Ganoderma lucidum polysaccharide Glps-II as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to ganoderma lucidum polysaccharide Glps-II as well as a preparation method and application thereof. Ganoderma lucidum in a certain ganoderma lucidum cultivation base in June County of Chat City is used as a raw material, dried, crushed and decolorized with an ethanol solution, and an ultrasonication extraction method is adopted to obtain a ganoderma lucidum crude sugar extracting solution; removing protein by combining an enzyme-Sevag method, and carrying out alcohol precipitation to obtain ganoderma lucidum crude polysaccharide; cellulose DEAE-52 anion exchange column chromatography is adopted to separate and purify the ganoderma lucidum crude polysaccharide, components under the characteristic elution condition are collected to obtain the ganoderma lucidum polysaccharide Glps-II, and compared with the ganoderma lucidum crude polysaccharide and the ganoderma lucidum polysaccharide Glps-I, the ganoderma lucidum polysaccharide Glps-II has the advantages that the purity of the ganoderma lucidum polysaccharide Glps-II is greatly improved; ganoderma lucidum polysaccharide Glps-III has better antioxidant activity, prostate cancer cell (LNCaP cell) proliferation inhibition effect and anti-inflammatory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a ganoderma lucidum polysaccharide Glps-II and a preparation method and application thereof. Background Art

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

[0003] Ganoderma lucidum, also known as red ganoderma, red ganoderma, purple ganoderma and black ganoderma, is a fruiting body of the Polyporaceae family. Modern pharmaceutical research shows that Ganoderma lucidum has the effects of anti-aging, anti-inflammatory and liver protection, improving human immunity, inhibiting tumors and anti-AIDS (HIV-1). It contains a variety of bioactive substances, the two most important of which are ganoderma triterpenic acid and ganoderma polysaccharide.

[0004] More than 200 types of Ganoderma polysaccharides have been isolated from Ganoderma lucidum. Relevant studies have shown that the configuration of Ganoderma polysaccharides is similar to DNA and RNA. It is a three-dimensional substance composed of three monosaccharide chains and has a helical shape. Studies have shown that the biological activity of polysaccharides is closely related to their configuration. The longer the main chain and the higher the frequency of side chains, the stronger the activity. There are differences in the biological activity of polysaccharides connected by α-type and β-type glycosidic bonds.

[0005] The Ganoderma lucidum polysaccharide obtained after extraction is a mixture of various polysaccharide components. There are obvious differences in the biological activities of different polysaccharide components. Therefore, it is of great significance to study the polysaccharide components with higher biological activities. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a Ganoderma lucidum polysaccharide Glps-Ⅱ and a preparation method and application thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing Ganoderma lucidum polysaccharide Glps-Ⅱ, comprising the following steps:

[0009] (1) crushing the fruiting body of Ganoderma lucidum and decolorizing it, and using ultrasonic crushing extraction to obtain Ganoderma lucidum crude sugar extract; using enzyme-Sevag method to remove protein, and obtaining Ganoderma lucidum crude polysaccharide after alcohol precipitation;

[0010] (2) The crude polysaccharide of Ganoderma lucidum was separated and purified by cellulose DEAE-52 anion exchange column chromatography to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ.

[0011] The second aspect of the present invention provides Ganoderma lucidum polysaccharide Glps-Ⅱ prepared by the preparation method described in the first aspect.

[0012] The third aspect of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of antioxidant drugs.

[0013] The fourth aspect of the present invention provides an antioxidant drug or pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

[0014] The fifth aspect of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of anti-inflammatory drugs.

[0015] The sixth aspect of the present invention provides an anti-inflammatory drug or pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

[0016] The seventh aspect of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of anti-tumor drugs.

