Ganoderma lucidum spore oil composite fungal polysaccharide anti-tumor oral preparation and application thereof

Abstract

The invention discloses a ganoderma lucidum spore oil composite fungal polysaccharide anti-tumor oral preparation and application thereof. The ganoderma lucidum spore oil composite fungal polysaccharide anti-tumor oral preparation comprises the following components in percentage by weight: 15-25% of ganoderma lucidum spore oil; 10%-15% of composite fungal polysaccharide; 1%-3% of coenzyme Q10; 1%-2% of a nano encapsulation carrier; the auxiliary materials comprise 3%-6% of phospholipid and 0.3%-0.8% of vitamin E; the balance is sterile water; the grifola frondosa polysaccharide activates macrophages, the schizophyllum commune polysaccharide enhances NK cytotoxicity, the agaricus blazei murill polysaccharide inhibits angiogenesis, and the cordyceps sinensis polysaccharide regulates immune balance, so that a triple synergistic network of direct killing, immune activation and blood vessel blocking is formed; by adopting a membrane emulsification technology of a sodium caseinate-chitosan composite carrier, the problem of co-delivery of fat-soluble and water-soluble components is solved; the positive charge characteristic of chitosan realizes targeted delivery; by means of component innovation, process optimization and mechanism breakthrough, inherent defects of existing fungus anti-tumor preparations are overcome, and a brand new solution is provided for efficient and low-toxicity tumor adjuvant therapy.

Description

Technical Field

[0001] This invention relates to the field of antitumor pharmaceutical preparations, specifically to an oral antitumor preparation of Ganoderma lucidum spore oil and fungal polysaccharides and its application. Background Technology

[0002] Current anti-tumor treatment faces multiple challenges: chemotherapy drugs generally have poor targeting and strong toxic side effects, such as the damage of cisplatin to the digestive tract and hematopoietic system; immune checkpoint inhibitors (such as PD-1 monoclonal antibodies) can activate the immune system, but are only effective in 20%-30% of patients and are prone to immune-related adverse reactions. In the field of natural medicines, fungal preparations have attracted attention due to their low toxicity, but existing technologies have obvious limitations: (1) Single component combination: most of the Ganoderma lucidum spore oil preparations on the market are used alone or only combined with a single fungal polysaccharide (such as Ganoderma lucidum polysaccharide), failing to form a multi-target synergistic effect; (2) Low bioavailability: the triterpenoid components in Ganoderma lucidum spore oil are highly lipid-soluble and are easily destroyed by gastric acid after oral administration, with a bioavailability of only 8%-12%; fungal polysaccharides are difficult to be absorbed through the intestine due to their large molecular weight (usually >100,000 Da), with a utilization rate of less than 15%. (3) Defects in process technology: Traditional extraction processes (such as ethanol hot reflux) will lead to the oxidative degradation of Ganoderma lucidum triterpenes, with a loss rate of more than 30%; ordinary preparations lack targeted delivery systems, and the amount of drug enriched in tumor tissues is only 1.5-2 times that in normal tissues. (4) Limitations in mechanism of action: Existing fungal preparations mostly focus on a single immune regulatory pathway (such as macrophage activation), and lack the ability to regulate immunosuppressive cells (such as MSCs and Treg cells) in the tumor microenvironment, making it difficult to break through the tumor immune escape barrier. Summary of the Invention

[0003] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0004] A reishi spore oil compound fungal polysaccharide oral antitumor preparation, comprising, by weight percentage:

[0005] Ganoderma lucidum spore oil: 15%-25%; compound fungal polysaccharides: 10%-15%; coenzyme Q10: 1%-3%; nano-encapsulation carrier: 1%-2%; excipients: including phospholipids: 3%-6%, vitamin E: 0.3%-0.8%; the balance is sterile water;

[0006] The Ganoderma lucidum spore oil contains ≥24.9% Ganoderma lucidum triterpenes and ≥70mg / 100g of ergosterol;

[0007] The compound fungal polysaccharide is composed of the following components: Grifola frondosa polysaccharide: 35%-40%, Schizophyllum commune polysaccharide: 25%-30%, Agaricus blazei polysaccharide: 18%-22%, Cordyceps sinensis polysaccharide: 12%-15%; the compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 150-250 nm.

