Preparation method and application of hericium erinaceus extract for regulating intestinal flora and metabolism

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a hericium erinaceus extract capable of regulating intestinal flora and metabolism and application of the hericium erinaceus extract. The preparation method of the hericium erinaceus alcohol extract comprises the following steps: taking hericium erinaceus as a raw material, cutting the hericium erinaceus into blocks, drying the blocks, crushing the blocks, and drying the crushed blocks; mixing the hericium erinaceus dry powder with an ethanol aqueous solution, and performing ultrasonic extraction at 50 + / -2 DEG C; centrifuging the obtained extracting solution, replacing the hericium erinaceus dry powder with the centrifuged residues, repeating the ultrasonic extraction, and then centrifuging the obtained extracting solution; and combining the supernate obtained by the two times of centrifugation, and then concentrating and freeze-drying to obtain the hericium erinaceus alcohol extract. The invention also provides application of the hericium erinaceus alcohol extract in preparation of products for regulating intestinal flora and metabolism.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a Hericium erinaceus extract for regulating intestinal flora and metabolism and an application thereof. Background Art

[0002] Hericium erinaceus, a member of the Odontaceae family, is named for its fruiting body, which resembles a monkey's head. It is a valuable edible and medicinal macrofungus and a specialty of our province. Its "Changshan Hericium" enjoys a high reputation nationwide. Rich in active ingredients such as polysaccharides, peptides, diterpenes, and sterols, Hericium erinaceus possesses significant medicinal value, showing significant therapeutic effects in treating gastrointestinal tumors, gastritis, gastric ulcers, duodenal ulcers, and abdominal distension. It can also treat gastric mucosal damage and chronic atrophic gastritis. However, the mechanism by which Hericium erinaceus prevents gastric ulcers remains unclear.

[0003] The invention of CN114634581A discloses a method for preparing Hericium erinaceus polysaccharide with the function of regulating intestinal flora, comprising the following steps: (1) drying the Hericium erinaceus fruiting body in a 50°C oven for 10 hours, crushing, and passing through a 50-mesh sieve to obtain Hericium erinaceus powder; (2) extracting the Hericium erinaceus powder with 95% ethanol for 24 hours, and then centrifuging for 15 minutes to remove the supernatant fat; (3) taking 100g of defatted Hericium erinaceus powder, adding deionized water according to a material-liquid ratio of 1:10 (w / v), extracting once with hot water at 90°C for 1 hour each time; centrifuging for 15 minutes to obtain a supernatant; (4) adding a certain amount of 95% ethanol in proportion to make the final ethanol concentration of 30%; (5) standing at room temperature for 12 hours, removing the lower layer of precipitate, re-dissolving, deproteinizing, and then dialyzing with a 3000Da dialysis bag for 3 days, rotary evaporating and concentrating the dialyzate until there is no organic solvent smell, and freeze-drying to obtain Hericium erinaceus crude polysaccharide with an ethanol concentration of 30%, named HEP30.

[0004] 16S rDNA high-throughput sequencing was performed on the sediment collected from the in vitro fermentation broth of HEP. The relative abundance changes of the intestinal flora at the phylum level were as follows: the relative abundance of Firmicutes, Bacteroidetes, and Actinobacteria increased compared to the blank culture medium, while the relative abundance of Proteobacteria decreased significantly compared to the blank culture medium. The relative abundance changes of the intestinal flora at the genus level were as follows: Bifidobacterium and Faecalibacterium increased significantly (P < 0.05), while the relative abundance of Escherichia coli-Shigella decreased significantly (P < 0.05). This shows that HEP can promote the growth of probiotics, especially Bifidobacterium, while inhibiting the growth of harmful bacteria, which can effectively improve the structure of the intestinal microbial flora and thus regulate host health.

