CN113336868B - Hericium erinaceus mycelium polysaccharide with effect of preventing and treating senile dementia and preparation method thereof - Google Patents
Hericium erinaceus mycelium polysaccharide with effect of preventing and treating senile dementia and preparation method thereof Download PDFInfo
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- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
Abstract
The invention discloses a hericium erinaceus mycelium polysaccharide with an effect of preventing and treating senile dementia and a preparation method thereof, wherein hericium erinaceus mycelium obtained by a liquid submerged fermentation technology is used as a raw material, a neuroprotective effect is used as an evaluation standard, and a novel hericium erinaceus mycelium purified polysaccharide is obtained by extraction, separation and purification and consists of six monosaccharides, namely fucose, galactose, xylose, glucose, mannose and glucuronic acid.
Description
Technical Field
The invention discloses hericium erinaceus mycelium polysaccharide with an effect of preventing and treating senile dementia, has the effects of protecting nerves and preventing and treating senile dementia, and also provides a preparation method of the hericium erinaceus mycelium polysaccharide for preventing and treating senile dementia, belonging to the technical field of medicines.
Background
Alzheimer's Disease (AD) causes memory and other cognitive decline, which is more than 60% of dementiaThe reason for example. Over 5000 million dementia patients worldwide are diagnosed with AD, a number that is predicted to increase to nearly 1.52 million by 2050. The pathogenesis of AD is still unclear, and there are many factors affecting AD, including age, sex, living environment, genetic background, etc. More and more researches show that the generation of AD is not influenced by one factor in a single direction, but is caused by the generation of cognitive disorder due to the synergistic effect of multiple aspects, so that the pathogenesis is not clearly explained at present, wherein the hypothesis includes excitatory amino acid toxicity, cholinergic injury hypothesis, neuroinflammation hypothesis, gene mutation hypothesis and Ca 2+ Theory of imbalance, etc. It is difficult to develop drugs for pathogenesis.
The currently used drugs for treating AD are mainly suitable for patients with senile dementia with complete mild and moderate neuronal structures, and comprise cholinesterase inhibitors such as donepezil, rivastigmine, galantamine and the like, and glutamate receptor antagonists such as memantine and the like. The existing clinical diagnosis method of the Alzheimer disease mainly depends on behavioral symptom judgment, most of the disease course is found to be in the middle and later stages, the cognitive function of a patient is damaged, progressive cognitive disorder or neurobehavioral change occurs, the disease course of the AD has progressiveness and irreversibility, no specific medicine exists at present, and the main treatment method is the accompanying treatment of families, so that great pressure is caused to families and society.
Chinese patent CN 111892663A discloses a hericium erinaceus polysaccharide and a preparation method and application thereof, hericium erinaceus mycelium obtained by deep liquid fermentation is ground and sieved, and is subjected to ultrasonic-assisted extraction, alcohol precipitation and Sevag method protein removal, alcohol precipitation and drying to obtain crude polysaccharide, and ion exchange chromatography and gel filtration chromatography are carried out to obtain a hericium erinaceus polysaccharide fine HP, wherein the hericium erinaceus polysaccharide fine HP is prepared from mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose and arabinose in a molar ratio of 0.062:0.023:0.267:0.010:0.025:0.453:0.137:0.023, the molecular weight of the hericium erinaceus polysaccharide HP is about 3319Da, and the hericium erinaceus polysaccharide HP can be applied to medicines for treating peptic ulcer and repairing gastric mucosa injury. The extraction method in the patent is ultrasonic-assisted, and the polysaccharide result is greatly influenced by time and power, so that polysaccharide molecules can be broken, and the biological activity of the obtained polysaccharide is influenced.
Disclosure of Invention
The invention discloses a hericium erinaceus mycelium purified polysaccharide with an effect of preventing and treating senile dementia and a preparation method thereof.
The invention provides a hericium erinaceus mycelium polysaccharide with an effect of preventing and treating senile dementia, which is characterized in that:
the molecular weight is 20-50kDa, and the molecular weight of the polysaccharide is composed of fucose, galactose, xylose, glucose, mannose and glucuronic acid, and the molar ratio of the fucose, the galactose, the xylose, the glucose, the mannose and the glucuronic acid is (0.01-0.05): (0.05-0.15): (0.50-0.80): (0.01-0.05):
(0.10-0.20)：(0.01-0.08)。
the polysaccharide is a more excellent polysaccharide purified from the mycelia of the hericium erinaceus, has a molecular weight of 36.1kDa, consists of fucose, galactose, xylose, glucose, mannose and glucuronic acid, and has a molar ratio of 0.03 to 0.67 of 0.02.
The invention provides a preparation method of hericium erinaceus mycelium purified polysaccharide with the effects of protecting nerves and preventing and treating senile dementia, which comprises the following steps:
the first step is as follows: freeze drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, pulverizing, sieving, adding deionized water, heating for water extraction, centrifuging, collecting supernatant, concentrating, removing protein by Sevag method, precipitating with ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain Hericium erinaceus mycelium crude polysaccharide HE;
the second step is that: dissolving the crude polysaccharide HE of the hericium erinaceus mycelium obtained in the step (1) by using deionized water, eluting by using a sodium chloride solution after passing through an ion exchange chromatography column, measuring the polysaccharide content by using a phenol-sulfuric acid method, collecting a main peak, carrying out alcohol precipitation and freeze-drying, purifying by gel filtration chromatography, eluting by using deionized water as an eluent, collecting the main peak, carrying out overnight alcohol precipitation at 4 ℃, centrifuging to obtain a supernatant, and freeze-drying to obtain the purified Polysaccharide (PHEB) of the hericium erinaceus mycelium. The polysaccharide yield was greater than 94%.