[0008] The nano-encapsulation carrier is selected from sodium caseinate-chitosan complex.

[0009] Preferably, the method for preparing the Ganoderma lucidum spore oil includes the following steps:

[0010] (1) The unbroken Ganoderma lucidum spore powder was purified with ethanol and then physically broken at a low temperature of -40℃ to -50℃.

[0011] (2) Supercritical CO2 extraction: Under the conditions of pressure 35-45MPa and temperature 45-55℃, the extraction time is 3-4 hours;

[0012] (3) Separation by stage: First stage separation pressure 10-12MPa, temperature 40-45℃; Second stage separation pressure 5-7MPa, temperature 35-40℃, to improve the purity of triterpenes.

[0013] Preferably, the extraction method of the compound fungal polysaccharide includes the following steps:

[0014] (1) The raw material is permeated with 1.2%-1.5% sodium chloride solution. Ultrasonic assistance is used during the permeation process. The ultrasonic assistance conditions are: 70-80℃, power 60-80%, 1.5h.

[0015] (2) The raw materials processed in step (1) are centrifuged, concentrated and precipitated with ethanol. The centrifugation speed is 3000-3500 r / min and the ethanol concentration in the precipitate is ≥80%.

[0016] (3) The raw material after step (2) was purified by dialysis, the molecular weight cutoff was 10000 Da, and the nano-sized polysaccharide was obtained by freeze drying.

[0017] Preferably, the nano-encapsulation employs membrane emulsification technology, including the following parameters: carrier to Ganoderma lucidum spore oil mass ratio 1:6-1:7; membrane pressure 1.5-2.0 bar; emulsification temperature 25-30℃; average particle size 120±10 nm; encapsulation efficiency ≥90%.

[0018] Preferably, the method for preparing the oral formulation includes:

[0019] (1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol to replace isopropanol and reduce the risk of residue.

[0020] (2) Dissolve the complex fungal polysaccharides and phospholipids in sterile water to form a continuous phase;

[0021] (3) The dispersed phase is pressed into the continuous phase through a microporous membrane at a homogenization pressure of 90-100 MPa;

[0022] (4) The solvent is removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of ≤0.5%.

[0023] The present invention also discloses the use of the above-mentioned oral preparation in the preparation of antitumor drugs, wherein the tumors to which the antitumor drugs are used are lung cancer, liver cancer, breast cancer or gastric cancer.

[0024] This formulation achieves its anti-tumor effect through the synergistic action of multiple components. Its pharmacological mechanism is based on a triple synergy of immune regulation, direct cytotoxicity, and microenvironment regulation.

[0025] (1) Targeted action of Ganoderma lucidum spore oil

[0026] ≥24.9% of Ganoderma lucidum spore oil contains ganoderic acids that can directly inhibit tumor cell proliferation: by downregulating the expression of Bcl-2 anti-apoptotic protein, it induces the activation of mitochondrial apoptosis pathways in lung cancer A549 and liver cancer HepG2 cells; ergosterol, on the other hand, inhibits cholesterol synthesis in tumor cells and disrupts cell membrane stability. The active ingredients preserved by the supercritical CO2 extraction process have 38% higher cytotoxic activity than those extracted by traditional ethanol.

[0027] (2) Immune enhancement network of complex fungal polysaccharides

[0028] Grifola frondosa polysaccharide: It activates macrophages through TLR4 receptors and promotes the secretion of cytokines such as IL-12 and TNF-α. Experiments show that it can increase the phagocytic rate of macrophages by 2.1 times.

[0029] Schizophyllum polysaccharide: specifically upregulates the expression of NKG2D receptor on the surface of NK cells, enhancing the recognition and killing of tumor cells;

[0030] Agaricus blazei polysaccharide: inhibits the migration of tumor vascular endothelial cells, reduces VEGF levels, and synergistically blocks tumor angiogenesis with Cordyceps sinensis polysaccharide;

[0031] Nanoscale particle size: Through targeted enrichment by the high permeability and retention effect (EPR) of tumor tissue, the utilization rate of polysaccharides is increased by 4.3 times compared with micron-scale.

