CN117064980A - Composition for preventing and treating liver cancer and preparation method thereof - Google Patents
Composition for preventing and treating liver cancer and preparation method thereof Download PDFInfo
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Abstract
The invention provides a composition for preventing and treating liver cancer and a preparation method thereof, belonging to the technical field of medicines. Extracting Saviae Miltiorrhizae radix, bulbus Iphigeniae Indicae, and Cordyceps militaris with water, filtering, collecting the residue, precipitating with ethanol to obtain active polysaccharide, and drying the filtrate to obtain water extract; obtaining selenium-enriched yeast extracellular polysaccharide and mixing the selenium-enriched yeast extracellular polysaccharide with active polysaccharide to prepare an organic selenium-active polysaccharide compound; adding the filter residue into water, inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis, fermenting, adding cysteine, fermenting again, lyophilizing to obtain fermentation product, and embedding water extract, organic selenium-active polysaccharide complex, fermentation product, curcumin and fructo-oligosaccharide. The composition for preventing and treating liver cancer prepared by the invention plays a good role in preventing liver cancer and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a composition for preventing and treating liver cancer and a preparation method thereof.
Background
The prognosis of liver cell cancer is poor, the cure rate is low, and the life cycle of a patient can be prolonged to the maximum extent by using the traditional Chinese medicine treatment, the life quality is improved, and the survival rate is improved. There are no specific drugs and specific treatments currently available. The western medicine operation can quickly cut off early and medium-term cancers, but is easy to recur; at the same time, for a patient suffering from liver cancer, the surgical procedure runs the risk of mortality, since he also suffers from a rather severe cirrhosis, in which case it is a better treatment if a good anticancer agent is used. Although radiation therapy can shrink and degenerate cancer cells, it can only treat locally, and the adaptive person only accounts for 20%; although chemotherapy can be used for systemic treatment, patients often cannot stay on due to serious toxic and side effects and adverse reactions.
The traditional Chinese medicine considers that liver cancer is a disease pattern of deficiency and excess with deficiency of qi and blood as the principal, and the syndrome of deficiency and excess with the accumulation of qi and blood damp-heat stasis and toxin as the principal, and the treatment should strengthen body resistance to eliminate pathogenic factors and treat both principal and secondary aspect of disease. The red sage root is a common anti-tumor drug, is originally found in Shennong Ben Cao Jing, and has the effects of activating blood circulation to remove blood stasis, nourishing blood to tranquilize mind, cooling blood and eliminating carbuncles. "Benjing" is: "pathogenic qi in the heart and abdomen, borborygmus, like water, cold and heat accumulation; break the symptoms, remove the obstruction, stop restlessness and fill, and benefit qi.
The Nanchang university 2011 'Inducing liver cancer cell SMMG-7721 apoptosis and mechanism research' discloses that tanshinone IIA can inhibit liver cancer cell growth by inhibiting cell cycle and inducing apoptosis, the inhibition effect is time and dose dependent, tanshinone IIA can promote activation of apoptosis protein caspase-3, shearing of PARP protein and down-regulating apoptosis inhibitor protein XIAP, but has no influence on expression of survivin mRNA.
Patent document CN101596202a, publication date 2009.12.09, discloses the application of tanshinone IIA emulsion in treating liver diseases, in particular, after intravenous administration of tanshinone IIA emulsion, the medicine can be rapidly targeted and distributed to the liver, and is used for treating liver diseases such as fatty hepatitis, viral hepatitis, liver fibrosis and liver cancer, etc., with good effect.
The paper published in the journal literature "Shizhen national medicine" 2011, 9 th stage, "research on the migration mechanism of icariin against liver cancer cells HepG 2", uses icariin with different concentrations to act on HepG2 cells for 24 hours, and then uses an adhesion experiment to detect the cell adhesion rate and a scratch damage experiment to detect the migration speed, so as to obtain the conclusion that icariin inhibits the adhesion and movement of the HepG2 cells and plays a role in inhibiting tumor metastasis.
Patent document CN106995829A, publication date 2017.08.01, discloses that icariin prepared by enzymatic conversion of epimedium total flavonoids has very remarkable proliferation inhibition effect on liver cancer, lung cancer, colon cancer and breast cancer.
In recent years, with the wide importance of traditional Chinese medicine for treating tumor diseases, the research of compatibility of components of traditional Chinese medicine compound for resisting tumor provides a new opportunity for treating malignant tumor. The compatibility of the effective components of the traditional Chinese medicine is superior to that of decoction pieces as a whole, and the interference of non-pharmacodynamic substances is eliminated due to the compatibility of the effective components.
Disclosure of Invention
The invention aims to provide a composition for preventing and treating liver cancer and a preparation method thereof, which have the functions of protecting mucous membrane barrier, reducing the toxicity of cancerogenic substances, reducing endotoxin level, restoring the immune response of an organism to be normal, relieving liver cell damage caused by inflammatory reaction, improving viral hepatitis symptoms, inhibiting further development of liver cirrhosis, promoting cancer cell apoptosis, playing a good role in preventing liver cancer and having wide application prospect.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a composition for preventing and treating liver cancer, which comprises the steps of extracting red sage root, indian iphigenia bulb and cordyceps militaris with water, filtering, reserving filter residues, precipitating with ethanol to obtain active polysaccharide, and drying filtrate to obtain a water extract; obtaining selenium-enriched yeast extracellular polysaccharide and mixing the selenium-enriched yeast extracellular polysaccharide with active polysaccharide to prepare an organic selenium-active polysaccharide compound; adding the filter residue into water, inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis, fermenting, adding cysteine, fermenting again, lyophilizing to obtain fermentation product, and embedding water extract, organic selenium-active polysaccharide complex, fermentation product, curcumin and fructo-oligosaccharide.
As a further improvement of the invention, the method comprises the following steps:
s1, water extraction: cleaning Saviae Miltiorrhizae radix, bulbus Iphigeniae Indicae, and Cordyceps militaris respectively, drying, pulverizing, adding into water, heating and boiling for extraction, repeating operation for 2-3 times, filtering, collecting residue, mixing filtrates, concentrating, precipitating with ethanol, centrifuging to collect solid, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
S2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting, culturing, centrifuging, adding a mixed solvent into supernatant, precipitating, centrifuging, adding ethanol into supernatant for precipitating, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
s3, preparing an organic selenium-active polysaccharide compound: adding the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into water, adding the active polysaccharide prepared in the step S1, stirring and mixing uniformly, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: respectively inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis into Gao's medium, and performing active culture to obtain strain seed solution;
s5, fermenting: adding the filter residues obtained in the step S1 into water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, fermenting for a first time period, adding an additive, and fermenting for a second time period to obtain a fermentation mixture;
s6, promoting glutathione synthesis and fermentation: adding cysteine into the fermentation mixture in the step S5, continuing to ferment and culture for a third time period, and freeze-drying to obtain a fermentation product;
s7, embedding: adding the water extract prepared in the step S1, the organic selenium-active polysaccharide compound prepared in the step S3, the fermentation product prepared in the step S6, curcumin, fructo-oligosaccharide, sodium alginate and sodium carboxymethyl cellulose into water, and uniformly mixing to prepare a water phase; adding water phase and lecithin into fish oil, emulsifying, dripping calcium chloride solution, solidifying at normal temperature, centrifuging, washing, and lyophilizing to obtain composition for preventing and treating liver cancer.
