Composition for relieving gastrointestinal reaction and enhancing appetite after chemotherapy in cancer patient

Abstract

The invention discloses a composition for relieving gastrointestinal reaction and enhancing appetite of cancer patients after chemotherapy, and relates to a composition for relieving gastrointestinal reaction and enhancing appetite. The invention aims to solve the problem of strong gastrointestinal reaction of cancer patients after chemotherapy. The composition for relieving gastrointestinal reaction and enhancing appetite after chemotherapy of cancer patients is prepared from ganoderma capense and coriolus versicolor polysaccharide fermentation products, phellinus igniarius polyphenol extracts and phellinus pini and ginseng flower water extracts according to the mass ratio of 1: 1: 1. The composition disclosed by the invention can be used for improving the anti-cancer effect in a mouse body. The traditional Chinese medicine composition not only can improve the immune function of the body and improve appetite, but also can change the immune microenvironment of the body and enhance the immune function of the body. The invention belongs to the technical field of relieving gastrointestinal reactions of cancer patients after chemotherapy.

Description

Technical Field

[0001] The invention relates to a composition for alleviating gastrointestinal reaction and enhancing appetite. Background Art

[0002] Anorexia and gastrointestinal reactions, such as bloating, abdominal distension, and nausea and vomiting, are common adverse reactions in malignant tumors and related anti-tumor treatments, which seriously reduce the quality of life of patients. Long-term anorexia, bloating, abdominal distension, and nausea and vomiting can cause patients to experience various physiological or psychological problems such as fatigue, anemia, hypoproteinemia, water and electrolyte disorders, fear, anxiety, and depression, which increase the physical and mental burden, affect the overall treatment, and thus affect the prognosis. Traditional Chinese medicine often classifies them into categories such as "poor appetite", "fullness", and "vomiting" based on symptoms, and believes that the tumor itself or various anti-tumor treatments will disrupt the balance of yin and yang in the human body and the rise and fall of qi. The spleen and stomach are the hubs of the middle jiao, the yangming stomach descends below, and the taiyin spleen rises above, so they can normally transport and transform water and grain; if the yin and yang of the spleen and stomach are unbalanced, the rise and fall of qi is disordered, the taiyin spleen does not rise, the yangming stomach does not descend, and the water and grain cannot be transformed, then anorexia, bloating, abdominal distension, and nausea and vomiting will occur. Active substances released by the tumor tissue itself, such as cytokines IL-1α, IL-6, TNF-α and GDF15, act on specific hypothalamic receptors by regulating the neurotransmitter cascade of the central nervous system, promote satiety through downstream neuropeptides, and cause loss of appetite or loss of appetite. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problem of strong gastrointestinal reaction in cancer patients after chemotherapy, and to provide a composition for alleviating gastrointestinal reaction and enhancing appetite in cancer patients after chemotherapy.

[0004] The composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy is composed of fermented polysaccharides of Ganoderma lucidum and Coriolus versicolor, mulberry linterus polyphenol extract, and pine needle porphyra and ginseng flower water extract in a mass ratio of 1:1:1;

[0005] The extraction method of the polysaccharide fermentation product of Ganoderma lucidum and Ganoderma versicolor:

[0006] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0007] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add complex enzyme, the amount of complex enzyme added is 0.5% to 1% of the total mass of the mixture, treat at 37 to 45°C for 2 to 4 hours, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0008] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5 to 1:2:1, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0009] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration to 30%, and after standing at room temperature for 36 hours, centrifuged, and the precipitate is collected to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration to 80%, and after standing at room temperature for 48 hours, centrifuged, and the precipitate is collected to obtain precipitate 2. After combining precipitate 1 and precipitate 2, freeze-drying is performed to obtain the fermentation product of Ganoderma lucidum and Ganoderma versicolor polysaccharide;

[0010] Preparation of DES: Choline chloride and oligofructose were mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which was cooled to room temperature to obtain DES.

[0011] Extraction method of mulberry linterus polyphenol extract:

[0012] According to the mass ratio of Phellinus lindens to DES of 1:25, the crushed Phellinus lindens fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus lindens polyphenol extract.

[0013] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0014] 10.0 g of pine needle pore fungus fruiting body and 1 g of ginseng flower are weighed, placed in an oven for drying at 50° C., crushed with a multifunctional Chinese medicine grinder, the crushed mixed powder is mixed with water in a ratio of 2:8 to form a suspension, sterilized at 121° C. by high pressure for 15 min, cooled to room temperature, and sterilized by ultraviolet in a biological safety cabinet for 30 min, and the composite fermentation liquid of the two bacteria is inoculated into the suspension at an inoculum amount of 5%, and cultured in a constant temperature shaker for 5 days; reflux extraction is performed twice with water in a ratio of 1 g:8 L of solid-liquid ratio, each time for 2 hours, the extracts are combined, and finally freeze-dried to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder.

[0015] The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1;

[0016] The preparation method of the Lactobacillus rhamnosus fermentation liquid:

[0017] The ampoule containing Lactobacillus rhamnosus CICC 20990 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule and shaken to mix. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum size of 5%. After anaerobically culturing at 37° C. for 18 h, the bacterial suspension was inoculated into MRS solid culture medium in three zones to purify the strain. The culture was anaerobically culturing at 37° C. for 18 h, and a single colony was picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid.

[0018] The preparation method of the Bifidobacterium breve fermentation broth:

[0019] The ampoule containing Bifidobacterium breve CICC 6182 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium was taken and placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%. After anaerobic culture at 37° C. for 18 h, three-zone lines of the bacterial suspension were inoculated into the brain heart infusion liquid culture medium to purify the strain, and the culture was anaerobically cultured at 37° C. for 18 h. A single colony was picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bifidobacterium breve fermentation liquid.