[0032] (3) Synergistic effect of coenzyme Q10

[0033] As a key component of the mitochondrial respiratory chain, coenzyme Q10 can alleviate oxidative stress in the tumor microenvironment and protect the activity of NK cells and T cells. At the same time, it can reduce the oxidative damage of Ganoderma lucidum triterpenes to normal cells by enhancing the activity of glutathione peroxidase, thereby reducing toxicity and enhancing efficacy.

[0034] (4) Nanoparticles formed by sodium caseinate-chitosan composite carrier have dual pharmacological value:

[0035] Improved bioavailability: The amphiphilic structure of the carrier promotes the co-delivery of lipid-soluble Ganoderma lucidum triterpenes and water-soluble polysaccharides. After oral administration, the transmembrane absorption efficiency in intestinal epithelial cells is increased by 172%, and the peak plasma concentration reaches 8.7 μg / mL.

[0036] Targeted delivery: The electrostatic interaction between the positive charge of chitosan and the negative charge on the surface of tumor cells increases the enrichment of the formulation in tumor tissue by 3.5 times compared to free drug;

[0037] Microenvironment regulation: The carrier degradation products can neutralize the acidic pH of the tumor microenvironment, inhibit the recruitment and activation of myeloid suppressor cells (MSCs), and relieve immunosuppression.

[0038] (5) The formulation improves the tumor immune microenvironment through the following pathways:

[0039] It inhibits the secretion of immunosuppressive factors such as IL-10 and TGF-β by MSCs, relieving functional inhibition of effector T cells; the antioxidant system composed of vitamin E and phospholipids reduces ROS levels in the tumor microenvironment and protects the activity of immune cells; the complex polysaccharide induces M2 macrophages to polarize towards M1, enhancing antigen presentation capacity; experiments show that CD8+ in tumor tissue... + T cell infiltration increased 2.3 times.

[0040] This invention utilizes Grifola frondosa polysaccharide to activate macrophages, Schizophyllum commune polysaccharide to enhance NK cell toxicity, Agaricus blazei polysaccharide to inhibit angiogenesis, and Cordyceps sinensis polysaccharide to regulate immune balance, forming a triple synergistic network of "direct killing + immune activation + angiogenesis blocking." The membrane emulsification technology using sodium caseinate-chitosan composite carriers solves the problem of co-delivering lipid-soluble and water-soluble components. The positive charge of chitosan allows for 4.5 times the enrichment of tumor tissue into normal tissue, achieving targeted delivery. Supercritical CO2 fractionation separation technology achieves a 92% retention rate of Ganoderma lucidum triterpenes, a 40% improvement compared to ethanol extraction. Nanoscale polysaccharide preparation combined with dialysis purification increases the intestinal absorption rate of polysaccharides to 58%, addressing the industry pain point of "difficult absorption of effective components." This solution, through component innovation, process optimization, and mechanistic breakthroughs, overcomes the inherent defects of existing fungal antitumor agents, providing a novel solution for highly effective and low-toxicity adjuvant cancer therapy. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown herein can generally be arranged and designed in various different configurations.

[0042] Example 1

[0043] A reishi spore oil compound fungal polysaccharide oral antitumor preparation, comprising, by weight percentage:

[0044] Ganoderma lucidum spore oil: 15%, of which Ganoderma lucidum triterpenes content is 25.1% and ergosterol is 72mg / 100g;

[0045] The compound fungal polysaccharide is 15% and is composed of the following components: Grifola frondosa polysaccharide: 35%, Schizophyllum commune polysaccharide: 30%, Agaricus blazei polysaccharide: 22%, Cordyceps sinensis polysaccharide: 13%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 150 nm.

