the technical solution of the present invention. Those skilled in the art can obtain other implementation modes within the protection scope of the present invention under the guidance of relevant technical knowledge, common general knowledge or common technical means in the technical field of the present invention.

[0053] Example 1

[0054] This example relates to a preparation method and structural analysis of low-molecular-weight Flammulina velutipes polysaccharide. The preparation method includes the following steps:

[0055] S1: Preparation of crude Flammulina velutipes polysaccharide:

[0056] The Flammulina velutipes is freeze-dried and ground into powder to obtain freeze-dried powder of Flammulina velutipes. Weigh 60 g of the freeze-dried powder of Flammulina velutipes, add an ethanol aqueous solution with a volume fraction of 85% according to a solid-liquid ratio of 1:15, soak overnight at room temperature to remove fat. After centrifuging at 5000 rpm for 12 min, take the precipitate. Add deionized water to the precipitate according to a solid-liquid ratio of 1:30, and perform ultrasonic extraction under the conditions of an ultrasonic power of 620 W, an ultrasonic extraction time of 50 min, and an ultrasonic extraction temperature of 45 °C. After the extraction is completed, centrifuge at 5000 rpm for 15 min and take the supernatant. Perform rotary evaporation on the supernatant, where the rotary evaporation vacuum is about 60 - 70 and the temperature is 55 °C, and rotary evaporate to 1 / 4 of the original volume. Add three times the volume of an ethanol aqueous solution with a volume fraction of 95% to the rotary-evaporated solution for alcohol precipitation, and keep warm at 4 °C overnight. Centrifuge the alcohol-precipitated solution and take the precipitate to obtain the alcohol-precipitated polysaccharide. Add deionized water to the alcohol-precipitated polysaccharide according to a solid-liquid ratio of 1:30 for re-dissolution, and then add Savage reagent (n-butanol: chloroform = 1:4) to the re-dissolved polysaccharide solution according to a solid-agent ratio of 1:0.2 to remove proteins, and stir magnetically for 30 min. Centrifuge at 5000 rpm for 15 min and take the supernatant. Perform rotary evaporation on the supernatant again, and freeze-dry the rotary-evaporated solution to obtain crude Flammulina velutipes polysaccharide.

[0057] S2: Ion exchange chromatography and gel purification

[0058] Load the DEAE Sepharose FF gel suspension into a chromatography column and let it stand still until it completely settles. Wash the chromatography column with ultrapure water at a flow rate of 250 cm / h until the gel surface reaches a stable state. Dissolve the crude polysaccharide sample of Flammulina velutipes in an appropriate amount of distilled water, after centrifugation, take the supernatant and filter it through a 0.45 μm filter membrane. Take a crude polysaccharide solution of Flammulina velutipes equivalent to 30% of the column volume and add it to the chromatography column. Elute the sample at an elution rate of 15 mL / min in the elution order of dH2O, 0.2 mol / L NaCl, 0.5 mol / L NaCl, and 1.0 mol / L NaCl. Collect 10 mL of eluate for each elution gradient as one tube. After collecting the solutions in each tube, use the anthrone-sulfuric acid method to detect the polysaccharide content in the tube solution. Measure the absorbance of each tube solution with an enzyme-labeled instrument at 630 nm. Take the tube number as the abscissa and the absorbance as the ordinate to plot the elution curve of the crude polysaccharide of Flammulina velutipes, as Figure 1 shown. Eluate collected from different elution peaks was loaded onto the chromatography column again for separation, and the operation was repeated multiple times. Finally, most of the solvent was removed from the collected eluate in each tube by vacuum concentration, and it was loaded into a dialysis bag (cut-off value 3.5 kDa) to remove small molecule impurities such as inorganic salts. The dialysis solution was freeze-dried in vacuo to obtain each group of components.

[0059] The obtained elution curve is as Figure 1 shown. Gradient elution was carried out successively with dH2O, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solutions. After measuring the polysaccharide content in each tube by the anthrone-sulfuric acid method, four components, namely FVP-N1, FVP-N2, FVP-N3, and FVP-N4, were obtained. The component eluted with dH2O is neutral polysaccharide, and the components eluted with 0.2, 0.5, and 1.0 M NaCl are acidic polysaccharides. Since the yield of the FVP-N1 component is the highest and the contents of the other three components are relatively low, FVP-N1 was selected for subsequent preparation.

[0060] Filter the Chromdex 75PG gel suspension to remove the liquid, wash it several times with 3 volumes of ultrapure water, transfer it to a beaker, add an appropriate amount of distilled water and stir well, and slowly add it to the chromatography column and let it stand still until it completely settles. Wash the chromatography column with ultrapure water at a flow rate of 30 cm / h until the gel surface reaches a stable state. Take an appropriate amount of distilled water to dissolve the Flammulina velutipes polysaccharide sample obtained after separation by the chromatography column. After centrifugation, take the supernatant and filter it through a 0.45 μm filter membrane. After filtration, take a sample equivalent to 1% of the column volume and add it to the gel filtration chromatography column. Elute with ultrapure water at a flow rate of 1.5 mL / min, collect the eluate, and take 5 mL of eluate as one tube. Combine with an on-line differential detector for detection. The results are as Figure 2As shown in the figure, collect the part of the same elution peak region with a relatively high absorption peak and good symmetry in the eluate, that is, collect the elution components in the 116 - 155 min region, and load them onto the gel filtration column chromatography again. Repeat the operation multiple times to enrich and purify the polysaccharide. Finally, remove most of the solvent from the collected eluate by vacuum concentration, load it into a dialysis bag (cut-off value 3.5 kDa) for dialysis, and vacuum freeze-dry to obtain the purified polysaccharide component, which is the low-molecular-weight Flammulina velutipes polysaccharide.

[0061] Figure 2 It is the liquid chromatography detection curve of the gel-purified component of the Flammulina velutipes polysaccharide sample. The result shows a single, uniform, and symmetric peak, indicating that the purified Flammulina velutipes polysaccharide is a homogeneous polysaccharide.

[0062] This example also relates to the determination of the relative molecular weight of the above-mentioned low-molecular-weight Flammulina velutipes polysaccharide. The molecular weight of the low-molecular-weight Flammulina velutipes polysaccharide is detected by high-performance gel permeation chromatography, and the specific steps are as follows:

[0063] ① Preparation of standard and sample solutions: Weigh 10 mg of dextran standard products with molecular weights of 1 kDa, 5 kDa, 12 kDa, 25 kDa, 50 kDa, 80 kDa, 150 kDa, 270 kDa, 410 kDa, and 670 kDa respectively, add 0.05 mol / L NaCl solution and make up the volume to 2 mL. After passing through a 0.22 μm filter membrane, transfer it to an injection vial for standby. Weigh 10 mg of the low-molecular-weight Flammulina velutipes polysaccharide sample, add 0.05 mol / L NaCl solution to the sample to make up the volume to 2 mL. After passing through a 0.22 μm filter membrane, transfer it to an injection vial for standby.