As a further improvement of the invention, in the step S1, the mass ratio of the red sage root, the Indian iphigenia bulb and the Cordyceps militaris is 3-5:1-3:5-7, the solid-liquid ratio of the mixed powder and the water is 1:5-7 g/mL, the heating boiling extraction time is 1-2h, the ethanol is added until the system ethanol content is 60-70wt%, and the precipitation time is 3-5h.
As a further improvement of the present invention, the formula of the medium in step S2 is: 20-40 parts of glucose, 3-5 parts of peptone and KH (KH) 2 PO 4 2-3 parts by weight of MgSO 4 ·7H 2 O1-2 parts by weight, vitamin B1.01-0.02 part by weight and water 1000 parts by weight, wherein the inoculation amount of selenium-enriched saccharomycetes is 2-3w/v%, the condition of fermentation culture is 50-55 ℃,50-70r/min, the time is 24-36h, the mixed solvent is a mixed solvent of chloroform and n-butanol, the volume ratio is 4:1, the volume ratio of supernatant and the mixed solvent is 3-5:1, the time of precipitation is 1-2h, the supernatant is added with ethanol until the system ethanol content is 60-70wt%, and the time of precipitation is 3-5h; and in the step S3, the mass ratio of the selenium-enriched yeast extracellular polysaccharide to the active polysaccharide is 5-7:3-5.
As a further improvement of the invention, the condition of the activation culture in the step S4 is 40-42 ℃,50-70r/min and the activation culture is carried out for 18-24 hours, and the bacterial seed liquid contains 10 percent of bacteria 8 -10 9 cfu/mL; in the step S5, the mass ratio of filter residues to water is 2-5:12-15, the inoculation amounts of saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are 2-3v/v%, 1-2v/v% and 2-3v/v%, the fermentation condition is 37-40 ℃,50-70r/min, the first time period is 24-36h, the second time period is 12-18h, the additive is a mixture of calcium chloride and vitamin B1, the mass ratio is 3-5:1-2, and the addition amount of the additive is 3-5wt% of the total mass of the system.
As a further improvement of the invention, the mass ratio of the fermentation mixture to the cysteine in the step S6 is 100-120:5-7, the condition of the fermentation culture is 37-40 ℃,50-70r/min, and the third time period is 18-24h.
As a further improvement of the invention, the mass ratio of the water extract, the organic selenium-active polysaccharide complex, the fermentation product, the curcumin, the fructo-oligosaccharide, the sodium alginate, the sodium carboxymethylcellulose and the water in the step S7 is 3-5:7-10:12-15:1-2:0.5-1:20-25:7-10:200-250, and the mass ratio of the water phase, the lecithin and the fish oil is 100-120:2-3: 200-250, wherein the concentration of the calcium chloride solution is 3-5wt%, and the normal temperature curing time is 30-40min.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, water extraction: cleaning 3-5 parts by weight of red sage root, 1-3 parts by weight of Indian iphigenia bulb and 5-7 parts by weight of Cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water with the solid-to-liquid ratio of 1:5-7 g/mL, heating, boiling and extracting for 1-2h, repeating the operation for 2-3 times, filtering, reserving filter residues, mixing filtrates, concentrating, adding ethanol until the ethanol content of the system is 60-70wt%, precipitating for 3-5h, centrifuging, collecting solids, drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
s2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 24-36h at the inoculum size of 2-3w/v%, at 50-55 ℃ and at 50-70r/min, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 3-5:1, precipitating for 1-2h, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 60-70wt%, precipitating for 3-5h, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
the formula of the culture medium is as follows: 20-40 parts of glucose, 3-5 parts of peptone and KH (KH) 2 PO 4 2-3 parts by weight of MgSO 4 ·7H 2 O1-2 weight portions, vitamin B1 0.01-0.02 weight portions and water 1000 weight portions;
The mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 5-7 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 3-5 parts by weight of the active polysaccharide prepared in the step S1, uniformly stirring and mixing, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: inoculating Saccharomyces cerevisiae, lactobacillus salivarius, and Bacillus subtilis into Gao's culture medium, respectively, and performing active culture at 40-42deg.C and 50-70r/min for 18-24 hr to obtain strain seed solution with a bacterial content of 10 8 -10 9 cfu/mL;
S5, fermenting: adding 20-50 parts by weight of filter residues obtained in the step S1 into 120-150 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2-3v/v%, 1-2v/v%, 2-3v/v%,37-40 ℃,50-70r/min, fermenting for 24-36h, and adding additives, wherein the additive amount is 3-5wt% of the total mass of the system, and fermenting for 12-18h to obtain a fermentation mixture;
the additive is a mixture of calcium chloride and vitamin B1, and the mass ratio is 3-5:1-2;
S6, promoting glutathione synthesis and fermentation: adding 5-7 parts by weight of cysteine into 100-120 parts by weight of the fermentation mixture obtained in the step S5, fermenting and culturing at 37-40 ℃ for 18-24 hours at 50-70r/min, and freeze-drying to obtain a fermentation product;
s7, embedding: adding 3-5 parts by weight of the water extract prepared in the step S1, 7-10 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 12-15 parts by weight of the fermentation product prepared in the step S6, 1-2 parts by weight of curcumin, 0.5-1 part by weight of fructo-oligosaccharide, 20-25 parts by weight of sodium alginate and 7-10 parts by weight of sodium carboxymethyl cellulose into 200-250 parts by weight of water, and uniformly mixing to prepare an aqueous phase; adding 100-120 parts by weight of water phase and 2-3 parts by weight of lecithin into 200-250 parts by weight of fish oil, emulsifying, dripping 50 parts by weight of 3-5wt% calcium chloride solution, solidifying at normal temperature for 30-40min, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
The invention further provides a composition for preventing and treating liver cancer, which is prepared by the preparation method.
The invention further provides application of the composition for preventing and treating liver cancer in preparing products for preventing and treating liver cancer, liver failure and hepatitis.
The invention has the following beneficial effects:
The red sage root is the dried root and rhizome of red sage root of Labiatae, has slightly cold nature, bitter taste, and is effective in promoting blood circulation, removing scar, dredging channels, relieving pain, clearing heart fire, relieving restlessness, cooling blood, and resolving carbuncle, and has antioxidant, antibacterial, antiinflammatory, microcirculation improving, pain relieving, and cardiovascular and cerebrovascular diseases resisting effects. The Pseudobulbus Cremastrae seu pleiones has antiinflammatory, antibacterial, antitumor, and antiangiogenic effects, and can be used for resisting cancer. The main active ingredient cordycepin in the cordyceps militaris is a special active ingredient in the cordyceps militaris, has obvious effects of calming, resisting fatigue and tumor, inhibiting cancer cells, promoting secretion of male hormone and the like, and can enhance the immune function of an organism.
The invention extracts the red sage root, the Indian iphigenia bulb and the Cordyceps militaris by water, and the obtained active components comprise active substances such as salvianolic acid B, cordycepin, alkaloid, saponin, flavonoids, cordyceps polysaccharide, red sage root polysaccharide, indian iphigenia bulb polysaccharide and the like, wherein the salvianolic acid B is polymerized by 3 molecules of salvianic acid A and 1 molecule of caffeic acid, is one component with highest content and strongest activity in the water-soluble active ingredient of the red sage root, and has the effect of inhibiting the growth of cancer cells in vitro. Salvianolic acid B can relieve acute liver injury by inhibiting TLR4/MyD88/NF- κB signaling, and TLR4/MyD88/NF- κB signaling is a key pathway widely existing in various cells and involved in inflammatory reaction, and is an important regulatory mechanism for mediating tumorigenesis. Salvianolic acid B can influence the composition of intestinal flora, increase the relative abundance of beneficial intestinal bacteria, reduce the relative abundance of harmful bacteria, and improve intestinal environment. The Pseudobulbus Cremastrae seu pleiones polysaccharide is a main bioactive component of Pseudobulbus Cremastrae seu pleiones, and can improve immunity, inhibit PI3K/AKT/mTOR activation, induce autophagy, inhibit angiogenesis, and promote tumor apoptosis. Cordycepin is a natural nucleoside antibiotic, is a natural anticancer antibiotic, namely 3-deadenosine, is a nucleic acid derivative of nitrogen-containing glycoside, belongs to purine alkaloids, has antiviral and antitumor effects, and particularly has strong inhibition effect on various solid malignant tumors.
