Application of flammulina velutipes extract in preparation of related products for relieving and / or treating drug-induced liver injury

Abstract

The invention discloses an application of a flammulina velutipes extract in preparation of related products for relieving and / or treating drug-induced liver injury. By constructing a drug-induced liver injury model, it is found that by means of the needle mushroom extract rich in needle mushroom polysaccharide in low dosage, liver pathological injuries such as cisplatin-induced hepatocyte swelling and necrosis can be remarkably relieved, the levels of serum liver function indexes of alanine transaminase and aspartate transaminase are reduced, and the liver injury resistance is improved. And meanwhile, the activity of superoxide dismutase and glutathione peroxidase in liver tissues is improved, the concentration of malonaldehyde is reduced, and excellent antioxidant characteristics are shown. In addition, the invention also discovers that the flammulina velutipes extract can improve the composition of intestinal microorganisms, promote colonization of beneficial bacteria, reduce the abundance of harmful bacteria and regulate the key metabolic pathway of the liver at the same time, and can provide a treatment strategy for the treatment of drug-induced liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of an enoki mushroom extract in preparing products related to alleviating and / or treating drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) refers to liver damage caused by drugs, traditional Chinese medicines, supplements, or their metabolites. DILI is a common adverse drug reaction in clinical practice, which can lead to abnormally elevated liver function tests, severe acute liver failure, and even death.

[0003] Currently, common drug-induced liver injury includes liver injury caused by cisplatin (CDDP) treatment and chemical liver injury induced by carbon tetrachloride (CCl4). Among them, CDDP is one of the most common cytotoxic drugs for treating cancer and is widely used to treat various solid tumors, including cervical cancer, ovarian cancer, prostate cancer, testicular cancer and colorectal cancer. It has become one of the most beneficial anticancer drugs for 40-80% of solid tumor patients. However, CDDP is also a toxic drug that causes drug-induced liver injury. CDDP interacts with the purine bases of DNA in cancer cells, causing DNA damage, disrupting DNA replication and transcription, and ultimately inhibiting the growth of these cancer cells. However, due to off-target effects, it has certain toxic effects on various tissues and organs of the body (such as ears, liver and kidneys). CCl4 is a common toxic drug that induces chemical liver injury in mice. However, unlike DILI caused by CDDP, CCl4 has a direct dissolving effect on liver cell membranes. Its metabolism generates trichloromethyl free radicals (CCl3·) and free radicals (Cl·), which can trigger lipid peroxidation, destroy liver cell membrane structure, and lead to hepatocellular necrosis. Treatment of CDDP liver injury should focus on inhibiting ferroptosis, combined with antioxidants, DNA protection, and metabolic regulation. For liver injury caused by CCl4, antioxidants (such as glutathione) can be used to directly bind to CCl4 metabolites to neutralize free radical toxicity. Hepatoprotective drugs can also be taken to promote liver cell regeneration and repair membrane structure.

[0004] Related studies have shown that regulating the gut-liver axis may provide new therapeutic strategies for treating liver diseases. The intestinal microbiome is involved in food digestion, vitamin synthesis, and the production of metabolites such as short-chain fatty acids and bile acids. These metabolites can enter the liver through the portal system, affecting the liver's metabolic and detoxification functions. With in-depth research on the relationship between the gut microbiome and DILI, it has been found that the gut microbiome can affect liver function by producing metabolites and affecting intestinal barrier function, thereby leading to drug-induced liver injury. This provides new insights into the treatment of DILI. Although research on the relationship between the gut microbiome and DILI has made some progress, our understanding of this area remains limited. Future research is needed to further explore the specific mechanisms by which the gut microbiome influences drug metabolism in the liver and to develop strategies for preventing and treating DILI based on the gut microbiome. Furthermore, given the differences in liver injury caused by different drugs, specific drug treatment options for DILI are currently limited. Therefore, there is an urgent need to identify new and effective drugs for the treatment of DILI. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of an extract of Flammulina velutipes in the preparation of products related to the alleviation and / or treatment of drug-induced liver injury. The present invention found that an extract of Flammulina velutipes rich in polysaccharides can significantly reduce the liver function and oxidative stress indicators of mice with liver damage caused by anticancer drugs (such as cisplatin), and at the same time help to change the intestinal microecological environment and liver metabolism of mice with liver damage caused by cisplatin, and has significant liver protection, antioxidant and intestinal flora regulation activities.

[0006] The present invention also provides a drug for treating drug-induced liver injury.

[0007] The first aspect of the present invention provides the use of an extract of Flammulina velutipes in the preparation of products for alleviating and / or treating drug-induced liver injury.

[0008] The application of the embodiments of the present invention has at least the following beneficial effects:

[0009] (1) The present invention successfully established a DILI mouse model using CDDP and found that a low dose of Flammulina velutipes extract had an excellent liver protective effect on DILI mice, significantly alleviating liver pathological damage (such as CDDP-induced hepatocyte swelling and necrosis), and significantly reducing the levels of serum liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST), while increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissue, downregulating the concentration of malondialdehyde (MDA), and having antioxidant properties.

[0010] (2) The present invention found that low-dose Flammulina velutipes extract can improve the intestinal microbial composition of mice with CDDP-induced liver injury, promote the colonization of beneficial bacteria, reduce the abundance of harmful bacteria, and regulate key metabolic pathways in the liver. Therefore, the use of Flammulina velutipes extract in the treatment of DILI with the chemotherapy drug CDDP may be an effective strategy for preventing and treating DILI.

[0011] (3) The present invention found that the use of low-dose Flammulina velutipes extract can improve the pathological symptoms of mice with CDDP-induced liver injury by regulating ABC transporters, cyanoamino acid metabolism, thiamine metabolism, phenylalanine / tyrosine / tryptophan biosynthesis, aminoacyl-tRNA biosynthesis, etc., which can provide a therapeutic strategy for the treatment of drug-induced liver injury.

[0012] In some embodiments of the present invention, the sugar content in the Flammulina velutipes extract is ≥70%.

[0013] In some embodiments of the present invention, the sugar content in the Enoki mushroom extract is ≥ 75%, for example, 76%, 78%, 80%, 85%, 90% or 95%.

[0014] In some embodiments of the present invention, the sugar in the Flammulina velutipes extract includes Flammulina velutipes polysaccharide (FVP).