[0064] ② Chromatographic conditions: Use a high-performance liquid chromatography system (HPLC) coupled with a refractive index detector (RID) for analysis. The chromatographic separation system consists of three polymer water-soluble chromatographic columns in series. The specific chromatographic column models and specifications are SB-803HQ (8 × 300 mm), SB-804HQ (8 × 300 mm), and SB-805HQ (8 × 300 mm) of the OHpak series. Use a 0.05 M NaCl solution as the mobile phase, set the flow rate to 0.65 mL / min, maintain the column temperature at 40 °C, and the injection volume is 30 μL.

[0065] ③ Establishment of the molecular weight calibration curve: Use the retention time as the abscissa and the logarithm of the standard product molecular weight as the ordinate for linear regression analysis to obtain the molecular weight calibration curve, as Figure 3 shown.

[0066] According to the dextran standard curve Log Mw = -0.1820T + 11.0623 (R 2The molecular weight of the low-molecular-weight Flammulina velutipes polysaccharide was calculated (= 0.9933). After calculation, the relative molecular weight of the low-molecular-weight Flammulina velutipes polysaccharide at 39.952 min was 6181 Da. The peak near 47.8 min was the mobile phase salt peak, and the peak after the salt peak might be substances such as small molecule oligosaccharides.

[0067] This example also involved the analysis of the monosaccharide composition of the above-mentioned low-molecular-weight Flammulina velutipes polysaccharide. The PMP pre-column derivation high performance liquid chromatography method was used to analyze the monosaccharide composition of the purified Flammulina velutipes polysaccharide, which specifically included the following steps:

[0068] (a) Standard preparation: Weigh 5 mg of a series of monosaccharide standards in turn, namely rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride and galactosamine hydrochloride. Weigh 10 mg of fucose, dissolve them respectively and make the volume up to 10 mL. After preparing the mother liquor, dilute it according to the gradient, filter through a 0.22 μm filter membrane and transfer it to an injection vial.

[0069] (b) Sample acid hydrolysis: Accurately weigh 5 mg of the low-molecular-weight Flammulina velutipes polysaccharide sample, add 1 mL of 2 M trifluoroacetic acid solution, and heat it in an oven at 121 °C for 2 h. After drying with a nitrogen blower, wash the sample with 3 mL of methanol, and then dry it with nitrogen. Repeat the above operation 3 times to remove the excess trifluoroacetic acid. Finally, add 5 mL of sterile water to dissolve the residue, collect the acid-hydrolyzed sample and transfer it to an injection vial for testing.

[0070] (c) Derivatization of the standard and the low-molecular-weight Flammulina velutipes polysaccharide sample: Take 200 μL of the monosaccharide standard solution or the low-molecular-weight Flammulina velutipes polysaccharide hydrolyzate in a centrifuge tube, add 200 μL of 0.5 M NaOH solution and 500 μL of PMP-methanol solution respectively. After mixing well on a vortex mixer, insert the centrifuge tube into a floating plate and place it in a water bath at 70 °C for 1 h. After the reaction, add 200 μL of 0.5 M hydrochloric acid to neutralize. Add 1 mL of chloroform to the centrifuge tube, vortex and let it stand for 10 min. Discard the chloroform layer, repeat the extraction 3 times, and take 300 μL of the solution and make the volume up to 1 mL with water.

[0071] (d) High performance liquid chromatography detection conditions: Use a Thermo high performance liquid chromatography system, equipped with an Agilent Eclipse XDB-C18 liquid chromatography column. The mobile phases A and B are acetonitrile and phosphate buffer solution (pH 6.8) respectively. The volume ratio of A and B is 17:83. The flow rate is set at 0.8 ml / min. The column temperature is maintained at 30 °C, the detection wavelength is 250 nm, and the injection volume is 10 μL.

[0072] Figure 4The HPLC chromatogram of the monosaccharide composition of the mixed standard and the low molecular weight Flammulina velutipes polysaccharide sample shows that the low molecular weight Flammulina velutipes polysaccharide (FVP) is mainly composed of mannose, glucose, and galactose, with a molar percentage of 0.60:98.868:0.531. Glucose accounts for the largest proportion, indicating that the low molecular weight Flammulina velutipes polysaccharide is a heteropolysaccharide with a glucan backbone.

[0073] In addition, infrared spectroscopy monitoring was carried out on the above-mentioned low molecular weight Flammulina velutipes polysaccharide, and the results are as Figure 5 shown. The low molecular weight Flammulina velutipes polysaccharide has a strong and broad absorption peak at 3396.79 cm -1 , which is a strong absorption peak of the O-H stretching vibration of intermolecular or intramolecular hydrogen bonds in the polysaccharide molecule; the medium-intensity sharp peak near 2920.28 cm -1 is the C-H stretching vibration absorption peak of methylene (-CH2); the absorption peak at 1650.50 cm -1 is attributed to the stretching vibration of C=O; the absorption peaks at 1454.93 cm -1 , 1413.62 cm -1 , and 1364.04 cm -1 are the bending vibration absorption peaks of C-H, and they and the stretching vibration of C-H constitute the characteristic absorption of the sugar ring; the absorption peak at 1157.46 cm -1 is the stretching vibration of C-O-C in pyranose; the three strong absorption peaks between 1200 - 1000 cm -1 , the absorption peaks at 1157.46 cm -1 , 1080.34 cm -1 , and 1016.99 cm -1 prove the existence of pyranoside again. The absorptions at these three places are the bending vibrations of the C-O bond in the C-O-H or C-O-C structure; the absorption peak at 926.09 cm -1 shows the asymmetric ring stretching vibration peak of the pyran ring, the absorption peak at 846.21 cm -1 shows the existence of α-glucosidic bond; the absorption peak at 763.58 cm -1 shows the symmetric ring stretching vibration peak of the pyran ring. In addition, there is no absorption peak in the 1720 cm -1 region, indicating that the low molecular weight Flammulina velutipes polysaccharide does not contain uronic acid, which is consistent with the monosaccharide composition detection results. The above results indicate that the low molecular weight Flammulina velutipes polysaccharide is a neutral polysaccharide and there is an α-type pyranoside.

[0074] Figure 7 Figures 1H NMR (A), 13C NMR (B), COSY (C), HSQC (D), HSQC-TOCSY (E), HMBC (F), and NOESY (G) of the above-mentioned low molecular weight Flammulina velutipes polysaccharide are shown. Table 2 shows the 1 H and13 Chemical shift assignment table of C.

