Anti-tumor composition and preparation method thereof

Abstract

The invention provides an anti-tumor composition and a preparation method thereof, and belongs to the technical field of medicines. Poria cocos and radix astragali are subjected to enzymolysis boiling water extraction and filtration, filter residues are subjected to fermentation, filtration and solid washing to obtain bacterial liquid, the bacterial liquid and filtrate are mixed, dried and embedded, and embedded microspheres are prepared; adding the washed solid into a culture medium, inoculating lactobacillus plantarum for fermentation, filtering, carrying out ethanol precipitation on filtrate, carrying out amination to obtain aminated selenium-rich extracellular polysaccharide, and reacting with carboxylated geraniol and dihydroartemisinin to obtain the active component selenium-rich extracellular polysaccharide. Uniformly mixing the water extract of the poria cocos and the astragalus membranaceus, the active components, namely the selenium-rich exopolysaccharide, the embedded microspheres, genipin and berberine, so as to obtain the anti-tumor composition. The traditional Chinese medicine composition has good anti-inflammatory, anti-oxidation and anti-tumor effects and is wider in application range.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an anti-tumor composition and a preparation method thereof. Background Art

[0002] Cancer cells have biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. They can invade surrounding normal tissues and even metastasize to other parts of the body through the circulatory system or lymphatic system. Worldwide, cancer has become the second largest killer of humans after cardiovascular disease. According to statistics, there were 19.3 million new cancer cases in the world in 2020, and about 10 million patients died from it.

[0003] Among them, liver cancer is one of the most common malignant tumors in the world, and it has many causes. The main cause of liver cancer in my country is chronic hepatitis B. In addition, smoking, alcohol, aflatoxin, etc. can also promote the occurrence and development of liver cancer. The incidence of liver cancer in my country is relatively high, and about half of the world's liver cancer occurs in China. Early liver cancer is mainly removed by surgery, and middle and late liver cancer can be treated with molecular targeted drugs, but the recurrence rate of liver cancer after treatment is still high. Liver cancer has the characteristics of high recurrence rate and high mortality rate. Modern Western medicine treats tumors mostly with chemotherapy or radiotherapy. While killing cancer cells, it also kills many normal cells in the human body, causing great pain and psychological pressure to patients.

[0004] Therefore, developing a therapeutic drug that is safer, more effective, has fewer side effects, and reduces the economic burden on patients will be of far-reaching significance. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-tumor composition and a preparation method thereof, which has excellent anti-inflammatory, antioxidant and anti-tumor effects, can improve the administration of active components, can deliver more drugs into cancer cells, tend to accumulate in tumor tissues, and have the effect of efficiently killing cancer cells, and has low systemic toxicity, few side effects, high safety, a wide source of raw materials, and low cost, which can reduce the economic burden of patients, thereby having a wider range of applications.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a method for preparing an anti-tumor composition. The method comprises the following steps: extracting poria cocos and astragalus through enzymatic hydrolysis and boiling water, filtering, fermenting the filter residue, filtering, washing solids to obtain bacterial liquid, mixing the solids with the filtrate, drying, embedding, and preparing embedded microspheres; adding the washed solids into a culture medium, inoculating lactobacillus plantarum for fermentation, filtering, precipitating the filtrate through ethanol, performing amination to obtain aminated selenium-rich extracellular polysaccharides, reacting the polysaccharides with carboxylated geraniol and dihydroartemisinin to obtain an active component-selenium-rich extracellular polysaccharides, and uniformly mixing the poria cocos and astragalus water extract, the active component-selenium-rich extracellular polysaccharides, the embedded microspheres, genipin, and berberine to prepare the anti-tumor composition.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Preparation of Poria cocos and Astragalus water extract: Poria cocos and Astragalus membranaceus were washed, dried, crushed, added to water, added with complex enzyme for enzymolysis, heated to boiling for extraction, filtered, and the residue was retained; the filtrate was dried to obtain Poria cocos and Astragalus membranaceus water extract;

[0010] S2. Preparation of Poria cocos and Astragalus fermentation product: The filter residue, nitrogen source and inorganic salt in step S1 are added to water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, fermented, filtered, the solids are washed to obtain a bacterial solution, mixed with the filtrate, freeze-dried, and a Poria cocos and Astragalus fermentation product is obtained, and the washed solids are retained;

[0011] S3. Preparation of a culture medium: The washed solids in step S2, sugar cane juice, yeast powder, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, vitamin B1, vitamin C, and a selenium source were added to water and sterilized to obtain a culture medium;

[0012] S4. Preparation of selenium-rich extracellular polysaccharides: inoculating the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, fermenting and culturing, filtering, and adding ethanol to the filtrate for precipitation to obtain selenium-rich extracellular polysaccharides;

[0013] S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve the selenium-enriched exopolysaccharide obtained in step S4 in water, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and adipic acid dihydrazide, adjust the pH value of the solution to 4-5, stir the reaction at room temperature, adjust the pH value of the solution to 6.9-7.1, dialyze, and freeze-dry the dialyzate to obtain amino-enriched selenium exopolysaccharide;

[0014] S6. Preparation of active component: Mix geraniol and dihydroartemisinin to obtain an active component;

[0015] S7. Preparation of carboxylated active component: The active component obtained in step S6, oxalic anhydride and the catalyst were stirred at room temperature under an inert gas, and the product was filtered, washed and dried to obtain a carboxylated active component;

[0016] S8. Preparation of active component - selenium-rich extracellular polysaccharide: The carboxylated active component prepared in step S7 is mixed with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride for activation, and the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 is added, stirred at room temperature for reaction, dialyzed, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide;

[0017] S9. Preparation of embedded microspheres: adding the fermentation product of Poria cocos and Astragalus membranaceus to water, adding sodium alginate and sodium hyaluronate, stirring and mixing to obtain an aqueous phase; adding the aqueous phase to the oil phase, adding lecithin, emulsifying, dropping calcium chloride solution, solidifying at room temperature, centrifuging, washing, and drying to obtain embedded microspheres;

[0018] S10. Preparation of an antitumor composition: The Poria cocos and Astragalus membranaceus water extract prepared in step S1, the active component - selenium-rich extracellular polysaccharide prepared in step S8, the embedded microspheres prepared in step S9, genipin and berberine are mixed evenly to prepare an antitumor composition.

[0019] As a further improvement of the present invention, the mass ratio of Poria cocos and Astragalus membranaceus described in step S1 is 12-15:7-10, the composite enzyme includes cellulase and hemicellulase, the mass ratio is 5-7:3-5, the addition amount of the composite enzyme is 1-2wt% of the total mass of the system, the temperature of the enzymolysis is 45-50°C, the time is 2-4h, and the time of the heating to boiling extraction is 1-3h; the mass ratio of the filter residue, nitrogen source, inorganic salt and water described in step S2 is 10-15:5-7:1-2:300-500, the nitrogen source is selected from at least one of fish bone powder, peptone and yeast powder, the inorganic salt is selected from at least one of sodium chloride, potassium chloride, magnesium sulfate, magnesium chloride, manganese sulfate, copper chloride, ferric chloride and calcium chloride, and the bacterial content of the Lactobacillus casei and Lactobacillus rhamnosus seed liquid is 10 8 -10 9 cfu / mL, the inoculation amounts were 1-3v / v% and 2-4v / v%, respectively, and the fermentation culture conditions were anaerobic conditions, 37-39°C, 100-200r / min, and fermentation culture for 48-56h.

[0020] As a further improvement of the present invention, the mass ratio of the washed solid, sugarcane juice, yeast powder, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, vitamin B1, vitamin C, selenium source, and water in step S3 is 10-15:20-25:5-7:2-4:1-2:0.5-1:0.2-0.5:0.1-0.2:0.3-0.5:0.5-0.7:300-500, the selenium source is sodium selenite or sodium selenate, and the sugar content of the sugarcane juice is 18-20°; the bacterial content of the plant lactobacillus seed liquid in step S4 is 10 8 -109 cfu / mL, the inoculation amount is 3-5v / v%, the fermentation culture conditions are anaerobic conditions, 37-39°C, 100-200r / min fermentation culture for 36-48h, the ethanol content added to the system is 80-85wt%, and the precipitation time is 3-5h.

[0021] As a further improvement of the present invention, the mass ratio of the selenium-rich extracellular polysaccharide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and adipic acid dihydrazide in step S5 is 10-12:2-4:7-10, the time of the room temperature stirring reaction is 2-3h, the dialysis bag pore size is 8000-10000D, and the time is 3-5h; the mass ratio of geraniol and dihydroartemisinin in step S6 is 10-12:7-10.

