Preparation with liver cancer therapeutic effect and preparation method thereof

Abstract

The present invention proposes a preparation with liver cancer treatment effect and a preparation method thereof, which belongs to the field of medical technology. Amino-disulfide bond modified nanospheres are prepared, folic acid is grafted on the surface, adriamycin and triptolide are loaded, and drug-loaded nanospheres are obtained; mistletoe, herbaceous cyperus, astragalus, nightshade, and rehmannia glutinosa are mixed and extracted, filtered, and the filtrate is dried to obtain a Chinese medicine extract, the filter residue is added to water, fermented by inoculation of fermentation bacteria, and a fermentation product is obtained, which is mixed and embedded with Chinese medicine extract, drug-loaded nanospheres, cordycepin, and ginsenoside Rh1 to obtain a preparation with liver cancer treatment effect, which can release drugs in a targeted manner, reduce the levels of serum tumor markers and serum inflammatory factors in patients, reduce inflammatory reactions, delay the condition, reduce adverse reactions of patients, protect liver and kidney functions, enhance patient physique, and help patients recover body functions as soon as possible.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation having a liver cancer treatment effect and a preparation method thereof. Background Art

[0002] Cancer, or malignant tumor, is one of the most difficult diseases to cure in the world. It is a general term for more than 100 related diseases. All organs in the human body are made up of cells. Cell growth and differentiation can meet the needs of the body. This orderly process can keep people healthy. When cells in the body mutate, they will continue to divide, out of the control of the body. These extra large numbers of cells form tumors and cancer. Malignant tumor cells can invade and destroy adjacent tissues and organs; moreover, cancer cells can pass through the tumor and enter the blood or lymphatic system. This is how cancer spreads from the primary site to other organs to form new tumors. This process is called cancer metastasis. Most cancers are named according to the organ or cell type where they start.

[0003] Primary liver cancer refers to tumors that occur in liver cells or intrahepatic bile duct cells. It is a highly malignant type of cancer. Clinically, it is mainly manifested by progressive liver enlargement and pain, jaundice, fever, ascites, bleeding, and positive alpha-fetoprotein. Currently, there is no effective method for the treatment of liver cancer. Most of them are surgery and postoperative radiotherapy and chemotherapy. For weak patients who have no chance of surgery, they can only take oral drugs and radiotherapy and chemotherapy. However, due to the large toxic side effects of radiotherapy and chemotherapy, the patient's body will be extremely weak, and the treatment effect is not ideal, making it difficult for patients to bear.

[0004] Although the occurrence of liver cancer is related to factors such as acute and chronic viral hepatitis, liver cirrhosis, aflatoxin in chemical carcinogens, and nitrosamines in some foods, factors such as oncogenes in the body, low immune function, and neurohumoral and metabolic disorders are also closely related to the occurrence and development of liver cancer. At present, anticancer drugs are mostly used to treat middle and late stage liver cancer. They are not very selective. While killing tumor cells, they also have a killing effect on normal cells, especially on cells with vigorous proliferation such as bone marrow. Therefore, side effects and toxic reactions are strong, causing low immune function, neurohumoral and metabolic disorders. Sometimes, it can make the condition of liver cancer patients worse. However, the use of traditional Chinese medicine treatment that promotes blood circulation and removes blood stasis, softens and disperses nodules, clears away heat and detoxifies, combined with the advantages of overall conditioning of traditional Chinese medicine, and implements individualized nursing, can complement each other, improve symptoms, reduce the toxic and side effects of chemotherapy, mobilize the body's immunity, and achieve the purpose of improving treatment effects, prolonging patients' lives, and improving patients' quality of life.

[0005] The application of traditional Chinese medicine in the treatment of liver cancer is specifically applicable to patients with obvious cirrhosis, small liver cancer nodules and diffuse throughout the liver. For liver cancer patients with obvious liver damage, Chinese medicine is used to improve symptoms first, and then other means of treatment are used. During surgery, radiation and chemotherapy, Chinese medicine is used as an adjuvant treatment. When advanced liver cancer is diagnosed, the best time for surgical treatment has generally been lost, and liver cancer is not sensitive to radiation and chemotherapy. As an important part of comprehensive treatment, traditional Chinese medicine has certain efficacy and unique advantages. It is the first choice for advanced liver cancer. The standard model of modern medicine for liver cancer treatment has changed. In the past, the evaluation criteria for solid tumors have changed from the pursuit of complete remission rate (CR) to the current RECIST standard: that is, the pursuit of disease remission time and survival time, the pursuit of quality of life and adverse reactions of drugs, and the combination of patient-centered health economics to benefit patients. This has enabled traditional Chinese medicine to be used in all stages of liver cancer, and more and more gratifying achievements have been made.

[0006] But so far, there has been no satisfactory Chinese medicine and measures for the treatment of liver cancer at home and abroad. Traditional Chinese medicine has great advantages in the treatment of residual jaundice caused by liver cancer. Therefore, there is an urgent need for drugs that can effectively enhance the body's immune function and promote liver cell proliferation. Drugs that can not only eliminate viruses and promote recovery, but also improve the patient's functional state, relieve pain, and prolong the patient's life. Summary of the invention

[0007] The purpose of the present invention is to propose a preparation with liver cancer therapeutic effect and a preparation method thereof, which can release drugs in a targeted manner, reduce the levels of serum tumor markers and serum inflammatory factors in patients, alleviate inflammatory reactions, delay the disease, reduce adverse reactions in patients, protect liver and kidney functions, enhance the physical fitness of patients, help patients recover their body functions as soon as possible, and promote patients to recover as soon as possible, and has broad application prospects.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The invention provides a preparation method of a preparation with liver cancer therapeutic effect. The method comprises the following steps: preparing amino-disulfide bond modified nanospheres by a template method, grafting folic acid on the surface of the nanospheres, adding the nanospheres to a doxorubicin and triptolide solution, volatilizing the solvent, and preparing drug-loaded nanospheres; extracting mistletoe, selfheal, astragalus, nightshade, and rehmannia root by mixing, filtering, and drying the filtrate to obtain a Chinese medicine extract; adding the filter residue to water, inoculating fermentation bacteria for fermentation, and preparing a fermentation product; and mixing and embedding the fermentation product, the Chinese medicine extract, the drug-loaded nanospheres, cordycepin, and ginsenoside Rh1 to prepare a preparation with liver cancer therapeutic effect.

[0010] As a further improvement of the present invention, the following steps are included:

[0011] S1. Preparation of amino-disulfide bond modified nanospheres: dissolving a cationic surfactant in water, adjusting the pH value of the solution to be alkaline, adding alkyl orthosilicate, aminosilane, 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and sodium salicylate, heating and stirring to react, standing and aging, centrifuging, washing with acidic ethanol and water in turn, centrifuging, and drying to obtain amino-disulfide bond modified nanospheres;

[0012] S2. Grafting of folic acid: Add folic acid, 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and N-hydroxysuccinimide to dimethyl sulfoxide, stir and mix evenly, add the amino-disulfide bond-modified nanospheres prepared in step S1, heat and stir to react in the dark, add the product to anhydrous ethanol, let stand, centrifuge, wash, and dry to obtain folic acid-grafted amino-disulfide bond-modified nanospheres;

[0013] S3. Drug loading: doxorubicin and triptolide were mixed and dissolved in an ethanol aqueous solution, and the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, stirred and mixed evenly, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0014] S4. Water extraction of Chinese medicine: The mistletoe, Prunella vulgaris, Astragalus, Solanum nigrum, and Rehmannia glutinosa were washed, dried, and crushed to obtain a Chinese medicine powder, added to an ethanol aqueous solution, heated under reflux for extraction, filtered, the solid residue was retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0015] S5. Fermentation: adding the solid residue in step S4 to water, sterilizing, inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, fermenting and culturing for a first period of time, adding the prebiotic composition, continuing the fermentation and culturing for a second period of time, filtering, washing the solid residue to obtain a bacterial liquid, combining the bacterial liquid and the filtrate, and freeze-drying to obtain a fermentation product;

[0016] S6. Preparation of additives: The cordycepin and ginsenoside Rh1 were mixed uniformly to obtain an additive;