[0075] In order to determine the linkage mode between monosaccharide residues in low molecular weight Flammulina velutipes polysaccharide, methylation analysis was carried out. The specific steps are as follows:

[0076] (A) Sample pretreatment: Weigh 1 mg of low molecular weight Flammulina velutipes polysaccharide sample and dissolve it in 1 ml of DMSO. Weigh 30 mg of solid NaOH and add it to the sample solution. React for 30 min. Add methyl iodide solution under dark conditions and react fully for 2 h. Add appropriate amount of water and dichloromethane and mix well with a vortex mixer. After centrifugation, wait for the two phases to separate, discard the aqueous phase, continue to wash with water and repeat the operation 3 times. Take the lower layer and dry it with nitrogen. Add 1 ml of TFA and react in an oven at 121 °C for 2 h. Dry it with a nitrogen blower. Add 1 mL of NaBD4 solution, stir magnetically at room temperature for 2.5 h, then add 300 μL of acetic acid to terminate the reaction, and dry it with a nitrogen blower. Dry it with 5% acetic acid methanol and pure methanol at 40 °C with nitrogen for 2 - 18 times. Add 1.5 mL of acetic anhydride, mix well with a vortex mixer and react at 100 °C for 2.5 h. Add water and dichloromethane and mix well with a vortex mixer. After centrifugation, discard the aqueous phase, wash with water 3 times and then let it stand for 10 min. Take the lower layer and detect it by GC-MS. The results are as Figure 6 shown.

[0077] (B) Chromatographic parameters: Use an Agilent gas chromatography system equipped with a capillary column. Its model specification is HP-5MS (30 m × 0.25 mm × 0.25 μm). The helium flow rate is set at 1.0 mL / min, the inlet temperature is 260 °C, the injection volume is 1 μL, and the solvent delay is 2.2 min. Programmed temperature rise: Keep at 50 °C for 1 min, rise to 130 °C at a rate of 50 °C / min, and then rise to 230 °C at a rate of 3 °C / min and keep for 2 min.

[0078] (C) Mass spectrometry parameters: Use an Aiglent quadrupole mass spectrometry detection system equipped with an electron impact ion source (EI) and a MassHunter workstation. The inlet temperature is 230 °C, the quadrupole temperature is 150 °C, and the electron energy is 70 eV. The scanning mode is full scan mode, and the mass scanning range (m / z): 30 - 600.

[0079] Table 1 Data table of methylation analysis of low molecular weight Flammulina velutipes polysaccharide

[0080]

[0081] Figure 6Figure 0 shows the total ion current chromatogram of the above-mentioned low molecular weight Flammulina velutipes polysaccharide, and Table 1 shows the data table of methylation analysis of the above-mentioned low molecular weight Flammulina velutipes polysaccharide. It can be seen that there are two residue types, glucose and galactose, in the low molecular weight Flammulina velutipes polysaccharide. Among them, the proportion of glucose residues is 99.3%, and there are 5 connection modes, from more to less in turn: 1,4-Glcp (67.20%), t-Glcp (18.75%), 1,4,6-Glcp (11.31%), 1,6-Glcp (1.27%) and 1,3,4-Glcp (0.77%); the proportion of galactose residues is 0.7%, and the connection mode is 1,4-Galp. It can be seen from this that the main derivative of the low molecular weight Flammulina velutipes polysaccharide is 1,4,5-tri-O-acetyl-2,3,6-tri-Omethyl glucitol, and the connection mode is 1,4-Glcp. Therefore, it is proposed that the low molecular weight Flammulina velutipes polysaccharide is a glucan, and (1→4)-linked-Glc is the main residue of the low molecular weight Flammulina velutipes polysaccharide. This inference is consistent with the above-mentioned monosaccharide composition detection results.

[0082] Nuclear magnetic resonance analysis was used to further clarify the structural characteristics of the low molecular weight Flammulina velutipes polysaccharide. A 600 MHz Bruker nuclear magnetic resonance spectrometer was used to measure the one-dimensional nuclear magnetic 1 1H-NMR, 13 13C-NMR and two-dimensional nuclear magnetic heteronuclear single quantum coherence spectrum (HSQC), correlation spectrum (COSY), heteronuclear-homonuclear mixing correlation spectrum (HSQC-TCOSY), nuclear Overhauser effect spectrum (NOESY) and heteronuclear multiple bond correlation spectrum (HMBC), as Figure 7 shown, while Table 2 shows the 1 1H and 13 13C chemical shift assignment table of each sugar residue of the above-mentioned low molecular weight Flammulina velutipes polysaccharide. Referring to Figure 7 and Table 2, it can be inferred that Figure 7 (A) The sugar residues A, B, C and D are →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →6)-α-D-Glcp-(1→ and α-D-Glcp-(1→ respectively; the sugar residue R α is →4)-α-D-Glcp, the sugar residue R β is →4)-β-D-Glcp, and the mutual connection order between each sugar residue can be further inferred.

[0083] Table 2 The 1 1H and 13 13C chemical shift assignment table of each sugar residue of the low molecular weight Flammulina velutipes polysaccharide

[0084]

[0085] Based on the analysis of the monosaccharide composition, polysaccharide methylation analysis results, and one-dimensional and two-dimensional nuclear magnetic information of the low-molecular-weight Flammulina velutipes polysaccharide sample, the structure of the low-molecular-weight Flammulina velutipes polysaccharide sample was preliminarily determined to be a glucan with →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ as the main chain and α-D-Glcp-(1→ as the side chain. Its molecular structure is shown below.

[0086]

[0087] Example 2

[0088] In this example, FITC was used to fluorescently label the low-molecular-weight Flammulina velutipes polysaccharide. A Caco-2 monolayer cell model was established in a Transwell chamber. The tightness of the Caco-2 cell monolayer membrane was verified through cell morphology observation, TEER value measurement, alkaline phosphatase measurement, and fluorescein sodium permeability experiment. The absorption and transport mechanism of the low-molecular-weight Flammulina velutipes polysaccharide in intestinal epithelial cells was studied using a bidirectional transmembrane experiment and transport inhibitors.

[0089] First, cell culture is required, including:

[0090] (1) Cell resuscitation: Take out the cell cryopreservation tube from the -80 °C refrigerator and quickly thaw it in a 37 °C water bath. After about 1 - 2 minutes, the liquid in the cryopreservation tube is completely dissolved. Transfer the cell suspension to a centrifuge tube containing 4 mL of pre-warmed complete medium, centrifuge at 900 rpm for 3 minutes, discard the medium, add 4 mL of fresh complete medium, pipette and mix evenly, transfer the cell suspension to a culture flask containing 4 mL of fresh medium, shake it crosswise, and place it in a 37 °C, 5% CO2 incubator for culture.

