New anti-tumor ganoderma lucidum variety, related product and preparation method and application of new anti-tumor ganoderma lucidum variety and related product

Abstract

The invention provides a new anti-tumor ganoderma lucidum variety, a related product as well as a preparation method and application of the new anti-tumor ganoderma lucidum variety, and belongs to the field of new ganoderma lucidum varieties. The new variety of ganoderma lucidum provided by the invention is classified and named as Ganoderma sichuanense Xianzhi No.13, and is preserved in the China Center for Type Culture Collection (CCTCC) on October 25, 2024, and the preservation number is CCTCC NO: M 20242347. Compared with the existing ganoderma lucidum variety, the new ganoderma lucidum variety provided by the invention has higher yield and higher content of effective components and anti-cancer active components, and the extract of the ganoderma lucidum has good anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of new varieties of Ganoderma lucidum, and relates to a new variety of Ganoderma lucidum with anti-tumor properties, related products, and a preparation method and application thereof. Background Art

[0002] Lingzhi, also known as Sanxiu (Chu Ci), Qu, Zhi (Er Ya), Lingzhicao (Yunnan Materia Medica), Mu Lingzhi (Hangzhou Medicinal Plants), and Jun Lingzhi (National Compendium of Chinese Herbal Medicines), is the fruiting body of the Ganoderma lucidum or Ganoderma sinensis fungus of the Polyporaceae family. It is a saprophytic fungus. Its cap is corky and stalked. Its cap is semicircular or kidney-shaped, 10-20 cm in diameter. The flesh is 1.5-2 cm thick, brownish-yellow or reddish-brown on the surface, fading to pale yellow at the edges with concentric rings, slightly wrinkled or smooth, with a lacquer-like luster and slightly dented edges. The flesh is milky white, light brown near the tube. The tube is 1 cm long, with a nearly circular opening. The stipe is cylindrical, lateral or lateral, occasionally mid-stalked. It is 10-19 cm long and 1.5-4 cm thick, similar in color to the cap. The hyphae within the cortex are rod-shaped, with a bulging tip. The mycelial system is trisomic: reproductive hyphae are transparent and thin-walled; skeletal hyphae are yellowish-brown with a nearly solid posterior wall; and entangled hyphae are colorless, with a curved posterior wall and uniformly branched. Spores are ovate, double-walled, truncated at the apex, with a transparent outer wall and a light brown inner wall with small spinules, measuring (9-11) μm x (6-7) μm. Wild Ganoderma lucidum often grows in the rhizosphere of sunny Fagaceae and Pinus plants or on dead wood.

[0003] Ganoderma lucidum's fruiting bodies, mycelium, and spores contain over 100 triterpenoids, most of which are highly oxidized lanostane derivatives. These include the ganoderic acid family, the ganoderic-22-enoic acid family, the ganoderic oxalic acid family, 2,2,2,3-dimethyleneganoderic oxalic acid, the monoganoderic acid family, ganoderic spore acid A, the danzhiol family, ganoderic alcohol A, ganoderic alcohol B, ganoderic terpene diols, ganoderic terpene triols, ganoderic terpene ketone diols, ganoderic terpene ketone alcohols, ganoderic aldehydes A and B, epoxyganoderic alcohols A, B, and C, ganoderic terpene ketones A and B, ganoderic spore lactones A and B, ganoderic sterones, and many more. Ganoderma lucidum also contains various polysaccharides. Traditional Chinese Medicine believes that Ganoderma lucidum is sweet and neutral, and enters the lung, heart, and spleen meridians. Ganoderma lucidum strengthens qi, nourishes the heart, and calms the mind. It is mainly used to treat weakness, loss of appetite, palpitations, insomnia, dizziness, fatigue, chronic cough and asthma, coronary heart disease, hypertension and hyperlipidemia.

[0004] Chinese patent application No. 201811387888.3 discloses the use of white-fleshed Ganoderma lucidum polysaccharide in the preparation of anti-tumor drugs. The preparation method of white-fleshed Ganoderma lucidum polysaccharide comprises the following steps: (1) extraction; (2) alcohol precipitation; (3) freeze-drying; and (4) separation and purification. This technology provides a new use for white-fleshed Ganoderma lucidum polysaccharide, which can effectively inhibit tumor growth and can be used to prepare anti-tumor drugs. It can also serve as a synergist for anti-tumor drugs such as paclitaxel, and has excellent medicinal value.

[0005] Chinese patent application number 202110744686.5 discloses a new Ganoderma lucidum strain, ZL167, which is deposited with the General Microbiology Center of the China Association for the Collection of Microorganisms under the accession number CGMCC No. 22443. The technology also provides Ganoderma polysaccharides extracted from this new strain, which increase ICAM-1 expression in endothelial cells by activating the NF-κB pathway and further enhance T cell tumor infiltration, exerting a tumor-suppressing effect. This Ganoderma lucidum strain and polysaccharide have excellent application value in the fields of food, health products, and pharmaceuticals.

[0006] Chinese patent application 201610058137.1 discloses a strain of Ganoderma lucidum (Levi's antlers) and its applications. This strain, designated NCPSLZ1, has a CCTCC deposit number of M2015796 and was deposited with the China Center for Type Culture Collection on December 29, 2015, at Wuhan University. The mycelium and fermentation broth of this strain can be used to prepare anti-tumor drugs.

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

[0008] Terms and Claims of the Present Invention: 1. As used herein, the articles "a", "an" or "the" include plural referents unless expressly limited to one or more referents in other ways.

[0009] 2. As used herein, numerical ranges: Unless expressly stated otherwise, all ranges or ratios disclosed herein are to be understood to include any and all subranges or subratios contained therein. For example, a range or ratio stated as 1 to 30 is to be considered inclusive of any and all subranges or subratios, integers, decimals, or subranges or subratios comprised therein, including the minimum value of 1 and the maximum value of 30.

[0010] In view of the problem that the existing technology cannot provide a new variety of Ganoderma lucidum with better anti-tumor effect, the purpose of the present invention is to provide a new variety of Ganoderma lucidum with anti-tumor effect, related products and their preparation method and application.

[0011] To achieve the above-mentioned purpose of the invention, in a first aspect, the present invention provides a red ganoderma lucidum, named Xianzhi No. 13 (also known as Kangai No. 1).

[0012] The Ganoderma lucidum strain was deposited in the China Center for Type Culture Collection (referred to as Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on October 25, 2024. The strain is named Xianzhi No. 13 (also known as Kangai No. 1) in Chinese and classified as Xianzhi No. 13, the preservation number is CCTCC NO: M 20242347.