[0005] The invention of CN118416115A discloses an efficient extraction method for Hericium erinaceus fruiting body polysaccharide rich in β-glucan: the Hericium erinaceus fruiting body is dried and then crushed and sieved through 40 mesh; then steam-exploded; 200g of Hericium erinaceus fruiting body powder after steam explosion is weighed, 3L of water is added for extraction at a temperature of 90°C, and the extraction is stirred for 2h; centrifuged, and the supernatant is retained; the residue is extracted once according to the above steps, and the supernatant is combined; the supernatant is evaporated using a rotary evaporator and the concentrated solution is collected, anhydrous ethanol is added until the volume concentration of ethanol in the solution reaches 75%, and then the addition of ethanol is stopped for alcohol precipitation, and the solution is allowed to stand for 24h and then centrifuged to retain the precipitate; the above precipitate is redissolved in water at 65°C and freeze-dried to obtain the required Hericium erinaceus fruiting body polysaccharide.

[0006] From the table of changes in intestinal flora at the genus level after 48 hours of fermentation of Hericium erinaceus fruiting body polysaccharide, it can be seen that at the genus level, HEW increased the abundance of beneficial bacteria Lactobacillus and Megasphaera compared with HEQ, and HEQ increased the abundance of beneficial bacteria Megamonas, Bifidobacterium, Collinsella, Prevotella and Bacillus compared with HEW. It can be concluded that Hericium erinaceus fruiting body polysaccharide (HEQ) treated with steam explosion wall showed better ability to increase the abundance of beneficial bacteria than untreated ones during in vitro fermentation. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a preparation method and application of Hericium erinaceus extract for regulating intestinal flora and metabolism.

[0008] In order to solve the above technical problems, the present invention provides a method for preparing an alcohol extract of Hericium erinaceus, comprising the following steps:

[0009] 1) Using Hericium erinaceus (fresh Hericium erinaceus) as raw material, cutting into pieces and then drying, then crushing and drying again to obtain Hericium erinaceus dry powder;

[0010] 2) Mix the Hericium erinaceus dry powder with 58-62% ethanol aqueous solution and extract at 50±2°C under ultrasonication for 30±1min;

[0011] The solid-liquid ratio of the Hericium erinaceus dry powder: ethanol aqueous solution = 1g: (15±0.5)mL;

[0012] 3) Centrifuging the extract obtained in step 2); Repeating step 2) with the residue obtained from the centrifugation instead of Hericium erinaceus dry powder, and then centrifuging the obtained extract;

[0013] 4) The supernatants obtained from the two centrifugations were combined, and then concentrated and freeze-dried to obtain the Hericium erinaceus alcohol extract.

[0014] As an improvement to the preparation method of the Hericium erinaceus alcohol extract of the present invention, the step 4) is: rotary evaporation concentration at 45±2°C to near dryness, and then freeze-drying at (-80±10)°C for (12±1) h.

[0015] As a further improvement to the preparation method of the Hericium erinaceus alcohol extract of the present invention, the pulverization and subsequent drying in step 1) are as follows: pulverizing until the extract passes through an 80-mesh sieve; and drying at (50±2)° C. to a constant weight.

[0016] As a further improvement to the method for preparing the Hericium erinaceus alcohol extract of the present invention, the step 1) of cutting into pieces and then drying is as follows: cutting the Hericium erinaceus into pieces and drying them at (50±2)° C. for (16±1) h.

[0017] As a further improvement to the preparation method of the Hericium erinaceus alcohol extract of the present invention, the centrifugation in step 3) is (4000±500) r·min -1 Centrifuge for (10±1) min.

[0018] As a further improvement to the method for preparing the Hericium erinaceus alcohol extract of the present invention, in step 2), the ultrasonic power is 600±100 W, and the ultrasonic frequency is 40±5 KHz.

[0019] Note: The amount of Hericium erinaceus dry powder is, for example, 2 g.

[0020] As a further improvement to the method for preparing the Hericium erinaceus alcohol extract of the present invention, step 2):

[0021] Using 60% ethanol aqueous solution;

[0022] Hericium erinaceus dry powder: ethanol aqueous solution = material-liquid ratio of 1g:15mL;

[0023] The extraction temperature was 50°C, the ultrasonic power was 600W, the ultrasonic frequency was 40KHz, and the ultrasonic treatment time was 30min.

[0024] The present invention also provides the use of the Hericium erinaceus alcohol extract prepared by any of the above methods in the preparation of products for regulating intestinal flora and metabolism.

