Agaricus blazei murill extract and application thereof in treating intestinal mucositis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an agaricus blazei murill extract and application thereof in treating intestinal mucositis. The invention provides application of an agaricus blazei murill extract FA-2-b-beta in preparation of a medicine for preventing, treating and / or relieving intestinal mucositis. The FA-2-b-beta can obviously improve 5-FU induced intestinal mucositis, obviously promote human intestinal epithelial cell proliferation, recover intestinal barriers and resist toxic and side effects of chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an extract of Agaricus blazei Murill and its use in treating intestinal mucositis, and more particularly to the use of the Agaricus blazei Murill extract FA-2-b-β in the prevention and / or treatment of 5-FU-induced intestinal mucositis. Background Art

[0002] 5-Fluorouracil (5-FU), as an anti-cancer drug, can be used to treat different types of cancers, such as colon cancer, pancreatic cancer, and breast cancer. It is one of the most commonly used antimetabolic chemotherapy drugs in recent decades. 5-FU not only inhibits the growth of cancer cells but also has a non-selective effect on normal cells in the bone marrow and digestive tract. The FDA states that 5-FU has a variety of serious side effects, and the most common one is mucositis. It is reported that 20%-50% of patients receiving 5-FU treatment suffer from chemotherapy-induced intestinal mucositis (CIM). The specific manifestations are nausea, fatigue, abdominal pain, and diarrhea, which are the main reasons for the decline in the quality of life, reduced survival rate, and early death of some chemotherapy patients. Clinically, the treatment of CIM mainly focuses on symptomatic and supportive treatment, and the treatment effect is poor. Therefore, there is an urgent need to find effective drugs for preventing and treating CIM induced by chemotherapy drugs such as 5-FU.

[0003] Kangzhen selenium-rich Agaricus blazei Murill, also known as Qinba selenium mushroom, Agaricus blazei Murill, Brazilian mushroom, and small Tricholoma matsutake, is a saprophytic fungus that grows in summer and autumn. Due to its low survival rate and short production season, it belongs to precious edible fungi and is known as "the true jade among mushrooms". Kangzhen selenium-rich Agaricus blazei Murill is a rare fungus that can be used both as medicine and food, rich in various nutrients such as polysaccharides, proteins, amino acids, vitamins, sterol compounds, and polyphenol compounds. At the same time, Kangzhen selenium-rich Agaricus blazei Murill is rich in selenium and thus also plays a key role in antioxidant defense. Selenium is an essential component of selenium-dependent enzymes such as GPX4. Kangzhen selenium-rich Agaricus blazei Murill may enhance the activity of GPX4, thereby helping to reduce the accumulation of lipid peroxides and inhibit ferroptosis.

[0004] Currently, there is no study on using an extract of Agaricus blazei Murill / Qinba selenium mushroom or its main components to treat 5-FU-induced intestinal mucositis. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in existing therapeutic drugs. Through a large number of repeated experimental tests, the Agaricus blazei Murill extract FA-2-b-β has a protective effect on 5-FU-induced intestinal mucositis.

[0006] To achieve the above technical purpose, the present invention proposes the following technical solutions:

[0007] In one aspect, the present invention provides the use of a plant extract in the preparation of a medicament for preventing, treating and / or alleviating intestinal mucositis, wherein the plant extract contains FA-2-b-β.

[0008] In some embodiments, the plant extract includes an extract of Agaricus blazei Murill.

[0009] In some preferred embodiments, the plant extract is a Kangzhen selenium-enriched Agaricus blazei Murill extract.

[0010] In some embodiments, the intestinal mucositis is chemotherapy-induced intestinal mucositis (CIM).

[0011] In some preferred embodiments, the intestinal mucositis is chemotherapy-induced intestinal mucositis caused by 5-fluorouracil (5-FU).

[0012] In some embodiments, the total selenium content of the plant extract is 80 mg / kg or more, 85 mg / kg or more, 90 mg / kg or more, or 95 mg / kg or more.