[0013] The aforementioned Ganoderma lucidum strain has the following biological characteristics: Xianzhi No. 13 exhibits typical Ganoderma lucidum morphology, with an umbrella-shaped fruiting body and a kidney-shaped cap, measuring 8-15 cm in diameter and 1-2 cm thick. The rind is hard, lustrous, and yellowish-brown, with annular ridges and radiating wrinkles. The margins are thin, flat, and slightly curled, with a yellowish-white underside.

[0014] Preferably, the weight content of Ganoderma lucidum polysaccharide in the mature dry fruiting body of Ganoderma lucidum is ≥1.01%, and the weight content of triterpene acid is ≥0.45%.

[0015] The "triterpene acids" mentioned above refer to triterpene compounds with carboxyl groups contained in higher fungi such as Ganoderma lucidum or Ganoderma lucidum, 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.

[0016] As an example of the present invention, the triterpene acid includes ganoderic acid TR.

[0017] In a second aspect, the present invention provides a method for cultivating the above-mentioned Ganoderma lucidum, comprising the following steps: S1. Inoculate the strain onto potato dextrose agar medium, seal it and culture it at a constant temperature of 27-30°C to obtain the immobilized strain; S2. Inoculate the immobilized bacterial strain obtained in step S1 into a liquid bacterial culture medium and culture at a constant temperature to obtain a liquid bacterial strain; S3, mixing the liquid spawn obtained in step S2 with bag material and sterile water, packaging, and dark culturing to obtain bacterial sticks; S4, the mushroom sticks obtained in step S3 are covered with soil for cultivation, and Ganoderma lucidum is managed to obtain Ganoderma lucidum.

[0018] More preferably, the composition of the liquid culture medium described in step S2 is: the solvent is water, 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 powder, 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.

[0019] More preferably, the composition of the liquid culture medium described in step S2 is: the solvent is water, and each liter of liquid culture medium contains 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, and the pH is 5.8.

[0020] More preferably, the raw materials of the bag material in step S3 are composed of the following components by weight: 40.5-56.5 parts of sawdust, 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.

[0021] More preferably, the raw materials of the bag material in step S3 are composed of the following components by weight: 50 parts of sawdust, 1 part of charcoal powder, 15 parts of soybean straw powder, 5 parts of corn cob powder, 0.5 parts of calcium carbonate powder, 0.5 parts of magnesium hydroxide powder, 3 parts of calcium chloride, 2 parts of sodium sulfate, 1 part of ammonium sulfate and 2 parts of sucrose.

[0022] Preferably, in step S4, the soil covering cultivation includes the following steps: Weed, sterilize and deinsect the site, dig pit beds, lay the mushroom sticks in the pit beds, cover with film to keep moist for 2-3 days, open the film, cover with soil, cover with film again, maintain the environmental humidity ≥90%, and remove the film for ventilation every day.

[0023] More preferably, in step S4, the soil covering cultivation comprises the following steps: Weed, sterilize, and deworm the site. Choose flat land with good water retention and easy drainage and irrigation. Dig a pit bed 12-15cm deep, 80-100cm wide, and of any length. Remove the film bags from the mushroom sticks and lay them horizontally in the pit bed. Cover with polypropylene film to retain moisture for 2-3 days, then remove the film. Cover with 1cm of crushed, sieved soil with a moisture content of 65%-70%, and cover again with polypropylene film. Maintain an ambient humidity of ≥90%. Remove the film daily to ventilate for 20-40 minutes, preferably 30 minutes.

[0024] Preferably, in step S4, the output management includes the following steps: After the mushrooms emerge, remove the film, adjust the temperature to 28-32℃, and the air humidity to 85%-95%. Cultivate until spores begin to spray and the edge of the cap turns from white to milky yellow to darker in color before harvesting.

[0025] More preferably, in step S4, the output management includes the following steps: After about 3-5 days of cultivation, remove the polypropylene film, adjust the temperature to 28-32℃, and the air humidity to 85%-95%. Cultivate until spores begin to spray and the edge of the cap turns from white to milky yellow to darker in color before harvesting.

[0026] In a third aspect, the present invention provides an anti-tumor Ganoderma lucidum extract prepared from the above-mentioned Ganoderma lucidum.

[0027] Preferably, in the Ganoderma lucidum extract, the content of ganoderinone triol is ≥5.0g / 100g, the content of ganoderic acid TR is ≥1.5g / 100g, the content of ganoderin alcohol F is ≥7.5g / 100g, and the content of ganoderic aldehyde A is ≥1.2g / 100g.

[0028] More preferably, in the Ganoderma lucidum extract, the content of ganoderinone triol is ≥5.52g / 100g, the content of ganoderic acid TR is ≥2.15g / 100g, the content of ganoderin alcohol F is ≥7.98g / 100g, and the content of ganoderic aldehyde A is 2.04g / 100g-2.52g / 100g.

[0029] More preferably, in the Ganoderma lucidum extract, the content of ganoderin triol is ≥6.33g / 100g, the content of ganoderic acid TR is ≥2.47g / 100g, the content of ganoderin F is ≥9.15g / 100g, and the content of ganoderic aldehyde A is 2.26g / 100g-2.44g / 100g.

[0030] Most preferably, and as a specific example of the present invention, in the Ganoderma lucidum extract, the content of ganoderin triol is 6.72g / 100g, the content of ganoderic acid TR is 2.62g / 100g, the content of ganoderin F is 9.71g / 100g, and the content of ganoderin aldehyde A is 2.40g / 100g.

[0031] In a fourth aspect, the present invention further provides a method for preparing a Ganoderma lucidum extract, comprising the following steps: (1) Ultrasonic extraction: Mix Ganoderma lucidum with solvent, sonicate, filter, repeat 2-3 times, and combine the filtrate to obtain ultrasonic extract; (2) Preparation of the adsorption solution: Concentrate the ultrasonic extract obtained in (1), add water and ethanol, and filter to obtain the adsorption solution; (3) Resin adsorption: The adsorbed solution obtained in (2) was loaded onto the column at a flow rate of 1-2 column volumes per hour (BV / h); (4) Elution: The resin loaded in (3) is eluted with 25%-35% by weight aqueous ethanol solution, 65%-70% by weight aqueous ethanol solution, and 95% by weight aqueous ethanol solution, respectively. The eluate obtained by elution with 95% by weight aqueous ethanol solution is collected, concentrated, and dried to obtain the Ganoderma lucidum extract.

[0032] Preferably, in step (1), the solvent is an ethanol aqueous solution, more preferably an 80%-99% ethanol aqueous solution, and even more preferably a 95% ethanol aqueous solution.