[0025] The Hericium erinaceus ethanol extract obtained by the present invention has the following properties:

[0026] 1. It can regulate intestinal flora and upregulate beneficial bacteria such as Prevotella, Lactobacillus, Anaerostipes, Alloprevotella and Lactococcus. These bacteria can produce short-chain fatty acids and have anti-inflammatory and antioxidant effects.

[0027] 2. Hericium erinaceus ethanol extract can alter metabolism. A metabolite, gramine (also known as gramine), was found in mouse serum. Its content was positively correlated with that of Anaerostipes, Alloprevotella, and Lactococcus. Gramine is known to dock with NF-κB p105 and inhibit its ubiquitination, preventing its processing into NF-κB p50. It also increases the expression of SOD and CAT mRNAs, exerting anti-inflammatory and antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0029] Figure 1 is the gastric mucosal injury index of each group in the acute alcohol gastric ulcer model;

[0030] Figure 2 is the inhibition index of gastric mucosal loss in the experimental group and the positive control group in the acute alcohol gastric ulcer model;

[0031] Figure 3 It is the main metabolite in rat serum;

[0032] Figure 4 Volcano plot of metabolite differences between the AE-H group and the model group;

[0033] Figure 5 The metabolite differences and KEGG enrichment between the experimental group and the model group in the acute alcoholic gastric ulcer model;

[0034] Figure 5 middle:

[0035] A: Butterfly plot of metabolite differences between the AE-H group and the model group in positive ion mode;

[0036] B: Butterfly plot of the differences in metabolites in negative ion mode between the AE-H group and the model group;

[0037] C: KEGG enrichment analysis bubble chart of significant differential metabolites in AE-H groups;

[0038] Figure 6 The differences in intestinal flora and COG and KEGG enrichment between the experimental and model groups in the acute alcoholic gastric ulcer model;

[0039] Figure 6 middle:

[0040] A: Lefse diagram of the significantly different bacterial communities between the AE-H group and the model group;

[0041] B: KEGG pathway enrichment differences between the AE-H group and the model group;

[0042] C: COG enrichment differences between the AE-H group and the model group;

[0043] Figure 7 The proteomic GO and KEGG enrichment of the experimental and model groups in the acute alcoholic gastric ulcer model;

[0044] Figure 7 middle:

[0045] A: GO annotation statistics of differentially expressed proteins between the AE-H group and the model group;

[0046] B: GO enrichment bubble map (BP) of differentially expressed proteins between the AE-H group and the model group;

[0047] C: GO enrichment bubble map (MF) of differentially expressed proteins between the AE-H group and the model group;

[0048] D: GO enrichment bubble map (CC) of differentially expressed proteins between the AE-H group and the model group;

[0049] E: KEGG pathway annotation and attribution bar chart of significantly different proteins between the AE-H group and the model group;

[0050] F: KEGG pathway enrichment butterfly plot of differentially expressed proteins between the AE-H group and the model group. DETAILED DESCRIPTION

[0051] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0052] Example 1: A Hericium erinaceus extract and its application, comprising the following steps:

[0053] 1) Select fresh Hericium erinaceus fruiting bodies as raw materials, cut into small pieces and dry at 50°C for 16 hours, then crush with a traditional Chinese medicine grinder, sieve through 80 mesh, and continue to dry at 50°C to constant weight as Hericium erinaceus dry powder for later use.

[0054] 2) Weigh a certain amount of Hericium erinaceus dry powder (about 2 g), add 60% ethanol (i.e., 60% ethanol aqueous solution by volume), set the material-liquid ratio to 1:15 (g / mL), extract at 50°C, power 600W for 30 min (ultrasonic frequency 40 kHz), and the resulting extract is 4000 r·min -1 The mixture was centrifuged for 10 min, the supernatant was collected, and the remaining residue was extracted once again according to the above conditions. The two supernatants were combined, concentrated by rotary evaporation at 45°C (-0.1 MPa) to near dryness (about 1 mL), and freeze-dried (freeze-dried at -80°C for 12 hours) to obtain the Hericium erinaceus alcohol extract.