[0013] In some preferred embodiments, the total selenium content of the plant extract can be 80 mg / kg, 81 mg / kg, 82 mg / kg, 83 mg / kg, 84 mg / kg, 85 mg / kg, 86 mg / kg, 87 mg / kg, 88 mg / kg, 89 mg / kg, 90 mg / kg, 91 mg / kg, 92 mg / kg, 93 mg / kg, 94 mg / kg, 95 mg / kg, 96 mg / kg, 97 mg / kg, 98 mg / kg, 99 mg / kg. Wherein mg is the mass of total selenium and kg is the mass of the fruiting body of the plant.

[0014] In some embodiments, the percentage of the organic selenium content in the total selenium content of the plant extract is 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more.

[0015] In some preferred embodiments, the percentage of the organic selenium content in the total selenium content of the plant extract is 99% or more.

[0016] In some more preferred embodiments, the percentage of the organic selenium content in the plant extract in the total selenium content can be 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%.

[0017] In some embodiments, the plant extract contains 90 mg / kg or more, 95 mg / kg or more, 100 mg / kg or more, 110 mg / kg or more, 120 mg / kg or more, 130 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, 200 mg / kg or more, 250 mg / kg or more, or 300 mg / kg or more of FA-2-b-β. Where mg is the mass of FA-2-b-β and kg is the mass of the fruiting body of the plant.

[0018] In some embodiments, the FA-2-b-β prevents, treats, and / or alleviates intestinal mucositis by at least one of the following methods:

[0019] (1) Promote the proliferation of human intestinal epithelial cells;

[0020] (2) Restore the intestinal barrier;

[0021] (3) Inhibit the ferroptosis of human intestinal epithelial cells;

[0022] (4) Increase the mRNA expression level of Occludin.

[0023] In yet another aspect, the present invention provides a pharmaceutical composition comprising a plant extract as a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the plant extract contains FA-2-b-β.

[0024] In some embodiments, the plant extract includes an Agaricus blazei extract.

[0025] In some preferred embodiments, the plant extract is a Kangzhen selenium-rich Agaricus blazei extract.

[0026] In some embodiments, the total selenium content of the plant extract is 80 mg / kg or more, 85 mg / kg or more, 90 mg / kg or more, or 95 mg / kg or more.

[0027] In some preferred embodiments, the total selenium content of the plant extract can be 80 mg / kg, 81 mg / kg, 82 mg / kg, 83 mg / kg, 84 mg / kg, 85 mg / kg, 86 mg / kg, 87 mg / kg, 88 mg / kg, 89 mg / kg, 90 mg / kg, 91 mg / kg, 92 mg / kg, 93 mg / kg, 94 mg / kg, 95 mg / kg, 96 mg / kg, 97 mg / kg, 98 mg / kg, 99 mg / kg. Wherein mg is the mass of total selenium and kg is the mass of the fruiting body of the plant.

[0028] In some embodiments, the percentage of the organic selenium content in the total selenium content of the plant extract is more than 80%, more than 85%, more than 90%, more than 95% or more than 99%.

[0029] In some preferred embodiments, the percentage of the organic selenium content in the total selenium content of the plant extract is more than 99%.

[0030] In some more preferred embodiments, the percentage of the organic selenium content in the total selenium content of the plant extract can be 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%.

[0031] In some embodiments, the plant extract contains more than 90 mg / kg, more than 95 mg / kg, more than 100 mg / kg, more than 110 mg / kg, more than 120 mg / kg, more than 130 mg / kg, more than 140 mg / kg, more than 150 mg / kg, more than 160 mg / kg, more than 170 mg / kg, more than 180 mg / kg, more than 190 mg / kg, more than 200 mg / kg, more than 250 mg / kg or more than 300 mg / kg of FA-2-b-β. Wherein mg is the mass of FA-2-b-β and kg is the mass of the fruiting body of the plant.

[0032] In some embodiments, the pharmaceutical composition is used for preventing, treating and / or relieving enteritis.

[0033] In some preferred embodiments, the pharmaceutical composition is used for preventing, treating and / or relieving chemotherapy-induced enteritis.

[0034] In some preferred embodiments, the pharmaceutical composition is used for preventing, treating and / or relieving chemotherapy-induced enteritis caused by 5-fluorouracil.

[0035] In some preferred embodiments, the pharmaceutical composition is used to promote the proliferation of human intestinal epithelial cells.

[0036] In some preferred embodiments, the pharmaceutical composition is used to restore the intestinal barrier.