[0033] Preferably, in step (1), the weight of the solvent is 10-20 times the weight of the Ganoderma lucidum, more preferably 15 times.

[0034] Preferably, in step (1), the ultrasonic time is 1-60 min, more preferably 10-45 min, and even more preferably 30 min. Preferably, in step (1), the number of repetitions is 3 times.

[0035] Preferably, in step (2), the concentration is vacuum concentration; More preferably, the temperature of the vacuum concentration is 60-70°C, and even more preferably 65°C.

[0036] Preferably, in step (2), the concentration is to concentrate the weight of the solution to 6-8 times the weight of the Ganoderma lucidum in step (1), more preferably 7 times the weight of the Ganoderma lucidum in step (1).

[0037] Preferably, in step (2), the adding of water and ethanol is specifically: Water is added to the concentrated solution until the weight concentration of ethanol accounts for 20%-35% of the total solution, and then diluted with ethanol of the same weight concentration until the total weight of the solution is 12-16 times the weight of the Ganoderma lucidum in step (1).

[0038] More preferably, in step (2), the adding of water and ethanol is specifically: Water is added to the concentrated solution until the weight concentration of ethanol accounts for 25% of the total solution, and then diluted with ethanol of the same weight concentration (25%) until the total weight of the solution is 14 times the weight of the Ganoderma lucidum described in step (1).

[0039] Preferably, in step (3), the resin is a macroporous resin.

[0040] Furthermore, the type of the macroporous resin is selected from one or more of the following: ADS-7, ADS-17, DM-130, D-101, AB-8, X-5.

[0041] As an example of the present invention, the model of the macroporous resin is D-101.

[0042] Preferably, in step (3), the resin is pretreated.

[0043] More preferably, the resin is pretreated by the following steps: Soak the resin in an ethanol-water solution, load the column, rinse with water until there is no alcohol smell, and rinse with an ethanol-water solution of the same weight concentration as in step (2).

[0044] Furthermore, the concentration of the ethanol aqueous solution is 90%-99%, and further 95%.

[0045] Furthermore, the column is packed with 0.5-1.5 times the weight of the Ganoderma lucidum in step (1), and further packed with 1 times the weight of the Ganoderma lucidum.

[0046] Furthermore, the flushing volume of the ethanol aqueous solution with the same weight concentration as that in step (2) is 1-2 column volumes (BV), and further 2 BV.

[0047] Preferably, in step (4), the amount of ethanol aqueous solution with a weight concentration of 25%-35% is 2-5 BV, more preferably 4 BV.

[0048] Preferably, in step (4), the amount of 65%-70% weight concentration of ethanol aqueous solution used is 7-10 BV, more preferably 8 BV.

[0049] Preferably, in step (4), the amount of 95% by weight ethanol aqueous solution used is 4-5 BV, more preferably 5 BV.

[0050] Preferably, in step (4), the drying is freeze-drying.

[0051] In a fifth aspect, the present invention also provides the use of the above-mentioned Ganoderma lucidum extract or the Ganoderma lucidum extract prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0052] The Ganoderma lucidum extract can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, etc., and suitable pharmaceutical carriers in the art can be selected for different dosage forms.

[0053] The pharmaceutical carrier used can be a solid, liquid or gas. 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.

[0054] When preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, ethanol, oils, alcohols, flavoring agents, preservatives, colorants, and the like can be used to form oral liquid formulations such as suspensions, elixirs, and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, and disintegrants can be used to form oral solid formulations 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 nonaqueous techniques.

[0055] Tablets containing the Ganoderma lucidum extract of the present invention can be prepared by tableting or molding, optionally with one or more auxiliary ingredients or adjuvants. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as a powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersant. Molded tablets can be molded in a suitable machine from a powdered mixture of the compound moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of the active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of the active ingredient. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to about 5 g of the active drug, mixed with an appropriate 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 the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0056] Pharmaceutical compositions suitable for parenteral administration of the present invention can be prepared as aqueous solutions or suspensions of the active compound. Suitable surfactants, such as hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and oil mixtures thereof. In addition, preservatives can be added to prevent the harmful growth of microorganisms.

[0057] The medicament prepared from the Ganoderma lucidum extract of the present invention can be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, powder, or the like. Furthermore, the medicament can be used in a suitable form for transdermal administration. These formulations can be prepared using the Chinese medicine composition of the present invention by conventional processing methods. For example, a cream or ointment having a desired consistency can be prepared by mixing a hydrophilic material and water with about 5 wt % to about 10 wt % of the compound.

[0058] The medicament prepared from the Ganoderma lucidum extract of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture is prepared into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a composition mixed with a softened or melted carrier, followed by cooling and shaping in a mold.

[0059] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants and flavoring agents, etc., may be added. The preparation is made isotonic with the blood of the intended recipient. The components containing the Chinese medicine composition of the present invention can also be prepared in the form of a powder or concentrate.

[0060] The tumors include, but are not limited to: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; adnexal carcinoma; basal cell carcinoma; extrahepatic bile duct carcinoma; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; childhood brainstem glioma, adult brain tumor; atypical teratoid central nervous system brain tumor, childhood rhabdoid tumor; central nervous system germinal tissue tumor; cerebellar astrocytoma; cerebral astrocytoma, malignant glioma; craniopharyngioma; ependymoma; ependymoma; medulloblastoma; medullary epithelioma; moderately differentiated pineal parenchymal tumor; supratentorial primitive neuroectodermal tumor and pinealoblastoma; optic pathway and hypothalamic glioma; brain and spinal cord tumor; breast cancer; bronchial tumor; Burkitt's lymphoma; benign CNS atypical teratoid rhabdoid tumor; CNS embryonal tumor; CNS lymphoma; cervical cancer; childhood chordoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disease; colon cancer; colorectal cancer; cutaneous lymphoma; esophageal cancer; Ewing's tumor; extragonadal malignant germ cell tumor; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; benign gastrointestinal tumor; gastrointestinal stromal tumor; extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; gestational trophoblastic tumor; glioma; childhood brainstem glioma; glioma, childhood cerebral astrocytoma; childhood optic tract and hypothalamic glioma; hairy cell leukemia; head and neck cancer; hepatocellular carcinoma carcinoma; Hodgkin lymphoma; hypopharyngeal carcinoma; hypothalamic and optic pathway gliomas; intraocular melanoma; pancreatic islet cell tumor; renal cell carcinoma of the kidney; 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 lymphoma; non-Hodgkin lymphoma; malignant fibrous histiocytoma and osteosarcoma of bone; medulloblastoma; melanoma; intraocular melanoma; Merkel cell carcinoma; mesothelioma; latent primary metastatic squamous neck cancer; multiple endocrine neoplasia syndrome in children; mycosis fungoides; myelodysplastic syndrome; myelodysplastic myeloproliferative disease; chronic myeloid leukemia; acute myeloid leukemia in adults childhood acute myeloid leukemia; chronic myeloproliferative disease; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma; ovarian cancer; ovarian epithelial cancer; ovarian germ cell cancer; ovarian low-grade carcinoma; pancreatic cancer; pancreatic islet cell carcinoma; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; moderately differentiated pineal parenchymal tumor; pinealoblastoma and supratentorial primitive neuroectodermal tumor; pituitary tumor; plasmacytoma, multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; respiratory tract cancers involving genes on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Ewing's sarcoma; Kaposi's sarcoma; uterine sarcoma;Sézary syndrome; skin cancer (non-melanoma); skin cancer (melanoma); Merkel cell skin cancer; small bowel cancer; soft tissue sarcoma; squamous cell carcinoma with latent primary metastasis; squamous neck cancer; gastric cancer; supratentorial primitive neuroectodermal tumor; testicular cancer; laryngeal cancer; thymic cancer; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic carcinoma; urethral cancer; endometrial cancer; vaginal cancer; vulvar cancer; Waldenstrom's macroglobulinemia and Wilms' tumor.

