CN116874622A - Armillariella mellea polysaccharide and preparation method and application thereof - Google Patents
Armillariella mellea polysaccharide and preparation method and application thereof Download PDFInfo
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Abstract
本发明具体公开了一种假蜜环菌多糖(AT‑P)及其制备方法和应用。将假蜜环菌子实体经热水浸提、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假蜜环菌多糖(AT‑P)。本发明还公开了假蜜环菌多糖(AT‑P)的组成：半乳糖、葡萄糖；半乳糖与葡萄糖残基的摩尔比为8:3，其中(1→6)‑半乳糖残基、(1→3,6)‑半乳糖残基和→1)‑半乳糖残基的摩尔比为3:3:2，(1→6)‑葡萄糖残基和→4)‑半乳糖残基的摩尔比为2:1。本发明制备的假蜜环菌多糖(AT‑P)具有显著的免疫调节活性，可应用在药品、保健品或食品中。该应用为假蜜环菌的开发提供了一种新的途径。
The invention specifically discloses a pseudo Armillaria polysaccharide (AT-P) and its preparation method and application. The fruiting bodies of Armillaria pseudomelillae were extracted with hot water, ethanol precipitation, protein removal, ion exchange column chromatography and dialysis, and finally concentrated to obtain purified Armillaria pseudo Armillaria polysaccharide (AT‑P). The invention also discloses the composition of pseudo Armillaria polysaccharide (AT‑P): galactose, glucose; the molar ratio of galactose and glucose residues is 8:3, in which (1→6)‑galactose residues, ( The molar ratio of 1→3,6)-galactose residues and →1)-galactose residues is 3:3:2, the molar ratio of (1→6)-glucose residues and →4)-galactose residues The ratio is 2:1. The pseudo Armillaria polysaccharide (AT‑P) prepared by the invention has significant immunomodulatory activity and can be used in medicines, health products or food. This application provides a new way for the development of Armillaria pseudobacteria.
Description
Technical Field
The invention relates to the technical field of application of fungus polysaccharide, in particular to Armillaria mellea polysaccharide, and a preparation method and application thereof.
Background
Edible fungi commonly called mushrooms are large fungi, and the fruiting bodies of the edible fungi are rich in nutrients such as proteins, vitamins, mineral elements, amino acids, polysaccharides and the like.
The edible fungus polysaccharide has the characteristics of resisting virus, resisting oxidation, resisting tumor, reducing blood fat, promoting proliferation and differentiation of immune cells, secretion of lymphokines, activating immune regulation of complement and the like, is safe and nontoxic, and is widely focused in the fields of health food, biological medicine and the like. The edible fungus polysaccharide is a nonspecific immunopotentiator, can improve the immunity of the organism through various ways, and has no side effect on the organism.
Armillariella mellea [ Armillariella tabescens (Scop. Ex Fr.) Sing ], also known as Armillariella tabescens, armillariella privet, and the like, belongs to the genus Armillariella, the order of Agaricales, the family of Leucopiaceae, and the genus Armillariella. Is mainly distributed in Sichuan, yunnan, guangxi and other places. Has physiological activities of treating hepatitis, resisting tumor, regulating immunity, etc.
The fruit body of the Armillariella mellea is brown, is clustered or singly grown on the earth's surface withered piles or roots in forests, and has isolated hyphae, branches, colorless and transparent and non-locking combination.
At present, the research of the Armillariella mellea is mainly focused on the optimization of the mycelium fermentation process, and the research of the Armillariella mellea polysaccharide is less. Ran Jing and the like can achieve 9.42 percent of yield of the polysaccharide of the armillaria mellea by microwave assistance, which is obviously higher than that of a hot water leaching method (5.93 percent), and the polysaccharide of the armillaria mellea has a certain antioxidant activity. Extracting, separating and purifying two polysaccharides from Armillariella mellea in Japanese Ji Baozhong to obtain water-soluble polysaccharide AT-HW composed of beta- (1- & gt 6) -linked D-glucopyranose and D-galactose; and water insoluble AT-AL based on (1- & gt 3) -alpha-D-glucan, and the AT-HW is found to influence macrophages, T cells and the like to participate in immunity, and the AT-AL mainly activates the macrophages to generate SOA and activates lysosomal enzyme so as to mediate an immunoregulation mechanism, thereby achieving the anti-tumor effect.