[0091] (2) Cell passage: Take out the cells to be passaged from the incubator, pour out the old medium, add 1 mL of PBS along the wall and rinse twice. Add 2 mL of trypsin to the culture flask, gently shake it to fully cover the bottom of the dish, place it in the incubator for digestion for 2 minutes, add 4 mL of pre-warmed complete medium to terminate cell digestion, gently pipette the cells at the bottom of the flask to make them completely detached, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 900 rpm for 3 minutes in a centrifuge. After centrifugation, discard the old medium, add 2 mL of pre-warmed fresh medium to resuspend, transfer the pipetted and mixed cell suspension to a culture flask containing fresh complete medium at a 1:2 ratio, and place it in the incubator for culture.

[0092] (3) Cell cryopreservation: Take out the cells to be cryopreserved from the incubator, pour out the old culture medium, and add 1 mL of PBS to wash twice. Add 2 mL of trypsin, gently shake to make it fully cover the bottom of the flask, quickly place it in the incubator for digestion for 2 min. After taking it out, gently shake the culture flask and observe with the naked eye that the cells have detached. Add 4 mL of preheated fresh complete culture medium to terminate cell digestion. Gently pipette the cells at the bottom of the flask to make them completely detached. Transfer the obtained cell suspension to a 15 mL centrifuge tube after repeated pipetting and mixing. Centrifuge at 900 rpm for 3 min, discard the culture medium, add the prepared cell cryopreservation solution (fetal bovine serum: DMSO = 9:1), gently pipette and mix, aspirate 1 mL of cell suspension and transfer it to a cryopreservation tube. After sealing, place it in a programmable cooling box and transfer it to a -80 °C refrigerator for storage.

[0093] Secondly, fluorescein isothiocyanate (FITC) is used for fluorescent labeling of low-molecular-weight Flammulina velutipes polysaccharide, which specifically includes the following steps:

[0094] A) Amination of low-molecular-weight Flammulina velutipes polysaccharide

[0095] Weigh 300 mg of low-molecular-weight Flammulina velutipes polysaccharide and dissolve it in 20 mL of phosphate solution with a pH of 8.0. Add 300 mg of tyramine and 112.5 mg of sodium cyanoborohydride, and react at 60 r / min in a shaker at 37 °C for 96 h. After the reaction, centrifuge at 6000 rpm for 10 min and take the supernatant. Precipitate with absolute ethanol three times, then dissolve the precipitate in distilled water and dialyze (cutoff value of Mw is 3.5 kDa) for 24 h. Freeze-dry to obtain the synthesized product of Flammulina velutipes polysaccharide-tyramine and name it Tyr-FVP.

[0096] B) FITC labeling of low-molecular-weight Flammulina velutipes polysaccharide

[0097] Dissolve 200 mg of Tyr-FVP in 10 mL of distilled water, and adjust the pH value of the solution to 8.5 with 1.0 mol / L NaHCO3. Add 25 mg of FITC at 25 °C and react overnight in the dark. Precipitate the conjugate several times with three volumes of absolute ethanol until there is no obvious fluorescent yellow in the ethanol. Dissolve the precipitate in distilled water and dialyze (cutoff value of Mw is 3.5 kDa) for 24 h. After freeze-drying, obtain the fluorescently labeled Flammulina velutipes polysaccharide, name it FFVP and store it. Dissolve FFVP into a solution of 100 μg / mL for ultraviolet-visible light scanning, with the scanning range of 300 - 800 nm, as Figure 8 shown. In the figure, the low-molecular-weight Flammulina velutipes polysaccharide has no ultraviolet absorption in the range of 300 - 800 nm, while FFVP has an obvious characteristic absorption peak at 490 nm, which is the characteristic absorption peak of FITC, indicating successful FITC labeling.

[0098] C) Determination of the fluorescence substitution degree of FFVP

[0099] Prepare a 1 μg / mL FITC solution. Take 0.5, 1.0, 1.5, 2.0, and 2.5 mL respectively and make up to 5 mL with distilled water. Use distilled water as the blank control. Measure its fluorescence intensity at an emission wavelength of 492 nm and an excitation wavelength of 518 nm using a multifunctional microplate reader. With the FITC concentration as the abscissa and the measured fluorescence intensity as the ordinate, plot the standard curve. As Figure 9 shown, the linear regression equation obtained is: y = 57.642x + 3.0037 (R 2 = 0.9939).

[0100] Prepare a 0.2 mg / mL FFVP solution. Take 1 mL of the solution and mix it with 3 mL of distilled water. Measure the fluorescence intensity using a microplate reader to get 20.3 au. Substitute the fluorescence intensity of FFVP into the standard curve of FITC to obtain the FITC concentration in FFVP, and calculate its fluorescence substitution degree to be 0.6%.

[0101] D) Cytotoxicity test

[0102] After the Caco-2 cells reach an attachment rate of 80% - 90% in the culture dish, discard the old medium, wash twice with PBS, add 2 mL of trypsin and place it in the incubator for digestion for 3 min. Add 2 mL of complete medium and gently pipette to make the cells detach. Transfer to a 15 mL centrifuge tube, centrifuge at 900 rpm for 3 min, discard the supernatant, add 4 mL of complete medium to resuspend, and adjust the cell concentration to 5×10 4 cells / mL and inoculate into a 96-well plate, 100 μL of cell suspension per well. Culture overnight in an incubator at 37°C with 5% CO2 to make the cells attach. Prepare FFVP solutions with concentrations of 10, 25, 50, 100, 200, and 400 μg / mL using blank DMEM medium. Add 100 μL of polysaccharide solutions with different mass concentrations to each well in the experimental group; add 100 μL of blank DMEM medium to each well in the control group; do not inoculate cells in the blank group and add 100 μL of blank DMEM medium to each well; set 5 replicates for each group. After culturing for 24 h in the incubator, discard the culture solution, add 10 μL of CCK-8 reagent to each well under light protection, place it back in the incubator for incubation for 1 h, measure the absorbance at a wavelength of 450 nm using a full-wavelength microplate reader, and calculate the cell survival rate. Figure 10 is a bar graph of the effects of different concentrations of FFVP on the viability of Caco-2 cells. It can be seen that FFVP has no toxic effect on Caco-2 cells within the concentration range of 10 - 400 μg / mL and can be used for subsequent absorption and transport studies.

[0103] E) Construction of Caco-2 cell absorption model

[0104] When the Caco-2 cells cover 80% - 90% of the bottom of the dish, discard the old medium, wash twice with PBS, add 2 mL of trypsin, place it in the incubator for digestion, then add 4 mL of complete medium to terminate the digestion. After pipetting and mixing evenly, transfer it to a 15 mL centrifuge tube, centrifuge at 900 rpm for 3 min, discard the supernatant, add 4 mL of complete medium to resuspend, and adjust the cell concentration to 2×10 5 cells / mL. Add 0.5 mL of the cell suspension to the apical side (AP) of a Transwell 12-well plate, and add 1.5 mL of complete medium to the basolateral side (BL). Culture in an incubator at 37 °C and 5% CO2. Replace the medium after 48 h. Change the medium every other day in the first week and daily starting from the second week. Each time the medium is changed, add 0.5 mL of medium to the AP side and 1.5 mL of medium to the BL side. Culture for 21 days until the Caco-2 cells form a dense cell monolayer for subsequent absorption and transport experiments.