[0037] In some preferred embodiments, the pharmaceutical composition is used for ferroptosis of human intestinal epithelial cells.

[0038] In some embodiments, the pharmaceutically active ingredient further comprises a second pharmaceutically active ingredient for preventing, treating, and / or alleviating chemotherapy-induced enteritis.

[0039] In the present invention, unless otherwise specified, the dosage form of the drug / drug composition is not particularly limited and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dripping pills, sprays, inhalants, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the administration method.

[0040] In the present invention, unless otherwise specified, the administration method of the drug or drug composition is not particularly limited and can be by subcutaneous injection, intravenous injection, oral administration, topical application, respiratory inhalation, topical application, sublingual administration, etc. The doctor can select the administration method according to the actual clinical needs.

[0041] In yet another aspect, the present invention provides the use of any of the foregoing pharmaceutical compositions in the preparation of a drug for preventing, treating, and / or alleviating enteritis.

[0042] In some embodiments, the enteritis is chemotherapy-induced enteritis.

[0043] In some preferred embodiments, the enteritis is chemotherapy-induced enteritis caused by 5-fluorouracil (5-FU).

[0044] Based on the activity characteristics of the RNA-protein complex FA-2-b-β (selenoamino acid protein) of Agaricus blazei Murill extract, the present invention screened out the best concentration and other analysis conditions through a large number of experiments, and studied that selenoamino acid protein has preventive and therapeutic effects on 5-FU-induced enteritis. The present invention mainly uses HE section, Western blot and other detections, and its method has high detection sensitivity and good stability, and can objectively, comprehensively and accurately evaluate the preventive and therapeutic effects of selenoamino acid protein on 5-FU-induced enteritis. Selenoamino acid protein FA-2-b-β can significantly improve 5-FU-induced enteritis, significantly promote the proliferation of human intestinal epithelial cells, restore the intestinal barrier, and resist the toxic and side effects of chemotherapy. Description of the Drawings

[0045] Figure 1The HE staining results after treatment with FA-2-b-β in different experimental groups are shown.

[0046] Figure 2 The changes in body weight after treatment with FA-2-b-β in different experimental groups are shown.

[0047] Figure 3 The statistical chart of the mRNA expression of Occludin detected by PCR after treatment with FA-2-b-β in different experimental groups is shown.

[0048] Figure 4 The bar chart showing the safe concentration of selenium amino acid protein FA-2-b-β for HIEC screened by in vitro experiments is shown.

[0049] Figure 5 and Figure 6 The WB band diagram and bar chart showing the upregulation effects of selenium amino acid protein FA-2-b-β on GPX4 and Nrf2 in HIEC are shown respectively. Detailed implementation manners

[0050] Unless otherwise defined, all technical terms and scientific and technical terms used in the present invention have the same meanings as those commonly used in the field to which the present invention belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0051] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, referring to "a cell" includes multiple such cells and equivalents known to those skilled in the art, and so on.

[0052] The term "about" used herein means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0053] The numerical ranges used herein should be understood to have enumerated all the numbers within that range. For example, the range of 1 to 20 should be understood to include any number, combination of numbers, or sub-range from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0054] As used herein, the term "comprises" or "comprising" means "including but not limited to". This term is intended to be open-ended, specifying the presence of any stated feature, element, integer, step or component, but not precluding the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of".

[0055] As used herein, the terms "optionally", "either", "any" or "any one" mean that the subsequent described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur. As used in the present invention, "a" and "an" are used in the present invention to refer to one or more than one grammatical object.

[0056] As used herein, the term "and / or" should be understood to mean any one of the alternatives or any combination of any two or more of the alternatives.

[0057] As used herein, "FA-2-b-β", "RNA-protein complex of Agaricus blazei Murill extract" or "selenium amino acid protein" can be used interchangeably, and all belong to the water-soluble active ingredient acidic RNA-protein complex of Agaricus blazei Murill polysaccharide. The nucleic acid has a molecular weight of 10,000, and the base components include A, Ap, Gp, Cp, Up and other modified bases (SUN Y Q, GUO T K, XI Y M, et al. World journal of gastroenterology, 2007, 13(31): 4185-4191.). The protein part consists of polysaccharide:protein = 40:60 w / w, and its sugar chain contains glucose, xylose, mannose, galactose, fucose, etc. (ZHANG J, WANG G, LI H, et al. Bioscience, biotechnology, and biochemistry, 1994, 58(7): 1195-1201.).