Therefore, the research on the fine structure of the Armillariella mellea polysaccharide and the application of the Armillariella mellea polysaccharide in the immunoregulatory activity are lacking in the prior art.
Disclosure of Invention
The invention overcomes the defects existing in the prior art and provides a Armillaria mellea polysaccharide and a preparation method and application thereof.
In a first aspect the invention provides a Armillariella mellea polysaccharide (AT-P) which is a heteropolysaccharide consisting of galactose and glucose, wherein the molar ratio of residues of galactose and glucose is 8:3.
Further, the chemical structure of the polysaccharide comprises (1- > 6) -galactose residues, (1- > 3, 6) -galactose residues, →1) -galactose residues, (1- > 6) -glucose residues and- > 4) -galactose residues.
In one embodiment of the invention, the polysaccharide consists of (1→6) -galactose residues, (1→3, 6) -galactose residues, →1) -galactose residues, (1→6) -glucose residues and→4) -glucose residues.
Further, the molar ratio of (1- > 6) -galactose residues, (1- > 3, 6) -galactose residues and → 1) -galactose residues is 3:3:2.
Further, the molar ratio of (1- > 6) -glucose residues to → 4) -galactose residues is 2:1.
Further, the weight average molecular weight of the polysaccharide is 5000-30000Da (such as 5000Da, 6000Da, 7000Da, 75007600Da, 7700Da, 7800Da, 7900Da, 8000Da, 8100Da, 8200Da, 8300Da, 8400Da, 8500Da, 9000Da, 10000Da, 15000Da, 16000Da, 16500Da, 17000Da, 17100Da, 17200Da, 17300Da, 17400Da, 17500Da, 1700 Da, 17800Da, 17900Da, 18000Da, 19000Da, 20000Da, 21000Da, 22000Da, 23000Da, 24000Da, 25000Da, 30000 Da), preferably 7000-25000Da, further preferably 8000-20000Da.
In one embodiment of the invention, the polysaccharide has a weight average molecular weight of 17600Da.
Further, the chemical structure of the polysaccharide comprises a main chain composed of (1- & gt 6) -galactose residues and (1- & gt 3, 6) -galactose residues and a branched chain composed of (1- & gt 6) -glucose residues and 4) -glucose residues, and the terminal sugar of the polysaccharide is composed of 6-O connection of the 1- & gt galactose residues and the 1- & gt 6) -glucose residues.
Further, the polysaccharide comprises the following structural formula:
where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably an integer from 2 to 15, more preferably an integer from 3 to 10.
Wherein Galp is galactose and Glcp is glucose.
In a second aspect, the present invention provides a method for preparing a polysaccharide from Armillariella mellea according to the first aspect, comprising the step of extracting fruit bodies from Armillariella mellea.
Further, the preparation method comprises the step of extracting crude polysaccharide by a water extraction and alcohol precipitation method.
Further, the preparation method comprises a step of purifying the crude polysaccharide (e.g., by ion exchange column chromatography).
In one embodiment of the invention, the preparation method comprises the following steps:
(1) Extracting Armillariella mellea fruiting body powder with hot water, concentrating the water extract, precipitating with ethanol, and oven drying to obtain crude polysaccharide;
(2) Subjecting the crude polysaccharide obtained in the step (1) to ion exchange column chromatography, 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 temperature of the leaching 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 feed liquid ratio (W/V, mg/mL) of the powder of the fruit body of the Armillariella mellea to water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in one embodiment of the invention, the feed to liquid ratio is 1:3.
Further, in step (1), the number of times of leaching is 1 or more (e.g., 2, 3, 4, 5); in one embodiment of the invention, the number of leaches is 3.