[0022] As a further improvement of the present invention, the mass ratio of the active component, oxalic anhydride, and catalyst in step S7 is 7-10:9-12:0.01-0.02, the catalyst is at least one of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and triethylenediamine, the time of the reaction under stirring at room temperature is 20-24 hours, and the mass ratio of the carboxylated active component, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and amino-selenium-enriched extracellular polysaccharide in step S8 is 5-7:8-10:10-15, the time of the reaction under stirring at room temperature is 18-20 hours, the time of the activation is 1-3 hours, the pore size of the dialysis bag is 8000-10000D, and the time is 3-5 hours.

[0023] As a further improvement of the present invention, the mass ratio of the Poria cocos and Astragalus fermentation product, sodium alginate, and sodium hyaluronate in step S9 is 10:7-10:3-5, the mass ratio of the aqueous phase, the oil phase, and the lecithin is 10-15:25-30:0.1-0.2, the concentration of the calcium chloride solution is 5-7wt%, and the time for room temperature curing is 20-30min; the mass ratio of the Poria cocos and Astragalus water extract, the active component-selenium-rich extracellular polysaccharide, the embedded microspheres, genipin, and berberine in step S10 is 15-20:27-32:7-10:0.5-1:0.2-0.4.

[0024] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0025] S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 12-15 parts by weight of Poria cocos and 7-10 parts by weight of Astragalus membranaceus were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1-2wt% of the total mass of the system, and the enzyme was hydrolyzed at 45-50°C for 2-4h, heated to boiling for extraction for 1-3h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract;

[0026] The complex enzyme comprises cellulase and hemicellulase in a mass ratio of 5-7:3-5;

[0027] S2. Preparation of Poria cocos and Astragalus fermentation product: 10-15 parts by weight of the filter residue in step S1, 5-7 parts by weight of a nitrogen source, 1-2 parts by weight of an inorganic salt are added to 300-500 parts by weight of water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, the inoculation amount is 1-3v / v% and 2-4v / v%, respectively, under anaerobic conditions, 37-39 ° C, 100-200r / min fermentation culture 48-56h, filtered, the solid washing to obtain a bacterial solution, mixed with the filtrate, freeze-dried, to obtain a Poria cocos and Astragalus fermentation product, the washed solids are retained;

[0028] The bacterial content of the Lactobacillus casei and Lactobacillus rhamnosus seed liquid is 10 8 -10 9 cfu / mL;

[0029] S3. Preparation of a culture medium: 10-15 parts by weight of the washed solid in step S2, 20-25 parts by weight of sugar cane juice having a sugar content of 18-20 °, 5-7 parts by weight of yeast powder, 2-4 parts by weight of sodium nitrate, 1-2 parts by weight of potassium dihydrogen phosphate, 0.5-1 parts by weight of magnesium sulfate, 0.2-0.5 parts by weight of calcium chloride, 0.1-0.2 parts by weight of vitamin B1, 0.3-0.5 parts by weight of vitamin C, 0.5-0.7 parts by weight of a selenium source are added to 300-500 parts by weight of water and sterilized to obtain a culture medium;

[0030] S4. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, and ferment and culture under anaerobic conditions at 37-39°C and 100-200r / min for 36-48h, filter, add ethanol to the filtrate until the ethanol content in the system is 80-85wt%, and precipitate for 3-5h to obtain selenium-rich extracellular polysaccharides;

[0031] The bacterial content of the plant lactobacillus seed liquid is 10 8 -10 9 cfu / mL;

[0032] S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve 10-12 parts by weight of the selenium-enriched exopolysaccharide obtained in step S4 in 500 parts by weight of water, add 2-4 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 7-10 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 4-5, stir at room temperature for 2-3 hours, adjust the pH value of the solution to 6.9-7.1, dialyze for 3-5 hours, the dialysis bag pore size of the dialysis is 8000-10000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide;

[0033] S6. Preparation of the active ingredient: 10-12 parts by weight of geraniol and 7-10 parts by weight of dihydroartemisinin were mixed to obtain an active ingredient;

[0034] S7. Preparation of carboxylated active component: 7-10 parts by weight of the active component obtained in step S6, 9-12 parts by weight of oxalic anhydride, 0.01-0.02 parts by weight of the catalyst are dissolved in 200 parts by weight of dichloromethane, and the reaction is stirred at room temperature under inert gas for 20-24h, the product is filtered, washed, and dried to obtain a carboxylated active component;

[0035] S8. Preparation of active component - selenium-rich extracellular polysaccharide: 5-7 parts by weight of the carboxylated active component prepared in step S7 and 8-10 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, and activated by stirring for 1-3 hours, and 10-15 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 are added, and the reaction is stirred at room temperature for 18-20 hours, and dialyzed for 3-5 hours, wherein the pore size of the dialysis bag is 8000-10000D, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide;

[0036] S9. Preparation of embedded microspheres: add 10 parts by weight of Poria cocos and Astragalus membranaceus fermentation product to 200 parts by weight of water, add 7-10 parts by weight of sodium alginate and 3-5 parts by weight of sodium hyaluronate, stir and mix evenly to obtain an aqueous phase; add 100-150 parts by weight of the aqueous phase to 250-300 parts by weight of the oil phase, add 1-2 parts by weight of lecithin, emulsify, add 20 parts by weight of 5-7wt% calcium chloride solution dropwise, solidify at room temperature for 20-30 minutes, centrifuge, wash, and dry to obtain embedded microspheres;

[0037] S10. Preparation of an antitumor composition: 15-20 parts by weight of the Poria cocos and Astragalus membranaceus water extract obtained in step S1, 27-32 parts by weight of the active component - selenium-rich extracellular polysaccharide obtained in step S8, 7-10 parts by weight of the embedded microspheres obtained in step S9, 0.5-1 parts by weight of genipin, and 0.2-0.4 parts by weight of berberine are mixed evenly to obtain an antitumor composition.

[0038] The present invention further protects an antitumor composition obtained by the above preparation method.

[0039] The present invention further protects the use of the above anti-tumor composition in the preparation of drugs for the treatment and auxiliary treatment of liver cancer.

[0040] The present invention has the following beneficial effects:

[0041] Astragalus contains rich astragalus polysaccharides, which have pharmacological effects such as regulating the body's immune function, improving blood sugar, and anti-tumor. It has high safety, multiple targets, and a wide range of pharmacological effects. It improves the tumor immune microenvironment and immunosuppression by regulating various immune cells in the body, inhibiting tumor growth. It improves the tumor immune microenvironment, reverses the immunosuppression state, enhances the immune response of tumor patients, and improves the body's immune cells' ability to kill tumor cells, effectively inhibiting tumor growth and metastasis.

[0042] The active components contained in Poria cocos, such as Poria cocos polysaccharide and Poria cocos triterpenes, have good anti-tumor effects. Among them, Poria cocos polysaccharide activates and enhances the body's immune cells, improves the body's immune response ability, thereby inhibiting the development of tumors, binding to the TLR4 receptors of macrophages and dendritic cells, activating the TRAF6 / NF-γB signaling pathway, promoting the maturation and activation of these cells, thereby improving their antigen presentation ability, while enhancing the effector function of immune cells, playing the role of immune enhancement and immune surveillance, increasing the phagocytic and killing ability of macrophages and natural killer cells, thereby clearing tumor cells, and promoting various immune cells to secrete and express a variety of cytokines with anti-tumor effects. Poria cocos triterpenes, including Pachycolic acid, Pachycod alcohol, and Poria cocos tetraketone, can inhibit the proliferation of human tumor cells by upregulating the expression of Bax, Caspase-3, Caspase-9 and PARP, downregulating the expression of Bcl-2, activating the mitochondria-mediated apoptosis pathway, inducing their apoptosis, and inhibiting the migration and invasion of tumor cells, thereby blocking the spread and metastasis of tumors.

[0043] Therefore, both Poria cocos and Astragalus membranaceus are good anti-tumor raw materials. The present invention promotes the rupture of plant cell walls of Poria cocos and Astragalus membranaceus under the action of composite enzymes, and promotes the dissolution of active components such as polysaccharides, triterpenes, and flavonoids, thereby obtaining a Poria cocos and Astragalus membranaceus water extract containing multiple anti-tumor active components.

[0044] The solid residue obtained by filtration still contains rich nitrogen sources such as cellulose, insoluble polysaccharides, fixed proteins, etc. Therefore, after inoculating fermentation bacteria including Lactobacillus casei and Lactobacillus rhamnosus for fermentation, on the one hand, the fermentation bacteria themselves produce substances such as short-chain fatty acids with anti-inflammatory and antioxidant activities, and on the other hand, promote the degradation and dissolution of other active components in the solid residue extracted from Poria cocos and Astragalus membranaceus, thereby obtaining components containing many proteins, amino acids, and oligosaccharides, which can bind to the Notch receptors of T cells, activate the Notch signaling pathway, promote the differentiation and proliferation of T cells, and improve their effector functions. It affects the differentiation and polarization of immune cells, induces Th1 immune response, inhibits the polarization of M1 macrophages, and can effectively eliminate tumor cells. At the same time, the fermentation bacteria Lactobacillus casei and Lactobacillus rhamnosus themselves can significantly reduce the effect of TNF-a produced by THP-1 cells, and have a significant inhibitory effect on the proliferation of liver cancer cells and breast cancer cells, with a synergistic effect. Therefore, after the fermentation bacteria are encapsulated, they can avoid the destruction of gastric acid and enter the intestine, play the role of probiotics, produce a large number of anti-inflammatory and antioxidant factors, and inhibit the occurrence and development of cancer.

