A new variety of antitumor red ganoderma, related products and preparation method and application thereof

Abstract

The present application provides a new variety of Ganoderma lucidum for resisting tumor, related products and a preparation method and application thereof, and belongs to the field of new varieties of Ganoderma lucidum. Ganoderma sichuanense The new variety of Ganoderma lucidum is named Xianzhi No. 13, which has been preserved in the China Center for Type Culture Collection on October 25, 2024, and the preservation number is CCTCC NO: M 20242347. Compared with the existing Ganoderma lucidum varieties, the new variety of Ganoderma lucidum provided by the present application has higher yield, higher content of effective components and anticancer active components, and the extract has good antitumor activity.

Description

Technical Field

[0001] This invention belongs to the technical field of new Ganoderma lucidum varieties, and relates to a new anti-tumor Ganoderma lucidum variety, related products, preparation methods and applications. Background Technology

[0002] Lingzhi, also known as Sanxiu (from *Chu Ci*), Bizhi (from *Erya*), Lingzhicao (from *Diannan Bencao*), Mulingzhi (from *Hangzhou Medicinal Plants*), and Junlingzhi (from *National Compendium of Chinese Herbal Medicine*), is the fruiting body of the fungus *Ganoderma lucidum* or *Ganoderma sinense*, belonging to the genus *Ganoderma* of the family Polyporaceae. It is a saprophytic fungus. It has a stalk and is corky. The cap is semi-circular or kidney-shaped, 10-20 cm in diameter. The cap flesh is 1.5-2 cm thick, brownish-yellow or reddish-brown on the surface, gradually turning pale yellow at the edge, with concentric rings, slightly wrinkled or smooth, with a glossy lacquer-like sheen, and a slightly blunt edge. The flesh is milky white, pale brown near the tubes. The tubes are 1 cm long. The pores are nearly circular. The stipe is cylindrical, lateral or eccentric, occasionally central. It is 10-19 cm long and 1.5-4 cm thick, similar in color to the cap. The hyphae in the pericarp are club-shaped, with enlarged tips. The mycelial system is triadic: reproductive hyphae are transparent and thin-walled; skeletal hyphae are yellowish-brown with a nearly solid posterior wall; and twining hyphae are colorless with a curved posterior wall and are evenly branched. Spores are oval, with double walls, a truncate apex, a transparent outer wall, and a pale brown inner wall with small spines, measuring (9-11) μm × (6-7) μm. Wild Ganoderma lucidum often grows on the roots of sun-loving plants in the Fagaceae and Pinaceae families, or on dead tree stumps.

[0003] The fruiting body, mycelium, and spores of Ganoderma lucidum contain over 100 triterpenoids, most of which are highly oxidized lanosterane derivatives. These include the ganoderic acid family, ganoderic-22-enoic acid family, ganoderic oxalic acid family, 22,23-dimethylene ganoderic acid, monoganoderic acid family, ganoderic spore acid A, ganoderol family, ganoderol A, ganoderol B, ganoderic terpenoid diol, ganoderic terpenoid triol, ganoderic terpenone diol, ganoderic terpenone alcohol, ganoderic aldehyde A, B, epoxyganoderol A, B, C, ganoderic terpenone A, B, ganoderic spore lactone A, B, ganoderic sterol, etc. Additionally, Ganoderma lucidum contains various polysaccharides. Traditional Chinese medicine considers Ganoderma lucidum to be sweet and neutral, and to enter the lung, heart, and spleen meridians. Ganoderma lucidum is believed to invigorate qi, strengthen the body, nourish the heart, and calm the mind. It is indicated for general weakness, loss of appetite, palpitations, insomnia, dizziness, fatigue, chronic cough and asthma, coronary heart disease, hypertension, and hyperlipidemia.

[0004] Chinese patent application 201811387888.3 discloses the use of Ganoderma lucidum polysaccharide in the preparation of antitumor drugs. The preparation method of the Ganoderma lucidum polysaccharide includes the following steps: (1) extraction; (2) alcohol precipitation; (3) freeze drying; and (4) separation and purification. This technology provides a new use for Ganoderma lucidum polysaccharide, which can effectively inhibit tumor growth and can be used to prepare antitumor drugs. It can also be used as a synergist for antitumor drugs such as paclitaxel, and has good medicinal value.

[0005] Chinese patent application 202110744686.5 discloses a novel Ganoderma lucidum strain, ZL167, which is a Ganoderma lucidum species deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.22443. This technology also provides Ganoderma lucidum polysaccharides extracted from the aforementioned novel strain, which can increase ICAM-1 expression in endothelial cells by activating the NF-κB pathway; furthermore, it can increase T-cell tumor infiltration, thus exerting a tumor-suppressive effect. This Ganoderma lucidum strain and its polysaccharides have excellent application value in the food, health product, and pharmaceutical fields.

[0006] Chinese patent application 201610058137.1 discloses a strain of Ganoderma lucidum and its applications. This strain is named NCPSLZ1, with accession number CCTCC No. M2015796; the depositary institution is the China Center for Type Culture Collection; the deposit date is December 29, 2015; and the deposit address is Wuhan University. The mycelium and fermentation broth of this strain can be used to prepare antitumor drugs.

[0007] However, existing technologies make it difficult to provide a new Ganoderma lucidum variety with higher content of active ingredients and better anti-tumor effects. Summary of the Invention

[0008] Terminology and Declarations of this Invention:

[0009] 1. As used in this text, the articles “a,” “a kind,” or “the” include plural objects unless otherwise explicitly specified as a single object.

[0010] 2. As used herein, numerical range: Unless otherwise expressly indicated, all ranges or ratios disclosed herein shall be understood to include any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between a minimum value of 1 and a maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0011] To address the problem that existing technologies cannot provide a new Ganoderma lucidum variety with better anti-tumor effects, the purpose of this invention is to provide a new anti-tumor Ganoderma lucidum variety, related products, its preparation method, and applications.

[0012] To achieve the above-mentioned objectives, in a first aspect, the present invention provides a Ganoderma lucidum, named Xianzhi No. 13 (also known as Kangai No. 1).

[0013] This Ganoderma lucidum strain was deposited on October 25, 2024, at the China Center for Type Culture Collection (Wuhan University Collection Center, located at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province). The strain is named Xianzhi 13 (also known as Kangai 1) in Chinese and is classified as follows: Xianzhi No. 13, with accession number CCTCC NO: M 20242347.

[0014] The aforementioned Ganoderma lucidum strains possess the following biological characteristics:

[0015] Xianzhi No. 13 exhibits typical Ganoderma lucidum morphological characteristics. The fruiting body is umbrella-shaped, with a kidney-shaped cap, 8-15 cm in diameter and 1-2 cm thick. The outer shell is hard, yellowish-brown, and glossy, with annular ridges and radial wrinkles. The edges are thin, flat, and slightly rolled, while the ventral side is yellowish-white.

[0016] Preferably, the mature dried fruiting body of Ganoderma lucidum contains ≥1.01% by weight of Ganoderma lucidum polysaccharide and ≥0.45% by weight of triterpenic acid.