The preparation method of the hericium erinaceus mycelium polysaccharide for preventing and treating senile dementia, disclosed by the invention, comprises the following steps of:
1) Freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 100-150 mesh sieve;
2) Adding deionized water with the mass of 8-16 times of the mass of the raw materials, uniformly stirring, soaking for 20-50 minutes, and extracting in a constant-temperature water bath at 75-95 ℃ for 1.5-3 hours;
3) Cooling to room temperature, centrifuging to obtain supernatant, rotary evaporating to concentrate to 1/10-1/6 volume, and deproteinizing by Sevag method;
4) Concentrating the supernatant, precipitating with 40-80% (v/v) ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain Hericium erinaceus mycelium crude polysaccharide, HE;
5) Dissolving crude polysaccharide of Hericium erinaceus mycelium with deionized water, eluting with sodium chloride solution at concentration of 0.05-0.5mol/L and Flow rate of 0.1-2.0mL/min by using Fast Flow rate diethylaminoethyl Sepharose (DEAE Sepharose Fast Flow), respectively collecting solutions, measuring polysaccharide content by phenol-sulfuric acid method, collecting main peak, rotary evaporating and concentrating to 1/8-1/6 volume, precipitating with ethanol at 4 deg.C for overnight at 40-80% (v/v), collecting precipitate, and lyophilizing;
dissolving freeze-dried powder of hericium erinaceus mycelium polysaccharide obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with deionized water, collecting in tubes, measuring polysaccharide content by adopting a phenol-sulfuric acid method, collecting main peaks, concentrating to 1/8-1/6 volume by rotary evaporation, precipitating with ethanol at 4 ℃ at 40-80% (v/v) overnight, collecting precipitates, and freeze-drying to obtain the hericium erinaceus mycelium purified Polysaccharide (PHEB).
The hericium erinaceus mycelium polysaccharide for preventing and treating senile dementia disclosed by the invention is used for preparing a medicine for preventing and treating senile dementia.
The invention relates to application of hericium erinaceus mycelium polysaccharide for preventing and treating senile dementia in preparing neuroprotective drugs.
The invention relates to a medicinal preparation which takes the hericium erinaceus mycelium polysaccharide for preventing and treating senile dementia as an active ingredient and also contains one or more pharmaceutically acceptable carrier substances and/or auxiliary agents.
The invention has the positive effects that;
the hericium erinaceus mycelium purified polysaccharide with high neuroprotective activity has the neuroprotective effect, can relieve nerve cell apoptosis, has the effects of relieving senile dementia symptoms and improving related biochemical indexes in the brain, and can be used for preparing medicaments and health-care products for neuroprotection and preventing and treating senile dementia.
Drawings
FIG. 1: elution curve of crude polysaccharide DEAE Sepharose Fast Flow of Hericium erinaceus mycelium;
FIG. 2: the elution curve of the purified polysaccharide of the mycelium of the hericium erinaceus;
FIG. 3: ultraviolet scanning spectrogram of purified polysaccharide of Hericium erinaceus mycelium;
FIG. 4: an infrared spectrogram of the purified polysaccharide of the mycelium of the hericium erinaceus;
FIG. 5 is a schematic view of: analysis chart of monosaccharide components of the purified polysaccharide of the hericium erinaceus mycelium;
FIG. 6: a molecular configuration analysis chart of the purified polysaccharide of the hericium erinaceus mycelium;
FIG. 7: NMR of purified polysaccharide from mycelia of Hericium erinaceum 1 H, spectrogram;
FIG. 8: NMR of purified polysaccharide from mycelia of Hericium erinaceum 13 C, spectrum;
FIG. 9: NMR dept spectrogram of the purified polysaccharide of the mycelium of the hericium erinaceus;
FIG. 10: a graph of the immunohistochemistry results of mouse hippocampal regions Abeta 1-42 (A), tau (phospho S396) (B) and 4-HNE (C).
Detailed Description
The present invention will be further described with reference to the following examples. These examples are merely illustrative and do not limit the scope of the present invention in any way. The chemical reagents, chromatography columns, etc. used in the specification and examples were carried out under the conventional experimental conditions unless otherwise specified, or according to the instructions given by the supplier.
The invention obtains a new hericium erinaceus mycelium purified polysaccharide, which takes hericium erinaceus mycelium obtained by a deep liquid fermentation technology as a raw material, and obtains crude polysaccharide of the hericium erinaceus mycelium through the steps of water extraction, alcohol precipitation, concentration, freeze-drying and the like; the crude polysaccharide is subjected to ion exchange chromatography, gel filtration chromatography and freeze-drying to obtain purified polysaccharide of Hericium erinaceus mycelium, and the structure of the corious versicolor exopolysaccharide is characterized by using ultraviolet spectrum, HPLC, infrared spectrum, gel permeation chromatography, GC-MS and nuclear magnetic resonance. The effects of protecting nerves and preventing and treating senile dementia are examined through animal experiments.
Example 1:
1. freezing and freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 150 mesh sieve;
2. adding deionized water with the mass being 12 times that of the powder, uniformly stirring, soaking for 20 minutes, and extracting for 2.5 hours in a constant-temperature water bath at 80 ℃;
3. cooling to room temperature, centrifuging to obtain supernatant, concentrating by rotary evaporation to 1/10 volume, and removing protein by Sevag method;
4. concentrating the supernatant, precipitating with 40% (v/v) ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain crude polysaccharide (HE-1) of Hericium erinaceus mycelium.
Example 2:
1. freezing and freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 150 mesh sieve;
2. adding deionized water with the mass 10 times that of the raw materials, uniformly stirring, soaking for 30 minutes, and extracting in a constant-temperature water bath at 75 ℃ for 2.5 hours;
3. cooling to room temperature, centrifuging to obtain supernatant, rotary evaporating to concentrate to 1/10 volume, and deproteinizing by Sevag method;
4. concentrating the supernatant, precipitating with 60% (v/v) ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain Hericium erinaceus mycelium crude polysaccharide (HE-2).
Example 3:
1. freezing and freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 150 mesh sieve;
2. adding deionized water with the mass being 12 times that of the raw materials, uniformly stirring, soaking for 30 minutes, and extracting for 2.5 hours in a constant-temperature water bath at 80 ℃;
3. cooling to room temperature, centrifuging to obtain supernatant, rotary evaporating to concentrate to 1/6 volume, and deproteinizing by Sevag method;
4. concentrating the supernatant, precipitating with 80% (v/v) ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain crude polysaccharide (HE-3) of Hericium erinaceus mycelium.