[0061] In the final aspect, the present invention further provides an anti-tumor drug, the active ingredient of which includes the above-mentioned Ganoderma lucidum extract or the Ganoderma lucidum extract prepared by the above-mentioned preparation method.

[0062] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a new Ganoderma lucidum strain Xianzhi No. 13 (also known as Kangai No. 1). Compared with existing Ganoderma lucidum strains, the Ganoderma lucidum strain provided by the present invention has the characteristics of short growth cycle, high yield, high content of effective ingredients Ganoderma lucidum polysaccharide, triterpenes and sterols, and high content of anti-tumor active ingredients.

[0063] (2) The present invention provides a method for preparing an extract of Xianzhi No. 13 (also known as Kangai No. 1). Using this method, a red ganoderma extract can be prepared. This red ganoderma extract can achieve a better tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is a bar graph showing the effect of gradient concentrations of GLe-1 on the activity of HCT116 cells.

[0065] Figure 2 This is a graph showing the anti-tumor activity of GLe-1 based on three-dimensional culture of HCT116 cells. The NucBlue channel represents the staining results of the total number of cells, and the PI channel represents the staining results of dead cells.

[0066] Figure 3 It is a line graph of the tumor therapeutic effect of the dried fruiting body powder suspension of Ganoderma lucidum / Ganoderma lucidum provided in Examples 1 to 5 and Comparative Example 5 on mice bearing HCT-116 heterogeneous tumors. DETAILED DESCRIPTION

[0067] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0068] The present invention will be further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all by weight. Unless otherwise specified, it is understood that the experiments were conducted at room temperature.

[0069] The sources of some raw materials used in the cultivation process of Ganoderma lucidum of the present invention are exemplified as follows.

[0070] Yeast powder: Adamas-life brand, product number 045717187. Sawdust was purchased from Langfang Bangke Biomass Energy Co., Ltd. Soybean straw powder was purchased from Zhengyang County Xintiandi Grass Industry Co., Ltd. Corn cob powder was purchased from Lingshou County Zhaochen Mineral Products Co., Ltd. Charcoal powder was purchased from Hebei Hengxun Mineral Products Processing Co., Ltd.

[0071] In the following examples, the strain Xianzhi No. 13 (also known as Kangai No. 1) used was deposited in the China Center for Type Culture Collection (referred to as Wuhan University Collection Center, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on October 25, 2024. The classification of this red ganoderma is named Xianzhi No. 13, with a deposit number of CCTCC NO: M 20242347. The Wuhan University Collection Center completed the viability test on Xianzhi No. 13 on November 1, 2024, and the result was viable.

[0072] The aforementioned Ganoderma lucidum strain has the following biological characteristics: Xianzhi No. 13 exhibits typical Ganoderma lucidum morphology, with an umbrella-shaped fruiting body and a kidney-shaped cap, measuring 8-15 cm in diameter and 1-2 cm thick. The rind is hard, lustrous, and yellowish-brown, with annular ridges and radiating wrinkles. The margins are thin, flat, and slightly curled, with a yellowish-white underside.

[0073] Example 1 A method for cultivating red ganoderma.

[0074] (1) Under sterile conditions, take the Xianzhi No. 13 (also known as Kangai No. 1) strain 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%. Cultivate it at constant temperature and humidity for 7 days until the colonies fill the culture dish and the fixed bacterial strain is obtained.

[0075] (2) Inoculate the immobilized bacterial strain obtained in (1) into a liquid bacterial culture medium and culture in a constant temperature shaker. The shaker culture temperature is 27-32°C and the shaking speed is 120-140 rpm. The preparation method of the liquid bacterial culture medium is as follows: Mix 8 g of maltose, 8 g of sucrose, 2 g of arabinose, 2 g of lactose, 2 g of yeast powder, 0.2 g of sodium tripolyphosphate, 1 g of chlorophyll, and 0.2 g of tryptophan with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH value to 5.8 with 0.1% acetic acid solution, make up to 1 L with water, sterilize with high temperature moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0076] After 7 days of shaking culture, liquid bacteria were obtained.

[0077] (3) Prepare cultivation bags and conduct dark cultivation.

[0078] Mix 50 parts of sawdust, 1 part of charcoal powder, 15 parts of soybean straw powder, 5 parts of corn cob powder, 0.5 parts of calcium carbonate powder and 0.5 parts of magnesium hydroxide powder by weight to obtain a solid matrix mixture. Mix 3 parts of calcium chloride, 2 parts of sodium sulfate, 1 part of ammonium sulfate, 2 parts of sucrose and an appropriate amount of water to form a suspension. Sprinkle the suspension evenly on the solid matrix mixture, stir, sterilize with high temperature and moist heat (121°C, 15 minutes), cool to room temperature and dry to obtain a bag material. Under sterile conditions, mix the above bag material, sterile water and the liquid culture medium obtained in (2) in a mass ratio of 4:6:0.25, package it into a cylindrical polypropylene film, tie the two ends tightly to form a cultivation bag. Each cultivation bag weighs about 1-1.5 kg. Puncture 20-40 small holes on the surface of the cultivation bag with a needle and culture it in the dark at 28°C. When the mycelium is densely distributed in the cultivation bag, a mushroom stick is obtained.

