CN110218264B - Lyophyllum nuciferum polysaccharide and preparation method and application thereof - Google Patents
Lyophyllum nuciferum polysaccharide and preparation method and application thereof Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 75
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- 241000218554 Lyophyllum Species 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
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- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 claims abstract description 11
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- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 description 1
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Images
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical Kinetics & Catalysis (AREA)
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- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
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Abstract
The invention discloses a polysaccharide of Lyophyllum nuciferum, a preparation method and application thereof. The polysaccharide is obtained by sequentially performing water extraction and alcohol precipitation, ion exchange column chromatography, dialysis, concentration, extraction and purification on Lyophyllum nuciferum fruiting body, and is heteropolysaccharide consisting of alpha-D-glucose and beta-D-galactose, wherein the molar ratio of residues of the alpha-D-glucose to the beta-D-galactose is 2: 1. The polysaccharide has significant immunoregulation activity and significant inhibitory activity on various tumor cells.
Description
Technical Field
The invention belongs to the technical field of application of fungal polysaccharide, and particularly relates to an aphrodisiac communis polysaccharide and a preparation method and application thereof.
Background
Most basidiomycotina fungi are edible and medicinal fungi, and the fungi are recorded as medicines in China thousands of years ago, such as lucid ganoderma recorded in Shennong Ben Cao Jing and poria cocos recorded in Ben Cao gang mu. The biological activity of the fungal polysaccharide is complex and various, and the fungal polysaccharide is concerned with high medical value, so that a large number of experiments prove that the fungal polysaccharide can resist oxidation aging, reduce blood pressure and blood fat, tonify qi and nourish spirit, resist inflammation and virus, enhance the anti-tumor capability of organisms, improve the immunity of the organisms and the like. The structure of the fungal polysaccharide greatly improves the analysis difficulty due to the connection mode and the branch of the monosaccharide, the variety of the formed monosaccharides, the derivative forms and the like, and is far less than the cognition degree of people on protein and nucleic acid.
Lyophyllum decastes, a name of Latin (Fr.: Fr.) Sing, also known as Clematis frigera, sheep, Nelumbo nucifera, and Tricholoma matsutake, is distributed in Sichuan, Yunnan, Qinghai, etc., has good taste and high nutritive value, and is called as Fried chicken mushroom in Western countries.
At present, no report is found on the fine structure and immunoregulatory activity research and application of the Lyophyllum japonicum polysaccharide LDS-1 in Xiaojin county of Sichuan province.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an aphrodisiac polysaccharide and a preparation method and application thereof.
The specific technical scheme is as follows:
the invention provides an aphrodisiac polysaccharide, which is a heteropolysaccharide composed of alpha-D-glucose and beta-D-galactose, wherein the molar ratio of residues of the alpha-D-glucose to the beta-D-galactose is 2: 1.
Further, the weight average molecular weight of the polysaccharide is 7000-10000Da (e.g., 7000, 8000, 8500, 9000, 9500, 10000 Da); in one embodiment of the invention, the polysaccharide has a weight average molecular weight of 8681 Da.
Further, the chemical structure of the polysaccharide comprises 1, 4-linked α -D-glucose residues and 1, 6-linked β -D-galactose residues.
Still further, the chemical structure of the polysaccharide comprises a 1, 4-linked α -D-glucose backbone and a side chain consisting of two 1, 6-linked β -D-galactose residues.
In one embodiment of the invention, the polysaccharide has the following structural formula:
wherein n is an integer of 5-10 (e.g., 5, 6, 7, 8, 9, 10).
The Lyophyllum decastes polysaccharide is obtained by sequentially extracting Lyophyllum decastes fruiting body with water, precipitating with ethanol, performing ion exchange column chromatography, dialyzing, concentrating, extracting and purifying.
The invention also provides a preparation method of the polysaccharide of the Lyophyllum nuciferum, which comprises the following steps:
(1) taking Lyophyllum decastes fruiting body, and extracting with water and precipitating 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.
Further, the water extraction step is hot water extraction, wherein the extraction temperature is 60-95 ℃ (such as 60, 70, 80, 85, 90, 95 ℃); in one embodiment of the invention, the leaching temperature is 90 ℃.
Further, the number of times of extraction in the water extraction step is 1 or more (e.g. 2, 3, 4, 5 times); in one embodiment of the present invention, the number of times of extraction is 3.
Further, in the alcohol precipitation step, the volume ratio of alcohol to crude sugar solution obtained by water extraction is 1-10:1 (such as 1:1, 3:1, 4:1, 5:1 and 10: 1); in one embodiment of the invention, the volume ratio is 4: 1.