Selenium can be involved in the occurrence and development of liver diseases of human or animals, can effectively prevent liver injury induced by various reasons, is a main component constituting glutathione peroxidase, and the glutathione peroxidase is an important antioxidant enzyme in the liver of human or animals. Meanwhile, selenium also eliminates various free radicals in the body together with superoxide dismutase, catalase and the like, thereby reducing the occurrence of organism diseases. Selenium also increases the levels of intra-hepatic glutathione to combat hepatocyte damage due to free radicals. Selenium addition can significantly reduce activation of hepatic stellate cells and expression of hepatic fibrosis related genes.
According to the invention, after selenium-enriched saccharomycetes are adopted for fermentation, thalli are removed through centrifugation, a Sevag method is adopted for removing protein to obtain selenium-enriched saccharomycetes extracellular polysaccharide liquid, and the selenium-enriched saccharomycetes extracellular polysaccharide liquid is mixed with active polysaccharide obtained through extraction of red sage root, indian iphigenia bulb and cordyceps militaris, and can form an organic selenium-active polysaccharide compound through chelation.
Under normal conditions, the colonised flora in the intestine forms a protective barrier against pathogens, which when damaged may lead to increased intestinal mucosa permeability, leading to translocation of intestinal bacteria. Dysbacteriosis in intestinal tract results in reduced colonisation resistance of the flora, reduced barrier protection, and massive invasion, colonisation and proliferation of pathogens in the intestinal tract. Intestinal bacteria shift can accelerate high power circulation state, increase portal vein pressure and increase intestinal mucosa injury, meanwhile, intestinal flora imbalance leads to reduced colonisation resistance of small intestine, causes overgrowth of small intestine bacteria, further secretes a great deal of proinflammatory cytokines, causes serious inflammatory reaction, damages microorganism barrier, finally causes bile acid metabolic disorder, leads to liver failure and further develops into liver cancer.
The probiotics can effectively inhibit apoptosis of intestinal mucosa epithelial cells, further protect mucosal barrier function, reduce toxicity of cancerogenic substances, reduce endotoxin level, improve intestinal flora disorder, maintain balance of microecology, restore immune response of organisms to be normal, relieve liver cell injury caused by inflammatory reaction, improve viral hepatitis symptoms, inhibit further development of liver cirrhosis, and play a good role in preventing liver cancer.
The added probiotics comprise saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis, wherein the lactobacillus salivarius has the effects of improving liver functions, protecting intestinal barriers and relieving liver fibrosis, can prevent or treat liver cirrhosis and liver failure, and metabolic products of the lactobacillus salivarius can protect liver functions, and play a positive role in preventing liver cancer, such as indopropionic acid, which is a product of tryptophan decomposed by bacteria, can inhibit NF-kappa B signal channels, reduce the level of pro-inflammatory cytokines, antagonize liver inflammation and liver injury, and can further improve the inherent immunity of the liver and intestinal tracts, further enhance the liver capacity of clearing hepatitis viruses, reduce liver inflammation and strengthen the intestinal barrier effect, thereby playing a role in preventing cancer. Saccharomyces cerevisiae can promote the growth of bifidobacteria (probiotics) in the intestinal flora while inhibiting the number of candida albicans (pathogenic bacteria); can also protect the mucosal barrier function by reducing apoptosis of intestinal mucosal epithelial cells, and prevent further development of liver cirrhosis by inhibiting release of inflammatory mediators such as TNF-alpha and IL-6. The bacillus subtilis can degrade aflatoxin, prevent liver cancer by reducing the toxicity of cancerogenic substances, effectively improve intestinal flora imbalance, restore the normal immune response of an organism to HBV, reduce the release of inflammatory mediators, reduce liver cell damage, further improve viral hepatitis symptoms, and simultaneously can effectively inhibit the Th17 cell level in tumors by stimulating the Proteus to secrete anti-inflammatory substances, wherein Th17 cells are an auxiliary T cell subgroup capable of secreting IL-17, inhibit tumor immunity and stimulate tumor angiogenesis, participate in the generation and development of tumors and play an anticancer role by inhibiting the Th17 cell level.
The probiotics not only can generate beneficial substances through self fermentation, but also can inhibit the colonization of harmful bacteria, improve viral hepatitis and liver cirrhosis disease conditions, strengthen inherent immunity of liver and intestines, reduce toxic carcinogenicity and the like, prevent liver cancer, but also can stimulate the secretion of anti-inflammatory substances, inhibit Th17 cell level in tumors and strengthen anti-tumor immune response to play an anticancer role, and has the advantages of safety, harmlessness, cheapness and the like, thereby being hopeful to become an emerging prevention and treatment means of liver cancer.
According to the invention, calcium chloride and vitamin B2 are supplemented in the fermentation process of probiotics, and the resistance of the probiotics is increased and the time period of the probiotics in the stationary phase is improved through the adjustment of trace elements, so that the probiotics are promoted to produce more beneficial substances.
Glutathione is an important antioxidant in vivo, can remove excessive free radicals in vivo, and the free radicals are combined to reduce the oxidative stress level in vivo, and simultaneously participate in glycolysis pathway and tricarboxylic acid cycle in vivo, so that the organism obtains higher energy, and activates various enzymes in vivo, thereby promoting synthesis and decomposition of saccharides, fat and protein, further improving the nutrition metabolism of cells, and further participating in various biochemical reactions in human or animal cells; by protecting integrity of liver cell membrane and increasing activity of related enzyme in liver. In addition, the glutathione has strong detoxification, and has good detoxification on substances harmful to organisms, such as organic solvents, carbon monoxide, alcohol, heavy metals, cancerogenic substances and the like.
The saccharomyces cerevisiae in the zymophyte can accumulate more glutathione in cells, so that the amount of the glutathione in the composition is greatly increased. Cysteine is an important synthetic raw material of glutathione, and the cysteine is added to promote the fermentation product glutathione of saccharomyces cerevisiae, so that the method plays a good role in protecting liver and plays a good role in preventing and treating liver cancer.
In addition, curcumin can be added to reduce liver injury by increasing the content of glutathione in the liver. Meanwhile, curcumin and fructo-oligosaccharide are good prebiotics, can directionally promote the proliferation of intestinal probiotics, thereby inhibiting the growth of harmful bacteria, regulating liver function through liver-intestinal axis, promoting the recovery of liver function, inhibiting the formation of fatty liver and protecting liver.