[0017] The eighth aspect of the present invention provides an anti-tumor drug or pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

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

[0019] The present invention belongs to the field of biomedicine technology, and specifically relates to a ganoderma lucidum polysaccharide Glps-Ⅱ, a preparation method thereof and an application thereof. The present invention uses ganoderma lucidum in a ganoderma lucidum breeding base in Guan County, Liaocheng as raw material, and after drying, crushing and decolorizing with an ethanol solution, an ultrasonic crushing extraction method is used to obtain a ganoderma lucidum crude sugar extract; an enzyme-Sevag method is used to remove protein, and a ganoderma lucidum crude polysaccharide is obtained after alcohol precipitation; a cellulose DEAE-52 anion exchange column chromatography method is used to separate and purify the ganoderma lucidum crude polysaccharide, and components under characteristic elution conditions are collected to obtain ganoderma lucidum polysaccharide Glps-Ⅱ. Compared with ganoderma lucidum crude polysaccharide and ganoderma lucidum polysaccharide Glps-Ⅰ, ganoderma lucidum polysaccharide Glps-Ⅲ has better antioxidant activity, inhibition of prostate cancer cell (LNCaP cell) proliferation effect and anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 1 is the elution curve in Example 1;

[0022] Figure 2 The DPPH free radical scavenging effect in Example 2;

[0023] Figure 3 The hydroxyl radical scavenging effect in Example 2;

[0024] Figure 4 This is the superoxide anion scavenging effect in Example 2. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] The first typical embodiment of the present invention provides a method for preparing Ganoderma lucidum polysaccharide Glps-Ⅱ, comprising the following steps:

[0028] (1) crushing the fruiting body of Ganoderma lucidum and decolorizing it, and using ultrasonic crushing extraction to obtain Ganoderma lucidum crude sugar extract; using enzyme-Sevag method to remove protein, and obtaining Ganoderma lucidum crude polysaccharide after alcohol precipitation;

[0029] (2) The crude polysaccharide of Ganoderma lucidum was separated and purified by cellulose DEAE-52 anion exchange column chromatography to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ.

[0030] In one or more embodiments, the method of decolorization comprises:

[0031] The crushed Ganoderma lucidum fruiting body powder is soaked in an ethanol solution, filtered and the residue is collected.

[0032] Preferably, the volume fraction of ethanol in the ethanol solution is 93-96%, preferably 95%;

[0033] Preferably, the mass ratio of the crushed Ganoderma lucidum fruiting body powder to the volume ratio of the ethanol solution is 1 g: (14-16) mL, preferably 1 g: 15 mL.

[0034] Preferably, the soaking time is 20 to 30 hours, preferably 24 hours.

[0035] In one or more embodiments, the ultrasonication extraction method comprises:

[0036] The decolorized Ganoderma lucidum fruiting body powder is dispersed in distilled water, mixed and then allowed to stand, and the first extraction is performed at a limited ultrasonic power. After stopping the ultrasonic crushing, the second extraction is performed at a limited temperature. After the extraction, the supernatant is collected by centrifugation and concentrated to obtain the Ganoderma lucidum crude sugar extraction solution.

[0037] Preferably, the volume ratio of the decolorized Ganoderma lucidum fruiting body powder to distilled water is 1 g: (18-22) mL, preferably 1 g: 20 mL.

[0038] Preferably, the standing time after mixing is 1 to 4 hours, preferably 2 hours.

[0039] Preferably, the power is limited to 380-450W, preferably 400W; the time for the first extraction is 20-30min, preferably 30min.

[0040] Preferably, the limited temperature is 80-90°C, preferably 85°C; the second extraction time is 1.5-3h, preferably 2h.

[0041] In one or more embodiments, when the enzyme-Sevag method is used in combination for protein removal, the enzyme used is papain; the enzymatic hydrolysis temperature is 55-60° C., preferably 60° C.; and the enzymatic hydrolysis time is 3.5-5 h, preferably 4 h.

[0042] In one or more embodiments, when the enzyme-Sevag method is used in combination for protein removal, the volume ratio of chloroform to n-butanol in the Sevag reagent is (3.8-4.2):1, preferably 4:1.

[0043] In one or more embodiments, during alcohol precipitation, the volume ratio of anhydrous ethanol to the Ganoderma lucidum crude sugar extract solution after protein removal is 1.8 to 1.9:1, preferably 1.86:1;

[0044] In one or more embodiments, the alcohol precipitation time is 20 to 30 hours, preferably 24 hours.