[0105] Then, the evaluation of the Caco-2 cell absorption model is required, including the following:

[0106] a) Cell morphology observation: Culture Caco-2 cells in a Transwell 12-well plate according to the above cell culture method. Observe the cell morphology under an inverted microscope every day and take pictures for record, as Figure 11 shown. As Figure 11 can be seen, the growth rate of the cells is slow in the first three days, the logarithmic growth phase is on the 4th day, the cells reach confluence on the 6th day, and when grown to 21 days, a cell monolayer that is uniformly dense, with clear boundaries between cells and no gaps in the tight junctions can be seen with the naked eye.

[0107] b) Measurement of transepithelial electrical resistance: Culture Caco-2 cells in a Transwell 12-well plate according to the above cell culture method. Measure the transepithelial electrical resistance value (TEER) of Caco-2 cells on the 3rd, 6th, 9th, 12th, 15th, 18th, and 21st days using a transepithelial electrical resistance meter. Before measuring the resistance value, immerse the electrode in 75% ethanol for disinfection for 15 min, and balance it in HBSS solution for 5 min after natural air drying. When measuring the transepithelial electrical resistance, insert the long end of the electrode vertically into the BL side and the short end into the AP side. After the resistance meter stabilizes, read the TEER value. Select 3 points at different positions in each well and measure 3 times in parallel. The average value is the resistance value of this well, and calculate the TEER value. Figure 12 shows the transepithelial electrical resistance values of the Caco-2 cell monolayer at different times. The TEER value of the cells stabilizes on the 21st day, reaching 616 Ω·cm2, indicating that the integrity of the cell monolayer is good and the model is successfully established.

[0108] c) Alkaline phosphatase activity assay: On the 2nd, 4th, 6th, 9th, 12th, 15th, 18th, and 21st days of Caco-2 cell culture, the culture media on the AP and BL sides were collected, and the enzyme activities on the AP and BL sides were detected using an alkaline phosphatase detection kit. The AKP activity ratio on the AP and BL sides was calculated according to the kit method. Figure 13 For the alkaline phosphatase activities on the apical (AP) and basolateral (BL) sides of the Caco-2 cell monolayer, on the 21st day of culture, the alkaline phosphatase on the AP side of the cell monolayer was significantly higher than that on the BL side, indicating that the distribution of alkaline phosphatase was extremely asymmetric. Most of the alkaline phosphatase was confined to the brush border side, and the cells had grown and differentiated into polarity, and had the carriers and enzymes required for absorption and transport.

[0109] d) Sodium fluorescein permeability experiment: Sodium fluorescein solutions with concentrations of 0, 0.25, 0.5, 1, 2, 4, and 10 μg / mL were prepared with HBSS. The absorbance was measured at 492 nm using an enzyme-linked immunosorbent assay (ELISA) reader. With the concentration (μg / mL) as the abscissa and the absorbance as the ordinate, a standard curve was plotted. As Figure 14 shown, the linear regression equation of sodium fluorescein was y = 0.0085x + 0.0396 (R 2 = 0.9996). Three wells of Caco-2 cells cultured for 21 days with a TEER value > 300 Ω·cm2 were selected as the experimental group, and a blank well without inoculated cells was used as the control group. The upper and lower chambers of the Transwell were rinsed 3 times with pre-warmed HBSS at 37°C. 0.5 mL of a sodium fluorescein solution with a concentration of 10 μg / mL was added to the AP side, and 1.5 mL of HBSS was added to the BL side. It was placed in an incubator at 37°C and 5% CO2 for 1 h. 500 μL of samples from the BL side were taken into EP tubes at 30 min, 60 min, 90 min, and 120 min, and wrapped with aluminum foil. The same volume of HBSS was immediately replenished after each sampling. 200 μL of the sample was aspirated from the EP tube, and the absorbance of the sample was measured at 492 nm using an ELISA reader to calculate the permeability of sodium fluorescein. Figure 15 For the permeability of sodium fluorescein through the Transwell polyester membrane and the Caco-2 cell monolayer at different time points, the permeability of sodium fluorescein in the cell monolayer was significantly lower than that of the blank group, and the permeability only increased from 0.2% to 1.6% within 2 h, indicating that the integrity of the cell monolayer was good and could be used for subsequent transport experiments.

[0110] Next, the transmembrane transport experiment of Caco-2 cells was carried out. The specific steps included:

[0111] After culturing Caco-2 for 21 days, select the group with TEER value and AKP ratio meeting the requirements. Wash the upper and lower chambers 3 times with HBSS, add 0.5 mL and 1.5 mL of HBSS to the AP side and BL side respectively, place it in the incubator to equilibrate for 30 min, and then aspirate and discard the equilibration solution for standby. Prepare a 1 mg / mL FFVP solution with PBS, and then dilute it into FFVP solutions with concentrations of 1, 2.5, 5, 10, 25, 50, and 100 μg / mL. Measure the fluorescence intensity of each concentration of polysaccharide solution with an enzyme-linked immunosorbent assay (ELISA) reader at an excitation wavelength of 492 nm and an emission wavelength of 518 nm. Plot a standard curve with concentration as the abscissa and fluorescence intensity as the ordinate. As Figure 16 shown, the standard curve equation of the FFVP fluorescence intensity is y = 51.43x + 3.6079 (R 2 = 0.9998).

[0112] The transport experiment is carried out in two directions: from the apical side to the basolateral side (AP→BL) and from the basolateral side to the apical side (BL→AP). AP→BL: Add 0.5 mL of Flammulina velutipes polysaccharide solution with different concentrations to the AP side of the Transwell chamber as the supply solution; add 1.5 mL of blank PBS to the BL side as the receiving solution. Incubate in a 37°C incubator. Collect 200 μL of sample solution from the BL side at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively. Immediately replenish the same volume of preheated blank PBS after each sampling, and set three replicates. BL→AP: Add 1.5 mL of Flammulina velutipes polysaccharide solution with different concentrations to the BL side of the Transwell chamber as the supply solution; add 0.5 mL of blank PBS to the AP side as the receiving solution. Incubate in a 37°C incubator. Collect 200 μL of sample solution from the AP side at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively. Immediately replenish the same volume of preheated blank PBS after each sampling, and set three replicates. After measuring the fluorescence intensity of the samples, calculate the FFVP concentration according to the FFVP fluorescence intensity standard curve, and calculate the apparent permeability coefficient (Papp) and efflux ratio (ER).