The composition for preventing and treating liver cancer prepared by the invention protects the barrier function of mucous membrane, reduces the toxicity of cancerogenic substances, reduces endotoxin level, enables the immune response of an organism to be recovered to be normal, reduces liver cell damage caused by inflammatory reaction, improves viral hepatitis symptoms, inhibits further development of liver cirrhosis, promotes apoptosis of cancer cells, plays a good role in preventing liver cancer, and has wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a graph showing the comparative viability of each group in test example 1 according to the present invention;
FIG. 2 is a graph showing the comparison of the release rates of each group in test example 1 according to the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Selenium-rich saccharomycetes, 100 hundred million cfu/g, purchased from Angel Yeast Co., ltd; saccharomyces cerevisiae, a high activity dry yeast for brewing wine, purchased from Angel Yeast Co., ltd; lactobacillus salivarius 3500 hundred million cfu/g, available from Guangzhou Huijian biotechnology Co., ltd; bacillus subtilis, 200 hundred million cfu/g, was purchased from North sea, wang Biotechnology Inc.
Example 1
The embodiment provides a preparation method of a composition for preventing and treating liver cancer, which specifically comprises the following steps:
s1, water extraction: cleaning 3 parts by weight of red sage root, 1 part by weight of Indian iphigenia bulb and 5 parts by weight of cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder into water, heating and boiling for extraction for 1h, repeating the operation for 2 times, filtering, reserving filter residues, combining the filtrates, concentrating to a relative density of 1.05, adding ethanol until the ethanol content of the system is 60wt%, precipitating for 3h, centrifuging, collecting solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
s2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 24 hours at 50 ℃ and 50r/min with the inoculum size of 2w/v%, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 3:1, precipitating for 1 hour, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 60wt%, precipitating for 3 hours, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
the formula of the culture medium is as follows: 20 parts by weight of glucose, 3 parts by weight of peptone and KH 2 PO 4 2 parts by weight of MgSO 4 ·7H 2 O1 weight portions, vitamin B1.01 weight portions and water 1000 weight portions;
the mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 5 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 3 parts by weight of the active polysaccharide prepared in the step S1, stirring and mixing for 20min, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: inoculating Saccharomyces cerevisiae, lactobacillus salivarius, and Bacillus subtilis into Gao's medium, respectively, and performing active culture at 40deg.C and 50r/min for 18 hr to obtain strain seed solution with a bacterial content of 10 8 cfu/mL;
S5, fermenting: adding 20 parts by weight of filter residues obtained in the step S1 into 120 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2v/v, 1v/v, 2v/v, 37 ℃,50r/min, fermenting for 24 hours, adding additives, and fermenting for 12 hours, wherein the additive amount is 3wt% of the total mass of the system, so as to obtain a fermentation mixture;
the additive is a mixture of calcium chloride and vitamin B1, and the mass ratio is 3:1;
S6, promoting glutathione synthesis and fermentation: adding 5 parts by weight of cysteine into 100 parts by weight of the fermentation mixture in the step S5, fermenting and culturing at 37 ℃ for 1 h at 50r/min, and freeze-drying to obtain a fermentation product;
s7, embedding: adding 3 parts by weight of the water extract prepared in the step S1, 7 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 12 parts by weight of the fermentation product prepared in the step S6, 1 part by weight of curcumin, 0.5 part by weight of fructo-oligosaccharide, 20 parts by weight of sodium alginate and 7 parts by weight of sodium carboxymethylcellulose into 200 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 100 parts by weight of water phase and 2 parts by weight of lecithin into 200 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 3wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Example 2
The embodiment provides a preparation method of a composition for preventing and treating liver cancer, which specifically comprises the following steps:
s1, water extraction: cleaning 5 parts by weight of red sage root, 3 parts by weight of Indian iphigenia bulb and 7 parts by weight of cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder into water, heating, boiling and extracting for 2 hours, repeating the operation for 3 times, filtering, reserving filter residues, merging filtrate, concentrating until the relative density is 1.12, adding ethanol until the ethanol content of the system is 70wt%, precipitating for 5 hours, centrifuging, collecting solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
S2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 36h at the inoculum size of 3w/v percent and at the temperature of 55 ℃ for 70r/min, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 5:1, precipitating for 2h, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 70wt%, precipitating for 5h, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
the formula of the culture medium is as follows: glucose 40 weight portions, protein5 parts by weight of peptone, KH 2 PO 4 3 parts by weight of MgSO 4 ·7H 2 O2, vitamin B1.02 and water 1000;
the mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 7 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 5 parts by weight of the active polysaccharide prepared in the step S1, stirring and mixing for 20min, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: inoculating Saccharomyces cerevisiae, lactobacillus salivarius, and Bacillus subtilis into Gao's culture medium, respectively, and performing active culture at 42deg.C and 70r/min for 24 hr to obtain strain seed solution with a bacterial content of 10 9 cfu/mL;
S5, fermenting: adding 50 parts by weight of filter residues obtained in the step S1 into 150 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 3v/v%, 2v/v%, 3v/v%,40 ℃,70r/min, fermenting for 36h, adding additives, and fermenting for 18h to obtain a fermentation mixture, wherein the additive amount is 5wt% of the total mass of the system;
the additive is a mixture of calcium chloride and vitamin B1, and the mass ratio is 5:2;
s6, promoting glutathione synthesis and fermentation: adding 7 parts by weight of cysteine into 120 parts by weight of the fermentation mixture in the step S5, fermenting and culturing at 40 ℃ for 24 hours at 70r/min, and freeze-drying to obtain a fermentation product;
s7, embedding: adding 5 parts by weight of the water extract prepared in the step S1, 10 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 15 parts by weight of the fermentation product prepared in the step S6, 2 parts by weight of curcumin, 1 part by weight of fructo-oligosaccharide, 25 parts by weight of sodium alginate and 10 parts by weight of sodium carboxymethylcellulose into 250 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 120 parts by weight of water phase and 3 parts by weight of lecithin into 250 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 5wt% calcium chloride solution, solidifying for 40min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Example 3
The embodiment provides a preparation method of a composition for preventing and treating liver cancer, which specifically comprises the following steps:
s1, water extraction: cleaning 4 parts by weight of red sage root, 2 parts by weight of Indian iphigenia bulb and 6 parts by weight of cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water into water, heating and boiling for extraction for 1.5 hours, repeating the operation for 3 times, filtering, reserving filter residues, combining the filtrates, concentrating until the relative density is 1.07, adding ethanol until the ethanol content of the system is 65wt%, precipitating for 4 hours, centrifuging and collecting solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
s2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 30 hours at the inoculum size of 2.5w/v percent and at the temperature of 52 ℃ for 60r/min, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 4:1, precipitating for 1.5 hours, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 65wt%, precipitating for 4 hours, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
the formula of the culture medium is as follows: 30 parts by weight of glucose, 4 parts by weight of peptone and KH 2 PO 4 2.5 parts by weight of MgSO 4 ·7H 2 1.5 parts of O, 0.015 part of vitamin B and 1000 parts of water;
the mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 6 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 4 parts by weight of the active polysaccharide prepared in the step S1, stirring and mixing for 20min, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis into Gao's culture medium, respectively, performing activation culture at 41 deg.C and 60r/min for 21 hr,preparing strain seed liquid with a bacterial content of 10 9 cfu/mL;
S5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2.5v/v, 1.5v/v, 2.5v/v, 38 ℃,60r/min, fermenting for 30h, adding an additive, wherein the additive amount is 4wt% of the total mass of the system, and fermenting for 15h to obtain a fermentation mixture;
the additive is a mixture of calcium chloride and vitamin B1, and the mass ratio is 4:1.5;
S6, promoting glutathione synthesis and fermentation: adding 6 parts by weight of cysteine into 110 parts by weight of the fermentation mixture in the step S5, fermenting and culturing at 38 ℃ for 21 hours at 60r/min, and freeze-drying to obtain a fermentation product;
s7, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 13.5 parts by weight of the fermentation product prepared in the step S6, 1.5 parts by weight of curcumin, 0.7 part by weight of fructo-oligosaccharide, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethyl cellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare an aqueous phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Example 4
The difference compared to example 3 is that the additive is a single calcium chloride.