[0058] As used herein, the term "pharmaceutical composition" generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered. The composition is sterile.

[0059] As used herein, the term "pharmaceutically acceptable" means that these structures, materials, compositions and / or dosage forms are suitable for contact with human and animal tissues within the scope of reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, and without excessive toxicity, irritation, allergic response or other problems or complications.

[0060] The term "pharmaceutically acceptable excipient" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining reagents, absorption delaying reagents, preservatives. For example, pH regulators include but are not limited to phosphate buffer solutions. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining reagents include but are not limited to sugars, NaCl and its analogs. Absorption delaying reagents include but are not limited to monostearates and gelatin. Diluents include but are not limited to water, aqueous buffer solutions (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and can stabilize the desired activity of the active ingredient in the drug, and include but are not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.

[0061] As used herein, terms such as "treatment", "prevention", etc. refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects can be preventive in terms of completely or partially preventing a disease or its symptoms, and / or can be therapeutic in terms of affecting the partial or complete cure of a disease and / or a disease symptom. As used in this application, "treatment" can include treating a disease or disorder (such as cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease or disease symptoms in a subject who is susceptible to the disease but has not been diagnosed as having the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing the regression of the disease. Treatment can refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making the disease disorder more tolerable to the patient; slowing the rate of deterioration or decline; or reducing the debilitating end point of deterioration. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes the administration of the antibodies or compositions or conjugates disclosed in this application to prevent or delay, alleviate or arrest or inhibit the development of symptoms or disorders associated with a disease (such as cancer). The term "treatment effect" refers to a reduction, elimination or prevention of a disease, a disease symptom or a disease side effect in a subject.

[0062] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to embodiments. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicated with the manufacturer are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning: A Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Press), Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0063] The Agaricus blazei Murill extract FA-2-b-β (selenium amino acid protein) of the present invention is obtained by the following method:

[0064] Provided by Kangzhen Biotechnology (Beijing) Co., Ltd., its ingredients and quality evaluation were detected by Beijing Qingxi Technology Research Institute and identified by PONY Testing. The total selenium content of this extract is 87 mg / kg, and the organic selenium is 86.8 mg / kg. The specific steps are as follows: The obtained Agaricus blazei extract was dried in a drying oven at 60 °C for 72 h and stored. When in use, the Kangzhen selenium-rich Agaricus blazei extract was added to a 70% methanol solution as the extraction reagent, and the supernatant of the Agaricus blazei extract solution obtained by centrifugation was used as the mother liquor (pH = 7.4, osmotic pressure = 300 mmol / L), which was stored in a -20 °C refrigerator and diluted to the corresponding experimental concentration with cell culture medium before use. The method for obtaining the Kangzhen selenium-rich Agaricus blazei can be referred to the inventor's prior application CN117581745A; the preparation method of the Agaricus blazei extract is as follows (only for exemplary description, other conventional extraction methods in the art can also be used in the present invention):

[0065]

[0066]

[0067] Some information related to the reagents involved in the present invention is as follows:

[0068] The HIEC cell line and its special culture medium were purchased from Gansu Weberxin Biotechnology Co., Ltd.: Fetal bovine serum (FBS) was purchased from Gansu Weberxin Biotechnology Co., Ltd.; C57 / BL6 mice were purchased from Spif (Beijing) Biotechnology Co., Ltd.; RIPA lysis buffer, SDS-PAGE gel preparation kit, antibody diluent, loading, electrophoresis, transfer membrane and rinsing buffer were purchased from Wuhan Sevier Biotechnology Co., Ltd.; GPX4 was purchased from Proteintech; The microplate reader was purchased from BioTek; The gel imaging analyzer was purchased from Bio-Rad.