[0045] The washed solids are further utilized to improve the resource utilization of raw materials. At the same time, the production of Lactobacillus plantarum extracellular polysaccharides is induced by adding sugarcane juice and yeast powder, which greatly increases the output of extracellular polysaccharides. At the same time, the process of converting inorganic selenium into organic selenium is promoted by adding a selenium source, and a selenium-rich extracellular polysaccharide is obtained. The selenium-rich element has a good antioxidant effect, which can reduce the generation of free radicals and oxidative damage to cells and tissues, reduce the risk of tumors by reducing oxidative stress, promote the DNA repair process, maintain the integrity of DNA, and increase the activity of T cells and NK cells, which helps the immune system to better identify and destroy cancer cells, promote apoptosis of tumor cells, and inhibit tumor angiogenesis, thereby limiting the blood supply to the tumor, making it difficult for it to obtain nutrients and oxygen, and limiting the growth of the tumor.

[0046] At the same time, the extracellular polysaccharides of Lactobacillus plantarum have the characteristics of low cytotoxicity and few side effects. They can cause differential expression of a large number of genes related to the cell cycle, induce cell arrest and thus avoid the occurrence of tumors. At the same time, they promote the expression of tissue inhibitor of metalloproteinase 3, thereby blocking the binding of vascular endothelial growth factor to its receptor to inhibit tumor angiogenesis, and ultimately play a role in inhibiting tumor formation. Antioxidants block the accumulation of reactive oxygen in the body to delay or even prevent the development of tumors.

[0047] Therefore, selenium-rich extracellular polysaccharide itself is a good anti-tumor substance. Furthermore, after the selenium-rich extracellular polysaccharide is aminated, it can react well with the carboxylated active component to obtain the active component - selenium-rich extracellular polysaccharide; the selenium-rich extracellular polysaccharide can also be used as a carrier. At the same drug concentration level, it can deliver more drugs to cancer cells, tends to accumulate in tumor tissues, and has lower systemic toxicity, thereby producing a stronger cell killing effect.

[0048] Geraniol has antioxidant, antibacterial, antiviral, neuroprotective and anticancer effects, and can significantly reduce liver damage and tumor formation, but geraniol has poor water solubility and administration. The peroxide bridge structure in the parent nucleus of dihydroartemisinin can generate highly active free radicals by ring-opening reaction under the action of divalent iron ions of tumor cell heme, and exerts antitumor activity through the mechanisms of action such as blocking tumor cell cycle, inhibiting tumor angiogenesis, inducing DNA damage and ferroptosis, and the administration of dihydroartemisinin is also poor. Therefore, the present invention is to react geraniol and dihydroartemisinin with amino-enriched selenium extracellular polysaccharides after carboxylation, and the obtained product has good antitumor, especially the effect of inhibiting the occurrence and development of liver cancer cells.

[0049] In addition, the anti-tumor composition of the present invention further adds active substances including genipin and berberine. The effects of genipin include anti-inflammatory, liver disease treatment, anti-tumor, etc. It can directly bind to the SH2 domain of STAT-3, inhibit the phosphorylation and dimerization of STAT-3 (Y705), and then inhibit the proliferation of liver cancer cells, angiogenesis, cell invasion, and reverse the transition (EMT) process of epithelial cells to mesenchymal cells. Berberine has anti-inflammatory, antibacterial, antiviral, and anti-tumor activities, destroys the mitochondrial structure of cancer cells, causes mitochondrial swelling, internal cristae reduction, vacuolization, and fragmentation, reduces mitochondrial copy number, inhibits the tricarboxylic acid cycle, and causes mitochondrial function loss, thereby preventing tumor cells from replicating in large quantities. The addition of the two also has a synergistic effect.

[0050] The anti-tumor composition of the present invention has excellent anti-inflammatory, antioxidant and anti-tumor effects, can improve the administration of active components, can deliver more drugs into cancer cells, tends to accumulate in tumor tissues, and has the effect of efficiently killing cancer cells. It also has low systemic toxicity, few side effects, high safety, a wide source of raw materials, and low cost, and can reduce the economic burden of patients, thereby having a wider range of applications. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Sodium hyaluronate, with an average molecular weight of 100,000, was purchased from Xi'an Ruilin Biotechnology Co., Ltd.

[0053] Cellulase, 4000 U / g, was purchased from Xiasheng Enzyme Biotechnology Co., Ltd.; hemicellulase, 6000 U / g, was purchased from Jiangxi Baiying Biotechnology Co., Ltd.

[0054] Lactobacillus casei, 10 billion cfu / g, was purchased from TaiZe (Guangzhou) Biotechnology Co., Ltd.; Lactobacillus rhamnosus, 10 billion cfu / g, was purchased from Youlike (Shanghai) Life Sciences Co., Ltd.; Lactobacillus plantarum, 10 billion cfu / g, was purchased from Shanghai Xuanke Biotechnology Co., Ltd.

[0055] The preparation method of Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus seed solution is as follows: the strains are inoculated into Gould's medium, and fermented and cultured for 24 hours at 37°C and 100 r / min under anaerobic conditions to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL.

[0056] Example 1

[0057] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0058] S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 12 parts by weight of Poria cocos and 7 parts by weight of Astragalus membranaceus were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1wt% of the total mass of the system, and the enzyme was hydrolyzed at 45°C for 2h, heated to boiling for 1h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract;

[0059] The complex enzyme includes cellulase and hemicellulase in a mass ratio of 5:3;

[0060] S2. Preparation of Poria cocos and Astragalus fermentation product: 10 parts by weight of the filter residue in step S1, 5 parts by weight of fish bone powder, 0.2 parts by weight of magnesium chloride, 0.2 parts by weight of manganese sulfate, 0.2 parts by weight of copper chloride, 0.4 parts by weight of sodium chloride were added to 300 parts by weight of water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, the inoculation amount was 1v / v% and 2v / v%, respectively, under anaerobic conditions, fermented and cultured at 37°C, 100r / min for 48h, filtered, the solid was washed to obtain a bacterial solution, mixed with the filtrate, freeze-dried, and a Poria cocos and Astragalus fermentation product was obtained, and the washed solid was retained;

[0061] S3. Preparation of a culture medium: 10 parts by weight of the washed solid in step S2, 20 parts by weight of sugar cane juice having a sugar content of 18°, 5 parts by weight of yeast powder, 2 parts by weight of sodium nitrate, 1 part by weight of potassium dihydrogen phosphate, 0.5 parts by weight of magnesium sulfate, 0.2 parts by weight of calcium chloride, 0.1 parts by weight of vitamin B1, 0.3 parts by weight of vitamin C, and 0.5 parts by weight of sodium selenite were added to 300 parts by weight of water and sterilized to obtain a culture medium;

[0062] S4. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, ferment and culture under anaerobic conditions at 37°C and 100 r / min for 36 h, filter, add ethanol to the filtrate until the ethanol content in the system is 80 wt%, and precipitate for 3 h to obtain selenium-rich extracellular polysaccharides;

[0063] S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve 10 parts by weight of the selenium-enriched exopolysaccharide obtained in step S4 in 500 parts by weight of water, add 2 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 7 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 4, stir at room temperature for 2 hours, adjust the pH value of the solution to 6.9, dialyze for 3 hours, the dialysis bag pore size of the dialysis is 8000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide;

[0064] S6. Preparation of the active ingredient: 10 parts by weight of geraniol and 7 parts by weight of dihydroartemisinin were stirred and mixed for 10 minutes to obtain an active ingredient;

[0065] S7. Preparation of carboxylated active component: 7 parts by weight of the active component obtained in step S6, 9 parts by weight of oxalic anhydride, 0.01 parts by weight of triethylenediamine were dissolved in 200 parts by weight of dichloromethane, and the reaction was stirred at room temperature under nitrogen for 20h, the product was filtered, washed and dried to obtain a carboxylated active component;

[0066] S8. Preparation of active component - selenium-rich extracellular polysaccharide: 5 parts by weight of the carboxylated active component prepared in step S7 and 8 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, stirred and activated for 1 hour, 10 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 were added, stirred and reacted at room temperature for 18 hours, dialyzed for 3 hours, the dialysis bag pore size of the dialysis was 8000D, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide;

[0067] S9. Preparation of embedded microspheres: 10 parts by weight of Poria cocos and Astragalus membranaceus fermentation product were added to 200 parts by weight of water, 7 parts by weight of sodium alginate and 3 parts by weight of sodium hyaluronate were added, and the mixture was stirred for 20 minutes to obtain an aqueous phase; 100 parts by weight of the aqueous phase were added to 250 parts by weight of fish oil, 1 part by weight of lecithin was added, emulsified at 10000 r / min for 15 minutes, 20 parts by weight of 5wt% calcium chloride solution were added dropwise, and the mixture was cured at room temperature for 20 minutes, centrifuged, washed, and dried to obtain embedded microspheres;

[0068] S10. Preparation of an antitumor composition: 15 parts by weight of the Poria cocos and Astragalus membranaceus water extract prepared in step S1, 27 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 7 parts by weight of the embedded microspheres prepared in step S9, 0.5 parts by weight of genipin, and 0.2 parts by weight of berberine were mixed for 20 minutes to prepare an antitumor composition.