Further, in the step (1), each leaching time is 1-10 hours (such as 1, 3,6, 8, 10 hours); in one embodiment of the invention, the time for each leaching is 6 hours.
In one embodiment of the invention, the leaching step in step (1) may comprise: mixing Armillariella mellea 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 concentrated solution of the alcohol and the water 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 4:1;
in an embodiment of the present invention, in the alcohol precipitation step, the alcohol is ethanol.
In one embodiment of the present invention, step (1) comprises: extracting Armillariella mellea fruiting body powder with hot water, collecting supernatant, concentrating, adding absolute 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, and a filler of the cellulose column is, for example, DEAE-52 cellulose.
Further, in the step (2), the eluent used for elution may be a NaCl solution; specifically, the concentration of the NaCl solution is 0.01-1.0mol/L (such as 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0).
Further, in the step (2), the elution may be 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).
In one embodiment of the present invention, step (2) includes: and (3) passing the aqueous solution of the crude polysaccharide obtained in the step (1) through a cellulose column, carrying out gradient elution, collecting eluent, and concentrating.
Further, in the step (3), the molecular weight cut-off of the dialysis bag is 5000-10000Da (such as 5000, 6000, 7000, 8000, 9000, 10000 Da); in one embodiment of the invention, the molecular weight cut-off is 7000Da.
In one embodiment of the present invention, step (3) includes: and (3) placing the eluent obtained in the step (2) in a dialysis bag for dialysis for two days.
In a third aspect the present invention provides a crude polysaccharide prepared by the process of the second aspect.
In a fourth aspect, the invention provides an application of the Armillariella mellea polysaccharide according to the first aspect in preparing health products and foods for enhancing immunity.
Further, in such applications, the polysaccharide may be used alone or in combination with other active ingredients.
The inventor of the invention obtains polysaccharide AT-P from the Armillaria mellea by separation and purification, and analyzes and identifies the molecular weight, monosaccharide composition, chemical structure and the like of the polysaccharide AT-P, and determines the weight average molecular weight and structural composition of the polysaccharide AT-P. Cell experiments show that the polysaccharide has remarkable immunoregulatory activity, and particularly has the highest proliferation rate of B cells at the concentration of 20 mug/mL; in particular, at a concentration of 10. Mu.g/mL, the T cell proliferation promoting rate is the highest; at a concentration of 2.5. Mu.g/mL, the proliferation rate of RAW264.7 cells was highest.
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 the armillaria pseudolaris.
Drawings
FIG. 1 shows the HPGPC chart of AT-P.
FIG. 2 shows the infrared spectrum of AT-P.
FIG. 3 shows the HLPC spectra of AT-P (wherein, (A-G) is monosaccharide standard, (H) is Armillariella mellea polysaccharide (AT-P)).
FIG. 4 shows the 1H NMR spectrum of AT-P.
FIG. 5 shows AT-P 13 C NMR spectrum.
FIG. 6 shows AT-P 1 H- 1 H-COSY spectrum.
FIG. 7 shows the HMQC spectrum of AT-P.
FIG. 8 shows the HMQC spectrum of AT-P.
FIG. 9 shows the chemical structure of AT-P.
FIG. 10 shows experimental results of the effect of AT-P on B cell proliferation.
FIG. 11 shows experimental results of the effect of AT-P on T cell proliferation.
FIG. 12 shows experimental results of the effect of AT-P on RAW264.7 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 relates.
In the present invention, "Armillariella mellea (Armillariella tabescens)" means fungi of the genus Armillariella of the order Agaricales, the phylum Basidiomycotina, the order Agaricales, the family Tricholomataceae, and the genus Armillariella, which comprises a fruiting body and a mycelium.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 isolation and extraction of Armillariella Mellea polysaccharide AT-P
1. Separation and extraction of Armillariella mellea polysaccharide AT-P
1.1 extracting Armillariella mellea crude polysaccharide by water extraction and alcohol precipitation
200g of dried Armillariella mellea fruiting body is weighed and crushed, the crushed Armillariella mellea fruiting body and distilled water are added into a beaker according to the ratio of 1:3, water bath is carried out for 6 hours at 98 ℃, the supernatant is collected and concentrated, the repeated steps are carried out for 3 times, and finally, the whole supernatant is concentrated to 200mL. Adding three times of anhydrous ethanol to precipitate, collecting precipitate, drying, and removing protein from the extractive solution to obtain Armillariella mellea crude polysaccharide.
