Composition for protecting liver and preparation method thereof

Abstract

The invention provides a liver protection composition and a preparation method thereof, and belongs to the technical field of medicines. Ganoderma lucidum, dendrobium officinale, tartary buckwheat, polygonum cuspidatum and dandelion are subjected to enzymolysis, fermentation with selenium-enriched saccharomycetes and zinc-enriched saccharomycetes and extraction and separation with ethyl acetate to prepare an extract, zymophyte is collected to prepare compound bacterium powder, brown algae and nostoc sphaeroides dry powder are subjected to enzymolysis and alcohol precipitation to prepare algae polysaccharide, and the algae polysaccharide is uniformly mixed with the compound bacterium powder and the extract to prepare the liver protection composition. The composition for protecting the liver, prepared by the invention, can be used for remarkably inhibiting oxidation of oxygen free radicals and liver injury, inhibiting inflammatory factors, reducing expression of inflammasome pathway related proteins, regulating mitochondrial injury and regulating apoptosis, has a very good protection effect on the liver and has a wide application prospect.

Description

Technical Field

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

[0002] The liver is the largest solid gland in the human body. It is a soft tissue composed of hepatocytes and filled with blood. It is composed of hepatocytes arranged in cables, with the liver lobule as the basic unit. The liver is the center of metabolism in the body and is known as the "largest chemical factory in the human body". It has very important and complex physiological functions. Alcoholic liver disease is the most common type of chemical liver damage. It is a liver damage disease caused by long-term and heavy drinking (alcoholism). In my country, the incidence of alcoholic liver disease caused by drinking has also shown a significant upward trend, and it has become a hidden killer that cannot be ignored. Excessive drinking can increase the burden on the liver, damage and degenerate liver cells, and eventually lead to cirrhosis. In Europe and the United States, alcoholic cirrhosis accounts for 50-90% of all cirrhosis. Long-term alcoholics also affect fat metabolism and can cause fatty liver. Alcohol can cause liver cell degeneration, necrosis, and fibrosis to cause cirrhosis. Alcoholic liver disease affects the synthesis and absorption of proteins and vitamins, causing malnutrition, which in turn becomes a secondary factor for further damage to liver cells. The mutual influence eventually leads to fat infiltration, inflammation, necrosis of liver cells, and the occurrence of cirrhosis. If allowed to develop, the occurrence of liver fibrosis and cirrhosis is inevitable, and many complications may also occur. These complications are often risk factors for death from alcoholic liver disease.

[0003] Chemical liver damage refers to liver damage caused by chemical hepatotoxic substances. These chemicals include alcohol, chemical toxins in the environment, and certain drugs. The liver, as an important detoxification organ in the human body, has dual blood supply from the hepatic artery and hepatic vein. Chemical substances can enter the liver through the portal vein of the gastrointestinal tract or the systemic circulation for transformation, so the liver is easily damaged by toxic substances in chemicals, causing varying degrees of liver cell necrosis, fat deformation, cirrhosis, and liver cancer.

[0004] The current methods of nourishing and protecting the liver have the following defects: 1. Long-term use of Western chemical medicines for nourishing and protecting the liver will produce large toxic side effects and strong dependence; 2. The efficacy of existing Chinese medicine prescriptions is not ideal; the prescription compatibility is not entirely reasonable; 3. The price is expensive.

[0005] Although Western and traditional Chinese medicine have made many efforts in prevention and clinical treatment in the modern medical community and have achieved remarkable results, there is no specific drug for liver care and protection, and the current situation of liver disease endangering human health has not been fundamentally changed. Given that patients with liver disease have the characteristics of repeated and continuous medication, the market is in urgent need of a drug with significant efficacy, few toxic and side effects, stable and controllable, convenient and suitable for long-term use. Summary of the invention

[0006] The purpose of the present invention is to provide a liver-protecting composition and a preparation method thereof, which can significantly inhibit the oxidative effect of oxygen free radicals and inhibit liver damage, inhibit inflammatory factors, reduce the expression of inflammasome pathway-related proteins, regulate mitochondrial damage and regulate apoptosis, have a good protective effect on the liver, and have broad application prospects.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a preparation method of a liver-protecting composition. The invention comprises the following steps: enzymolysis of ganoderma lucidum, dendrobium officinale, tartary buckwheat, knotweed and dandelion, fermentation with selenium-enriched yeast and zinc-enriched yeast, extraction and separation with ethyl acetate to obtain an extract, collecting the fermented bacteria to obtain composite bacterial powder, enzymolysis of brown algae and kudzu vine dry powder, alcohol precipitation to obtain algal polysaccharide, and uniformly mixing the algal polysaccharide with the composite bacterial powder and the extract to obtain the liver-protecting composition.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Wash, dry and disperse the Ganoderma lucidum, Dendrobium officinale, Tartary buckwheat, Polygonum cuspidatum and Taraxacum officinale respectively to obtain Chinese medicine powder;

[0011] S2. Mix betaine and maltose evenly, heat to react, add sterile water to prepare a deep eutectic solvent;