[0113] Figure 17 is the apparent permeability of different concentrations of FFVP from the AP side to the BL side at different times, Figure 18 is the apparent permeability of different concentrations of F-F from the BL side to the AP side at different times. It can be seen that the Papp values of the three concentrations of FFVP solution in the absorption direction of AP→BL side are all greater than 1×10 -6 cm / s and less than 1×10 -5cm / s, indicating that the low-molecular-weight Flammulina velutipes polysaccharide is a medium-absorbing substance. Table 3 shows the apparent permeability and efflux rate of different concentrations of FFVP. The ER of the three concentrations of FFVP is less than 1, indicating that the low-molecular-weight Flammulina velutipes polysaccharide is not transported by P-glycoprotein (P-gp) efflux and is not a P-gp substrate. The ratio of the AP→BL side to the BL→AP side is greater than 1.5, further indicating that the low-molecular-weight Flammulina velutipes polysaccharide may have energy-consuming active transport during the transport process in Caco-2 cells.

[0114] Table 3 Apparent permeability and efflux rate of different concentrations of FFVP

[0115]

[0116] Finally, the absorption and transport mechanism of the low-molecular-weight Flammulina velutipes polysaccharide in the Caco-2 model was studied, which specifically includes the following contents:

[0117] After establishing the cell absorption model, discard the culture solutions in the upper and lower chambers, and wash the upper and lower chambers 3 times with pre-warmed HBSS. Add 0.5 mL and 1.5 mL of cell transport-related inhibitors or promoters to the AP side and BL side of the experimental group, respectively, namely chlorpromazine (endocytosis inhibitor, 20 μM), genistein (endocytosis inhibitor, 60 μM), amiloride (endocytosis inhibitor, 100 μM), verapamil (P-glycoprotein inhibitor, 100 μM). Add 0.5 mL and 1.5 mL of HBSS solution to the AP side and BL side of the control group, respectively, and incubate in the incubator for 30 min and then aspirate. Add Flammulina velutipes polysaccharide solutions with different concentrations to the AP side of each group, and add 1.5 mL of HBSS solution to the BL side. After incubating in the incubator for 150 min, sample from the BL side. Set three replicates for each group, and calculate the polysaccharide content and apparent permeability coefficient on the BL side.

[0118] Figure 19 The effects of four transport inhibitors on the apparent permeability of FFVP. Compared with the control group, after adding amiloride, the apparent permeability of FFVP decreased by 55%, indicating that FFVP is mainly absorbed by cells through the endocytosis pathway mediated by macropinocytosis; after adding verapamil, chlorpromazine, and genistein, the P app values of each concentration of FFVP in the AP→BL direction had no significant difference from those of the control group, indicating that FFVP is effluxed through P-gp, and its transport also does not pass through the endocytosis pathways mediated by clathrin and caveolin.

[0119] Example 3

[0120] In this example, an inflammation model was constructed by inducing RAW264.7 cells with LPS. The anti-inflammatory effect of low-molecular-weight Flammulina velutipes polysaccharide on RAW264.7 cells was detected by measuring the levels of inflammatory mediators (NO, IL-1β, IL-6, TNF-α), and the related mechanism of low-molecular-weight Flammulina velutipes polysaccharide mediating anti-inflammatory effects independent of the gut microbiota was elucidated based on metabolomics.

[0121] First of all, cell culture also needs to be carried out, including:

[0122] (1) Cell resuscitation: Take out the RAW264.7 cell cryopreservation tube from the -80 °C refrigerator and quickly thaw it in a 37 °C water bath for about 1 min. Quickly transfer the cell suspension in the tube to a centrifuge tube containing 4 mL of pre-warmed complete medium, centrifuge at 900 rpm for 3 min, discard the medium, add 1 mL of pre-warmed fresh complete medium to resuspend, gently pipette and mix evenly, then transfer the cell suspension to a T25 culture flask, shake it crosswise, and place it in a 37 °C, 5% CO2 incubator for culture.

[0123] (2) Cell passage: Take out the cells with an adhesion rate of about 90% from the incubator, discard the old medium, add 1 mL of PBS to wash twice, add 2 mL of complete medium, gently pipette the adherent cells down, and transfer them to a T25 culture flask containing 4 mL of fresh medium at a ratio of one to two, and place it in the incubator for culture.

[0124] (3) Cell cryopreservation: Take out the cells to be cryopreserved from the incubator, pour out the old medium, add 1 mL of PBS to wash twice, add 2 mL of complete medium to gently pipette the adherent cells down, transfer them to a 15 mL centrifuge tube, centrifuge at 900 rpm for 3 min, discard the medium, add the prepared cell cryopreservation solution (fetal bovine serum: DMSO = 9:1), gently pipette and mix evenly with a pipette gun, aspirate 1 mL of cell suspension and transfer it to a cryopreservation tube, seal it and then put it into a programmable freezing box, and transfer it to a -80 °C refrigerator for storage.

[0125] Secondly, a cytotoxicity test needs to be carried out, and the specific content includes:

[0126] Select RAW264.7 cells with good growth status, make them into a cell suspension, and adjust the cell concentration to 5×10 5Cells were inoculated into 96-well plates at a density of 5×10^5 cells / mL, with 100 μL of cell suspension in each well, and cultured overnight in an incubator to allow them to adhere. The 96-well plates were taken out, and the culture medium was discarded. Low molecular weight Flammulina velutipes polysaccharide solutions with concentrations of 10, 25, 50, 100, 200, 400, and 800 μg / mL were prepared using high-glucose DMEM medium. In the experimental groups, 100 μL of low molecular weight Flammulina velutipes polysaccharide solutions at different concentrations were added to each well; in the control group, 100 μL of blank DMEM medium was added to each well; in the blank group, cells were not inoculated, and 100 μL of blank high-glucose DMEM medium was added to each well. Five replicate wells were set up in each group. After culturing for 24 h in an incubator, the culture medium was discarded. Under light-proof conditions, 10 μL of CCK-8 reagent was added to each well, and then the plates were returned to the incubator for incubation for 1 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and the cell survival rate was calculated. Figure 20 The effects of low molecular weight Flammulina velutipes polysaccharide at different concentrations on the viability of RAW264.7 cells are shown. It can be seen that low molecular weight Flammulina velutipes polysaccharide has no toxicity to RAW264.7 cells in the concentration range of 10-50 μg / mL, and has a certain toxic effect on cells when the concentration is above 100 μg / mL. Therefore, concentrations of 10, 25, and 50 μg / mL of FVP were selected for subsequent anti-inflammatory experiments.