[0017] S7. Preparation of a preparation having a therapeutic effect on liver cancer: the drug-loaded nanospheres prepared in step S3, the Chinese medicine extract prepared in step S4, the fermentation product prepared in step S5 and the additive prepared in step S6 are mixed and added into water, an emulsifier, sodium alginate and sodium carboxymethyl cellulose are added, the mixture is stirred and mixed evenly, rapid membrane emulsification is performed, a calcium chloride solution is added dropwise, the mixture is solidified at room temperature, centrifuged, washed and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0018] As a further improvement of the present invention, the cationic surfactant in step S1 is selected from at least one of dodecyltrimethylammonium chloride, dodecyltrimethyl sodium bromide, dodecyldimethylbenzylammonium chloride, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, hexadecyltrimethyl sodium bromide, hexadecyldimethylbenzylammonium chloride, octadecyltrimethylammonium bromide, and octadecyltrimethylammonium chloride, the pH value of the adjusted solution is 11-12, and the mass ratio of the cationic surfactant, alkyl orthosilicate, aminosilane, 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and sodium salicylate is 1-2:7-1 2:3-5:5-7:0.5-1, the heating temperature is 70-80°C, the reaction time is 2-4h, the aging time is 2-4h, the acidic ethanol is a mixture of ethanol and concentrated hydrochloric acid in a volume ratio of 10:2-3, and the aminosilane is selected from at least one of γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-β (aminoethyl) -γ-aminopropyltrimethoxysilane, N-β (aminoethyl) -γ-aminopropyltriethoxysilane, N-β (aminoethyl) -γ-aminopropylmethyldimethoxysilane, N-β (aminoethyl) -γ-aminopropylmethyldiethoxysilane, and diethylenetriaminopropyltrimethoxysilane.

[0019] As a further improvement of the present invention, the mass ratio of folic acid, 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and amino-disulfide bond-modified nanospheres in step S2 is 4-5:8-10:4-6:15-17, the temperature of the heating, stirring, and light-proof reaction is 25-32°C, the time is 10-12h, the amount of anhydrous ethanol added is 5-7 times the total volume of the system, and the standing time is 5-7h.

[0020] As a further improvement of the present invention, the mass ratio of doxorubicin, triptolide, and folic acid grafted amino-disulfide bond modified nanospheres in step S3 is 3-5:2-4:17-20, and the concentration of the ethanol aqueous solution is 40-50wt%.

[0021] As a further improvement of the present invention, the mass ratio of mistletoe, selfheal, astragalus, nightshade and rehmannia root in step S4 is 15-20:3-5:3-5:2-4:1-3, the solid-liquid ratio of the traditional Chinese medicine powder and the ethanol aqueous solution is 1:5-10 g / mL, the concentration of the ethanol aqueous solution is 50-60wt%, and the heating reflux extraction time is 3-5h.

[0022] As a further improvement of the present invention, the mass ratio of the solid residue to water in step S5 is 15-20:100; the inoculation amounts of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solutions are 2-3 v / v% and 1-2 v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL; the fermentation culture conditions are 37-39°C, 50-70r / min, 5-7% CO2, 3-5% O2, and the balance is nitrogen, wherein % is the volume percentage, the first time period is 36-48h, the second time period is 24-36h, the prebiotic composition includes inulin and arabinose in a mass ratio of 5-7:3-5, and the added amount is 3-5wt% of the total mass of the system.

[0023] As a further improvement of the present invention, the mass ratio of cordycepin and ginsenoside Rh1 in step S6 is 3-5:7-10; the mass ratio of the drug-loaded nanospheres, Chinese medicine extracts, fermentation products, additives, emulsifiers, sodium alginate and sodium carboxymethyl cellulose in step S7 is 10-15:3-5:2-4:0.7-1.2:0.5-1:12-15:3-5, the membrane pore size of the rapid membrane emulsification is 1-2 μm, the concentration of the calcium chloride solution is 3-5wt%, the time of room temperature curing is 20-40min, and the emulsifier is selected from at least one of lecithin, betaine, sorbitan fatty acid esters, polysorbate fatty acid esters, and dienoylglycerol fatty acid esters.

[0024] The present invention further protects a preparation having liver cancer treatment effect obtained by the above preparation method.

[0025] The present invention further protects the use of the above-mentioned preparation having liver cancer treatment effect in the preparation of drugs for treating and assisting the treatment of liver cancer.

[0026] The present invention has the following beneficial effects:

[0027] The synthesis and modification methods of the inorganic nano drug delivery system are simple and have high stability. The silica nano drug delivery system prepared by the template method of the present invention can be orally administered, avoids the influence of hydrophobic drugs in the digestive tract, can tolerate lower pH values, and has adjustable pore size, large specific surface area, high drug loading, high cell permeability, and low cytotoxicity. However, the poor biodegradability and incomplete drug release of ordinary silica nano drug delivery systems can also lead to the accumulation of toxic substances and weakened anti-cancer effects.

[0028] In the preparation process of the silicon dioxide nano drug delivery system, aminosilane and 1,2-bis(trimethoxysilyl)-propyl tetrasulfide are added. Under the action of the structure-directing agent sodium salicylate, the surface of the prepared amino-disulfide bond-modified nanospheres is distributed with amino groups. At the same time, the structure of the silicon dioxide spheres is in the form of -O-Si-CCSSSSCC-Si-O-, so that the prepared amino-disulfide bond-modified nanospheres can stably exist in disulfide bonds under normal physiological conditions and the drugs are not released. After entering the cells, the disulfide bonds are broken under the stimulation of glutathione, breaking the dense structure of the carrier, and the drugs loaded therein are quickly released.

[0029] Furthermore, the present invention reacts the prepared amino-disulfide bond modified nanospheres with folic acid, and the amino groups and hydroxyl groups on the surface of the nanospheres can react with folic acid, so that the folic acid is loaded on the surface of the amino-disulfide bond modified nanospheres to become a targeting group. The folic acid receptor is a glycoprotein on the surface of the cell membrane, which is overexpressed on the surface of many tumor cells. Therefore, the prepared drug-loaded nanospheres can be targeted and delivered to liver cancer tumor cells, broken in the cells, releasing the drugs, causing the tumor cells to die, and inhibiting the development of the tumor.

[0030] Triptolide is a diterpenoid triepoxide, which has extremely effective anti-cancer effects on various cancers including liver cancer. Adriamycin has a significant therapeutic effect on liver cancer. The synergistic effect of the two can effectively reduce the levels of serum tumor markers and serum inflammatory factors in patients, reduce inflammatory reactions, and delay the disease. In addition, the targeted release of the drug by the present invention makes the safety of the drug within an acceptable range.

[0031] Mistletoe is a traditional Chinese medicine that is bitter and drying, and sweet and nourishing. Its extracts mainly contain high-molecular compounds such as flavonoids, alkaloids, terpenes, and sugars, which play a role in anti-arrhythmia, prevention and treatment of acute myocardial infarction, inhibition of platelet aggregation, enhancement of immune function, improvement of microcirculation, and anti-tumor. Among them, the anti-tumor active components include mistletoe alkaloids, mistletoe lectins, and mistletoe toxic peptides. Among them, mistletoe lectins are highly active ribosome inhibitory proteins that can inhibit protein synthesis at the ribosome level, thereby inducing apoptosis of cancer cells, and also promote cellular immunity and humoral immunity. Mistletoe toxic peptides work by destroying the cell membrane structure. The membrane bilayer is destroyed, and the membrane contents leak out, causing cell death and inhibiting the proliferation of malignant tumor cells. Total mistletoe alkaloids can inhibit the expression of telomerase activity in precancerous liver lesions by inhibiting the overexpression of apoptosis-related gene nuclear factor (NF)-κB, increasing the expression of IκB-α, reducing telomerase activity, inhibiting the synthesis of atypical hepatocyte telomeres, and blocking the cancer-promoting stage of the carcinogenesis process. Total flavonoids in Prunella vulgaris extract inhibit tumor autophagy and promote cell apoptosis by activating the PI3K / Akt / mTOR signaling pathway, and have a significant anti-cancer effect.

[0032] With the synergistic effect of other Chinese medicinal ingredients (astragalus, Solanum nigrum, and Rehmannia glutinosa), it can reduce the adverse reactions of liver cancer chemotherapy patients, protect liver and kidney functions, reduce the degree of vomiting, enhance the patient's appetite, strengthen the patient's physical fitness, improve the efficacy of radiotherapy, and help the patient recover the body function as soon as possible. It can also strengthen the body and remove blood stasis, help quickly remove residual tumor cells in the body, and further promote the patient's recovery.