[0012] S3. adding the Chinese medicine powder to a deep eutectic solvent, adding an immobilized enzyme, performing enzymolysis, inoculating a selenium-enriched yeast and a zinc-enriched yeast seed solution, fermenting and culturing, separating the immobilized enzyme with a magnet, washing, recycling, filtering, washing the solid, collecting the bacterial solution, freeze-drying, obtaining a composite bacterial powder, and retaining the filtrate;

[0013] S4. The filtrate was extracted with ethyl acetate, the organic phase was collected, and the solvent was removed under reduced pressure to obtain an extract;

[0014] S5. Add brown algae and dried algae powder to water, add immobilized enzyme, perform enzymolysis, separate the immobilized enzyme with a magnet, wash, recover the immobilized enzyme, filter, add ethanol to the filtrate for precipitation, centrifuge, wash, and dry to obtain algae polysaccharide;

[0015] S6. Evenly mix the composite bacterial powder, extract and algae polysaccharide to prepare a liver-protecting composition.

[0016] As a further improvement of the present invention, in step S1, the mass ratio of Ganoderma lucidum, Dendrobium officinale, Buckwheat, Polygonum cuspidatum and Taraxacum officinale is 10:8-12:3-5:2-4:1-3.

[0017] As a further improvement of the present invention, the molar ratio of betaine to maltose in step S2 is 3-5:1, the temperature of the heating reaction is 55-65°C, the time is 1-3h, and sterile water is added until the water content of the system is 15-25wt%.

[0018] As a further improvement of the present invention, the mass ratio of the Chinese medicine powder, the immobilized enzyme, and the deep eutectic solvent in step S3 is 10:5-7:70-100, the temperature of the enzymatic hydrolysis is 35-45°C, the time is 1-2h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL, the inoculation amounts of the selenium-enriched yeast and zinc-enriched yeast seed solutions are 1-3v / v% and 0.5-1.5v / v%, respectively, and the fermentation culture conditions are 28-32°C, 100-200r / min, and the fermentation culture is 24-36h.

[0019] As a further improvement of the present invention, in step S5, the mass ratio of brown algae, scutellaria baicalensis and immobilized enzyme is 10-15:5-7:7-10, the enzymatic hydrolysis temperature is 35-45°C, the time is 1-2h, ethanol is added until the ethanol content of the system is 75-85wt%, and the precipitation is 1-2h.

[0020] As a further improvement of the present invention, the mass ratio of the composite bacterial powder, the extract and the algae polysaccharide in step S6 is 3-5:12-15:7-10.

[0021] As a further improvement of the present invention, the preparation method of the immobilized enzyme is as follows:

[0022] T1. Dissolve ferric nitrate, ferrous nitrate, cobalt nitrate and nickel nitrate in water, add ammonia water under inert gas protection, heat and crystallize, filter, wash, dry and calcine to obtain a magnetic composite layered metal oxide;

[0023] T2. Add cellulase and neutral protease to water, add NHS and EDC, stir to activate, then add silane coupling agent with amino group, stir to react, dialyze, freeze-dry, and obtain modified composite enzyme;

[0024] T3. Add the modified composite enzyme and the magnetic composite layered metal oxide into ethanol, heat and stir to react, separate with a magnet, wash, and dry to obtain an immobilized enzyme.

[0025] As a further improvement of the present invention, the molar ratio of ferric nitrate, ferrous nitrate, cobalt nitrate and nickel nitrate in step T1 is 2:1:0.5-1.5:1-2, the pH value of the solution is adjusted to 9-10 by dropping ammonia water, the temperature of the heating crystallization is 85-95°C, the time is 4-6h, the temperature of the calcination is 400-500°C, and the time is 1-2h; the mass ratio of cellulase, neutral protease, NHS, EDC and silane coupling agent with amino group in step T2 is 10:3-5:4-6:3-5:2-4, and the silane coupling agent with amino group is selected from γ-aminopropyltrimethoxysilane, γ-aminopropyltrimethoxysilane, At least one of ethoxysilane, N-β (aminoethyl) -γ-aminopropyltrimethoxysilane, N-β (aminoethyl) -γ-aminopropyltriethoxysilane, N-β (aminoethyl) -γ-aminopropylmethyldimethoxysilane, N-β (aminoethyl) -γ-aminopropylmethyldiethoxysilane, and diethylenetriaminepropyltrimethoxysilane, the stirring reaction time is 10-12h, and the dialysis bag pore size used in the dialysis is 10-20KDa; the mass ratio of the modified composite enzyme and the magnetic composite layered metal oxide in step T3 is 3-5:10, the temperature of the heating and stirring reaction is 35-45°C, and the time is 1-3h.

[0026] The present invention further protects a liver-protecting composition prepared by the above-mentioned preparation method.

[0027] The present invention has the following beneficial effects:

[0028] The invention prepares an immobilized enzyme. A magnetic composite layered metal oxide is prepared by a coprecipitation method. The magnetic composite layered metal oxide has the properties of large specific surface area, rich hydroxyl groups on the surface and the like, and can load a large amount of enzymes. The composite enzyme (cellulase and neutral protease) is activated, reacted with a silane coupling agent with an amino group, thereby connecting the silane coupling agent, and then reacting and coupling with the magnetic composite layered metal oxide, thereby preparing the immobilized enzyme. The immobilized enzyme has the advantages of easy separation, large enzyme loading amount, multiple reaction sites, high catalytic activity and the like, and also solves the disadvantage of high temperature enzyme inactivation after the enzyme works. The invention directly separates the enzyme with a magnet, can be reused, reduces the cost, and simplifies the process steps.