[0055] The Hericium erinaceus alcohol extract was separated using an ACQUITY UPLC™ I-Class and Xevo G3XS liquid chromatography-mass spectrometry system on a Waters BEH T3 1.8 μm (2.1 mm × 150 mm) column. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) in water, with a gradient elution condition: 5% B (0–5 min); 5%–30% B (5–15 min); 30%–45% B (15–20 min); 45%–80% B (20–30 min); 80%–95% B (30–45 min); and 95%–5% B (45.10–55 min). The flow rate was 0.3 mL / min. The injection volume was 10 μL, and the column temperature was 40°C. Detection was performed in positive ion mode. The acquisition mass range was 50–1200 Da. Other parameters were set as follows: capillary voltage, 3 kV; ion source temperature, 100°C; nebulizer temperature, 280°C; nebulizer flow rate, 800 L / h; cone voltage, 40 V; and collision energy, low energy, 6 V; high energy, 20–30 V. Data processing software was UNIFI 1.9.2, and the database was Waters traditional medicine.

[0056] A total of 215 components were detected in the alcohol extract of Hericium erinaceus, including 64 terpenes, 21 alkaloids, 20 fatty acids, 13 phenols, 11 phenolphthales, 9 ketones, 8 carboxylic acids, 8 flavonoids, 8 steroids, 7 coumarins, 6 lignans, 5 heterocyclic compounds, 5 saponins, 4 amino compounds, 4 fatty amides and 3 quinones, 2 each of aromatic hydrocarbons, fatty aldehydes, nucleosides, lactones and glycosides, 1 each of glycolipids, ethers, glycerolipids, vine lactones, resin acids, lipids, fatty alcohols, fatty esters and organic acids, as shown in Table 1.

[0057] Table 1 Composition of Hericium erinaceus ethanol extract

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Experiment 1: Using the Hericium erinaceus ethanol extract obtained in Example 1:

[0068] Adult male SPF Sprague-Dawley rats (weighing 200 ± 20 g) were housed under standard laboratory conditions (25.0 ± 0.5°C, 50 ± 5% relative humidity, and a 12-h light-dark cycle). Rats were given free access to food and water and acclimated to the experimental environment for one week before the experiment. Five groups of six rats were randomly assigned to the experimental group: a blank control (CK), a model group (Modle), a positive control (ranitidine group, PC), and two experimental groups (a low-concentration Hericium erinaceus ethanol extract group, AE-L, and a high-concentration Hericium erinaceus ethanol extract group, AE-H). The experimental groups received the corresponding extracts daily by oral gavage at a dose of 150 mg / kg for the low-concentration extract and 250 mg / kg for the high-concentration extract. The positive control group received 30 mg / kg of ranitidine, a clinical drug for treating gastric mucosal lesions, by oral gavage at a dose of 10 mL / kg. Both the blank and model groups received the same dose of distilled water daily. The gavage lasted for 21 days. After the last administration, all animals were strictly fasted but not watered for 24 hours. Modeling began. Except for the blank control group, all experimental groups were given 5.0 mL / kg of anhydrous ethanol and sacrificed by spinal dislocation 1 hour later.

[0069] Gastric lesions were histologically evaluated in accordance with the standards of the "Technical Guidelines for Functional Testing and Evaluation of Health Foods (2023 Edition)". The complete stomach was exposed, the pylorus was ligated, the weight was recorded, and an appropriate amount of 10% formaldehyde solution was perfused. After fixation for 20 minutes, the stomach was cut open along the greater curvature, the gastric contents were washed with distilled water, the gastric mucosa was unfolded, and the bleeding points and the length and width of the bleeding band were measured with a vernier caliper under the naked eye for scoring. Scoring criteria: 1 point for each bleeding point; 1 point for the length of the bleeding band 0-1 mm, 2 points for 1-2 mm, 3 points for 2-3 mm, 4 points for 3-4 mm, and 5 points for greater than 5 mm; 1 point for the width of the bleeding band 1-2 mm, and 2 points for greater than 2 mm. Total injury score = bleeding point score + length score + width score.

[0070] like Figure 1 As shown in the results, compared with the CK group, the Modle group had more severe bleeding and a significantly increased gastric mucosal lesion index, indicating a successful model establishment. Low and high concentrations of Hericium erinaceus ethanol extract and drug intervention significantly reduced the gastric mucosal lesion index in rats. The inhibitory rates of gastric tissue lesions in rats were calculated to be 56.32%, 55.06%, and 67.72%, respectively. Figure 2 ).