[0046] Coenzyme Q10: 0.5%;

[0047] Nano-encapsulation carrier: 1%, selected from sodium caseinate-chitosan complex;

[0048] Excipients: 68.5%, including 3% phospholipids, 0.8% vitamin E, and 64.7% sterile water.

[0049] (1) Ganoderma lucidum spore oil extraction process:

[0050] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-40℃); (1.2) Supercritical CO2 extraction: pressure 35MPa, temperature 45℃, extraction time 3 hours; (1.3) Separation conditions: first separation pressure 10MPa, temperature 40℃; second separation pressure 5MPa, temperature 35℃.

[0051] (2) Preparation method of compound fungal polysaccharide:

[0052] (2.1) The raw material was permeated with 1.2% sodium chloride solution and extracted with ultrasonic assistance (70℃, 60% power, 1.5h); (2.2) After centrifugation (3000r / min), the material was concentrated and precipitated with 80% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0053] (3) Nano-encapsulation process:

[0054] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:6; (3.2) The membrane pressure was 1.5 bar, the emulsification temperature was 25℃, and the average particle size of the obtained nanoparticles was 110 nm with an encapsulation efficiency of 91%.

[0055] (4) Formulation preparation method:

[0056] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (30 kHz, 500 W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.2 μm microporous membrane at a homogenization pressure of 90 MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.3%.

[0057] Example 2

[0058] A reishi spore oil compound fungal polysaccharide oral antitumor preparation, comprising, by weight percentage:

[0059] Ganoderma lucidum spore oil: 25%, of which Ganoderma lucidum triterpenes content is 24.9% and ergosterol is 70mg / 100g;

[0060] The compound fungal polysaccharide is 10% and is composed of the following components: Grifola frondosa polysaccharide: 40%, Schizophyllum commune polysaccharide: 25%, Agaricus blazei polysaccharide: 18%, Cordyceps sinensis polysaccharide: 17%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 250 nm.

[0061] Coenzyme Q10: 5%;

[0062] Nano-encapsulation carrier: 3%, selected from sodium caseinate-chitosan complex;

[0063] Excipients: 57%, including 6% phospholipids, 0.3% vitamin E, and 50.7% sterile water.

[0064] (1) Ganoderma lucidum spore oil extraction process:

[0065] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-50℃); (1.2) Supercritical CO2 extraction: pressure 45MPa, temperature 55℃, extraction time 4 hours; (1.3) Separation conditions: first separation pressure 12MPa, temperature 45℃; second separation pressure 7MPa, temperature 40℃.

[0066] (2) Preparation method of compound fungal polysaccharide:

[0067] (2.1) The raw material was permeated with 1.5% sodium chloride solution and extracted with ultrasonic assistance (80℃, 80% power, 1.5h); (2.2) After centrifugation (3500r / min), the material was concentrated and precipitated with 85% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0068] (3) Nano-encapsulation process:

[0069] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:7; (3.2) The membrane pressure was 2.0 bar, the emulsification temperature was 30℃, and the average particle size of the obtained nanoparticles was 130 nm with an encapsulation efficiency of 93%.

[0070] (4) Formulation preparation method:

[0071] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (40kHz, 500W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.5μm microporous membrane at a homogenization pressure of 100MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.4%.

[0072] Example 3

[0073] A reishi spore oil compound fungal polysaccharide oral antitumor preparation, comprising, by weight percentage:

[0074] Ganoderma lucidum spore oil: 20%, of which Ganoderma lucidum triterpenes content is 25.0% and ergosterol is 71mg / 100g;

[0075] The compound fungal polysaccharide is 12% and is composed of the following components: Grifola frondosa polysaccharide: 38%, Schizophyllum commune polysaccharide: 28%, Agaricus blazei polysaccharide: 20%, Cordyceps sinensis polysaccharide: 14%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 200 nm.

[0076] Coenzyme Q10: 2%;

[0077] Nano-encapsulation carrier: 2%, selected from sodium caseinate-chitosan complex;

[0078] Excipients: 64%, including 4% phospholipids, 0.5% vitamin E, and 59.5% sterile water.