In one embodiment of the present invention, in the alcohol precipitation step, the alcohol is ethanol.
Further, the step (1) comprises: oven drying the fruiting body of Lyophyllum decastes, pulverizing, extracting with hot water to obtain polysaccharide crude extract, adding anhydrous ethanol, centrifuging, collecting precipitate, and oven drying to obtain crude polysaccharide.
Further, the ion exchange column is a cellulose column and/or an agarose gel column.
In one embodiment of the present invention, the filler of the cellulose column is DEAE cellulose-52.
In one embodiment of the invention, the packing of the sepharose column is DEAE-sepharose FF.
Further, the step (2) comprises: and (2) sequentially passing the crude polysaccharide obtained in the step (1) through a cellulose column, eluting, collecting eluent, passing through an agarose gel column, eluting, and collecting eluent.
Further, the eluent used for elution is water.
Further, the cut-off molecular weight of the dialysis bag is 5000-; in one embodiment of the invention, the dialysis bag has a molecular weight cut-off of 7000 Da.
Further, the step (3) comprises: and (3) dialyzing and concentrating the eluent obtained in the step (2) in ultrapure water by using a dialysis bag.
The invention also provides application of the polysaccharide of the Lyophyllum nuciferum in preparing medicines, health-care products and foods for enhancing immunity.
The invention also provides application of the polysaccharide of the Lyophyllum nuciferum in preparing medicines, health products and foods for preventing and/or treating tumors.
In the above applications, the polysaccharides of Lyophyllum nuciferum can be used alone or in combination with other active ingredients.
The polysaccharide LDS-1 is obtained by separation and purification from Lyophyllum nuciferum and has obvious immunoregulation activity, for example, the proliferation rate of T cells can reach 47.95% by adopting 10 mu g/mL LDS-1, and the proliferation rate of B cells can reach 53.86% by adopting 15 mu g/mL LDS-1; and LDS-1 has significant inhibitory activity to various tumor cells, for example, when the mass concentration of LDS-1 is 20 mug/mL, the inhibition rate to MFC cells can reach 45.01%.
Drawings
FIG. 1 shows an HPLC-RID spectrum of LDS-1;
FIG. 2 shows an IR spectrum of LDS-1;
FIG. 3 shows a HLPC profile of the acidic hydrolysate of LDS-1;
FIG. 4 shows LDS-11H NMR spectrum;
FIG. 5 shows LDS-113C NMR spectrum;
FIG. 6 shows a spectrum of HH-COSY of LDS-1;
FIG. 7 shows the HMQC spectrum of LDS-1;
FIG. 8 shows an HMBC spectrum of LDS-1;
FIG. 9 shows the structure of LDS-1;
FIG. 10 shows the experimental results of the effect of LDS-1 on T cell proliferation;
FIG. 11 shows the experimental results of the effect of LDS-1 on B cell proliferation;
FIG. 12 shows the experimental results of the effect of LDS-1 on the proliferation of RAW264.7 cells.
FIG. 13 shows the experimental results of the effect of LDS-1 on the proliferation of CT26.WT cells.
FIG. 14 shows the experimental results of the effect of LDS-1 on MFC cell proliferation.
FIG. 15 shows the experimental results of the effect of LDS-1 on S180 cell proliferation.
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.
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Example 1 separation and extraction of Lyophyllum Inophyllum polysaccharide LDS-1
1.1 separation and extraction of Lyophyllum Inophyllum polysaccharide LDS-1
1.1.1 extraction of Lyophyllum Inophyllum polysaccharide by Hot Water extraction
Taking 200g of Lyophyllum decastes fruiting body, cleaning, drying, pulverizing, and extracting with distilled water at 90 deg.C for 3 times to obtain crude extractive solution of Lyophyllum decastes polysaccharide. Precipitating the polysaccharide crude extract with 4 parts of absolute ethyl alcohol and 1 part of crude sugar concentrate, centrifuging, collecting precipitate, and oven drying.
1.1.2 DEAE-cellulose column chromatography for separating and purifying crude polysaccharide of Lyophyllum Inophyllum
Weighing 40g of DEAE cellulose-52 cellulose, dissolving the DEAE cellulose-52 cellulose in ultrapure water until no particles exist, standing and precipitating for 6h, removing ultrapure water, soaking the DEAE cellulose-52 cellulose in 0.5mol/L NaOH for 6h to activate the dehydrated DEAE cellulose-52 cellulose, washing the DEAE cellulose-52 cellulose with ultrapure water to be neutral, soaking the DEAE cellulose-52 cellulose in 0.5mol/L HCL for 6h, washing the DEAE cellulose with ultrapure water to be neutral, finally soaking the DEAE cellulose-52 cellulose in 0.5mol/L NaOH for 6h, washing the DEAE cellulose-52 cellulose with ultrapure water to be neutral, and completing the activation of the DEAE cellulose-52 cellulose.