[0029] In the present invention, the effective ingredients of Ganoderma lucidum, Dendrobium officinale, Tartary buckwheat, Polygonum cuspidatum and Taraxacum officinale include flavonoids, alkaloids, polyphenols, saponins, triterpenes, etc., and the mechanisms of their liver protection are mainly: first, anti-oxidation, scavenging oxygen free radicals and reducing liver ROS levels; second, anti-inflammatory, inhibiting inflammatory factors, and reducing the expression of proteins related to the inflammasome pathway; third, regulating mitochondrial damage and regulating apoptosis.

[0030] Deep eutectic solvents have the designability, environmental friendliness, non-volatility and non-flammability of ionic liquids, as well as the unique advantages of simple and inexpensive synthesis. They are composed of a mixture of two or more substances. The freezing point of the mixture is significantly reduced due to the eutectic effect, and it can exist in liquid form at room temperature, playing its role as a solvent. By adding immobilized enzymes, it can significantly improve the enzyme catalytic performance, enhance the operational stability of the immobilized enzyme, etc., and at the same time greatly improve the extraction of plant active ingredients, effectively promote the dissolution or dispersion of substrates, avoid local accumulation of products, and greatly improve the extraction rate of active substances. After fermentation by selenium-rich yeast and zinc-rich yeast, on the one hand, it promotes the rupture of plant cell walls and the dissolution of active substances. On the other hand, a large number of active components containing organic selenium and organic zinc are synthesized, which also promotes the growth and proliferation of yeast.

[0031] Selenium has a protective effect on the liver and is known as an "anti-liver necrosis protective factor". Selenium has a selective killing and inhibitory effect on liver cancer cells, but has no significant effect on normal liver cells. Selenium supplementation can be an effective measure to prevent liver cancer and prevent liver disease. Selenium can also reduce the damage to the liver caused by harmful substances in the environment, protect the structural integrity of liver cells through antioxidant effects, remove harmful substances, and accelerate the decomposition of lipid peroxides, thereby protecting the liver. Zinc is directly involved in various life metabolic processes in the body, such as cell growth and development, reproduction, and tissue repair, and is essential for maintaining the normal functioning of the immune function and antioxidant system. Therefore, the two have a synergistic effect.

[0032] Brown algae polysaccharides and kudzu vine polysaccharides have multiple biological activities such as lowering blood sugar, anti-coagulation, anti-virus, regulating immunity and protecting the liver. They can significantly inhibit the oxidative effect of oxygen free radicals and inhibit liver damage, and have a good protective effect on the liver. Enzymatic hydrolysis greatly promotes the rupture of the thick cell wall of the algae. The method is simple, efficient and economical, and can be realized in large-scale industrial production.

[0033] The liver-protecting composition prepared by the present invention can significantly inhibit the oxidative effect of oxygen free radicals and the effect of inhibiting liver damage, inhibit inflammatory factors, reduce the expression of proteins related to the inflammasome pathway, regulate mitochondrial damage and regulate apoptosis, has a good protective effect on the liver, and has broad application prospects. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Selenium-enriched yeast, 20 billion cfu / g, was purchased from Angel Yeast Co., Ltd.; zinc-enriched yeast, 10 billion cfu / g, was purchased from Fufeng Snort Biotechnology Co., Ltd. Cellulase, 50,000 U / g, neutral protease, 110,000 U / g, were purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0036] The preparation method of selenium-enriched yeast and zinc-enriched yeast seed liquid is as follows: Selenium-enriched yeast and zinc-enriched yeast are inoculated into Gao's medium respectively, and activated and cultured at 30°C, 150r / min, for 24h to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of selenium-enriched yeast and zinc-enriched yeast seed liquid.

[0037] Preparation Example 1 Preparation of immobilized enzyme

[0038] Here’s how:

[0039] T1. Dissolve 20mmol of ferric nitrate, 10mmol of ferrous nitrate, 5mmol of cobalt nitrate and 10mmol of nickel nitrate in 200mL of water. Under nitrogen protection, add ammonia water to adjust the pH value of the solution to 9. Heat to 85°C, crystallize for 4h, filter, wash, dry and calcine at 400°C for 1h to obtain a magnetic composite layered metal oxide.

[0040] T2. 1 g of cellulase and 0.3 g of neutral protease were added to 100 mL of water, 0.4 g of NHS and 0.3 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.2 g of diethylenetriaminepropyltrimethoxysilane was added, and the mixture was stirred for reaction for 10 h, and dialyzed for 12 h using a dialysis bag with a pore size of 10 kDa, and freeze-dried to obtain a modified composite enzyme;

[0041] T3. Add 0.3 g of the modified composite enzyme and 1 g of the magnetic composite layered metal oxide into 100 mL of ethanol, heat to 35°C, stir to react for 1 h, separate with a magnet, wash, and dry to obtain the immobilized enzyme.