[0071] Gastric tissue lesion inhibition rate = (lesion index of gastric mucosa in the model group - lesion index of gastric mucosa in the experimental group) / lesion index of gastric mucosa in the model group.

[0072] According to the above Experiment 1, it can be known that the Hericium erinaceus ethanol extract of the present invention has excellent performance in preventing and treating acute gastric ulcer induced by ethanol.

[0073] Experiment 2

[0074] The experimental groups were set up in the same manner as in Experiment 1. After the rats were sacrificed, blood was collected from the orbits to obtain serum.

[0075] Through metabolomics, all metabolites identified in each group of serum (combined with metabolites identified by positive and negative ions) were classified and counted according to their chemical classification information. The proportion of each type of metabolite was, for example, Figure 3 As shown. Lipids and lipid molecules, organic acids and their derivatives, organic heterocyclic compounds, benzene compounds, and organic oxygen compounds are the main components of metabolites. Among them, lipids and lipid molecules account for the highest proportion, reaching 28.256%. Based on univariate analysis, differential analysis was performed on all detected metabolites. Metabolites that met FC>1.5 or FC<0.67 and p<0.05 calculated by T test were regarded as differential metabolites, and were visualized in the form of volcano plots. The results are shown as follows Figure 4 shown.

[0076] Compared with the model group, the high concentration Hericium erinaceus ethanol extract group had:

[0077] In the positive ion mode, the upregulated significant metabolites mainly included: decamethylcyclopentasiloxane, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, platelet activating factor, cytosine, gramine, 4-aminopyridine, 1-oleoyl-SN-glycero-3-phosphocholine; the downregulated significant metabolites mainly included: phenylalanine-bound chenodeoxycholic acid, pyridoxal, ursodeoxycholic acid, N-glycolylneuraminic acid, 2-methylpyrrolidine, proline-tryptophan, leucine, indole-3-pyruvate, 2,4-dihydroxybenzophenone, pyridine, 3-methylhistidine ( Figure 5 A).

[0078] In negative ion mode, the upregulated significant differential metabolites included phenyllactic acid and 2-naphthalenesulfonic acid; the downregulated significant differential metabolites mainly included: 2',6'-dihydroxy 4'-methoxydihydrochalcone, 2-bromophenol butan-6-ol, biocytin, andrastin D, anacardic acid, evergreen gelsinine, linoleic acid, lysine, 3-hydroxybutyric acid, rauwolfia, mestranol, guanyltaurine, phenylpyruvic acid, eicosenoic acid, 12(S)-hydroxy(5Z,8Z,10E,14Z)-eicosatetraenoic acid, cinchonine, azelaic acid, glycine, L-pyroglutamic acid, decitabine, canrenone and hydroquinidine ( Figure 5 B).

[0079] Gramine, a metabolite with anti-inflammatory and antioxidant properties, can dock with NF-κB p105 and inhibit its ubiquitination, preventing its processing into NF-κB p50. It can also increase the mRNA expression of SOD and CAT. Compared with the model group, the high-concentration Hericium erinaceus alcohol extract group showed a significant upregulation of gramine in the serum metabolites of rats, demonstrating that the Hericium erinaceus alcohol extract of the present invention has anti-inflammatory and antioxidant effects.

[0080] In the KEGG pathways of the high-concentration Hericium erinaceus ethanol extract group, protein digestion and absorption, aminoacyl-tRNA biosynthesis, D-amino acid metabolism, mineral absorption, biotin metabolism, amino acid biosynthesis, mTOR signaling pathway, ABC transporter, tumor central carbon metabolism and glutathione metabolism pathways were significantly enriched, such as Figure 5 C. Aminoacyl-tRNA biosynthesis, amino acid biosynthesis, and glutathione metabolism are associated with gastric mucosal protection. Glutathione, in particular, as a major antioxidant, has a protective effect against ethanol-induced gastric ulcers. Among the differentially expressed metabolites, glycine and L-pyroglutamate were both downregulated. Glycine is added to γ-glutamylcysteine ​​by glutathione synthetase to form glutathione. L-pyroglutamate, catalyzed by 5-oxoprolinase, consumes ATP to generate glutamate, which is then synthesized into glutathione. The downregulation of both glycine and L-pyroglutamate may be due to their synthesis into glutathione through the glutathione metabolic pathway. The sulfhydryl group of glutathione can directly react with ROS, terminating free radical chain reactions. Furthermore, it can repair protein sulfhydryl oxidation caused by oxidative stress through thiol-disulfide exchange reactions.