[0045] In one or more embodiments, the method of separating and purifying the crude Ganoderma lucidum polysaccharide by cellulose DEAE-52 anion exchange column chromatography to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ comprises:

[0046] The crude polysaccharide of Ganoderma lucidum was dissolved in distilled water to prepare a 100 mg / mL aqueous solution of the crude polysaccharide of Ganoderma lucidum, and added to a cellulose DEAE-52 anion exchange column, and gradient eluted with distilled water and a NaCl solution of gradient concentrations in sequence; wherein the concentration of the NaCl solution was 0.2 mol / L, 0.4 mol / L and 0.6 mol / L; and the elution rate was 2 mL / min;

[0047] The eluted fractions from 0.2 mol / L NaCl solution were collected, dialyzed and freeze-dried to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ.

[0048] The second typical embodiment of the present invention provides Ganoderma lucidum polysaccharide Glps-Ⅱ prepared by the preparation method described in the first aspect.

[0049] A third typical embodiment of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of antioxidant drugs.

[0050] A fourth typical embodiment of the present invention provides an antioxidant drug or pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

[0051] A fifth typical embodiment of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of anti-inflammatory drugs.

[0052] A sixth typical embodiment of the present invention provides an anti-inflammatory drug or pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

[0053] A seventh typical embodiment of the present invention provides the use of the Ganoderma lucidum polysaccharide Glps-II described in the second aspect in the preparation of anti-tumor drugs.

[0054] Preferably, the tumor is a prostate tumor.

[0055] An eighth typical embodiment of the present invention provides an anti-tumor drug or a pharmaceutical composition, comprising the Ganoderma lucidum polysaccharide Glps-Ⅱ described in the second aspect.

[0056] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0057] Example 1

[0058] Preparation of Ganoderma lucidum polysaccharide Glps-Ⅱ:

[0059] (1) Using Ganoderma lucidum from a Ganoderma lucidum breeding base in Guan County, Liaocheng as raw material, the Ganoderma lucidum fruiting body was dried and then crushed, and the crushed powder was passed through a 200-mesh sieve. Take 10g of the crushed Ganoderma lucidum fruiting body powder, add 150mL of anhydrous ethanol (volume fraction of 95%), soak for 24h, filter and collect the residue. Repeat the above soaking and filtering process 3 times, and dry the residue in an oven at 80°C for use.

[0060] (2) Take 2 g of the decolorized and dried Ganoderma lucidum fruiting body powder in step (1), disperse it in 40 mL of distilled water, stir and mix evenly, let it stand and soak for 2 hours, and place it in an ultrasonic device with an ultrasonic power of 400 W for ultrasonic extraction for 30 minutes; after stopping ultrasonic crushing, place it in a water bath pot at 85°C, stir (300 rpm) and extract for 2 hours, centrifuge the extracted solid-liquid mixture (5000 rpm for 8 minutes), and collect the supernatant; subject the solid precipitate to the above-mentioned ultrasonic crushing extraction and water bath heating extraction process again, combine the supernatants after the two extractions, and use a rotary evaporator to concentrate to 15 mL to obtain a Ganoderma lucidum crude sugar extraction solution.

[0061] (3) Add 1% papain by mass to the Ganoderma lucidum crude sugar extract solution obtained in step (2), enzymolysis at 60°C for 4h, place the entire reaction system in boiling water at 100°C to inactivate papain, centrifuge (5000rpm for 8min) after taking out, collect the supernatant, and concentrate it to 30mL using a rotary evaporator. Add 10mL sevage reagent (the volume ratio of chloroform to n-butanol is 4:1) to the concentrated supernatant, shake it thoroughly for 30min, remove the lower water layer and the protein at the junction, repeat this operation 5 times, and concentrate it to 10mL using a rotary evaporator. Add 18.6ml 95% anhydrous ethanol to the concentrated liquid, precipitate overnight, centrifuge (5000rpm for 8min), collect the precipitate, freeze-dry the precipitate, and obtain Ganoderma lucidum crude polysaccharide.