[0042] Preparation Example 2 Preparation of immobilized enzyme

[0043] Here’s how:

[0044] T1. Dissolve 20mmol of ferric nitrate, 10mmol of ferrous nitrate, 15mmol of cobalt nitrate and 20mmol of nickel nitrate in 200mL of water, add ammonia water under nitrogen protection to adjust the pH value of the solution to 10, heat to 95°C, crystallize for 6h, filter, wash, dry, and calcine at 500°C for 2h to obtain a magnetic composite layered metal oxide;

[0045] T2. 1 g of cellulase and 0.5 g of neutral protease were added to 100 mL of water, 0.6 g of NHS and 0.5 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.4 g of N-β (aminoethyl) -γ-aminopropyltrimethoxysilane was added, and the mixture was stirred for reaction for 12 h, and dialyzed for 12 h using a dialysis bag with a pore size of 20 KDa, and freeze-dried to obtain a modified composite enzyme;

[0046] T3. Add 0.5 g of the modified composite enzyme and 1 g of the magnetic composite layered metal oxide into 100 mL of ethanol, heat to 45°C, stir and react for 3 h, separate with a magnet, wash, and dry to obtain the immobilized enzyme.

[0047] Preparation Example 3 Preparation of immobilized enzyme

[0048] Here’s how:

[0049] T1. Dissolve 20mmol of ferric nitrate, 10mmol of ferrous nitrate, 10mmol of cobalt nitrate and 15mmol of nickel nitrate in 200mL of water. Under nitrogen protection, add ammonia water to adjust the pH value of the solution to 9.5. Heat to 90°C, crystallize for 5h, filter, wash, dry and calcine at 450°C for 1.5h to obtain a magnetic composite layered metal oxide.

[0050] T2. 1 g of cellulase and 0.4 g of neutral protease were added to 100 mL of water, 0.5 g of NHS and 0.4 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.3 g of γ-aminopropyltriethoxysilane was added, and the mixture was stirred for reaction for 11 h, and dialyzed for 12 h using a dialysis bag with a pore size of 15 KDa, and freeze-dried to obtain a modified composite enzyme;

[0051] T3. Add 0.4 g of the modified composite enzyme and 1 g of the magnetic composite layered metal oxide into 100 mL of ethanol, heat to 40°C, stir and react for 2 h, separate with a magnet, wash, and dry to obtain the immobilized enzyme.

[0052] Comparative Preparation Example 1

[0053] Compared with Preparation Example 3, the difference is that no cellulase is added in step T2.

[0054] The details are as follows:

[0055] T1. Dissolve 20mmol of ferric nitrate, 10mmol of ferrous nitrate, 10mmol of cobalt nitrate and 15mmol of nickel nitrate in 200mL of water. Under nitrogen protection, add ammonia water to adjust the pH value of the solution to 9.5. Heat to 90°C, crystallize for 5h, filter, wash, dry and calcine at 450°C for 1.5h to obtain a magnetic composite layered metal oxide.

[0056] T2. 1.4 g of neutral protease was added to 100 mL of water, 0.5 g of NHS and 0.4 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.3 g of γ-aminopropyltriethoxysilane was added, and the mixture was stirred for reaction for 11 h, and dialyzed for 12 h using a dialysis bag with a pore size of 15 KDa, and freeze-dried to obtain a modified enzyme;

[0057] T3. Add 0.4 g of modified enzyme and 1 g of magnetic composite layered metal oxide into 100 mL of ethanol, heat to 40°C, stir and react for 2 h, separate with a magnet, wash and dry to obtain the immobilized enzyme.

[0058] Comparative Preparation Example 2

[0059] Compared with Preparation Example 3, the difference is that no neutral protease is added in Step T2.

[0060] The details are as follows:

[0061] T1. Dissolve 20mmol of ferric nitrate, 10mmol of ferrous nitrate, 10mmol of cobalt nitrate and 15mmol of nickel nitrate in 200mL of water. Under nitrogen protection, add ammonia water to adjust the pH value of the solution to 9.5. Heat to 90°C, crystallize for 5h, filter, wash, dry and calcine at 450°C for 1.5h to obtain a magnetic composite layered metal oxide.

[0062] T2. 1.4 g of cellulase was added to 100 mL of water, 0.5 g of NHS and 0.4 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.3 g of γ-aminopropyltriethoxysilane was added, and the mixture was stirred for reaction for 11 h, and dialyzed for 12 h using a dialysis bag with a pore size of 15 KDa, and freeze-dried to obtain a modified enzyme;

[0063] T3. Add 0.4 g of modified enzyme and 1 g of magnetic composite layered metal oxide into 100 mL of ethanol, heat to 40°C, stir and react for 2 h, separate with a magnet, wash and dry to obtain the immobilized enzyme.

[0064] Comparative Preparation Example 3

[0065] Compared with Preparation Example 3, the difference is that cobalt nitrate is not added in step T1.

