阅读说明：本技术 西藏灵芝多糖glp-3及其制备方法与应用 (Tibetan ganoderma lucidum polysaccharide G L P-3 and preparation method and application thereof ) 是由 高雄 穆静静 谢意珍 胡惠萍 莫伟鹏 于 2020-04-08 设计创作，主要内容包括：本发明公开了西藏灵芝多糖GLP-3及其制备方法与应用,所述西藏灵芝多糖GLP-3提取于西藏灵芝粉末,其多糖含量为98.8％,重均分子量为159.7kDa,不含蛋白,主要由葡萄糖、阿拉伯糖、木糖、甘露糖、半乳糖组成,其中葡萄糖的摩尔占比为92.7％,具有-OH、C-H、C＝O以及α-异构吡喃糖的特征吸收峰。本发明首次从西藏灵芝中分离制备出西藏灵芝多糖GLP-3,制备方法加入超滤步骤,直接按照多糖的分子量进行初步分离,降低了后续纯化的难度,且得到的多糖纯度较高；得到的多糖GLP-3可显著促进巨噬细胞RAW264.7吞噬中性红的能力,显著促进巨噬细胞RAW264.7产生NO、细胞因子和趋化因子,在免疫调节活性方面具有很好的食用和药用价值。(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.)

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:

(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, 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;

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.

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;

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 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.

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.