[0017] The term "triterpenoid acid" refers to triterpenoid compounds containing carboxyl groups found in higher fungi such as Ganoderma lucidum or Ganoderma sinense, such as ganoderic acid A, ganoderic acid B, ganoderic acid C, ganoderic acid D, ganoderic acid E, ganoderic acid F, ganoderic acid G, ganoderic acid I, ganoderic acid L, ganoderic acid ma, ganoderic acid md, ganoderic acid mg, ganoderic acid TR, etc.

[0018] As an example of the present invention, the triterpenic acid includes ganoderic acid (TR).

[0019] Secondly, the present invention provides a method for cultivating the above-mentioned Ganoderma lucidum, comprising the following steps:

[0020] S1. Inoculate the strain onto potato dextrose agar medium and seal it for constant temperature culture at 27-30℃ to obtain immobilized strains.

[0021] S2. Inoculate the immobilized bacterial strain obtained in step S1 into the liquid bacterial culture medium and incubate at a constant temperature to obtain the liquid bacterial strain.

[0022] S3. Mix the liquid inoculum obtained in step S2 with the bag material and sterile water, package, and incubate in the dark to obtain the inoculum.

[0023] S4. Cover the mushroom sticks obtained in step S3 with soil for cultivation, manage the growth of Ganoderma lucidum, and obtain Ganoderma lucidum.

[0024] More preferably, the liquid culture medium in step S2 consists of the following components: water as the solvent, and each liter of liquid culture medium contains 7.5-8.5g of maltose, 7.5-8.5g of sucrose, 1.5-2.5g of arabinose, 1.5-2.5g of lactose, 1.5-2.5g of yeast extract, 0.15-0.25g of sodium tripolyphosphate, 1.5-2.5g of chlorophyll, 0.15-0.25g of tryptophan, and 0.8-1.2g of agar.

[0025] More preferably, the liquid culture medium in step S2 consists of the following components: water as the solvent, 8g of maltose, 8g of sucrose, 2g of arabinose, 2g of lactose, 2g of yeast powder, 0.2g of sodium tripolyphosphate, 2g of chlorophyll, 0.2g of tryptophan, and 1g of agar per liter of liquid culture medium, with a pH of 5.8.

[0026] More preferably, the raw materials for the bag material in step S3 are composed of the following components by weight: 40.5-56.5 parts sawdust, 0.5-1.5 parts charcoal powder, 10-20 parts soybean straw powder, 3-10 parts corn cob powder, 0.3-0.7 parts calcium carbonate powder, 0.3-0.7 parts magnesium hydroxide powder, 2.5-3.5 parts calcium chloride, 1.5-2.5 parts sodium sulfate, 0.5-1.5 parts ammonium sulfate, and 1.5-2.5 parts sucrose.

[0027] More preferably, the raw materials for the bag material in step S3 are composed of the following components by weight: 50 parts sawdust, 1 part charcoal powder, 15 parts soybean straw powder, 5 parts corn cob powder, 0.5 parts calcium carbonate powder, 0.5 parts magnesium hydroxide powder, 3 parts calcium chloride, 2 parts sodium sulfate, 1 part ammonium sulfate, and 2 parts sucrose.

[0028] Preferably, in step S4, the soil-covered cultivation includes the following steps:

[0029] Remove weeds, bacteria, and pests from the site. Dig pit beds, place the mushroom sticks horizontally in the pit beds, cover with a film to keep them moist for 2-3 days, then uncover the film, cover with soil, and cover with the film again to maintain an environmental humidity of ≥90%. Uncover the film daily for ventilation.

[0030] More preferably, in step S4, the soil-covered cultivation includes the following steps:

[0031] Weeding, sterilization, and pest control are performed on the site. Select a flat area with good water retention and easy irrigation / drainage. Dig pits 12-15cm deep, 80-100cm wide, and of unlimited length. Remove the film bags from the mushroom logs and place them horizontally in the pits. Cover with a polypropylene film to maintain moisture for 2-3 days, then remove the film. Cover with 1cm of crushed, sieved soil with a moisture content of 65%-70%, then cover again with the polypropylene film. Maintain an ambient humidity of ≥90%. Ventilate the site daily for 20-40 minutes, preferably 30 minutes.

[0032] Preferably, in step S4, the outgoing management includes the following steps:

[0033] After the mushrooms emerge, remove the film, adjust the temperature to 28-32℃ and the air humidity to 85%-95%, and cultivate until they begin to spray spores and the edges of the cap turn from white to milky yellow, and the tender parts turn darker in color. They are then ready for harvesting.

[0034] More preferably, in step S4, the outgoing management includes the following steps:

[0035] After about 3-5 days of cultivation, remove the polypropylene film, adjust the temperature to 28-32℃ and the air humidity to 85%-95%, and harvest when the mushroom begins to spray spores, the edge of the cap turns from white to milky yellow, and the tender parts darken in color.

[0036] Thirdly, the present invention provides an anti-tumor Ganoderma lucidum extract, which is prepared from the aforementioned Ganoderma lucidum.

[0037] Preferably, the content of ganoderic acid triol in the Ganoderma lucidum extract is ≥5.0g / 100g, the content of ganoderic acid TR is ≥1.5g / 100g, the content of ganoderic acid F is ≥7.5g / 100g, and the content of ganoderic aldehyde A is ≥1.2g / 100g.

[0038] More preferably, the content of ganoderic acid triol in the Ganoderma lucidum extract is ≥5.52g / 100g, the content of ganoderic acid TR is ≥2.15g / 100g, the content of ganoderic acid F is ≥7.98g / 100g, and the content of ganoderic aldehyde A is 2.04g / 100g-2.52g / 100g.

[0039] More preferably, the Ganoderma lucidum extract contains ≥6.33g / 100g of ganoderic triol, ≥2.47g / 100g of ganoderic acid TR, ≥9.15g / 100g of ganoderic alcohol F, and 2.26g / 100g-2.44g / 100g of ganoderic aldehyde A.

[0040] Most preferably, and as a specific example of the present invention, the Ganoderma lucidum extract contains 6.72 g / 100 g of ganoderic triol, 2.62 g / 100 g of ganoderic acid TR, 9.71 g / 100 g of ganoderic alcohol F, and 2.40 g / 100 g of ganoderic aldehyde A.

[0041] Fourthly, the present invention also provides a method for preparing Ganoderma lucidum extract, comprising the following steps:

[0042] (1) Ultrasonic extraction: Mix Ganoderma lucidum with solvent, sonicate, filter, repeat 2-3 times, and combine the filtrates to obtain ultrasonic extract;

[0043] (2) Preparation of the adsorption solution: Concentrate the ultrasonic extract obtained in (1), add water and ethanol, filter, and obtain the adsorption solution;

[0044] (3) Resin adsorption: The solution to be adsorbed obtained in (2) is loaded onto the column at a flow rate of 1-2 times the column volume per hour (BV / h);

[0045] (4) Elution: The resin after loading onto the column in (3) was eluted with 25%-35% ethanol aqueous solution, 65%-70% ethanol aqueous solution and 95% ethanol aqueous solution respectively. The eluent obtained by elution with 95% ethanol aqueous solution was collected, concentrated and dried to obtain Ganoderma lucidum extract.