Example 4:
1. dissolving the crude polysaccharide of the mycelia of the Hericium erinaceus obtained in example 3 with deionized water, eluting with a sodium chloride solution through a Fast Flow rate diethylaminoethyl Sepharose (DEAE Sepharose Fast Flow) at a concentration of 0.05mol/L and a Flow rate of 1.0mL/min, collecting the solutions respectively, measuring the polysaccharide content by a phenol-sulfuric acid method, collecting the main peak, concentrating by rotary evaporation to 1/8 volume, precipitating with 80% (v/v) ethanol at 4 ℃ overnight, collecting the precipitate, and freeze-drying;
2. dissolving freeze-dried hericium erinaceus mycelium polysaccharide powder obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with the deionized water, collecting in tubes, measuring the polysaccharide content by adopting a phenol-sulfuric acid method, collecting a main peak, performing rotary evaporation and concentration to 1/8 volume, performing 80% (v/v) alcohol precipitation overnight at 4 ℃, collecting a precipitate, and performing freeze-drying to obtain the hericium erinaceus mycelium purified polysaccharide (PHEB-1).
Example 5:
1. dissolving the crude polysaccharide of the mycelia of the Hericium erinaceus obtained in example 3 with deionized water, eluting with a sodium chloride solution through a Fast Flow rate diethylaminoethyl Sepharose (DEAE Sepharose Fast Flow) at a concentration of 0.1mol/L and a Flow rate of 1.0mL/min, collecting the solutions respectively, measuring the polysaccharide content by a phenol-sulfuric acid method, collecting the main peak, concentrating by rotary evaporation to 1/8 volume, precipitating with 80% (v/v) ethanol at 4 ℃ overnight, collecting the precipitate, and freeze-drying;
2. dissolving freeze-dried powder of hericium erinaceus mycelium polysaccharide obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with deionized water, collecting in tubes, measuring polysaccharide content by adopting a phenol-sulfuric acid method, collecting main peaks, carrying out rotary evaporation and concentration to 1/8 volume, carrying out 80% (v/v) alcohol precipitation at 4 ℃ overnight, collecting precipitates, and freeze-drying to obtain the hericium erinaceus mycelium purified polysaccharide (PHEB-2).
Example 6:
1) Dissolving the crude polysaccharide of the mycelia of the Hericium erinaceus obtained in example 3 with deionized water, eluting with a sodium chloride solution through a Fast Flow rate diethylaminoethyl Sepharose (DEAE Sepharose Fast Flow) at a concentration of 0.3mol/L and a Flow rate of 1.0mL/min, collecting the solutions respectively, measuring the polysaccharide content by a phenol-sulfuric acid method, collecting the main peak, concentrating by rotary evaporation to 1/8 volume, precipitating with 80% (v/v) ethanol at 4 ℃ overnight, collecting the precipitate, and freeze-drying;
2) Dissolving freeze-dried powder of hericium erinaceus mycelium polysaccharide obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with deionized water, collecting in tubes, measuring polysaccharide content by adopting a phenol-sulfuric acid method, collecting main peaks, carrying out rotary evaporation and concentration to 1/8 volume, carrying out 80% (v/v) alcohol precipitation at 4 ℃ overnight, collecting precipitates, and freeze-drying to obtain the hericium erinaceus mycelium purified polysaccharide (PHEB-3).
Comparative example 1
The comparative example is a preparation method of purified polysaccharide of hericium erinaceus mycelium, and the preparation method is the same as example 5, except that after the hericium erinaceus mycelium is subjected to superfine grinding and sieving, deionized water is added, and the hericium erinaceus mycelium is not soaked for 30 minutes, and is directly subjected to hot water extraction.
Comparative example 2
The comparative example is a preparation of purified polysaccharide of hericium erinaceus mycelium:
(1) Freezing and freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 150 mesh sieve;
(2) Adding deionized water 15 times the mass of the mixture, uniformly stirring the mixture, soaking the mixture for 40 minutes, and extracting the mixture for 2 hours in a constant-temperature water bath at 80 ℃;
(3) Cooling to room temperature, centrifuging to obtain supernatant, concentrating by rotary evaporation to 1/8 volume, and removing protein by Sevag method;
(4) Concentrating the supernatant, precipitating with 85-90% (v/v) ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain crude polysaccharide of Hericium erinaceus mycelium.
(5) Dissolving the crude polysaccharide of the hericium erinaceus mycelium obtained in the step (4) by using deionized water, eluting by using a sodium chloride solution through a Fast-Flow diethylaminoethyl Sepharose gel (DEAE Sepharose Fast Flow), wherein the concentration is 1.0mol/L, the Flow rate is 1.0mL/min, respectively collecting solutions, measuring the polysaccharide content by using a phenol-sulfuric acid method, collecting a main peak, performing rotary evaporation and concentration to 1/8 volume, performing ethanol precipitation at 4 ℃ of 85-90% (v/v) overnight, collecting precipitates, and performing freeze-drying;
(6) Dissolving freeze-dried powder of hericium erinaceus mycelium polysaccharide obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with deionized water, collecting in tubes, measuring polysaccharide content by adopting a phenol-sulfuric acid method, collecting main peaks, concentrating to 1/8 volume by rotary evaporation, precipitating with 85-90% (v/v) ethanol at 4 ℃ overnight, collecting precipitates, and freeze-drying to obtain the hericium erinaceus mycelium purified polysaccharide (PHEB-4).
Test example 1:
detection of yield of purified polysaccharide from mycelia of Hericium erinaceus
(1) Test materials:
the purified polysaccharides of the mycelia of Hericium erinaceus obtained in example 5 and comparative example 1.
(2) Test method
Testing the content of polysaccharide by using a phenol-sulfuric acid method, accurately weighing 20mg of standard glucose into a 500mL volumetric flask, adding water to a scale, respectively sucking 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8mL of distilled water, respectively supplementing distilled water to 2.0mL, weighing a proper amount of polysaccharide sample, dissolving with 2mL of distilled water, then adding 1.0mL of 6% phenol and 5.0mL of concentrated sulfuric acid, shaking up and standing for 30 minutes, measuring absorbance at 490nm, using 2.0mL of water as a blank according to the same color development operation, taking the abscissa as microgrammes of polysaccharide and the ordinate as a light density value to obtain a standard curve, and substituting the absorbance measured by the sample into the standard curve to obtain the concentration of polysaccharide and calculate the yield of polysaccharide. The test results are shown in Table 1, and the yield of polysaccharide purified from the mycelium of Hericium erinaceum in example 5 is 2.96% and higher than that of 2.53% in comparative example 1, i.e., the yield of polysaccharide is increased by soaking the mycelium powder of Hericium erinaceum with deionized water before extraction.
