阅读说明：本技术 一种黄白侧耳多糖及其制备方法和应用 (Pleurotus citrinopileatus polysaccharide and preparation method and application thereof ) 是由 丁祥 侯怡铃 唐贤 朱淼 宋志强 陈茜 杨彤 朱洪庆 于 2020-07-27 设计创作，主要内容包括：本发明具体公开了一种黄白侧耳多糖(PC-1)及其制备方法和应用。将黄白侧耳子实体经热水浸提、乙醇醇沉、除蛋白、离子交换柱层析和透析,最后浓缩得到纯化的黄白侧耳多糖(PC-1)。本发明还公开了黄白侧耳多糖(PC-1)的组成：α-D-木糖、α-D-半乳糖、α-D-葡萄糖和β-D-葡萄糖；其中α-D-木糖、α-D-半乳糖与葡萄糖(包括α-D-葡萄糖与β-D-葡萄糖)的残基的摩尔比为2:2:8,α-D-葡萄糖与β-D-葡萄糖的残基的摩尔比为7:1。本发明制备的黄白侧耳多糖(PC-1)具有显著的免疫调节活性,可应用在药品、保健品或食品中。该应用为黄白侧耳的开发提供了一种新的途径。(The invention particularly discloses pleurotus citrinopileatus polysaccharide (PC-1) and a preparation method and application thereof. The pleurotus citrinopileatus polysaccharide (PC-1) is obtained by extracting pleurotus citrinopileatus sporocarp with hot water, precipitating with ethanol, removing protein, performing ion exchange column chromatography and dialysis, and finally concentrating. The invention also discloses the composition of pleurotus citrinopileatus polysaccharide (PC-1): alpha-D-xylose, alpha-D-galactose, alpha-D-glucose and beta-D-glucose; wherein the molar ratio of the residues of alpha-D-xylose, alpha-D-galactose and glucose (including alpha-D-glucose and beta-D-glucose) is 2:2:8, and the molar ratio of the residues of alpha-D-glucose and beta-D-glucose is 7: 1. The pleurotus citrinopileatus polysaccharide (PC-1) prepared by the invention has obvious immunoregulation activity and can be applied to medicines, health-care products or foods. The application provides a new way for the development of Pleurotus cornucopiae.)

1. A polysaccharide which is a heteropolysaccharide consisting of α -D-xylose, α -D-galactose, α -D-glucose and β -D-glucose, wherein the molar ratio of the residues of α -D-xylose, α -D-galactose and glucose is 2:2: 8.

2. The polysaccharide of claim 1, wherein the polysaccharide comprises α -D-xylose, α -D-galactose, α -D-glucose and β -D-glucose, wherein the molar ratio of the residues of α -D-glucose to β -D-glucose is 7: 1.

3. The polysaccharide of claim 1, wherein the polysaccharide comprises 1, 6-linked α -D-glucose residues, 1,2, 6-linked α -D-galactose residues, 1-linked α -D-xylose residues, 4-linked β -D-glucose residues.

4. The polysaccharide of claim 1, wherein the polysaccharide comprises the structure:

wherein n is an integer of 3 to 10.

5. Polysaccharide according to any one of claims 1 to 4, characterized in that the weight average molecular weight of the polysaccharide is 8000-20000Da, preferably 14992 Da.

6. The method for producing polysaccharides according to any one of claims 1 to 4, which comprises the step of extracting Pleurotus cornucopiae fruit bodies:

preferably, the preparation method comprises the following steps:

(1) taking pleurotus citrinopileatus sporocarp powder, extracting with water, and precipitating the obtained water extract with ethanol to obtain crude polysaccharide;

(2) performing ion exchange column chromatography on the crude polysaccharide obtained in the step (1), eluting, and collecting eluent;

(3) and (3) dialyzing and concentrating the eluent obtained in the step (2) by using a dialysis bag.

7. The preparation method according to claim 6, wherein in the step (1), the leaching temperature is 80-100 ℃, preferably 98 ℃;

preferably, the material-liquid ratio of the pleurotus citrinopileatus sporocarp powder to water is 1: 1-10;

preferably, the leaching times are 1-5 times, and each leaching time is 1-10 hours;

preferably, the volume ratio of the concentrated solution of the alcohol to the aqueous extract is 1-10: 1;

preferably, the alcohol is ethanol.

