CN111410699A

CN111410699A - Tibetan ganoderma lucidum polysaccharide G L P-3 and preparation method and application thereof

Info

Publication number: CN111410699A
Application number: CN202010267013.0A
Authority: CN
Inventors: 高雄; 穆静静; 谢意珍; 胡惠萍; 莫伟鹏
Original assignee: Guangdong Yuewei Biotechnology Co ltd; Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
Current assignee: Guangdong Yuewei Biotechnology Co ltd; Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
Priority date: 2020-04-08
Filing date: 2020-04-08
Publication date: 2020-07-14
Anticipated expiration: 2040-04-08
Also published as: CN111410699B

Abstract

The invention discloses a Tibetan ganoderan G L P-3 and a preparation method and application thereof, wherein the Tibetan ganoderan G L P-3 is extracted from Tibetan ganoderan powder, the content of the polysaccharide is 98.8%, the weight-average molecular weight is 159.7kDa, the Tibetan ganoderan G L P-3 does not contain proteins, and the Tibetan ganoderan mainly comprises glucose, arabinose, xylose, mannose and galactose, wherein the molar ratio of the glucose is 92.7%, and the Tibetan ganoderan G L P-3 has characteristic absorption peaks of-OH, C-H, C ═ O and α -isopyranan.

Description

Tibetan ganoderma lucidum polysaccharide G L P-3 and preparation method and application thereof

Technical Field

The invention relates to the field of research on ganoderma lucidum polysaccharides, and in particular relates to Tibetan ganoderma lucidum polysaccharide G L P-3 and a preparation method and application thereof.

Background

Ganoderma lucidum (Ganoderma lucidum Karst), also known as Lingzhongling and Qiongzhen, is the fruiting body of Ganoderma lucidum of Polyporaceae, and belongs to Eumycota, Basidiomycotina, Hymenomycetes, Aphyllophorales, Gamodemataceae and Ganoderma. Ganoderma lucidum has been used in China for over 2000 years, and is regarded as a miraculous treasure by physicians of all generations for nourishing and strengthening body, strengthening body resistance and consolidating constitution. In 2000, about 100 species of Ganoderma (Ganoderma) fungi, most widely distributed as Ganoderma lucidum (g.lucidum), Ganoderma sinensis (g.japonicum), Ganoderma applanatum (g.applanatum), Ganoderma tsugae (g.tsugae), and Ganoderma capense (g.capense) were known for pharmaceutical use. Through a large number of clinical researches, the ganoderma lucidum has different degrees of curative effects on neurasthenia, hyperlipoidemia, coronary heart disease, arrhythmia, keshan disease, altitude sickness, hepatitis, hemorrhagic fever, dyspepsia, tracheitis and the like.

The classification history of ganoderma is long and different, and the ancient books such as Ben Cao gang mu classify ganoderma into six colors: green Ganoderma, Ganoderma lucidum, yellow Ganoderma, white Ganoderma, black Ganoderma, and purple Ganoderma. In 2015, Litaihui et al, published in Mycoscience, a journal of mycology, announced that they discovered a new species of Ganoderma lucidum, Ganoderma leucoctextum, in the Tibet Linzhi region. Therefore, systematic research is urgently needed for the edible and medicinal values of Tibetan lucid ganoderma.

From the 50 s of the 20 th century, polysaccharides have attracted wide attention due to their unique physiological activities and their safe and nontoxic characteristics, and have gradually become hot spots of research as spectral immune enhancers in the 60 s to now, more than 300 kinds of polysaccharide compounds have been extracted and structurally identified and used as drugs for adjuvant therapy or direct therapy.

However, in the prior art, the research results on new ingredients of Tibetan ganoderan are few, and the isolation of Tibetan ganoderan by methods such as ultrafiltration and the like and reports on the immunoregulation effect of Tibetan ganoderan are not yet available.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide Tibetan ganoderma polysaccharide G L P-3 and a preparation method and application thereof, wherein Tibetan ganoderma is taken as a research object, and polysaccharide with the molecular weight of more than 100kDa is subjected to separation and purification and biological activity research to find the structure of Tibetan ganoderma polysaccharide (>100kDa) and the edible and medicinal values thereof in the aspect of immunoregulation.