Example 5
The difference compared to example 3 is that the additive is a single vitamin B1.
Comparative example 1
In comparison with example 3, the difference is that no radix Salviae Miltiorrhizae was added in step S1.
The method comprises the following steps:
S1, water extraction: cleaning 2 parts by weight of Indian iphigenia bulb and 6 parts by weight of Cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water into water, heating and boiling for extraction for 1.5 hours, repeating the operation for 3 times, filtering, reserving filter residues, merging the filtrates, concentrating until the relative density is 1.07, adding ethanol until the ethanol content of the system is 65wt%, precipitating for 4 hours, centrifuging and collecting solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract.
Comparative example 2
The difference from example 3 is that no edible tulip is added in step S1.
The method comprises the following steps:
s1, water extraction: cleaning 4 parts by weight of red sage root and 6 parts by weight of cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water into water, heating and boiling for extraction for 1.5 hours, repeating the operation for 3 times, filtering, reserving filter residues, merging filtrate, concentrating until the relative density is 1.07, adding ethanol until the ethanol content of the system is 65wt%, precipitating for 4 hours, centrifuging to collect solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract.
Comparative example 3
In comparison with example 3, the difference is that no cordyceps militaris was added in step S1.
The method comprises the following steps:
s1, water extraction: cleaning 4 parts by weight of red sage root and 2 parts by weight of Indian iphigenia bulb respectively, drying, crushing to prepare mixed powder, adding the mixed powder and water into water, heating and boiling for extraction for 1.5h, repeating the operation for 3 times, filtering, reserving filter residues, merging filtrate, concentrating until the relative density is 1.07, adding ethanol until the ethanol content of the system is 65wt%, precipitating for 4h, centrifuging to collect solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract.
Comparative example 4
In comparison with example 3, the difference is that steps S2 and S3 are not performed, and the organic selenium-active polysaccharide complex in step S7 is replaced by the active polysaccharide produced in step Sl.
The method comprises the following steps:
s7, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the active polysaccharide prepared in the step S1, 13.5 parts by weight of a fermentation product, 1.5 parts by weight of curcumin, 0.7 part by weight of fructo-oligosaccharide, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Comparative example 5
In contrast to example 3, step S5 was not inoculated with Saccharomyces cerevisiae and step S6 was not performed.
The method comprises the following steps:
s5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculation amounts of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2.5v/v, 1.5v/v, 2.5v/v, 38 ℃,60r/min, fermenting for 30h, and adding an additive, wherein the addition amount of the additive is 4wt% of the total mass of the system, and fermenting for 15h to obtain a fermentation mixture.
Comparative example 6
The difference compared to example 3 is that lactobacillus salivarius was not inoculated in step S5.
The method comprises the following steps:
s5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae and bacillus subtilis strain seed liquid are respectively 2.5v/v%, 4v/v%,38 ℃,60r/min, fermenting for 30h, and adding an additive, wherein the additive amount is 4wt% of the total mass of the system, and fermenting for 15h to obtain a fermentation mixture.
Comparative example 7
The difference compared with example 3 is that in step S5, bacillus subtilis is not inoculated.
The method comprises the following steps:
s5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae and lactobacillus salivarius strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae and lactobacillus salivarius strain seed liquid are 2.5v/v%, 4v/v%,38 ℃,60r/min respectively, fermenting for 30h, and adding an additive, wherein the additive amount is 4wt% of the total mass of the system, and fermenting for 15h to obtain a fermentation mixture.
Comparative example 8
The difference from example 3 is that lactobacillus salivarius and bacillus subtilis seed solution were not inoculated in step S5.
The method comprises the following steps:
s5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae strain seed liquid obtained in the step S4, fermenting for 30 hours at 38 ℃ with the inoculum size of 2.5v/v% respectively, and adding an additive with the additive amount of 4wt% of the total mass of the system, and fermenting for 15 hours to obtain a fermentation mixture.
Comparative example 9
In comparison with example 3, the difference is that no additive is added in step S5.
The method comprises the following steps:
s5, fermenting: adding 35 parts by weight of filter residues obtained in the step S1 into 135 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2.5v/v%, 1.5v/v%, 2.5v/v%,38 ℃, and fermenting for 45 hours at the temperature of 60r/min to obtain a fermentation mixture.
Comparative example 10
In comparison with example 3, the difference is that steps S4, S5 and S6 are not performed.
The method comprises the following steps:
s1, water extraction: cleaning 4 parts by weight of red sage root, 2 parts by weight of Indian iphigenia bulb and 6 parts by weight of cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water into water, heating and boiling for extraction for 1.5 hours, repeating the operation for 3 times, filtering, reserving filter residues, combining the filtrates, concentrating until the relative density is 1.07, adding ethanol until the ethanol content of the system is 65wt%, precipitating for 4 hours, centrifuging and collecting solids, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
s2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 30 hours at the inoculum size of 2.5w/v percent and at the temperature of 52 ℃ for 60r/min, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 4:1, precipitating for 1.5 hours, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 65wt%, precipitating for 4 hours, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
The formula of the culture medium is as follows: 30 parts by weight of glucose, 4 parts by weight of peptone and KH 2 PO 4 2.5 parts by weight of MgSO 4 ·7H 2 1.5 parts of O, 0.015 part of vitamin B and 1000 parts of water;
the mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 6 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 4 parts by weight of the active polysaccharide prepared in the step S1, stirring and mixing for 20min, and drying to prepare an organic selenium-active polysaccharide compound;
s4, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 1.5 parts by weight of curcumin, 0.7 part by weight of fructo-oligosaccharides, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethyl cellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Comparative example 11
In comparison with example 3, the difference is that cysteine is not added in step S6.
The method comprises the following steps:
s6, re-fermentation: and (3) fermenting and culturing the fermentation mixture obtained in the step (S5) at 38 ℃ for 21 hours at 60r/min, and freeze-drying to obtain a fermentation product.
Comparative example 12
In comparison with example 3, the difference is that no embedding is performed in step S7, which is a simple mixing between the components.
The method comprises the following steps:
s7, embedding: 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 13.5 parts by weight of the fermentation product prepared in the step S6, 1.5 parts by weight of curcumin and 0.7 part by weight of fructo-oligosaccharide are mixed for 20min to prepare the composition for preventing and treating liver cancer.
Comparative example 13
In comparison with example 3, the difference is that curcumin is not added in step S7.
The method comprises the following steps:
s7, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 13.5 parts by weight of the fermentation product prepared in the step S6, 2.2 parts by weight of fructo-oligosaccharides, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Comparative example 14
In comparison with example 3, the difference is that fructooligosaccharides are not added in step S7.
The method comprises the following steps:
s7, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 13.5 parts by weight of the fermentation product prepared in the step S6, 2.2 parts by weight of curcumin, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Comparative example 15
In comparison with example 3, the difference is that curcumin and fructo-oligosaccharide are not added in step S7.