[0046] Preferably, in step (1), the solvent is an aqueous ethanol solution, more preferably an 80%-99% aqueous ethanol solution, and even more preferably a 95% aqueous ethanol solution.

[0047] Preferably, in step (1), the weight of the solvent is 10-20 times the weight of the Ganoderma lucidum, more preferably 15 times.

[0048] Preferably, in step (1), the ultrasound duration is 1-60 min, more preferably 10-45 min, and even more preferably 30 min.

[0049] Preferably, in step (1), the repetition is performed 3 times.

[0050] Preferably, in step (2), the concentration is vacuum concentration;

[0051] More preferably, the temperature of the vacuum concentration is 60-70°C, and even more preferably 65°C.

[0052] Preferably, in step (2), the concentration is to concentrate the weight of the solution to 6-8 times the weight of Ganoderma lucidum in step (1), more preferably 7 times the weight of Ganoderma lucidum in step (1).

[0053] Preferably, in step (2), the addition of water and ethanol specifically refers to:

[0054] Add water to the concentrated solution until the weight concentration of ethanol is 20%-35% of the total solution, and then dilute with ethanol of the same weight concentration until the total weight of the solution is 12-16 times the weight of Ganoderma lucidum mentioned in step (1).

[0055] More preferably, in step (2), the addition of water and ethanol specifically refers to:

[0056] Add water to the concentrated solution until the weight concentration of ethanol accounts for 25% of the total solution. Then dilute with ethanol of the same weight concentration (25%) until the total weight of the solution is 14 times the weight of Ganoderma lucidum described in step (1).

[0057] Preferably, in step (3), the resin is a macroporous resin.

[0058] Furthermore, the macroporous resin is selected from one or more of the following:

[0059] ADS-7, ADS-17, DM-130, D-101, AB-8, X-5.

[0060] As an example of the present invention, the macroporous resin is of type D-101.

[0061] Preferably, in step (3), the resin is pretreated.

[0062] More preferably, the resin undergoes a pretreatment step as follows:

[0063] Soak the resin in an ethanol aqueous solution, pack it into the column, rinse with water until there is no alcohol odor, and rinse with an ethanol aqueous solution of the same weight concentration as in step (2).

[0064] Furthermore, the concentration of the ethanol aqueous solution is 90%-99%, and even more specifically 95%.

[0065] Furthermore, the column packing is 0.5-1.5 times the weight of Ganoderma lucidum in step (1), and even more specifically, 1 times the weight of Ganoderma lucidum.

[0066] Furthermore, the rinsing volume using the same weight concentration of ethanol aqueous solution as in step (2) is 1-2 column volumes (BV), and even more specifically, 2 BV.

[0067] Preferably, in step (4), the amount of 25%-35% ethanol aqueous solution used is 2-5 BV, more preferably 4 BV.

[0068] Preferably, in step (4), the amount of 65%-70% ethanol aqueous solution used is 7-10 BV, more preferably 8 BV.

[0069] Preferably, in step (4), the amount of 95% ethanol aqueous solution used is 4-5 BV, more preferably 5 BV.

[0070] Preferably, in step (4), the drying is freeze drying.

[0071] Fifthly, the present invention also provides the application of the above-mentioned Ganoderma lucidum extract or the Ganoderma lucidum extract prepared by the above-mentioned preparation method in the preparation of antitumor drugs.

[0072] The Ganoderma lucidum extract can be prepared into various dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, and lozenges. For different dosage forms, suitable drug carriers in this field can be selected.

[0073] The drug carrier used can be solid, liquid, or gaseous. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0074] In preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, ethanol, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used to form oral liquid dosage forms, such as suspensions, solutions, and granules; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulators, emulsifiers, lubricants, binders, and disintegrants, can be used to form oral solid dosage forms, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or non-aqueous techniques.

[0075] Tablets containing the Ganoderma lucidum extract of this invention can be prepared by compression or molding, optionally using one or more excipients or adjuvants. The active ingredient can be compressed in a free-flowing form (e.g., powder or granules) in a suitable machine, optionally mixed with binders, lubricants, inert diluents, surfactants, or dispersants. Molded tablets can be molded in a suitable machine, i.e., a mixture of powdered compounds moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, formulations intended for oral administration to humans may contain about 0.5 mg to about 5 g of active ingredient, mixed with a suitable and convenient carrier material, which may comprise about 5% to 95% of the total composition. Unit dosage forms typically contain about 1 mg to about 2 g of active ingredient, typically in doses of 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0076] The pharmaceutical compositions of this invention suitable for parenteral administration can be prepared as aqueous solutions or suspensions of the active compound. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersions can also be prepared in mixtures of glycerol, liquid polyethylene glycol, and their oils. Furthermore, preservatives may be added to prevent harmful microbial growth.

[0077] The medicines prepared from the Ganoderma lucidum extract of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, or the like. Furthermore, the medicines can be used in suitable forms for transdermal drug delivery devices. These formulations can be prepared using the traditional Chinese medicine compositions of the present invention through conventional processing methods. For example, a cream or ointment with the desired consistency can be prepared by mixing a hydrophilic material and water, and about 5 wt% to about 10 wt% of the compound.

[0078] The medicine prepared from the Ganoderma lucidum extract of the present invention can be in a form suitable for rectal administration, wherein the carrier is solid. It is preferable to formulate the mixture into unit-dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a mixture containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0079] In addition to the carrier components described above, the pharmaceutical formulations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients may be added, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants, and flavoring agents, etc., to make the formulation isotonic with the blood of the intended receptor. Components containing the traditional Chinese medicine composition of this invention can also be prepared in powder or concentrated form.