8. The production method according to claim 6, wherein in the step (2), the ion exchange column is a cellulose column, and a filler thereof is DEAE-cellulose;

preferably, the eluent is a NaCl solution;

preferably, the elution is a gradient elution with an eluent concentration of 0.01-1.0 mol/L.

9. The method according to claim 6, wherein in the step (3), the cut-off molecular weight of the dialysis bag is 5000-10000Da, preferably 7000 Da.

10. Use of a polysaccharide according to any one of claims 1 to 5 in the preparation of immune enhancing health products and food products.

Technical Field

The invention belongs to the technical field of application of fungal polysaccharide, and relates to pleurotus citrinopileatus polysaccharide (PC-1) and a preparation method and application thereof.

Background

The edible fungus is commonly called mushroom, is a large fungus, and the fruiting body of the edible fungus is rich in nutrient substances such as protein, vitamins, mineral elements, amino acid, polysaccharide and the like.

The edible fungi polysaccharide has the characteristics of antivirus, antioxidation, antitumor, blood fat reduction, immunity regulation and other biological activities such as promotion of proliferation and differentiation of immune cells, secretion of lymphokines, activation of complement and the like, is safe and nontoxic, and has wide attention in the fields of health food, biological medicine and the like. And the edible fungus polysaccharide is a non-specific immunopotentiator, and can improve the immune function of the organism through various ways without side effects on the organism.

Pleurotus cornucopiae (Pleurotus cornucopiae), also known as Pleurotus geesteranus, Pleurotus cornucopiae and the like, belongs to the subphylum, Hymenomycetes, Agaricales, Pleurotaceae and Pleurotus, has delicious taste and rich nutrition, contains eight essential amino acids for human body, has the functions of resisting cancer and oxidation, improving immunity, lowering blood pressure and cholesterol, and is a low-calorie low-fat edible fungus which is commercially planted in China, Korea and Japan. Pleurotus albus was introduced from hong Kong in 1978, and cultivation tests were successively conducted in Guangzhou, Fujian, Shanxi, Jilin, etc. of our country and widely spread.

The mycelium of Pleurotus cornucopiae is white, has locked combination, and has thin, flat and stretched colony appearance. The fruiting body grows singly or in clusters, medium to large, the diameter of the pileus can reach 3 cm-12 cm, the surface is in light tan, and the edge of the pileus is in wave shape after the pileus is mature; the fungus folds and the fungus flesh are white, and the fungus folds grow and are unequal in length; the stipe is lateral, white, the base is slightly thin, the thickness is 0.6 cm-1.5 cm, and the length is 2 cm-6 cm. The mature pleurotus citrinopileatus sporocarp is completely stretched in pileus, has no obvious difference with the common oyster mushroom in appearance, but has low fibration degree of the pleurotus citrinopileatus stipe on the quality and the taste, is fine, smooth and soft compared with other pleurotus varieties, is relatively crisp and has fragrant and rich mushroom flavor. And the raw mushroom stem is easy to peel off, so that people like the raw mushroom stem. The pleurotus citrinopileatus has the characteristics of simple cultivation process, regular fruiting, short cultivation period, strong stress resistance, high yield and good market, and is very suitable for industrial cultivation.