[0069] Example 2

[0070] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0071] S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 15 parts by weight of Poria cocos and 10 parts by weight of Astragalus membranaceus were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 2wt% of the total mass of the system, and the enzyme was hydrolyzed at 50°C for 4h, heated to boiling for extraction for 3h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract;

[0072] The complex enzyme includes cellulase and hemicellulase in a mass ratio of 7:5;

[0073] S2. Preparation of Poria cocos and Astragalus fermentation product: 15 parts by weight of the filter residue in step S1, 7 parts by weight of yeast powder, 1.7 parts by weight of potassium chloride, 0.3 parts by weight of magnesium sulfate were added to 500 parts by weight of water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, the inoculation amount was 3v / v% and 4v / v%, respectively, under anaerobic conditions, fermented and cultured at 39°C, 200r / min for 56h, filtered, the solid was washed to obtain the bacterial solution, mixed with the filtrate, freeze-dried, and the Poria cocos and Astragalus fermentation product was obtained, and the washed solid was retained;

[0074] S3. Preparation of a culture medium: 15 parts by weight of the washed solid in step S2, 25 parts by weight of sugar cane juice having a sugar content of 20°, 7 parts by weight of yeast powder, 4 parts by weight of sodium nitrate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of magnesium sulfate, 0.5 parts by weight of calcium chloride, 0.2 parts by weight of vitamin B1, 0.5 parts by weight of vitamin C, and 0.7 parts by weight of sodium selenate were added to 500 parts by weight of water and sterilized to obtain a culture medium;

[0075] S4. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, ferment and culture under anaerobic conditions at 39°C and 200r / min for 48h, filter, add ethanol to the filtrate until the ethanol content in the system is 85wt%, precipitate for 5h, and obtain selenium-rich extracellular polysaccharides;

[0076] The bacterial content of the plant lactobacillus seed liquid is 10 9 cfu / mL;

[0077] S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve 12 parts by weight of the selenium-enriched exopolysaccharide obtained in step S4 in 500 parts by weight of water, add 4 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 10 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 5, stir and react at room temperature for 3 hours, adjust the pH value of the solution to 7.1, dialyze for 5 hours, the dialysis bag pore size of the dialysis is 10000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide;

[0078] S6. Preparation of the active ingredient: 12 parts by weight of geraniol and 10 parts by weight of dihydroartemisinin were stirred and mixed for 10 minutes to obtain an active ingredient;

[0079] S7. Preparation of carboxylated active component: 10 parts by weight of the active component obtained in step S6, 12 parts by weight of oxalic anhydride, 0.02 parts by weight of 1,8-diazabicyclo[5.4.0]undec-7-ene were dissolved in 200 parts by weight of dichloromethane, and the reaction was stirred at room temperature under nitrogen for 24 hours. The product was filtered, washed, and dried to obtain a carboxylated active component;

[0080] S8. Preparation of active component - selenium-rich extracellular polysaccharide: 7 parts by weight of the carboxylated active component prepared in step S7 and 10 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, stirred and activated for 3 hours, 15 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 were added, stirred and reacted at room temperature for 20 hours, dialyzed for 5 hours, the dialysis bag pore size of the dialysis was 10000D, freeze-dried, and the active component - selenium-rich extracellular polysaccharide was obtained;

[0081] S9. Preparation of embedded microspheres: 10 parts by weight of Poria cocos and Astragalus membranaceus fermentation product were added to 200 parts by weight of water, 10 parts by weight of sodium alginate and 5 parts by weight of sodium hyaluronate were added, and the mixture was stirred for 20 minutes to obtain an aqueous phase; 150 parts by weight of the aqueous phase were added to 300 parts by weight of fish oil, 2 parts by weight of lecithin were added, emulsified at 10000 r / min for 15 minutes, 20 parts by weight of 7wt% calcium chloride solution were added dropwise, and the mixture was cured at room temperature for 30 minutes, centrifuged, washed, and dried to obtain embedded microspheres;

[0082] S10. Preparation of an antitumor composition: Mix 20 parts by weight of the Poria and Astragalus water extract obtained in step S1, 32 parts by weight of the active component - selenium-rich extracellular polysaccharide obtained in step S8, 10 parts by weight of the embedded microspheres obtained in step S9, 1 part by weight of genipin, and 0.4 parts by weight of berberine for 20 minutes to obtain an antitumor composition.

[0083] Example 3

[0084] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0085] S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 3 parts by weight of Poria cocos and 8 parts by weight of Astragalus membranaceus were washed, dried, and crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1.5wt% of the total mass of the system, and the enzyme was hydrolyzed at 47°C for 3h, heated to boiling for 2h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract;

[0086] The complex enzyme includes cellulase and hemicellulase in a mass ratio of 6:4;

[0087] S2. Preparation of Poria cocos and Astragalus fermentation product: 12 parts by weight of the filter residue in step S1, 6 parts by weight of peptone, 1 part by weight of sodium chloride, 0.2 parts by weight of magnesium sulfate, 0.3 parts by weight of calcium chloride were added to 400 parts by weight of water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, the inoculation amount was 2v / v% and 3v / v%, respectively, under anaerobic conditions, 38°C, 150r / min fermentation culture for 52h, filtered, the solid was washed to obtain a bacterial solution, mixed with the filtrate, freeze-dried, and the Poria cocos and Astragalus fermentation product was obtained, and the washed solid was retained;

[0088] S3. Preparation of a culture medium: 12 parts by weight of the washed solid in step S2, 22 parts by weight of sugar cane juice having a sugar content of 19°, 6 parts by weight of yeast powder, 3 parts by weight of sodium nitrate, 1.5 parts by weight of potassium dihydrogen phosphate, 0.7 parts by weight of magnesium sulfate, 0.35 parts by weight of calcium chloride, 0.15 parts by weight of vitamin B1, 0.4 parts by weight of vitamin C, and 0.6 parts by weight of sodium selenite were added to 400 parts by weight of water and sterilized to obtain a culture medium;

[0089] S4. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, ferment and culture under anaerobic conditions at 38°C and 150r / min for 42h, filter, add ethanol to the filtrate until the ethanol content in the system is 82wt%, precipitate for 4h, and obtain selenium-rich extracellular polysaccharides;

[0090] S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve 11 parts by weight of the selenium-enriched exopolysaccharide obtained in step S4 in 500 parts by weight of water, add 3 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 8.5 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 4.5, stir at room temperature for 2.5 hours, adjust the pH value of the solution to 7, dialyze for 4 hours, the dialysis bag pore size of the dialysis is 9000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide;

[0091] S6. Preparation of the active ingredient: 11 parts by weight of geraniol and 8.5 parts by weight of dihydroartemisinin were stirred and mixed for 10 min to obtain an active ingredient;

[0092] S7. Preparation of carboxylated active component: 8.5 parts by weight of the active component obtained in step S6, 10 parts by weight of oxalic anhydride, 0.015 parts by weight of 4-dimethylaminopyridine were dissolved in 200 parts by weight of dichloromethane, and the reaction was stirred at room temperature under nitrogen for 22h, the product was filtered, washed, and dried to obtain a carboxylated active component;

[0093] S8. Preparation of active component - selenium-rich extracellular polysaccharide: 6 parts by weight of the carboxylated active component prepared in step S7 and 9 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, stirred and activated for 2 hours, 12 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 were added, stirred and reacted at room temperature for 19 hours, dialyzed for 4 hours, the dialysis bag pore size of the dialysis was 9000D, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide;

[0094] S9. Preparation of embedded microspheres: 10 parts by weight of Poria cocos and Astragalus membranaceus fermentation product were added to 200 parts by weight of water, 8.5 parts by weight of sodium alginate and 4 parts by weight of sodium hyaluronate were added, and the mixture was stirred for 20 minutes to obtain an aqueous phase; 125 parts by weight of the aqueous phase were added to 270 parts by weight of fish oil, 1.5 parts by weight of lecithin were added, emulsified at 10000 r / min for 15 minutes, 20 parts by weight of 6wt% calcium chloride solution were added dropwise, and the mixture was cured at room temperature for 25 minutes, centrifuged, washed, and dried to obtain embedded microspheres;

[0095] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract prepared in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 8.5 parts by weight of the embedded microspheres prepared in step S9, 0.7 parts by weight of genipin, and 0.3 parts by weight of berberine were mixed for 20 minutes to prepare an antitumor composition.