[0066] The details are as follows:

[0067] T1. 30mmol of ferric nitrate, 10mmol of ferrous nitrate and 15mmol of nickel nitrate were dissolved in 200mL of water. Under nitrogen protection, ammonia water was added dropwise to adjust the pH value of the solution to 9.5. The solution was heated to 90°C, crystallized for 5h, filtered, washed, dried and calcined at 450°C for 1.5h to obtain a magnetic composite layered metal oxide.

[0068] T2. 1 g of cellulase and 0.4 g of neutral protease were added to 100 mL of water, 0.5 g of NHS and 0.4 g of EDC were added, and the mixture was stirred for activation for 30 min, and then 0.3 g of γ-aminopropyltriethoxysilane was added, and the mixture was stirred for reaction for 11 h, and dialyzed for 12 h using a dialysis bag with a pore size of 15 KDa, and freeze-dried to obtain a modified composite enzyme;

[0069] T3. Add 0.4 g of the modified composite enzyme and 1 g of the magnetic composite layered metal oxide into 100 mL of ethanol, heat to 40°C, stir and react for 2 h, separate with a magnet, wash, and dry to obtain the immobilized enzyme.

[0070] Test Example 1

[0071] The specific surface areas of the immobilized enzymes prepared in Preparation Examples 1-3 and Comparative Preparation Examples 1-3 were measured using a 3-FLEX 3500 multi-station high-throughput gas adsorption instrument. The results are shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] It can be seen from the above table that the immobilized enzymes prepared in Preparation Examples 1-3 of the present invention have a larger specific surface area.

[0076] Example 1

[0077] This embodiment provides a method for preparing a liver-protecting composition, comprising the following steps:

[0078] S1. Wash 10g of Ganoderma lucidum, 8g of Dendrobium officinale, 3g of Tartary Buckwheat, 2g of Polygonum cuspidatum, and 1g of Taraxacum officinale, dry them, and disperse them to obtain Chinese medicine powder;

[0079] S2. 3 mol of betaine and 1 mol of maltose were stirred and mixed for 10 min, heated to 55 ° C, reacted for 1 h, and sterile water was added to the system until the water content was 15 wt % to obtain a deep eutectic solvent;

[0080] S3. Add 10g of Chinese medicine powder to 70g of deep eutectic solvent, add 5g of immobilized enzyme prepared in Preparation Example 1, enzymolysis at 35°C for 1h, sterilize, and inoculate with a bacterial count of 10 8 -10 9 cfu / mL of selenium-enriched yeast and zinc-enriched yeast seed liquid, inoculated at 1v / v% and 0.5v / v%, respectively, at 28°C, 100r / min, fermented for 24h, separated the immobilized enzyme with a magnet, washed, recycled, filtered, washed the solid, collected the bacterial liquid, freeze-dried, prepared the composite bacterial powder, and retained the filtrate;

[0081] S4. The filtrate was extracted with ethyl acetate, the organic phase was collected, and the solvent was removed under reduced pressure to obtain an extract;

[0082] S5. Add 10 g of brown algae and 5 g of dried algae powder to 500 mL of water, add 7 g of the immobilized enzyme prepared in Preparation Example 1, perform enzymolysis at 35 ° C for 1 h, separate the immobilized enzyme with a magnet, wash, recover the immobilized enzyme, filter, add ethanol to the filtrate until the ethanol content of the system is 75 wt%, precipitate for 1 h, centrifuge, wash, and dry to obtain algae polysaccharide;

[0083] S6. Stir and mix 3g of composite bacterial powder, 12g of extract, and 7g of algae polysaccharide for 15 minutes to prepare a liver-protecting composition.

[0084] Example 2

[0085] This embodiment provides a method for preparing a liver-protecting composition, comprising the following steps:

[0086] S1. 10g of Ganoderma lucidum, 12g of Dendrobium officinale, 5g of Tartary Buckwheat, 4g of Polygonum cuspidatum, and 3g of Taraxacum were washed, dried, and dispersed to obtain Chinese medicine powder;

[0087] S2. 5 mol of betaine and 1 mol of maltose were stirred and mixed for 10 min, heated to 65 ° C, reacted for 3 h, and sterile water was added to the system until the water content was 25 wt % to obtain a deep eutectic solvent;

[0088] S3. Add 10g of Chinese medicine powder to 100g of deep eutectic solvent, add 7g of immobilized enzyme prepared in Preparation Example 2, enzymolysis at 45°C for 2h, sterilize, and inoculate with a bacterial count of 10 8 -10 9 cfu / mL of selenium-enriched yeast and zinc-enriched yeast seed liquid, inoculated at 3v / v% and 1.5v / v%, respectively, at 32°C, 200r / min, fermented for 36h, separated the immobilized enzyme with a magnet, washed, recycled, filtered, washed the solid, collected the bacterial liquid, freeze-dried, prepared the composite bacterial powder, and retained the filtrate;

[0089] S4. The filtrate was extracted with ethyl acetate, the organic phase was collected, and the solvent was removed under reduced pressure to obtain an extract;

[0090] S5. 15 g of brown algae and 7 g of dried algae powder were added to 500 mL of water, 10 g of the immobilized enzyme prepared in Preparation Example 2 was added, and the enzyme was hydrolyzed at 45 ° C for 2 h. The immobilized enzyme was separated by a magnet, washed, and the immobilized enzyme was recovered. The filtrate was added with ethanol until the ethanol content of the system was 85 wt%, and precipitated for 2 h, centrifuged, washed, and dried to obtain algae polysaccharide;

[0091] S6. Stir and mix 5 g of the composite bacterial powder, 15 g of the extract, and 10 g of algae polysaccharide for 15 minutes to prepare a liver-protecting composition.