[0080] The tumors mentioned include, but are not limited to: acute lymphoblastic tumors; acute myeloid leukemia; adrenocortical carcinoma; pediatric adrenocortical carcinoma; adnexal carcinoma; basal cell carcinoma; extrahepatic bile duct carcinoma; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; pediatric brainstem gliomas, adult brain tumors; atypical teratoid central nervous system brain tumors; pediatric rod tumors; central nervous system phylogenetic histiocytomas; cerebellar astrocytomas; cerebral astrocytomas; malignant gliomas; craniopharyngiomas; ependymoblastomas; ependymoblastomas; medulloblastomas; moderately differentiated pineal parenchymal tumors; supratentorial primitive neuroectodermal tumors and pinealocyte gliomas; visual pathway and hypothalamic gliomas; brain and spinal cord tumors; breast cancer; bronchomas; Burkitt lymphomas; benign Sexual tumors; atypical teratoma-like rhabdomyosarcoma of the central nervous system; embryonal tumors of the central nervous system; lymphoma of the central nervous system; cervical cancer; chordoma of children; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous lymphoma; esophageal cancer; Ewing's tumor; extragonadal malignant germ cell tumors; intraocular melanoma; retinoblastoma of the eye; gallbladder cancer; benign tumors of the gastrointestinal tract; gastrointestinal stromal tumors; extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; gestational trophoblastoma; glioma; pediatric brainstem glioma; glioma; pediatric cerebral astrocytoma; pediatric visual pathway and hypothalamic glioma; hairy cell leukemia; head and neck cancer; hepatocellular carcinoma. Cancer; Hodgkin's lymphoma; hypopharyngeal cancer; hypothalamic and optic pathway glioma; intraocular melanoma; islet cell tumor; renal cell carcinoma; Langerhans cell histiocytosis; laryngeal cancer; acute lymphoblastic leukemia; acute myeloid leukemia; lip and oral cavity cancer; liver cancer; non-small cell lung cancer; small cell lung cancer; AIDS-related lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; malignant fibrous histiocytoma and osteosarcoma of bone; medulloblastoma; melanoma; intraocular melanoma; Merkel cell carcinoma; mesothelioma; latent primary and metastatic squamous cell carcinoma of the neck; childhood multiple endocrine adenoma syndrome; mycosis fungoides; myelodysplastic syndrome; myelodysplastic syndrome; chronic myeloid leukemia; adult acute myeloid leukemia Acute myeloid leukemia in children; chronic myeloproliferative disorders; nasal and paranasal sinus carcinoma; nasopharyngeal carcinoma; neuroblastoma; ovarian cancer; ovarian epithelial carcinoma; ovarian germ cell carcinoma; low-grade ovarian cancer; pancreatic cancer; pancreatic islet cell carcinoma; papilloma; parathyroid carcinoma; penile cancer; pharyngeal carcinoma; pheochromocytoma; moderately differentiated pineal parenchymal tumor; pineoblastoma and supratentorial primitive neuroectodermal tumor; pituitary adenoma; plasmacytoma; multiple myeloma; pleural pulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; respiratory tract cancer involving genes on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland carcinoma; Ewing's sarcoma; Kaposi's sarcoma; uterine sarcoma;Cézari syndrome; non-melanoma skin cancer; melanoma skin cancer; Merkel cell skin cancer; small bowel cancer; soft tissue sarcoma; squamous cell carcinoma; latent primary and metastatic squamous neck cancer; gastric cancer; supratentorial primitive neuroectodermal tumor; testicular cancer; laryngeal cancer; thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic carcinoma; urethral cancer; endometrial cancer; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia and nephroblastoma.

[0081] Finally, the present invention also provides an anti-tumor drug, the effective ingredient of which includes the above-mentioned Ganoderma lucidum extract or the Ganoderma lucidum extract prepared by the above-mentioned preparation method.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] (1) This invention provides a novel Ganoderma lucidum strain, Xianzhi No. 13 (also known as Kangai No. 1). Compared with existing Ganoderma lucidum strains, the Ganoderma lucidum strain provided by this invention has the characteristics of short growth cycle, high yield, high content of effective ingredient Ganoderma lucidum polysaccharide, high content of triterpenes and sterols, and high content of anti-tumor active ingredients.

[0084] (2) This invention provides a method for preparing an extract of Ganoderma lucidum No. 13 (also known as Kangai No. 1). Using this method, a Ganoderma lucidum extract can be prepared. This Ganoderma lucidum extract can achieve better tumor treatment effects. Attached Figure Description

[0085] Figure 1 This is a bar chart showing the effect of gradient concentrations of GLe-1 on the activity of HCT116 cells.

[0086] Figure 2 This is a graph showing the antitumor activity of GLe-1 cells based on three-dimensional culture of HCT116 cells. The NucBlue channel represents the total cell count staining result, and the PI channel represents the dead cell staining result.

[0087] Figure 3 This is a line graph showing the therapeutic effect of dried fruiting body powder suspensions of Ganoderma lucidum / Reishi mushroom provided in Examples 1-5 and Comparative Example 5 on tumors in mice carrying HCT-116 heterogeneous tumors. Detailed Implementation

[0088] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0089] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0090] The sources of some of the raw materials used in the cultivation of Ganoderma lucidum in this invention are illustrated below.

[0091] Yeast powder: Brand Adamas-life, Product No. 045717187. Wood chips purchased from Langfang Bangke Biomass Energy Co., Ltd. Soybean straw powder purchased from Zhengyang County Xintiandi Grass Industry Co., Ltd. Corn cob powder purchased from Lingshou County Zhaochen Mineral Products Co., Ltd. Charcoal powder purchased from Hebei Hengxun Mineral Products Processing Co., Ltd.

[0092] In the following examples, the *Ganoderma lucidum* strain 13 (also known as *Kangai* 1) used was deposited on October 25, 2024, at the China Center for Type Culture Collection (Wuhan University Collection Center, located at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province). This *Ganoderma lucidum* strain is classified as... Xianzhi No. 13, with accession number CCTCC NO: M 20242347. The Wuhan University Collection Center completed the viability test of Xianzhi No. 13 strain on November 1, 2024, and the result was that it was viable.

[0093] The aforementioned Ganoderma lucidum strains possess the following biological characteristics:

[0094] Xianzhi No. 13 exhibits typical Ganoderma lucidum morphological characteristics. The fruiting body is umbrella-shaped, with a kidney-shaped cap, 8-15 cm in diameter and 1-2 cm thick. The outer shell is hard, yellowish-brown, and glossy, with annular ridges and radial wrinkles. The edges are thin, flat, and slightly rolled, while the ventral side is yellowish-white.

[0095] Example 1

[0096] A method for cultivating Ganoderma lucidum.

[0097] (1) Under sterile conditions, take the strain of Xianzhi No. 13 (also known as Kangai No. 1) and inoculate it on PDA medium (potato dextrose agar medium). Seal it and place it in an incubator with a temperature of 27-30℃ and an air humidity of 50%-80%. Incubate it at constant temperature and humidity for 7 days until the colonies fill the culture dish and obtain the immobilized strain.

[0098] (2) Inoculate the immobilized bacterial strain obtained in (1) into a liquid bacterial culture medium and incubate on a constant temperature shaker. The shaker incubation temperature is 27-32℃, and the shaking speed is 120-140 rpm. The preparation method of the liquid bacterial culture medium is as follows:

[0099] Mix 8g maltose, 8g sucrose, 2g arabinose, 2g lactose, 2g yeast powder, 0.2g sodium tripolyphosphate, 1g chlorophyll, and 0.2g tryptophan with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with 0.1% acetic acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0100] After 7 days of culture on a shaker, a liquid bacterial culture was obtained.

[0101] (3) Prepare cultivation bags and carry out dark culture.

[0102] Mix 50 parts sawdust, 1 part charcoal powder, 15 parts soybean straw powder, 5 parts corn cob powder, 0.5 parts calcium carbonate powder, and 0.5 parts magnesium hydroxide powder by weight to obtain a solid matrix mixture. Mix 3 parts calcium chloride, 2 parts sodium sulfate, 1 part ammonium sulfate, 2 parts sucrose, and an appropriate amount of water to form a suspension. Sprinkle the suspension evenly on the solid matrix mixture, stir, sterilize by high-temperature moist heat (121℃, 15min), cool to room temperature, and dry to obtain bag material. Under aseptic conditions, mix the above bag material, sterile water, and the liquid culture medium obtained in (2) at a mass ratio of 4:6:0.25, package it into a cylindrical polypropylene film, and tie both ends tightly to form a cultivation bag. Each cultivation bag weighs about 1-1.5kg. Puncture 20-40 small holes on the surface of the cultivation bag and incubate in the dark at 28℃. When the mycelium in the cultivation bag is dense, the mycelium is obtained.