[0020] Phellinus pini, also known as pine white rot fungus, belongs to the phylum Basidiomycetes, class Agaricaceae, order Pucciniformis, family Pucciniformis, genus Phellinus. Also known as pine wood phylum fungus, pine white rot fungus, yellow ganoderma, pine needle ganoderma, red edge tree tongue, etc. It is distributed in Northeast China, Hebei, Shanxi, Gansu, Sichuan and other places. It often grows on the trunks or rotten wood of coniferous trees. Studies have shown that it has pharmacological effects such as anti-tumor, immune regulation, and hypoglycemic. Pine needle phylum fungus has a long history of medicinal use in my country. "Shennong's Herbal Classic" once recorded: Yellow ganoderma, sweet and flat, mainly treats five evils of the heart and abdomen, benefits the spleen, calms the mind, is loyal and harmonious, and long-term consumption makes the body light and prolongs life.

[0021] The composition of the present invention can improve the anti-cancer effect in mice, not only improve the immune function of the body and increase appetite, but also change the immune microenvironment of the body and enhance the immune function of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the box plot of mouse body weight in Experiment 1;

[0023] Figure 2 is a box plot of tumor weights of mice in Experiment 1;

[0024] Figure 3This is the result of the serum inflammatory factor IL-6 test in Experiment 1;

[0025] Figure 4 This is the result of the serum inflammatory factor IL-10 test in Experiment 1;

[0026] Figure 5 It is the result of the test of serum inflammatory factor TNF-α in experiment 1;

[0027] Figure 6 This is the result of the serum inflammatory factor IFN-γ test in Experiment 1;

[0028] Figure 7 This is a graph showing the response of different doses to cell viability in Experiment 1. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0030] Specific implementation method 1: The composition for reducing gastrointestinal reactions and enhancing appetite of cancer patients after chemotherapy in this implementation method is composed of fermented polysaccharides of Ganoderma lucidum and Coriolus versicolor, mulberry linterus polyphenol extract, and pine needle pore mushroom ginseng flower water extract in a mass ratio of 1:1:1;

[0031] The extraction method of the polysaccharide fermentation product of Ganoderma lucidum and Ganoderma versicolor:

[0032] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0033] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add complex enzyme, the amount of complex enzyme added is 0.5% to 1% of the total mass of the mixture, treat at 37 to 45°C for 2 to 4 hours, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0034] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5 to 1:2:1, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0035] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration to 30%, and after standing at room temperature for 36 hours, centrifuged, and the precipitate is collected to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration to 80%, and after standing at room temperature for 48 hours, centrifuged, and the precipitate is collected to obtain precipitate 2. After combining precipitate 1 and precipitate 2, freeze-drying is performed to obtain the fermentation product of Ganoderma lucidum and Ganoderma versicolor polysaccharide;

[0036] Preparation of DES: Choline chloride and oligofructose were mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which was cooled to room temperature to obtain DES.

[0037] Extraction method of mulberry linterus polyphenol extract:

[0038] According to the mass ratio of Phellinus lindens to DES of 1:25, the crushed Phellinus lindens fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus lindens polyphenol extract.

[0039] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0040] 10.0 g of pine needle pore fungus fruiting body and 1 g of ginseng flower are weighed, placed in an oven for drying at 50° C., crushed with a multifunctional Chinese medicine grinder, the crushed mixed powder is mixed with water in a ratio of 2:8 to form a suspension, sterilized at 121° C. by high pressure for 15 min, cooled to room temperature, and sterilized by ultraviolet in a biological safety cabinet for 30 min, and the composite fermentation liquid of the two bacteria is inoculated into the suspension at an inoculum amount of 5%, and cultured in a constant temperature shaker for 5 days; reflux extraction is performed twice with water in a ratio of 1 g:8 L of solid-liquid ratio, each time for 2 hours, the extracts are combined, and finally freeze-dried to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder.

[0041] The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1;

[0042] The preparation method of the Lactobacillus rhamnosus fermentation liquid:

[0043] The ampoule containing Lactobacillus rhamnosus CICC 20990 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule and shaken to mix. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum size of 5%. After anaerobically culturing at 37° C. for 18 h, the bacterial suspension was inoculated into MRS solid culture medium in three zones to purify the strain. The culture was anaerobically culturing at 37° C. for 18 h, and a single colony was picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid.

[0044] The preparation method of the Bifidobacterium breve fermentation broth:

[0045] The ampoule containing Bifidobacterium breve CICC 6182 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium was taken and placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%. After anaerobic culture at 37° C. for 18 h, three-zone lines of the bacterial suspension were inoculated into the brain heart infusion liquid culture medium to purify the strain, and the culture was anaerobically cultured at 37° C. for 18 h. A single colony was picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bifidobacterium breve fermentation liquid.

[0046] Specific embodiment 2: This embodiment is different from the specific embodiment 1 in that the extraction method of the polysaccharide fermentation product of Ganoderma lucidum and Ganoderma versicolor is as follows:

[0047] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0048] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add complex enzyme, the amount of complex enzyme added is 0.5% to 1% of the total mass of the mixture, treat at 37 to 45°C for 2 to 4 hours, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0049] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5 to 1:2:1, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0050] 3. Combine the treated liquid 1 and the supernatant 2, and concentrate the combined filtrate under reduced pressure to 30% to 50% of the original mass, then use graded alcohol precipitation, add ethanol to make the ethanol volume concentration 30%, stand at room temperature for 36 hours, centrifuge, collect the precipitate to obtain precipitate 1, collect the supernatant and concentrate it in vacuum at 60°C, add ethanol to make the ethanol volume concentration 80%, stand at room temperature for 48 hours, centrifuge, collect the precipitate to obtain precipitate 2, combine precipitate 1 and precipitate 2, and freeze-dry to obtain the fermentation product of Ganoderma lucidum and Ganoderma versicolor polysaccharide. The rest is the same as the first embodiment.

[0051] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that: 1. Preparation of DES: Choline chloride and oligofructose are mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which is cooled to room temperature to obtain DES;

[0052] 2. According to the mass ratio of Phellinus linteus to DES of 1:25, the crushed Phellinus linteus fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus linteus polyphenol extract. The rest is the same as the specific implementation method one or two.