The method comprises the following steps:
s7, embedding: adding 4 parts by weight of the water extract prepared in the step S1, 8.5 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 13.5 parts by weight of the fermentation product prepared in the step S6, 22 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethyl cellulose into 225 parts by weight of water, and mixing for 20 minutes to prepare a water phase; adding 110 parts by weight of water phase and 2.5 parts by weight of lecithin into 225 parts by weight of fish oil, emulsifying for 10min at 10000r/min, dripping 50 parts by weight of 4wt% calcium chloride solution, solidifying for 35min at normal temperature, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
Test example 1 sustained and controlled Release test
1g of the composition for preventing and treating liver cancer prepared in examples 1 to 3 and comparative example 12 of the present invention was added to 9mL of artificial simulated gastric fluid and 9mL of artificial simulated intestinal fluid, respectively, and reacted at 37℃under 50r/min for 2 hours and 3 hours, respectively, and in addition, an equal amount of the composition for preventing and treating liver cancer was added to 9mL of artificial simulated gastric fluid, reacted at 37℃under 50r/min for 2 hours, centrifuged, and then added to 9mL of artificial simulated intestinal fluid, and the reaction was continued at 37℃under 50r/min for 3 hours. Probiotic colony cell counts were performed after the end of the reaction. Survival was calculated according to the following formula:
survival (%) =n t /N 0 ×100%
Wherein N is t For the concentration of probiotics (cfu/g) surviving after a certain incubation time, N 0 Is the initial probiotic raw concentration (cfu/g).
The release rate was calculated according to the following formula:
release rate (%) = (W) t -W 0 )/W 0 ×100%
In which W is t Initial weight for sample; w (W) 0 The samples were incubated for a certain time before weighing.
The results are shown in FIGS. 1 and 2.
As can be seen from the graph, the composition for preventing and treating liver cancer prepared in the embodiments 1-3 has better integrity in artificial simulated gastric fluid, and releases active substances after being transferred into artificial simulated intestinal fluid, so that the composition for preventing and treating liver cancer has the characteristics of pH responsiveness and gastric acid resistance, and has the characteristics of targeted delivery and controlled release of active components in intestinal tracts.
Test example 2 inhibition of cell proliferation
The hepatoma-preventing and treating compositions prepared in examples 1 to 5 and comparative examples 1 to 15 were added to water, and stirred at 37℃for 10 hours to prepare a 50. Mu.g/mL sample solution.
Taking mouse liver cancer H22 cells in logarithmic phase, preparing cell suspension by RPMI1640 culture solution, and adjusting cell density to 10 4 mu.L per well, 100. Mu.L per mL, was seeded in 96-well plates. The sample solution was placed in 20 groups of 5 wells each, 50. Mu.L of the sample solution was added to each well, and the mixture was placed at 37℃with 5v/v% CO 2 Culturing in a saturated humidity cell culture box for 24h, adding 5 mug/mL MTT solution, incubating in the culture box for 4h, centrifuging, sucking off the liquid in the hole, adding 150 mug DMSO, shaking for 10min, and measuring the absorbance OD value at the wavelength of 490nm by using an enzyme-labeled instrument. Each group was repeated 3 times.
Inhibition = (1-test well OD value/control well OD value) ×100%
The results are shown in Table 1.
TABLE 1
As shown in the table above, the compositions for preventing and treating liver cancer prepared in examples 1-3 of the present invention have good effect of inhibiting proliferation of liver cancer cells in vitro.
Test example 3
Collecting liver cancer H22 cell strain in test example 1, centrifuging for 3 times at 10000r/rain for 5min, and adjusting cell density to 1×10 6 0.2mL of cell (survival rate is more than or equal to 95%) suspension is injected into the right armpit of a BALB/c mouse subcutaneously, and the whole process is strictly aseptic. After 24h, the samples were randomly divided into 22 groups, namely a model group, cyclophosphamide group, examples 1-5, and comparative examples 1-15. Meanwhile, the group I is a normal group without inoculating tumor strains, 20mg/kg cyclophosphamide is administered to the cyclophosphamide group, physiological saline with the same volume is administered to the normal group and the model group in a lavage way, 1g/kg of the composition for preventing and treating liver cancer prepared from the corresponding groups is administered to each group in a lavage way, the composition is continuously administered for 14d, the weight of the mice is weighed before and after the test, the mice are anesthetized on the 15 th day, blood is taken from the orbit, serum is separated for standby, the mice are killed after marrow breaking, tumor bodies are peeled off, and the mice are weighed.
Weight gain (%) = (dosing group weight gain-model group weight gain)/model group weight gain x 100%;
tumor inhibition rate (%) = (average tumor weight of model group-average tumor weight of administration group)/average tumor weight of model group x 100%).
Serum IL-2 and TNF- α detection: the mice of each group were bled, centrifuged without anticoagulant, assayed by ELISA, and the procedure was performed as indicated by the kit.
The results are shown in Table 2.
TABLE 2
Annotation: * P < 0.05 compared with the blank group; # is P < 0.05 compared to model group.
From the above table, the composition for preventing and treating liver cancer prepared in examples 1-3 has obvious effect of inhibiting liver cancer cell growth, does not damage the constitution of mice, and has the effects of protecting and improving the damage of immune indexes.
Examples 4 and 5 compare with example 3 in which the additive was single calcium chloride or vitamin B1. Comparative example 9 in contrast to example 3, no additive was added in step S5. The inhibition rate of cell proliferation is slightly reduced, the tumor inhibition rate is slightly reduced, and the IL-2 and TNF-alpha contents are reduced. According to the invention, calcium chloride and vitamin B2 are supplemented in the fermentation process of probiotics, and the resistance of the probiotics is increased and the time period of the probiotics in the stationary phase is improved through the adjustment of trace elements, so that the probiotics are promoted to produce more beneficial substances.
Comparative examples 1, 2 and 3 in comparison with example 3, no radix Salviae Miltiorrhizae, pseudobulbus Cremastrae seu pleiones or Cordyceps militaris was added in step S1. The inhibition rate of cell proliferation is reduced, the tumor inhibition rate is reduced, the weight growth rate is reduced, and the IL-2 and TNF-alpha contents are reduced. The invention extracts the red sage root, the Indian iphigenia bulb and the Cordyceps militaris by water, and the obtained active components comprise active substances such as salvianolic acid B, cordycepin, alkaloid, saponin, flavonoids, cordyceps polysaccharide, red sage root polysaccharide, indian iphigenia bulb polysaccharide and the like, wherein the salvianolic acid B is polymerized by 3 molecules of salvianic acid A and 1 molecule of caffeic acid, is one component with highest content and strongest activity in the water-soluble active ingredient of the red sage root, and has the effect of inhibiting the growth of cancer cells in vitro. Salvianolic acid B can relieve acute liver injury by inhibiting TLR4/MyD88/NF- κB signaling, and TLR4/MyD88/NF- κB signaling is a key pathway widely existing in various cells and involved in inflammatory reaction, and is an important regulatory mechanism for mediating tumorigenesis. Salvianolic acid B can influence the composition of intestinal flora, increase the relative abundance of beneficial intestinal bacteria, reduce the relative abundance of harmful bacteria, and improve intestinal environment. The Pseudobulbus Cremastrae seu pleiones polysaccharide is a main bioactive component of Pseudobulbus Cremastrae seu pleiones, and can improve immunity, inhibit angiogenesis and promote apoptosis of tumor cells by inhibiting activation of P13K/AKT/mTOR. Cordycepin is a natural nucleoside antibiotic, is a natural anticancer antibiotic, namely 3-deadenosine, is a nucleic acid derivative of nitrogen-containing glycoside, belongs to purine alkaloids, has antiviral and antitumor effects, and particularly has strong inhibition effect on various solid malignant tumors.