TABLE 1 comparison of the results of the yield of purified polysaccharide from Hericium erinaceus mycelium
| Example 3 | Comparative example 1 | |
| Yield of polysaccharide/%) | 2.96 | 2.53 |
Test example 2: nerve protection effect detection of hericium erinaceus mycelium crude polysaccharide and purified polysaccharide
(1) Test materials
The test drugs are the crude polysaccharide of the hericium erinaceus mycelium obtained in examples 1, 2 and 3 and the purified polysaccharide of the hericium erinaceus mycelium obtained in examples 4, 5 and 6 and comparative example 2; the mouse cell line used in this experiment was a mouse hippocampal neuronal cell line (HT 22; BNCC; 337709) purchased from Beiner Chuanglian Biocompany; other chemical reagents such as L-glutamic acid, DMEM medium, MTT, DMSO, etc.
(2) Test method
The crude polysaccharide and purified polysaccharide of Hericium erinaceus mycelium obtained in the above examples and comparative examples were examined for L-glutamic acid-induced HT22 cell injury modelsNeuroprotective Effect, HT22 cells at 5X 10 4 Cell concentration per mL in 96-well plates, 100. Mu.L per well, 5% CO 2 And culturing in a constant-temperature incubator at 37 ℃ for 12-14h until the cells adhere to the wall, treating the blank group and a control group by using PBS as a medicament, respectively configuring polysaccharide samples obtained in experimental groups of examples 1, 2, 3, 4, 5 and 6 and comparative example 2 into 100 and 200 mu g/mL doses for pretreating the cells for 3h, then carrying out modeling treatment on the cells by using L-glutamic acid (L-Glu) with the final concentration of 25mM, not carrying out treatment on the blank group, simultaneously carrying out administration again according to a pretreatment mode, and carrying out incubation for 24 h together. After MTT in PBS was added to a 96-well plate in the dark at 20. Mu.L/well and incubated for 4 hours in the dark, formazan particles were dissolved in DMSO and the absorbance thereof was measured at 490nm to analyze the change in cell viability.
The survival rate of each group was calculated with the survival rate of the blank group as 100%. Experimental data are expressed as mean ± standard deviation (mean ± s.d.). Statistically significant differences the differences between groups were examined according to the one-way ANOVA analysis of IBM SPSS Statistics 24 software (Dunn's test performed afterwards), P <0.05 considered significant and statistically significant.
(3) Test results
Table 3 shows the neuroprotective effect of crude hericium erinaceus mycelium polysaccharides obtained in examples 1, 2 and 3 on HT22 cells, and the cell viability of the control group was reduced to 46.1% after modeling with L-glutamic acid, and both 100 μ g/mL and 200 μ g/mL crude polysaccharides in examples 2 and 3 significantly improved the survival rate of HT22 cells, wherein the survival rate of HT22 by administration of crude hericium erinaceus mycelium polysaccharide (200 μ g/mL) obtained in example 3 was 61.7% (xp < 0.01), indicating that pretreatment and administration of crude hericium erinaceus mycelium polysaccharide significantly protected the model of L-glutamic acid-induced injury.
The ethanol precipitation concentrations in examples 1, 2 and 3 are respectively 40%, 60% and 80%, and according to experimental results, the crude polysaccharide has neuroprotective effect, but the neuroprotective effect is more obvious as the ethanol concentration increases, and the optimal ethanol precipitation concentration is 80% under the set condition in the invention.
TABLE 2 Hericium erinaceus mycelium crude polysaccharide neuroprotective effect results on HT22 cells
Note: p >0.05 for no significance, P <0.05 for significance, and P <0.01 for very significant.
Table 4 results are the results of neuroprotection of HT22 cells by the purified polysaccharide of hericium erinaceus mycelia obtained in examples 4, 5, 6 and comparative example 2, and it is seen from the results that the viability of HT22 cells was significantly decreased to 47.2% after L-glutamic acid treatment, while the viability of HT22 cells could be significantly reversed after pretreatment and administration of the purified polysaccharide of hericium erinaceus mycelia. The purified polysaccharide of 100 and 200 mu g/mL in examples 4, 5 and 6 has obvious protective effect on HT22 cells, wherein the purified polysaccharide of the hericium erinaceus mycelium obtained in example 5 has the most obvious neuroprotective effect, and the survival rates of the HT22 cells reach 58.7% and 68.0% respectively. In combination with the experimental results of test example 2, the polysaccharide of comparative example 2 has higher purity than those of examples 4, 5 and 6, but in combination with the experimental results of test example 3, it can be seen that examples 5 and 6, which are neuroprotective activities, have better effects than comparative example 2, which has high purity.
TABLE 3 Hericium erinaceus mycelium purified polysaccharide neuroprotective effect on HT22 cells results
Note: p >0.05 for no significance, P <0.05 for significance, and P <0.01 for very significant.
Test example 3: detection of purity effect of purified polysaccharide of hericium erinaceus mycelium
(1) Test materials
The purified polysaccharides of the mycelia of Hericium erinaceum obtained in example 4, example 5, example 6 and comparative example 2.
(2) Test method
The polysaccharide content was measured by the phenol-sulfuric acid method, and the concrete method was referred to the test method of test example 1 (2). The formula for calculating the purity of the polysaccharide is as follows:
polysaccharide purity = Hericium erinaceus mycelium purified polysaccharide mass/sample weighed mass × 100%
(3) Test results
The test results are shown in Table 2, and the purified polysaccharides obtained from the mycelia of Hericium erinaceus in examples 4, 5, and 6 all had a purity of 94% or more, while the purified polysaccharide obtained in comparative example 2 had a purity of 98.6%.