[0127] Then, an inflammation model needs to be established. The specific steps are as follows:

[0128] A) Effects of different concentrations of LPS on the viability of RAW264.7 cells: RAW264.7 cells with good growth status and an adhesion rate of about 90% were selected, made into a cell suspension, and the cell concentration was adjusted to 5×10^5 5 cells / mL, and inoculated into 96-well plates, with 100 μL of cell suspension in each well, and cultured overnight in an incubator to allow the cells to adhere. The 96-well plates were taken out, and the culture medium was discarded. LPS solutions with concentrations of 0.2, 0.5, 1.0, 1.5, and 2 μg / mL were prepared using DMEM medium. In the experimental groups, 100 μL of LPS solutions with different mass concentrations were added to each well; in the control group, 100 μL of blank DMEM medium was added to each well; in the blank group, cells were not inoculated, and 100 μL of blank high-glucose DMEM medium was added to each well. Six replicate wells were set up in each group. After culturing for 24 h in an incubator, the culture medium was discarded. Under light-proof conditions, 10 μL of CCK-8 reagent was added to each well, and then the plates were returned to the incubator for incubation for 1 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and the cell survival rate was calculated. Figure 21 The effects of different concentrations of LPS on the viability of RAW264.7 cells are shown. It can be seen that when using LPS to stimulate the inflammatory response, the concentration of LPS used should be lower than the maximum non-toxic dose of 1.5 μg / mL.

[0129] B) Effects of LPS at different concentrations on NO production in RAW264.7 cells: RAW264.7 cells with good growth status and an adhesion rate of about 90% were selected, made into cell suspensions, the cell concentration was adjusted to 5×105 cells / mL, and inoculated into 24-well plates, 500 μL of cell suspension per well. After overnight adhesion in the incubator, the culture medium was discarded. LPS solutions with concentrations of 0.2, 0.5, 1.0, and 1.5 μg / mL were prepared with DMEM medium. 500 μL of LPS solutions at different concentrations were added to each well in the experimental group; 500 μL of blank DMEM medium was added to each well in the control group. Each group was set with 4 replicates. After culturing for 24 h in the incubator, the supernatant was taken, and the NO content in the cell supernatant was detected according to the instructions of the NO kit. Figure 22 For the effects of LPS at different concentrations on NO production in RAW264.7 cells, it can be seen that the NO release amount is the highest when the concentration is 0.5 μg / mL. Therefore, 0.5 μg / mL was finally selected as the working concentration of LPS to induce inflammation.

[0130] Next, it was the study on the anti-inflammatory effect of low-molecular-weight Flammulina velutipes polysaccharide, that is, the effects of low-molecular-weight Flammulina velutipes polysaccharide on the production of NO, IL-1β, IL-6, and TNF-α in RAW264.7 cells. The specific contents included:

[0131] When RAW264.7 cells reached 90% confluence at the bottom of the flask, the cell suspension was collected, the cell concentration was adjusted to 5×10 5 cells / mL and inoculated into 6-well plates, 2 mL of cell suspension per well. After overnight adhesion in the incubator, the culture medium was discarded. 2 mL of blank DMEM was added to each well in the normal group and the model group, and 2 mL of low-molecular-weight Flammulina velutipes polysaccharide solutions with concentrations of 10, 25, and 50 μg / mL were added to each well in the experimental group. Each group was set with 6 replicates. After culturing in the incubator for 4 h, LPS solution was added to each well in the model group and the experimental group to make the final concentration 0.5 μg / mL, and then continued to culture in the incubator for 24 h. The supernatant was extracted for standby. The production amount of NO was detected with the NO kit, and the production amounts of IL-1β, IL-6, and TNF-α were detected according to the instructions of the ELISA kit. Figure 23 For the effects of low-molecular-weight Flammulina velutipes polysaccharide at different concentrations on NO (A), IL-1β (B), IL-6 (C), and TNF-α (D) induced by LPS in RAW264.7 cells, it can be seen that at a concentration of 50 μg / mL, low-molecular-weight Flammulina velutipes polysaccharide has strong anti-inflammatory potential for LPS-stimulated inflammation by inhibiting the secretion levels of NO, TNF-α, IL-1β, and IL-6.

[0132] Finally, cell metabolomics research was carried out. The specific contents included:

[0133] a) Cell sample preparation: Cells were cultured according to the above method. In the drug administration group, 2 mL of low-molecular-weight Flammulina velutipes polysaccharide solution with a concentration of 50 μg / mL was added to each well. In the model group and the experimental group, LPS solution was added to each well to make the final concentration 0.5 μg / mL. Then, the cells were placed in an incubator and cultured for 24 h, and the cells were collected for subsequent metabolomics analysis.

[0134] b) Sample pretreatment: 50 mg of cell samples were weighed into 2 mL centrifuge tubes, and grinding beads were added. 400 μL of extraction solution (methanol-water mixed solution with a volume ratio of 4:1) was prepared, and 0.02 mg / mL of L-2-chlorophenylalanine was added as an internal standard. The metabolites of the cells were extracted using this extraction solution. The sample solution was ground in a tissue grinder at -10°C for 6 min, and then ultrasonically extracted at low temperature for 30 min. The sample was left standing at -20°C for 30 min, centrifuged at 13000 rpm for 15 min, and the supernatant was transferred to an injection vial for subsequent on-machine analysis.

[0135] c) LC-MS / MS analysis conditions: Chromatographic conditions: Chromatographic column HSS T3 (100 mm × 2.1 mm, 1.8 μm); mobile phase A was water (95%) and acetonitrile (5%, containing 0.1% formic acid); mobile phase B was acetonitrile (47.5%), isopropanol (47.5%) and water (5%, containing 0.1% formic acid); flow rate was 0.40 mL / min, the column temperature was maintained at 40°C, and the injection volume was 3 μL. Mass spectrometry conditions: Positive and negative ion scanning modes were used, and the mass scanning range was 70 - 1050 m / z. The sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the auxiliary gas heating temperature was 425°C, and the capillary temperature was 325°C; in the positive mode, the ion spray voltage was set to 3500 V; in the negative mode, the ion spray voltage was set to -3500 V. The normalized collision energy was set with a cyclic collision energy of 20 - 40 - 60 eV. The resolution of the first-stage mass spectrometry was 60000, and the resolution of the second-stage mass spectrometry was 7500. Data was collected using the data-dependent acquisition (DDA) mode.

[0136] d) Data processing and analysis: The original data was imported into the metabolomics processing software Progenesis QI for baseline filtering, peak identification, integration, retention time correction, etc., to obtain a data matrix of retention time, mass-to-charge ratio, and peak intensity. The data matrix was uploaded to the Majorbio Cloud Platform (cloud.majorbio.com) for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Differential metabolites were screened according to variable importance in projection (VIP) ≥ 1.5 and p < 0.05, and the differential metabolites were identified based on the HMDB database. Pathway enrichment analysis of the differential metabolites was performed according to KEGG.