Comparative example 4 in comparison with example 3, steps S2 and S3 were not performed, and the organic selenium-active polysaccharide complex in step S7 was replaced by the active polysaccharide prepared in step Sl. The inhibition rate of cell proliferation is reduced, the tumor inhibition rate is reduced, the weight growth rate is reduced, and the IL-2 and TNF-alpha contents are reduced. According to the invention, after selenium-enriched saccharomycetes are adopted for fermentation, thalli are removed through centrifugation, a Sevag method is adopted for removing protein to obtain selenium-enriched saccharomycetes extracellular polysaccharide liquid, and the selenium-enriched saccharomycetes extracellular polysaccharide liquid is mixed with active polysaccharide obtained through extraction of red sage root, indian iphigenia bulb and cordyceps militaris, and can form an organic selenium-active polysaccharide compound through chelation.
Comparative example 5 in contrast to example 3, no Saccharomyces cerevisiae was inoculated in step S5, and step S6 was not performed. The inhibition rate of cell proliferation is reduced, the tumor inhibition rate is reduced, the weight growth rate is reduced, and the IL-2 and TNF-alpha contents are reduced. The saccharomyces cerevisiae in the fermentation bacteria can promote the growth of bifidobacteria (probiotics) in intestinal flora, and inhibit the number of candida albicans (pathogenic bacteria); the intestinal mucosa epithelial cells can be reduced to protect the mucosa barrier function, and simultaneously, the release of inflammatory mediators such as TNF-alpha, IL-6 and the like can be inhibited to prevent the further development of liver cirrhosis, and the saccharomyces cerevisiae can accumulate more glutathione in cells, so that the amount of the glutathione in the composition is greatly increased.
Comparative examples 6 and 7 in comparison with example 3, lactobacillus salivarius or bacillus subtilis was not inoculated in step S5. Comparative example 8 in comparison with example 3, lactobacillus salivarius and bacillus subtilis seed solution were not inoculated in step S5. Comparative example 10 compared to example 3, steps S4, S5 and S6 were not performed. The inhibition rate of cell proliferation is reduced, the tumor inhibition rate is reduced, the weight growth rate is reduced, and the IL-2 and TNF-alpha contents are reduced. The lactobacillus salivarius added in the invention has the effects of improving liver function, protecting intestinal tract barrier and relieving liver fibrosis, can prevent or treat liver cirrhosis and liver failure, and the metabolite of the lactobacillus salivarius can protect liver function, plays a positive role in preventing liver cancer, such as indopropionic acid, which is a product of tryptophan decomposed by bacteria, can inhibit NF- κB signal path, reduce the level of proinflammatory cytokines, antagonize liver inflammation and liver injury, and can further enhance the liver's ability of clearing hepatitis virus and lighten liver inflammation by improving the inherent immunity of liver and intestinal tract, and meanwhile strengthen the barrier effect of intestinal tract, thereby playing the role of preventing cancer. The bacillus subtilis can degrade aflatoxin, prevent liver cancer by reducing the toxicity of cancerogenic substances, effectively improve intestinal flora imbalance, recover the normal immune response of an organism to HBV, reduce the release of inflammatory mediators, reduce liver cell damage, further improve viral hepatitis symptoms, and simultaneously can effectively inhibit the Th17 cell level in tumors by stimulating the Proteus to secrete anti-inflammatory substances, wherein Th17 cells are an auxiliary T cell subgroup capable of secreting IL-17 characteristically, inhibit tumor immunity and stimulate tumor angiogenesis, participate in the generation and development of tumors, play an anticancer role by inhibiting the Th17 cell level, and have a synergistic effect.
In comparative example 11, in contrast to example 3, no cysteine was added in step S6. The tumor inhibition rate is reduced, the weight gain rate is reduced, and the IL-2 and TNF-alpha contents are reduced. Cysteine is an important synthetic raw material of glutathione, and the cysteine is added to promote the fermentation product glutathione of saccharomyces cerevisiae, so that the method plays a good role in protecting liver and plays a good role in preventing and treating liver cancer.
Comparative example 12, in contrast to example 3, no embedding was performed in step S7, and the components were simply mixed. The inhibition rate of cell proliferation is reduced, the tumor inhibition rate is reduced, the weight growth rate is reduced, and the IL-2 and TNF-alpha contents are reduced. The resistance of probiotics to gastric acid can be improved through embedding, the polysaccharide active component is prevented from being oxidized and decomposed, and the method has the characteristics of targeted delivery and controlled release of the active component in intestinal tracts, so that the activity of the active component is greatly improved.
In comparative examples 13 and 14, curcumin or fructo-oligosaccharide was not added in step S7, as compared with example 3. Comparative example 15 in contrast to example 3, no curcumin and fructooligosaccharides were added in step S7. The inhibition rate of cell proliferation is reduced, and the tumor inhibition rate is reduced. Curcumin also added in the invention can reduce liver injury by increasing glutathione content in liver. Meanwhile, curcumin and fructo-oligosaccharide are good prebiotics, can directionally promote the proliferation of intestinal probiotics, thereby inhibiting the growth of harmful bacteria, regulating liver function through liver-intestinal axis, promoting the recovery of liver function, inhibiting the formation of fatty liver and protecting liver.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A preparation method of a composition for preventing and treating liver cancer is characterized by extracting Saviae Miltiorrhizae radix, pseudobulbus Cremastrae seu pleiones and Cordyceps militaris with water, filtering, retaining residue, precipitating with ethanol to obtain active polysaccharide, and drying the filtrate to obtain water extract; obtaining selenium-enriched yeast extracellular polysaccharide and mixing the selenium-enriched yeast extracellular polysaccharide with active polysaccharide to prepare an organic selenium-active polysaccharide compound; adding the filter residue into water, inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis, fermenting, adding cysteine, fermenting again, lyophilizing to obtain fermentation product, and embedding water extract, organic selenium-active polysaccharide complex, fermentation product, curcumin and fructo-oligosaccharide.
2. The method of manufacturing according to claim 1, comprising the steps of:
s1, water extraction: cleaning Saviae Miltiorrhizae radix, bulbus Iphigeniae Indicae, and Cordyceps militaris respectively, drying, pulverizing, adding into water, heating and boiling for extraction, repeating operation for 2-3 times, filtering, collecting residue, mixing filtrates, concentrating, precipitating with ethanol, centrifuging to collect solid, and drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
S2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting, culturing, centrifuging, adding a mixed solvent into supernatant, precipitating, centrifuging, adding ethanol into supernatant for precipitating, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
s3, preparing an organic selenium-active polysaccharide compound: adding the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into water, adding the active polysaccharide prepared in the step S1, stirring and mixing uniformly, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: respectively inoculating Saccharomyces cerevisiae, lactobacillus salivarius and Bacillus subtilis into Gao's medium, and performing active culture to obtain strain seed solution;
s5, fermenting: adding the filter residues obtained in the step S1 into water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, fermenting for a first time period, adding an additive, and fermenting for a second time period to obtain a fermentation mixture;
s6, promoting glutathione synthesis and fermentation: adding cysteine into the fermentation mixture in the step S5, continuing to ferment and culture for a third time period, and freeze-drying to obtain a fermentation product;
s7, embedding: adding the water extract prepared in the step S1, the organic selenium-active polysaccharide compound prepared in the step S3, the fermentation product prepared in the step S6, curcumin, fructo-oligosaccharide, sodium alginate and sodium carboxymethyl cellulose into water, and uniformly mixing to prepare a water phase; adding water phase and lecithin into fish oil, emulsifying, dripping calcium chloride solution, solidifying at normal temperature, centrifuging, washing, and lyophilizing to obtain composition for preventing and treating liver cancer.