[0053] Specific embodiment 4: This embodiment is different from the specific embodiments 1 to 3 in that the extraction method of the pine needle phyllostachys ginseng flower water extract is:

[0054] Weigh 10.0g of pine needle pore fungus fruiting body and 1g of ginseng flower, place in an oven to dry at 50°C, crush with a multifunctional Chinese medicine grinder, mix the crushed mixed powder with water in a ratio of 2:8 to form a suspension, sterilize at 121°C under high pressure for 15min, cool to room temperature, sterilize under ultraviolet in a biological safety cabinet for 30min, inoculate the two bacteria composite fermentation liquid into the suspension at an inoculum amount of 5%, and culture in a constant temperature shaker for 5d; reflux extract twice with water in a ratio of 1g:8L to solid-liquid, each time for 2h, combine the extracts, and finally freeze-dry to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder;

[0055] The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1;

[0056] The preparation method of the Lactobacillus rhamnosus fermentation liquid:

[0057] The ampoule containing Lactobacillus rhamnosus CICC 20990 was placed in a biological safety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum of 5%, and after anaerobic culture at 37° C. for 18 h, the bacterial suspension was inoculated into MRS solid culture medium by three-zone lines to purify the strain, and anaerobic culture was carried out at 37° C. for 18 h. A single colony was picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid.

[0058] The preparation method of the Bifidobacterium breve fermentation broth:

[0059] The ampoule containing Bifidobacterium breve CICC 6182 is placed in a biological safety cabinet, disinfected with an alcohol cotton ball, 1 ml of brain heart infusion liquid culture medium is placed in the ampoule, shaken and mixed, the bacterial suspension is inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%, and after anaerobic culture at 37°C for 18 hours, the bacterial suspension is inoculated into the brain heart infusion liquid culture medium in three zones to purify the strain, and anaerobic culture is carried out at 37°C for 18 hours, and a single colony is picked and cultured in a constant temperature shaker at 180r / min at 37°C for 24 hours to obtain Bifidobacterium breve fermentation liquid. The rest is the same as one of the specific embodiments one to three.

[0060] Specific embodiment 5: This embodiment is different from specific embodiments 1 to 4 in that the amount of the complex enzyme added in step 2 is 0.5% of the total mass of the mixture, and the mixture is treated at 37° C. for 2 h. The rest is the same as specific embodiments 1 to 4.

[0061] Specific embodiment 6: This embodiment is different from specific embodiments 1 to 5 in that in step 3, the treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 40% of the original mass. The rest is the same as specific embodiments 1 to 5.

[0062] Specific implementation method 7: This implementation method is different from specific implementation methods 1 to 6 in that the microwave power is 300 W. The rest is the same as specific implementation methods 1 to 6.

[0063] The following examples are used to verify the effects of the present invention:

[0064] Embodiment 1:

[0065] Extraction method of polysaccharide fermentation products of Ganoderma lucidum and Coriolus versicolor:

[0066] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0067] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add the compound enzyme, the amount of the compound enzyme added is 0.5% of the total mass of the mixture, treat at 37°C for 2h, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0068] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0069] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration of 30%, and the mixture is allowed to stand at room temperature for 36 hours, and then centrifuged to collect the precipitate to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration of 80%. The mixture is allowed to stand at room temperature for 48 hours, and then centrifuged to collect the precipitate to obtain precipitate 2. After combining precipitate 1 and precipitate 2, lyophilization is performed to obtain the fermentation products of Ganoderma lucidum and Ganoderma versicolor polysaccharides.

[0070] Embodiment 2:

[0071] Extraction method of polysaccharide fermentation products of Ganoderma lucidum and Coriolus versicolor:

[0072] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Coriolus versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated solution, extract the pre-treated solution by ultrasonic at 6000 Hz for 30 minutes, centrifuge the extracted turbid solution at 8000 rpm for 5 minutes, take the supernatant and obtain the treated solution 1;

[0073] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add the compound enzyme, the amount of the compound enzyme added is 0.5% of the total mass of the mixture, treat at 37°C for 2h, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0074] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0075] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration of 30%, and the mixture is allowed to stand at room temperature for 36 hours, and then centrifuged to collect the precipitate to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration of 80%. The mixture is allowed to stand at room temperature for 48 hours, and then centrifuged to collect the precipitate to obtain precipitate 2. After combining precipitate 1 and precipitate 2, lyophilization is performed to obtain the fermentation products of Ganoderma lucidum and Ganoderma versicolor polysaccharides.

[0076] Example 3

[0077] Extraction method of polysaccharide fermentation products of Ganoderma lucidum and Coriolus versicolor:

[0078] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0079] 2. Add 10 times the amount of water to the residue obtained in step 1, extract with steam reflux for 1 hour, then centrifuge and filter to obtain supernatant 2;

[0080] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration of 30%, and the mixture is allowed to stand at room temperature for 36 hours, and then centrifuged to collect the precipitate to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration of 80%. The mixture is allowed to stand at room temperature for 48 hours, and then centrifuged to collect the precipitate to obtain precipitate 2. After combining precipitate 1 and precipitate 2, lyophilization is performed to obtain the fermentation products of Ganoderma lucidum and Ganoderma versicolor polysaccharides.

[0081] Embodiment 4:

[0082] Extraction method of polysaccharide fermentation products of Ganoderma lucidum and Coriolus versicolor:

[0083] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0084] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add the compound enzyme, the amount of the compound enzyme added is 0.5% of the total mass of the mixture, treat at 37°C for 2h, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0085] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0086] 3. Combine the treated liquid 1 and the supernatant 2, and concentrate the combined filtrate under reduced pressure to 30% to 50% of the original mass. Then add ethanol to make the ethanol volume concentration 80%. After standing at room temperature for 36 hours, centrifuge and collect the precipitate to obtain precipitate 2. After combining precipitate 1 and precipitate 2, freeze-dry them to obtain the fermentation products of Ganoderma lucidum and Ganoderma versicolor polysaccharides.