[0015] Flammulina velutipes polysaccharide is a biologically active natural polysaccharide extracted from Flammulina velutipes. Studies have shown that it has anti-cancer effects, inhibiting tumor cell proliferation and inducing apoptosis. However, there are few reports on its use in treating liver-related diseases. Some studies have found that Flammulina velutipes polysaccharide has a protective effect against acute liver injury, but in practice, it has been found that higher dosages are required to achieve a good protective effect (such as against acute liver injury caused by CCl4), which greatly limits its application. The present invention finds that a low dose of Flammulina velutipes extract can achieve excellent therapeutic effects in treating CDDP-induced liver injury.

[0016] In some embodiments of the present invention, the method for preparing the Flammulina velutipes extract comprises:

[0017] The powder of Flammulina velutipes is mixed with water, and the mixture is subjected to hot water extraction, protein removal and ethanol precipitation treatment to obtain the product.

[0018] In some embodiments of the present invention, the method for obtaining the enoki mushroom powder comprises: drying fresh enoki mushrooms and crushing the powder to obtain the powder.

[0019] In some embodiments of the present invention, the variety of the enoki mushroom is Gutian enoki mushroom.

[0020] In some embodiments of the present invention, the drying method comprises oven drying or vacuum freeze drying.

[0021] In some embodiments of the present invention, the oven drying temperature is 40-70°C.

[0022] In some embodiments of the present invention, the volume ratio of the Enoki mushroom powder to water is 1:20-40.

[0023] In some embodiments of the present invention, the temperature of the hot water extraction is 90-110°C.

[0024] In some embodiments of the present invention, the hot water extraction time is 1-4 hours.

[0025] In some embodiments of the present invention, the hot water extraction is performed 1-4 times, for example, 2, 3 or 4 times.

[0026] In some embodiments of the present invention, the hot water extraction comprises: mixing the Enoki mushroom powder with the water, subjecting the mixture to hot water extraction at 90-110° C. for 1-4 hours, and then collecting the filtrate and concentrating the mixture to obtain the product.

[0027] In some embodiments of the present invention, the volume after concentration is 1 / 4-1 / 15 of the volume of the filtrate.

[0028] In some embodiments of the present invention, the protein removal treatment comprises mixing the product after hot water extraction with a protein removal agent, reacting, and concentrating after centrifugation.

[0029] In some embodiments of the present invention, the deproteinization reagent comprises chloroform and / or n-butanol.

[0030] In some embodiments of the present invention, the deproteinization reagent comprises chloroform and n-butanol.

[0031] In some embodiments of the present invention, in the deproteinization reagent, the volume ratio of chloroform to n-butanol is 3-5:1.

[0032] In some embodiments of the present invention, in the deproteinization reagent, the volume ratio of chloroform to n-butanol is 3-4:1.

[0033] In some embodiments of the present invention, the volume ratio of the product after hot water extraction to the protein removal agent is 1:1-3. For example, it can be 1:1, 1:2 or 1:3.

[0034] In some embodiments of the present invention, during the protein removal process, the centrifugal speed is 3000-5000 r / min.

[0035] In some embodiments of the present invention, during the protein removal process, the centrifugation time is 8-20 minutes.

[0036] In some embodiments of the present invention, the deproteinization treatment is repeated 6-20 times. For example, the deproteinization treatment can be repeated 6, 8, 10, 12, 15, 18, 20 times, etc.

[0037] In some embodiments of the present invention, the ethanol precipitation treatment comprises: mixing the product after the deproteinization treatment with an ethanol solution, collecting the precipitate after centrifugation, and drying it to obtain the product.

[0038] In some embodiments of the present invention, the volume ratio of the deproteinized product to the ethanol solution is 1:3 to 5. For example, the volume ratio can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0039] In some embodiments of the present invention, the ethanol solution is an aqueous solution of ethanol.

[0040] In some embodiments of the present invention, the ethanol content in the ethanol solution is greater than 95%, for example, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

[0041] In some embodiments of the present invention, during the ethanol precipitation treatment, the mixing treatment includes: adding the ethanol solution to the product after the deproteinization treatment, mixing, and standing at 0-4° C. for 8-12 hours.

[0042] In some embodiments of the present invention, during the ethanol precipitation process, the centrifugal speed is 4000-8000 r / min.

[0043] In some embodiments of the present invention, during the ethanol precipitation process, the centrifugation time is 10-20 minutes.

[0044] In some embodiments of the present invention, the ethanol precipitation treatment further includes dialysis treatment.

[0045] In some embodiments of the present invention, the method for preparing the Flammulina velutipes extract comprises:

[0046] S1. Drying fresh Enoki mushrooms and crushing them to obtain Enoki mushroom powder;

[0047] S2, mixing the Enoki mushroom powder and water in a volume ratio of 1:20-30, extracting the mixture in hot water at 90-110° C. for 1-4 hours, collecting the filtrate, and concentrating the mixture to obtain a hot water extract;

[0048] S3, mixing the hot water extract with a protein removal agent, reacting, repeating the protein removal step 6-12 times after centrifugation, and concentrating under reduced pressure to obtain a crude extract;

[0049] S4. The crude extract is mixed with an ethanol solution, and the precipitate is collected after centrifugation, dried, and collected; the precipitate is then dissolved with a solvent and dialyzed to obtain the product.

[0050] The preparation method of the Flammulina velutipes extract of the present invention can retain the active ingredients in Flammulina velutipes to a large extent, such as Flammulina velutipes polysaccharides, flavonoids, organic acids, etc., among which Flammulina velutipes polysaccharides are one of its main biologically active ingredients, which has multiple functions such as antioxidant, immunomodulatory, and anti-tumor, can enhance the body's immunity, promote intestinal health, and help fight free radicals and delay aging; flavonoids can help eliminate free radicals in the body, reduce oxidative stress, and protect cells from damage; organic acids (such as malic acid, citric acid, etc.) help promote metabolism and improve the health of the digestive system.

[0051] In some embodiments of the present invention, in step S4, the solvent includes water.

[0052] In some embodiments of the present invention, in step S4, the mass volume ratio of the precipitate to the solvent is 1 g:8-12 mL.

[0053] In some embodiments of the present invention, the dialysis treatment time is 24-72 hours.

[0054] In some embodiments of the present invention, the molecular weight cut-off for the dialysis treatment is 2.5 to 3.5 kDa, and specifically, can be 3 kDa.

[0055] The monosaccharide composition of Flammulina velutipes polysaccharide mainly consists of three monosaccharides: mannitol (7.74%), glucose (70.41%) and galactose (16.38%).