[0096] Example 4

[0097] Compared with Example 3, the difference is that the composite enzyme is a single cellulase.

[0098] Example 5

[0099] Compared with Example 3, the difference is that the complex enzyme is a single hemicellulase.

[0100] Comparative Example 1

[0101] Compared with Example 3, the difference is that no Poria cocos is added in step S1.

[0102] The details are as follows:

[0103] S1. Preparation of Astragalus membranaceus aqueous extract: 11 parts by weight of Astragalus membranaceus were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1.5wt% of the total mass of the system, and the enzyme was hydrolyzed at 47°C for 3h, heated to boiling for 2h, filtered, and the residue was retained; the filtrate was dried to obtain an Astragalus membranaceus aqueous extract;

[0104] The complex enzyme comprises cellulase and hemicellulase in a mass ratio of 6:4.

[0105] Comparative Example 2

[0106] Compared with Example 3, the difference is that no astragalus is added in step S1.

[0107] The details are as follows:

[0108] S1. Preparation of Poria cocos water extract: 11 parts by weight of Poria cocos were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1.5wt% of the total mass of the system, and the enzyme was hydrolyzed at 47°C for 3h, heated to boiling for 2h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos water extract;

[0109] The complex enzyme comprises cellulase and hemicellulase in a mass ratio of 6:4.

[0110] Comparative Example 3

[0111] Compared with Example 3, the difference is that no complex enzyme is added in step S1.

[0112] The details are as follows:

[0113] S1. Preparation of Poria cocos and Astragalus membranaceus aqueous extract: 3 parts by weight of Poria cocos and 8 parts by weight of Astragalus membranaceus were washed, dried, and crushed, respectively, added to 200 parts by weight of water, heated to boiling and extracted for 2 hours, filtered, and the residue was retained; the filtrate was dried to obtain the Poria cocos and Astragalus membranaceus aqueous extract.

[0114] Comparative Example 4

[0115] Compared with Example 3, the difference is that the Lactobacillus casei seed liquid is not inoculated in step S2.

[0116] The details are as follows:

[0117] S2. Preparation of Poria cocos and Astragalus fermentation product: 12 parts by weight of the filter residue in step S1, 6 parts by weight of peptone, 1 part by weight of sodium chloride, 0.2 parts by weight of magnesium sulfate, and 0.3 parts by weight of calcium chloride are added to 400 parts by weight of water, sterilized, inoculated with Lactobacillus rhamnosus seed solution, the inoculation amount is 5 v / v%, and fermented and cultured at 38°C and 150 r / min for 52 h under anaerobic conditions, filtered, washed the solid to obtain the bacterial solution, mixed with the filtrate, and freeze-dried to obtain the Poria cocos and Astragalus fermentation product, and the washed solid is retained.

[0118] Comparative Example 5

[0119] Compared with Example 3, the difference is that the Lactobacillus rhamnosus seed liquid is not inoculated in step S2.

[0120] The details are as follows:

[0121] S2. Preparation of Poria cocos and Astragalus fermentation product: 12 parts by weight of the filter residue in step S1, 6 parts by weight of peptone, 1 part by weight of sodium chloride, 0.2 parts by weight of magnesium sulfate, and 0.3 parts by weight of calcium chloride are added to 400 parts by weight of water, sterilized, inoculated with Lactobacillus casei seed solution, the inoculation amount is 5 / v%, and fermented and cultured at 38°C, 150r / min for 52h under anaerobic conditions, filtered, and the solid is washed to obtain a bacterial solution, mixed with the filtrate, and freeze-dried to obtain a Poria cocos and Astragalus fermentation product, and the washed solid is retained.

[0122] Comparative Example 6

[0123] Compared with Example 3, the difference is that step S2 is not performed.

[0124] This embodiment provides a method for preparing an antitumor composition, which specifically comprises the following steps:

[0125] S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 3 parts by weight of Poria cocos and 8 parts by weight of Astragalus membranaceus were washed, dried, and crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1.5wt% of the total mass of the system, and the enzyme was hydrolyzed at 47°C for 3h, heated to boiling for 2h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract;

[0126] The complex enzyme includes cellulase and hemicellulase in a mass ratio of 6:4;

[0127] S2. Preparation of a culture medium: 12 parts by weight of the filter residue in step S1, 22 parts by weight of sugar cane juice having a sugar content of 19°, 6 parts by weight of yeast powder, 3 parts by weight of sodium nitrate, 1.5 parts by weight of potassium dihydrogen phosphate, 0.7 parts by weight of magnesium sulfate, 0.35 parts by weight of calcium chloride, 0.15 parts by weight of vitamin B1, 0.4 parts by weight of vitamin C, and 0.6 parts by weight of sodium selenite were added to 400 parts by weight of water and sterilized to obtain a culture medium;

[0128] S3. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S2, and ferment and culture under anaerobic conditions at 38°C and 150r / min for 42h, filter, add ethanol to the filtrate until the ethanol content in the system is 82wt%, and precipitate for 4h to obtain selenium-rich extracellular polysaccharides;

[0129] S4. Preparation of amino-enriched selenium exopolysaccharide: dissolve 11 parts by weight of the selenium-enriched exopolysaccharide obtained in step S3 in 500 parts by weight of water, add 3 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 8.5 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 4.5, stir at room temperature for 2.5 hours, adjust the pH value of the solution to 7, dialyze for 4 hours, the dialysis bag pore size of the dialysis is 9000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide;

[0130] S5. Preparation of the active ingredient: 11 parts by weight of geraniol and 8.5 parts by weight of dihydroartemisinin were stirred and mixed for 10 min to obtain an active ingredient;

[0131] S6. Preparation of carboxylated active component: 8.5 parts by weight of the active component obtained in step S5, 10 parts by weight of oxalic anhydride, 0.015 parts by weight of 4-dimethylaminopyridine were dissolved in 200 parts by weight of dichloromethane, and the reaction was stirred at room temperature under nitrogen for 22h, the product was filtered, washed and dried to obtain a carboxylated active component;

[0132] S7. Preparation of active component - selenium-rich extracellular polysaccharide: 6 parts by weight of the carboxylated active component prepared in step S6 and 9 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, stirred and activated for 2 hours, 12 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S4 were added, stirred and reacted at room temperature for 19 hours, dialyzed for 4 hours, the dialysis bag pore size of the dialysis was 9000D, freeze-dried, and the active component - selenium-rich extracellular polysaccharide was obtained;

[0133] S8. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract prepared in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 0.7 parts by weight of genipin, and 0.3 parts by weight of berberine were mixed for 20 minutes to prepare an antitumor composition.

[0134] Comparative Example 7

[0135] Compared with Example 3, the difference is that sodium selenite is not added in step S3.

[0136] The details are as follows:

[0137] S3. Preparation of culture medium: 12 parts by weight of the washed solid in step S2, 22 parts by weight of sugar cane juice with a sugar content of 19°, 6 parts by weight of yeast powder, 3 parts by weight of sodium nitrate, 1.5 parts by weight of potassium dihydrogen phosphate, 0.7 parts by weight of magnesium sulfate, 0.35 parts by weight of calcium chloride, 0.15 parts by weight of vitamin B1, and 0.4 parts by weight of vitamin C are added to 400 parts by weight of water and sterilized to obtain a culture medium.

[0138] Comparative Example 8

[0139] Compared with Example 3, the difference is that geraniol is not added in step S6.

[0140] The details are as follows:

[0141] S6. Preparation of active component: 19.5 parts by weight of dihydroartemisinin was stirred and mixed for 10 minutes to obtain an active component.

[0142] Comparative Example 9

[0143] Compared with Example 3, the difference is that dihydroartemisinin is not added in step S6.

[0144] The details are as follows:

[0145] S6. Preparation of active component: 19.5 parts by weight of geraniol were stirred and mixed for 10 minutes to obtain an active component.

[0146] Comparative Example 10

[0147] Compared with Example 3, the difference is that the Poria cocos and Astragalus membranaceus fermentation product is not embedded in step S9.

[0148] The details are as follows:

[0149] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria and Astragalus water extract prepared in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 8.5 parts by weight of the Poria and Astragalus fermentation product prepared in step S2, 0.7 parts by weight of genipin, and 0.3 parts by weight of berberine are mixed for 20 minutes to prepare an antitumor composition.