[0087] Comparative Example 1:

[0088] Extraction method of polysaccharide fermentation products of Ganoderma lucidum and Coriolus versicolor:

[0089] After mixing the powder of Ganoderma lucidum and crushed fruiting bodies of Coriolus versicolor in a mass ratio of 1:1, add 20 times the amount of water for immersion for 8 hours to obtain a pre-treated solution, and then use the water vapor reflux method, reflux for 2 hours each time, and reflux twice. Obtain the extract, centrifuge at 8000rpm for 5 minutes. Take the supernatant to obtain the treated solution. Use ethanol precipitation, add ethanol to make the ethanol volume concentration of 80%, stand at room temperature for 36 hours, centrifuge, collect the precipitate to obtain the precipitate, and freeze-dry to obtain the fermentation product of Ganoderma lucidum and Coriolus versicolor polysaccharide.

[0090] Determination of polysaccharide content:

[0091] Polysaccharide determination method:

[0092] Preparation of standard curve: Use 2 mL of glucose solution of different concentrations as the standard, add 50 μL of phenol with a mass concentration of 80% and 3 mL of concentrated sulfuric acid respectively, mix well, cool to room temperature, and measure the absorbance at 490 nm.

[0093] Sample determination: Weigh 10 mg of sample, add 1 mL of 72% H2SO4, stir at room temperature for 30 min to completely dissolve the sample, dilute to 0.2 mg / mL, take 2 mL of diluent, add 50 μL of 80% phenol solution and 3 mL of concentrated sulfuric acid, mix well, cool to room temperature and measure the absorbance, and calculate the total sugar purity in the sample according to the following formula:

[0094] Total sugar purity (%) = C × V / m × 100

[0095] Where: C is the total sugar concentration (mg / mL) obtained from the standard curve; V is the volume of the sample during measurement (mL); m is the mass of the sample (mg).

[0096] The calculation formula of polysaccharide yield is as follows: Polysaccharide yield (%) = m1×C1 / m×100

[0097] Table 1 Polysaccharide yield

[0098] Example Polysaccharide yield (%) Example 1 26.4 Example 2 16.6 Example 3 18.4 Example 4 19.4 Comparative Example 1 9.3

[0099] It can be seen from the polysaccharide yield that the polysaccharide yield obtained in Example 1 is higher than that in other groups.

[0100] Embodiment 5:

[0101] Deep eutectic solvents (DES) are fluid systems composed of eutectic mixtures of several natural ingredients, which have attracted wide attention due to their many advantages (easily synthesized, inexpensive, and environmentally friendly). In recent years, DES have been widely used to extract natural products from plants.

[0102] Preparation of DES: In this embodiment, the deep eutectic solvent is made of choline chloride (ChCl) and oligofructose. Choline chloride (ChCl) and oligofructose are mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which is cooled to room temperature to obtain DES.

[0103] Extraction method of mulberry linterus polyphenol extract:

[0104] According to the mass ratio of Phellinus lindens to DES of 1:25, the crushed Phellinus lindens fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus lindens polyphenol extract.

[0105] Embodiment 6:

[0106] Preparation of DES: In this embodiment, the deep eutectic solvent is made of choline chloride (ChCl) and urea. Choline chloride (ChCl) and urea are mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3-4 hours to obtain a colorless transparent liquid, which is cooled to room temperature to obtain DES.

[0107] Extraction method of mulberry linterus polyphenol extract:

[0108] According to the mass ratio of Phellinus lindens to DES of 1:25, the crushed Phellinus lindens fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus lindens polyphenol extract.

[0109] Embodiment 7:

[0110] Preparation of DES: In this embodiment, the deep eutectic solvent is made of choline chloride (ChCl) and urea. Choline chloride (ChCl) and urea are mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which is cooled to room temperature to obtain DES.

[0111] Extraction method of mulberry linterus polyphenol extract:

[0112] According to the mass ratio of Phellinus linteus to DES of 1:25, the crushed Phellinus linteus fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus linteus polyphenol extract.

[0113] Embodiment 8:

[0114] Preparation of DES: In this embodiment, the deep eutectic solvent is made of choline chloride (ChCl) and citric acid. Choline chloride (ChCl) and citric acid are mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which is cooled to room temperature to obtain DES.

[0115] Extraction method of mulberry linterus polyphenol extract:

[0116] According to the mass ratio of Phellinus linteus to DES of 1:25, the crushed Phellinus linteus fruiting bodies were weighed, and then DES was added, and then placed in an ultrasonic-microwave reactor, the microwave power was set to 200-400w, the extraction temperature was 40°C, extraction, filtration, centrifugation at 6000r / min, and the supernatant was collected to obtain the Phellinus linteus polyphenol extract.

[0117] Comparative Example 2: Traditional ethanol extraction method

[0118] According to the mass ratio of Phellinus lindens to 75% ethanol by volume of 1:25, weigh the crushed Phellinus lindens fruiting bodies, add 75% ethanol by volume, place in an ultrasonic-microwave reactor, set the microwave power to 200-400w, the extraction temperature to 40°C, extract, filter, centrifuge at 6000r / min, collect the supernatant, and obtain the Phellinus lindens polyphenol extract.

[0119] Take 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of 1.0mg / mL gallic acid standard solution respectively, place in a 15mL colorimetric tube, add 1.0mL of Folin-phenol color developer and 3mL of 20% Na2CO3 solution, mix well, and react in a 50℃ water bath for 30min. Measure the absorbance at a wavelength of 765nm. Draw a standard curve of polyphenol content with gallic acid concentration as the horizontal axis and absorbance as the vertical axis.

[0120] Table 2 Content of mulberry linterus polyphenols

[0121] Example Polyphenol content (mg / ml) Example 5 26.8 Example 6 18.56 Example 7 17.56 Example 8 18.14

[0122] Example 5: Higher content of mulberry linterus polyphenols

[0123] Phellinus pini, also known as pine white rot fungus, belongs to the phylum Basidiomycetes, class Agaricaceae, order Pucciniformis, family Pucciniformis, genus Phellinus. Also known as pine wood phylum fungus, pine white rot fungus, yellow ganoderma, pine needle ganoderma, red edge tree tongue, etc. It is distributed in Northeast China, Hebei, Shanxi, Gansu, Sichuan and other places. It often grows on the trunks or rotten wood of coniferous trees. Studies have shown that it has pharmacological effects such as anti-tumor, immune regulation, and hypoglycemic. Pine needle phylum fungus has a long history of medicinal use in my country. "Shennong's Herbal Classic" once recorded: Yellow ganoderma, sweet and flat, mainly treats five evils of the heart and abdomen, benefits the spleen, calms the mind, is loyal and harmonious, and long-term consumption makes the body light and prolongs life.