1.2 separating and purifying Armillariella mellea crude polysaccharide by DEAE-52 cellulose column chromatography
50g of DEAE cellulose was weighed out precisely and dissolved in 1L of ultrapure water, and stirring was stopped if no macroscopic cellulose particles were present. Standing for 24h, and discarding supernatant for later use. Preparing 0.5mol/L NaOH, soaking cellulose for 6 hours, washing with ultrapure water to neutrality, discarding the supernatant, adding 0.5mol/L HCl, soaking for 6 hours, washing with distilled water to neutrality, discarding the supernatant; adding 0.5mol/L NaOH, soaking for 6 hours again, washing with distilled water to neutrality, and standing for use.
And (3) loading the activated cellulose into a column, and balancing for 24 hours through a distilled water pressure column to separate and purify the crude polysaccharide. The supernatant (5 mL) after dilution of the crude polysaccharide was applied to a DEAE-cellulose column and eluted with different concentrations of NaCl (0.01 mol/L,0.05mol/L,0.1 mol/L). The polysaccharide was measured by the sulfuric acid-phenol method. The eluate was concentrated to 5mL and the sample was purified on a cellulose column. Dialyzing with dialysis bag (Mw is greater than or equal to 7 kDa) for 48 hr, and lyophilizing to obtain Armillariella mellea polysaccharide, designated AT-P.
2. Structural identification of Armillariella mellea polysaccharide AT-P
And carrying out structural analysis on the Armillaria mellea polysaccharide (AT-P) by using acid hydrolysis, methylation analysis, high performance gel permeation chromatography, high performance liquid chromatography, gas chromatography and mass spectrometry combined technology, infrared spectrum technology and nuclear magnetic resonance technology.
2.1 determination of molecular weight
10mg of Armillariella pseudoalternifolia polysaccharide AT-P was sampled with 1mL ddH 2 O was dissolved and sonicated for 5min for HPGPC analysis.
2.2 Infrared Spectrometry of Armillariella Mellea polysaccharide AT-P
Tabletting 2mg AT-P and KBr, scanning 4000cm by infrared spectrophotometer -1 -400cm -1 Range.
2.3 analysis of monosaccharide composition of Armillariella Mellea polysaccharide AT-P
The 7 standards and AT-P samples after TFA acid hydrolysis were dissolved in mobile phase (75% acetonitrile) and subjected to HLPC analysis.
2.4 Nuclear magnetic resonance analysis of Armillariella Mellea polysaccharide AT-P
50mg of AT-P sample was taken and dissolved in 0.6mL of heavy water (D 2 O), loading into a nuclear magnetic resonance tube, and detecting on a nuclear magnetic resonance apparatus.
2.5 methylation and silylation derivatization of Armillariella Mellea polysaccharide AT-P followed by GC-MS analysis
A20 mg sample of AT-P was weighed, the beaker was sealed, 2mL of DMSO (dimethyl sulfoxide) was added to the sealed beaker, and the beaker was gently shaken to allow the AT-P to dissolve sufficiently. Excess NaOH was then added until just the NaOH did not dissolve and it was left to oscillate in the shaker for 1h at room temperature. After the completion of the shaking, 1.5mL of methyl iodide was added, the reaction was carried out in a dark place for 1 hour, and after the reaction, water was added to terminate the reaction. Extracting the product with chloroform, and drying to obtain methylated polysaccharide. And (3) after the methylated polysaccharide is subjected to TFA complete acid hydrolysis, washing with water for three times to obtain a methylated complete acid hydrolysis product.