[0033] Using the solid residue of traditional Chinese medicine as the fermentation matrix and inoculating probiotics for fermentation can, on the one hand, break the plant cell walls and promote the dissolution of most of the active components of the traditional Chinese medicine, so that the fermentation products contain richer active components. At the same time, the polysaccharides and proteins in the traditional Chinese medicine components can also be used and proliferated by the fermentation bacteria, promoting the rapid growth of the fermentation bacteria and producing a large number of beneficial active substances, including short-chain fatty acids.

[0034] Among them, fermented probiotics can regulate the balance of intestinal flora, inhibit inflammatory response, and enhance host immunity. Lactobacillus rhamnosus can reduce the expression of glutathione transferase, reduce the exposure of intestinal aflatoxin, and increase the excretion of fecal aflatoxin B1 to reduce the incidence of non-alcoholic fatty liver and liver cancer; Bifidobacterium longum and Lactobacillus can modify helper T cells 17 and secrete interleukin 17 to regulate the immune mechanism of liver cancer. At the same time, they have a protective effect on the intestinal mucosa, can repair damaged intestinal mucosa and reduce its permeability, prevent the specific binding of bacterial lipopolysaccharide to TOLL-like receptors, reduce the release of inflammatory factors, and inhibit the occurrence and development of liver cancer.

[0035] Short-chain fatty acids are the main metabolites of fermented probiotics, including butyrate, acetate and propionate, which play an important role in protecting the intestinal barrier and preventing bacteria from entering the blood circulation. They can increase the expression of claudin-1 and Occludens-1 and the redistribution of occludin to enhance the intestinal barrier function, reduce lipopolysaccharide translocation, inhibit macrophage activation, proinflammatory cytokine production and neutrophil infiltration, inhibit TNF-α, IL-6 and myeloperoxidase activity, thereby reducing liver damage and even the progression of liver cancer.

[0036] The prebiotics added in the fermentation process of the present invention can promote the proliferation of fermentation probiotics in a directional manner, thereby accelerating the fermentation process, promoting the large-scale reproduction of fermentation probiotics, and obtaining highly active fermentation products in a short time.

[0037] The additives added in the present invention include cordycepin and ginsenoside Rh1. Cordycepin can induce tumor cell apoptosis through the Caspase pathway to inhibit tumor development, promote the expression of reactive oxygen ROS in tumor cells, and inhibit the proliferation of tumor cells; ginsenoside Rh1 can induce autophagy of liver cancer cells by enhancing the expression of Beclin1, resulting in decreased drug sensitivity, inhibiting the formation of tumor angiogenesis, thereby inhibiting the occurrence and development of tumors. At the same time, it can also enhance the body's immunity and improve the body's ability to fight tumor cells. The two have a synergistic effect.

[0038] Furthermore, the present invention can improve the gastric acid resistance of the preparation after composite encapsulation of the obtained fermentation products, Chinese medicine extracts, drug-loaded nanospheres, cordycepin, and ginsenoside Rh1 through sodium alginate and sodium carboxymethyl cellulose, promote the sustained release of the preparation in the intestine, effectively avoid the inactivation of probiotics in the gastric acid environment and reduce the effect, and also avoid the inactivation of protein peptide active substances under acidic conditions, thereby significantly improving the overall efficacy of the preparation.

[0039] The preparation with liver cancer treatment effect prepared by the present invention can release drugs in a targeted manner, reduce the levels of patient serum tumor markers and serum inflammatory factors, alleviate inflammatory reactions, delay the disease, reduce adverse reactions of patients, protect liver and kidney functions, enhance the patient's physical fitness, help patients recover their body functions as soon as possible, and promote patients' recovery as soon as possible, and has broad application prospects. DETAILED DESCRIPTION

[0040] 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.

[0041] Lactobacillus rhamnosus, 10 billion cfu / g, Bifidobacterium longum, 10 billion cfu / g, were purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.

[0042] The preparation method of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution is as follows: Lactobacillus rhamnosus and Bifidobacterium longum are inoculated into Gao's medium respectively, and activated and cultured for 24 hours at 38°C, 70r / min, and micro-anoxic conditions to obtain a culture medium with a bacterial content of 10 9 cfu / mL of bacterial seed solution.

[0043] In the above and following examples, the micro-anoxic condition is 5% CO2, 4% O2, and the balance is nitrogen, wherein % is the volume percentage.

[0044] Example 1

[0045] This embodiment provides a method for preparing a preparation having a liver cancer treatment effect, comprising the following steps:

[0046] S1. Preparation of amino-disulfide bond modified nanospheres: 1 part by weight of tetradecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11, 7 parts by weight of methyl orthosilicate, 3 parts by weight of N-β (aminoethyl) -γ-aminopropyltrimethoxysilane, 5 parts by weight of 1,2-bis (trimethoxysilyl) -propyl tetrasulfide, 0.5 parts by weight of sodium salicylate were added, heated to 70 ° C, stirred for reaction for 2 hours, allowed to stand for 2 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0047] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2;

[0048] S2. Grafting of folic acid: 4 parts by weight of folic acid, 8 parts by weight of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and 4 parts by weight of N-hydroxysuccinimide are added to 100 parts by weight of dimethyl sulfoxide, stirred and mixed evenly, 15 parts by weight of the amino-disulfide bond-modified nanospheres prepared in step S1 are added, heated to 25°C, stirred and reacted in the dark for 10 hours, 5 times the total volume of anhydrous ethanol of the system is added to the product, and allowed to stand for 5 hours, centrifuged with deionized water, washed, and dried to obtain folic acid-grafted amino-disulfide bond-modified nanospheres;

[0049] S3 drug loading: 3 parts by weight of doxorubicin and 2 parts by weight of triptolide were mixed and dissolved in 100 parts by weight of a 40wt% ethanol aqueous solution, 17 parts by weight of the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, the mixture was stirred and mixed, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0050] S4. Water extraction of Chinese medicine: 15 parts by weight of mistletoe, 3 parts by weight of Prunella vulgaris, 3 parts by weight of Astragalus, 2 parts by weight of Solanum nigrum, and 1 part by weight of Rehmannia glutinosa were washed, dried, and crushed to obtain Chinese medicine powder, added to a 50wt% ethanol aqueous solution, the solid-liquid ratio of the Chinese medicine powder and the ethanol aqueous solution was 1:5g / mL, heated and refluxed for extraction for 3h, filtered, the solid residue was dried and retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0051] S5. Fermentation: adding 15 parts by weight of the solid residue in step S4 to 100 parts by weight of deionized water, sterilizing, inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, wherein the inoculation amount of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution is 2v / v% and 1v / v%, respectively, at 37°C, 50r / min, under micro-anoxic conditions, fermenting and culturing for 36h, adding the prebiotic composition, the addition amount is 3wt% of the total mass of the system, continuing the fermentation and culturing for 24h, filtering, washing the solid residue to obtain a bacterial liquid, combining the bacterial liquid and the filtrate, and freeze-drying to obtain a fermentation product;

[0052] The prebiotic composition comprises inulin and arabinose in a mass ratio of 5:3;

[0053] S6. Preparation of additives: 3 parts by weight of cordycepin and 7 parts by weight of ginsenoside Rh1, stirred and mixed for 15 min to obtain an additive;

[0054] S7. Preparation of a preparation having a therapeutic effect on liver cancer: 10 parts by weight of drug-loaded nanospheres prepared in step S3, 3 parts by weight of the Chinese medicine extract prepared in step S4, 2 parts by weight of the fermentation product prepared in step S5 and 0.7 parts by weight of the additive prepared in step S6 are mixed into deionized water, 0.5 parts by weight of sorbitan fatty acid ester, 12 parts by weight of sodium alginate and 3 parts by weight of sodium carboxymethyl cellulose are added, the mixture is stirred for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1 μm, 10 parts by weight of 3wt% calcium chloride solution is added dropwise, the mixture is cured at room temperature for 20 minutes, centrifuged, washed with deionized water and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0055] Example 2

[0056] This embodiment provides a method for preparing a preparation having a liver cancer treatment effect, comprising the following steps:

[0057] S1. Preparation of amino-disulfide bond modified nanospheres: 2 parts by weight of hexadecyltrimethyl sodium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 12, 12 parts by weight of ethyl orthosilicate, 5 parts by weight of γ-aminopropyltrimethoxysilane, 7 parts by weight of 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and 1 part by weight of sodium salicylate were added, heated to 80°C, stirred for reaction for 4 hours, allowed to stand for 4 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0058] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:3;