In order to achieve the purpose, the invention provides the following technical scheme:

the Tibetan ganoderan G L P-3 has the following basic repeating unit structure:

preferably, the Tibetan ganoderma lucidum polysaccharide G L P-3 has the polysaccharide content of 98.8 percent and the weight-average molecular weight of 159.7 kDa.

Preferably, the Tibetan ganoderma lucidum polysaccharide G L P-3 does not contain protein and mainly comprises glucose, arabinose, xylose, mannose and galactose, wherein the molar ratio of the glucose is 92.7%.

Preferably, the Tibetan ganoderan G L P-3 has characteristic absorption peaks of-OH, C-H, C ═ O and α -isomeric pyranose.

The invention also provides a preparation method of the Tibetan ganoderma lucidum polysaccharide G L P-3, which comprises the following steps:

(1) defatting Tibetan Ganoderma powder to obtain defatted Tibetan Ganoderma powder;

(2) adding water into the defatted powder of Tibetan Ganoderma, extracting for several times to obtain water extract, and concentrating under reduced pressure to obtain concentrated solution;

(3) precipitating the concentrated solution, removing protein, dialyzing, and freeze-drying to obtain crude polysaccharide;

(4) preparing crude polysaccharide into crude polysaccharide solution, ultrafiltering the crude polysaccharide solution, and lyophilizing to obtain Tibetan Ganoderma polysaccharide CG L P-3;

(5) preparing Tibetan ganoderan CG L P-3 solution from Tibetan ganoderan CG L P-3, and separating and eluting Tibetan ganoderan CG L P-3 solution with ion exchange column;

(6) and (3) decompressing and concentrating the eluted solution, separating by using a molecular sieve, eluting by using first-grade water, detecting the content of polysaccharide by using a phenol-sulfuric acid method, combining 10-17 tubes of solution with peaks, concentrating, dialyzing and freeze-drying the combined solution to obtain the Tibetan ganoderma polysaccharide G L P-3.

Preferably, step (1) comprises: adding 95% ethanol into Tibetan Ganoderma powder, extracting at 75 deg.C for 2 hr according to the material-liquid ratio of 1:20, repeating the operation once, and oven drying Tibetan Ganoderma to obtain Tibetan Ganoderma defatted powder;

the step (2) comprises the following steps: adding water into defatted powder of Tibetan Ganoderma, extracting at 90 deg.C for 2 hr at a material-to-liquid ratio of 1:20, filtering to obtain water extract, repeating the operation for 2 times, and concentrating under reduced pressure at 60 deg.C to obtain concentrated solution;

the step (3) comprises the following steps: adding 4 times volume of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 16 hr to precipitate polysaccharide, centrifuging at 6000rpm for 10min, and dissolving the precipitate in first-stage water to obtain polysaccharide solution; adding 1/3 times volume of Sevage reagent into the polysaccharide solution, wherein the Sevage reagent is chloroform, n-butyl alcohol is 4:1, violently shaking for 30min, centrifuging at 4 ℃ at 6000rpm for 10min, taking supernatant, and repeating the operation until protein is completely removed; the deproteinized polysaccharide solution was then dialyzed using a 5000Da dialysis bag for 72h, followed by lyophilization to give crude polysaccharide.