[0062] (4) Take 1 g of the crude polysaccharide of Ganoderma lucidum prepared in step (3), dissolve it in distilled water to prepare a crude polysaccharide aqueous solution with a concentration of 100 mg / mL, add it to a cellulose DEAE-52 anion exchange column, and gradient elute with distilled water and gradient concentration NaCl solution in sequence; wherein the concentration of the NaCl solution is 0.2 mol / L, 0.4 mol / L and 0.6 mol / L; the elution rate is 2 mL / min.

[0063] The elution curve is Figure 1 As shown, from Figure 1 It can be seen that after elution, the crude Ganoderma lucidum polysaccharide was separated into three polysaccharide components. Ganoderma lucidum polysaccharide Glps-Ⅰ was obtained under the elution of distilled water, Ganoderma lucidum polysaccharide Glps-Ⅱ was obtained under the elution of 0.2 mol / L NaCl solution, and Ganoderma lucidum polysaccharide Glps-Ⅲ was obtained under the elution of 0.6 mol / L NaCl solution.

[0064] The above elution fractions were collected, concentrated using a rotary evaporator, dialyzed for 48 hours, and freeze-dried to obtain Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ and Ganoderma lucidum polysaccharide Glps-Ⅲ.

[0065] Composition analysis of each monosaccharide, according to the gas chromatography results after hydrolysis and acetylation of Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ and Ganoderma lucidum polysaccharide Glps-Ⅲ, showed that Ganoderma lucidum polysaccharide Glps-Ⅰ and Ganoderma lucidum polysaccharide Glps-Ⅲ were composed of glucose, arabinose, mannose and xylose, while Ganoderma lucidum polysaccharide Glps-Ⅱ was composed of glucose, galactose, arabinose, mannose and xylose.

[0066] The molecular weight of Ganoderma lucidum polysaccharide Glps-Ⅰ is 2.96×10 5 Da, the molecular weight of Ganoderma lucidum polysaccharide Glps-Ⅱ is 3.74×10 4 Da, the molecular weight of Ganoderma lucidum polysaccharide Glps-Ⅲ is 8.23×10 3 Da.

[0067] Example 2

[0068] Antioxidant test:

[0069] The Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and crude Ganoderma lucidum polysaccharide prepared in Example 1 were dissolved in distillation to prepare polysaccharide solutions with gradient concentrations of 1000 μg / mL to 62.5 μg / mL, specifically 1000 μg / mL, 750 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL and 62.5 μg / mL.

[0070] The following DPPH free radical scavenging experiments, hydroxyl free radical scavenging experiments and superoxide anion scavenging experiments all used VC as a positive control.

[0071] (1) DPPH free radical scavenging experiment:

[0072] Experimental group: Take 1 mL of the prepared sample solution (i.e., Ganoderma lucidum polysaccharide Glps-Ⅰ solution, Ganoderma lucidum polysaccharide Glps-Ⅱ solution, Ganoderma lucidum polysaccharide Glps-Ⅲ solution, and Ganoderma lucidum crude polysaccharide solution), add 1 mL of 0.2 mmol / LDPPH ethanol solution, mix well, and react for 40 min in the dark, measure the absorbance at a wavelength of 510 nm, and record it as A. a ; Control group: 1 mL of anhydrous ethanol solution was used to replace the DPPH ethanol solution in the previous experimental group, and the reaction was carried out under the same conditions. The absorbance value was measured at a wavelength of 510 nm and recorded as A b ; Blank control: 1 mL of anhydrous ethanol solution was used to replace the sample solution in the above experimental group, and the reaction was carried out under the same conditions. The absorbance value was measured at a wavelength of 510 nm and recorded as A0. Three parallel experiments were performed and the average value was taken to obtain the DPPH free radical scavenging rate.

[0073] The formula for DPPH free radical scavenging rate (I (%)) is:

[0074] I(%)=[1-(A a -A b ) / A0]×100%;

[0075] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide all have good DPPH free radical scavenging rate, and the removal rate increases with the increasing concentration of each component. Among them, the DPPH free radical scavenging rate of Ganoderma lucidum polysaccharide Glps-Ⅱ is better than that of Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide.