[0059] S2. Grafting of folic acid: 5 parts by weight of folic acid, 10 parts by weight of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and 6 parts by weight of N-hydroxysuccinimide are added to 100 parts by weight of dimethyl sulfoxide, stirred and mixed evenly, 17 parts by weight of the amino-disulfide bond-modified nanospheres prepared in step S1 are added, heated to 32° C., stirred and reacted in the dark for 12 hours, 7 times the total volume of the system of anhydrous ethanol was added to the product, and allowed to stand for 7 hours, centrifuged with deionized water, washed, and dried to obtain folic acid-grafted amino-disulfide bond-modified nanospheres;

[0060] S3 drug loading: 5 parts by weight of doxorubicin and 4 parts by weight of triptolide were mixed and dissolved in 100 parts by weight of a 50wt% ethanol aqueous solution, 20 parts by weight of the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, the mixture was stirred and mixed, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0061] S4. Water extraction of Chinese medicine: 20 parts by weight of mistletoe, 5 parts by weight of Prunella vulgaris, 5 parts by weight of Astragalus, 4 parts by weight of Solanum nigrum, and 3 parts by weight of Rehmannia glutinosa were washed, dried, and crushed to obtain Chinese medicine powder, added to a 60wt% ethanol aqueous solution, the solid-liquid ratio of the Chinese medicine powder and the ethanol aqueous solution was 1:10g / mL, heated and refluxed for extraction for 5h, filtered, the solid residue was dried and retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0062] S5. Fermentation: adding 20 parts by weight of the solid residue in step S4 to 100 parts by weight of deionized water, sterilizing, inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, wherein the inoculation amount of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution is 3v / v% and 2v / v%, respectively, at 39°C, 70r / min, under micro-anoxic conditions, fermenting and culturing for 48h, adding the prebiotic composition, the addition amount is 5wt% of the total mass of the system, continuing the fermentation and culturing for 36h, filtering, washing the solid residue to obtain a bacterial liquid, combining the bacterial liquid and the filtrate, and freeze-drying to obtain a fermentation product;

[0063] The prebiotic composition comprises inulin and arabinose in a mass ratio of 7:5;

[0064] S6. Preparation of additives: 5 parts by weight of cordycepin and 10 parts by weight of ginsenoside Rh1, stirred and mixed for 15 min to obtain an additive;

[0065] S7. Preparation of a preparation having a therapeutic effect on liver cancer: 15 parts by weight of drug-loaded nanospheres prepared in step S3, 5 parts by weight of the traditional Chinese medicine extract prepared in step S4, 4 parts by weight of the fermentation product prepared in step S5 and 1.2 parts by weight of the additive prepared in step S6 are mixed and added into deionized water, 1 part by weight of polysorbate, 15 parts by weight of sodium alginate and 5 parts by weight of sodium carboxymethyl cellulose are added, stirred and mixed for 20 minutes, rapid membrane emulsification, the membrane pore size is 2 μm, 15 parts by weight of 5wt% calcium chloride solution is added dropwise, solidified at room temperature for 40 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0066] Example 3

[0067] This embodiment provides a method for preparing a preparation having a liver cancer treatment effect, comprising the following steps:

[0068] S1. Preparation of amino-disulfide bond modified nanospheres: 1.5 parts by weight of hexadecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11.5, 10 parts by weight of ethyl orthosilicate, 4 parts by weight of γ-aminopropyltriethoxysilane, 6 parts by weight of 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and 0.7 parts by weight of sodium salicylate were added, heated to 75°C, stirred for reaction for 3 hours, allowed to stand for 3 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0069] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2.5;

[0070] S2. Grafting of folic acid: 4.5 parts by weight of folic acid, 9 parts by weight of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and 5 parts by weight of N-hydroxysuccinimide are added to 100 parts by weight of dimethyl sulfoxide, stirred and mixed evenly, 16 parts by weight of the amino-disulfide bond-modified nanospheres prepared in step S1 are added, heated to 28°C, stirred and reacted in the dark for 11 hours, 6 times the total volume of the system of anhydrous ethanol is added to the product, and the mixture is allowed to stand for 6 hours, centrifuged with deionized water, washed, and dried to obtain folic acid-grafted amino-disulfide bond-modified nanospheres;

[0071] S3 drug loading: 4 parts by weight of doxorubicin and 3 parts by weight of triptolide were mixed and dissolved in 100 parts by weight of a 45wt% ethanol aqueous solution, 18.5 parts by weight of the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, the mixture was stirred and mixed uniformly, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0072] S4. Water extraction of Chinese medicine: 17 parts by weight of mistletoe, 4 parts by weight of Prunella vulgaris, 4 parts by weight of Astragalus, 3 parts by weight of Solanum nigrum, and 2 parts by weight of Rehmannia glutinosa were washed, dried, and crushed to obtain Chinese medicine powder, added to a 55wt% ethanol aqueous solution, the solid-liquid ratio of the Chinese medicine powder to the ethanol aqueous solution was 1:7g / mL, heated under reflux for 4h, filtered, the solid residue was dried and retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0073] S5. Fermentation: 17 parts by weight of the solid residue in step S4 was added to 100 parts by weight of deionized water, sterilized, inoculated with activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, the inoculation amount of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution was 2.5 v / v% and 1.5 v / v%, respectively, at 38°C, 60 r / min, under micro-anoxic conditions, fermented and cultured for 42 h, and a prebiotic composition was added in an amount of 4 wt% of the total mass of the system, and the fermentation culture was continued for 30 h, filtered, and the solid residue was washed to obtain a bacterial liquid, the bacterial liquid and the filtrate were combined, freeze-dried, and a fermentation product was obtained;

[0074] The prebiotic composition comprises inulin and arabinose in a mass ratio of 6:4;

[0075] S6. Preparation of additives: 4 parts by weight of cordycepin and 8.5 parts by weight of ginsenoside Rh1 were stirred and mixed for 15 min to obtain an additive;

[0076] S7. Preparation of a preparation having a therapeutic effect on liver cancer: 12 parts by weight of drug-loaded nanospheres prepared in step S3, 4 parts by weight of traditional Chinese medicine extract prepared in step S4, 3 parts by weight of fermentation product prepared in step S5 and 1 part by weight of additive prepared in step S6 are mixed and added into deionized water, 0.7 parts by weight of lecithin, 13.5 parts by weight of sodium alginate and 4 parts by weight of sodium carboxymethyl cellulose are added, stirred and mixed for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1.5 μm, 12 parts by weight of 4wt% calcium chloride solution is added dropwise, solidified at room temperature for 30 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0077] Example 4

[0078] Compared with Example 3, the difference is that in step S5, no activated Lactobacillus rhamnosus seed solution is inoculated, and the inoculation amount of activated Bifidobacterium longum seed solution is 4 v / v%.

[0079] Example 5

[0080] Compared with Example 3, the difference is that in step S5, the activated Bifidobacterium longum seed solution is not inoculated, and the inoculation amount of the activated Lactobacillus rhamnosus seed solution is 4 v / v%.

[0081] Comparative Example 1

[0082] Compared with Example 3, the difference is that γ-aminopropyltriethoxysilane is not added in step S1.

[0083] The details are as follows:

[0084] S1. Preparation of amino-disulfide bond modified nanospheres: 1.5 parts by weight of hexadecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11.5, 10 parts by weight of ethyl orthosilicate, 10 parts by weight of 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and 0.7 parts by weight of sodium salicylate were added, and the mixture was heated to 75° C., stirred for reaction for 3 h, allowed to stand for 3 h, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0085] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2.5.

[0086] Comparative Example 2

[0087] Compared with Example 3, the difference is that 1,2-bis(trimethoxysilyl)-propyl tetrasulfide is not added in step S1.

[0088] The details are as follows:

[0089] S1. Preparation of amino-disulfide bond modified nanospheres: 1.5 parts by weight of hexadecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11.5, 10 parts by weight of ethyl orthosilicate, 10 parts by weight of γ-aminopropyltriethoxysilane, and 0.7 parts by weight of sodium salicylate were added, and the mixture was heated to 75°C, stirred for reaction for 3 hours, allowed to stand for 3 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0090] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2.5.

[0091] Comparative Example 3

[0092] Compared with Example 3, the difference is that step S2 is not performed.