[0103] (4) Soil covering cultivation.

[0104] Cultivation is carried out using conventional soil-covering cultivation techniques. Details are as follows:

[0105] Cover the mushroom logs with soil for cultivation. Weed, sterilize, and control pests at the site. Select a flat area with good water retention and easy irrigation / drainage. Dig pits 12-15cm deep, 80-100cm wide, and of unlimited length. Remove the film bags from the mushroom logs and lay them horizontally in the pits. Cover with polypropylene film to maintain moisture for 2-3 days, then remove the film. Cover with 1cm of crushed, sieved soil with a moisture content of 65%-70%, then cover again with polypropylene film, maintaining an ambient humidity of ≥90%. Ventilate for 30 minutes daily by uncovering the film. After approximately 3-5 days of cultivation, once the mushrooms have emerged, remove the polypropylene film. Adjust the temperature to 28-32℃ and the air humidity to 85%-95%. Harvest when the mushrooms begin to release spores, the edges of the caps turn white to milky yellow, and the tender parts darken in color.

[0106] Example 2

[0107] Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed as follows, and the rest of the steps are the same.

[0108] Mix 7.5g maltose, 8.5g sucrose, 1.5g arabinose, 2.5g lactose, 1.5g yeast powder, 0.25g sodium tripolyphosphate, 2.5g chlorophyll, and 0.15g tryptophan with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with 0.1% acetic acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0109] Example 3

[0110] Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed as follows, and the rest of the steps are the same.

[0111] Mix 8.5g maltose, 7.5g sucrose, 2.5g arabinose, 1.5g lactose, 2.5g yeast powder, 0.15g sodium tripolyphosphate, 1.5g chlorophyll, and 0.25g tryptophan with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with an acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0112] Example 4

[0113] Compared with Example 1, the only difference is that in step (3), the method for preparing the cultivation bag is changed as follows:

[0114] Mix 40.5 parts by weight of sawdust, 0.5 parts of charcoal powder, 20 parts of soybean straw powder, 10 parts of corn cob powder, 0.3 parts of calcium carbonate powder, and 0.7 parts of magnesium hydroxide powder to obtain a solid matrix mixture. Mix 3.5 parts of calcium chloride, 1.5 parts of sodium sulfate, 1.5 parts of ammonium sulfate, 1.5 parts of sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0115] Example 5

[0116] Compared with Example 1, the only difference is that in step (3), the method for preparing the cultivation bag is changed as follows:

[0117] Mix 56.5 parts by weight of sawdust, 1.5 parts of charcoal powder, 10 parts of soybean straw powder, 3 parts of corn cob powder, 0.7 parts of calcium carbonate powder, and 0.3 parts of magnesium hydroxide powder to obtain a solid matrix mixture. Mix 2.5 parts of calcium chloride, 2.5 parts of sodium sulfate, 0.5 parts of ammonium sulfate, 2.5 parts of sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0118] Comparative Example 1

[0119] Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed as follows, and the rest of the steps are the same.

[0120] Mix 8g maltose, 8g sucrose, 2g arabinose, 2g lactose, 2g yeast powder, 0.4g sodium tripolyphosphate, and 1g chlorophyll with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with 0.1% acetic acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0121] Comparative Example 2

[0122] Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed as follows, and the rest of the steps are the same.

[0123] Mix 8g maltose, 8g sucrose, 2g arabinose, 2g lactose, 2g yeast powder, 0.2g sodium tripolyphosphate, 1g chlorophyll, and 0.2g lysine with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with 0.1% acetic acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0124] Comparative Example 3

[0125] Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed as follows, and the rest of the steps are the same.

[0126] Mix 8g maltose, 8g sucrose, 2g arabinose, 2g lactose, 2g yeast powder, 0.2g sodium tripolyphosphate, 1g magnesium sulfate, and 0.2g tryptophan with an appropriate amount of water until completely dissolved. Mix 1g agar with an appropriate amount of water until completely dissolved. Mix the two solutions thoroughly, adjust the pH to 5.8 with 0.1% acetic acid solution, and bring the volume to 1L with water. Sterilize by high-temperature moist heat (121℃, 15min), and cool to room temperature to obtain liquid bacterial culture medium.

[0127] Comparative Example 4

[0128] Compared with Example 1, the only difference is that in step (3), the method for preparing the cultivation bag is changed as follows:

[0129] Mix 50 parts by weight of sawdust, 1 part by weight of charcoal powder, 15 parts by weight of soybean straw powder, 5 parts by weight of corn cob powder, 0.5 parts by weight of calcium carbonate powder, and 0.5 parts by weight of magnesium hydroxide powder to obtain a solid matrix mixture. Mix 3 parts by weight of calcium chloride, 3 parts by weight of sodium sulfate, 2 parts by weight of sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0130] Comparative Example 5

[0131] Compared with Example 1, the only difference is that the Xanzhi 13 strain was replaced with an equal amount of Hu Nong Lingzhi 1 strain, and all other aspects are the same.

[0132] In Comparative Example 5, Hu Nong Lingzhi No. 1 is a Lingzhi variety known to those skilled in the art, and it is also consistent with the Hu Nong Lingzhi No. 1 recorded in existing technologies such as the local standard DB45 / T 2918-2024 "Cultivation Technical Regulations of Hu Nong Lingzhi No. 1". It is a variety certified by Hu Nong Product Certification Edible Fungus (2009) No. 003.

[0133] Example of effect 1

[0134] Evaluation of the culture methods in Examples 1-5 and Comparative Examples 1-5.

[0135] The production cycle, yield, and polysaccharide, triterpenoid, and sterol content of the Ganoderma lucidum / Ganoderma lucidum fruiting bodies produced by the cultivation methods provided in Examples 1-5 and Comparative Examples 1-5 were tested.

[0136] The production cycle is defined as the time from the inoculation of the strain (Xianzhi 13 / Hunong Lingzhi 1) onto PDA medium to the harvesting of Ganoderma lucidum / Reishi mushroom, expressed in days. Yield is defined as the mass of dried Ganoderma lucidum / Reishi mushroom fruiting bodies that can be produced from one kilogram of bagged material within one production cycle, expressed in g / kg.

[0137] The polysaccharide content in the produced Ganoderma lucidum / Reishi mushroom fruiting bodies was tested. Specifically, according to the detection method for polysaccharide content in Ganoderma lucidum recorded in the Pharmacopoeia of the People's Republic of China (2020 edition, Part I), the content of Ganoderma lucidum polysaccharides, triterpenes, and sterols in the dried and mature fruiting bodies of Xianzhi 13 / Hu Nong Ganoderma lucidum 1 in each example and comparative example was tested. The content of Ganoderma lucidum polysaccharides was calculated on a dried basis, expressed as anhydrous glucose (C6H4O3). 12 O6) is expressed as a percentage of mass (%).