The sample was reacted with 2mL hexamethyldisilazane, 1mL trimethylchlorosilane, and 2mL anhydrous pyridine, and the mixture was subjected to water bath at 50℃for 20min, centrifuged at 12000rpm/min with a low temperature high speed centrifuge at 4℃for 10min, the precipitate was discarded, and the filtrate was filtered with a 0.22 μm filter, and the supernatant was used for GC-MS analysis.
3. Results
3.1 basic Property results of Armillariella Mellea polysaccharide AT-P
The HPGPC chart of PC-1 is shown in FIG. 1, and shows that AT-P has a weight average molecular weight of 17600Da.
3.2 FTIR spectroscopic analysis of Armillariella Mellea polysaccharide AT-P
The AT-P was characterized by the primary structure using Fourier infrared spectroscopy, and the result is shown in FIG. 2, the wave number is 3425cm -1 ，2928cm -1 1400-1200 cm -1 Typical polysaccharide absorption peaks and no other impurity peaks exist in the parts, which indicates that the separated and purified AT-P is a polysaccharide substance. Table 4-1 shows AT-PFunctional group analysis table. 4000-1250 cm -1 Is characterized by a peak area of 3.425.88 cm -1 The broad and intense absorption peak at which occurs is designated as the O-H stretching vibration. 2928.49cm -1 The signal peak of (2) is designated as-CH 2 Stretching vibration peak at 1632.92cm -1 And 1.404.47 cm -1 The signal peaks at these are designated as c=o stretching vibration peak and C-H bending vibration peak, respectively. At 1250-400 cm -1 1077.59cm -1 The signal peak is designated as C-O stretching vibration peak. The above indicates that AT-P contains pyranose. Furthermore, at 1730cm -1 The absence of an absorption peak in the vicinity indicates that AT-P does not contain uronic acid.
3.3 analysis of monosaccharide composition of Armillariella Mellea polysaccharide AT-P
After AT-P was completely hydrolyzed, monosaccharide composition analysis was performed by HPLC, and as shown in FIG. 3, the off-time of each monosaccharide standard was Rha (4.838 min), xyl (5.638 min), ara (6.363 min), fru (7.120 min), man (8.704 min), glc (8.386 min), gal (8.949 min), respectively. The HPLC results after complete hydrolysis of AT-P had two retention times 7.934 and 8.486min, designated Glc, gal, respectively, as compared to the peak time of the monosaccharide standard. It was shown that AT-P consisted of Glc and Gal, with a peak area ratio of about 3:8.
3.4 NMR Spectroscopy analysis of Armillariella mellea polysaccharide AT-P
AT-P 1 The H NMR results are shown in FIG. 4. The results show that AT-P has five anomeric hydrogen signals, respectively: δ5.24, δ5.01, δ4.94, δ4.86 and δ4.38, and the integrated area ratio is 2.52:2.03:3.52:2.65:0.61. signals between delta 3.0 and 4.2 are attributed to the hydrogen signals of C2-C6 in the sugar residues.
AT-P 13 The C NMR results are shown in FIG. 5, where AT-P has five anomeric carbon signals AT delta 102.64, delta 101.61, delta 99.27, delta 98.21 and delta 97.83. Signals between delta 60 and 78 are attributed to the C2-C6 carbon signals in the sugar residues.
AT-P 1 H- 1 The H-COSY spectrum is shown in FIG. 6, from which the coupling relationship between adjacent hydrogen nuclei can be identified. The signals of the A part H1/H2 and the D part H1/H2 are respectively delta 5.24/3.53, delta 5.01/3.81 and delta 4.94/3.66, respectivelyThe signal of part E H1/H2 is δ4.38/3.18, with the number δ4.86/3.71.
All chemical shifts of hydrogen are summarized in table 1.
The HMQC spectrum of AT-P is shown in FIG. 7, from which proximity-dependent can be identified 1 H and 13 coupling relationship between C. The signals of the A part H1/C1 are delta 5.24/99.27, the B part H1/C1 are delta 5.01/98.21, the C part H1/C1 are delta 4.94/101.61, the D part H1/C1 are delta 4.86/97.83, and the E part H1/C1 are delta 4.38/102.64.