[0079] (1) Ganoderma lucidum spore oil extraction process:

[0080] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-45℃); (1.2) Supercritical CO2 extraction: pressure 40MPa, temperature 50℃, extraction time 3.5 hours; (1.3) Separation conditions: first separation pressure 11MPa, temperature 42℃; second separation pressure 6MPa, temperature 37℃.

[0081] (2) Preparation method of compound fungal polysaccharide:

[0082] (2.1) The raw material was permeated with 1.3% sodium chloride solution and extracted with ultrasonic assistance (75℃, 70% power, 1.5h); (2.2) After centrifugation (3200r / min), the material was concentrated and precipitated with 82% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0083] (3) Nano-encapsulation process:

[0084] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:6.5; (3.2) The membrane pressure was 1.8 bar, the emulsification temperature was 28℃, and the average particle size of the obtained nanoparticles was 120 nm with an encapsulation efficiency of 92%.

[0085] (4) Formulation preparation method:

[0086] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (35 kHz, 500 W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.3 μm microporous membrane at a homogenization pressure of 95 MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.35%.

[0087] II. Comparative Example

[0088] Comparative Example 1 (lacking Schizophyllum commune polysaccharide)

[0089] This comparative example was prepared using the following raw materials in parts by weight: Ganoderma lucidum spore oil: 20%, of which Ganoderma lucidum triterpenes content was 25.0% and ergosterol was 71 mg / 100g;

[0090] The compound fungal polysaccharide, 12%, is composed of the following components: Grifola frondosa polysaccharide: 40%, Trametes versicolor polysaccharide: 30%, Agaricus blazei polysaccharide: 20%, Cordyceps sinensis polysaccharide: 10%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 200 nm.

[0091] Coenzyme Q10: 2%;

[0092] Nano-encapsulation carrier: 2%, selected from sodium caseinate-chitosan complex;

[0093] Excipients: 64%, including 4% phospholipids, 0.5% vitamin E, and 59.5% sterile water.

[0094] (1) Ganoderma lucidum spore oil extraction process:

[0095] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-45℃); (1.2) Supercritical CO2 extraction: pressure 40MPa, temperature 50℃, extraction time 3.5 hours; (1.3) Separation conditions: first separation pressure 11MPa, temperature 42℃; second separation pressure 6MPa, temperature 37℃.

[0096] (2) Preparation method of compound fungal polysaccharide: completely consistent with Example 3.

[0097] (2.1) The raw material was permeated with 1.3% sodium chloride solution and extracted with ultrasonic assistance (75℃, 70% power, 1.5h); (2.2) After centrifugation (3200r / min), the material was concentrated and precipitated with 82% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0098] (3) Nano-encapsulation process:

[0099] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:6.5; (3.2) The membrane pressure was 1.8 bar, the emulsification temperature was 28℃, and the average particle size of the obtained nanoparticles was 120 nm with an encapsulation efficiency of 92%.

[0100] (4) Formulation preparation method:

[0101] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (35 kHz, 500 W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.3 μm microporous membrane at a homogenization pressure of 95 MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.35%.

[0102] Comparative Example 2 (Traditional Media)

[0103] This comparative example was prepared using the following raw materials in parts by weight: Ganoderma lucidum spore oil: 20%, of which Ganoderma lucidum triterpenes content was 25.0% and ergosterol was 71 mg / 100g;

[0104] The compound fungal polysaccharide is 12% and is composed of the following components: Grifola frondosa polysaccharide: 38%, Schizophyllum commune polysaccharide: 28%, Agaricus blazei polysaccharide: 20%, Cordyceps sinensis polysaccharide: 14%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 200 nm.

[0105] Coenzyme Q10: 2%;

[0106] Nano-encapsulation carrier: 2%, selected from chitosan alone;

[0107] Excipients: 64%, including 4% phospholipids, 0.5% vitamin E, and 59.5% sterile water.

[0108] (1) Ganoderma lucidum spore oil extraction process:

[0109] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-45℃); (1.2) Supercritical CO2 extraction: pressure 40MPa, temperature 50℃, extraction time 3.5 hours; (1.3) Separation conditions: first separation pressure 11MPa, temperature 42℃; second separation pressure 6MPa, temperature 37℃.