[0076] (2) Hydroxyl free radical scavenging experiment:

[0077] Control group: To 1.0mL 0.75mol / L o-phenanthroline anhydrous ethanol solution, add 2.0mL 0.20 mol / L phosphate buffer (pH 7.4) and 1.0mL distilled water in sequence, mix well, add 1.0mL of newly prepared 0.75mmol / L ferrous sulfate solution, mix well again, add 1.0mL of newly prepared 0.01% H2O2, place the entire reaction system in a 37℃ water bath and heat for 60min, then measure the absorbance at 510nm, recorded as A2; Blank control group: Use 1.0mL distilled water to replace 1.0mL of newly prepared 0.01% H2O2 in the control group H2O2, reacted under the same conditions, measured the absorbance at 510nm wavelength, recorded as A1; experimental group: 1.0mL of sample solution (i.e. Ganoderma lucidum polysaccharide Glps-Ⅰ solution, Ganoderma lucidum polysaccharide Glps-Ⅱ solution, Ganoderma lucidum polysaccharide Glps-Ⅲ solution and Ganoderma lucidum crude polysaccharide solution) was used to replace 1.0mL of distilled water in the control group, reacted under the same conditions, measured the absorbance at 510nm wavelength, recorded as A3. Three parallel experiments were performed, and the average value was taken to calculate the hydroxyl radical scavenging rate.

[0078] The formula for hydroxyl radical scavenging rate is:

[0079] Hydroxyl radical scavenging rate = (A3-A2)(A1-A2)×100%;

[0080] The results are as follows Figure 3 As shown, from Figure 3It can be seen that Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide all have good hydroxyl free radical scavenging rates, and the scavenging rates increase with the increasing concentrations of each component. Among them, the scavenging rate of Ganoderma lucidum polysaccharide Glps-Ⅱ on hydroxyl free radicals is better than that of Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide.

[0081] (3) Superoxide anion scavenging experiment:

[0082] Experimental group: 2.0 mL of the prepared sample solutions (i.e., Ganoderma lucidum polysaccharide Glps-Ⅰ solution, Ganoderma lucidum polysaccharide Glps-Ⅱ solution, Ganoderma lucidum polysaccharide Glps-Ⅲ solution, and Ganoderma lucidum crude polysaccharide solution) and 0.4 mL of 5.00 mmol / L o-catechol were added to 2.0 mL of 50.00 mmol / L Tris-HCl buffer solution (pH 8.2), mixed well and reacted for 4 min, and then 0.1 mL of hydrochloric acid solution (8.00 mol / L) was added to terminate the reaction. The absorbance was measured at 420 nm and recorded as A. 样品 ; Control group: 2.0 mL of distilled water was used to replace the sample solution in the experimental group. Other conditions were the same as those in the experimental group. The absorbance value was measured at 420 nm and recorded as A 对照 ; Blank group: distilled water was used to replace the pyrogallol in the experimental group. Other conditions were the same as those in the experimental group. The absorbance was measured at 420 nm and recorded as A 空白 ; Three parallel experiments were performed, and the average value was taken to calculate the superoxide anion clearance rate.

[0083] Superoxide anion clearance rate = (A 对照 -A 样品 )(A 对照 -A 空白 )×100%;

[0084] The results are as follows Figure 4 As shown, from Figure 4 It can be seen that Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide all have good superoxide anion scavenging rates, and the scavenging rates increase with the increasing concentrations of each component. Among them, Ganoderma lucidum polysaccharide Glps-Ⅱ has a better superoxide anion scavenging rate than Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide.

[0085] Example 3

[0086] Cell culture and cell proliferation experiments:

[0087] Prostate cancer cells (LNCaP cells) were cultured in RPMI 1640 medium containing 10% calf serum in a 5% CO2, 37°C cell culture incubator. Cell growth was observed regularly and subcultured, and cell proliferation experiments were performed using the MTT method to screen different Ganoderma lucidum polysaccharide components.