[0092] Example 3

[0093] This embodiment provides a method for preparing a liver-protecting composition, comprising the following steps:

[0094] S1. 10 g of Ganoderma lucidum, 10 g of Dendrobium candidum, 4 g of Tartary buckwheat, 3 g of Polygonum cuspidatum, and 2 g of Taraxacum officinale were washed, dried, and dispersed to obtain Chinese medicine powder;

[0095] S2. 4 mol of betaine and 1 mol of maltose were stirred and mixed for 10 min, heated to 60 ° C, reacted for 2 h, and sterile water was added to the system until the water content was 20 wt % to obtain a deep eutectic solvent;

[0096] S3. Add 10g of Chinese medicine powder to 85g of deep eutectic solvent, add 6g of immobilized enzyme prepared in Preparation Example 3, enzymolysis at 40°C for 1.5h, sterilize, and inoculate with a bacterial count of 10 8 -10 9 cfu / mL of selenium-enriched yeast and zinc-enriched yeast seed liquid, inoculated at 2v / v% and 1v / v%, respectively, at 30°C, 150r / min, fermented for 30h, separated the immobilized enzyme with a magnet, washed, recycled, filtered, washed the solid, collected the bacterial liquid, freeze-dried, prepared the composite bacterial powder, and retained the filtrate;

[0097] S4. The filtrate was extracted with ethyl acetate, the organic phase was collected, and the solvent was removed under reduced pressure to obtain an extract;

[0098] S5. 12 g of brown algae and 6 g of dried algae powder were added to 500 mL of water, and 8 g of the immobilized enzyme prepared in Preparation Example 3 was added. The enzyme was hydrolyzed at 40 ° C for 1.5 h, and the immobilized enzyme was separated by a magnet, washed, and the immobilized enzyme was recovered. The filtrate was added with ethanol until the ethanol content of the system was 80 wt%, and precipitated for 1.5 h, centrifuged, washed, and dried to obtain algae polysaccharide;

[0099] S6. Stir and mix 4 g of composite bacterial powder, 13 g of extract, and 8 g of algae polysaccharide for 15 minutes to prepare a liver-protecting composition.

[0100] Comparative Example 1

[0101] Compared with Example 3, the difference is that the immobilized enzyme is prepared by Comparative Preparation Example 1.

[0102] Comparative Example 2

[0103] Compared with Example 3, the difference is that the immobilized enzyme is prepared by Comparative Preparation Example 2.

[0104] Comparative Example 3

[0105] Compared with Example 3, the difference is that the immobilized enzyme is prepared by Comparative Preparation Example 3.

[0106] Comparative Example 4

[0107] Compared with Example 3, the difference is that in step S3, the deep eutectic solvent is replaced by an equal amount of sterile water.

[0108] The details are as follows:

[0109] S3. Add 10g of Chinese medicine powder to 85g of sterile water, add 6g of the immobilized enzyme prepared in Preparation Example 3, enzymolysis at 40°C for 1.5h, sterilize, and inoculate with a bacterial count of 10 8 -10 9 cfu / mL of selenium-enriched yeast and zinc-enriched yeast seed liquid, inoculation amounts of 2v / v% and 1v / v%, respectively, at 30°C, 150r / min, fermentation culture for 30h, separation of immobilized enzyme by magnet, washing, recycling, filtration, solid washing, collection of bacterial liquid, freeze-drying, preparation of composite bacterial powder, and retention of filtrate.

[0110] Comparative Example 5

[0111] Compared with Example 3, the difference is that no selenium-enriched yeast seed liquid is inoculated in step S3.

[0112] The details are as follows:

[0113] S3. Add 10g of Chinese medicine powder to 85g of deep eutectic solvent, add 6g of immobilized enzyme prepared in Preparation Example 3, enzymolysis at 40°C for 1.5h, sterilize, and inoculate with a bacterial count of 10 8 -10 9 cfu / mL of zinc-rich yeast seed liquid, inoculation amount of 3v / v%, 30℃, 150r / min, fermentation culture for 30h, separation of immobilized enzyme by magnet, washing, recycling, filtering, solid washing, collection of bacterial liquid, freeze-drying, preparation of composite bacterial powder, and retention of filtrate.

[0114] Comparative Example 6

[0115] Compared with Example 3, the difference is that the zinc-enriched yeast seed liquid is not inoculated in step S3.

[0116] The details are as follows:

[0117] S3. Add 10g of Chinese medicine powder to 85g of deep eutectic solvent, add 6g of immobilized enzyme prepared in Preparation Example 3, enzymolysis at 40°C for 1.5h, sterilize, and inoculate with a bacterial count of 10 8 -10 9cfu / mL of selenium-enriched yeast seed liquid, inoculation amount of 3v / v%, 30℃, 150r / min, fermentation culture for 30h, separation of immobilized enzyme by magnet, washing, recycling, filtration, solid washing, collection of bacterial liquid, freeze-drying, preparation of composite bacterial powder, and retention of filtrate.