[0079] (4) Covering the soil for cultivation.

[0080] Conventional soil covering cultivation technology is used for cultivation. The details are as follows: Cover the mushroom sticks with soil for cultivation. Weed, sterilize, and deworm the site. Choose a flat land with good water retention and easy drainage and irrigation, and dig a pit bed with a depth of 12-15cm, a width of 80-100cm, and an unlimited length. Remove the film bags from the mushroom sticks, lay them in the pit bed, cover them with polypropylene film to keep them moist for 2-3 days, and then remove the film. Cover them with 1cm thick crushed and sieved soil with a moisture content of 65%-70%, and cover them with polypropylene film again. Maintain the ambient humidity ≥90%, and uncover the film for ventilation for 30 minutes every day. After about 3-5 days of cultivation, remove the polypropylene film, adjust the temperature to 28-32℃, and the air humidity to 85%-95%. Cultivate until spores begin to spray and the edge of the cap turns from white to milky yellow and the tender part becomes darker in color before harvesting.

[0081] Example 2 Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed to the following, and the remaining steps are the same.

[0082] Mix 7.5 g of maltose, 8.5 g of sucrose, 1.5 g of arabinose, 2.5 g of lactose, 1.5 g of yeast powder, 0.25 g of sodium tripolyphosphate, 2.5 g of chlorophyll, and 0.15 g of tryptophan with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH value to 5.8 with 0.1% acetic acid solution, make up to 1 L with water, sterilize with high temperature moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0083] Example 3 Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed to the following, and the remaining steps are the same.

[0084] Mix 8.5 g of maltose, 7.5 g of sucrose, 2.5 g of arabinose, 1.5 g of lactose, 2.5 g of yeast powder, 0.15 g of sodium tripolyphosphate, 1.5 g of chlorophyll, and 0.25 g of tryptophan with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH to 5.8 with an acid solution, make up to 1 L with water, sterilize with high temperature and moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0085] Example 4 Compared with Example 1, the only difference is that in step (3), the cultivation bag preparation method is changed to the following: Mix 40.5 parts sawdust, 0.5 parts charcoal powder, 20 parts soybean straw powder, 10 parts corncob powder, 0.3 parts calcium carbonate powder, and 0.7 parts magnesium hydroxide powder by weight to form a solid matrix mixture. Mix 3.5 parts calcium chloride, 1.5 parts sodium sulfate, 1.5 parts ammonium sulfate, 1.5 parts sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0086] Example 5 Compared with Example 1, the only difference is that in step (3), the cultivation bag preparation method is changed to the following: Mix 56.5 parts sawdust, 1.5 parts charcoal powder, 10 parts soybean straw powder, 3 parts corncob powder, 0.7 parts calcium carbonate powder, and 0.3 parts magnesium hydroxide powder by weight to form a solid matrix mixture. Mix 2.5 parts calcium chloride, 2.5 parts sodium sulfate, 0.5 parts ammonium sulfate, 2.5 parts sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0087] Comparative Example 1 Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed to the following, and the remaining steps are the same.

[0088] Mix 8 g of maltose, 8 g of sucrose, 2 g of arabinose, 2 g of lactose, 2 g of yeast powder, 0.4 g of sodium tripolyphosphate, and 1 g of chlorophyll with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH value to 5.8 with 0.1% acetic acid solution, make up to 1 L with water, sterilize with high temperature moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0089] Comparative Example 2 Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed to the following, and the remaining steps are the same.

[0090] Mix 8 g of maltose, 8 g of sucrose, 2 g of arabinose, 2 g of lactose, 2 g of yeast powder, 0.2 g of sodium tripolyphosphate, 1 g of chlorophyll, and 0.2 g of lysine with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH value to 5.8 with 0.1% acetic acid solution, make up to 1 L with water, sterilize with high temperature moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0091] Comparative Example 3 Compared with Example 1, the only difference is that in step (2), the preparation method of the liquid culture medium is changed to the following, and the remaining steps are the same.

[0092] Mix 8 g of maltose, 8 g of sucrose, 2 g of arabinose, 2 g of lactose, 2 g of yeast powder, 0.2 g of sodium tripolyphosphate, 1 g of magnesium sulfate, and 0.2 g of tryptophan with an appropriate amount of water until completely dissolved. Mix 1 g of agar with an appropriate amount of water until completely dissolved. Mix the two solutions evenly, adjust the pH value to 5.8 with 0.1% acetic acid solution, make up to 1 L with water, sterilize with high temperature moist heat (121°C, 15 min), and cool to room temperature to obtain a liquid culture medium.

[0093] Comparative Example 4 Compared with Example 1, the only difference is that in step (3), the cultivation bag preparation method is changed to the following: Mix 50 parts sawdust, 1 part charcoal powder, 15 parts soybean straw powder, 5 parts corncob powder, 0.5 part calcium carbonate powder, and 0.5 part magnesium hydroxide powder by weight to form a solid matrix mixture. Mix 3 parts calcium chloride, 3 parts sodium sulfate, 2 parts sucrose, and an appropriate amount of water to form a suspension. The remaining steps are the same.

[0094] Comparative Example 5 Compared with Example 1, the only difference is that the Xianzhi No. 13 strain is replaced by an equal amount of the Shanghai Agricultural Ganoderma Lucidum No. 1 strain, and the rest are the same.

[0095] In Comparative Example 5, Shanghai Agricultural Lingzhi No. 1 is a Lingzhi variety known to those skilled in the art, and is consistent with the Shanghai Agricultural Lingzhi No. 1 recorded in existing technologies such as the local standard DB45 / T 2918-2024 "Shanghai Agricultural Lingzhi No. 1 Cultivation Technical Regulations", and is a recognized variety in Shanghai Agricultural Products Recognition of Edible Fungi (2009) No. 003.

[0096] Effect Example 1 Evaluation of the culture methods in Examples 1 to 5 and Comparative Examples 1 to 5.

[0097] The production cycle, yield, and polysaccharide content, triterpenoid and sterol content in the Ganoderma lucidum / Ganoderma lucidum fruiting bodies of the cultivation methods provided in Examples 1 to 5 and Comparative Examples 1 to 5 were tested.

[0098] The production cycle is defined as the time from inoculation of the strain (Xianzhi No. 13 / Shanghai Nong Lingzhi No. 1) on PDA culture medium to harvesting of Ganoderma lucidum / Ganoderma lucidum, measured in days. Yield is defined as the mass of dry Ganoderma lucidum / Ganoderma lucidum fruiting bodies produced per kilogram of bagged material within a production cycle, measured in g / kg.