[0124] Embodiment 9:

[0125] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0126] 10.0 g of pine needle pore fungus fruiting body and 1 g of ginseng flower are weighed, placed in an oven for drying at 50° C., crushed with a multifunctional Chinese medicine grinder, the crushed mixed powder is mixed with water in a ratio of 2:8 to form a suspension, sterilized at 121° C. by high pressure for 15 min, cooled to room temperature, and sterilized by ultraviolet in a biological safety cabinet for 30 min, and the composite fermentation liquid of the two bacteria is inoculated into the suspension at an inoculum amount of 5%, and cultured in a constant temperature shaker for 5 days; reflux extraction is performed twice with water in a ratio of 1 g:8 L of solid-liquid ratio, each time for 2 hours, the extracts are combined, and finally freeze-dried to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder.

[0127] The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1;

[0128] The preparation method of the Lactobacillus rhamnosus fermentation liquid:

[0129] An ampoule containing Lactobacillus rhamnosus CICC 20990 (Lactobacillus rhamnosus CICC 20990 can be purchased commercially) is placed in a biological safety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium is placed in the ampoule, and the mixture is shaken and mixed. The bacterial suspension is inoculated into the MRS liquid culture medium at an inoculum size of 5%, and after anaerobically culturing at 37° C. for 18 h, the bacterial suspension is inoculated into MRS solid culture medium in three zones to purify the strain, and anaerobically cultured at 37° C. for 18 h, and a single colony is picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid.

[0130] The preparation method of the Bifidobacterium breve fermentation broth:

[0131] An ampoule containing Bifidobacterium breve CICC 6182 (Bifidobacterium breve CICC 6182 can be purchased commercially) is placed in a biological safety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium is placed in the ampoule and shaken to mix. The bacterial suspension is inoculated into the brain heart infusion liquid culture medium at an inoculum size of 5%, and after anaerobic culture at 37° C. for 18 h, three-zone lines of the bacterial suspension are inoculated into the brain heart infusion liquid culture medium to purify the strain, and anaerobic culture is performed at 37° C. for 18 h. A single colony is picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bifidobacterium breve fermentation liquid.

[0132] Embodiment 10:

[0133] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0134] Weigh 10.0g of pine needle pore fungus fruiting body and 1g of ginseng flower. Place in an oven to dry at 50°C, crush with a multifunctional Chinese medicine grinder, mix the crushed mixed powder with water in a ratio of 2:8 to form a suspension, sterilize at 121°C for 15min, cool to room temperature, sterilize with ultraviolet light in a biosafety cabinet for 30min, inoculate Lactobacillus casei fermentation liquid in the suspension at an inoculum amount of 5%, and culture in a constant temperature shaker for 5d; extract twice with water in a ratio of 1g:10L to solid-liquid ratio, each time for 2h, combine the extracts, and finally freeze-dry to obtain freeze-dried powder of pine needle pore fungus ginseng flower water extract.

[0135] Embodiment 11:

[0136] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0137] Weigh 10.0g of the fruiting body of the pine needle layer fungus and 1g of ginseng flower. Place in an oven to dry at 50°C, crush with a multifunctional Chinese medicine grinder, mix the crushed mixed powder with water in a ratio of 2:8 to form a suspension, sterilize at 121°C for 15min, cool to room temperature, sterilize with ultraviolet light in a biological safety cabinet for 30min, inoculate the breve fermentation liquid into the suspension at an inoculum amount of 5%, and shake and culture in a constant temperature shaker for 5d; reflux extract twice with water in a ratio of 1g:10L to the solid-liquid ratio, each time for 2h, combine the extracts, and finally freeze-dry to obtain the freeze-dried powder of the pine needle layer fungus and ginseng flower water extract.

[0138] Comparative Example 3:

[0139] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0140] 10.0g of pine needle pore fungus fruiting body and 1g of ginseng flower. Add 100mL of deionized water, reflux extraction at 100℃ for 1h, extract twice, combine the two extracts, centrifuge at 6000r / min for 10min, take the supernatant to obtain the pine needle pore fungus ginseng flower water extract, concentrate and freeze-dry for later use. After freeze-drying, the pine needle pore fungus ginseng flower water extract freeze-dried powder is obtained.

[0141] Determination of total triterpenes:

[0142] Drawing of standard curve: Using oleanolic acid as standard, the total triterpene content was determined by vanillin-acetic acid-perchloric acid spectrophotometry. The absorbance was measured at a wavelength of 550 nm. The standard curve was drawn with the mass x of oleanolic acid as the horizontal coordinate and the absorbance y as the vertical coordinate. The regression equation was: Y = 6.3045x + 0.1672, r 2 =0.9998. w = af / m × 100% where w is the total triterpenoid extraction rate, %;

[0143] a——the mass of triterpenoid in the sample to be tested, mg;

[0144] f——dilution multiple;

[0145] m——sample mass, mg.

[0146] Example Total triterpenoid yield% Example 9 18.62％ Example 10 9.56％ Embodiment 11 9.41％ Comparative Example 3 4.29％

[0147] The results showed that under the same crude drug dosage of P. pine fruiting body + ginseng flower, the total triterpenoid extraction rate of fermented fungus was significantly increased compared with that of P. pine fruiting body and ginseng flower water extract (P<0.01).

[0148] The reason is speculated to be that the mixed strains promote the decomposition of cell walls during the fermentation process, thereby promoting the dissolution of total triterpenoids from the fruiting bodies of Pinus truncatula + ginseng flowers.