[0069] Example 1: Selenium amino acid protein FA-2-b-β alleviates 5-FU-induced intestinal mucositis in mice

[0070] Construction of the intestinal mucositis mouse model:

[0071] The Animal Experiment Center of Gansu University of Chinese Medicine followed all the protocols for the ethical treatment of animals used in the study established by the Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health (NIH). Male C57BL / 6 mice from SPF Biotechnology Co., Ltd., from Beijing were housed in a controlled environment with a temperature range of 23 ± 2 degrees Celsius, a humidity level between 50% and 70%, and a 12-hour light-dark cycle, with the lights turned on at 8:00 in the morning. There were six groups of mice, six in each group.

[0072] The following doses of FA-2-b-β were used: 1× dose for the normal group, 100 mg / kg for the FA-2-b-β low-dose group and the preventive low-dose group, and 200 mg / kg for the FA-2-b-β high-dose group and the preventive high-dose group. Among them, the preventive administration groups (the preventive low-dose group and the preventive high-dose group) were administered three days before modeling until the end of the experiment, and the FA-2-b-β low-dose group and high-dose group were administered after modeling until the end of the experiment. Within seven days, mice were intraperitoneally injected with 50 mg / kg body weight of 5-FU (purchased from Selleck), and intestinal mucositis occurred. These injections were divided into four groups: the 5-FU group, the 5-FU + FA-2-b-β low-dose group, the 5-FU + FA-2-b-β preventive low-dose group, the 5-FU + FA-2-b-β high-dose group, and the 5-FU + FA-2-b-β preventive high-dose group.

[0073] From the first day of administration, body weight was monitored daily. Mice in the low-dose group, preventive group, and high-dose group were orally administered FA-2-b-β, which was provided by Kangzhen Biotechnology (Beijing) Co., Ltd. Fasting was performed on the 8th day, and the mice were euthanized on the 9th day for histopathological tissue analysis. The results showed that after 5-FU treatment, the intestinal villi became shorter, the integrity of the villi was damaged, the intestinal crypt depressions decreased, and the body weight decreased significantly, while selenium amino acid protein effectively reversed these changes ( Figure 1 - Figure 2 ).

[0074] Example 2: PCR detection of Occludin mRNA expression

[0075] Occludin is a tight junction protein between apical epithelia, which can prevent the destruction of intestinal microvilli and the parietal membrane structure and is indispensable for the intestinal tight junction structure and barrier function.

[0076] The construction of the intestinal mucositis mouse model and experimental grouping were the same as in Example 1.

[0077] 30 mg of fresh ileal tissue was mixed with 1 mL of lysis buffer LB using a tissue homogenizer set to high-speed and low-temperature mode. Then the mixture was homogenized. After that, the mixture was centrifuged at 12,000 revolutions per minute for seven minutes in a centrifuge to separate the liquid and solid. After obtaining the total RNA, cDNA was prepared using a reverse transcription kit. To measure the mRNA expression level of each gene, real-time polymerase chain reaction was performed. The Occludin expression level was detected using primers developed and manufactured by Beijing Ruiboxing Biotechnology Co., Ltd. and purified by ULTRAPAGE. GAPDH was used as an internal reference. The results showed that selenium amino acid protein FA-2-b-β could restore the mRNA expression of Occludin ( Figure 3 ).

[0078] Example 3: Selenium Amino Acid Protein FA-2-b-β Promotes the Proliferation of HIEC

[0079] The construction of the intestinal mucositis mouse model and the experimental grouping were the same as in Example 1.

[0080] The HIEC cell line (normal human intestinal epithelial cells) was inoculated into a 96-well plate at a density of 100 μL 5×10 3 / mL. After attachment for 24 h, different concentrations of selenium amino acid protein were added and the cells were treated for 48 h. After incubating the cells with CCK-8 reagent, the optical density in the wells of the microplate was measured at 450 nm to determine cell viability. The results showed that selenium amino acid protein could promote the proliferation of HIEC cells within a certain concentration range ( Figure 4 ).