Preferably, the step (4) comprises collecting 1.5g crude polysaccharide, preparing into solution with concentration of 3mg/m L, ultrafiltering with 100kDa ultrafiltration membrane, and lyophilizing to obtain Tibetan Ganoderma polysaccharide CG L P-3 with molecular weight of >100 kDa;

step (5) comprises taking Tibetan ganoderan CG L P-360 mg, preparing into 10mg/m L solution, separating with DEAE cellulose ion exchange column, eluting with NaCl solution with concentration of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 mol/L sequentially at elution flow rate of 2m L/min and elution time of 4 min/tube, collecting with full-automatic partial collector, and detecting polysaccharide content with phenol-sulfuric acid method;

and (6) carrying out decompression concentration on the solution eluted by 0.1 mol/L NaCl at 60 ℃, then separating by using a Sephacryl S-300 molecular sieve, eluting by using first-stage water, wherein the elution flow rate is 1m L/min, the elution time is 8 min/tube, then collecting by using a full-automatic partial collector, detecting the polysaccharide content by using a phenol-sulfuric acid method, finally merging 10-17 tubes with peaks, and concentrating, dialyzing and freeze-drying to obtain the Tibetan ganoderma polysaccharide G L P-3.

In the above method, the step (5) is a separation based on the charge of the polysaccharide, and the step (6) is a separation based on the molecular weight of the polysaccharide. The existing polysaccharide separation methods are many and comprise ion exchange columns or molecular sieve separation, but the polysaccharide obtained by a single separation method is often not uniform enough. The ultrafiltration step is added before the ion exchange column and the molecular sieve, so that the approximate molecular weight of the polysaccharide can be estimated, and the method has a key effect on the selection of subsequent molecular sieve fillers.

The invention also provides application of the Tibetan ganoderan G L P-3 in preparing an immunomodulator medicament.

The invention also provides application of the Tibetan ganoderan G L P-3 in preparing an immunomodulator medicament with the effect of promoting phagocytosis of macrophages and/or increasing the generation amount of macrophage NO and/or increasing the generation amount of cytokines and chemokines of the macrophages.

The immunomodulator is recognized by the natural immunoreceptor T L R2 and exerts its immunoregulatory function by promoting the production of NO and/or cytokines and/or chemokines by macrophages.

Preferably, the cytokines include TNF- α, I L-6, GM-CSF, I L-1 α, I L-1 β and I L-10, and the chemokines include CXC L5, MIP-2 and MCP-1.

Compared with the prior art, the invention has the following advantages:

1. the invention separates the Tibetan ganoderma lucidum polysaccharide G L P-3 from the Tibetan ganoderma lucidum for the first time and determines the specific activity application of the Tibetan ganoderma lucidum polysaccharide;

2. the weight average molecular weight of the Tibetan ganoderma polysaccharide G L P-3 is 159.7kDa through GPC measurement, the Tibetan ganoderma polysaccharide G L P-3 has no protein through ultraviolet absorption spectrum analysis, the G L P-3 mainly consists of glucose (92.7 percent) through GC-MS analysis, and a small amount of arabinose, xylose, mannose and galactose, through infrared spectrum analysis, the G L P-3 has characteristic absorption peaks of polysaccharides such as-OH, C-H, C ═ O, α -isopyraose and the like, and the chemical structure of the G L P-3 is analyzed by combining methylation and nuclear magnetic analysis, so that the Tibetan ganoderma polysaccharide G L P-3 is determined to be a new substance;

3. the Tibetan ganoderan G L P-3 has the concentration range of 50-200 mug/m L, can remarkably promote the phagocytic capacity of macrophage RAW264.7 to phagocytose neutral red compared with a control group, can remarkably promote macrophage RAW264.7 to generate NO, can remarkably increase the generation amount of macrophage RAW264.7 cytokines (TNF- α, I L0-6, GM-CSF, I L1-1 α, I L2-1 β and I L-10) and chemokines (C XC L5, MIP-2 and MCP-1) and the like, can be recognized by a natural immune receptor T L R2 when the concentration of G L P-3 is 200 mug/m L, and can play an immune regulation function by promoting the generation of NO, cytokines and chemokines of macrophages;

4. compared with the existing preparation method, the extraction method of the Tibetan ganoderma lucidum polysaccharide G L P-3 has the advantages that an ultrafiltration step is added, separation is directly carried out according to the molecular weight of the polysaccharide, the difficulty of subsequent purification is reduced, and the purity of the obtained polysaccharide is high and reaches 98.8%.