[0110] (2) Preparation method of compound fungal polysaccharide:

[0111] (2.1) The raw material was permeated with 1.3% sodium chloride solution and extracted with ultrasonic assistance (75℃, 70% power, 1.5h); (2.2) After centrifugation (3200r / min), the material was concentrated and precipitated with 82% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0112] (3) Nano-encapsulation process:

[0113] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:6.5; (3.2) The membrane pressure was 1.8 bar, the emulsification temperature was 28℃, and the average particle size of the obtained nanoparticles was 120 nm with an encapsulation efficiency of 92%.

[0114] (4) Formulation preparation method:

[0115] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (35 kHz, 500 W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.3 μm microporous membrane at a homogenization pressure of 95 MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.35%.

[0116] Comparative Example 3 (without nano-encapsulation)

[0117] This comparative example was prepared using the following raw materials in parts by weight: Ganoderma lucidum spore oil: 20%, of which Ganoderma lucidum triterpenes content was 25.0% and ergosterol was 71 mg / 100g;

[0118] The compound fungal polysaccharide is 12% and is composed of the following components: Grifola frondosa polysaccharide: 38%, Schizophyllum commune polysaccharide: 28%, Agaricus blazei polysaccharide: 20%, Cordyceps sinensis polysaccharide: 14%. The compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 200 nm.

[0119] Coenzyme Q10: 2%;

[0120] Excipients: 66%, including 4% phospholipids, 0.5% vitamin E, and 61.5% sterile water.

[0121] (1) Ganoderma lucidum spore oil extraction process:

[0122] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-45℃); (1.2) Supercritical CO2 extraction: pressure 40MPa, temperature 50℃, extraction time 3.5 hours; (1.3) Separation conditions: first separation pressure 11MPa, temperature 42℃; second separation pressure 6MPa, temperature 37℃.

[0123] (2) Preparation method of compound fungal polysaccharide:

[0124] (2.1) The raw material was permeated with 1.3% sodium chloride solution and extracted with ultrasonic assistance (75℃, 70% power, 1.5h); (2.2) After centrifugation (3200r / min), the material was concentrated and precipitated with 82% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0125] (4) Formulation preparation method:

[0126] (4.1) Ganoderma lucidum spore oil and coenzyme Q10 were directly mixed with compound fungal polysaccharide, phospholipid, vitamin E and sterile water; (4.2) After ultrasonic dispersion (35 kHz, 500 W), the homogenization pressure was 95 MPa; (4.3) The solid powder was obtained by freeze drying.

[0127] Comparative Example 4 (Traditional Extraction Process)

[0128] The composition of this comparative formulation is completely identical to that of Example 3.

[0129] (1) Ganoderma lucidum spore oil extraction process:

[0130] (1.1) Unbroken Ganoderma lucidum spore powder was purified with ethanol and physically broken at low temperature (-45℃); (1.2) Ethanol hot reflux extraction was performed: 60℃, extraction time 3h, ethanol concentration 90%; (1.3) Ganoderma lucidum spore oil was obtained by vacuum distillation to recover ethanol (Ganoderma lucidum triterpenoid content 18.2%, ergosterol 51mg / 100g).

[0131] (2) Preparation method of compound fungal polysaccharide:

[0132] (2.1) The raw material was permeated with 1.3% sodium chloride solution and extracted with ultrasonic assistance (75℃, 70% power, 1.5h); (2.2) After centrifugation (3200r / min), the material was concentrated and precipitated with 82% ethanol; (2.3) The material was purified by dialysis (molecular weight cutoff 10000Da) and freeze-dried to obtain nano-sized polysaccharides.

[0133] (3) Nano-encapsulation process:

[0134] (3.1) Membrane emulsification technology was used, with a carrier to Ganoderma lucidum spore oil mass ratio of 1:6.5; (3.2) The membrane pressure was 1.8 bar, the emulsification temperature was 28℃, and the average particle size of the obtained nanoparticles was 120 nm with an encapsulation efficiency of 92%.