[0056] In some embodiments of the present invention, the efficacy of the product for alleviating and / or treating drug-induced liver injury includes at least one of the following:

[0057] A) Improve the symptoms of weight loss caused by drug-induced liver injury;

[0058] B) reduce serum alanine aminotransferase and aspartate aminotransferase levels in patients with drug-induced liver injury;

[0059] C) Relieve symptoms of liver swelling, congestion and / or necrosis in patients with drug-induced liver injury;

[0060] D) Alleviate the liver oxidative stress level and reduce liver oxidative damage in patients with drug-induced liver injury;

[0061] E) Improve the intestinal microbial composition of patients with drug-induced liver injury and promote the colonization of beneficial bacteria;

[0062] F) Improving liver injury in patients with drug-induced liver injury by regulating any one of the following pathways: ABC transporters, cyanoamino acid metabolism, thiamine metabolism, phenylalanine / tyrosine / tryptophan biosynthesis, and aminoacyl-tRNA biosynthesis.

[0063] In some embodiments of the present invention, the drug-induced liver injury includes cisplatin-induced liver injury.

[0064] Cisplatin (CDDP) is one of the most common cytotoxic drugs for treating cancer. It is widely used to treat various solid tumors, including cervical cancer, ovarian cancer, prostate cancer, testicular cancer, and colorectal cancer. It has become one of the most effective anticancer drugs for 40-80% of solid tumor patients. The clinical manifestations of cisplatin-induced liver injury mainly include a significant increase in transaminase levels (such as ALT and AST) and liver cell necrosis. The present invention found that the use of a low dose of Flammulina velutipes extract can help significantly alleviate these symptoms. It can also improve the intestinal microbial composition of animal models of cisplatin-induced liver injury, promote the colonization of beneficial bacteria, reduce the abundance of harmful bacteria, and regulate key metabolic pathways in the liver. This can provide a therapeutic strategy for the treatment of drug-induced liver injury.

[0065] In some embodiments of the invention, the product comprises a pharmaceutical.

[0066] The second aspect of the present invention provides a drug for treating drug-induced liver injury, the active ingredient of which comprises the Flammulina velutipes extract described in the first aspect.

[0067] In some embodiments of the present invention, the drug for treating drug-induced liver injury further comprises a pharmaceutically acceptable excipient.

[0068] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0069] In some embodiments of the invention, the excipient comprises water.

[0070] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0071] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0072] In some embodiments of the invention, the humectant comprises glycerin.

[0073] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0074] In some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0075] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0076] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0077] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0078] In some embodiments of the present invention, the drug for treating drug-induced liver injury further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is generally recognized for this purpose and serves as an inactive ingredient in the medicament. A compilation of pharmaceutically acceptable carriers can be found in reference books such as the Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).

[0079] In some embodiments of the present invention, the dosage of the drug for treating drug-induced liver injury can be adjusted according to actual conditions.

[0080] In some embodiments of the present invention, the drug for treating drug-induced liver injury can be administered via the gastrointestinal tract and / or parenteral routes.

[0081] In some embodiments of the present invention, the non-gastrointestinal administration route is selected from injection, respiratory tract administration, skin administration, mucosal administration or cavity administration.

[0082] In some embodiments of the present invention, the pharmaceutical dosage form for parenteral administration is selected from injections, sprays, aerosols, patches, and the like.

[0083] In some embodiments of the present invention, the pharmaceutical dosage form for gastrointestinal administration is selected from tablets, capsules, powders, granules, pills, solutions, emulsions or syrups.

[0084] In some embodiments of the present invention, the drug is in the form of an oral preparation, an injection or a topical preparation.

[0085] In some embodiments of the present invention, the oral preparations include tablets, capsules, pills, powders, granules, syrups or solutions; the injections include injection liquid dosage forms or lyophilized powder injection dosage forms; topical preparations include creams, ointments, sprays, aerosols, gels, papules or patches.

[0086] In some embodiments of the present invention, carriers that can be used in the preparation of oral formulations may include conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin, various chemically modified cyclodextrins, sucrose, and stearates. Freeze-dried powder injections may be prepared by methods such as sterile spray drying, low-temperature vacuum drying, and freeze drying. The subsequent preparation processes and equipment for each formulation are conventional in the pharmaceutical field and are not limited thereto by the present invention. Other features and advantages of the present invention will be described in the subsequent description. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0088] Figure 1 This is the standard curve for the sugar content detection of Flammulina velutipes extract;

[0089] Figure 2 The effect of Flammulina velutipes extract on the body weight and liver index of mice, where A is the average body weight of mice in each group before intervention, B is the average body weight of mice in each group on the 7th day after intervention, C is the average body weight of mice in each group on the 10th day after intervention, D is the average body weight change of mice in each group during the administration period, and E is the statistical result of liver index of mice in each group;

[0090] Figure 3 The effect of Flammulina velutipes extract on liver function indexes in mice, where A is the statistical result of alanine aminotransferase (ALT) content, and B is the statistical result of aspartate aminotransferase (AST) content;

[0091] Figure 4 The effect of Flammulina velutipes extract on the morphology and pathology of mouse liver, where A is the staining result of liver tissue of mice in each group, and B is the morphology of liver tissue of mice in each group;

[0092] Figure 5 This is the Suzuki score result of mouse liver;

[0093] Figure 6 The results of the effect of Flammulina velutipes extract on the oxidative stress level in mouse liver, where A is the statistical result of superoxide dismutase (SOD), B is the statistical result of glutathione peroxidase (GSH-Px), and C is the statistical result of malondialdehyde (MDA);

[0094] Figure 7Shannon and Simpson dilution curves of mouse intestinal flora samples at the OTU level, where A is the Shannon dilution curve and B is the Simpson dilution curve;

[0095] Figure 8 The results of the effect of Flammulina velutipes extract on the β diversity of intestinal flora in mice, where A is the visualization diagram of PLS-DA analysis and B is the Anosim analysis result;

[0096] Figure 9 The results of the effect of Flammulina velutipes extract on the species composition of the intestinal flora of mice at the order level, where A is the statistical result of the species difference at the order level of each group of mice, and B is the statistical result of the relative abundance of Peptostreptococcales-Tissierellales and Clostridia_UCG-014;

[0097] Figure 10 The results of the effect of Flammulina velutipes extract on the species composition of the intestinal flora of mice at the genus level, where A is the statistical result of species differences at the genus level of each group of mice, and B is the statistical result of the relative abundance of Escherichia-Shigella;

[0098] Figure 11 The following are the results of differential analysis of the dominant bacterial flora in the mouse intestine, where A is the species taxonomic branch diagram and B is the statistical results of the LDA value distribution histogram of significantly different species;

[0099] Figure 12 The following are the quality control charts of mouse liver tissue metabolomics data, where A is the data quality control chart and B is the cluster analysis result;