[0088] LNCaP cells in the logarithmic growth phase were inoculated in a 96-well plate and placed in a 5% CO2, 37°C cell culture incubator to allow the cells to adhere to the wall and grow. RPMI 1640 medium was used to prepare 50 mg / mL sample solutions of Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide. The control group was only added with RPMI 1640 medium for 24 hours. 20 μL of 5 mg / mL MTT solution was added to each well and cultured for 4 hours. The culture supernatant in the well was carefully aspirated and discarded. 200 μL of dimethyl sulfoxide was added to each well. After shaking for 10 minutes, the optical density of each well was measured at a wavelength of 490 nm using an ELISA reader. The cell proliferation inhibition rates of Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide and the control group were calculated, and the results are shown in Table 1.

[0089] Cell proliferation inhibition rate = (absorbance value of experimental group - absorbance value of control group) / absorbance value of control group × 100%.

[0090] Table 1 Cell proliferation inhibition rate

[0091] Group Cell proliferation inhibition rate Control group 0.00±0.00 Ganoderma lucidum polysaccharide Glps-Ⅰ 19.87±4.96 Ganoderma lucidum polysaccharide Glps-Ⅱ 80.32±3.75 Ganoderma lucidum polysaccharide Glps-Ⅲ 36.17±6.48 Ganoderma lucidum polysaccharide 50.28±3.45

[0092] As can be seen from Table 1, Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide can effectively inhibit the proliferation of LNCaP cells, among which Ganoderma lucidum polysaccharide Glps-Ⅱ has the best effect.

[0093] Example 4

[0094] Anti-inflammatory experiment:

[0095] Experimental group: Mouse mononuclear macrophage RAW264.7 was inoculated in a 96-well plate and cultured in a cell culture incubator at 5% CO2 and 37°C. After the cells adhered to the wall, the DMEM medium of the 96-well plate was removed. Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide were dissolved in DMEM culture medium to prepare a 50 mg / mL sample solution. 100 μL of sample solution was added to mouse mononuclear macrophage RAW264.7 respectively. After incubation for 1 hour, LPS solution (final concentration of 10 μg / mL) was added. Each experimental group was placed in an incubator and continued to be cultured for 24 hours. Then, 100 μL of the supernatant of each experimental group was taken in a new 96-well plate, and then 100 μL of Griess reagent with a concentration of 40 mg / mL was added to each well and mixed by the cross method. The absorbance of each well was measured and recorded at a wavelength of 540 nm to calculate the NO inhibition rate. Indomethacin was used as the positive control group, and the blank control group was not treated. IC was calculated using GraphPad Prism 5.0 software 50 The experiment was repeated three times and the results are shown in Table 2.

[0096] Inhibition rate (%) = (A m -A n ) / (A m -A0)×100%

[0097] Among them, A0 is the absorbance of the blank control group measured at 540nm, A n is the absorbance of each experimental group measured at 540 nm, A m The absorbance of the negative (LPS added) control group was measured at 540 nm.

[0098] The results are shown in Table 2.

[0099] Table 2 Effects of polysaccharides on NO production rate of RAW264.7 cell line (IC 50 , μM)

[0100] Group <![CDATA[Anti-inflammatory (IC 50 , μM)]]> Positive control group 43.56±1.23 Ganoderma lucidum polysaccharide Glps-Ⅰ 28.76±0.48 Ganoderma lucidum polysaccharide Glps-Ⅱ 64.87±0.75 Ganoderma lucidum polysaccharide Glps-Ⅲ 16.14±0.51 Ganoderma lucidum polysaccharide 35.03±1.35

[0101] As can be seen from Table 2, Ganoderma lucidum polysaccharide Glps-Ⅰ, Ganoderma lucidum polysaccharide Glps-Ⅱ, Ganoderma lucidum polysaccharide Glps-Ⅲ and Ganoderma lucidum crude polysaccharide all have certain anti-inflammatory effects, and Ganoderma lucidum polysaccharide Glps-Ⅱ has the best effect.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing Ganoderma lucidum polysaccharide Glps-Ⅱ, characterized in that: The steps include: (1) crushing the fruiting body of Ganoderma lucidum and decolorizing it, and using ultrasonic crushing extraction to obtain Ganoderma lucidum crude sugar extract; using enzyme-Sevag method to remove protein, and obtaining Ganoderma lucidum crude polysaccharide after alcohol precipitation; (2) The crude polysaccharide of Ganoderma lucidum was separated and purified by cellulose DEAE-52 anion exchange column chromatography to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ.