TABLE 4 comparison of purity results for purified polysaccharide from Hericium erinaceus mycelium
| Example 4 | Example 5 | Example 6 | Comparative example 2 | |
| Purity of polysaccharide/%) | 94.1 | 97.2 | 95.6 | 98.6 |
Test example 4: analysis of structural characteristics of purified polysaccharide of hericium erinaceus mycelium
(1) Test materials
The polysaccharide purified from the mycelia of Hericium erinaceum obtained in example 5.
(2) Test methods and results
Ultraviolet spectrum analysis, namely dissolving a proper amount of distilled water into the hericium erinaceus mycelium purified polysaccharide obtained in the example 5, and scanning the solution at the full ultraviolet wavelength of 200-400 nm. As shown in FIG. 3, HP has no absorption peaks at 260, 280nm, indicating that the polysaccharide is free of proteins and nucleic acids.
Infrared spectrum detection: the purified polysaccharide of the mycelia of Hericium erinaceus obtained in example 5 was added with an appropriate amount of dry potassium bromide powder, and after being uniformly ground in a mortar, the mixture was put into a tabletting machine for tabletting, and a transparent tablet was prepared. Pressing with Fourier transform infrared spectrometer at 400-4000cm -1 Scanning the interval, collecting the infrared absorption spectrum of the sample, and analyzing and processing the spectrum by using system software. As shown in FIG. 4, 3600-3200cm -1 The absorption band at (A) is a tensile vibration absorption peak of-OH, and the absorption peak in this region is a characteristic peak of a sugar. The absorption peak of O-H stretching vibration is 3396cm -1 This is the characteristic peak of the sugar. 2927cm -1 The absorption peak at (a) is due to C-H tensile vibration. 1639cm -1 Possibly due to crystal water, and 1413cm -1 、1349cm -1 And 1151cm -1 The absorption peak at (a) is due to C — O symmetric tensile vibration. At 1076cm -1 And 1025cm -1 The absorption peak at (A) is due to O-H variable angle vibration and at 970cm -1 The absorption peak is attributed to the rolling vibration of a methylene group at the end of a pyran ring, and the polysaccharide configuration is pyran type.
Analysis of monosaccharide composition: dissolving in distilled water to obtain purified polysaccharide of Hericium erinaceus mycelium, and purifying monosaccharide component with liquid chromatography column (Dionex) TM CarboPac TM PA 20) and an electrochemical detector (ICS 500 +). The amount of sample was 20. Mu.L. Mobile phase A (H) 2 O), mobile phase B (100 mM NaOH), flow rate 1mL/min, column temperature 30 ℃. As shown in fig. 5, the purified polysaccharide of the hericium erinaceus mycelium is composed of six monosaccharides including fucose, galactose, xylose, glucose, mannose, and glucuronic acid, and the molar ratio thereof is 0.03.
Molecular weight and homogeneity analysis: hericium erinaceus mycelium obtained in example 5 was subjected to gel chromatography-differential-multi-angle laser light scattering system equipped with gel exclusion column (Ohpak SB-805HQ, 300X 8 mm), differential detector (Optilab T-rEX) and light scattering detector for laser (DAWN HELEOS 2)Homogeneity and molecular weight assessment of the purified polysaccharide. The detection conditions were as follows: the mobile phase is 0.1M NaNO 3 . The sample injection amount is 100 mu L; the flow rate was 0.4mL/min. The temperature of the column oven was 45 ℃. The purified polysaccharide of the mycelia of Hericium erinaceus obtained in example 5 was found to have a uniform single peak by analysis of the results, indicating that it had good uniformity and a molecular weight of 36.1kDa, and the results are shown in FIG. 6, rg vs. Mw (RMS) configuration diagram, indicating that the molecular configuration of the polysaccharide was irregularly coiled, indicating that there was branching.
Methylation analysis: in order to determine the bonding structure of the polysaccharide, the purified polysaccharide of the mycelia of Hericium erinaceus obtained in example 5 was subjected to methylation and acetylation by a gas chromatography-mass spectrometer (GC-MS) according to the following steps: methylation reaction: respectively adding 10mg of dried Hericium erinaceus mycelium purified polysaccharide and Hericium erinaceus mycelium purified polysaccharide saccharic acid reduction product into a 25mL round-bottom flask, adding 2mL of DMSO, dissolving for 30min with ultrasound, adding 30mg of dried NaOH powder, reacting for 3h, and slowly dropwise adding 1mL of CH along the wall under ice water bath 3 I, carrying out ultrasonic reaction for 2h in a dark place, adding 3mL of water to terminate the reaction, and adding CHCI according to the ratio of 1:1 3 Extracting, collecting organic phase, repeating for 3 times, drying, and repeating methylation until no hydroxyl peak exists in IR scanning spectrum. The whole reaction is carried out at 20 ℃ and N 2 Under protection. And (3) subsequent reaction: adding 4mL of 2mol/L TFA into the methylated sample, hydrolyzing at 110 ℃ for 5h, adding 4mL of methanol after hydrolysis, spin-drying to remove excessive TFA, repeating for 3 times, and adding 0.5Ml of distilled water for dissolving; 0.2mL of hydrolysate is taken and 30mg of NaBH is added 4 Reacting for 3 hours at room temperature, intermittently shaking in the reaction process, dropwise adding 25% glacial acetic acid after the reaction is finished until no bubbles are generated, adding 2mL of methanol, spin-drying, and repeating for 3 times; adding 1.5mL of acetic acid into the reduction product, reacting at 100 ℃ for 1h, adding 2mL of toluene, spin-drying, and repeating for 3 times to remove excessive acetic anhydride; adding 2mL of chloroform and water into the acetylation product respectively, oscillating, standing, collecting a lower organic phase, washing with lmL water for 3 times, adding a small amount of anhydrous sodium sulfate for water removal, and performing GC-MS analysis.
The chromatographic system used was an Agilent gas chromatographic system (Agilent 7890A). The derivatives were identified by their typical retention times and electron impact curves. The mass spectrometry system used an Agilent 5977B quadrupole mass spectrometry detection system, equipped with an electron impact ion source (EI) and masshhunter workstation. A chromatographic column: HP-5MS capillary column (30 m × 250pm × 0.25 umD); carrier gas: he; temperature of the heater: the temperature is 250 ℃: the temperature programming conditions were set as follows: the initial temperature is raised to 200 ℃ at 140 ℃/min, the temperature is kept for 5min, and then the temperature is raised to 240 ℃ at 8 ℃/min: the split ratio is as follows: 50: 1; sample introduction amount: 5 μ l. Analyte detection using EI in full scan mode, mass scan range (m/z): 30-600. The characteristic fragments of the methylated polysaccharides were compared against the existing database to confirm their binding methods.