[0138] The method provided in Example 1 of Chinese Invention Patent CN118817912A was used to detect the content of ganoderic acid (TR) in the produced Ganoderma lucidum / Ganoderma lucidum fruiting bodies, and the unit is mass percentage (%).

[0139] The evaluation results of the culture methods provided in Examples 1-5 and Comparative Examples 1-5 are summarized in Table 1 below:

[0140] Table 1

[0141]

[0142] Example 6

[0143] A method for preparing Ganoderma lucidum extract. The steps are as follows:

[0144] (1) Ultrasonic extraction.

[0145] Mix Ganoderma lucidum with 15 times its weight of 95% ethanol aqueous solution, sonicate for 30 min per cycle, and filter. Repeat 3 times. Combine the filtrates to obtain the ultrasonic extract.

[0146] (2) Prepare the adsorption solution.

[0147] The ultrasonic extract obtained in (1) was concentrated under vacuum (65°C) until the total weight of the solution was 7 times that of Ganoderma lucidum in (1). Water was added to the concentrated solution until the weight concentration of ethanol accounted for 25% of the total solution, and then diluted with 25% ethanol until the total weight of the solution was 14 times that of Ganoderma lucidum in step (1). The solution was filtered to obtain the adsorption solution.

[0148] (3) Resin adsorption.

[0149] The macroporous resin D-101 was soaked in a 95% (w / w) ethanol aqueous solution and packed into the column using one times the weight of Ganoderma lucidum from step (1). The column was rinsed with purified water until no alcohol odor was detected, and then rinsed with 2 BV of 25% (w / w) ethanol aqueous solution. The adsorbent obtained in (2) was loaded onto the column at a flow rate of 2 column volumes per hour (BV / h).

[0150] (4) Washing.

[0151] The resin after loading onto the column in (3) was eluted with 4 BV of 30% ethanol aqueous solution, 8 BV of 70% ethanol aqueous solution, and 5 BV of 95% ethanol aqueous solution, respectively. The eluent obtained by elution with 95% ethanol aqueous solution was collected, concentrated, and freeze-dried to obtain Ganoderma lucidum extract.

[0152] The Ganoderma lucidum / Ganoderma lucidum fruiting bodies obtained in Examples 1-5 and Comparative Examples 1-5 were extracted using the extraction method described in Example 6. The resulting Ganoderma lucidum extracts were named GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4 and GLc-5, respectively.

[0153] Example 7

[0154] Isolation and identification of antitumor components in Ganoderma lucidum extract.

[0155] Referring to the experimental method provided in Example 1 of Chinese Invention Patent CN108181396A, the content of the antitumor component ganoderic acid triol (compound I) in the Ganoderma lucidum extracts GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4 and GLc-5 obtained in Example 6 was detected sequentially. Referring to the experimental method provided in Example 1 of Chinese Invention Patent CN118817912A, the content of the antitumor components ganoderic acid TR (compound II), ganoderic acid F (compound III) and ganoderic acid A (compound IV) in the Ganoderma lucidum extracts GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4 and GLc-5 obtained in Example 6 was detected sequentially.

[0156] The chemical structural formula of ganoderic triol is:

[0157] .

[0158] The chemical structural formula of ganoderic acid TR is:

[0159] .

[0160] The chemical structural formula of ganoderic acid F is:

[0161] .

[0162] The chemical structural formula of ganoderic acid A is:

[0163] .

[0164] The extraction amounts of compounds I, II, III, and IV per 100 grams of Ganoderma lucidum extract in each group were statistically analyzed, and the results are shown in Table 2.

[0165] Table 2

[0166]

[0167] It is evident that the extract of Ganoderma lucidum No. 13 provided by this invention contains higher levels of compounds I-IV, which have anti-tumor effects, and these levels are significantly higher than those of existing Ganoderma lucidum strains (Comparative Example 5).

[0168] Example 2

[0169] Evaluation of the antitumor activity of Ganoderma lucidum extract

[0170] 1. Evaluation of the antitumor effect on in vitro two-dimensional (2D) tumor cells

[0171] 1.1 Activity inhibition assay against HCT116 human colon cancer cells

[0172] Remove HCT116 cells in logarithmic growth phase from the incubator and discard the original culture medium. Add 2 mL of PBS to rinse the cells and discard the PBS. Add 1 mL of 0.25% trypsin to digest the cells. After the cells detach from the flask wall, add 5 mL of culture medium and pipette to form a single-cell suspension. Mix 10 μL of the cell suspension with 10 μL of PBS in a new EP tube, pipette to a cell counting chamber, insert the chamber into the cell counter, click "capture count," and record the number of viable cells. Based on the counting results, add culture medium to adjust the cell density to 1 × 10⁻⁶ cells / cells. 5 Cells / mL were seeded into 96-well plates (excluding edge wells), with 100 μL of cell suspension added to each well. PBS was added to each edge well, 100 μL, and the plates were incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0173] Weigh the Ganoderma lucidum extracts GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 prepared in Example 7 using an analytical balance and place them into EP tubes. Add DMSO to prepare a sample stock solution with a concentration of 100 mg / mL. After the sample is fully dissolved, sterilize it by filtering it through a 0.22 μm filter membrane and aliquot it for storage at -20℃. In experiments, dilute the samples to appropriate concentrations using McCoy's 5A medium to obtain gradient concentration solutions of the Ganoderma lucidum extracts GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 prepared in Example 7.

[0174] After plating, HCT116 cells were co-cultured with gradient concentration solutions of Ganoderma lucidum extracts GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 prepared in Example 7 for 24 hours. The original culture medium was then discarded, and tumor cell viability was detected using a CCK-8 assay kit. The specific steps were as follows:

[0175] Add 90 μL of fresh culture medium and 10 μL of CCK-8 solution to each well. Simultaneously, add 90 μL of fresh culture medium and 10 μL of CCK-8 solution to cell-free wells as a background control (background group). Incubate at 37°C in the dark for 2 h, and measure the absorbance (OD) at 450 nm using a multi-mode microplate reader. Calculate the relative cell viability based on the OD values ​​of each well using the following formula:

[0176]

[0177] In the formula, "experimental group" refers to the well containing solutions with different gradient concentrations of Ganoderma lucidum extract, and "control group" refers to the well containing solutions without Ganoderma lucidum extract.

[0178] The effects of different concentrations of Glu-2 on the activity of HCT116 colon cancer cells, such as Figure 1 As shown.

[0179] Calculate IC using Graphpad Prism software 50 Value. The IC50 values ​​of Ganoderma lucidum extracts GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 against HCT116 colon cancer cells. 50 The values ​​are shown in Table 3.

[0180] Table 3

[0181]

[0182] It is evident that GLe-1 exhibits the best inhibitory effect on HCT116 colon cancer cells; the extract of Ganoderma lucidum 13 shows a significantly better inhibitory effect on HCT116 colon cancer cells than existing Ganoderma lucidum strains (Comparative Example 5).