The HMBC spectral diagram of AT-P is shown in FIG. 8, from which remote correlation can be identified 1 H and 13 coupling relationship between C. The signals of H2/C3 of the A residue are delta 3.53/77.05, H3/C4 of the B residue are delta 3.96/68.33, H3/C5 of the C residue are delta 3.91/72.84, H1/C3 of the D residue are delta 4.86/66.55, H4/C5 are delta 3.54/71.77, H5/C6 are delta 3.75/68.19, and H2/C1 of the E residue are delta 3.18/102.64.
All chemical shifts of carbon are summarized in table 2.
TABLE 1AT-P 1 Chemical shift of H
TABLE 2AT-P 13 Chemical shift of C
3.5 gas chromatography and Mass Spectrometry analysis of Armillariella Mellea polysaccharide AT-P
The methylation results are shown in Table 3, and indicate that the main repeating structural unit of AT-P is composed of (1- > 6) -galactose residues, (1- > 3, 6) -galactose residues as the main chain, (1- > 6) -glucose residues, →4) -glucose residues as the branched chain, and →1) -galactose as the terminal sugar. From the above, it can be primarily inferred that the structure of AT-P is shown in FIG. 9.
TABLE 3 analysis of AT-P methylation results
Example 2: research on immunomodulatory activity of Armillariella mellea polysaccharide AT-P
The immunomodulatory activity of Armillariella mellea polysaccharide AT-P was determined in vitro using two CCK-8 methods.
1. Reagent(s)
CCK-8 kit, RPIM1640, FBS, DMSO, diabody and the like are all commercial products.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument; cell incubator.
3. Method of
Effect of AT-P on proliferation of immune cells (B cells, T cells and RAW264.7 cells)
The effect of Armillariella mellea polysaccharide (AT-P) on proliferation of T cells, B cells and RAW264.7 cells was determined by a cell counting kit (CCK-8) method. Culturing T cells, B cells and RAW264.7 cells in vitro to logarithmic phase, counting by cell counting plate, diluting cell suspension to 1×10 with new culture solution 5 mu.L of each well of a 96-well plate, into which the cell suspension was put, was added to each well of 100. Mu.L of the cell suspension, and the 96-well plate was put into CO 2 Culturing in an incubator for 24 hours. After 24h, AT-P solutions of different mass concentrations (final mass concentrations 2.5, 5, 10, 20. Mu.g/mL) were added to the experimental group, 100. Mu.L LPS solution (final mass concentration 10. Mu.g/mL) was added to the positive control, and 100. Mu.L cell culture solution was added to the blank group. In CO 2 After culturing in an incubator for 24 hours, 10. Mu.l of CCK-8 was added to each well, and CO was added 2 After incubation for 2h in the incubator, absorbance values were detected (450 nm) on a microplate reader and images were taken.
4. Results
4.1 effect of AT-P on proliferation of B cells
As a result, as shown in FIG. 10, the AT-P was able to significantly (P < 0.05) stimulate the growth of B cells in the range of 1.25 to 20. Mu.g/mL of the final concentration of AT-P, and the proliferation rates increased with the increase of the drug concentration, and the proliferation rates were 26.33%, 34.54%, 51.96% and 70.16%, respectively. Wherein, when AT-P is 20 mug/mL, the proliferation rate reaches the highest value 70.16%, which is far higher than 42.58% of that of positive control group LPS (10 mug/mL).
4.2 effect of AT-P on proliferation of T cells
As shown in FIG. 11, the AT-P significantly promoted cell proliferation in the final concentration range of 1.25 to 40. Mu.g/mL, and the difference was statistically significant (P < 0.05) compared with the blank. When the final concentration of AT-P is 10 mug/mL, the proliferation efficiency of T cells is maximum, the proliferation rate is 38.28% (P < 0.01), and the proliferation rate is 40.84% close to that of positive control LPS (10 mug/mL).