[0149] The following experiments were used to verify the effect of the present invention:

[0150] Experiment 1:

[0151] A composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy, comprising fermented products of Gastrodia elata and Coriolus versicolor polysaccharides, mulberry linterus polyphenol extract, and a fermented composition in a mass ratio of 1:1:1;

[0152] The extraction method of the polysaccharide fermentation product of Ganoderma lucidum and Ganoderma versicolor:

[0153] 1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1;

[0154] 2. Add 10 times the amount of water to the residue obtained in step 1, and then add the compound enzyme, the amount of the compound enzyme added is 0.5% of the total mass of the mixture, treat at 37°C for 2h, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2;

[0155] The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g;

[0156] 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated to 30% of the original mass under reduced pressure, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration to 30%, and after standing at room temperature for 36 hours, centrifuged, and the precipitate is collected to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration to 80%, and after standing at room temperature for 48 hours, centrifuged, and the precipitate is collected to obtain precipitate 2. After combining precipitate 1 and precipitate 2, freeze-drying is performed to obtain the fermentation product of Ganoderma lucidum and Ganoderma versicolor polysaccharide;

[0157] Preparation of DES: Choline chloride and oligofructose were mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3 to 4 hours to obtain a colorless transparent liquid, which was cooled to room temperature to obtain DES.

[0158] Extraction method of mulberry linterus polyphenol extract:

[0159] According to the mass ratio of Phellinus lindens to DES of 1:25, the crushed Phellinus lindens fruiting bodies were weighed, and then DES was added. The mixture was placed in an ultrasonic-microwave reactor, and the microwave power was set to 200w and the extraction temperature to 40°C. The mixture was extracted, filtered, and centrifuged at 6000r / min. The supernatant was collected to obtain the Phellinus lindens polyphenol extract.

[0160] Extraction method of pine needle phyllostachys ginseng flower water extract:

[0161] 10.0 g of pine needle pore fungus fruiting body and 1 g of ginseng flower are weighed, placed in an oven for drying at 50° C., crushed with a multifunctional Chinese medicine grinder, the crushed mixed powder is mixed with water in a ratio of 2:8 to form a suspension, sterilized at 121° C. by high pressure for 15 min, cooled to room temperature, and sterilized by ultraviolet in a biological safety cabinet for 30 min, and the composite fermentation liquid of the two bacteria is inoculated into the suspension at an inoculum amount of 5%, and cultured in a constant temperature shaker for 5 days; reflux extraction is performed twice with water in a ratio of 1 g:8 L of solid-liquid ratio, each time for 2 hours, the extracts are combined, and finally freeze-dried to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder.

[0162] The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1;

[0163] The preparation method of the Lactobacillus rhamnosus fermentation liquid:

[0164] The ampoule containing Lactobacillus rhamnosus CICC 20990 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule and shaken to mix. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum size of 5%. After anaerobically culturing at 37° C. for 18 h, the bacterial suspension was inoculated into MRS solid culture medium in three zones to purify the strain. The culture was anaerobically culturing at 37° C. for 18 h, and a single colony was picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid.

[0165] The preparation method of the Bifidobacterium breve fermentation broth:

[0166] The ampoule containing Bifidobacterium breve CICC 6182 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium was taken and placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%. After anaerobic culture at 37° C. for 18 h, three-zone lines of the bacterial suspension were inoculated into the brain heart infusion liquid culture medium to purify the strain, and the culture was anaerobically cultured at 37° C. for 18 h. A single colony was picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bifidobacterium breve fermentation liquid.

[0167] 1. Experimental Animals

[0168] 300 5-week-old SPF-grade C57BL / 6 (about 20g) male mice were raised in the Animal Experiment Center of Harbin Medical College. According to the "Standards for Laboratory Animal Environment and Facilities of the Ministry of Health of the People's Republic of China", the experimental conditions for experimental animals were specified: discontinuous lighting, 12 hours of day and night. Cages, bedding, feed and other materials entering the animal house were sterilized with high temperature and high pressure. Ultraviolet disinfection was frequently performed. The behavior, mental state, diet, and defecation of rats in each group were observed.

[0169] 2. Experimental Methods

[0170] 2.1 Cell culture and passaging

[0171] 1) MC38 mouse colon cancer cell line (MC38) was purchased from Wuhan Pronocell Biotechnology Co., Ltd. and transported to the laboratory using a liquid nitrogen tank, taken out and quickly thawed in a 37°C water bath, sterilized with an alcohol spray bottle, and then transferred to an ultra-clean workbench;

[0172] 2) Use a pipette to transfer the dissolved cell suspension to a 15 mL centrifuge tube, wash the cryotube with DMEM / F12 complete culture medium, transfer it to a centrifuge tube, add 10 mL of culture medium, and centrifuge at 1000 rpm for 5 minutes;

[0173] 3) Discard the supernatant, add 1 mL of culture medium, stir evenly with a pipette, then transfer to DMEM / F12 culture medium containing 4 mL of DMEM / F12 complete culture medium, shake gently to make it evenly distributed; then place it in an incubator at 37°C with 5% carbon dioxide for culture.

[0174] 2.2 Cell passaging

[0175] 1) Transfer the cell culture flask to the clean bench and pour out the culture medium;

[0176] 2) Add 2 mL of 0.25% EDTA-trypsin solution (so that the digestion solution can submerge the bottom of the bottle), let it stand for 2 minutes to allow the adherent cells to detach in a spherical state, and add 5 mL of DMEM / F12 complete culture medium to end the enzymatic hydrolysis;

[0177] 3) Blow the bottle wall several times to obtain a cell suspension, transfer it to a centrifuge tube, and centrifuge it in a centrifuge at 1000 rpm for 5 minutes;

[0178] 4) discard the supernatant, add DMEM / F12 complete culture medium to DMEM / F12 complete culture medium, use a pipette to make it into a suspension, and then refill it into a new culture bottle filled with DMEM / F12 complete culture medium;

[0179] 5) After marking the culture medium, place it in an incubator at 37°C and 5% carbon dioxide for culture.