[0183] 1.2 Live / death staining assay for HCT116 colon cancer cells

[0184] HCT116 cells in the logarithmic growth phase were seeded in 6-well plates, with 5 × 10⁶ cells per well. 4 Cells were cultured for 24 hours to allow them to adhere and grow. After 24 hours, the culture medium was replaced with fresh medium containing 200 μg / mL of GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5, and incubated for 6 hours, 8 hours, and 12 hours, respectively. Cells were then collected by pipetting into centrifuge tubes and centrifuged at 1000 rpm for 4 minutes. The collected cells were stained using the Calcein-AM / PI live / dead cell double staining kit. After staining, the staining solution was removed by centrifugation, and the cells were resuspended in serum-free McCoy's 5A medium at 37°C, adjusting the cell concentration to 5 × 10⁶ cells / mL. 4 Cells were resuspended at a density of 1 cell / mL and plated in a confocal dish. The cells were allowed to settle for 15 minutes. Confocal images were collected, and the number of red and green fluorescent cells was counted, calculating the percentage. Red fluorescent cells represent dead cells stained with PI, and green fluorescent cells represent live cells stained with Calcein AM. The experimental results are shown in Table 4.

[0185] Table 4

[0186]

[0187] It is evident that, compared to existing Ganoderma lucidum strains, the extract of Ganoderma lucidum No. 13 exhibits a stronger killing effect on HCT116 cells.

[0188] 1.3 Inhibition assay of A549 human non-small cell lung cancer cell line and HepG2 human liver cancer cell line

[0189] Compared to section 1.1, the only difference in the experimental procedure was that the culture medium for the A549 human non-small cell lung cancer cell line was changed from McCoy's 5A medium to F-12K medium, and the culture medium for the HepG2 human liver cancer cell line was changed from McCoy's 5A medium to EMEM medium. The extracts of Ganoderma lucidum (GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 showed IC50 effects on the above two cancer cell lines. 50 The values ​​are shown in Table 5.

[0190] Table 5

[0191]

[0192] It is evident that, compared to existing Ganoderma lucidum strains, the Ganoderma lucidum strain 13 provided by this invention has a better effect on killing lung cancer and liver cancer.

[0193] 2. Evaluation of the anti-tumor effect of in vitro three-dimensionally grown (3D) tumor cells

[0194] Assay of antitumor activity of extracts from HCT116 cells in three-dimensional (3D) culture

[0195] Transfer the microfluidic plate to the biosafety cabinet, remove the lid, and add 50 μL of PBS to each of the three observation wells using a multichannel or reusable pipette.

[0196] After HCT116 cells were digested with trypsin to detach from the flask wall, 5 ml of culture medium was added to stop the digestion. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. Cell counting was then performed.

[0197] Thaw the Matrigel gel overnight at 4°C. After cell counting, transfer a portion of the cells to a 1.5 ml EP tube, centrifuge again, discard the supernatant, and pre-chill the EP tube containing the cells on ice. Resuspend the cells in a certain amount of ECM (cell density 2 × 10⁻⁶). 7(cells / ml). Before adding the cells, pre-cool the pipette tip in the ECM. Add 2 μL of ECM cell solution (400 μm plate) to each chip, using the pipette tip along the empty channel 1 in the microfluidic chip to allow the gel to enter the channel. Observe the gel formation using a microscope through the "viewing window" 3 wells. If the gel does not completely fill the channel, an additional droplet of 1 μL can be added.

[0198] After cell gelation, the cells were incubated at 37°C in a CO2 incubator for 15 min. 25 μL of culture medium was then added to the four outlet channels, and the cells were incubated at 37°C in a CO2 incubator for 45 min. Subsequently, 100 μL of culture medium was added to the two outlet channels. For perfusion culture, the plates were placed on the matching rocker platform (set to a 7-degree angle, 8-min shaking interval) to obtain a continuous bidirectional average flow rate of 1 μL / min. For static culture, the culture plates were placed horizontally in a cell culture incubator. On days 1 and 2 after seeding, the culture plates were observed under a microscope to confirm cell status and viability. The culture medium was changed every 3 days. On day 7, bright-field photography was used to observe and compare cell proliferation and spheroid formation under perfusion and static culture conditions.

[0199] After the cells were seeded on the organ-on-a-chip and cultured for 7 days, the HCT116 cell line was exposed to different extract solutions at a concentration of 100 μg / mL and cultured for 48 h. The cells were then detected using a high-throughput imaging system based on the Molecular Devices Image XPress® MicroConfocal confocal high-content imaging system.

[0200] After treatment, the culture medium was aspirated from the culture plate, and NucBlue and propidium iodide (PI) were added to fresh complete culture medium, followed by staining with an organ-on-a-chip. The culture plate was incubated for 1 h in a cell culture incubator under either shaking or horizontal still conditions. Subsequently, the cell culture plate was observed and photographed using a high-content imaging system. The number of PI-positive cells (dead cells) was quantified, and the survival rate was calculated by determining the ratio of live cells (total nuclei minus PI-positive cells) to the total cell count.

[0201] Tumor cell inhibition rate = 1 - survival rate.

[0202] Among them, the experimental results of the GLe-1 group are as follows: Figure 2 As shown, this component has a strong inhibitory effect on the growth of HCT116 3D cultured cells. The tumor cell inhibition rate can reach 32.11%.

[0203] Furthermore, the inhibitory effect was tested in other groups. The test results are summarized in Table 6.

[0204] Table 6

[0205]

[0206] It is evident that, compared to existing Ganoderma lucidum strains, the extract GLe-1 from Ganoderma lucidum strain 13 provided by this invention has a better inhibitory effect on three-dimensional HCT116 tumor tissue.

[0207] 3. Animal experiments

[0208] 3.1 Laboratory Animals

[0209] Five-week-old female Balb-c / nu mice (purchased from Vital River) were housed in an SPF-grade standard environment for one week to acclimatize. HCT-116 human colon cancer cells were cultured to the logarithmic growth phase, collected, centrifuged, and counted. 5 × 10⁵ cells were subcutaneously injected into the flank of the mice. 6 1 HCT-116 cells. Continue culturing until the tumor volume reaches 85-100 mm. 3 This is considered a successful model establishment.

[0210] Tumor volume = 0.5 × a 2 ×b.

[0211] Where a is the tumor width and b is the tumor length, both in mm.

[0212] After successful modeling, the mice were randomly divided into groups of 5 mice each. The specific grouping details are as follows:

[0213] Blank control group: Unmodeled mice were given 0.1 mL of deionized water by gavage daily.

[0214] Modeling control group: Mice that successfully modeled the mice were given 0.1 mL of deionized water by gavage daily.

[0215] Example group / comparative group: Mice that successfully modeled the mice were administered the actual powder suspension of each example / comparative example by gavage daily (100 mg / kg mouse body weight / day, approximately 20 g / L suspension administered by gavage 0.1 mL daily).