[0118] Comparative Example 7

[0119] Compared with Example 3, the difference is that the selenium-enriched yeast and zinc-enriched yeast seed liquid are not inoculated in step S3.

[0120] The details are as follows:

[0121] S3. Add 10 g of Chinese medicine powder to 85 g of deep eutectic solvent, add 6 g of immobilized enzyme prepared in Preparation Example 3, perform enzymolysis at 40° C. for 1.5 h, sterilize, separate the immobilized enzyme with a magnet, wash, recycle, filter, and retain the filtrate.

[0122] Comparative Example 8

[0123] Compared with Example 3, the difference is that no brown algae is added in step S5.

[0124] The details are as follows:

[0125] S5. Add 18g of dry powder of Miscanthus sphaerocephalus into 500mL of water, add 8g of the immobilized enzyme prepared in Preparation Example 3, perform enzymolysis at 40°C for 1.5h, separate the immobilized enzyme with a magnet, wash, recover the immobilized enzyme, filter, add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 1.5h, centrifuge, wash, and dry to obtain algae polysaccharide.

[0126] Comparative Example 9

[0127] Compared with Example 3, the difference is that no Myriophyllum puerariae is added in step S5.

[0128] The details are as follows:

[0129] S5. Add 18 g of brown algae dry powder into 500 mL of water, add 8 g of the immobilized enzyme prepared in Preparation Example 3, perform enzymolysis at 40°C for 1.5 h, separate the immobilized enzyme with a magnet, wash, recover the immobilized enzyme, filter, add ethanol to the filtrate until the ethanol content of the system is 80 wt%, precipitate for 1.5 h, centrifuge, wash, and dry to obtain algae polysaccharide.

[0130] Comparative Example 10

[0131] Compared with Example 3, the difference is that algae polysaccharide is not added in step S6.

[0132] The details are as follows:

[0133] S6. Stir and mix 4 g of the composite bacterial powder and 13 g of the extract for 15 minutes to prepare a liver-protecting composition.

[0134] Test Example 2

[0135] The liver protection composition prepared in Examples 1-3 or Comparative Examples 1-10 was used to prepare a test solution with a mass concentration of 0.05 mg / mL and a 0.05 mg / mL vitamin C solution as a positive control group. 2 mL of 0.2 mg / mL DPPH 50% ethanol solution and scavengers of different concentrations were respectively placed in stoppered test tubes, shaken, and reacted in the dark for 30 minutes. The absorbance A was measured at 517 nm using anhydrous ethanol as a reference. i ; Same method as A i Operation, measure the absorbance A after 2mL DPPH 50% ethanol solution is mixed with anhydrous ethanol c ; Same method as A i Operation, measure the absorbance A after mixing 2mL of different concentrations of scavenger and 50% ethanol solution j The clearance rate was calculated according to the formula. The clearance rate is used to represent the scavenging ability of the scavenger. The larger the clearance rate, the stronger the antioxidant activity. The results are shown in Table 2.

[0136] Calculated using the following formula:

[0137] DPPH·scavenging rate (%) = 1-(A i -A j ) / A c ×100%

[0138] in:

[0139] A i : absorbance of DPPH system when scavenger is added; A j : absorbance value of the scavenger; A c : Absorbance value of DPPH system without adding scavenger.

[0140] Table 2

[0141] Group DPPH·Scavenging rate (%) Vitamin C 90.25 Example 1 95.67 Example 2 95.82 Example 3 96.07 Comparative Example 1 91.24 Comparative Example 2 90.47 Comparative Example 3 93.21 Comparative Example 4 84.67 Comparative Example 5 85.52 Comparative Example 6 87.60 Comparative Example 7 80.29 Comparative Example 8 82.24 Comparative Example 9 81.59 Comparative Example 10 78.57

[0142] It can be seen from the above table that the liver protection compositions prepared in Examples 1-3 of the present invention have good antioxidant capacity.

[0143] Test Example 3

[0144] After the Kunming mice were adaptively raised for 1 week, they were randomly divided into a normal control group, a model group, an embodiment 1-3 group, a comparative example 1-10 group, and a bifendate group, with 10 mice in each group. Except for the normal control group, the mice in the other groups were gavaged with 0.2wt% carbon tetrachloride olive oil solution, and the gavage dose was 0.1mL / 10g mouse body weight. At 24h, 48h, and 71h after modeling, the corresponding liver protection and liver protection composition was gavaged at 2.0g / kg in the embodiment 1-3 group and the comparative example 1-10 group; the bifendate group was gavaged with bifendate dripping pills 4mg / kg; the normal control group and the model group were gavaged with an equal volume of physiological saline for a total of 3 times. After 72h, blood was collected from the orbits of all animals, centrifuged, and serum was separated to determine alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The results are shown in Table 3.