2. The preparation method according to claim 1, characterized in that Methods of decolorization include: Soaking the crushed Ganoderma lucidum fruiting body powder in an ethanol solution, filtering and collecting the residue; Preferably, the volume fraction of ethanol in the ethanol solution is 93-96%, preferably 95%; Preferably, the mass ratio of the crushed Ganoderma lucidum fruiting body powder to the volume ratio of the ethanol solution is 1 g: (14-16) mL, preferably 1 g: 15 mL; Preferably, the soaking time is 20 to 30 hours, preferably 24 hours.

3. The preparation method according to claim 1, characterized in that: Ultrasonication extraction methods include: The decolorized Ganoderma lucidum fruiting body powder is dispersed in distilled water, mixed and then allowed to stand, and the first extraction is performed at a limited ultrasonic power, and the second extraction is performed at a limited temperature after stopping the ultrasonic crushing, and the supernatant is collected by centrifugation after extraction, and concentrated to obtain a Ganoderma lucidum crude sugar extraction solution; Preferably, the mass ratio of the decolorized Ganoderma lucidum fruiting body powder to the volume of distilled water is 1 g: (18-22) mL, preferably 1 g: 20 mL; Preferably, the standing time after mixing is 1 to 4 hours, preferably 2 hours; Preferably, the power is limited to 380-450W, preferably 400W; the first extraction time is 20-30min, preferably 30min; Preferably, the limited temperature is 80-90°C, preferably 85°C; the second extraction time is 1.5-3h, preferably 2h.

4. The preparation method according to claim 1, characterized in that: When the enzyme-Sevag method is used to remove protein, the enzyme used is papain; the enzymatic hydrolysis temperature is 55-60°C, preferably 60°C; the enzymatic hydrolysis time is 3.5-5h, preferably 4h; Or, the volume ratio of chloroform to n-butanol in the Sevag reagent is (3.8-4.2):1, preferably 4:1; Or, during the alcohol precipitation, the volume ratio of anhydrous ethanol to the Ganoderma lucidum crude sugar extract solution after protein removal is 1.8-1.9:1, preferably 1.86:1; Alternatively, the alcohol precipitation time is 20 to 30 hours, preferably 24 hours.

5. The preparation method according to claim 1, characterized in that: The method of separating and purifying the crude polysaccharide of Ganoderma lucidum by using cellulose DEAE-52 anion exchange column chromatography to obtain the polysaccharide Glps-Ⅱ of Ganoderma lucidum comprises: The crude polysaccharide of Ganoderma lucidum was dissolved in distilled water to prepare a 100 mg / mL aqueous solution of the crude polysaccharide of Ganoderma lucidum, and added to a cellulose DEAE-52 anion exchange column, and gradient eluted with distilled water and a NaCl solution of gradient concentrations in sequence; wherein the concentration of the NaCl solution was 0.2 mol / L, 0.4 mol / L and 0.6 mol / L; and the elution rate was 2 mL / min; The eluted fractions from 0.2 mol / L NaCl solution were collected, dialyzed and freeze-dried to obtain Ganoderma lucidum polysaccharide Glps-Ⅱ.

6. Ganoderma lucidum polysaccharide Glps-Ⅱ, characterized in that: Prepared by the preparation method described in any one of 1 to 5 above.

7. Use of the Ganoderma lucidum polysaccharide Glps-II as claimed in claim 6 in the preparation of antioxidant drugs.

8. Use of the Ganoderma lucidum polysaccharide Glps-II as claimed in claim 6 in the preparation of anti-inflammatory drugs.

9. Use of the Ganoderma lucidum polysaccharide Glps-II as claimed in claim 6 in the preparation of anti-tumor drugs.

10. A product, characterized in that The polysaccharide Glps-Ⅱ of claim 6; Preferably, the product is a medicament or a pharmaceutical composition.

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