[0081] Example 4: Selenium Amino Acid Protein FA-2-b-β Inhibits Ferroptosis in Human Intestinal Epithelial Cells

[0082] The HIEC cell line was inoculated into a 6-well plate at a density of 1.5 mL 1×10 6 / mL. The suspended cells were collected, RIPA lysis buffer was added, and the mixture was placed on ice for 15 min. After centrifugation at 4°C and 15,000 r / min for 10 min, the protein concentration was determined using the BCA method. 10 μL of the protein sample was taken for SDS-PAGE and transferred to a PVDF membrane using the wet transfer method. The membrane was placed in 5% skim milk and blocked at room temperature for 2 h; the primary antibodies were diluted with the antibody dilution solution at 1:2000 (GPX4) and 1:1000 (β-actin), incubated overnight at 4°C, washed 3 times with TBST, 10 min each time; the secondary antibody (goat anti-rabbit IgG, 1:4000) was incubated at room temperature for 40 min; washed 3 times with TBST, 10 min each time; when detecting on the machine, 400 μL of ECL luminescent solution was added. After exposure, the gel imaging analyzer was used to observe and take pictures, and the gray value of the protein band was analyzed using Image J software.

[0083] Compared with the control group, the expression levels of GPX4 and Nrrf2 were downregulated and upregulated after using the drug. Ferroptosis is a cell death mechanism induced by the disruption of glutathione (GSH) synthesis or the inhibition of glutathione peroxidase 4 (GPX4), and the nuclear factor E2-related factor 2 (Nrf2) / GPX4 signaling pathway plays an important role in ferroptosis. These results indicate that selenium amino acid protein inhibits ferroptosis in HIEC cells ( Figure 5 - Figure 6 ).

[0084] Based on the activity characteristics of selenium amino acid protein FA-2-b-β, the present invention screened out the optimal concentration and other analysis conditions through a large number of experiments, and studied the protective effect of the selenium amino acid protein of the Agaricus blazei extract on 5-FU-induced intestinal mucositis.

[0085] The present invention mainly uses detections such as CCK8, Western blot, HE staining, etc. The detection method has high sensitivity and good stability, and can objectively, comprehensively and accurately evaluate the protective effect of selenium amino acid protein on 5-FU-induced enteritis. The FA-2-b-β extract of Conzenn's Agaricus blazei can improve 5-FU-induced enteritis, promote the proliferation of human intestinal epithelial cells, restore the intestinal barrier, and resist the toxic and side effects of chemotherapy.

[0086] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a plant extract in the preparation of a medicament for preventing, treating and / or alleviating intestinal mucositis, characterized in that: The plant extract comprises FA-2-b-β, preferably Agaricus blazei Murrill extract.

2. The use according to claim 1, characterized in that The intestinal mucositis is chemotherapy-induced intestinal mucositis, preferably chemotherapy-induced intestinal mucositis caused by 5-fluorouracil.

3. The use according to claim 1, characterized in that The total selenium content of the plant extract is above 80 mg / kg, above 85 mg / kg, above 90 mg / kg or above 95 mg / kg.

4. The use according to claim 1, characterized in that The percentage of organic selenium content in the plant extract to total selenium content is more than 80%, more than 85%, more than 90%, more than 95% or more than 99%.

5. The use according to any one of claims 1 to 4, characterized in that: The plant extract contains 90 mg / kg or more, 95 mg / kg or more, 100 mg / kg or more, 110 mg / kg or more, 120 mg / kg or more, 130 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, 200 mg / kg or more, 250 mg / kg or more, or 300 mg / kg or more of FA-2-b-β.

6. A pharmaceutical composition, characterized in that The invention comprises a plant extract as a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the plant extract comprises FA-2-b-β, and preferably an Agaricus blazei Murrill extract.

7. The pharmaceutical composition according to claim 6, characterized in that The total selenium content of the plant extract is above 80 mg / kg, above 85 mg / kg, above 90 mg / kg or above 95 mg / kg.

8. The pharmaceutical composition according to claim 6, characterized in that The percentage of organic selenium content in the plant extract to total selenium content is more than 80%, more than 85%, more than 90%, more than 95% or more than 99%.

9. The pharmaceutical composition according to claim 6, characterized in that The plant extract contains 90 mg / kg or more, 95 mg / kg or more, 100 mg / kg or more, 110 mg / kg or more, 120 mg / kg or more, 130 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, 200 mg / kg or more, 250 mg / kg or more, or 300 mg / kg or more of FA-2-b-β.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of a medicament for preventing, treating and / or alleviating intestinal mucositis, characterized in that: The intestinal mucositis is preferably chemotherapy-induced intestinal mucositis, more preferably chemotherapy-induced intestinal mucositis caused by 5-fluorouracil.

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