[0100] Figure 13 The PCA and OPLS-DA analysis results of mouse liver metabolites, where A is the PCA analysis result, B is the OPLS-DA analysis result of the Control group and the Model group, and C is the OPLS-DA analysis result of the Model group and the LFVP group;

[0101] Figure 14 The results of the effect of Flammulina velutipes extract on the metabolite composition of mouse liver, where A is the statistical result of the differential metabolites in each group, and B is the Venn diagram analysis result;

[0102] Figure 15 These are enriched pathways in liver metabolism after CDDP treatment in mice;

[0103] Figure 16 The enriched pathway in liver metabolism after the intervention of Flammulina velutipes extract in mice;

[0104] Figure 17This is a heat map of the correlation between mouse intestinal flora and liver metabolites at the level of the eye;

[0105] Figure 18 Figure 2 is a heat map of the correlation between mouse gut microbiota and liver metabolites at the genus level. DETAILED DESCRIPTION

[0106] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0107] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0108] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0109] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0110] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0111] In an embodiment of the present invention, the enoki mushrooms used are Gutian enoki mushrooms, which were purchased in Fuzhou, Fujian.

[0112] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0113] Example 1:

[0114] This embodiment provides a method for preparing a Flammulina velutipes extract, which specifically comprises the following steps:

[0115] (1) Place 100 g of fresh Enoki mushrooms in a vacuum freeze dryer. After freeze drying and dehydration, use a grinder to crush the dried Enoki mushrooms to obtain Enoki mushroom powder.

[0116] (2) The above-mentioned Enoki mushroom powder was mixed with distilled water in a mass-to-volume ratio of 1:30, and the mixture was extracted with boiling water under reflux for 2 h, with the extraction number being 1; the Enoki mushroom powder residue was then filtered out with a filter, and the filtrate was collected and concentrated with a rotary evaporator at 60°C to obtain a concentrated extract with a volume of 1 / 4 to 1 / 5 of the original filtrate, i.e., a hot water extract.

[0117] (3) The hot water extract was deproteinized using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method of deproteinization by the Sevag method is as follows:

[0118] The hot water extract was mixed with Sevag reagent in a 1:1 volume ratio, placed on a shaker, and shaken at 270 rpm for 2 hours at 25°C. The mixture was then centrifuged at 3500 rpm for 10 minutes at 4°C, and the supernatant was removed. The shaking and centrifugation steps were repeated 10 times, and the supernatant was collected and concentrated under reduced pressure (60°C) to 1 / 10 of its volume to obtain a crude extract solution after protein removal.

[0119] (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal, and let it stand at 4°C overnight; then centrifuge at 3500 r / min for 10 minutes, collect the precipitate, dissolve the precipitate with an appropriate amount of ultrapure water at a ratio of 1 g:10 mL, and dialyze at 4°C for 48 hours. The molecular weight cutoff of the dialysis treatment is 3 kDa. Then, after vacuum freeze-drying, the enoki mushroom extract is obtained and set aside.

[0120] Example 2:

[0121] This embodiment provides a method for preparing a Flammulina velutipes extract, which specifically comprises the following steps:

[0122] (1) 100 g of fresh enoki mushrooms were dried at 70 °C for 0.5 h, and then crushed using a grinder to obtain enoki mushroom powder.

[0123] (2) The above-mentioned Enoki mushroom powder was mixed with distilled water in a mass-to-volume ratio of 1:20, and extracted with hot water at 90°C for 2 hours, and the extraction times were 3 times; the extract was collected and centrifuged at 4000 r / min for 15 minutes, and the supernatant was collected and concentrated under reduced pressure using a rotary evaporator at 60°C to obtain a concentrated extract with a volume of 1 / 10 of the original filtrate, i.e., the hot water extract.

[0124] (3) The hot water extract was deproteinized using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method of deproteinization by the Sevag method is as follows:

[0125] The above hot water extract was mixed with Sevag reagent in a volume ratio of 1:1, placed on a shaker, and shaken at 270 rpm for 20 min at 25°C. Subsequently, centrifuged at 4°C and 3000 r / min for 10 min, and the upper layer solution was removed. The shaking and centrifugation steps were repeated 10 times. The upper layer solution was collected and concentrated under reduced pressure at 60°C to obtain a crude extract solution after deproteinization.

[0126] (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal, and let it stand at 4°C overnight; then centrifuge at 8000 r / min for 10 minutes, collect the precipitate, dissolve the precipitate in ultrapure water at a ratio of 1 g:10 mL, and dialyze at 4°C for 48 hours. The molecular weight cutoff of the dialysis treatment is 3 kDa. Then, after vacuum freeze-drying, the enoki mushroom extract is obtained and set aside.

[0127] Example 3:

[0128] This embodiment provides a method for preparing a Flammulina velutipes extract, which specifically comprises the following steps:

[0129] (1) 100 g of fresh enoki mushrooms were dried at 60 °C for 0.5 h, and then crushed using a grinder to obtain enoki mushroom powder.

[0130] (2) The above-mentioned Enoki mushroom powder was mixed with distilled water in a mass-to-volume ratio of 1:30, and extracted with hot water at 100°C for 4 h, with the extraction times being 3 times; the extract was collected and centrifuged at 8000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation at 60°C under reduced pressure to obtain a concentrated extract with a volume of 1 / 10 of the original filtrate, i.e., the hot water extract.

[0131] (3) The hot water extract was deproteinized using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method of deproteinization by the Sevag method is as follows:

[0132] The hot water extract was mixed with Sevag reagent in a 1:1 volume ratio. The mixture was shaken at 270 rpm for 20 minutes at 25°C. The mixture was then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was removed. The shaking and centrifugation steps were repeated 10 times. The supernatant was collected and concentrated under reduced pressure at 60°C to obtain the deproteinized crude extract.

[0133] (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal, and let it stand at 4°C overnight; then centrifuge at 4000 r / min for 15 minutes, collect the precipitate, dissolve the precipitate in ultrapure water at a ratio of 1 g:10 mL, and dialyze at 4°C for 48 hours. The molecular weight cutoff of the dialysis treatment is 3 kDa. Then, after vacuum freeze-drying, the enoki mushroom extract is obtained and set aside.

[0134] Test Example 1: Determination of sugar content in Flammulina velutipes extract

[0135] This test example measures the sugar content of the Flammulina velutipes extracts in Examples 1 to 3 above. The specific method is as follows:

[0136] The sugar content in the Flammulina velutipes extract was determined using the phenol-sulfuric acid method. A standard curve was drawn with the concentration of glucose in the standard solution as the horizontal axis and the absorbance of the glucose solution at 490 nm as the vertical axis.