[0093] The details are as follows:

[0094] S1. Preparation of amino-disulfide bond modified nanospheres: 1.5 parts by weight of hexadecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11.5, 10 parts by weight of ethyl orthosilicate, 4 parts by weight of γ-aminopropyltriethoxysilane, 6 parts by weight of 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and 0.7 parts by weight of sodium salicylate were added, heated to 75°C, stirred for reaction for 3 hours, allowed to stand for 3 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0095] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2.5;

[0096] S2. Drug loading: 4 parts by weight of doxorubicin and 3 parts by weight of triptolide were mixed and dissolved in 100 parts by weight of a 45wt% ethanol aqueous solution, 18.5 parts by weight of the amino-disulfide bond-modified nanospheres prepared in step S1 were added, the mixture was stirred and mixed, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0097] S3. Water extraction of Chinese medicine: 17 parts by weight of mistletoe, 4 parts by weight of Prunella vulgaris, 4 parts by weight of Astragalus, 3 parts by weight of Solanum nigrum, and 2 parts by weight of Rehmannia glutinosa were washed, dried, and crushed to obtain Chinese medicine powder, added to a 55wt% ethanol aqueous solution, the solid-liquid ratio of the Chinese medicine powder and the ethanol aqueous solution was 1:7g / mL, heated and refluxed for 4h, filtered, the solid residue was dried and retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0098] S4. Fermentation: 17 parts by weight of the solid residue in step S3 was added to 100 parts by weight of deionized water, sterilized, inoculated with activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, the inoculation amount of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution was 2.5 v / v% and 1.5 v / v%, respectively, at 38°C, 60 r / min, under micro-anoxic conditions, fermented for 42 h, added a prebiotic composition, added in an amount of 4 wt% of the total mass of the system, continued fermentation for 30 h, filtered, washed with solid residue to obtain a bacterial liquid, the bacterial liquid and the filtrate were combined, freeze-dried, and a fermentation product was obtained;

[0099] The prebiotic composition comprises inulin and arabinose in a mass ratio of 6:4;

[0100] S5. Preparation of additives: 4 parts by weight of cordycepin and 8.5 parts by weight of ginsenoside Rh1, stirred and mixed for 15 min to obtain an additive;

[0101] S6. Preparation of a preparation having a therapeutic effect on liver cancer: 12 parts by weight of drug-loaded nanospheres prepared in step S2, 4 parts by weight of the traditional Chinese medicine extract prepared in step S3, 3 parts by weight of the fermentation product prepared in step S4 and 1 part by weight of the additive prepared in step S5 are mixed and added into deionized water, 0.7 parts by weight of lecithin, 13.5 parts by weight of sodium alginate and 4 parts by weight of sodium carboxymethyl cellulose are added, the mixture is stirred for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1.5 μm, 12 parts by weight of 4wt% calcium chloride solution is added dropwise, the mixture is cured at room temperature for 30 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0102] Comparative Example 4

[0103] Compared with Example 3, the difference is that doxorubicin is not added in step S3.

[0104] The details are as follows:

[0105] S3. Drug loading: 7 parts by weight of triptolide are mixed and dissolved in 100 parts by weight of 45 wt% ethanol aqueous solution, 18.5 parts by weight of folic acid grafted amino-disulfide bond modified nanospheres obtained in step S2 are added, the mixture is stirred and mixed evenly, and the solvent is evaporated to obtain drug-loaded nanospheres.

[0106] Comparative Example 5

[0107] Compared with Example 3, the difference is that triptolide is not added in step S3.

[0108] The details are as follows:

[0109] S3. Drug loading: 7 parts by weight of doxorubicin were mixed and dissolved in 100 parts by weight of 45 wt% ethanol aqueous solution, 18.5 parts by weight of folic acid grafted amino-disulfide bond modified nanospheres obtained in step S2 were added, the mixture was stirred and mixed evenly, and the solvent was evaporated to obtain drug-loaded nanospheres.

[0110] Comparative Example 6

[0111] Compared with Example 3, the difference is that no heating extraction with ethanol aqueous solution is performed in step S4.

[0112] The details are as follows:

[0113] S4. Preparation of Chinese herbal powder: 17 parts by weight of mistletoe, 4 parts by weight of selfheal, 4 parts by weight of astragalus, 3 parts by weight of nightshade, and 2 parts by weight of rehmannia were washed, dried, and crushed to obtain Chinese herbal powder for preparing fermentation medium.

[0114] Comparative Example 7

[0115] Compared with Example 3, the difference is that no prebiotic composition is added in step S5.

[0116] The details are as follows:

[0117] S5. Fermentation: 17 parts by weight of the solid residue in step S4 is added to 100 parts by weight of deionized water, sterilized, and inoculated with activated Lactobacillus rhamnosus and Bifidobacterium longum seed liquid, wherein the inoculation amounts of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed liquid are 2.5 v / v% and 1.5 v / v%, respectively, and the fermentation is cultured at 38°C, 60 r / min, and slightly anoxic conditions for 72 hours, filtered, and the solid residue is washed to obtain a bacterial liquid, the bacterial liquid and the filtrate are combined, and freeze-dried to obtain a fermentation product.

[0118] Comparative Example 8

[0119] Compared with Embodiment 3, the difference is that step S5 is not performed.

[0120] The details are as follows:

[0121] S1. Preparation of amino-disulfide bond modified nanospheres: 1.5 parts by weight of hexadecyltrimethylammonium bromide was dissolved in 200 parts by weight of deionized water, the pH value of the solution was adjusted to 11.5, 10 parts by weight of ethyl orthosilicate, 4 parts by weight of γ-aminopropyltriethoxysilane, 6 parts by weight of 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and 0.7 parts by weight of sodium salicylate were added, heated to 75°C, stirred for reaction for 3 hours, allowed to stand for 3 hours, centrifuged, washed with acidic ethanol and deionized water in turn, centrifuged, and dried to obtain amino-disulfide bond modified nanospheres;

[0122] The acidic ethanol is a mixture of anhydrous ethanol and concentrated hydrochloric acid (12 mol / L) in a volume ratio of 10:2.5;

[0123] S2. Grafting of folic acid: 4.5 parts by weight of folic acid, 9 parts by weight of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and 5 parts by weight of N-hydroxysuccinimide are added to 100 parts by weight of dimethyl sulfoxide, stirred and mixed evenly, 16 parts by weight of the amino-disulfide bond-modified nanospheres prepared in step S1 are added, heated to 28°C, stirred and reacted in the dark for 11 hours, 6 times the total volume of the system of anhydrous ethanol is added to the product, and the mixture is allowed to stand for 6 hours, centrifuged with deionized water, washed, and dried to obtain folic acid-grafted amino-disulfide bond-modified nanospheres;

[0124] S3 drug loading: 4 parts by weight of doxorubicin and 3 parts by weight of triptolide were mixed and dissolved in 100 parts by weight of a 45wt% ethanol aqueous solution, 18.5 parts by weight of the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, the mixture was stirred and mixed uniformly, and the solvent was evaporated to obtain drug-loaded nanospheres;

[0125] S4. Water extraction of Chinese medicine: 17 parts by weight of mistletoe, 4 parts by weight of Prunella vulgaris, 4 parts by weight of Astragalus, 3 parts by weight of Solanum nigrum, and 2 parts by weight of Rehmannia glutinosa were washed, dried, and crushed to obtain Chinese medicine powder, added to a 55wt% ethanol aqueous solution, the solid-liquid ratio of the Chinese medicine powder to the ethanol aqueous solution was 1:7g / mL, heated under reflux for 4h, filtered, the solid residue was dried and retained, and the filtrate was dried to obtain a Chinese medicine extract;

[0126] S5. Preparation of additives: 4 parts by weight of cordycepin and 8.5 parts by weight of ginsenoside Rh1, stirred and mixed for 15 min to obtain an additive;

[0127] S6. Preparation of a preparation having a therapeutic effect on liver cancer: 12 parts by weight of drug-loaded nanospheres prepared in step S3, 4 parts by weight of the traditional Chinese medicine extract prepared in step S4 and 1 part by weight of the additive prepared in step S5 are mixed and added into deionized water, 0.7 parts by weight of lecithin, 13.5 parts by weight of sodium alginate and 4 parts by weight of sodium carboxymethyl cellulose are added, the mixture is stirred for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1.5 μm, 12 parts by weight of 4wt% calcium chloride solution is added dropwise, the mixture is cured at room temperature for 30 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0128] Comparative Example 9

[0129] Compared with Example 3, the difference is that cordycepin is not added in step S6.