[0135] (4) Formulation preparation method:

[0136] (4.1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol (35 kHz, 500 W); (4.2) Compound fungal polysaccharides and phospholipids were dissolved in sterile water to form a continuous phase; (4.3) The dispersed phase was pressed into the continuous phase through a 0.3 μm microporous membrane at a homogenization pressure of 95 MPa; (4.4) The solvent was removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of 0.35%.

[0137] Comparative Example 5 (Commercially Available Control Product)

[0138] This comparative example was prepared according to the following parts by weight of raw materials: Ganoderma lucidum spore oil: 20%, of which Ganoderma lucidum triterpenes content was 19.5% and ergosterol was 62mg / 100g; Ganoderma lucidum polysaccharide: 10% (non-nano-sized, average particle size 5000nm); excipients: 70%, including 50% vegetable oil, 0.2% vitamin E and 19.8% magnesium stearate.

[0139] (1) Ganoderma lucidum spore oil extraction process:

[0140] (1.1) Ganoderma lucidum spore oil is extracted by pressing;

[0141] (2) Preparation method of Ganoderma lucidum polysaccharide:

[0142] (2.1) Water extraction and alcohol precipitation method was used (without nano-processing);

[0143] (4) Formulation preparation method:

[0144] (4.1) All ingredients are directly mixed and then filled into capsules without nano-encapsulation process.

[0145] III. Experimental Data and Results Analysis

[0146] (1) Anti-tumor effect (lung cancer A549 cell model)

[0147] Group Tumor inhibition rate (48h, %) MSC activity inhibition rate (%) NK cell killing rate (%) Example 1 68.2±3.1 72.5±2.8 65.3±2.5 Example 2 71.5±2.9 75.1±3.0 68.7±2.3 Example 3 73.8±2.5 78.3±2.2 70.5±2.1 Comparative Example 1 52.6±3.5 51.2±3.3 50.8±2.7 Comparative Example 2 58.4±3.2 60.3±2.9 56.2±2.6 Comparative Example 3 45.7±4.0 48.5±3.6 47.3±3.0 Comparative Example 4 50.3±3.8 55.7±3.1 52.1±2.8 Comparative Example 5 42.1±4.2 40.2±3.8 43.5±3.2

[0148] Conclusion: The tumor inhibition rate of Example 3 was 39% higher than that of Comparative Example 1 (Schizophyllum commune), demonstrating the synergistic effect of Schizophyllum commune polysaccharide; it was 26% higher than that of Comparative Example 2 (traditional carrier), demonstrating the advantages of composite carrier; and it was 61% higher than that of Comparative Example 3 (unencapsulated), verifying the necessity of nanoencapsulation.

[0149] (2) Ingredient stability (stored at 4℃ for 30 days)

[0150] Group Ganoderma lucidum triterpenoid retention rate (%) Polysaccharide activity retention rate (%) Example 3 92.3±1.5 90.7±1.8 Comparative Example 2 78.5±2.2 75.3±2.5 Comparative Example 3 65.2±3.1 60.8±3.0 Comparative Example 5 58.7±3.5 52.4±3.3

[0151] Conclusion: The component stability of Example 3 was significantly better than that of the control group, and the composite carrier + nano-encapsulation improved the triterpene retention rate by 41%.

[0152] (3) Bioavailability (SD rat gavage test)

[0153]

[0154] Conclusion: Nanoencapsulation improved bioavailability by 172% and shortened the time to peak concentration by 52%, verifying the synergistic effect of membrane emulsification technology.

[0155] IV. Summary

[0156] Experimental data show that: (1) the combination of Schizophyllum commune polysaccharide and a specific ratio of compound fungal polysaccharides can significantly enhance antitumor activity; (2) sodium caseinate-chitosan composite carrier + membrane emulsification technology is the key to improving stability and bioavailability; (3) supercritical CO2 fractionation separation process can effectively retain active ingredients. The above results fully demonstrate the significant advantages of this scheme in terms of component synergy, process innovation and efficacy enhancement.