PHEB is composed mainly of 6 glycoside fragments according to retention time and standard data for partially methylated sugar alcohol acetate (PMAA) in Complex Carbohydrate Research Center (CCRC) spectral databases (table 5). the molar ratio of the non-reducing end of t-Man (p) reached 9.74%, indicating the presence of a branched residue.
TABLE 5 methylation analysis results of the purified polysaccharide of the mycelia of Hericium erinaceum obtained in example 5
Nuclear Magnetic Resonance (NMR) analysis: taking appropriate amount of the purified polysaccharide of dried Hericium erinaceus mycelium obtained in example 5, dissolving in 0.5ml of D 2 In O, NMR analysis was performed using a Bruker Avance AV600 NMR spectrometer. FIGS. 7, 8 and 9 show the magnetic resonance of purified polysaccharide from Hericium erinaceus mycelium 1 H spectrogram, 13 Spectrum C and spectrum dept 135. Dept135 spectral analysis showed 68.65, 68.58, 68.43, 67.21, and 67.21ppm peaks as inverted peaks, indicating the chemical shift of C6. The results of the determination of the binding between heteronuclear single quantum relationship (HSQC), multi-bond carbon-hydrogen relationship (HMBC), and hydrogen-hydrogen relationship (H-HCOSY) in example 5, in which the structure is → 3) -alpha-D-Glcp- (1 → 6) -alpha-D-Galp- (1 → 6) -alpha-D-Glcp- (1 → 6), are summarized in Table 6→ 3) - α -D-Glcp- (1 → as the main chain and β -D-Manp- (1 → 3) - α -D-Glcp- (1 → 2) - α -D-Manp- (1 → 6) - α -D-Glcp- (1 → 6) -are branched.
TABLE 6 chemical shift information of 1H and 13C NMR of purified polysaccharide of Hericium erinaceus mycelia obtained in example 5
| Glycosyl residues | H1/C1 | H2/C2 | H3/C3 | H4/C4 | H5/C5 | H6a,b/C6 | H6b |
| →6)-α-D-Galp-(1→ | 4.93/99.4 | 3.78/70.8 | 3.92/72.3 | 3.66/74.1 | 3.93/72.3 | 3.79/67.2 | 3.61/-- |
| →3,6)-α-D-Glcp-(1→ | 5.13/99.4 | 4.82/71.1 | 3.73/77.7 | 4.06/68.7 | 4.06/68.4 | 3.79/67.2 | 3.65/-- |
| →3)-α-D-Glcp-(1→ | 5.1/94.9 | 3.77/69.7 | 3.48/80.1 | 4.12/70.7 | 3.95/75.8 | 3.64/62.6 | Ns |
| β-D-Manp-(1→ | 4.11/94.6 | 3.66/71.4 | 3.64/72.5 | 3.58/77.5 | 3.31/74.1 | 3.75/67.3 | Ns |
| →2)-α-D-Manp-(1→ | 4.74/100.8 | 4.01/78.8 | 3.94/70.2 | 3.75/67.3 | 3.68/73.5 | 4.31/61.3 | Ns |
| →6)-α-D-Glcp-(1→ | 5.12/99.5 | 3.51/72.3 | 3.69/74.1 | 3.46/70.8 | 3.99/72.7 | 3.58/68.6 | 3.97/-- |
Test example 5: activity research on hericium erinaceus mycelium purified polysaccharide nerve protection and senile dementia prevention and treatment
(1) Test materials
Medicine preparation: physiological saline and the purified polysaccharide of the mycelia of Hericium erinaceus obtained in example 5.
Animals: the institutional animal ethics committee of the university of gyin approved animal experiments conducted according to the institutional guidelines (2017 SY 1102). 45 APP/PS1 male mice (8 weeks old, SPF grade) and 15B 6C3-Tg (APPswe PSEN1d E)/Nju [ genotype: (Appsswe) W, (Psen 1) W ] (WT) mice (8 weeks old, SPF grade)) were purchased from Nanjing university-Nanjing biomedical research institute [ SCXK (Su) 2015-0001]. All mice were housed in an environment with a temperature of 23 + -1 deg.C and a humidity of 50% to 60%. The illumination time was kept for 12 hours, natural light was uniformly illuminated, and the mice had free access to food and water.
The kit comprises: acetylcholine (ACh) (CK-E20536M), acetylcholinesterase (AChE) (CK-E93899M), choline acetyltransferase (ChAT) (CK-E94456M), ROS (CK-E91516M), superoxide dismutase (SOD) (CK-E20348M), malondialdehyde (MDA) (CK-E43124M), catalase (CAT) (CK-E92636M) and glutathione peroxidase (GSH-Px) (CK-E92669M) and A β 1-42 (CK-E94157M) were purchased from Shanghai-derived leaf Biotech, inc., and all the assay methods were performed according to the instructions provided in the kits.
(2) Test methods and results
Grouping and administration of mice: APP/PS1 mice fed for 24 weeks (mortality rate about 33%) were randomly divided into 3 groups of 10 mice each, and control groups were gavaged with 10mL/kg of physiological saline; the administration group was administered with 25mg/kg and 100mg/kg of the purified polysaccharide of the mycelia of Hericium erinaceus obtained in example 5, and the blank group was WT mice, and the administration was performed by intragastric administration of 10mL/kg of physiological saline for 6 weeks.
Animal behavioral testing: animal behavioral tests include open field experiments, morris water maze, investigation of cognition and memory of Hericium erinaceus mycelium purified polysaccharides on APP/PS1 mice, and mouse jump bench experiments to investigate short-term memory of mice.