[0130] The details are as follows:

[0131] S6. Preparation of additives: Ginsenoside Rh1 is an additive.

[0132] Comparative Example 10

[0133] Compared with Example 3, the difference is that ginsenoside Rh1 is not added in step S6.

[0134] The details are as follows:

[0135] S6. Preparation of additives: Cordycepin is an additive.

[0136] Comparative Example 11

[0137] Compared with Example 3, the difference is that no additive is added in step S7.

[0138] The details are as follows:

[0139] S7. Preparation of a preparation having a therapeutic effect on liver cancer: 12 parts by weight of drug-loaded nanospheres prepared in step S3, 4 parts by weight of the traditional Chinese medicine extract prepared in step S4, and 3 parts by weight of the fermentation product prepared in step S5 are mixed and added into deionized water, 0.7 parts by weight of lecithin, 13.5 parts by weight of sodium alginate and 4 parts by weight of sodium carboxymethyl cellulose are added, the mixture is stirred for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1.5 μm, 12 parts by weight of 4wt% calcium chloride solution is added dropwise, the mixture is cured at room temperature for 30 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0140] Comparative Example 12

[0141] Compared with Example 3, the difference is that no drug-loaded nanospheres are added in step S7.

[0142] The details are as follows:

[0143] S7. Preparation of a preparation having a therapeutic effect on liver cancer: 4 parts by weight of the Chinese herbal extract prepared in step S4, 3 parts by weight of the fermentation product prepared in step S5 and 1 part by weight of the additive prepared in step S6 are mixed and added into deionized water, 0.7 parts by weight of lecithin, 13.5 parts by weight of sodium alginate and 4 parts by weight of sodium carboxymethyl cellulose are added, the mixture is stirred for 20 minutes, rapid membrane emulsification is performed, the membrane pore size is 1.5 μm, 12 parts by weight of 4wt% calcium chloride solution is added dropwise, the mixture is cured at room temperature for 30 minutes, centrifuged, washed with deionized water, and dried to obtain a preparation having a therapeutic effect on liver cancer.

[0144] Test Example 1

[0145] 20 mg of the drug-loaded nanospheres prepared in Examples 1-3 of the present invention and Comparative Examples 1-3 (first completely crushed and then the absorbance of the filtrate at 480 nm was measured to determine the total content of doxorubicin) were dispersed in 100 mL of PBS buffer solution at pH = 7, stirred and released for 2 hours, filtered, the absorbance of the filtrate at 480 nm was tested, and the release rate of doxorubicin was calculated; then the nanospheres were added to a fresh 100 mL of PBS buffer solution at pH = 7, GSH was added to make its final concentration 3 mmol / L, stirred and released for 2 hours, filtered, the absorbance of the filtrate at 480 nm was tested, and the release rate of doxorubicin was calculated. The results are shown in Table 1.

[0146] Table 1

[0147] Group Release rate of doxorubicin without GSH addition Release rate of doxorubicin after adding GSH Example 1 0.09 92.12 Example 2 0.11 92.31 Example 3 0.07 92.59 Comparative Example 1 0.13 91.34 Comparative Example 2 0.12 0.19 Comparative Example 3 0.10 91.02

[0148] It can be seen from the above table that the drug-loaded nanospheres prepared in Examples 1-3 of the present invention can quickly release the drug in the presence of GSH, but almost no drug is released in the absence of GSH.

[0149] Test Example 2

[0150] After the rabbit was anesthetized with chloral hydrate, blood was collected from the heart, and the collected blood was placed in a centrifuge tube coated with heparin, centrifuged, and the supernatant was discarded. The remaining red blood cells were diluted with physiological saline to form a 2% red blood cell suspension by volume, and the 2% red blood cell suspension was mixed with an equal volume of physiological saline solution containing the drug-loaded nanospheres prepared in Examples 1-3 of the present invention and Comparative Examples 1-3, so that the final concentration of the drug-loaded nanospheres was 2000 μg / mL, and the red blood cell samples added with physiological saline and distilled water were used as negative controls and positive controls, respectively. After the mixture was allowed to stand for 4 hours, it was centrifuged, and the absorbance A of the supernatant solution was measured at a wavelength of 541 nm. The results are shown in Table 2.

[0151] Hemolysis percentage (%) = (A 样品 -A 阴性对照 ) / (A 阳性对照 -A 阴性对照 )×100%

[0152] Table 2

[0153]

[0154]

[0155] As can be seen from the above table, the drug-loaded nanospheres prepared in Examples 1-3 of the present invention have a low hemolysis rate and good biocompatibility. Amino modification and folic acid modification on the surface of the nanospheres can significantly reduce the reactive oxygen free radicals ROS generated by the silanol groups on the surface of silica and the electrostatic interaction between silica and membrane proteins, thereby reducing the hemolysis rate.

[0156] Test Example 3

[0157] 10 mg of the drug-loaded nanospheres prepared in Examples 1-3 of the present invention and Comparative Examples 1-3 were dispersed in 5 mL of a PBS buffer solution at pH = 7, and then 5 mL of a 0.1 g / L bovine serum albumin (BSA) solution was added. The mixture was placed on a 37°C constant temperature shaker, shaken at 100 r / min for 4 hours, centrifuged, and the supernatant was collected. The mixture was stained with a Coomassie brilliant blue solution, and the absorbance was measured at a wavelength of 595 nm. The results are shown in Table 3.

[0158] The calculation formula of the amount of BSA adsorbed on the surface of nanospheres is:

[0159] q=(C i -C f )V / m

[0160] Among them C f and C iare the BSA concentrations of the solution before and after adsorption, respectively; V is the total volume of the solution; and m is the weight of the nanospheres.

[0161] Table 3

[0162] Group BSA adsorption amount (%) Example 1 0.32 Example 2 0.30 Example 3 0.27 Comparative Example 1 12.19 Comparative Example 2 0.52 Comparative Example 3 17.47

[0163] As can be seen from the above table, the drug-loaded nanospheres prepared in Examples 1-3 of the present invention can significantly reduce the amount of BSA adsorbed to the surface, reduce its clearance rate in the body, and increase its circulation time in the body.

[0164] Test Example 4

[0165] Modeling: H22 cells with good growth status were taken and cell suspension (1×10 7 The H22 tumor-inoculated mice of the third generation with obvious abdominal bulge were killed by cervical dislocation. The peritoneal tumor fluid was obtained, diluted, centrifuged, and the supernatant was discarded to obtain tumor cells. Cell suspension (1.0×10 7 The model was successfully established when a hard lump about the size of a mung bean was felt under the mouse's armpit on the 4th day.

[0166] The successfully modeled mice were randomly divided into a model group, a cisplatin group, an embodiment 1-5 group, and a comparative example 1-12 group, 10 mice in each group, and 10 normal mice were selected as the normal group. The mice in the embodiment 1-5 group and the comparative example 1-12 group were gavaged with 1 g / kg body weight of the preparation with liver cancer therapeutic effect, once a day, and the normal group and the model group were gavaged with the same volume of normal saline every day. The cisplatin group was gavaged with 2.5 mg / kg cisplatin, once every 3 days, and each group was continuously intervened for 15 days.

[0167] 24 hours after the last administration, the blood from the orbital venous plexus of mice in each group was centrifuged, and the supernatant was taken to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) according to the instructions of the kit.

[0168] After blood was collected from mice, they were killed by cervical dislocation, the tumors were removed and weighed using an analytical balance, and the tumor volume and tumor inhibition rate of each group of mice were calculated.

[0169] Tumor inhibition rate (%) = (average tumor weight of model group - average tumor weight of drug-treated group) / average tumor weight of model group × 100%.

[0170] The liver tissues of mice in each group were separated, rinsed with ice saline, dried with filter paper, weighed and the liver index was calculated, liver index = (liver weight / body weight) × 100.

[0171] The results are shown in Tables 4 and 5.