[0157] The present invention and its embodiments have been described above. This description is not restrictive. In short, if a person skilled in the art is inspired by this description and designs a similar structure and embodiment without departing from the spirit of the present invention, such design should fall within the protection scope of the present invention.

Claims

1. An oral preparation of Ganoderma lucidum spore oil combined with fungal polysaccharides for antitumor treatment, characterized in that, Included by weight percentage: Ganoderma lucidum spore oil: 15%-25%; compound fungal polysaccharides: 10%-15%; coenzyme Q10: 1%-3%; nano-encapsulation carrier: 1%-2%; excipients: including phospholipids: 3%-6%, vitamin E: 0.3%-0.8%; the balance is sterile water; The Ganoderma lucidum spore oil contains ≥24.9% Ganoderma lucidum triterpenes and ≥70mg / 100g of ergosterol; The compound fungal polysaccharide is composed of the following components: Grifola frondosa polysaccharide: 35%-40%, Schizophyllum commune polysaccharide: 25%-30%, Agaricus blazei polysaccharide: 18%-22%, Cordyceps sinensis polysaccharide: 12%-15%; the compound fungal polysaccharide is a nano-sized polysaccharide with an average particle size of 150-250 nm. The nano-encapsulation carrier is selected from sodium caseinate-chitosan complex.

2. The oral antitumor preparation of Ganoderma lucidum spore oil and fungal polysaccharides according to claim 1, characterized in that, The method for preparing the Ganoderma lucidum spore oil includes the following steps: (1) The unbroken Ganoderma lucidum spore powder was purified with ethanol and then physically broken at a low temperature of -40℃ to -50℃. (2) Supercritical CO2 extraction: Under the conditions of pressure 35-45MPa and temperature 45-55℃, the extraction time is 3-4 hours; (3) Separation by stage: First stage separation pressure 10-12MPa, temperature 40-45℃; Second stage separation pressure 5-7MPa, temperature 35-40℃, to improve the purity of triterpenes.

3. The oral antitumor preparation of Ganoderma lucidum spore oil and fungal polysaccharides according to claim 1, characterized in that, The extraction method of the complex fungal polysaccharide includes the following steps: (1) The raw material is permeated with 1.2%-1.5% sodium chloride solution. Ultrasonic assistance is used during the permeation process. The ultrasonic assistance conditions are: 70-80℃, power 60-80%, 1.5h. (2) The raw materials processed in step (1) are centrifuged, concentrated and precipitated with ethanol. The centrifugation speed is 3000-3500 r / min and the ethanol concentration in the precipitate is ≥80%. (3) The raw material after step (2) was purified by dialysis, the molecular weight cutoff was 10000 Da, and the nano-sized polysaccharide was obtained by freeze drying.

4. The oral antitumor preparation of Ganoderma lucidum spore oil and fungal polysaccharides according to claim 1, characterized in that, The nano-encapsulation employs membrane emulsification technology, including the following parameters: carrier to Ganoderma lucidum spore oil mass ratio 1:6-1:7; membrane pressure 1.5-2.0 bar; emulsification temperature 25-30℃; average particle size 120±10 nm; encapsulation efficiency ≥90%.

5. The use of the oral formulation according to any one of claims 1-4 in the preparation of an antitumor drug, characterized in that: The tumor is lung cancer, liver cancer, breast cancer, or stomach cancer.

6. A method for preparing the oral formulation according to claim 1, characterized in that... include: (1) Ganoderma lucidum spore oil, coenzyme Q10 and nano-encapsulation carrier were ultrasonically dispersed in ethanol to replace isopropanol and reduce the risk of residue. (2) Dissolve the complex fungal polysaccharides and phospholipids in sterile water to form a continuous phase; (3) The dispersed phase is pressed into the continuous phase through a microporous membrane at a homogenization pressure of 90-100 MPa; (4) The solvent is removed by rotary evaporation and freeze-dried to obtain a solid powder with a solvent residue of ≤0.5%.

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