Drawings

FIG. 1 is DEAE cellulose column chromatography elution diagram of Tibetan ganoderan G L P-3;

FIG. 2 is the Sephacryl S-300 molecular sieve elution pattern of Tibetan ganoderan G L P-3;

FIG. 3 is the UV absorption spectrum of Tibetan Ganoderma polysaccharides G L P-3;

FIG. 4 is a GPC chart of Tibetan ganoderan G L P-3;

FIG. 5 is a monosaccharide composition diagram of Tibetan ganoderan G L P-3;

FIG. 6 is an infrared spectrum of Tibetan ganoderan G L P-3;

FIG. 7 shows the preparation of Tibetan ganoderan G L P-3¹³A C NMR spectrum;

FIG. 8 shows the preparation of Tibetan ganoderan G L P-3¹H NMR spectrum;

FIG. 9 is an HH-COSY map of Tibetan ganoderan G L P-3;

FIG. 10 is the HSQC map of Tibetan ganoderan G L P-3;

FIG. 11 is an HMBC profile of Tibetan ganoderan G L P-3;

FIG. 12 is a graph of the cytotoxic effect of Tibetan ganoderan G L P-3 on macrophage RAW 264.7;

FIG. 13 is a graph of the effect of Tibetan ganoderan G L P-3 on the phagocytosis of neutral red by macrophage RAW 264.7;

FIG. 14 is a graph of the effect of Tibetan ganoderan G L P-3 on the production of NO by macrophage RAW 264.7;

FIG. 15 is a graph of the effect of Tibetan ganoderan G L P-3 on the production of cytokines by macrophage RAW 264.7;

FIG. 16 is a graph of the effect of Tibetan ganoderan G L P-3 on the production of chemokines by macrophage RAW 264.7;

FIG. 17 is a graph of the effect of T L R2 and T L R4 antibodies on NO production by Tibetan ganoderan G L P-3-induced macrophage RAW 264.7;

FIG. 18 is a graph of the effect of T L R2 and T L R4 antibodies on TNF- α production by Tibetan ganoderan G L P-3 induced macrophage RAW 264.7;

FIG. 19 is a graph of the effect of T L R2 and T L R4 antibodies on I L-6 production by Tibetan ganoderan G L P-3 induced macrophage RAW 264.7.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, equipment and methods employed in the present invention are all reagents, equipment and methods conventionally available in the art and conventionally used methods, unless otherwise specified.

Example 1

One) preparation of Tibetan ganoderan G L P-3

Adding 95% ethanol into Tibetan Ganoderma powder, extracting at 75 deg.C for 2 hr according to the material-liquid ratio of 1:20, repeating the operation once, and oven drying Tibetan Ganoderma to obtain Tibetan Ganoderma defatted powder; adding water into the defatted powder of Tibetan Ganoderma (the ratio of defatted powder of Tibetan Ganoderma to water is 1:20), extracting at 90 deg.C for 2 hr, filtering to obtain water extract, repeating the operation for 2 times, and concentrating at 60 deg.C under reduced pressure to obtain concentrated solution;

adding 4 times volume of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 16 hr to precipitate polysaccharide, centrifuging at 4 deg.C to collect precipitate, centrifuging at 6000rpm for 10min, and dissolving the precipitate in first-stage water to obtain polysaccharide solution;

adding 1/3 times volume of Sevage reagent into the polysaccharide solution, wherein the Sevage reagent is chloroform, n-butyl alcohol is 4:1, violently shaking for 30min, centrifuging at 4 ℃ at 6000rpm for 10min, taking supernatant, and repeating the operation until protein is completely removed; then dialyzing the polysaccharide solution after protein removal for 72 hours by adopting a 5000Da dialysis bag; then freeze-drying to obtain crude polysaccharide;

taking 1.5g of crude polysaccharide, preparing into solution with concentration of 3mg/m L, performing ultrafiltration with 100kDa ultrafiltration membrane, and lyophilizing to obtain Tibetan Ganoderma polysaccharide CG L P-3 with molecular weight of more than 100 kDa;