[0150] Comparative Example 11

[0151] Compared with Example 3, the difference is that the active component - selenium-rich extracellular polysaccharide is not added in step S10.

[0152] The details are as follows:

[0153] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract obtained in step S1, 8.5 parts by weight of the embedded microspheres obtained in step S9, 0.7 parts by weight of genipin, and 0.3 parts by weight of berberine were mixed for 20 minutes to obtain an antitumor composition.

[0154] Comparative Example 12

[0155] Compared with Example 3, the difference is that the active component - selenium-rich extracellular polysaccharide in step S10 is replaced by an equal amount of active components.

[0156] The details are as follows:

[0157] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract prepared in step S1, 30 parts by weight of the active ingredient prepared in step S6, 8.5 parts by weight of the embedded microspheres prepared in step S9, 0.7 parts by weight of genipin, and 0.3 parts by weight of berberine were mixed for 20 minutes to prepare an antitumor composition.

[0158] Comparative Example 13

[0159] Compared with Example 3, the difference is that no genipin is added in step S10.

[0160] The details are as follows:

[0161] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract prepared in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 8.5 parts by weight of the embedded microspheres prepared in step S9, and 1 part by weight of berberine were mixed for 20 minutes to prepare an antitumor composition.

[0162] Comparative Example 14

[0163] Compared with Example 3, the difference is that berberine is not added in step S10.

[0164] The details are as follows:

[0165] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria and Astragalus water extract prepared in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide prepared in step S8, 8.5 parts by weight of the embedded microspheres prepared in step S9, and 1 part by weight of genipin are mixed for 20 minutes to prepare an antitumor composition.

[0166] Comparative Example 15

[0167] Compared with Example 3, the difference is that genipin and berberine are not added in step S10.

[0168] The details are as follows:

[0169] S10. Preparation of an antitumor composition: 17 parts by weight of the Poria cocos and Astragalus membranaceus water extract obtained in step S1, 30 parts by weight of the active component - selenium-rich extracellular polysaccharide obtained in step S8, and 8.5 parts by weight of the embedded microspheres obtained in step S9 were mixed for 20 minutes to obtain an antitumor composition.

[0170] Test Example 1 In vitro activity experiment on human liver cancer cell HepG2

[0171] The antitumor compositions prepared in Examples 1-5 and Comparative Examples 1-15 were subjected to the following experiments.

[0172] Cell viability assay:

[0173] 3×10 cells / 100 μL were inoculated into a 96-well plate. 4 HepG2 cells in the logarithmic phase were cultured until adherent, and 100 μL of drug-containing culture medium was added to each group, so that the final working concentration of the anti-tumor composition in each well was 100 μg / L, and the medium without drug was used as a control. Each group was repeated 5 times, and 24 hours after administration, the culture medium was discarded, and after washing, 100 μL of CCK-8 reagent and serum-free medium mixture was added to each well (the mass ratio of CCK-8 reagent and serum-free medium was 1:10). Incubate at 37°C for 2 hours, measure the OD value at 450nm, and calculate the cell viability.

[0174] Cell apoptosis experiment:

[0175] The drug administration was carried out in the same manner as in the cell activity experiment. After 24 hours of drug administration, the culture medium was discarded, the cells were fixed, TUNEL detection solution was added, DAPI (4',6-diamidino-2-phenylindole) was used for staining, and fluorescence microscopy was used to image the cells and calculate the cell apoptosis rate.

[0176] The results are shown in Table 1.

[0177] Table 1

[0178]

[0179]

[0180] It can be seen from the above table that the anti-tumor compositions prepared in Examples 1-3 of the present invention can significantly reduce the survival rate of HepG2 cells and promote cell apoptosis.

[0181] Test Example 2

[0182] Mouse hepatoma H22 cells in the logarithmic phase were used, and the tumor cell suspension was diluted with sterile saline. The cell number was 1.2×10 7 / mL, inoculated into the peritoneal cavity of mice. After 5-7 days, when the abdominal cavity of mice becomes larger and soft, ascites is extracted through the peritoneal cavity under sterile conditions and inoculated into the peritoneal cavity of normal mice. The in vivo passage is repeated 3 times and collected for modeling. The mouse ascites cells are extracted and the cell concentration is adjusted to 1×10 7 / mL of cell suspension was inoculated subcutaneously at the junction of the right scapula and neck of mice at 0.2mL / mouse. When nodules could be felt 5-7 days after inoculation, 3 mice were randomly selected for pathological examination. When the nodules were tumor tissues, the inoculation was successful.

[0183] Then the mice were randomly divided into groups, each group had 6 mice, the experimental group was divided into Example 1-5 and Comparative Example 1-15 groups, each mouse was gavaged with 0.5 mL (the anti-tumor composition prepared in the corresponding group with a concentration of 10 mg / mL); the model group was given 0.5 mL of distilled water; the positive drug group was given cisplatin 2 mg / kg, intraperitoneal injection, once every 3 days, for 10 consecutive days. Routine feeding. On the next day after drug withdrawal, all mice were killed by cervical dislocation, the tumor mass was removed, weighed, and the tumor inhibition rate (%) was calculated.

[0184] Tumor inhibition rate (%) = (average tumor weight of model group - average tumor weight of experimental group) / average tumor weight of model group × 100%

[0185] The thymus and spleen of mice were removed and weighed, and the thymus index and spleen index were calculated.

[0186] Thymus index (mg / g) = thymus weight (mg) / body weight (g); spleen index (mg / g) = spleen weight (mg) / body weight (g).

[0187] The results are shown in Table 2.

[0188] Table 2

[0189]

[0190]

[0191] Note: *Compared with the model group, P < 0.05.

[0192] As can be seen from the above table, the anti-tumor composition prepared in Examples 1-3 of the present invention can significantly improve the tumor inhibition rate, and the thymus index and spleen index are not significantly increased, and there is no toxicity to the thymus and spleen. However, the spleen index of the cisplatin group is significantly increased, which may be related to the toxic damage of cisplatin to the spleen, which is related to the inhibition of bone marrow by cisplatin to activate extramedullary spleen hematopoiesis.

[0193] Compared with Example 3, in Examples 4 and 5, the composite enzyme is a single cellulase or hemicellulase. Compared with Example 3, in Comparative Example 3, no composite enzyme is added in step S1. The survival rate of HepG2 cells is improved, the apoptosis rate is reduced, and the tumor inhibition rate is reduced. Under the action of the composite enzyme, the present invention promotes the rupture of the plant cell walls of Poria cocos and Astragalus membranaceus, and promotes the dissolution of active components such as polysaccharides, triterpenes, and flavonoids, thereby obtaining a Poria cocos and Astragalus membranaceus water extract containing multiple anti-tumor active components.

[0194] Comparative Examples 1 and 2 are compared with Example 3, and no Poria or Astragalus is added in step S1. The survival rate of HepG2 cells is improved, the apoptosis rate is decreased, and the tumor inhibition rate is decreased. Poria and Astragalus are both good anti-tumor raw materials. Astragalus contains rich astragalus polysaccharides, which have the pharmacological effects of regulating the body's immune function, improving blood sugar, and anti-tumor. It has high safety, multiple targets, and extensive pharmacological effects. By regulating the body's various immune cells, the tumor immune microenvironment is improved, the immunosuppressive state is improved, and tumor growth is inhibited. By regulating the immune cells in the tumor immune microenvironment, such as macrophages, dendritic cells, myeloid suppressor cells, T cells, regulatory T cells, natural killer cells, and B cells, the tumor immune microenvironment is improved, the immunosuppressive state is reversed, the immune response ability of tumor patients is enhanced, and the killing ability of the body's immune cells to tumor cells is improved, and the growth and metastasis of tumors are effectively inhibited. The active components contained in Poria cocos, such as Poria cocos polysaccharide and Poria cocos triterpenes, have good anti-tumor effects. Among them, Poria cocos polysaccharide activates and enhances the body's immune cells, improves the body's immune response ability, thereby inhibiting the development of tumors, binding to the TLR4 receptors of macrophages and dendritic cells, activating the TRAF6 / NF-γB signaling pathway, promoting the maturation and activation of these cells, thereby improving their antigen presentation ability, while enhancing the effector function of immune cells, playing the role of immune enhancement and immune surveillance, increasing the phagocytic and killing ability of macrophages and natural killer cells, thereby clearing tumor cells, and promoting various immune cells to secrete and express a variety of cytokines with anti-tumor effects. Poria cocos triterpenes, including pachymic acid, pachymanol, and pachyman tetraketone, can inhibit the proliferation of human tumor cells by upregulating the expression of Bax, Caspase-3, Caspase-9 and PARP, downregulating the expression of Bcl-2, activating the mitochondria-mediated apoptosis pathway, inducing their apoptosis, and inhibiting the migration and invasion of tumor cells, thereby blocking the spread and metastasis of tumors. The addition of the two has a synergistic effect.