[0137] Figure 1 The standard curve obtained by fitting is: y = 3.52598x + 0.0261005, r 2 =0.99956(r 2 ≥0.99). The absorbance of the Flammulina velutipes extract solution prepared in Example 1 was measured using the phenol-sulfuric acid method, and the absorbance was 1.373. Substituting the absorbance into the standard curve equation, the sugar concentration in the Flammulina velutipes extract was 0.382 mg / L. Further calculation showed that the sugar content of the Flammulina velutipes extract could reach 76.4%, indicating that the Flammulina velutipes polysaccharide (FVP) in the Flammulina velutipes extract prepared in Example 1 of the present invention was of high purity and could be used for subsequent research.

[0138] The average absorbance of the Flammulina velutipes extract solution prepared in Example 2 was 1.956, and the sugar content was 67.9%;

[0139] The average absorbance value of the Flammulina velutipes extract solution prepared in Example 3 was 1.989, and the sugar content was 69.8%.

[0140] Test Example 2: Experiment on the Effect of Flammulina velutipes Extract on Cisplatin-Induced Liver Damage

[0141] This test example takes the Flammulina velutipes extract prepared in Example 1 as an example, and tests the effect of the Flammulina velutipes extract on a mouse cisplatin (CDDP) liver damage model, specifically including the following contents.

[0142] 1. Establishment of CDDP liver damage model in mice

[0143] Fifty six-week-old male C57BL / 6J mice were obtained (purchased from the Guangdong Provincial Animal Experimental Center) and housed in an SPF-grade animal room at a temperature of 22-25°C and a relative humidity of 55-70%. The mice were maintained under a regular 12-hour light / 12-hour dark cycle and had free access to water and feed. All animal experiments designed in this study adhered to the guidelines of the national standard "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T35892-2018).

[0144] After one week of adaptive feeding, the mice were randomly divided into five groups (10 mice per group), namely the control group (Control group), the model group (Model group), the low-dose FVP group (L-FVP, 50 mg / kg), the medium-dose FVP group (M-FVP, 100 mg / kg) and the high-dose FVP group (H-FVP, 200 mg / kg), and the dosage was measured with the Flammulina velutipes extract. Among them, the mice in the L-FVP group, the M-FVP group and the H-FVP group were gavaged with the corresponding dose of FVP solution (i.e., the Flammulina velutipes extract prepared in Example 1) every day, while the mice in the Control and Model groups were gavaged with the same amount of sterile double distilled water for 10 consecutive days. On the seventh day of the experiment, the mice in the Model group and the FVP intervention group were intraperitoneally injected with cisplatin (CDDP, 30 mg / kg), and the mice in the Control group were intraperitoneally injected with the same amount of normal saline. After 72 hours, all mice were anesthetized with tribromoethanol (400 mg / kg), blood was collected through the orbital cavity and euthanasia was performed.

[0145] 2. Weight changes and liver index testing

[0146] The weight changes of mice were recorded during the experiment. The mice were killed 10 days after the intervention. The liver tissues were collected, photographed and weighed, and the liver index was calculated. The liver index was calculated as follows:

[0147] Liver index = liver weight (g) / body weight (g).

[0148] The results of the effects of Flammulina velutipes extract on the body weight and liver index of mice are as follows Figure 2 As shown in the figure, A is the average body weight of mice in each group before intervention, B is the average body weight of mice in each group on the 7th day after intervention, C is the average body weight of mice in each group on the 10th day after intervention, D is the average body weight change of mice in each group during the administration period, and E is the liver index of mice in each group.

[0149] The results showed that after one week of adaptive feeding, the body weight of mice was concentrated between 19.69±0.76g (e.g. Figure 2 As shown in A in Figure 2), the trend of weight changes in mice (as shown in Figure 2) Figure 2 The body weights of mice in each group increased during the first 7 days, and there was no statistical difference in the body weights of mice in each group on the 7th day (as shown in D). Figure 2 (As shown in B). However, after intraperitoneal injection of CDDP on day 7, the model group and each FVP-treated group experienced a rapid weight loss, with the model group experiencing a greater weight loss than the FVP-treated group. The weight change trend during the intervention period suggests that FVP can alleviate the weight loss caused by CDDP. After 10 days of intervention, the model group had a significant weight loss compared to the control group (P < 0.001, as shown in B). Figure 2 As shown in Figure C, FVP intervention can increase the body weight of mice to a certain extent, with statistically significant differences in body weight in the L-FVP group, with the most significant increase (P < 0.05). These results indicate that FVP intervention significantly improves the body weight loss caused by CDDP in mice.

[0150] The statistical results of liver index of mice in each group are as follows Figure 2 As shown in Figure E, the liver index of the Model group mice was significantly increased compared with the Control group mice (P<0.01). Compared with the Model group mice, the liver index of the FVP intervention groups was significantly decreased (P<0.05), and the decrease was most significant in the L-FVP group, indicating the most obvious intervention effect.

[0151] The above results indicate that the FVP-rich Flammulina velutipes extract of the present invention has a significant improvement effect on weight loss caused by CDDP and helps maintain the health of the liver.

[0152] 3. Plasma liver function index detection

[0153] After 10 days of treatment, mice were euthanized and blood was collected from each group. The blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 10 minutes. The supernatant was extracted and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a Chemray 240 automatic analyzer.

[0154] The results of the effects of Enoki mushroom extract on liver function indicators in mice are as follows Figure 3As shown in the results, the plasma ALT and AST levels of mice in the Model group were significantly higher than those in the Control group (P<0.05). FVP intervention can reduce the serum ALT and AST levels of mice, among which L-FVP intervention has the best effect (P<0.05), and the ALT and AST levels of mice in the L-FVP group are close to those in the Control group.

[0155] The above results indicate that the Flammulina velutipes extract of the present invention has the effect of improving the liver function impairment in mice caused by CDDP.

[0156] 4. Liver histopathological examination and Suzuki score

[0157] Liver tissues of mice in each group were collected, fixed in paraformaldehyde (4%), embedded in conventional paraffin, sliced, and then stained with hematoxylin-eosin (H&E). Changes were observed under a microscope.