[0180] 2.3 Experimental animal grouping and model establishment

[0181] 2.3.1 Experimental Animal Grouping

[0182] After 7 days of adaptive feeding, the mice were randomly divided into 3 groups, namely, a decoction composition (ZHWS) (mix the powder of Ganoderma lucidum, fruiting body of Versicolor versicolor, fruit of Phellinus lintei, fruiting body of Polyporus pine needles and Panax ginseng flower in a mass ratio of 1:1:1:1:1, add 3 times the amount of water, and boil for 1 hour to obtain the decoction composition), a yang-opposite medicine (YD), and a composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy (ZHW), 20 mice in each group.

[0183] 2.3.2 Establishment of experimental animal model

[0184] 2.3.2.1 Tumor acquisition

[0185] Culture colon cancer cells, prepare 10 mice, inject 1 mL subcutaneously in the armpit, and inoculate 1x10 cells 6 After 2 weeks, the tumor volume was about 100 mm 3 The mice were euthanized, and the tumors were removed and weighed. Tumor tissue blocks were cut into 1 mm 3 .

[0186] 2.3.2.2 Tumor transplantation

[0187] The mice were anesthetized and disinfected, and the abdominal cavity was entered through a midline incision. The cecum was pulled out and part of the serosal tissue was scraped off. 3 Place the tumor of different sizes on the damaged serosa, drip medical anastomosis glue into the tumor to fix the tumor. Put the cecum into the stomach, and disinfect it after suturing.

[0188] 2.4 Measurement of mouse body weight

[0189] After the last administration, the mice were fasted for 12 hours. The mice were weighed and killed the next day, and a box plot of the mouse weight was drawn ( Figure 1 ).

[0190] 2.5 Collection of serum

[0191] The eyeballs were removed and blood was collected. The blood was allowed to stand for 1.5 h and then centrifuged at 4°C and 3000 r / min for 15 min. The upper serum was aspirated and frozen at -80°C. The blood was used for detection according to the kit instructions.

[0192] 2.6 Measurement of mouse tumor weight

[0193] After the last oral administration, the mice were fasted for 12 hours. The mice were weighed the next day, and the tumors were removed and weighed. A box plot of the tumor weight was drawn. The tumor tissue was divided into two portions with scissors, each 1 cm 3 They were placed in POM fixative, placed in EP tubes, frozen in liquid nitrogen, and refrigerated at -80°C.

[0194] 2.7 Detection of inflammatory factors

[0195] The eyeballs were removed and blood was collected. The blood was allowed to stand for 1.5 hours and then centrifuged at 3000 r / min for 15 minutes at 4°C. The upper serum was aspirated and stored at -80°C. The blood was then used for testing according to the instructions of the kit. Serum inflammatory factors (IFN-γ, IL-10, IL-6, TNF-α) were tested strictly according to the instructions of the enzyme-linked immunosorbent assay. The test results were as follows: Figure 3-Figure 6 .

[0196] 2.8 Cell viability assay

[0197] In order to observe the effects of each group of drugs on MC38 cells, the cells were divided into five groups (ZHW), a composition for reducing gastrointestinal reactions and enhancing appetite after chemotherapy for cancer patients, a water extract of pine needle pore fungus and ginseng flowers (SS-PE), a decoction composition (ZHWS), a fermentation product of polysaccharides of Glechoma longituba and Coriolus versicolor (SY-PE), and a polyphenol extract of Morinda igniarius (SH). The CCK-8 method was used to detect the effects of the composition, the water extract of pine needle pore fungus and ginseng flowers, the decoction composition, the fermentation product of polysaccharides of Glechoma longituba and Coriolus versicolor, and the polyphenol extract of Morinda igniarius 15 μmol / L on MC38 cells. MC38 cells were cultured at 1×10 6 The cells were inoculated into a 96-well plate at a density of 10 cells / well. After culturing for 24 hours, the culture medium was replaced with a series of drugs diluted with the corresponding culture medium. After incubation for 48 hours, 10 μL of CCK-8 solution (Biyuntian, Shanghai, China) was added to each well. After culturing in a 37°C incubator for 2 hours, the absorbance value (OD value) of each well was measured at 450 nm on a microplate reader. There were 3 replicate wells in each group. Cell viability (%) was calculated based on the OD value = [A (drug added) - A (blank)] / [A (drug added) - A (blank)] × 100%, and a graph of different doses and cell viability responses was drawn ( Figure 7 ).

[0198] Conclusion: Colorectal cancer can cause weight loss in mice. After drug administration, the weight of mice in the ZHW, YD, and ZHWS groups showed a trend of correction. The tumor weight of mice decreased after drug administration, and the effect of the ZHW group was the most obvious. Interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were used as evaluation criteria, and the three groups were used as research objects to explore the material basis of their efficacy. Compared with the ZHW group, the content of IFN-γ, IL-6, and TNF-α in each group was significantly reduced. Compared with ZHW, the level of IL-10 in each group was significantly increased. There was a significant difference between the ZHW groups. The present invention explores the therapeutic effects of various therapeutic drugs on colorectal cancer by establishing an orthotopic implantation model of MC38 mouse colorectal cancer. The composition used to reduce gastrointestinal reactions and enhance appetite in cancer patients after chemotherapy can significantly improve the anti-tumor effect of colorectal cancer patients, and CD8 in tumor tissue + The number of T lymphocytes increased significantly, accompanied by an increase in IFN-γ, IL-6, IL-10 and TNF-α. After using each composition on tumor-bearing mice, it was found that the growth of the tumor tended to slow down, among which the composition used to reduce gastrointestinal reactions and enhance appetite in cancer patients after chemotherapy had a more obvious effect.