[0137] Figure 24PCA score plots of the control group (C), model group (M), and drug administration group (F) indicate good experimental repeatability. The model group and the control group can be clearly separated, demonstrating the successful establishment of the LPS-induced RAW264.7 cell inflammation model. Meanwhile, the drug administration group and the model group are clearly separated and approach the control group, indicating that low molecular weight Flammulina velutipes polysaccharide has a certain regulatory effect on cell inflammation.

[0138] Figure 25 A to Figure 25 D are respectively the OPLS-DA score plots of the model group and the control group, the 200 permutation test plots of the model group and the control group, the OPLS-DA score plots of the drug administration group and the model group, and the 200 permutation test plots of the drug administration group and the model group. This also shows the successful establishment of the LPS-induced RAW264.7 cell inflammation model. Low molecular weight Flammulina velutipes polysaccharide has a certain regulatory effect on LPS-induced cell inflammation. At the same time, the OPLS-DA model has a low overfitting risk and reliability.

[0139] VIP≥1.5 and p<0.05 were used as the screening criteria to screen for differential metabolites. The overall distribution of differential metabolites is as Figure 26 shown in the volcano plot, where Figure 26 A corresponds to the control group and the model group, Figure 26 B corresponds to the drug administration group and the model group. Table 4 shows the table of differential metabolites common to the model group, control group, and drug administration group. It can be seen that compared with the control group, the levels of 8 lipid metabolites and histamine in the model group are upregulated, indicating that these metabolites may be key metabolites for macrophages to produce inflammation. After administration of low molecular weight Flammulina velutipes polysaccharide, the levels of 9 metabolites are significantly reversed, indicating that the therapeutic effect of low molecular weight Flammulina velutipes polysaccharide on LPS-induced macrophage inflammation is closely related to its effect on metabolite changes.

[0140] Table 4 Table of differential metabolites common to the model group, control group, and drug administration group

[0141]

[0142] To study the potential correlation between the altered metabolites and inflammatory mediators, Pearson correlation analysis was performed. As Figure 27 shown (different colors represent the magnitude of the correlation coefficient r between attributes, and asterisks represent the magnitude of the significance p value, *0.01 < p ≤ 0.05, **0.001 < p ≤ 0.01, ***p ≤ 0.001), the analysis shows that the changes in metabolites are closely related to the changes in biochemical parameters such as IL-1β, IL-6, TNF-ɑ, and NO, providing evidence for understanding the pathological mechanism of LPS-induced inflammation.

[0143] Perform relevant pathway analysis on the 9 screened differential metabolites. The darker the bubble color, the more metabolites are involved in this pathway. The larger the bubble radius, the greater the impact value, indicating that the role of this pathway is more significant. Figure 28 As shown, the pathways mainly involved in the anti-inflammatory effect of low-molecular-weight Flammulina velutipes polysaccharide are histidine metabolism and glycerophospholipid metabolism.

Claims

1. A preparation method of low-molecular-weight Flammulina velutipes polysaccharide, characterized in that, The preparation method includes the following steps: S1: Preparation of Flammulina velutipes crude polysaccharide: The freeze-dried powder of Flammulina velutipes is defatted with ethanol, and the precipitate is obtained by centrifugation; deionized water is added to the precipitate for ultrasonic extraction, and the supernatant is obtained by centrifugation; the supernatant is first rotary evaporated and then ethanol-precipitated, and the precipitate is obtained by centrifugation; deionized water is added to the precipitate for re-dissolution, and Savage reagent is added for protein removal, stirred, and the supernatant is obtained by centrifugation; after rotary evaporation of the supernatant again, it is freeze-dried to obtain Flammulina velutipes crude polysaccharide; S2: Isolation and purification: The Flammulina velutipes crude polysaccharide is dissolved in deionized water, and the supernatant is obtained by centrifugation. It is passed through a DEAE Sepharose FF chromatography column and eluted and separated with dH2O. This is repeated multiple times, and the eluates are combined, concentrated, dialyzed, and freeze-dried; it is separated and purified multiple times with a Chromdex 75PG gel chromatography column to obtain low-molecular-weight Flammulina velutipes polysaccharide.

2. The preparation method of a low-molecular-weight Flammulina velutipes polysaccharide according to claim 1, characterized in that, The ethanol defatting includes: adding an ethanol aqueous solution with a volume fraction of 85% to the freeze-dried powder of Flammulina velutipes at a solid-liquid ratio of 1:15, and soaking at room temperature for 8 - 24 h; The ultrasonic extraction includes: adding deionized water to the precipitate at a solid-liquid ratio of 1:30, and performing ultrasonic extraction at a ultrasonic power of 620 W, an ultrasonic extraction time of 50 min, and an ultrasonic extraction temperature of 45 °C.

3. The preparation method of a low-molecular-weight Flammulina velutipes polysaccharide according to claim 1, characterized in that, The vacuum degree of the rotary evaporation is 60 - 70, the temperature is 55 °C, and it is rotary evaporated to 1 / 4 of the original volume; The ethanol precipitation includes: adding three volumes of an ethanol aqueous solution with a volume fraction of 95% to the solution after rotary evaporation, and keeping it at 4 °C for 8 - 24 h.

4. The preparation method of a low molecular weight Flammulina velutipes polysaccharide according to claim 1, characterized in that, The rotation speed of the centrifugation is 5000 rpm, and the centrifugation time is 12 - 15 min; during re-dissolution, deionized water is added at a solid-liquid ratio of 1:30 for re-dissolution; the Savage reagent is prepared from n-butanol and chloroform in a volume ratio of 1:4 and added to the solution at a reagent ratio of 1:0.2; the stirring time is 30 min.

5. A low-molecular-weight Flammulina velutipes polysaccharide, characterized in that, The low-molecular-weight Flammulina velutipes polysaccharide is prepared by the preparation method of the low-molecular-weight Flammulina velutipes polysaccharide as described in any one of claims 1 - 4.

6. A low molecular weight Flammulina velutipes polysaccharide according to claim 5, characterized in that, The low-molecular-weight Flammulina velutipes polysaccharide is a neutral polysaccharide, with α-pyranoside, and is a glucan with →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ as the main chain and α-D-Glcp-(1→ as the side chain, and the relative molecular mass is 6,181 Da.

7. Use of the low molecular weight Flammulina velutipes polysaccharide as described in claim 5 or 6 in an anti-inflammatory drug, characterized in that, By down-regulating the levels of dimethylphosphatidylethanolamine and histamine, it exerts an anti-inflammatory effect.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the low-molecular-weight Flammulina velutipes polysaccharide as described in claim 5 or 6.

9. A functional article, characterized in that, The functional product contains the low-molecular-weight Flammulina velutipes polysaccharide as described in claim 5 or 6.

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