Pleurotus cornucopiae (paul. ex. per) Roll polysaccharide research (Ministry of Gansu science, 1993, 5(3):6-13.) by Liudong et al reported that Pleurotus cornucopiae (Pleurotus cornucopiae) fruiting body is extracted with hot water and precipitated with ethanol to obtain crude polysaccharide product P_Ⅲ(ii) a Then, protein is removed, dialysis and DEAF-SephadexA-25 purification are carried out to obtain P_FⅢ-1、P_FⅢ-2、P_FⅢ-3Three polysaccharide fractions. The ratio of the amounts is about 146:2: 1. P_FⅢ-1The gel chromatography of SephadexG-200 and the electrophoresis of cellulose acetate film prove that the two are integrated. Average molecular weightSpecific rotation degree [ a ]]²⁰ _D＝+168°(C＝0.0l,H₂O) analysis by P.L.C and G.L.C showed that P_FⅢ-1Is a heteropolysaccharide containing fructose. Consists of glucose, fructose and mannose in the molar ratio of l to 0.76 to 0.286. Elemental analysis showed P_FⅢ-1Contains no N, 39.97 percent of C and 6.13 percent of H, and is in the range of 3300-^-1，1645cm^-1.1195cm^-1There is infrared absorption. Animal pharmacological experiments show that: p_Ⅲ、P_FⅢ-1Has effects in enhancing immunity, and can be used for treating Lewis lung cancer and Lewis lung cancer C₂₇Colon cancer, Hep liver cancer, etc.

Zhang et al, in Fractionation, partial characterization of aromatic bioactivities of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus, ([ J ]. International Journal of biological macromolecules,2011,48:5-12.) reported the isolation of polysaccharides with high antitumor activity from Pleurotus xanthus fruit bodies.

Therefore, the fine structure of the pleurotus eryngii polysaccharide and the application of the pleurotus eryngii polysaccharide in the immunoregulatory activity are lacked in the prior art.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides pleurotus citrinopileatus polysaccharide (PC-1) and a preparation method and application thereof.

The specific technical scheme is as follows:

the invention provides pleurotus citrinopileatus polysaccharide (PC-1), which is heteropolysaccharide consisting of alpha-D-xylose, alpha-D-galactose, alpha-D-glucose and beta-D-glucose, wherein the molar ratio of the xylose to the galactose to the glucose (including the alpha-D-glucose to the beta-D-glucose) residues is 2:2:8, and the molar ratio of the alpha-D-glucose to the beta-D-glucose residues is 7: 1.

Further, the weight average molecular weight of the above polysaccharide is 8000-20000 Da; in one embodiment of the invention, the weight average molecular weight of the polysaccharide is 14992 Da.

Further, the chemical structure of the above polysaccharide comprises 1, 6-linked α -D-glucose residues, 1,4, 6-linked α -D-glucose residues, 1,2, 6-linked α -D-galactose residues, 1-linked α -D-xylose residues and 4-linked β -D-glucose residues.

Still further, the chemical structure of the above polysaccharide comprises a main chain consisting of 1, 6-linked α -D-glucose and side chains consisting of 1,4, 6-linked α -D-glucose residues, 1,2, 6-linked α -D-galactose residues, 1-linked α -D-xylose residues and 4-linked β -D-glucose residues.

Further, the above polysaccharide comprises the following structural formula:

wherein n is an integer of 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10).

The invention also provides a preparation method of the polysaccharide, which comprises the step of extracting pleurotus citrinopileatus sporocarp.

Further, the preparation method comprises the step of extracting crude polysaccharide by a water extraction and alcohol precipitation method.

Further, the above production method may further comprise a step of purifying the crude polysaccharide (for example, by ion exchange column chromatography).

In one embodiment of the present invention, the above preparation method comprises the steps of:

(1) taking pleurotus citrinopileatus sporocarp powder, extracting with hot water, and sequentially concentrating, precipitating with ethanol and drying the obtained water extract to obtain crude polysaccharide;

(2) performing ion exchange column chromatography on the crude polysaccharide obtained in the step (1), eluting, and collecting eluent;

(3) and (3) dialyzing and concentrating the eluent obtained in the step (2) by using a dialysis bag.

Preferably, (4) freeze-drying the liquid in the dialysis bag after the completion of step (3).

Further, in step (1), the leaching temperature may be 80-100 ℃ (e.g. 80, 85, 90, 95, 100 ℃); in one embodiment of the invention, the leaching temperature is 98 ℃.

Further, in the step (1), the material-to-liquid ratio (W/V, mg/mL) of the pleurotus eryngii fruiting body powder to the water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:5, 1:8 and 1: 10); in one embodiment of the invention, the feed to liquid ratio is 1: 3.

Further, in the step (1), the leaching times are 1 or more (such as 2, 3, 4 and 5); in one embodiment of the present invention, the number of leaching times is 3.