And (3) loading the activated DEAE cellulose-52 into a glass column, taking a DEAE cellulose-52 chromatographic column as a stationary phase and distilled water as a mobile phase, and separating and purifying to obtain the Lyophyllum decastes polysaccharide solution. Separating and purifying the Lyophyllum decastes polysaccharide again by using a DEAE-FF gel chromatographic column, wherein the DEAE-FF gel chromatographic column is used as a stationary phase, and distilled water is used as a mobile phase. Dialyzing the concentrated Lyophyllum decastes polysaccharide solution in ultrapure water by using a 7000Da dialysis bag to further obtain purified Lyophyllum decastes polysaccharide, hereinafter abbreviated as LDS-1.
1.2 structural identification of Lyophyllum nuciferum polysaccharide LDS-1
The structure of the polysaccharide LDS-1 of the Lyophyllum decastes is analyzed by hydrolysis, methylation analysis, gas chromatography-mass spectrometry, infrared spectroscopy, nuclear magnetic resonance and high performance liquid chromatography.
1.2.1 determination of molecular weight
The molecular weight of the polysaccharide LDS-1 of the Lyophyllum nuciferum is measured by high performance gel permeation chromatography (HPLC-RID).
1.2.2 Infrared Spectroscopy of Lyophyllum Inophyllum polysaccharide LDS-1
LDS-1 sample 2mg and KBr mixed tablet, scanning 4000cm by infrared spectrophotometer-1-400cm-1And (3) a range.
1.2.3 high performance liquid chromatography analysis of Lyophyllum Inophyllum polysaccharide LDS-1
The standard and LDS-1 sample after TFA acid hydrolysis were dissolved with mobile phase (75% acetonitrile) for HLPC analysis.
1.2.4 Nuclear magnetic resonance analysis of Lyophyllum Inophyllum polysaccharide LDS-1
10mg of LDS-1 sample was dissolved in 0.5mL of heavy water (D)2O), loading into a nuclear magnetic tube, and detecting on a nuclear magnetic resonance instrument.
1.2.5 methylation and silanization of Lyophyllum nuciferum polysaccharide LDS-1 followed by GC-MS analysis
Weighing 40mg of LDS-1 sample, adding 5mL of saturated sodium hydroxide solution to fully dissolve the sample, then gradually adding dimethyl sulfate to react, and adding water to stop the reaction after the reaction. Extracting the product with chloroform, and drying to obtain methylated polysaccharide. After the methylated polysaccharide is completely hydrolyzed by TFA, the methylated polysaccharide is washed by water for three times to obtain a product of completely hydrolyzed methylated acid.
1mg of the sample was added with anhydrous pyridine (0.2mL), hexamethyldisilazane (0.2mL) and trimethylchlorosilane (0.1mL) in this order, reacted at 50 ℃ for 20min, centrifuged (10000rpm, 20min), and the upper layer solution was taken for GC-MS analysis.
1.3 results
1.3.1 basic Property results of Lyophyllum nuciferum polysaccharide LDS-1
The HPLC-RID spectrum of LDS-1 is shown in FIG. 1, which shows that LDS-1 has a weight average molecular weight of 8681 Da.
1.3.2 FTIR Spectroscopy of Lyophyllum Inophyllum polysaccharide LDS-1
The infrared spectrum of LDS-1 is shown in FIG. 2, which shows that the purified LDS-1 is at 4000-400cm-1The peak appearance of (1). Wavelength 3439.552cm-13750-3000cm in O-H telescopic vibration region-1Within the range of wave number 2930.597cm-13000-2700cm in C-H telescopic vibration zone of saturated hydrocarbon-1Within the range of wave number 1655.970cm-11900-1650cm in the carbonyl stretching vibration area-1Within the range of wave number 1397.761cm-11300cm in the C-H bending vibration region (in-plane) 1475--1Within the range of wave number 1070.900cm-1Is C-O stretching vibration with wave number of 617.910cm-1Is the C-H rocking vibration peak. As explained above, LDS-1 has O-H, C-H, carbonyl and C-O functional groups and has the characteristic of polysaccharide infrared spectrum.
1.3.3 high performance liquid chromatography analysis of Lyophyllum nuciferum polysaccharide LDS-1
Analysis of the acidic hydrolysate of LDS-1 by HPLC gave a monosaccharide composition analysis of LDS-1, as shown in FIG. 3. The results show that LDS-1 polysaccharide mainly comprises alpha-D-glucose and beta-D-galactose, and the composition ratio is 2: 1.