CG L P-360 mg is taken to prepare solution with the concentration of 10mg/m L, the solution is separated by a DEAE cellulose ion exchange column, the solution is eluted by NaCl solutions with the concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 mol/L in sequence, the elution flow rate is 2m L/min, the elution time is 4 min/tube, the elution curve is shown in figure 1, the solution is collected by a full-automatic partial collector after elution, the polysaccharide content is detected by a phenol-sulfuric acid method, the solution eluted by 0.1 mol/L NaCl is reduced and concentrated at 60 ℃, the solution is separated by a Sephacryl S-300 molecular sieve, the solution is eluted by primary water, the elution flow rate is 1m L/min, the elution time is 8 min/tube, the elution curve is shown in figure 2, the full-automatic partial collector is used for collection after elution, the polysaccharide content is detected by the phenol-sulfuric acid method, the 10-17 tubes with peaks are combined, and the P-L in the Tibetan medicine is obtained by concentration, dialysis and freeze drying.

II) ultraviolet spectrum analysis of Tibetan ganoderan G L P-3

A certain amount of G L P-3 is dissolved in primary water to prepare a solution with the concentration of 1mg/m L, and the solution is scanned and analyzed on a U-2910 spectrophotometer, the wavelength range is 200-400nm, and the result is shown in figure 3, and the characteristic absorption peaks do not exist at 260nm and 280nm, which indicates that the G L P-3 does not contain protein and nucleic acid.

Thirdly) determination of molecular weight of Tibetan ganoderan G L P-3

The average relative molecular weight of G L P-3 was determined by Waters ACQUITY APC, the system being equipped with Waters ACQUITY APC AQ 900 and ACQUITY APC AQ 450 columns (2.5 μm × 4.6.6 mm × 150mm, particle size 2.5 μm, size 4.6mm × 150mm), column temperature 35 ℃ and mobile phase NaNO₃(100nM), flow rate 0.4m L/min, molecular weight of G L P-3 estimated from calibration curves obtained from dextran standards of different molecular weights (5.2, 11.6, 23.8, 48.6, 148, 273, 410, 668kDa), the results are shown in FIG. 4.

IV) monosaccharide composition analysis of Tibetan ganoderan G L P-3

Taking 2mg of polysaccharide, hydrolyzing with 1M L of 2M trifluoroacetic acid for 90min, evaporating to dryness by using a rotary evaporator, adding 2M L of double distilled water into residues, reducing by using 100mg of sodium borohydride, adding glacial acetic acid for neutralization, performing rotary evaporation, drying in a 110 ℃ oven, adding 1M L of acetic anhydride, performing acetylation at 100 ℃ for 1h, cooling, adding 3M L of methylbenzene, performing reduced pressure concentration and evaporation to dryness, repeating for 4-5 times to remove redundant acetic anhydride, dissolving the acetylated product by using 3M L of chloroform, transferring the dissolved product to a separating funnel, adding a small amount of distilled water, fully shaking, removing an upper aqueous solution, repeating for 5 times, drying a chloroform layer by using a proper amount of anhydrous sodium sulfate, fixing the volume to 10M L, and analyzing by using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer to determine an acetylated product sample;

GC-MS conditions are that RXI-5SI L MS chromatographic column 30m × 0.25mm × 0.25.25 μm, the programmed temperature rise condition is that the initial temperature is 120 ℃, the temperature rises to 250 ℃/min at 3 ℃/min, the temperature is kept for 5min, the injection port temperature is 250 ℃, the detector temperature is 250 ℃/min, the carrier gas is helium, and the flow rate is 1m L/min.

Wherein the standard substance sequence is as follows: rhamnose, fucose, arabinose, xylose, mannose, glucose, galactose. The results are shown in FIG. 5.