[0158] The results of the effects of Flammulina velutipes extract on the morphology and pathology of mouse liver are as follows Figure 4 As shown, the livers of mice in the model group were significantly larger and more congested than those in the control group. Their glossiness decreased, their edges darkened, their surfaces were rough, and they were covered with white spots, indicating localized necrosis and hardening of the livers in the model group. Compared with the model group, the livers of mice in the FVP intervention group showed reduced swelling and congestion, a translucent red appearance, a smooth and shiny surface without spots or nodules, transparent edges, and a soft texture, indicating that the liver morphology of the mice recovered after FVP intervention.

[0159] Furthermore, compared with the control group, H&E sections of the liver tissue of mice treated with CDDP showed obvious pathological damage, with hepatocytes swollen and necrotic, disordered hepatic cords, and congested hepatic sinusoids. After FVP intervention, the arrangement of hepatocytes in mice was restored, the congestion of hepatic sinusoids disappeared, and no obvious hepatocyte necrosis was found. Among them, L-FVP intervention had the best effect, and the Suzuki score of the mouse liver was as follows: Figure 5 shown.

[0160] The above results indicate that the intervention with the FVP-rich Flammulina velutipes extract of the present invention can effectively alleviate CDDP-induced hepatocyte swelling and necrosis.

[0161] 5. Oxidative stress index detection

[0162] Accurately weigh 50 mg of mouse liver tissue from each group and add 9 volumes of ice-cold physiological saline at a ratio of 1:9. A 10% tissue homogenate was prepared using a cryo-grinding apparatus. The homogenate was then centrifuged at 2500 rpm and 4°C for 10 minutes. The supernatant was collected and assayed for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels using the corresponding assay kits.

[0163] Oxidative stress index test results such as Figure 6 As shown in the results, compared with the control group, the levels of SOD and GSH-Px in the liver tissue of mice in the Model group were significantly reduced, and the level of MDA was significantly increased, and the differences were statistically significant (P<0.001). FVP intervention can significantly increase the levels of SOD and GSH-Px in the liver tissue of mice and reduce the content of MDA (P<0.05).

[0164] The above results indicate that the FVP-rich Flammulina velutipes extract of the present invention can alleviate the oxidative stress level in the liver of mice, thereby reducing liver oxidative damage.

[0165] Test Example 3: Effect of Flammulina velutipes extract on intestinal flora in a cisplatin-induced liver injury model

[0166] This test case examined the effect of Enoki mushroom extract on the intestinal flora in a cisplatin-induced liver injury model. The construction and intervention methods of the cisplatin-induced liver injury model were referenced to the above-mentioned test case 2, specifically including the following contents.

[0167] 1. Experimental methods

[0168] In this case, the 16s rRNA sequencing of mouse intestinal flora was commissioned by Guangzhou Kidio Biotechnology Co., Ltd. The specific procedures included the following:

[0169] First, physical disruption and chemical treatment were used to extract microbial DNA from fecal samples of each group of mice, and then the DNA was purified by centrifugation and washing. Specific primers were then designed for the V3-V4 target region of the 16s rRNA gene of the intestinal flora for PCR amplification. The primers contained barcodes to distinguish different samples during sequencing. The primer sequences were 341F (CCTACGGGNGGCWGCAG, SEQ ID NO.1) and 806R (GGACTACHVGGGTWTCTAAT, SEQ ID NO.2). Where N is unknown, W is A or T, H is A or C, and V is A or G.

[0170] PCR product quality was assessed using 2% agarose gel electrophoresis and purified and quantified using AMPure XP Beads and Qubit 3.0, respectively. Sequencing libraries were constructed using the Illumina DNA Prep Kit and quality tested using the ABI StepOnePlus Real-Time PCR System. Sequencing was then performed using the Novasek 6000 platform in PE250 mode. Following sequencing, raw sequencing data were processed using bioinformatics tools, including quality control (removal of low-quality reads and adapter sequences), read assembly, OTU clustering, species annotation, and diversity and abundance analysis.

[0171] 2. Experimental results

[0172] (1) Detection results of the impact on species composition of mouse intestinal flora

[0173] The results of Shannon and Simpson dilution curves of mouse intestinal flora samples at the OTU level are as follows: Figure 7 As shown, the ends of the dilution curves of the Shannon index and Simpson index of the intestinal flora of each group of mice tend to be flat, indicating that the sequencing depth is qualified and can cover the diversity information of most of the flora in the sample.

[0174] Furthermore, the beta diversity analysis of the mouse intestinal flora was performed, and the partial least squares discriminant analysis (PLS-DA) method based on Bray-Curtis distance was used for visualization and mapping to examine the distribution of intestinal flora samples in each group of mice. Figure 8 As shown in Figure 2, there are clear boundaries between the Control group, Model group, and LFVP group, indicating that there are differences in the intestinal flora composition of the three groups of mice (e.g. Figure 8 Compared with the Model group, the L-FVP group was closer to the Control group, indicating that the intestinal flora composition of the L-FVP group was closer to that of the Control group. Figure 8 Figure B is the result of Anosim analysis, where P = 0.013 < 0.05, R = 0.4028. Therefore, it can be considered that there are significant differences in the intestinal microbiota of mice in the Control group, Model group and LFVP group. This result further verifies that CDDP treatment and low-dose FVP intervention can change the intestinal flora of mice.

[0175] The above results indicate that FVP can change the intestinal flora of mice in the CDDP-induced liver injury model.

[0176] (2) Effects on the species composition of mouse intestinal flora

[0177] Effects of Flammulina velutipes extract on species composition of intestinal flora at the order level in mice Figure 9 As shown in the figure, at the order level, the intestinal flora with relatively high abundance include Enterobacterales, Bacteroidales, Staphylococcales, Lactobacillales, Verrucomicrobiales, Saccharimonadales, Oscillospirales, Lachnospirales, Clostridia_UCG-014 and Erysipelotrichales (e.g. Figure 9 Compared with the Control group, the relative abundance of Peptostreptococcales-Tissierellale and Clostridium-UCG-014 in the intestinal flora of the Model group mice treated with CDDP increased (P<0.01), while FVP intervention reduced the relative abundance of Peptostreptococcales-Tissierellale and Clostridium-UCG-014 (P<0.01). Figure 9 (as shown in B in the figure).

[0178] Furthermore, the species composition of the mouse intestinal flora at the genus level was analyzed, and the results were as follows: Figure 10 As shown in Figure 2, at the genus level, the top ten intestinal bacterial groups with the highest relative abundance include Escherichia-Shigella, Proteus, Enterococcus, Lactobacillus, Staphylococcus, Akkermansia, Candidatus_Saccharimonas, Enterobacter, Alloprevotella, and Bacteroides (e.g., Figure 10 Compared with the control group, CDDP treatment increased the relative abundance of Escherichia-Shigella in the intestinal flora of mice (P<0.001), while FVP intervention reduced the relative abundance of Escherichia-Shigella (as shown in Figure 2A). Figure 10 (as shown in B in the figure).