4.3 effect of AT-P on proliferation of RAW264.7 cells
As a result, as shown in FIG. 12, the cell proliferation rate was significantly improved and the difference was significant (P < 0.05) when the AT-P final concentration was 1.25 to 20. Mu.g/mL, as compared with the blank group. When the AT-P concentration is 2.5 mug/mL, the proliferation of RAW264.7 cells can be remarkably promoted (P < 0.0001), and the proliferation rate reaches the highest 49.65%.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.
Claims (10)
1. A polysaccharide which is a heteropolysaccharide consisting of galactose and glucose, wherein the molar ratio of galactose to glucose residues is 8:3.
2. The polysaccharide according to claim 1, wherein the polysaccharide comprises (1→6) -galactose residues, (1→3, 6) -galactose residues, →1) -galactose residues, (1→6) -glucose residues and→4) -glucose residues.
3. The polysaccharide according to claim 2, wherein the molar ratio of (1→6) -galactose residues, (1→3, 6) -galactose residues and →1) -galactose residues is 3:3:2 and the molar ratio of (1→6) -glucose residues and →4) -galactose residues is 2:1.
4. The polysaccharide of claim 1, wherein the polysaccharide comprises the structure:
wherein n is an integer of 1 to 20.
5. The polysaccharide according to any one of claims 1 to 4, wherein the weight average molecular weight of the polysaccharide is 5000-30000Da, preferably 7000-25000Da, further preferably 8000-20000Da, particularly preferably 17600Da.
6. The method for producing a polysaccharide according to any one of claims 1 to 4, comprising the step of extracting fruit bodies of Armillariella mellea:
preferably, the preparation method comprises the following steps:
(1) Extracting Armillariella mellea fruiting body powder with water, and precipitating the obtained water extract with ethanol to obtain crude polysaccharide;
(2) Subjecting the crude polysaccharide obtained in the step (1) to ion exchange column chromatography, eluting, and collecting eluent;
(3) And (3) dialyzing and concentrating the eluent obtained in the step (2) by using a dialysis bag.
7. The process according to claim 6, wherein in step (1), the leaching temperature is 80 to 100 ℃,
preferably 98 ℃;
preferably, the feed liquid ratio of the powder of the fruit bodies of the Armillariella mellea 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 alcohol to the concentrated solution of the water extract is 1-10:1;
preferably, the alcohol is ethanol.
8. The process of claim 6, wherein in step (2), the ion exchange column is a cellulose column, and the filler is DEAE-52 cellulose;
preferably, the eluent used for elution is NaCl solution;
preferably, the elution is gradient elution, and the concentration of the eluent is 0.01-1.0mol/L.
9. The method according to claim 6, wherein in step (3), the dialysis bag has a molecular weight cut-off of 5000-10000Da, preferably 7000Da.
10. Use of the polysaccharide according to any one of claims 1-5 for the preparation of an immune enhancing health product and food.
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| CN118203597A (en) * | 2024-04-28 | 2024-06-18 | 安徽医科大学第一附属医院 | Application of armillarisin in preparing medicine for treating chemotherapy-induced intestinal mucosa injury |
| CN118852475A (en) * | 2024-06-21 | 2024-10-29 | 西华师范大学 | A kind of smoke-colored red mushroom polysaccharide and its preparation method and application |
| CN118852476A (en) * | 2024-06-26 | 2024-10-29 | 西华师范大学 | A kind of thrombin polysaccharide and its preparation method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118203597A (en) * | 2024-04-28 | 2024-06-18 | 安徽医科大学第一附属医院 | Application of armillarisin in preparing medicine for treating chemotherapy-induced intestinal mucosa injury |
| CN118852475A (en) * | 2024-06-21 | 2024-10-29 | 西华师范大学 | A kind of smoke-colored red mushroom polysaccharide and its preparation method and application |
| CN118852476A (en) * | 2024-06-26 | 2024-10-29 | 西华师范大学 | A kind of thrombin polysaccharide and its preparation method and application |
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