From the results of GC-MS, the monosaccharide composition type and ratio of the tibetan ganoderan G L P-3 were arabinose, xylose, mannose, glucose, galactose, 2.4:3.3:0.8:92.7: 0.8.

Fifthly) infrared spectroscopic analysis of Tibetan ganoderma lucidum polysaccharide G L P-3

Adding 100mg dry potassium bromide powder into 2mg polysaccharide, mixing, grinding, placing into a tabletting mold, tabletting for 30s, placing in a sample scanning window at 4000-^-1As shown in fig. 6, it can be seen that: characteristic absorption peaks of the polysaccharide appear. At 3394.61cm^-1There is a strong and broad absorption peak indicating the tensile vibration of the polysaccharide chain-OH. On the other hand, the weak absorption peak was 2930.12cm due to C-H stretching vibration of free sugar^-1，1649.58cm^-1The band at (b) corresponds to the valence vibration of C ═ O. In addition, the length of the probe is 1155.46 cm, 1080.71 cm and 1023.40cm^-1Is a characteristic peak of α -isomeric pyranose.

Sixthly) methylation analysis of Tibetan ganoderan G L P-3

Placing a polysaccharide sample in a reaction bottle, adding DMSO (dimethyl sulfoxide), quickly adding NaOH powder, sealing, dissolving under the action of ultrasound, adding methyl iodide for reaction, finally adding water into the mixture to terminate methylation reaction, taking methylated polysaccharide, hydrolyzing with 1M L of 2M trifluoroacetic acid for 90min, evaporating to dryness by using a rotary evaporator, adding 2M L of double distilled water into residues, reducing 60mg of sodium borohydride for 8 hours, adding glacial acetic acid for neutralization, performing rotary evaporation, drying in a 101 ℃ oven, adding 1M L of acetic anhydride for reaction at 100 ℃, cooling, adding 3M L of toluene, performing reduced pressure concentration and evaporation to dryness, repeating for 4-5 times to remove redundant acetic anhydride, dissolving the acetylated product by using 3M L of chloroform, transferring to a separating funnel, adding a small amount of distilled water, fully shaking, removing an upper aqueous solution, repeating for 4 times, drying a chloroform layer by using a proper amount of anhydrous sodium sulfate, fixing the volume to 10M L, and analyzing by using a Shimadzu GCMS-2010 gas chromatography-QP combined QP analyzer to determine the acetylated product;

GC-MS conditions are that RXI-5SI L MS chromatographic column 30m × 0.25mm × 0.25.25 μm, the programmed temperature rise condition is that the initial temperature is 120 ℃, the temperature rises to 280 ℃/min at 4 ℃/min, the temperature is kept for 5min, the injection port temperature is 250 ℃, the detector temperature is 250 ℃/min, the carrier gas is helium, and the flow rate is 1m L/min.

The results are shown in table 1 below.

TABLE 1 GC-MS analysis of methylated sugar residues of G L P-3, Tibetan Ganoderma polysaccharides

Methylated product	Type of glycosidic bond	Molar ratio of	Principal Mass Spectroscopy fragment (m/z)
				2,3,4,6-Me₄-Gl	Glcp-(1→	0.050	43,71,87,101,117,129,145,161,205
2,3,6-Me₃-Glcp	→4)-Glcp-(	0.875	43,87,99,101,113,117,129,131,161,173
				2,3-Me₂-Glcp	→	4,6)-Glcp-	0.075	43,71,85,87,99,101,117,127,159,161,2

Seventhly) nuclear magnetic resonance analysis of Tibetan ganoderan G L P-3

45mg of G L P-3 were dissolved in 0.5m L D₂And (3) detecting the oxygen, performing nuclear magnetic resonance analysis on the Tibetan ganoderma polysaccharide G L P-3, wherein the results are shown in figures 7-11, and attributing chemical shift values of each carbon and hydrogen of each residue according to nuclear magnetic spectra of figures 7-11, and the attribution results are shown in the following table 2.