[0199] Secondly, the tumor volume of mice in the composition group used to reduce gastrointestinal reactions and enhance appetite after chemotherapy for cancer patients was significantly smaller than that in the Yangdui medicine and composition decoction groups, and the difference in the composition group was more significant. The tumor immune microenvironment-related factors were analyzed by ELISA, and it was found that the IFN-gamma and IL-2 levels in the tumors of each group of mice were significantly increased, indicating that the body's immune response to tumors was improved. Interferon-γ (IFN-γ) is an important cell-mediated immune response that can promote antigen recognition of allogeneic T cells. Interferon-γ (IFN-γ), as a multi-effect cytokine, plays an important role in the immune editing process of tumors. It can be seen from this that the composition of the present invention can improve the anti-cancer effect in mice. It can not only improve the body's immune function and increase appetite, but also change the body's immune microenvironment and enhance the body's immune function.

Claims

1. A composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy, characterized in that The composition for alleviating gastrointestinal reactions and enhancing appetite of cancer patients after chemotherapy is composed of fermented polysaccharides of Gastrodia elata and Coriolus versicolor, mulberry linterus polyphenol extract, and pine needle pore fungus and ginseng flower water extract in a mass ratio of 1:1:

1.

2. The composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy according to claim 1, characterized in that The extraction method of the polysaccharide fermentation product of Ganoderma lucidum and Ganoderma versicolor:

1. Mix the powder of Ganoderma lucidum and crushed fruiting body of Versicolor versicolor in a mass ratio of 1:1, add 20 times the amount of water and soak for 8 hours to obtain a pre-treated liquid, extract the pre-treated liquid by ultrasonic at 6000 Hz for 30 minutes, then put it in a pressure steam cooker and extract it with high-temperature steam at 90°C for 20 minutes, then centrifuge the extracted turbid liquid at 8000 rpm for 5 minutes, take the supernatant and obtain treatment liquid 1; 2. Add 10 times the amount of water to the residue obtained in step 1, and then add complex enzyme, the amount of complex enzyme added is 0.5% to 1% of the total mass of the mixture, treat at 37 to 45°C for 2 to 4 hours, then inactivate the enzyme, centrifuge, and filter to obtain supernatant 2; The complex enzyme is composed of hemicellulase, pectinase and xylanase in a mass ratio of 1:1:0.5 to 1:2:1, and the enzyme activity units of the hemicellulase, pectinase and xylanase are 10,000 to 200,000 U / g; 3. The treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 30% to 50% of the original mass, and then graded alcohol precipitation is performed, ethanol is added to make the ethanol volume concentration of 30%, and the mixture is allowed to stand at room temperature for 36 hours, and then centrifuged to collect the precipitate to obtain precipitate 1. The supernatant is collected and concentrated under vacuum at 60°C, and ethanol is added to make the ethanol volume concentration of 80%. The mixture is allowed to stand at room temperature for 48 hours, and then centrifuged to collect the precipitate to obtain precipitate 2. After combining precipitate 1 and precipitate 2, lyophilization is performed to obtain the fermentation products of Ganoderma lucidum and Ganoderma versicolor polysaccharides.

3. The composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy according to claim 1, characterized in that The extraction method of the mulberry linterus polyphenol extract:

1. Preparation of DES: Choline chloride and oligofructose were mixed in a molar ratio of 1:1, heated to 85°C, and magnetically stirred for 3-4 hours to obtain a colorless transparent liquid, which was cooled to room temperature to obtain DES; 2. According to the mass ratio of Phellinus linteus to DES of 1:25, weigh the crushed Phellinus linteus fruiting bodies, then add DES, and then place them in an ultrasonic-microwave reactor, set the microwave power to 200-400w, the extraction temperature to 40℃, extract, filter, centrifuge at 6000r / min, collect the supernatant, and obtain the Phellinus linteus polyphenol extract.

4. The composition for alleviating gastrointestinal reactions and enhancing appetite in cancer patients after chemotherapy according to claim 1, characterized in that The extraction method of the pine needle phyllostachys ginseng flower water extract: Weigh 10.0g of pine needle pore fungus fruiting body and 1g of ginseng flower, place in an oven to dry at 50°C, crush with a multifunctional Chinese medicine grinder, mix the crushed mixed powder with water in a ratio of 2:8 to form a suspension, sterilize at 121°C under high pressure for 15min, cool to room temperature, sterilize under ultraviolet in a biological safety cabinet for 30min, inoculate the two bacteria composite fermentation liquid into the suspension at an inoculum amount of 5%, and culture in a constant temperature shaker for 5d; reflux extract twice with water in a ratio of 1g:8L to solid-liquid, each time for 2h, combine the extracts, and finally freeze-dry to obtain pine needle pore fungus ginseng flower water extract freeze-dried powder; The two-bacteria composite fermentation liquid is composed of Lactobacillus rhamnosus fermentation liquid and Bifidobacterium breve fermentation liquid in a mass ratio of 1:1; The preparation method of the Lactobacillus rhamnosus fermentation liquid: The ampoule containing Lactobacillus rhamnosus CICC 20990 was placed in a biological safety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum of 5%, and after anaerobic culture at 37° C. for 18 h, the bacterial suspension was inoculated into MRS solid culture medium by three-zone lines to purify the strain, and anaerobic culture was carried out at 37° C. for 18 h. A single colony was picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus rhamnosus fermentation liquid. The preparation method of the Bifidobacterium breve fermentation broth: The ampoule containing Bifidobacterium breve CICC 6182 was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium was taken and placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%. After anaerobic culture at 37° C. for 18 h, three-zone lines of the bacterial suspension were inoculated into the brain heart infusion liquid culture medium to purify the strain, and the culture was anaerobically cultured at 37° C. for 18 h. A single colony was picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bifidobacterium breve fermentation liquid.

5. The method for extracting polysaccharides from fermented products of Ganoderma lucidum and Coriolus versicolor according to claim 2, characterized in that In step 2, the amount of the complex enzyme added is 0.5% of the total mass of the mixture, and the mixture is treated at 37° C. for 2 h.

6. The method for extracting polysaccharides from fermented products of Ganoderma lucidum and Coriolus versicolor according to claim 2, characterized in that In step 3, the treated liquid 1 and the supernatant 2 are combined, and the combined filtrate is concentrated under reduced pressure to 40% of the original mass.

7. The method for extracting the mulberry linterinary polyphenol extract according to claim 3, characterized in that The microwave power is 300w.

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