[0195] Compared with Example 3, Lactobacillus casei strain seed liquid or Lactobacillus rhamnosus strain seed liquid were not inoculated in step S2 in comparative example 4 and 5. Compared with Example 3, step S2 was not performed in comparative example 6. Compared with Example 3, in step S9, Poria cocos and astragalus fermentation product was not embedded. The survival rate of HepG2 cells was improved, and the apoptosis rate decreased, and the tumor inhibition rate decreased. Fermentation bacteria Lactobacillus casei and Lactobacillus rhamnosus themselves can significantly reduce the TNF-a effect produced by THP-1 cells, and have a significant inhibitory effect on the proliferation of liver cancer cells and breast cancer cells, and have a synergistic effect. Therefore, after the fermentation bacteria are embedded, the destruction of gastric acid can be avoided, thereby entering the intestinal tract, and the probiotic effect is played, and a large amount of anti-inflammatory and antioxidant factors are produced to inhibit the occurrence and development of cancer.

[0196] Comparative Example 7 Compared with Example 3, sodium selenite was not added in step S3. The survival rate of HepG2 cells was improved, the apoptosis rate was reduced, the tumor inhibition rate was reduced, and the thymus index and spleen index were improved. The present invention promotes the process of converting inorganic selenium into organic selenium by adding a selenium source, and obtains a selenium-rich extracellular polysaccharide, which is rich in selenium and has a good antioxidant effect, can reduce the generation of free radicals and oxidative damage to cells and tissues, reduce tumors by reducing oxidative stress, promote DNA repair process, maintain DNA integrity, increase the activity of T cells and NK cells, help the immune system to better identify and destroy cancer cells, promote tumor cell apoptosis, inhibit tumor angiogenesis, thereby limiting the blood supply to the tumor, making it difficult to obtain nutrients and oxygen, and limiting the growth of the tumor.

[0197] Comparative Examples 8 and 9 Compared with Example 3, geraniol or dihydroartemisinin was not added in step S6. Comparative Example 11 Compared with Example 3, active component-selenium-rich extracellular polysaccharide was not added in step S10. The survival rate of HepG2 cells was improved, the apoptosis rate decreased, the tumor inhibition rate decreased, and the thymus index and spleen index increased. Geraniol has antioxidant, antibacterial, antiviral, neuroprotective and anticancer effects, which can significantly reduce liver damage and tumor formation, but the water solubility and administration of geraniol are poor. The peroxide bridge structure in the dihydroartemisinin parent nucleus can generate highly active free radicals by ring-opening reaction under the action of divalent iron ions of tumor cell heme, and exerts antitumor activity through mechanisms such as blocking tumor cell cycle, inhibiting tumor angiogenesis, inducing DNA damage and ferroptosis. Similarly, the administration of dihydroartemisinin is not good. Therefore, the present invention is to react geraniol and dihydroartemisinin with amino-enriched selenium-rich extracellular polysaccharides after carboxylation, and the obtained product has a good anti-tumor effect, especially inhibiting the occurrence and development of liver cancer cells.

[0198] Comparative Example 12 Compared with Example 3, the active component-selenium-rich extracellular polysaccharide in step S10 is replaced by an equal amount of active components. The survival rate of HepG2 cells is improved, the apoptosis rate is reduced, and the tumor inhibition rate is reduced. Selenium-rich extracellular polysaccharide itself is a very good anti-tumor substance. Furthermore, after the selenium-rich extracellular polysaccharide is aminated, it can react well with the carboxylated active component to obtain the active component-selenium-rich extracellular polysaccharide; the selenium-rich extracellular polysaccharide can also be used as a carrier. At the same drug concentration level, it can deliver more drugs to cancer cells, tend to accumulate in tumor tissues, and has lower systemic toxicity, thereby producing a stronger cell killing effect.

[0199] Compared with Example 3, in Comparative Examples 13 and 14, genipin and berberine were not added in step S10. Compared with Example 3, in Comparative Example 15, genipin and berberine were not added in step S10. The survival rate of HepG2 cells was improved, the apoptosis rate was reduced, and the tumor inhibition rate was reduced. Active substances including genipin and berberine are also added to the anti-tumor composition of the present invention. The effects of genipin include anti-inflammatory, treatment of liver disease, anti-tumor, etc., and can directly bind to the SH2 domain of STAT-3, inhibit the phosphorylation and dimerization of STAT-3 (Y705), and then inhibit liver cancer cell proliferation, angiogenesis, cell invasion, and reverse the transition (EMT) process of epithelial cells to mesenchymal cells. Berberine has anti-inflammatory, antibacterial, antiviral, and anti-tumor activities. It destroys the mitochondrial structure of cancer cells, causing mitochondrial swelling, reduced internal cristae, vacuolation, and fragmentation. It also reduces the mitochondrial copy number, inhibits the tricarboxylic acid cycle, and causes mitochondrial dysfunction, thereby preventing tumor cells from replicating and proliferating in large numbers. The addition of the two also has a synergistic effect.

[0200] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an antitumor composition, characterized in that: The poria and astragalus are subjected to enzymatic hydrolysis and boiling water extraction, filtered, the filter residue is fermented, filtered, the solid is washed to obtain a bacterial liquid, mixed with the filtrate, dried, and embedded to obtain embedded microspheres; the washed solid is added to a culture medium, inoculated with Lactobacillus plantarum for fermentation, filtered, the filtrate is ethanol precipitated, and amino-treated to obtain amino-enriched selenium extracellular polysaccharide, which is reacted with carboxylated geraniol and dihydroartemisinin to obtain an active component-selenium-enriched extracellular polysaccharide; the poria and astragalus water extract, the active component-selenium-enriched extracellular polysaccharide, the embedded microspheres, genipin, and berberine are evenly mixed to obtain an anti-tumor composition.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of Poria cocos and Astragalus water extract: Poria cocos and Astragalus membranaceus were washed, dried, crushed, added to water, added with complex enzyme for enzymolysis, heated to boiling for extraction, filtered, and the residue was retained; the filtrate was dried to obtain Poria cocos and Astragalus membranaceus water extract; S2. Preparation of Poria cocos and Astragalus fermentation product: The filter residue, nitrogen source and inorganic salt in step S1 are added to water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, fermented, filtered, the solids are washed to obtain a bacterial solution, mixed with the filtrate, freeze-dried, and a Poria cocos and Astragalus fermentation product is obtained, and the washed solids are retained; S3. Preparation of a culture medium: The washed solids in step S2, sugar cane juice, yeast powder, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, vitamin B1, vitamin C, and a selenium source were added to water and sterilized to obtain a culture medium; S4. Preparation of selenium-rich extracellular polysaccharides: inoculating the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, fermenting and culturing, filtering, and adding ethanol to the filtrate for precipitation to obtain selenium-rich extracellular polysaccharides; S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve the selenium-enriched exopolysaccharide obtained in step S4 in water, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and adipic acid dihydrazide, adjust the pH value of the solution to 4-5, stir the reaction at room temperature, adjust the pH value of the solution to 6.9-7.1, dialyze, and freeze-dry the dialyzate to obtain amino-enriched selenium exopolysaccharide; S6. Preparation of active component: Mix geraniol and dihydroartemisinin to obtain an active component; S7. Preparation of carboxylated active component: The active component obtained in step S6, oxalic anhydride and the catalyst were stirred at room temperature under an inert gas, and the product was filtered, washed and dried to obtain a carboxylated active component; S8. Preparation of active component - selenium-rich extracellular polysaccharide: The carboxylated active component prepared in step S7 is mixed with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride for activation, and the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 is added, stirred at room temperature for reaction, dialyzed, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide; S9. Preparation of embedded microspheres: adding the fermentation product of Poria cocos and Astragalus membranaceus to water, adding sodium alginate and sodium hyaluronate, stirring and mixing to obtain an aqueous phase; adding the aqueous phase to the oil phase, adding lecithin, emulsifying, dropping calcium chloride solution, solidifying at room temperature, centrifuging, washing, and drying to obtain embedded microspheres; S10. Preparation of an antitumor composition: The Poria cocos and Astragalus membranaceus water extract prepared in step S1, the active component - selenium-rich extracellular polysaccharide prepared in step S8, the embedded microspheres prepared in step S9, genipin and berberine are mixed evenly to prepare an antitumor composition.