TABLE 2 assignment of hydrogen and carbon signals for each sugar residue in G L P-3 of Tibetan Ganoderma polysaccharides

The basic repeating unit structure of G L P-3 is shown by combining monosaccharide composition, infrared spectrum, methylation and nuclear magnetic resonance analysis as follows:

the analysis result shows that G L P-3 has less branched structure, the main chain is connected in a → 4) - α -D-Glcp- (1 → 4) - α -D-Glcp- (1 → 4,6) - β -D-Glcp- (1 → glycosidic bond, and the end group β -D-Glcp- (1 → is connected on the main chain through an O-4 bond.

Example 2

One) toxicity test of Tibetan ganoderan G L P-3 on macrophage RAW264.7

Take RAW264.7 cells (1 × 10)⁵One/well) in 96-well plates at 37 ℃ with 5% CO₂Culturing in incubator for 24 hr, discarding old culture solution, adding G L P-3(50, 100, 200, 400 μ G/m L) with different concentrations, culturing for 24 hr, taking out 96-well plate, discarding old culture solutionNutrient medium, 200 μ L CCK-8(200 μ L serum-free medium/3 μ L CCK-8 stock solution as control group), 5% CO at 37 deg.C₂After the culture in the incubator for 1h, the absorbance value of 450nm is measured by a microplate reader, and the result is shown in figure 12. from figure 12, G L P-3 has no toxicity to macrophage RAW264.7 within the concentration range of 50-400 mug/m L.

II) neutral red phagocytosis experiment of Tibetan ganoderan G L P-3 on macrophage RAW264.7

Take RAW264.7 cells (1 × 10)⁵Pieces/m L) were inoculated in 96-well plates at 37 ℃ with 5% CO₂Culturing in incubator for 4 hr, discarding old culture solution, adding G L P-3(50, 100, 200 μ G/m L) and L PS (1 μ G/m L) with different concentrations, culturing for 24 hr, discarding old culture medium, adding 100 μ L neutral red solution (0.1%), and adding 5% CO at 37 deg.C₂The cells were incubated in an incubator for 1 hour, the neutral red solution was discarded, the cells were washed with DPBS for 3 times, 100. mu. L ethanol acetate solution (1: 1 acetate: ethanol as a control) was added, the cells were lysed at room temperature for 2 hours, and then the absorbance at 540nm was measured with a microplate reader, as shown in FIG. 13. As seen from FIG. 13, G L P-3 significantly promoted the ability of macrophage RAW264.7 to phagocytose neutral red compared to the control, in the concentration range of 50-200. mu.g/m L.

Thirdly) the effect of Tibetan ganoderan G L P-3 on the release of NO, cytokines and chemokines from macrophage RAW264.7

Take RAW264.7 cells (1 × 10)⁵One/well) in 96-well plates at 37 ℃ with 5% CO₂Culturing in incubator for 24h, discarding old culture solution, adding G L P-3(50, 100, 200 μ G/m L) and L0 PS (1 μ G/m L) with different concentrations, culturing for 24h with control group only adding culture medium, collecting cell culture supernatant for measuring the production of NO, cytokine and chemotactic factor, measuring NO with Griess reagent, taking 100 μ L supernatant culture solution to new plate, adding Griess reagent 100 μ L to new plate, measuring 542nm absorbance with microplate reader, the result is shown in FIG. 14, the production of cytokine and chemotactic factor is measured with MI L L IP L EX MAP mouse high sensitivity T cell magnetic bead panel on L uminex MagPix, the result is shown in FIGS. 15 and 16, GLP-3 is in 50-200 μ G/mL concentration range, and compared with control group, it can remarkably promote giant cell growthThe macrophage cell RAW264.7 can generate NO, and can remarkably increase the production of macrophage RAW264.7 cytokine (TNF- α, I L-6, GM-CSF, I L-1 α, I L-1 β, I L-10) and chemotactic factor (CXC L5, MIP-2, MCP-1) and the like.