[0172] Table 4

[0173] Group ALT(U / L) AST(U / L) LDH (U / L) Normal group 5.17±0.42 7.82±0.81 27.89±1.82 Model Group 14.38±1.49* 22.18±2.49* 59.10±6.04* Cisplatin group 13.89±1.42# 21.57±2.15# 60.23±5.77# Example 1 6.67±0.75# 11.49±1.42# 34.59±2.11# Example 2 6.59±0.69# 11.35±1.39# 34.42±2.19# Example 3 6.52±0.71# 11.31±1.44# 34.25±2.05# Example 4 8.27±0.63 13.27±1.32 36.77±2.16 Example 5 8.15±0.74 13.08±1.45 36.42±1.94 Comparative Example 1 6.89±0.70 11.67±1.47 34.82±1.91 Comparative Example 2 6.82±0.56 11.74±1.31 34.70±2.01 Comparative Example 3 6.94±0.66 11.92±1.29 34.95±1.96 Comparative Example 4 6.94±0.74 11.84±1.46 34.82±2.07 Comparative Example 5 6.90±0.73 11.79±1.44 34.79±2.14 Comparative Example 6 9.24±0.68 15.82±1.37 39.22±2.37 Comparative Example 7 8.10±0.62 13.01±1.36 36.11±2.25 Comparative Example 8 9.12±0.73 15.59±1.28 38.95±2.08 Comparative Example 9 7.82±0.66 12.88±1.38 35.82±1.96 Comparative Example 10 7.90±0.72 12.94±1.43 35.78±2.02 Comparative Example 11 8.05±0.58 13.01±1.40 36.97±1.92 Comparative Example 12 6.88±0.64 11.77±1.37 34.74±1.88

[0174] Note: * compared with the normal group, P < 0.05; # compared with the model group, P < 0.05.

[0175] It can be seen from the above table that the preparations with liver cancer treatment effect prepared in Examples 1-3 of the present invention can significantly improve the liver function of mice with liver cancer.

[0176] Table 5

[0177] Group Tumor inhibition rate (%) Liver index Normal group / 4.45±0.22 Model Group / 12.74±1.24* Cisplatin group 88.17 15.17±1.32# Example 1 82.11 6.10±0.29# Example 2 83.24 6.02±0.31# Example 3 83.75 5.94±0.33# Example 4 77.22 7.10±0.37 Example 5 78.45 7.05±0.32 Comparative Example 1 73.52 6.22±0.36 Comparative Example 2 74.17 6.19±0.35 Comparative Example 3 69.02 6.31±0.38 Comparative Example 4 70.11 6.17±0.41 Comparative Example 5 68.59 6.14±0.39 Comparative Example 6 79.29 8.04±0.26 Comparative Example 7 79.07 7.01±0.42 Comparative Example 8 70.19 8.21±0.33 Comparative Example 9 76.23 7.12±0.36 Comparative Example 10 75.84 7.23±0.40 Comparative Example 11 73.27 7.51±0.31 Comparative Example 12 60.29 6.11±0.35

[0178] Note: * compared with the normal group, P < 0.05; # compared with the model group, P < 0.05.

[0179] It can be seen from the above table that the preparations with liver cancer treatment effect prepared in Examples 1-3 of the present invention can significantly improve the tumor inhibition rate of liver cancer mice and have a good protective effect on the liver.

[0180] Compared with Example 3, Example 4 and 5 did not inoculate activated Lactobacillus rhamnosus strain seed solution or activated Bifidobacterium longum strain seed solution in step S5. Compared with Example 3, Comparative Example 8 did not perform step S5, the anti-tumor effect decreased, the liver index increased, and the liver function improvement effect decreased. The fermented probiotics of the present invention can regulate the balance of intestinal flora, inhibit inflammatory response, and enhance host immunity. Lactobacillus rhamnosus can reduce the expression of glutathione transferase, reduce the exposure of intestinal aflatoxin, and increase the excretion of fecal aflatoxin B1 to reduce the incidence of non-alcoholic fatty liver and liver cancer; Bifidobacterium longum and Lactobacillus can secrete interleukin 17 by modifying helper T cells 17 to regulate the immune mechanism of liver cancer, and at the same time have a protective effect on the intestinal mucosa, can repair damaged intestinal mucosa and reduce its permeability, prevent the specific binding of bacterial lipopolysaccharide to TOLL-like receptors, reduce the release of inflammatory factors, and inhibit the occurrence and development of liver cancer.

[0181] Comparative Example 2 Compared with Example 3, 1,2-bis(trimethoxysilyl)-propyl tetrasulfide is not added in step S1, and the drug cannot be quickly released in the presence of GSH, and the anti-tumor effect is reduced. In the preparation process of the silica nano drug delivery system, aminosilane and 1,2-bis(trimethoxysilyl)-propyl tetrasulfide are also added. Under the action of the structure-directing agent sodium salicylate, the surface of the prepared amino-disulfide bond-modified nanospheres is distributed with amino groups. At the same time, the structure of the silica spheres exists in the form of -O-Si-CCSSSSCC-Si-O-, so that the prepared amino-disulfide bond-modified nanospheres can stably exist under normal physiological conditions, and the drug is not released; after entering the cell, under the stimulation of glutathione, the disulfide bond breaks, breaking the dense structure of the carrier, and the drug loaded therein is quickly released.

[0182] Compared with Example 3, γ-aminopropyltriethoxysilane was not added in step S1 of Comparative Example 1. Compared with Example 3, step S2 was not performed in Comparative Example 3. The hemolysis rate and BSA adsorption amount were increased, and the anti-tumor effect was reduced. The present invention reacts the prepared amino-disulfide bond modified nanospheres with folic acid, and the amino and hydroxyl groups on the surface of the nanospheres can react with folic acid, so that folic acid is loaded on the surface of the amino-disulfide bond modified nanospheres to become a targeting group. The folic acid receptor is a glycoprotein on the surface of the cell membrane, which is overexpressed on the surface of a variety of tumor cells. Therefore, the prepared drug-loaded nanospheres can be targeted and delivered to liver cancer tumor cells, broken in the cells, and the drugs are released, so that the tumor cells die and the development of the tumor is inhibited. At the same time, the loading of folic acid can improve the biocompatibility of the nanospheres to a certain extent, reduce their clearance rate in the body, and increase their circulation time in the body.

[0183] Compared with Example 3, in Comparative Examples 4 and 5, no doxorubicin or triptolide was added in step S3. In Comparative Example 12, compared with Example 3, no drug-loaded nanospheres were added in step S7, and the anti-tumor effect decreased. Triptolide is a diterpenoid triepoxide, which has a very effective anti-cancer effect on a variety of cancers including liver cancer. Doxorubicin has a significant therapeutic effect on liver cancer. Under the synergistic effect of the two, it can effectively reduce the levels of serum tumor markers and serum inflammatory factors in patients, reduce inflammatory reactions, delay the disease, and through the targeted release of the drug of the present invention, the safety of the drug is within an acceptable range.

[0184] Comparative Example 6 Compared with Example 3, the ethanol aqueous solution was not heated for extraction in step S4, the anti-tumor effect decreased, the liver index increased, and the liver function improvement effect decreased. Mistletoe is a bitter and dry, sweet and tonic Chinese medicine, and the anti-tumor active components of its extract include mistletoe alkaloids, mistletoe lectins, mistletoe toxic peptides, etc. Among them, mistletoe lectins are highly active ribosome inhibitory proteins that can inhibit protein synthesis at the ribosome level, thereby inducing cancer cell apoptosis, and also promoting cellular immunity and humoral immunity. Mistletoe toxic peptides work by destroying the cell membrane structure, the membrane bilayer is destroyed, the membrane contents leak out, and the cells die, inhibiting the proliferation of malignant tumor cells. Total alkaloids of mistletoe can inhibit the expression of telomerase activity in precancerous lesions of liver. It inhibits the overexpression of apoptosis-related gene nuclear factor (NF)-κB, increases the expression of IκB-α, reduces the activity of telomerase, inhibits the synthesis of atypical hepatocyte telomeres, and has a blocking effect on the cancer-promoting stage of the carcinogenesis process. Total flavonoids in Prunella vulgaris extract inhibit tumor autophagy and promote cell apoptosis by activating the PI3K / Akt / mTOR signaling pathway, and have a significant anti-cancer effect. Under the synergistic effect of other Chinese medicine components (astragalus, Solanum nigrum, Rehmannia glutinosa), it can reduce the adverse reactions of patients undergoing chemotherapy for liver cancer, protect liver and kidney function, reduce the degree of vomiting, enhance the patient's appetite, enhance the patient's physical fitness, improve the efficacy of radiotherapy, and help patients recover their body functions as soon as possible. It can also strengthen the body and remove blood stasis, help the body quickly remove the remaining tumor cells, and further help the patient's recovery.