Further, in the step (1), the leaching time is 1-10 hours (such as 1, 3, 6, 8 and 10 hours) for each time; in one embodiment of the invention, the leaching time is 6 hours per time.

In one embodiment of the present invention, the leaching step in step (1) may comprise: mixing Pleurotus cornucopiae fruiting body powder with water, and boiling in water bath.

Further, in the step (1), in the alcohol precipitation step, the volume ratio of the alcohol to the concentrated solution of the aqueous extract is 1-10:1 (such as 1:1, 3:1, 4:1, 5:1, 10: 1); in one embodiment of the invention, the volume ratio is 3: 1;

in an embodiment of the invention, in the above alcohol precipitation step, the alcohol is ethanol.

In one embodiment of the present invention, step (1) comprises: extracting Pleurotus cornucopiae fruiting body powder with hot water, collecting supernatant, concentrating, adding anhydrous ethanol, collecting precipitate, oven drying, and removing protein to obtain crude polysaccharide.

Further, in the step (2), the ion exchange column may be a cellulose column packed with a filler such as DEAE-cellulose.

Further, in the step (2), the eluent used for elution may be a NaCl solution; specifically, the NaCl solution has a concentration of 0.01-1.0mol/L (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0) mol/L.

Further, in step (2), the elution may be a gradient elution, and the concentration of the eluent may be 0.01-1.0mol/L (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0) mol/L.

In one embodiment of the present invention, the step (2) comprises: and (2) passing the aqueous solution of the crude polysaccharide obtained in the step (1) through a cellulose column, performing gradient elution, collecting eluent, and concentrating.

Further, in the step (3), the cut-off molecular weight of the dialysis bag is 5000-; in one embodiment of the invention, the molecular weight cut-off is 7000 Da.

In one embodiment of the present invention, step (3) comprises: and (3) putting the eluent obtained in the step (2) into a dialysis bag for dialysis for 3 days and dialyzing for two days.

The invention also provides the crude polysaccharide prepared by the method.

The invention also provides application of the polysaccharide in preparing health-care products and foods for enhancing immunity.

In the above applications, the above polysaccharides may be used alone or in combination with other active ingredients.

The inventor of the invention separates and purifies pleurotus cornucopiae to obtain the polysaccharide PC-1, analyzes and identifies the molecular weight, monosaccharide composition, chemical structure and the like of the polysaccharide PC-1, and determines the weight average molecular weight and the structural composition of the polysaccharide PC-1. Cell experiments show that the polysaccharide has obvious immunoregulation activity, and particularly, the proliferation rate of T cells is the highest at the concentration of 15 mu g/mL; particularly, the proliferation promoting rate of B cells and RAW264.7 cells is the highest when the concentration is 10 mu g/mL;

the polysaccharide can promote B cell to secrete immunoglobulin. Based on the polysaccharide, the polysaccharide can be used for preparing health care products and foods for enhancing immunity, has better application prospect and commercial value, and can also improve the utilization value of pleurotus nebrodensis.

Drawings

FIG. 1 shows the HPGPC spectrum of PC-1;

FIG. 2 shows an IR spectrum of PC-1;

FIG. 3 shows the HLPC spectrum of PC-1;

FIG. 4 shows PC-1¹H NMR spectrum;

FIG. 5 shows a schematic representation of PC-1¹³C NMR spectrum;

FIG. 6 shows PC-1¹H-¹H-COSY spectrum;

FIG. 7 shows the HMQC spectrum of PC-1;

FIG. 8 shows the HMQC spectrum of PC-1;

FIG. 9 shows the chemical structure of PC-1;

FIG. 10 shows experimental results of the effect of PC-1 on B cell proliferation;

FIG. 11 shows experimental results of the effect of PC-1 on T cell proliferation;

FIG. 12 shows the experimental results of the effect of PC-1 on the proliferation of RAW264.7 cells.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

In the present invention, "Pleurotus cornucopiae" refers to a fungus of the genus Pleurotus of the family Pleurotaceae, the class Hymenomycetes, the order Agaricales, the subdivision Basidiomycotina, which contains both fruit bodies and mycelia.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.