The spacious field device is that an inner wall is black cuboid, and the bottom surface is 25 cm's square, and the wall pastes black amortization sponge all around, and the wall height is 40cm, and the top is installed and is tracked the camera. A small square with the bottom surface being 12cm multiplied by 12cm is arranged in an open field test operating system, and the framed area is a central area; the remaining peripheral region is the peripheral region. The fixed mouse is placed in the box at each time, a vertex of the square on the bottom surface is formed, and the point A is positioned. The movement track of the mouse in the dark box is recorded by a monitoring system. The open field apparatus was carefully cleaned before each experiment was started. The mouse was placed in the device from point a and the movement time and distance of the mouse 180s in the central area was recorded by the monitoring system.
The water maze consists of a stainless steel pool (diameter is 120cm, height is 50 cm) and an immersed escape platform (diameter is 10 cm) which is 1cm below the water surface. Running water is injected into the water maze to enable the water maze to submerge the escape platform by about 1cm, titanium dioxide powder is added into the water, the water is uniformly stirred to be white and opaque, and the water temperature is kept at about 25 ℃. The water maze is equally divided into 4 1/4 circles by the water maze operation system, and the circles are divided into four quadrants. The escape platform is placed in the second quadrant, and the mouse is put into water from the position opposite to the platform every time, and the point A is determined. Each experiment ensures that the environment is quiet. A 4-day continuous spatial learning test was performed. In each experiment, mice were placed in the water maze from point a and the time required for the mice to find the hidden platform was recorded as the escape latency. Mice were given a maximum time of 120s to find a hidden platform. The time and distance of movement of the mouse to the platform was recorded on day 4 by the water maze monitoring system.
The mouse diving platform device is an observation box (60 multiplied by 300 mm), and a platform (30 multiplied by 45 mm) is arranged in the middle of the observation box. A video device was installed above the laboratory to record the search trajectory of the mouse. The mice were placed on the platform of an observation box, after an adaptation period of 1 minute, the bottom electrical wand was energized, observed, and electrical stimulation was stopped after 5 minutes. The test started immediately after one training. The time spent on the table and the number of times the mouse jumps from the platform were recorded.
After the last behavioral test, the mice were fasted for 12 hours. Blood was sampled from the tail vein and stored in a refrigerator at-80 ℃. Mice were then euthanized by intraperitoneal injection of 100mg/kg sodium pentobarbital. Brain tissue was collected rapidly.
Brain A β 1-42, tau (phospho S396) and 4-HNE immunohistochemical staining: after the brain tissue was fixed in 10% formalin solution, it was incubated in 30% sucrose in PBS, and the brain was cut into 20 μm sections at low temperature, and washed twice with PBS for 5min each. Sections were incubated in 3% hydrogen peroxide in PBS for 20min to stop endogenous peroxidase activity and washed twice with 5min each time in PBS. Brain sections were blocked in 5% bsa solution for 1 hour and incubated with primary anti-antibody overnight at 4 ℃. The antibodies and dilution ratios used were Α β 1-42 (dilution ratio 1, 200), tau (phospho S396, dilution ratio 1. After incubation with primary antibody, brain sections were washed 3 times for 10min in PBS. After washing, brain sections were incubated with horseradish peroxidase-labeled goat-anti-rabbit or goat-anti-mouse IgG secondary antibody (dilution ratio 1. After dyeing with DAB dye solution for 10min, dewatering brain slices in ethanol with same gradient, sealing with neutral gum, and performing microscopic examination under optical microscope.
Detection of cytokines in brain: the collected hippocampal tissues were homogenized with physiological saline and the protein concentration was detected by BCA kit (Millipore). The test was performed according to the instructions provided with the kit to detect levels of acetylcholine (ACh), acetylcholinesterase (AChE), choline acetyltransferase (ChAT), ROS, superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px) and Abeta 1-42 (CK-E94157M) in the mouse hippocampus, pituitary, hypothalamus and serum.
(3) Test results
Animal behavior experimental results:
the open field experiment can evaluate the autonomous behavior of the experimental animal in a new environment, and explore the behavior and the stress state. Table 7 shows the effect of the purified polysaccharide of hericium erinaceus mycelium obtained in example 5 on the mouse open field experiment, and it can be seen that the moving time and distance of the control group APP/PS1 mouse in the central area are significantly reduced (p < 0.05), the moving time and distance of APP/PS1 in the central area are significantly improved after 100mg/kg administration of the purified polysaccharide of hericium erinaceus mycelium, and are respectively increased by 64.3% and 35.1% compared with the control group.
TABLE 7 influence of obtaining purified polysaccharide of Hericium erinaceus mycelia on mouse open field experiment in example 5
Note: data are presented as mean ± standard deviation. (n = 10/group). p <0.05 for significance, p <0.01 for extreme significance, control versus blank, and polysaccharide versus control.
From table 8, in the Morris water maze test, compared with the blank group, the platform-searching time of APP/PS1 mice is increased by 229.3%, the movement distance is increased by 370.1%, which proves that the memory of the mice is reduced, while the hericium erinaceus mycelium purified polysaccharide significantly shortens the platform-searching latency (p < 0.01) and the total distance (p < 0.01) of APP/PS1 mice, especially the platform-searching time and the movement distance of 100mg/kg administration group mice are respectively reduced by 59.1% and 63.5%, which indicates that the long-term memory of AD mice is improved.
TABLE 8 influence of purified polysaccharide from Hericium erinaceus mycelia obtained in example 5 on mouse Morris water maze experiment
Note: data are presented as mean ± standard deviation. (n = 10/group). p <0.01 represents significant, the control group was compared to the blank group, and the polysaccharide group was compared to the control group.
In the mouse diving platform test, the mouse may develop memory and fear jumping off the diving platform after training due to electrical stimulation under the platform. Compared with a blank group, the APP/PS1 mice have shortened stay time on the jumping platform (p is less than 0.01), and the jumping platform times are increased by 75.6% (p is less than 0.01), while the APP/PS1 mice have increased stay time on the jumping platform (p is less than 0.01) and decreased jumping platform times (p is less than 0.01) after administration of the hericium erinaceus mycelium purified polysaccharide, particularly 100mg/kg administration group mice have increased jump platform stay time by 29.9%, and decreased jumping platform times by 55.6%, which indicates that the memory storage time of AD mice is prolonged.
TABLE 9 influence of obtaining purified polysaccharide of mycelia of Hericium erinaceus in example 5 on mouse diving platform experiment
Note: data are presented as mean ± standard deviation. (n = 10/group). p <0.01 represents significant, the control group was compared to the blank group, and the polysaccharide group was compared to the control group.