3. The preparation method according to claim 2, characterized in that: The mass ratio of Poria cocos and Astragalus membranaceus described in step S1 is 12-15:7-10, the composite enzyme includes cellulase and hemicellulase, the mass ratio is 5-7:3-5, the addition amount of the composite enzyme is 1-2wt% of the total mass of the system, the temperature of the enzymolysis is 45-50°C, the time is 2-4h, and the time of heating to boiling extraction is 1-3h; the mass ratio of the filter residue, nitrogen source, inorganic salt and water described in step S2 is 10-15:5-7:1-2:300-500, the nitrogen source is selected from at least one of fish bone powder, peptone and yeast powder, the inorganic salt is selected from at least one of sodium chloride, potassium chloride, magnesium sulfate, magnesium chloride, manganese sulfate, copper chloride, ferric chloride and calcium chloride, and the bacterial content of the Lactobacillus casei and Lactobacillus rhamnosus seed liquid is 10 8 -10 9 cfu / mL, the inoculation amounts were 1-3v / v% and 2-4v / v%, respectively, and the fermentation culture conditions were anaerobic conditions, 37-39°C, 100-200r / min, and fermentation culture for 48-56h.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the washed solid, sugarcane juice, yeast powder, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, vitamin B1, vitamin C, selenium source and water in step S3 is 10-15:20-25:5-7:2-4:1-2:0.5-1:0.2-0.5:0.1-0.2:0.3-0.5:0.5-0.7:300-500, the selenium source is sodium selenite or sodium selenate, and the sugar content of the sugarcane juice is 18-20°; the bacterial content of the plant lactobacillus seed liquid in step S4 is 10 8 -10 9 cfu / mL, the inoculation amount is 3-5v / v%, the fermentation culture conditions are anaerobic conditions, 37-39°C, 100-200r / min fermentation culture for 36-48h, the ethanol content added to the system is 80-85wt%, and the precipitation time is 3-5h.

5. The preparation method according to claim 2, characterized in that: The mass ratio of selenium-rich extracellular polysaccharide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and adipic acid dihydrazide in step S5 is 10-12:2-4:7-10, the time of the reaction under room temperature stirring is 2-3h, the dialysis bag pore size is 8000-10000D, and the time is 3-5h; the mass ratio of geraniol and dihydroartemisinin in step S6 is 10-12:7-10.

6. The preparation method according to claim 2, characterized in that: The mass ratio of the active component, oxalic anhydride and catalyst in step S7 is 7-10:9-12:0.01-0.02, the catalyst is at least one of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene and triethylenediamine, the reaction time under stirring at room temperature is 20-24h, and the mass ratio of the carboxylated active component, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and amino-enriched selenium extracellular polysaccharide in step S8 is 5-7:8-10:10-15, the reaction time under stirring at room temperature is 18-20h, the activation time is 1-3h, the dialysis bag pore size is 8000-10000D, and the time is 3-5h.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the Poria cocos and Astragalus fermentation product, sodium alginate, and sodium hyaluronate in step S9 is 10:7-10:3-5, the mass ratio of the aqueous phase, the oil phase, and the lecithin is 10-15:25-30:0.1-0.2, the concentration of the calcium chloride solution is 5-7wt%, and the time for room temperature curing is 20-30min; the mass ratio of the Poria cocos and Astragalus water extract, the active component-selenium-rich extracellular polysaccharide, the embedded microspheres, genipin, and berberine in step S10 is 15-20:27-32:7-10:0.5-1:0.2-0.

4.

8. The preparation method according to claim 2, characterized in that: The specific steps include: S1. Preparation of Poria cocos and Astragalus membranaceus water extract: 12-15 parts by weight of Poria cocos and 7-10 parts by weight of Astragalus membranaceus were washed, dried, crushed, added to 200 parts by weight of water, and a composite enzyme was added, wherein the amount of the composite enzyme added was 1-2wt% of the total mass of the system, and the enzyme was hydrolyzed at 45-50°C for 2-4h, heated to boiling for extraction for 1-3h, filtered, and the residue was retained; the filtrate was dried to obtain a Poria cocos and Astragalus membranaceus water extract; The complex enzyme comprises cellulase and hemicellulase in a mass ratio of 5-7:3-5; S2. Preparation of Poria cocos and Astragalus fermentation product: 10-15 parts by weight of the filter residue in step S1, 5-7 parts by weight of a nitrogen source, 1-2 parts by weight of an inorganic salt are added to 300-500 parts by weight of water, sterilized, inoculated with Lactobacillus casei and Lactobacillus rhamnosus seed solution, the inoculation amount is 1-3v / v% and 2-4v / v%, respectively, under anaerobic conditions, 37-39 ° C, 100-200r / min fermentation culture 48-56h, filtered, the solid washing to obtain a bacterial solution, mixed with the filtrate, freeze-dried, to obtain a Poria cocos and Astragalus fermentation product, the washed solids are retained; The bacterial content of the Lactobacillus casei and Lactobacillus rhamnosus seed liquid is 10 8 -10 9 cfu / mL; S3. Preparation of a culture medium: 10-15 parts by weight of the washed solid in step S2, 20-25 parts by weight of sugar cane juice having a sugar content of 18-20 °, 5-7 parts by weight of yeast powder, 2-4 parts by weight of sodium nitrate, 1-2 parts by weight of potassium dihydrogen phosphate, 0.5-1 parts by weight of magnesium sulfate, 0.2-0.5 parts by weight of calcium chloride, 0.1-0.2 parts by weight of vitamin B1, 0.3-0.5 parts by weight of vitamin C, 0.5-0.7 parts by weight of a selenium source are added to 300-500 parts by weight of water and sterilized to obtain a culture medium; S4. Preparation of selenium-rich extracellular polysaccharides: inoculate the Lactobacillus plantarum seed solution into the culture medium prepared in step S3, and ferment and culture under anaerobic conditions at 37-39°C and 100-200r / min for 36-48h, filter, add ethanol to the filtrate until the ethanol content in the system is 80-85wt%, and precipitate for 3-5h to obtain selenium-rich extracellular polysaccharides; The bacterial content of the plant lactobacillus seed liquid is 10 8 -10 9 cfu / mL; S5. Preparation of amino-enriched selenium exopolysaccharide: dissolve 10-12 parts by weight of the selenium-enriched exopolysaccharide obtained in step S4 in 500 parts by weight of water, add 2-4 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 7-10 parts by weight of adipic acid dihydrazide, adjust the pH value of the solution to 4-5, stir at room temperature for 2-3 hours, adjust the pH value of the solution to 6.9-7.1, dialyze for 3-5 hours, the dialysis bag pore size of the dialysis is 8000-10000D, and the dialysate is freeze-dried to obtain amino-enriched selenium exopolysaccharide; S6. Preparation of the active ingredient: 10-12 parts by weight of geraniol and 7-10 parts by weight of dihydroartemisinin were mixed to obtain an active ingredient; S7. Preparation of carboxylated active component: 7-10 parts by weight of the active component obtained in step S6, 9-12 parts by weight of oxalic anhydride, 0.01-0.02 parts by weight of the catalyst are dissolved in 200 parts by weight of dichloromethane, and the reaction is stirred at room temperature under inert gas for 20-24h, the product is filtered, washed, and dried to obtain a carboxylated active component; S8. Preparation of active component - selenium-rich extracellular polysaccharide: 5-7 parts by weight of the carboxylated active component prepared in step S7 and 8-10 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are mixed and dissolved in 200 parts by weight of N,N-dimethylformamide, and activated by stirring for 1-3 hours, and 10-15 parts by weight of the amino-enriched selenium-rich extracellular polysaccharide prepared in step S5 are added, and the reaction is stirred at room temperature for 18-20 hours, and dialyzed for 3-5 hours, wherein the pore size of the dialysis bag is 8000-10000D, and freeze-dried to obtain the active component - selenium-rich extracellular polysaccharide; S9. Preparation of embedded microspheres: add 10 parts by weight of Poria cocos and Astragalus membranaceus fermentation product to 200 parts by weight of water, add 7-10 parts by weight of sodium alginate and 3-5 parts by weight of sodium hyaluronate, stir and mix evenly to obtain an aqueous phase; add 100-150 parts by weight of the aqueous phase to 250-300 parts by weight of the oil phase, add 1-2 parts by weight of lecithin, emulsify, add 20 parts by weight of 5-7wt% calcium chloride solution dropwise, solidify at room temperature for 20-30 minutes, centrifuge, wash, and dry to obtain embedded microspheres; S10. Preparation of an antitumor composition: 15-20 parts by weight of the Poria cocos and Astragalus membranaceus water extract obtained in step S1, 27-32 parts by weight of the active component - selenium-rich extracellular polysaccharide obtained in step S8, 7-10 parts by weight of the embedded microspheres obtained in step S9, 0.5-1 parts by weight of genipin, and 0.2-0.4 parts by weight of berberine are mixed evenly to obtain an antitumor composition.

9. An antitumor composition obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the anti-tumor composition according to claim 9 in preparing drugs for treating and assisting in treating liver cancer.

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