IV) antibody inhibition experiment of Tibetan ganoderan G L P-3 on macrophage RAW264.7

Take RAW264.7 cells (1 × 10)⁵One/well) was inoculated into a 96-well plate, pretreated with 5. mu.g/m L T L R2, T L0R 4, T L2R 2 and T L3R 4 or isotype control antibody for 1 hour, then 200. mu.g/m L4G L5P-3 was added to the plate for 24 hours, cell culture supernatant was collected for determination of NO, TNF-L1, I L6-6. NO was measured using Griess reagent, 100. mu. L supernatant was taken to the plate, Griess reagent 100. mu. L was added to the plate, absorbance value at 542nm was measured using microplate reader, TNF- α, I L-6 was measured using E L ISA kit, the results are shown in FIGS. 17-19, G L P-3 was recognized by natural immunoreceptors T L R2 at 200. mu.g/m L concentration, and was expressed by the production of chemokine, by promoting the production of NO, cell growth, and macrophage function.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.

Claims

1. A Tibetan ganoderan G L P-3 is characterized in that the basic repeating unit has the following structure:

2. the Tibetan ganoderma polysaccharide G L P-3, according to claim 1, wherein the polysaccharide content is 98.8%, and the weight average molecular weight is 159.7 kDa.

3. The Tibetan ganoderan G L P-3 according to claim 1, characterized in that it is protein-free and mainly consists of glucose, arabinose, xylose, mannose, galactose, wherein the molar ratio of glucose is 92.7%.

4. The Tibetan ganoderan G L P-3 according to claim 1, characterized in that it has characteristic absorption peaks for-OH, C-H, C ═ O and α -isomeric pyranoses.

5. A method for preparing Tibetan ganoderan G L P-3 according to any one of claims 1 to 4, comprising the steps of:

(6) and (3) decompressing and concentrating the eluted solution, separating by using a molecular sieve, eluting by using first-grade water, detecting the content of polysaccharide by using a phenol-sulfuric acid method, combining 10-17 tubes of solution, concentrating, dialyzing and freeze-drying the combined solution to obtain the Tibetan ganoderan G L P-3.

6. The method for preparing Tibetan lucid ganoderma polysaccharide G L P-3 as claimed in claim 5, wherein the step (1) comprises adding 95% ethanol into Tibetan lucid ganoderma powder, extracting for 2h at 75 ℃ according to a material-liquid ratio of 1:20, repeating the operation once, and finally drying Tibetan lucid ganoderma to obtain Tibetan lucid ganoderma defatted powder;

7. The preparation method of Tibetan ganoderan G L P-3 according to claim 5, wherein the step (4) comprises taking 1.5G of crude ganoderan, preparing into solution with concentration of 3mg/m L, performing ultrafiltration with 100kDa ultrafiltration membrane, and lyophilizing to obtain Tibetan ganoderan CG L P-3 with molecular weight >100 kDa;

and (6) carrying out decompression concentration on the solution eluted by 0.1 mol/L NaCl at 60 ℃, then separating by using a Sephacryl S-300 molecular sieve, eluting by using first-stage water, wherein the elution flow rate is 1m L/min, the elution time is 8 min/tube, then collecting by using a full-automatic partial collector, detecting the polysaccharide content by using a phenol-sulfuric acid method, finally combining 10-17 tubes, and carrying out concentration, dialysis and freeze drying to obtain the Tibetan ganoderan G L P-3.

8. The use of the Tibetan ganoderan G L P-3 according to any one of claims 1 to 4 in the preparation of an immunomodulator drug.

9. Use of the Tibetan ganoderan G L P-3 according to any one of claims 1 to 4 in the preparation of an immunomodulator medicament having the efficacy of promoting phagocytosis of macrophages and/or increasing the production of NO by macrophages and/or increasing the production of cytokines and chemokines by macrophages.

10. The use of claim 9, wherein the cytokine comprises TNF- α, I L-6, GM-CSF, I L-1 α, I L-1 β, I L-10, and the chemokine comprises CXC L5, MIP-2, MCP-1.