[0185] Comparative Example 7 Compared with Example 3, no prebiotic composition was added in step S5, the anti-tumor effect decreased, the liver index increased, and the liver function improvement effect decreased. The prebiotics added during the fermentation process of the present invention can promote the proliferation of fermented probiotics in a directional manner, thereby accelerating the fermentation process, promoting the large-scale reproduction of fermented probiotics, and obtaining highly active fermentation products in a short time.

[0186] Compared with Example 3, in Comparative Examples 9 and 10, cordycepin or ginsenoside Rh1 was not added in step S6, the anti-tumor effect decreased, the liver index increased, and the liver function improvement effect decreased. Compared with Example 3, in Comparative Example 11, no additive was added in step S7. The additives added in the present invention include cordycepin and ginsenoside Rh1. Cordycepin can induce tumor cell apoptosis through the Caspase pathway to inhibit tumor development, promote the expression of reactive oxygen ROS in tumor cells, and inhibit the proliferation of tumor cells; ginsenoside Rh1 can induce autophagy of liver cancer cells by enhancing the expression of Beclin1, resulting in decreased drug sensitivity, inhibiting the formation of tumor angiogenesis, thereby inhibiting the occurrence and development of tumors. At the same time, it can also enhance the body's immunity and improve the body's ability to fight tumor cells. The two have a synergistic effect.

[0187] 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 a preparation having a liver cancer therapeutic effect, characterized in that: The amino-disulfide bond modified nanospheres are prepared by a template method, and after folic acid is grafted on the surface, they are added to a solution of doxorubicin and triptolide, and the solvent is volatilized to obtain drug-loaded nanospheres; mistletoe, selfheal, astragalus, nightshade, and rehmannia root are mixed and extracted, filtered, and the filtrate is dried to obtain a Chinese medicine extract, the filter residue is added to water, and fermented by inoculating fermentation bacteria to obtain a fermentation product, and the fermentation product, the Chinese medicine extract, the drug-loaded nanospheres, cordycepin, and ginsenoside Rh1 are mixed and embedded to obtain a preparation with a liver cancer treatment effect; the steps include: S1. Preparation of amino-disulfide bond modified nanospheres: dissolving a cationic surfactant in water, adjusting the pH value of the solution to be alkaline, adding alkyl orthosilicate, aminosilane, 1,2-bis(trimethoxysilyl)-propyl tetrasulfide, and sodium salicylate, heating and stirring to react, standing and aging, centrifuging, washing with acidic ethanol and water in turn, centrifuging, and drying to obtain amino-disulfide bond modified nanospheres; S2. Grafting of folic acid: Add folic acid, 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, and N-hydroxysuccinimide to dimethyl sulfoxide, stir and mix evenly, add the amino-disulfide bond-modified nanospheres prepared in step S1, heat and stir to react in the dark, add the product to anhydrous ethanol, let stand, centrifuge, wash, and dry to obtain folic acid-grafted amino-disulfide bond-modified nanospheres; S3. Drug loading: doxorubicin and triptolide were mixed and dissolved in an ethanol aqueous solution, and the folic acid grafted amino-disulfide bond-modified nanospheres obtained in step S2 were added, stirred and mixed evenly, and the solvent was evaporated to obtain drug-loaded nanospheres; S4. Water extraction of Chinese medicine: The mistletoe, Prunella vulgaris, Astragalus, Solanum nigrum, and Rehmannia glutinosa were washed, dried, and crushed to obtain a Chinese medicine powder, added to an ethanol aqueous solution, heated under reflux for extraction, filtered, the solid residue was retained, and the filtrate was dried to obtain a Chinese medicine extract; S5. Fermentation: adding the solid residue in step S4 to water, sterilizing, inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum seed solution, fermenting and culturing for a first period of time, adding the prebiotic composition, continuing the fermentation and culturing for a second period of time, filtering, washing the solid residue to obtain a bacterial liquid, combining the bacterial liquid and the filtrate, and freeze-drying to obtain a fermentation product; S6. Preparation of additives: The cordycepin and ginsenoside Rh1 were mixed uniformly to obtain an additive; S7. Preparation of a preparation having a therapeutic effect on liver cancer: the drug-loaded nanospheres prepared in step S3, the Chinese medicine extract prepared in step S4, the fermentation product prepared in step S5 and the additive prepared in step S6 are mixed and added into water, an emulsifier, sodium alginate and sodium carboxymethyl cellulose are added, the mixture is stirred and mixed evenly, rapid membrane emulsification is performed, a calcium chloride solution is added dropwise, the mixture is solidified at room temperature, centrifuged, washed and dried to obtain a preparation having a therapeutic effect on liver cancer.

2. The preparation method according to claim 1, characterized in that: In step S1, the cationic surfactant is selected from at least one of dodecyltrimethylammonium chloride, dodecyltrimethyl sodium bromide, dodecyldimethylbenzyl ammonium chloride, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, hexadecyltrimethyl sodium bromide, hexadecyldimethylbenzyl ammonium chloride, octadecyltrimethylammonium bromide and octadecyltrimethylammonium chloride, the pH value of the adjustment solution is 11-12, and the mass ratio of the cationic surfactant, alkyl orthosilicate, aminosilane, 1,2-bis(trimethoxysilyl)-propyl tetrasulfide and sodium salicylate is 1-2:7-12:3-5: 5-7:0.5-1, the heating temperature is 70-80°C, the reaction time is 2-4h, the aging time is 2-4h, the acidic ethanol is a mixture of ethanol and concentrated hydrochloric acid in a volume ratio of 10:2-3, and the aminosilane is selected from at least one of γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-β (aminoethyl)-γ-aminopropyltrimethoxysilane, N-β (aminoethyl)-γ-aminopropyltriethoxysilane, N-β (aminoethyl)-γ-aminopropylmethyldimethoxysilane, N-β (aminoethyl)-γ-aminopropylmethyldiethoxysilane, and diethylenetriaminopropyltrimethoxysilane.

3. The preparation method according to claim 1, characterized in that: The mass ratio of folic acid, 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and amino-disulfide bond-modified nanospheres in step S2 is 4-5: 8-10: 4-6: 15-17, the temperature of the heated stirring and light-proof reaction is 25-32° C., the time is 10-12 h, the amount of anhydrous ethanol added is 5-7 times the total volume of the system, and the standing time is 5-7 h.

4. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of doxorubicin, triptolide, and folic acid grafted amino-disulfide bond modified nanospheres is 3-5:2-4:17-20, and the concentration of the ethanol aqueous solution is 40-50wt%.

5. The preparation method according to claim 1, characterized in that: In step S4, the mass ratio of mistletoe, selfheal, astragalus, nightshade and rehmannia root is 15-20:3-5:3-5:2-4:1-3, the solid-liquid ratio of the traditional Chinese medicine powder and the ethanol aqueous solution is 1:5-10 g / mL, the concentration of the ethanol aqueous solution is 50-60wt%, and the heating reflux extraction time is 3-5h.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the solid residue to water in step S5 is 15-20:100; the inoculation amounts of the activated Lactobacillus rhamnosus and Bifidobacterium longum seed solutions are 2-3 v / v% and 1-2 v / v%, respectively; the bacterial content of the seed solution is 10 8 -10 9 cfu / mL; the fermentation culture conditions are 37-39°C, 50-70r / min, 5-7% CO2, 3-5% O2, and the balance is nitrogen, wherein % is the volume percentage, the first time period is 36-48h, the second time period is 24-36h, the prebiotic composition includes inulin and arabinose in a mass ratio of 5-7:3-5, and the added amount is 3-5wt% of the total mass of the system.

7. The preparation method according to claim 1, characterized in that: The mass ratio of cordycepin to ginsenoside Rh1 in step S6 is 3-5:7-10; the mass ratio of drug-loaded nanospheres, Chinese herbal extracts, fermentation products, additives, emulsifiers, sodium alginate and sodium carboxymethyl cellulose in step S7 is 10-15:3-5:2-4:0.7-1.2:0.5-1:12-15:3-5, the membrane pore size of the rapid membrane emulsification is 1-2 μm, the concentration of the calcium chloride solution is 3-5wt%, the room temperature curing time is 20-40min, and the emulsifier is selected from at least one of lecithin, betaine, sorbitan fatty acid esters, polysorbate fatty acid esters, and dienoylglycerol fatty acid esters.

8. A preparation having a liver cancer therapeutic effect obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the preparation having liver cancer treatment effect as claimed in claim 8 in the preparation of drugs for treating and assisting the treatment of liver cancer.

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