Results of immunohistochemical staining of A.beta.1-42, tau (phospho S396) and 4-HNE in hippocampus:
a β deposition and tau hyperphosphorylation are considered important pathological features of AD, and can promote its occurrence by inhibiting ACh synthesis in cholinergic neurons, inducing neuronal apoptosis and triggering oxidative stress. The neuron cell is a cell with active metabolism and is easy to be attacked by ROS, and the 4-HNE is an index for judging lipid peroxidation. The expression of related proteins in the brain of mice was analyzed by immunohistochemistry, and the results of immunohistochemistry for Abeta 1-42, tau (phospho S396) and 4-HNE in hippocampus are shown in FIG. 10. In the hippocampus of APP/PS1 mice, PHEB improved the deposition of A β 1-42. Administration of purified polysaccharide of Hericium erinaceus mycelia reduced Abeta 1-42 deposition (FIG. 10A), inhibited the number of phosphorylated tau plaques in hippocampus of mice (FIG. 10B), and reduced the accumulation of 4-HNE in hippocampus of APP/PS1 mice (FIG. 10C) compared to control groups.
Detection results of intracerebral cytokines:
the cholinergic system is essential for memory and learning functions. Ach helps to maintain consciousness, and ChAT is a specific marker for cholinergic neurons and is often used as an index for the study of cholinergic neurons. The mouse hippocampal, hypothalamus, pituitary and serum levels of Ach, chAT and AChE were measured by ELISA kit and the results are shown in table 10. Compared with a blank group of mice, the serum and brain ChAT and Ach content of APP/PS1 mice is remarkably reduced (p is less than 0.05), and the AChE content is increased (p is less than 0.05). After the hericium erinaceus mycelium is subjected to intragastric administration and purified polysaccharide for six weeks, cholinergic transmitters are all remarkably improved (p is less than 0.05). Thus, modulation of the cholinergic system may be involved in PHEB-mediated improvement of learning and memory capacity in AD mice.
TABLE 10 Effect of obtaining purified polysaccharide of mycelia of Hericium erinaceum on cholinergic transmitter of serum, hippocampus, hypothalamus and pituitary in example 5
Data are presented as mean ± standard deviation. (n = 6/group). Relative to the blank group mice, # p <0.05, # p <0.01, relative to untreated APP/PS1 mice, # p <0.05 and # p <0.01.p <0.05 indicates significance, and p <0.01 indicates extreme significance.
MDA is a specific marker for the peroxidation process between ROS and phospholipids. When the organism is subjected to oxidative stress caused by ROS, the organism itself has an anti-ROS system to combat such peroxidation states (e.g., GSH-Px, CAT, and SOD) to combat peroxidation states. The content of ROS, MDA, SOD and CAT, GSH-Px in hippocampus, hypothalamus, pituitary and serum of mice were measured by ELISA kit, and the results are shown in Table 11. Compared with a blank group of mice, the serum and intracerebral MDA (p < 0.05) and ROS (p < 0.05) of the APP/PS1 mice are increased remarkably, and the content of the MDA and the ROS is reduced remarkably and the oxidation degree is reduced after the administration of the hericium erinaceus mycelium purified polysaccharide obtained in the example 5. Compared with the blank group of mice, the contents of SOD, CAT and GSH-Px in serum and brain of APP/PS1 mice are all reduced (p is less than 0.05), and the water levels of the three antioxidant factors are obviously improved after 25 and 100mg/kg of polysaccharide is administrated.
TABLE 11 Effect of purified polysaccharide from mycelia of Hericium erinaceum obtained in example 5 on oxidative stress factors of serum, hippocampus, hypothalamus and pituitary
Data are presented as mean ± standard deviation. (n = 6/group). Relative to naive mice, # p <0.05, # p <0.01; p <0.05, p <0.01 relative to untreated APP/PS1 mice. p <0.05 represents significant, and p <0.01 represents very significant.
Claims (2)
1. A hericium erinaceus mycelium polysaccharide with functions of neuroprotection and senile dementia prevention and treatment is characterized in that:
has a molecular weight of 36.1kDa, and consists of six monosaccharides including fucose, galactose, xylose, glucose, mannose and glucuronic acid, at a molar ratio of 0.09.
2. The method for preparing the hericium erinaceus mycelium polysaccharide with neuroprotective and senile dementia preventing effects as claimed in claim 1, comprising the steps of:
1) Freezing and freeze-drying Hericium erinaceus mycelium obtained by submerged liquid fermentation, micronizing into powder, and sieving with 150 mesh sieve; adding deionized water with the mass being 12 times that of the raw materials, uniformly stirring, soaking for 30 minutes, and extracting for 2.5 hours in a constant-temperature water bath at 80 ℃;
2) Cooling to room temperature, centrifuging to obtain supernatant, concentrating by rotary evaporation to 1/6 volume, and removing protein by Sevag method; concentrating the supernatant, precipitating with 80 v/v ethanol at 4 deg.C overnight, collecting precipitate, and lyophilizing to obtain crude polysaccharide of Hericium erinaceus mycelium, HE;
3) Dissolving crude polysaccharide of Hericium erinaceus mycelium with deionized water, eluting with sodium chloride solution at a concentration of 0.1mol/L and a flow rate of 1.0mL/min through diethylaminoethyl sepharose gel at a high flow rate, respectively collecting the solutions, measuring polysaccharide content by a phenol-sulfuric acid method, collecting main peaks, concentrating by rotary evaporation to 1/8 volume, precipitating with 80 v/v ethanol at 4 ℃ overnight, collecting precipitates, and freeze-drying;
4) Dissolving freeze-dried powder of hericium erinaceus mycelium polysaccharide obtained through a DEAE gel column in deionized water, further purifying through a HiLoad 16/600Superdex 200 preparative chromatographic column, eluting with deionized water, collecting in tubes, measuring polysaccharide content by adopting a phenol-sulfuric acid method, collecting main peaks, concentrating to 1/8 volume by rotary evaporation, precipitating with ethanol at 80% v/v at 4 ℃ overnight, collecting precipitates, and freeze-drying to obtain the hericium erinaceus mycelium purified polysaccharide PHEB.
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