[0099] The polysaccharide content in the produced Ganoderma lucidum / Ganoderma lucidum fruiting bodies was detected. 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 Ganoderma lucidum polysaccharide, triterpenoid and sterol contents in the dried and mature Xianzhi No. 13 / Shanghai Agricultural Ganoderma lucidum No. 1 fruiting bodies in each embodiment and comparative example were detected. The content of Ganoderma lucidum polysaccharide was calculated based on the dry product. The content of Ganoderma lucidum polysaccharide was determined by anhydrous glucose (C6H 12 O6), the unit is mass percentage (%).

[0100] The method provided in Example 1 of the prior art Chinese invention patent CN118817912A was used to detect the ganoderic acid TR content in the produced Ganoderma lucidum / Ganoderma lucidum fruiting bodies, and the unit is mass percentage (%).

[0101] The evaluation results of the culture methods provided in Examples 1 to 5 and Comparative Examples 1 to 5 are summarized in Table 1 below: Table 1

[0102] Example 6 A method for preparing a Ganoderma lucidum extract. The steps are as follows: (1) Ultrasonic extraction.

[0103] Mix Ganoderma lucidum with 15 times its weight of 95% ethanol in water, sonicate for 30 minutes each time, and filter. Repeat three times. Combine the filtrates to obtain the ultrasonic extract.

[0104] (2) Prepare the adsorption solution.

[0105] The ultrasonic extract obtained in step (1) was vacuum concentrated (65°C) to a total weight of 7 times the weight of the Ganoderma lucidum in step (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 the weight of the Ganoderma lucidum in step (1). Filter to obtain the adsorption solution; (3) Resin adsorption.

[0106] Soak the macroporous resin D-101 in a 95% ethanol solution by weight and load the column with 1 times the weight of the Ganoderma lucidum obtained in step (1). Rinse the column with purified water until the alcohol smell is gone, then rinse the column with 2 BV of a 25% ethanol solution by weight. Load the adsorbent obtained in step (2) onto the column at a flow rate of 2 column volumes per hour (BV / h).

[0107] (4) Elution.

[0108] The resin loaded in (3) was eluted with 4 BV of 30% by weight aqueous ethanol solution, 8 BV of 70% by weight aqueous ethanol solution, and 5 BV of 95% by weight aqueous ethanol solution, respectively. The eluate obtained by elution with 95% by weight aqueous ethanol solution was collected, concentrated, and freeze-dried to obtain the Ganoderma lucidum extract.

[0109] The Ganoderma lucidum / Ganoderma lucidum fruiting bodies obtained in Examples 1 to 5 and Comparative Examples 1 to 5 were extracted using the extraction method described in Example 6, and the obtained Ganoderma lucidum extracts were respectively designated as GLe-1, GLe-2, GLe-3, GLe-4, GLe-5, GLc-1, GLc-2, GLc-3, GLc-4 and GLc-5.

[0110] Example 7 Isolation and identification of antitumor components from Ganoderma lucidum extract.

[0111] With reference to the experimental method provided in Example 1 of Chinese invention patent CN108181396A, the contents of the anti-tumor component ganoderinonetriol (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 were detected respectively; with reference to the experimental method provided in Example 1 of Chinese invention patent CN118817912A, the contents of the anti-tumor components ganoderin acid TR (compound II), ganoderin alcohol F (compound III) and ganoderin aldehyde 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 were detected respectively.

[0112] The chemical structural formula of Ganoderma lucidum ketone triol is: .

[0113] The chemical structure of Ganoderic acid TR is: .

[0114] The chemical structural formula of Ganoderma lucidum alcohol F is: .

[0115] The chemical structure of Ganoderma lucidum aldehyde A is: .

[0116] The extraction amounts of compound I, compound II, compound III and compound IV per 100 grams of Ganoderma lucidum extract in each group were statistically analyzed, and the results are shown in Table 2.

[0117] Table 2

[0118] It can be seen that in the Xianzhi No. 13 extract provided by the present invention, the extraction amount of Compound I to Compound IV with anti-tumor effect is higher and significantly higher than that of the existing Ganoderma lucidum strain (Comparative Example 5).

[0119] Effect Example 2 Evaluation of the antitumor activity of Ganoderma lucidum extract 1. Evaluation of anti-tumor effects on in vitro two-dimensional (2D) tumor cells 1.1. Activity inhibition test on HCT116 human colon cancer cells Remove HCT116 cells in the logarithmic growth phase from the incubator and discard the original culture medium. Add 2 mL PBS to rinse the cells and discard the PBS. Add 1 mL 0.25% trypsin to digest the cells. After the cells are detached from the bottle wall, add 5 mL culture medium and use a pipette to blow the cells into a single cell suspension. Pipette 10 μL of cell suspension + 10 μL PBS in a new EP tube to mix well, draw 10 μL and add it to the cell counting plate, insert the counting plate into the cell counter, click to capture the count, and record the number of live cells. Add culture medium according to the counting results and adjust the cell density to 1×10 5 cells / mL, the cells were seeded in a 96-well plate (except the edge wells), 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to the edge wells, and the plates were cultured in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0120] Weigh GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 of the Ganoderma lucidum extract prepared in Example 7 using an analytical balance and place into EP tubes. Add DMSO to prepare a sample stock solution at a concentration of 100 mg / mL. After the sample is fully dissolved, sterilize it through a 0.22 μm filter and store it in aliquots at -20°C. During the experiment, dilute the sample to the appropriate concentration using McCoy's 5A medium to obtain gradient concentration solutions of GLe-2, GLe-3, GLe-4, GLe-5, GLe-6, GLc-1, GLc-2, GLc-3, GLc-4, and GLc-5 of the Ganoderma lucidum extract prepared in Example 7.

[0121] After plating, HCT116 cells were co-cultured with the 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 discarded and the tumor cell activity was detected using a CCK-8 kit. The specific steps are as follows: 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 wells without cells as a background control (background control). Incubate at 37°C in the dark for 2 h. Measure the absorbance (OD) at 450 nm using a multifunctional microplate reader. Calculate relative cell viability based on the OD value of each well using the following formula:

[0122] Wherein, the "experimental group" refers to the wells to which solutions of Ganoderma lucidum extract with different gradient concentrations were added, and the "control group" refers to the wells to which solutions of Ganoderma lucidum extract were not added.

[0123] Effects of different concentrations of GLe-2 on the activity of HCT116 colon cancer cells Figure 1 shown.