[0081] According to the above Experiment 2, it can be known that the Hericium erinaceus ethanol extract of the present invention has the ability to regulate metabolites and exert anti-inflammatory and antioxidant properties.

[0082] Experiment 3

[0083] The experimental groups were set up in the same way as in Experiment 1. According to conventional methods, the colon contents of the rats were obtained after sacrifice; then the 16S rDNA of the intestinal flora was sequenced and lefse analysis was performed, such as Figure 6 As shown in A; at the genus level, the number of 8 genera increased significantly in the high-concentration Hericium erinaceus ethanol extract group compared with the model group, including: Prevotella, Lactobacillus, Anaerostipes, Alloprevotella, NK4A214_group, Bacillus, Lactococcus and DNF00809. The number of 13 genera decreased significantly, including: Muribaculaceae, Allobaculum, Dubosiella, Clostridia_UCG_014, Blautia, Gastranaerolophilales, Parasutterella, Alistipes, Coriobacteriaceae_UCG_002, Rothia, Defluviitaleaceae_UCG_011, Enterococcus and Macellibacteroides. Among the genera upregulated in the AE-H group, short-chain fatty acid-producing genera (Prevotella, Lactobacillus, Anaerostipes, Alloprevotella and Lactococcus) can produce short-chain fatty acids and play an antioxidant role. In KEGG, glycolysis, gluconeogenesis, fructose and mannose metabolism, cell cycle of Clostridium, drug metabolism, glutathione metabolism, streptomycin biosynthesis, mismatch repair, and purine metabolism pathways were significantly increased ( Figure 6 B) Replication, recombination and repair, nucleotide transport and metabolic pathways are significantly improved in COG ( Figure 6 C).

[0084] According to the above Experiment 3, it can be known that the Hericium erinaceus ethanol extract of the present invention has the performance of regulating beneficial intestinal flora such as Prevotella, Lactobacillus, Anaerostipes, Alloprevotella and Lactococcus and exerting anti-inflammatory and antioxidant effects.

[0085] Experiment 4

[0086] The experimental groups were set up in the same way as in Experiment 1. According to conventional methods, gastric tissues of rats were obtained after sacrifice and protein extraction was performed. Through proteomics, Blast2Go (https: / / www.blast2go.com / ) software was used to perform GO functional annotations on all significantly differentially expressed proteins, significantly upregulated differential proteins, and significantly downregulated differential proteins. There were 49 significantly differentially expressed proteins in the high-concentration Hericium erinaceus ethanol extract group and the model group. Compared with the model group, the AE-H group had 25 significantly upregulated differential proteins and 24 significantly downregulated differential proteins. Among them, the expression levels of Calpain-8 protein and copper-transporting ATPase were both upregulated. Calpain-8 protein can participate in the membrane transport of mucous cells on the gastric surface, and may participate in the membrane transport of mucous cells through interaction with membrane proteins to resist gastric mucosal damage caused by stress. Copper-transporting ATPase is involved in copper metabolism. Copper is an important component of antioxidant enzymes and may affect oxidative stress. Chemokine CXCL7 and sodium-hydrogen exchanger were significantly downregulated. Chemokine CXCL7 recruits inflammatory cells and exacerbates inflammatory damage, while sodium-hydrogen exchanger promotes the release of proinflammatory factors. 461 differentially expressed proteins were associated with GO functional annotations. Macromolecular methylation, membrane raft organization, and endocrine processes were significantly enriched in BP (P < 0.05). Figure 7 B. N-methyltransferase activity, N-acetyllactosamine beta-1,3-N-acetylglucosaminyltransferase activity, and protein lysine N-methyltransferase activity were significantly enriched in MF (P<0.05). Figure 7 C. Early endosomes, early endosomal membranes, and endosomal membranes were significantly enriched in CC (P<0.05) Figure 7 D. In the KEGG pathway enrichment, the high-concentration Hericium erinaceus ethanol extract group significantly upregulated the JAK-STAT signaling pathway, terpenoid skeleton biosynthesis, mineral absorption, platinum drug resistance, and human T-cell leukemia virus type 1 infection; while the complement and coagulation cascade, the interaction between viral proteins and cytokines and their receptors, cytokine-cytokine receptor interaction, collecting duct acid secretion, and neuroactive ligand-receptor interaction were significantly downregulated. Figure 7 F.