3. The preparation method according to claim 2, wherein in the step S1, the mass ratio of the red sage root, the edible tulip and the cordyceps militaris is 3-5:1-3:5-7, the solid-to-liquid ratio of the mixed powder and the water is 1:5-7 g/mL, the heating boiling extraction time is 1-2h, the ethanol is added until the system ethanol content is 60-70wt%, and the precipitation time is 3-5h.
4. The method according to claim 2, wherein the formula of the medium in step S2 is: 20-40 parts of glucose, 3-5 parts of peptone and KH (KH) 2 PO 4 2-3 parts by weight of MgSO 4 ·7H 2 O1-2 parts by weight, vitamin B1.01-0.02 part by weight and water 1000 parts by weight, wherein the inoculation amount of selenium-enriched saccharomycetes is 2-3w/v%, the condition of fermentation culture is 50-55 ℃,50-70r/min, the time is 24-36h, the mixed solvent is a mixed solvent of chloroform and n-butanol, the volume ratio is 4:1, the volume ratio of supernatant and the mixed solvent is 3-5:1, the time of precipitation is 1-2h, the supernatant is added with ethanol until the system ethanol content is 60-70wt%, and the time of precipitation is 3-5h; and in the step S3, the mass ratio of the selenium-enriched yeast extracellular polysaccharide to the active polysaccharide is 5-7:3-5.
5. The method according to claim 2, wherein the conditions for the activation culture in step S4 are 40-42℃and 50-70r/min, and the culture is carried out for 18-24 hours, and the seed solution of the strain has a bacterial content of 10 8 -10 9 cfu/mL; in the step S5, the mass ratio of filter residues to water is 2-5:12-15, the inoculation amounts of saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are 2-3v/v%, 1-2v/v% and 2-3v/v%, the fermentation condition is 37-40 ℃,50-70r/min, the first time period is 24-36h, the second time period is 12-18h, the additive is a mixture of calcium chloride and vitamin B1, the mass ratio is 3-5:1-2, and the addition amount of the additive is 3-5wt% of the total mass of the system.
6. The method according to claim 2, wherein the mass ratio of the fermentation mixture to cysteine in step S6 is 100-120:5-7, the conditions of the fermentation culture are 37-40 ℃,50-70r/min, and the third period of time is 18-24 hours.
7. The preparation method according to claim 2, wherein the mass ratio of the water extract, the organic selenium-active polysaccharide complex, the fermentation product, the curcumin, the fructo-oligosaccharide, the sodium alginate, the sodium carboxymethyl cellulose and the water in the step S7 is 3-5:7-10:12-15:1-2:0.5-1:20-25:7-10:200-250, the mass ratio of the water phase, the lecithin and the fish oil is 100-120:2-3:200-250, the concentration of the calcium chloride solution is 3-5wt%, and the curing time at the normal temperature is 30-40min.
8. The preparation method according to claim 2, characterized by comprising the following steps:
s1, water extraction: cleaning 3-5 parts by weight of red sage root, 1-3 parts by weight of Indian iphigenia bulb and 5-7 parts by weight of Cordyceps militaris respectively, drying, crushing to obtain mixed powder, adding the mixed powder and water with the solid-to-liquid ratio of 1:5-7 g/mL, heating, boiling and extracting for 1-2h, repeating the operation for 2-3 times, filtering, reserving filter residues, mixing filtrates, concentrating, adding ethanol until the ethanol content of the system is 60-70wt%, precipitating for 3-5h, centrifuging, collecting solids, drying to obtain active polysaccharide; recovering ethanol from the filtrate, and drying to obtain water extract;
s2, preparing selenium-enriched yeast extracellular polysaccharide: inoculating selenium-enriched saccharomycetes into a culture medium, fermenting and culturing for 24-36h at the inoculum size of 2-3w/v%, at 50-55 ℃ and at 50-70r/min, centrifuging, adding a mixed solvent into supernatant, wherein the volume ratio of the supernatant to the mixed solvent is 3-5:1, precipitating for 1-2h, centrifuging, adding ethanol into the supernatant until the ethanol content of the system is 60-70wt%, precipitating for 3-5h, and drying to obtain selenium-enriched saccharomycetes extracellular polysaccharide;
the formula of the culture medium is as follows: 20-40 parts of glucose, 3-5 parts of peptone and KH (KH) 2 PO 4 2-3 parts by weight of MgSO 4 ·7H 2 O1-2 weight portions, vitamin B1 0.01-0.02 weight portions and water 1000 weight portions;
the mixed solvent is a mixed solvent of chloroform and n-butanol, and the volume ratio is 4:1;
s3, preparing an organic selenium-active polysaccharide compound: adding 5-7 parts by weight of the selenium-enriched yeast extracellular polysaccharide prepared in the step S2 into 100 parts by weight of water, adding 3-5 parts by weight of the active polysaccharide prepared in the step S1, uniformly stirring and mixing, and drying to prepare an organic selenium-active polysaccharide compound;
s4, activating strains: inoculating Saccharomyces cerevisiae, lactobacillus salivarius, and Bacillus subtilis into Gao's culture medium, respectively, and performing active culture at 40-42deg.C and 50-70r/min for 18-24 hr to obtain strain seed solution with a bacterial content of 10 8 -10 9 cfu/mL;
S5, fermenting: adding 20-50 parts by weight of filter residues obtained in the step S1 into 120-150 parts by weight of water, sterilizing, inoculating the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid obtained in the step S4, wherein the inoculum sizes of the saccharomyces cerevisiae, lactobacillus salivarius and bacillus subtilis strain seed liquid are respectively 2-3v/v%, 1-2v/v%, 2-3v/v%,37-40 ℃,50-70r/min, fermenting for 24-36h, and adding additives, wherein the additive amount is 3-5wt% of the total mass of the system, and fermenting for 12-18h to obtain a fermentation mixture;
The additive is a mixture of calcium chloride and vitamin B1, and the mass ratio is 3-5:1-2;
s6, promoting glutathione synthesis and fermentation: adding 5-7 parts by weight of cysteine into 100-120 parts by weight of the fermentation mixture obtained in the step S5, fermenting and culturing at 37-40 ℃ for 18-24 hours at 50-70r/min, and freeze-drying to obtain a fermentation product;
s7, embedding: adding 3-5 parts by weight of the water extract prepared in the step S1, 7-10 parts by weight of the organic selenium-active polysaccharide compound prepared in the step S3, 12-15 parts by weight of the fermentation product prepared in the step S6, 1-2 parts by weight of curcumin, 0.5-1 part by weight of fructo-oligosaccharide, 20-25 parts by weight of sodium alginate and 7-10 parts by weight of sodium carboxymethyl cellulose into 200-250 parts by weight of water, and uniformly mixing to prepare an aqueous phase; adding 100-120 parts by weight of water phase and 2-3 parts by weight of lecithin into 200-250 parts by weight of fish oil, emulsifying, dripping 50 parts by weight of 3-5wt% calcium chloride solution, solidifying at normal temperature for 30-40min, centrifuging, washing, and freeze-drying to obtain the composition for preventing and treating liver cancer.
9. A composition for preventing and treating liver cancer prepared by the preparation method of any one of claims 1 to 8.
10. Use of the composition for preventing and treating liver cancer according to claim 9 for preparing products for preventing and treating liver cancer, liver failure and hepatitis.
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