1.3.4 NMR Spectroscopy of Lyophyllum Inophyllum polysaccharide LDS-1
Of LDS-11The H NMR spectrum is shown in FIG. 4. As can be seen from FIG. 4, LDS-1 contains four anomeric hydrogen signals, wherein δ 5.03, δ 4.96 and δ 24.89 belong to δ 0 type pyranose, and δ 34.40 belongs to δ 1 type pyranose. Integral analysis found that the ratio between delta 5.03, delta 4.96, delta 4.89 and delta 4.40 was 1:2:1:2 and the ratio of alpha to beta pyranose was 2: 1. Delta 4.09-delta 3.19 are the hydrogen signal regions of C2-C6.
Of LDS-113The C NMR spectrum is shown in FIG. 5. As can be seen from FIG. 5, LDS-1 contains three anomeric carbons, i.e., δ 102.96, δ 101.69, δ 098.28. Delta 2101.69 and delta 398.28 in LDS-1 are in the range of delta 90-delta 102 and are alpha configuration anomeric glycosyl; delta 102.96 is in the range of delta 102-delta 112, and is anomeric glycosyl in delta 1 configuration. Delta 78.23-delta 66.76 is the carbon signal region of C2-C6.
Of LDS-11H-1The H COSY spectrum is shown in FIG. 6, wherein the anomeric hydrogen signals are of four types (A, B, C, D), the signal H1/H2 (delta 5.03/delta 3.85) of the A part, the signal H1/H2 (delta 4.96/delta 3.67) of the B part, the signal H1/H2 (delta 4.89/delta 3.73) of the C part, and the signal H1/H2 (delta 4.40/delta 3.21) of the D part.
The HMQC spectrum of LDS-1 is shown in FIG. 7, and the HMBC spectrum of LDS-1 is shown in FIG. 8. The H/C related chemical shifts were determined and checked according to fig. 7. The chemical shifts for all hydrogens are summarized in table 1 and the chemical shifts for all carbons are summarized in table 2.
TABLE 1 LDS-11Nuclear magnetic signal attribution of H
TABLE 2 LDS-11Nuclear magnetic signal attribution of H
1.3.5 GC-MS spectrum analysis result of Lyophyllum nuciferum polysaccharide LDS-1
The methylation results of LDS-1 are shown in Table 3, which indicates that LDS-1 has (1 → 4) linked α -D-glucopyranose and (1 → 4,6) - α -D-glucopyranose in the repeating unit, branching to (6 → 1) - β -D-galactopyranose residues. In conclusion, the structure of LDS-1 can be preliminarily deduced, as shown in FIG. 9.
TABLE 3 LDS-1 methylation analysis
2. Research on immunoregulatory activity of Lyophyllum nuciferum polysaccharide LDS-1
In vitro two CCK-8 methods were used to determine the in vitro immunomodulatory and antitumor activities of P.nucifera polysaccharide LDS-1, including the proliferative effects on T, B, RAW 264.7.7, CT26.WT, MFC, S180 cells.
2.1 reagents
CCK-8 kit, RPIM1640, FBS, DMSO, double antibody and the like, which are all commercial products.
2.2 instruments
A microplate reader.
2.3 method
Proliferation Effect of LDS-1 on immune cells (T, B, RAW264.7, CT26.WT, MFC, S180 cells)
The effect of LDS-1 on the proliferation of T, B, RAW 264.7.7, CT26.WT, MFC, S180 cells was determined by the CCK-8 method. A blank control group, an LPS (lipopolysaccharide) positive control group and 6 LDS-1 drug groups are arranged in a 96-well plate, and each group comprises 6 wells. 5mL of the suspension cell solution was mixed well and added to a 96-well plate in an amount of 100. mu.L per well for culture for 24 hours. The cell culture solution, 5. mu.g/mL LPS solution, 2.5, 5, 10, 15, 20, 25. mu.g/mL LDS-1 solution at a mass concentration of 100. mu.L/well were added in this order. After 24h of culture, 5 mu L of CCK-8 is added into each well, after 3h, the OD value is measured by a microplate reader, the data are processed by Excel, the data are analyzed by one-way ANOVA (one-way ANOVA) in SPSS19.0, and the OD value is converted into the proliferation rate.
2.4 results
2.4.1 proliferative Effect of LDS-1 on T cells
As shown in FIG. 10, it was found that the proliferation rate of T cells was the highest at a concentration of 10. mu.g/mL in LDS-1 compared with the blank control, and was 47.95%, while the proliferation rate of T cells was stabilized at about 21.00% at LDS-1 concentrations of 15, 20 and 25. mu.g/mL.