[0087] According to the above experiment 4, it can be known that the Hericium erinaceus ethanol extract of the present invention has the performance of upregulating the anti-inflammatory protein Calpain-8 protein and copper-transporting ATPase, and downregulating the pro-inflammatory protein chemokine CXCL7 and sodium-hydrogen exchanger.

[0088] During the invention process, the following comparative experiments were conducted: "60% ethanol concentration" was replaced with "80% ethanol concentration" or "40% ethanol concentration," with the remainder being the same as in Example 1. The resulting Hericium erinaceus alcohol extract was tested according to Experiment 2 above, and it was found that Gramine, a metabolite in rat serum, was not significantly upregulated in the high-concentration group compared to the model group.

[0089] During the invention process, the following comparative experiment was conducted: the "extraction temperature of 50°C, ultrasonic treatment at 600W power for 30 minutes" in Example 1 was changed to "extraction temperature of 70°C, extraction time of 2 hours," with all other conditions remaining the same as in Example 1. The resulting Hericium erinaceus alcohol extract was tested according to Experiment 2 above, revealing that Gramine, a metabolite in rat serum, was not significantly upregulated in the high-concentration group compared to the model group.

[0090] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing Hericium erinaceus alcohol extract, characterized in that The following steps are involved: 1) Using Hericium erinaceus as raw material, cutting into pieces and then drying, then crushing and drying again to obtain Hericium erinaceus dry powder; 2) Mix the Hericium erinaceus dry powder with 58-62% ethanol aqueous solution and extract at 50±2°C under ultrasonication for 30±1min; The solid-liquid ratio of the Hericium erinaceus dry powder: ethanol aqueous solution = 1g: (15±0.5)mL; 3) Centrifuging the extract obtained in step 2); Repeating step 2) with the residue obtained from the centrifugation instead of Hericium erinaceus dry powder, and then centrifuging the obtained extract; 4) The supernatants obtained from the two centrifugations were combined, and then concentrated and freeze-dried to obtain the Hericium erinaceus alcohol extract.

2. The method for preparing the Hericium erinaceus alcohol extract according to claim 1, characterized in that The step 4) comprises: concentrating by rotary evaporation at 45±2° C. to near dryness, and then freeze-drying at -80±10° C. for 12±1 hours.

3. The method for preparing the Hericium erinaceus alcohol extract according to claim 2, characterized in that The step 1) of pulverizing and then drying comprises: pulverizing until the powder passes through an 80-mesh sieve; and drying at 50±2° C. to a constant weight.

4. The method for preparing the Hericium erinaceus alcohol extract according to claim 3, characterized in that The step 1) of cutting into pieces and then drying is as follows: cutting the Hericium erinaceus into pieces and drying them at 50±2° C. for 16±1 h.

5. The method for preparing the Hericium erinaceus alcohol extract according to claim 4, characterized in that The centrifugation in step 3) is 4000±500 r·min -1 Centrifuge for 10±1 min.

6. The method for preparing the Hericium erinaceus alcohol extract according to any one of claims 1 to 5, characterized in that In the step 2), the ultrasonic power is 600±100W and the ultrasonic frequency is 40±5KHz.

7. The method for preparing the Hericium erinaceus alcohol extract according to any one of claim 6, characterized in that Step 2): Using 60% ethanol aqueous solution; Hericium erinaceus dry powder: ethanol aqueous solution = material-liquid ratio of 1g:15mL; The extraction temperature was 50°C, the ultrasonic power was 600W, the ultrasonic frequency was 40KHz, and the ultrasonic treatment time was 30min.

8. Use of the Hericium erinaceus alcohol extract prepared by the method of any one of claims 1 to 7 in the preparation of products for regulating intestinal flora and metabolism.

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