[0124] IC was calculated using Graphpad Prism software. 50 IC 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.

[0125] Table 3

[0126] It can be seen that GLe-1 has the best inhibitory effect on HCT116 colon cancer cells; the inhibitory effect of Xianzhi No. 13 extract on HCT116 colon cancer cells is significantly better than that of existing Ganoderma lucidum strains (Comparative Example 5).

[0127] 1.2 Live-dead staining assay for HCT116 colon cancer cells HCT116 cells in the logarithmic growth phase were plated in 6-well plates with 5×10 cells per well. 4 The cells were cultured for 24 hours to allow them to adhere to the wall and grow. After 24 hours, fresh culture 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 was replaced and incubated for 6 hours, 8 hours and 12 hours respectively. The cells were blown up with a pipette and collected in a centrifuge tube. The cells were centrifuged at 1000 rpm for 4 minutes to collect the cells. The collected cells were stained with Calcein-AM / PI live / dead cell double staining kit. After staining, the dye was removed by centrifugation and the cells were resuspended in McCoy's 5A culture medium at 37°C without serum to adjust the cell concentration to 5×10 4 The resuspended cells were plated onto a confocal dish and allowed to settle for 15 minutes. Confocal images were collected, and the number of cells with red and green fluorescent signals was counted and calculated as percentages. Red fluorescent cells represent dead cells stained with PI, while green fluorescent cells represent live cells stained with Calcein AM. The experimental results are shown in Table 4.

[0128] Table 4

[0129] It can be seen that compared with existing Ganoderma lucidum strains, the extract of Xianzhi No. 13 has a stronger killing ability against HCT116 cells.

[0130] 1.3. Activity inhibition test on A549 human non-small cell lung cancer cell line and HepG2 human liver cancer cell line Compared with Section 1.1, the only difference in the experimental process is that the culture medium of A549 human non-small cell lung cancer cell line was changed from McCoy's 5A medium to F-12K medium, and the culture medium of HepG2 human liver cancer cell line was changed from McCoy's 5A medium to EMEM medium. IC values 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 on the above two cancer cells were obtained. 50 The values are shown in Table 5.

[0131] Table 5

[0132] It can be seen that compared with existing Ganoderma lucidum strains, the Xianzhi No. 13 provided by the present invention has a better effect in killing lung cancer and liver cancer.

[0133] 2. Evaluation of the anti-tumor effect on in vitro three-dimensional (3D) tumor cells Antitumor activity assay of extracts based on three-dimensional (3D) culture of HCT116 cells Transfer the microfluidic plate to a biosafety cabinet, remove the lid, and use a multichannel or repeating pipette to add 50 μL of PBS to each of the three observation wells.

[0134] After trypsinizing HCT116 cells to remove them from the flask wall, add 5 ml of culture medium to terminate digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Count the cells and set aside.

[0135] Thaw Matrigel gel at 4℃ overnight and wait for use. After counting the cells, aspirate some cells into a 1.5 ml EP tube and centrifuge again to discard the supernatant. Place the EP tube containing cells on ice for pre-cooling and add a certain amount of ECM to resuspend the cells (cell density is 2×10 7 Before adding the gel to the microfluidic chip, pre-cool the pipette tip in ECM. Add 2 μL of ECM-containing cell solution (400 μm slab) to each chip and pipette the tip along an empty channel 1 in the microfluidic chip to allow the gel to enter the channel. Observe gel formation using a microscope through the "observation window" 3 channel. If the gel does not completely fill the channel, add an additional 1 μL droplet.

[0136] After cell gelation, the cells were incubated in a 37°C CO2 incubator for 15 minutes. 25 μL of culture medium was then pipetted into the fourth outlet channel and incubated in a 37°C CO2 incubator for 45 minutes. Subsequently, 100 μL of culture medium was pipetted into the second outlet channel. For perfusion culture, the plate was placed on a rocker platform (set at a 7-degree angle and an 8-minute rocking interval) to achieve a continuous bidirectional average flow rate of 1 μL / min. For static culture, the plate was placed horizontally in the cell culture incubator. On days 1 and 2 after seeding, the plates were observed microscopically to confirm cell status and viability. The culture medium was changed every 3 days. On day 7 of culture, brightfield photography was performed to compare cell proliferation and spheroid formation under perfusion and static culture conditions.

[0137] After the cells were seeded into the organ chip and cultured for 7 days, the HCT116 cell line was exposed to different groups of extract solutions at a concentration of 100 μg / mL and cultured for 48 hours respectively. High-throughput detection was performed using the Molecular Devices Image XPress® MicroConfocal confocal high-content imaging system.

[0138] After treatment, the culture medium was aspirated from the plate, and NucBlue and propidium iodide (PI) were added to fresh complete culture medium before the plate was added to the organ chip for staining. The plate was incubated in a cell culture incubator on a rocking platform or in a horizontal, stationary state for 1 hour. The plate was then visualized and photographed on a high-content imaging system. The number of PI-positive (dead) cells was quantified, and the viability was calculated by calculating the ratio of live cells (total nuclei minus PI-positive cells) to the total cell count.

[0139] Tumor cell inhibition rate = 1-survival rate.

[0140] Among them, the experimental effects 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, with a tumor cell inhibition rate of up to 32.11%.

[0141] Furthermore, the inhibitory effect of other groups was tested. The test results are summarized in Table 6.

[0142] Table 6

[0143] It can be seen that compared with existing Ganoderma lucidum strains, the extract GLe-1 of Xianzhi No. 13 provided by the present invention has a better inhibitory effect on three-dimensional HCT116 tumor tissue.

[0144] 3. Animal Experiments 3.1 Experimental Animals Five-week-old Balb-c / nu female mice (purchased from Vital River) were housed in a standard SPF enclosure for one week to acclimate to the environment. HCT-116 human colon cancer cell lines were cultured to the logarithmic growth phase, harvested, centrifuged, and counted. 5×10 6 HCT-116 cells were cultured until the tumor volume reached 85-100 mm 3 , the model is considered to be successfully established.

[0145] Tumor volume = 0.5 × a 2 ×b.

[0146] Wherein, a is the width of the tumor, b is the length of the tumor, both in mm.

[0147] After successful modeling, the mice with successful modeling were randomly divided into groups, with 5 mice in each group. The specific groupings are as follows: Blank control group: mice without modeling were gavaged with 0.1 mL of deionized water daily.

[0148] Modeling control group: Mice with successful modeling were gavaged with 0.1 mL of deionized water daily.