2.4.2 proliferative Effect of LDS-1 on B cells
The results are shown in FIG. 11, which shows that when the concentration of LDS-1 is 15 μ g/mL, the proliferation effect of B cells is the best, and the proliferation rate is 53.86% when the LDS-1 drug group is compared with the blank control group differently.
2.4.3 proliferative Effect of LDS-1 on RAW264.7 cells
The results are shown in fig. 12, which shows that when the mass concentration of LDS-1 is 2.5, 5, 10, 15, 20, 25 μ g/mL, the proliferation of macrophages can be promoted, and the difference is very significant (P < 0.01). When the mass concentration of LDS-1 is 15 mug/mL, the proliferation effect on RAW264.7 cells is the best, and the proliferation rate can reach 127.15%.
2.4.4 proliferative Effect of LDS-1 on CT26.WT cells
As shown in FIG. 13, the inhibition rate of the LDS-1 was 39.11% when the concentration was 20. mu.g/mL, compared with the blank control. The result shows that LDS-1 has the effect of inhibiting the growth of CT26.WT cells, and the inhibition rate is gradually increased along with the increase of the mass concentration of LDS-1, so that the inhibition rate has a certain trend.
2.4.5 proliferative Effect of LDS-1 on MFC cells
The conditions in which LDS-1 stimulation affected the growth of MFC cells for proliferation are shown in FIG. 14. Compared with a blank control group, the inhibition rate of the MFC cells can reach 45.01% when the mass concentration of LDS-1 is 20 mu g/mL. The LDS-1 has the effect of inhibiting the growth of MFC cells, and the inhibition rate is gradually increased along with the increase of the mass concentration of the LDS-1, so that the inhibition rate has a certain trend.
2.4.6 proliferative Effect of LDS-1 on S180 cells
The result is shown in fig. 15, when the mass concentration of LDS-1 is 20 μ g/mL, the significant difference is larger than that of the blank control group, and the inhibition rate of the LDS-1 on S180 cells can reach 28.82%. The result shows that the LDS-1 has the effect of inhibiting the growth of mouse S180 sarcoma cells, and the inhibition rate is gradually increased along with the increase of the mass concentration of the LDS-1, so that the LDS-1 has a certain trend.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (11)
1. An aphanidermia nucifera polysaccharide is heteropolysaccharide composed of alpha-D-glucose and beta-D-galactose, wherein the molar ratio of the residues of alpha-D-glucose and beta-D-galactose is 2: 1.
2. The polysaccharide of claim 1, wherein the chemical structure of said polysaccharide comprises 1, 4-linked α -D-glucose residues and 1, 6-linked β -D-galactose residues.
3. The polysaccharide of claim 1, wherein the chemical structure of said polysaccharide comprises a 1, 4-linked α -D-glucose backbone and a side chain consisting of two 1, 6-linked β -D-galactose residues.
4. The polysaccharide of claim 1, wherein the polysaccharide has a weight average molecular weight of 7000-10000 Da.
5. The polysaccharide of claim 1, wherein the polysaccharide has a weight average molecular weight of 8681 Da.
6. The polysaccharide of claim 1, wherein the polysaccharide has the following structural formula:
wherein n is an integer of 5 to 10.
7. A process for the preparation of the polysaccharides of Lyophyllum nuciferum according to any one of claims 1 to 6, comprising the steps of:
(1) taking Lyophyllum decastes fruiting body, and extracting with water and precipitating 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.
8. The method of claim 7, wherein the water extraction step is hot water extraction, wherein the extraction temperature is 60-95 ℃;
in the alcohol precipitation step, the volume ratio of alcohol to crude sugar liquid obtained by water extraction is 1-10: 1;
the ion exchange column is a cellulose column and/or an agarose gel column;
the cut-off molecular weight of the dialysis bag is 5000-.
9. The method of claim 8, wherein step (2) comprises: and (2) passing the crude polysaccharide obtained in the step (1) through a cellulose column in sequence, eluting, collecting eluent, passing through an agarose gel column, eluting, and collecting eluent.
10. The method according to claim 9, wherein the filler of the cellulose column is DEAE cellulose-52;
the filler of the sepharose column is DEAE-sepharose FF;
the eluent is water;
the cut-off molecular weight of the dialysis bag is 7000 Da.
11. Use of the polysaccharide of Lyophyllum nuciferum according to any one of claims 1 to 6 for the preparation of a medicament, health product and food for enhancing immunity, preventing and/or treating tumors.
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