[0179] The above results indicate that FVP helps restore the intestinal flora of CDDP-treated Model group mice to normal relative abundance.

[0180] (3) Effects on the dominant flora in the intestinal flora of mice

[0181] The LEfSe method was used to screen out bacterial communities with linear discriminant analysis values ​​(LDA) greater than 3 (LDA>3) and detect the specific dominant bacterial communities in each group. The larger the LDA value, the more significant the difference between the microbial colonies. Figure 11 As shown in the figure, 9, 3, and 2 dominant bacterial groups were screened out in the control group, model group, and LFVP group, respectively. Among them, Lachnospiraceae, A2, and Lachnospiraceae_NK4A136_group were the main dominant bacterial groups in the control group, Enterobacteriaceae and Escherichia-Shigella were the main dominant bacterial groups in the model group, and Vampirivibrionia and Gastran-erophilales were mainly enriched in the LFVP group.

[0182] The above results indicate that the FVP-rich Flammulina velutipes extract of the present invention can improve the composition of intestinal microorganisms and promote the colonization of beneficial bacteria while reducing the abundance of harmful bacteria.

[0183] Test Example 4: Effect on Mouse Liver Metabolism

[0184] This test case examined the effect of Flammulina velutipes extract on liver metabolism in mice with a cisplatin-induced liver injury model. The construction and intervention methods of the cisplatin-induced liver injury model were based on the above-mentioned test case 2, specifically including the following contents.

[0185] 1. Experimental methods

[0186] This study investigated the regulatory effects of FVP on mouse liver metabolism. Non-targeted metabolomics sequencing of mouse liver tissue was commissioned by Guangzhou Kidio Biotechnology Co., Ltd. The specific methods are as follows:

[0187] 80 mg of liver samples from each treatment group were collected and ground using a tissue grinder with 200 μL of water and five grinding beads. The homogenate was then added with 800 μL of a methanol / acetonitrile mixture (1:1, v / v) and centrifuged at 14,000 g at 4°C for 15 min. The supernatant was collected and concentrated to dryness. Prior to analysis, the dried samples were reconstituted in 100 μL of an acetonitrile / water solution (1:1, v / v). Metabolomics analysis was performed on an Agilent 1290 Infinity liquid chromatography (LC) system and an AB SCIEX Triple TOF 6600 triple quadrupole high-resolution time-of-flight mass spectrometer (MS). Samples were analyzed using an ACQUIY UPLC BEH column (2.1 mm × 100 mm, 1.7 μm). The LC / MS raw data were converted into mzXML files using Proteo Wizard MSconventer software (version 3.0.8789), and then the data were processed using XCMS online software.

[0188] 2. Experimental results

[0189] (1) Metabolomics data quality control

[0190] First, quality control (QC) samples were used to assess data quality to ensure the stability and reproducibility of the metabolomics method. Figure 12 As shown, the Pearson correlation coefficient between the QC samples is close to 1, indicating that the instrument is in good condition during the testing process, the detection method is highly stable, and the obtained data is reliable. Next, principal component analysis (PCA) was performed on the obtained QC sample data matrix to further monitor system stability. The results showed that the QC samples were well clustered and densely distributed, confirming that the sample analysis process was good.

[0191] (2) Effects on metabolites and metabolic pathways in mouse liver tissue

[0192] PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to determine the metabolite differences between and within groups. The PCA and OPLS-DA analysis results of mouse liver metabolites are shown in Figure 2. Figure 13 As shown, the three groups of samples can be clearly separated, indicating that there are differences in metabolites among the groups.

[0193] Furthermore, differential metabolites were screened based on the threshold VIP>1.0, P<0.05, and the results were as follows Figure 14As shown in the figure, 95, 111, and 112 differential metabolites were screened between the Control group and the Model group, between the Model group and the LFVP group, and between the Control group and the LFVP group, respectively. Furthermore, 20 potential biomarkers were screened using a Venn diagram, including 2-methoxy-5-nitrophenol, 3,7-dimethyluric acid, γ-aminobutyric acid, 4,4-dihydroxydiphenyl sulfone, 1,2,3,4-tetrahydro-7-nitro-2,3-dioxo-6-quinoxalinecarbonitrile, 7-methyluric acid, D-alanine, D-aspartic acid, D-ornithine, D-proline, DL-glutamic acid, DL-phenylalanine, DL-serine, L-threonine, DL-tyrosine, glutamic acid, glycine, humulone, indole, and propyl gallate.

[0194] The detailed information of the above 20 potential biomarkers is shown in Table 1.

[0195] Table 1:

[0196]

[0197] The results showed that the levels of metabolites such as γ-aminobutyric acid, glutamic acid, glycine, indole, and propyl gallate decreased in the Model group and increased in the LFVP group.

[0198] The screened differential metabolites were further compared with the KEGG database and enrichment pathway analysis was performed. The results showed that drug-induced liver injury (DILI) caused by CDDP significantly changed 25 metabolic pathways (such as Figure 15 The results showed that the changes in the metabolism pathways of DILI mice were significant (P<0.05), including biosynthesis of amino acids, central carbon metabolism in cancer, cyanoamino acid metabolism, protein digestion and absorption, and phenylalanine metabolism. FVP intervention caused significant changes in the five metabolic pathways in the liver tissue of DILI mice (such as Figure 16 The differential metabolites in these metabolic pathways were 9, 4, 3, 3 and 3, respectively, as shown in Table 2 .

[0199] Table 2:

[0200]

[0201] Two of the aforementioned differential metabolites were found in the ABC transporter pathway: DL-threonine and glycine, accounting for 22.2% (2 / 9). The cyanoamino acid metabolic pathway contained four metabolites: DL-glutamate, DL-tyrosine, glutamate, and glycine, accounting for 100% (4 / 4). The thiamine metabolic pathway contained two metabolites: DL-tyrosine and glycine, accounting for 66.7% (2 / 3). The phenylalanine, tyrosine, and tryptophan biosynthesis pathways contained two metabolites: DL-tyrosine and indole, accounting for 66.7% (2 / 3). The aminoacyl-tRNA biosynthesis pathway contained three metabolites: DL-threonine, DL-tyrosine, and glycine, accounting for 100% (3 / 3). Furthermore, glycine was enriched in all four of the aforementioned metabolic pathways.

[0202] These results indicate that FVP can improve CDDP-induced liver injury by regulating the levels and metabolic pathways of the above-mentioned differential metabolites.