[0216] The preparation method of Ganoderma lucidum fruiting body powder suspension is as follows:

[0217] Take dried Ganoderma lucidum fruiting bodies, pulverize them using a traditional Chinese medicine pulverizer, grind them in a mortar and pestle, and pass them through a 200-mesh sieve to obtain Ganoderma lucidum fruiting body powder. Mix the Ganoderma lucidum fruiting body powder with deionized water at a ratio of 20 g / L, and sonicate until homogeneous to obtain a Ganoderma lucidum fruiting body powder suspension.

[0218] Starting from the first day of oral administration of the Ganoderma lucidum fruiting body powder suspension, the size and volume of mouse tumors were measured and calculated daily. Daily tumor size was summarized to evaluate the efficacy of the tumor treatment experiment. Daily weight gain was summarized to reflect the survival status of the mice.

[0219] Results of mouse tumor size experiment as follows Figure 3 As shown in Table 7, the tumor volume sizes of Examples 1-5, Comparative Example 5, and the modeling control group on days 13 and 15 after drug administration are shown in Table 7 (mean ± standard deviation, unit mm). 3 ).

[0220] Table 7

[0221]

[0222] Table 7 compares data in the same column. This indicates that each example / comparative example group had a significant difference compared to the modeling control group, with p < 0.0001. This indicates that each example / comparative example group had a significant difference compared to the modeling control group, with p < 0.001. This indicates that each example / comparative example group had a significant difference compared to the modeling control group, with p < 0.01. This indicates that each example / comparative example group had a significant difference compared to the modeling control group, and p < 0.05; #### This indicates that Example 1 group had a significant difference compared to other Example / Comparative Example groups, with p < 0.0001. ## This indicates that Example 1 group had a significant difference compared to other Example / Comparative Example groups, with p < 0.01. # This indicates that Example 1 group had significant differences compared to other Example / Comparative groups, with p < 0.05.

[0223] It can be seen that, by Figure 3 As shown in Table 7, the Ganoderma lucidum provided by this invention has a significantly better therapeutic effect on colorectal cancer compared with the Ganoderma lucidum powder of Hu Nong Ganoderma No. 1 variety in Comparative Example 5. Moreover, the Ganoderma lucidum fruiting body powder provided in Example 1 has a better therapeutic effect.

[0224] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-tumor red mushroom, characterized in that, Classification name is Ganoderma sichuanense Xianzhi No. 13 has been preserved in China Center for Type Culture Collection on October 25, 2024, and the preservation number is CCTCC NO: M 20242347.

2. The red mushroom according to claim 1, characterized in that, The weight content of Ganoderma lucidum polysaccharide in the mature dried fruiting body of the Ganoderma lucidum is greater than or equal to 1.01%, and the weight content of triterpene acid is greater than or equal to 0.45%.

3. The cultivation method of Ganoderma lucidum according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: S1, inoculating Ganoderma lucidum spores on a potato glucose agar medium, sealing and culturing at a constant temperature of 27-30°C to obtain immobilized spores; S2, inoculating the immobilized spores obtained in step S1 into a liquid spore culture medium and culturing at a constant temperature to obtain liquid spores; S3, mixing the liquid spores obtained in step S2 with bag material and sterile water, packaging, and dark culturing to obtain a fungus stick; S4, covering and cultivating the fungus stick obtained in step S3, and managing the Ganoderma lucidum to obtain Ganoderma lucidum. In step S2, the components of the liquid spore culture medium are as follows: the solvent is water, and each liter of the liquid spore culture medium contains 7.5-8.5 g of maltose, 7.5-8.5 g of sucrose, 1.5-2.5 g of arabinose, 1.5-2.5 g of lactose, 1.5-2.5 g of yeast powder, 0.15-0.25 g of sodium tripolyphosphate, 1.5-2.5 g of chlorophyll, 0.15-0.25 g of tryptophan, and 0.8-1.2 g of agar.

4. The culture method according to claim 3, characterized by, In step S3, the raw materials of the bag material consist of the following components by weight: 40.5-56.5 parts of wood chips, 0.5-1.5 parts of charcoal powder, 10-20 parts of soybean straw powder, 3-10 parts of corn cob powder, 0.3-0.7 parts of calcium carbonate powder, 0.3-0.7 parts of magnesium hydroxide powder, 2.5-3.5 parts of calcium chloride, 1.5-2.5 parts of sodium sulfate, 0.5-1.5 parts of ammonium sulfate, and 1.5-2.5 parts of sucrose. In step S4, the covering and cultivating comprises the following steps: weeding, sterilizing, and de-insecting the site, digging a pit bed, laying the fungus stick in the pit bed, covering with a film to maintain humidity for 2-3 days, opening the film, covering with soil, covering with the film again, maintaining an environmental humidity of greater than or equal to 90%, and uncovering the film to ventilate every day; 5. An anti-tumor extract of Ganoderma lucidum, characterized in that, The Ganoderma lucidum management comprises the following steps: after the Ganoderma lucidum is harvested, removing the film, adjusting the temperature to 28-32°C and the air humidity to 85%-95%, and harvesting after spores are sprayed, the edge of the cap is white, and the color of the tender part is dark.

6. The Ganoderma lucidum extract of claim 5, characterized in that, The Ganoderma lucidum is extracted from the Ganoderma lucidum obtained by the culture method according to any one of claims 1-2 or any one of claims 3-4.

7. The preparation method of the Ganoderma lucidum extract according to any one of claims 5-6, characterized in that, In the Ganoderma lucidum extract, the content of ganoderma triol is greater than or equal to 5.0 g / 100 g, the content of ganoderic acid TR is greater than or equal to 1.5 g / 100 g, the content of ganoderol F is greater than or equal to 7.5 g / 100 g, and the content of ganodermic aldehyde A is greater than or equal to 1.2 g / 100 g. The method comprises the following steps: (1) ultrasonic extraction: mixing Ganoderma lucidum with a solvent, ultrasonic extraction, filtering, repeating 2-3 times, and combining the filtrates to obtain an ultrasonic extraction liquid; (2) preparing an adsorption liquid: concentrating the ultrasonic extraction liquid obtained in (1), adding water and ethanol for blending, filtering to obtain an adsorption liquid; (3) resin adsorption: loading the adsorption liquid obtained in (2) onto a column at a flow rate of 1-2 column volumes per hour; (4) Elution: the resin after column loading in (3) is eluted with 25%-35% by weight of ethanol aqueous solution, 65%-70% by weight of ethanol aqueous solution and 95% by weight of ethanol aqueous solution in sequence, respectively, the eluate obtained by elution with 95% by weight of ethanol aqueous solution is collected, concentrated, dried to obtain the Ganoderma lucidum extract.

8. Use of the Ganoderma lucidum of any one of claims 1-2 or the Ganoderma lucidum obtained by the culture method of any one of claims 3-4 or the Ganoderma lucidum extract of any one of claims 5-6 or the Ganoderma lucidum extract prepared by the preparation method of claim 7 in the preparation of an antitumor drug.

9. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The effective component of the antitumor drug comprises the Ganoderma lucidum of any one of claims 1-2 or the Ganoderma lucidum obtained by the culture method of any one of claims 3-4 or the Ganoderma lucidum extract of any one of claims 5-6 or the Ganoderma lucidum extract prepared by the preparation method of claim 7.

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