[0149] Example group / comparative example group: The mice with successful modeling were gavaged daily with the solid powder suspension of each example / comparative example (100 mg / kg mouse body weight / day, equivalent to 0.1 mL of suspension of approximately 20 g / L per day).

[0150] The preparation method of Ganoderma lucidum fruiting body powder suspension is as follows: Take dried Ganoderma lucidum fruiting body, crush it with a traditional Chinese medicine grinder, grind it in a mortar, and pass it 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 20g / L and ultrasonically homogenize to obtain a Ganoderma lucidum fruiting body powder suspension.

[0151] Starting from the first day of gavage with Ganoderma lucidum fruiting body powder suspension, the size of the mouse tumors was measured and the tumor volume was calculated daily. Tumor size was summarized daily to evaluate the efficacy of the tumor treatment experiment. Body weight gain was also summarized daily to reflect the survival status of the mice.

[0152] The results of mouse tumor size experiments are as follows Figure 3 Specifically, the tumor volumes of Examples 1 to 5, Comparative Example 5, and the modeling control group on the 13th and 15th days after administration are shown in Table 7 (mean ± standard deviation, unit mm 3 ).

[0153] Table 7

[0154] In Table 7, the data in the same column are compared. Indicates that each example / comparative example group has significant difference compared with the modeling control group and p<0.0001, Indicates that each example / comparative example group has significant difference compared with the modeling control group and p<0.001, Indicates that each example / comparative example group has significant difference compared with the modeling control group and p<0.01, Indicates that each Example / Comparative Example group has significant difference compared with the modeling control group and p<0.05; #### Indicates that the Example 1 group has significant differences compared with the other Example / Comparative Example groups and p < 0.0001, ## Indicates that the Example 1 group has significant differences compared with the other Example / Comparative Example groups and p < 0.01, # This indicates that the Example 1 group has significant differences compared with the other Example / Comparative Example groups and p<0.05.

[0155] It can be seen that Figure 3 As can be seen from the data in Table 7, the Ganoderma lucidum provided by the present invention has a significantly better effect on the treatment of colon cancer tumors than the Ganoderma lucidum powder of Shanghai Agricultural Ganoderma Lucidum No. 1 variety in Comparative Example 5, and the Ganoderma lucidum fruiting body powder provided by Example 1 has a better therapeutic effect.

[0156] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A red ganoderma lucidum, characterized in that: Classification named Ganoderma sichuanense Xianzhi No. 13 was deposited in the China Center for Type Culture Collection on October 25, 2024, with the deposit number CCTCC NO: M 20242347.

2. The red ganoderma according to claim 1, characterized in that The weight content of ganoderma lucidum polysaccharide in the mature dry fruiting body of Ganoderma lucidum is ≥1.01%, and the weight content of triterpene acid is ≥0.45%.

3. The method for cultivating red ganoderma according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Inoculate the Ganoderma lucidum strain on a potato dextrose agar medium, seal it and culture it at a constant temperature of 27-30°C to obtain the immobilized strain; S2. Inoculate the immobilized bacterial strain obtained in step S1 into a liquid bacterial culture medium and culture at a constant temperature to obtain a liquid bacterial strain; S3, mixing the liquid spawn obtained in step S2 with bag material and sterile water, packaging, and dark culturing to obtain bacterial sticks; S4, the mushroom sticks obtained in step S3 are covered with soil for cultivation, and Ganoderma lucidum is managed to obtain Ganoderma lucidum.

4. The culture method according to claim 3, wherein In step S2, the liquid culture medium comprises the following components: the solvent is water, and each liter of the liquid 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; In step S3, the raw materials of the bag material are composed of the following components by weight: 40.5-56.5 parts of sawdust, 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.

5. The culture method according to claim 3, wherein In step S4, the soil covering cultivation includes the following steps: weeding, sterilizing, and deworming the site, digging a pit bed, laying the mushroom sticks in the pit bed, covering with a film to keep them moist for 2-3 days, opening the film, covering with soil, and covering with a film again, maintaining the ambient humidity ≥90%, and uncovering the film for ventilation every day; The mushroom management includes the following steps: after the mushrooms are grown, the film is removed, the temperature is adjusted to 28-32°C, and the air humidity is adjusted to 85%-95%. The mushrooms are cultured until they begin to spray spores and the white to milky yellow tender part of the cap edge turns darker in color before they can be harvested.

6. An anti-tumor Ganoderma lucidum extract, characterized in that: The invention is extracted from the Ganoderma lucidum described in any one of claims 1-2 or the Ganoderma lucidum obtained by the cultivation method described in any one of claims 3-5.

7. The red ganoderma extract according to claim 6, characterized in that In the Ganoderma lucidum extract, the content of ganoderin triol is ≥5.0g / 100g, the content of ganoderic acid TR is ≥1.5g / 100g, the content of ganoderin alcohol F is ≥7.5g / 100g, and the content of ganoderic aldehyde A is ≥1.2g / 100g.

8. The method for preparing the Ganoderma lucidum extract according to any one of claims 6 to 7, characterized in that: The following steps are involved: (1) Ultrasonic extraction: Mix Ganoderma lucidum with solvent, sonicate, filter, repeat 2-3 times, and combine the filtrate to obtain ultrasonic extract; (2) Preparation of the adsorption liquid: Concentrate the ultrasonic extraction liquid obtained in (1), add water and ethanol to mix, and filter to obtain the adsorption liquid; (3) Resin adsorption: The adsorbed solution obtained in (2) is loaded onto the column at a flow rate of 1-2 column volumes per hour; (4) Elution: The resin loaded in (3) is eluted with 25%-35% by weight aqueous ethanol solution, 65%-70% by weight aqueous ethanol solution, and 95% by weight aqueous ethanol solution, respectively. The eluate obtained by elution with 95% by weight aqueous ethanol solution is collected, concentrated, and dried to obtain the Ganoderma lucidum extract.

9. Use of the Ganoderma lucidum according to any one of claims 1-2, or the Ganoderma lucidum obtained by the cultivation method according to any one of claims 3-5, or the Ganoderma lucidum extract according to any one of claims 6-7, or the Ganoderma lucidum extract obtained by the preparation method according to claim 8 in the preparation of anti-tumor drugs.

10. An anti-tumor drug, characterized in that: The effective ingredient of the anti-tumor drug includes the Ganoderma lucidum described in any one of claims 1-2, or the Ganoderma lucidum obtained by the cultivation method described in any one of claims 3-5, or the Ganoderma lucidum extract described in any one of claims 6-7, or the Ganoderma lucidum extract obtained by the preparation method described in claim 8.

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