[0203] (3) Correlation analysis between mouse liver metabolic profile and intestinal flora

[0204] The Pearson correlation analysis method was used to perform correlation analysis between the screened intestinal flora biomarkers and liver differential metabolites, and the correlation heat map was used to display them.

[0205] Among them, the heat map of the correlation between mouse intestinal flora and liver metabolites at the level of the subject is as follows Figure 17 As shown, at the mesh level, Clostridium-UCG-014 was negatively correlated with glycine, 2-methoxy-5-nitrophenol, 3,7-dimethyluric acid, 4,4-dihydroxydiphenyl sulfone, propyl gallate, 1,2,3,4-tetrahydro-7-nitro-2,3-dioxo-6-quinoxalinecarbonitrile, 7-methyluric acid, D-alanine, D-aspartic acid, D-ornithine, D-proline, DL-glutamic acid, DL-phenylalanine, DL-serine, and DL-tyrosine, but positively correlated with DL-threonine and indole. Peptostreptococcus-Tittlesella was negatively correlated with glycine, 2-methoxy-5-nitrophenol, 3,7-dimethyluric acid, 4,4-dihydroxydiphenyl sulfone, 7-methyluric acid, D-aspartic acid, D-ornithine, D-proline, DL-glutamic acid, DL-phenylalanine, and propyl gallate, and positively correlated with DL-threonine. Furthermore, Oscillospirales was positively correlated with DL-threonine.

[0206] The heat map of the correlation between mouse intestinal flora and liver metabolites at the genus level is shown in Figure 2. Figure 18As shown, at the genus level, Isoprevotella was positively correlated with DL-serine. Intestinimonas was positively correlated with glutamate and DL-serine. Rikenellaceae_RC9_gut_group was positively correlated with DL-threonine. Lachnoclostridium was positively correlated with D-proline, glutamate, 2-methoxy-5-nitrophenol, and DL-phenylalanine. Coriobacteriaceae_UCG-002 was positively correlated with D-proline, glutamate, 2-methoxy-5-nitrophenol, 4,4-dihydroxydiphenyl sulfone, and glycine. Eubacterium_brachy_group was negatively correlated with D-aspartic acid, 7-methyluric acid, and propyl gallate, and positively correlated with DL-threonine. Lachnospiraceae_UCG_006 was positively correlated with 1,2,3,4-tetrahydro-7-nitro-2,3-dioxo-6-quinoxalinecarbonitrile. Faecalibaculum was positively correlated with glutamate. UBA1819 was positively correlated with DL-serine, D-ornithine, glutamate, DL-phenylalanine, 2-methoxy-5-nitrophenol, D-alanine, and 1,2,3,4-tetrahydro-7-nitro-2,3-dioxo-6-quinoxalinecarbonitrile. Therefore, the gut microbiota may ameliorate CDDP-induced DILI by affecting hepatic metabolism.

[0207] The above results indicate that the FVP-rich Flammulina velutipes extract of the present invention can improve drug-induced liver injury caused by CDDP by regulating key metabolic pathways in the liver.

[0208] In summary, the present invention provides the use of Flammulina velutipes extract in the preparation of products related to the alleviation and / or treatment of drug-induced liver injury. The present invention successfully established a DILI mouse model using CDDP. Further studies found that Flammulina velutipes extract has a liver-protective effect on DILI mice, can alleviate liver pathological damage, significantly reduce the levels of serum liver function indicators ALT and AST, while increasing the activity of SOD and GSH-Px in liver tissue, and downregulating MDA concentration, showing antioxidant properties. In addition, the present invention found that Flammulina velutipes extract can improve the composition of intestinal microorganisms, promote the colonization of beneficial bacteria, reduce the abundance of harmful bacteria, and regulate key metabolic pathways in the liver. Therefore, when treated with the chemotherapy drug CDDP, the use of Flammulina velutipes extract can become an effective strategy for the prevention and treatment of DILI.

[0209] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of Flammulina velutipes extract in the preparation of products for alleviating and / or treating drug-induced liver injury.

2. The use according to claim 1, characterized in that The sugar content in the Flammulina velutipes extract is ≥70%.

3. The use according to claim 1, characterized in that The preparation method of the Flammulina velutipes extract comprises: The powder of Flammulina velutipes is mixed with water, and the mixture is subjected to hot water extraction, protein removal and ethanol precipitation treatment to obtain the product.

4. The use according to claim 3, characterized in that The volume ratio of the enoki mushroom powder to water is 1:20-40; and / or the temperature of the hot water extraction is 90-110° C.; and / or the time of the hot water extraction is 1-4 hours; and / or the number of hot water extractions is 1-4 times.

5. The use according to claim 3, characterized in that The protein removal treatment comprises mixing the product after hot water extraction with a protein removal agent, reacting, and concentrating after centrifugation; Preferably, the deproteinizing agent comprises chloroform and / or n-butanol; Preferably, the centrifugal speed is 3000-5000 r / min; Preferably, the centrifugation time is 8-20 min; Preferably, the deproteinization treatment is repeated 6-20 times.

6. The use according to claim 5, characterized in that The ethanol precipitation treatment comprises: mixing the product after the protein removal treatment with an ethanol solution, collecting the precipitate after centrifugation, and drying the precipitate.

7. The use according to claim 6, characterized in that The ethanol precipitation treatment further includes dialysis treatment.

8. The use according to claim 1, characterized in that The efficacy of the product for alleviating and / or treating drug-induced liver injury includes at least one of the following: A) Improve the symptoms of weight loss caused by drug-induced liver injury; B) reduce serum alanine aminotransferase and aspartate aminotransferase levels in patients with drug-induced liver injury; C) Relieve symptoms of liver swelling, congestion and / or necrosis in patients with drug-induced liver injury; D) Alleviate the liver oxidative stress level and reduce liver oxidative damage in patients with drug-induced liver injury; E) Improve the intestinal microbial composition of patients with drug-induced liver injury and promote the colonization of beneficial bacteria; F) Improving liver injury in patients with drug-induced liver injury by regulating any one of the following pathways: ABC transporters, cyanoamino acid metabolism, thiamine metabolism, phenylalanine / tyrosine / tryptophan biosynthesis, and aminoacyl-tRNA biosynthesis.

9. The use according to any one of claims 1 to 8, characterized in that The drug-induced liver injury includes cisplatin-induced liver injury.

10. A drug for treating drug-induced liver injury, characterized in that: The active ingredient comprises the Flammulina velutipes extract according to any one of claims 1 to 9.

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