[0145] Table 3

[0146] Group ALT content (U / L) AST content (U / L) Normal control group 32.98±3.28 30.17±3.18 Model Group 138.45±13.19* 89.92±9.27* Bifendate group 63.32±6.83# 43.29±5.76# Example 1 62.17±5.48# 41.59±4.82# Example 2 61.89±4.82# 41.21±5.09# Example 3 61.10±4.10# 41.02±4.47# Comparative Example 1 72.63±6.52 49.67±6.75 Comparative Example 2 75.11±5.98 51.12±6.23 Comparative Example 3 70.10±6.02 47.84±5.88 Comparative Example 4 80.52±6.28 53.29±6.09 Comparative Example 5 76.69±5.78 52.10±5.72 Comparative Example 6 74.14±6.13 51.77±6.31 Comparative Example 7 82.18±6.59 54.82±6.27 Comparative Example 8 75.55±6.47 52.19±6.65 Comparative Example 9 76.09±5.84 52.03±5.97 Comparative Example 10 81.30±6.55 53.85±6.12

[0147] Note: * compared with the normal control group, P < 0.05; # compared with the model group, P < 0.05.

[0148] It can be seen from the above table that the liver-protecting compositions prepared in Examples 1-3 of the present invention can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice.

[0149] Weigh 0.4 g of liver tissue, add 9 times of cold physiological saline, homogenize, remove the supernatant after centrifugation, and determine superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) according to the instructions of the kit. The results are shown in Table 4.

[0150] Table 4

[0151]

[0152]

[0153] Note: * compared with the normal control group, P < 0.05; # compared with the model group, P < 0.05.

[0154] It can be seen from the above table that the liver-protecting compositions prepared in Examples 1-3 of the present invention can significantly increase the levels of SOD, CAT, and GSH, and reduce the content of MDA.

[0155] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a liver-protecting composition, characterized in that: Ganoderma lucidum, Dendrobium officinale, buckwheat, Polygonum cuspidatum and dandelion are enzymatically hydrolyzed, fermented with selenium-rich yeast and zinc-rich yeast, and separated by ethyl acetate extraction to obtain an extract, the fermented bacteria are collected to obtain a composite bacterial powder, brown algae and kudzu vine dry powder are enzymatically hydrolyzed, and alcohol precipitation is used to obtain algal polysaccharide, which is evenly mixed with the composite bacterial powder and the extract to obtain a liver-protecting composition.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Wash, dry and disperse the Ganoderma lucidum, Dendrobium officinale, Tartary buckwheat, Polygonum cuspidatum and Taraxacum officinale respectively to obtain Chinese medicine powder; S2. Mix betaine and maltose evenly, heat to react, add sterile water to prepare a deep eutectic solvent; S3. adding the Chinese medicine powder to a deep eutectic solvent, adding an immobilized enzyme, performing enzymolysis, inoculating a selenium-enriched yeast and a zinc-enriched yeast seed solution, fermenting and culturing, separating the immobilized enzyme with a magnet, washing, recycling, filtering, washing the solid, collecting the bacterial solution, freeze-drying, obtaining a composite bacterial powder, and retaining the filtrate; S4. The filtrate was extracted with ethyl acetate, the organic phase was collected, and the solvent was removed under reduced pressure to obtain an extract; S5. Add brown algae and dried algae powder to water, add immobilized enzyme, perform enzymolysis, separate the immobilized enzyme with a magnet, wash, recover the immobilized enzyme, filter, add ethanol to the filtrate for precipitation, centrifuge, wash, and dry to obtain algae polysaccharide; S6. Evenly mix the composite bacterial powder, extract and algae polysaccharide to prepare a liver-protecting composition.

3. The preparation method according to claim 2, characterized in that: In step S1, the mass ratio of ganoderma lucidum, dendrobium officinale, buckwheat, knotweed and dandelion is 10:8-12:3-5:2-4:1-3.

4. The preparation method according to claim 2, characterized in that: In step S2, the molar ratio of betaine to maltose is 3-5:1, the temperature of the heating reaction is 55-65° C., the time is 1-3 h, and sterile water is added until the water content of the system is 15-25 wt %.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass ratio of the Chinese medicine powder, the immobilized enzyme, and the deep eutectic solvent is 10:5-7:70-100, the temperature of the enzymatic hydrolysis is 35-45°C, the time is 1-2h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL, the inoculation amounts of the selenium-enriched yeast and zinc-enriched yeast seed solutions are 1-3v / v% and 0.5-1.5v / v%, respectively, and the fermentation culture conditions are 28-32°C, 100-200r / min, and the fermentation culture is 24-36h.

6. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of brown algae, scutellaria baicalensis and immobilized enzyme is 10-15:5-7:7-10, the temperature of enzymolysis is 35-45°C, the time is 1-2h, ethanol is added until the ethanol content of the system is 75-85wt%, and precipitation is 1-2h.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the composite bacterial powder, the extract and the algae polysaccharide in step S6 is 3-5:12-15:7-10.

8. The preparation method according to claim 2, characterized in that: The preparation method of the immobilized enzyme is as follows: T1. Dissolve ferric nitrate, ferrous nitrate, cobalt nitrate and nickel nitrate in water, add ammonia water under inert gas protection, heat and crystallize, filter, wash, dry and calcine to obtain a magnetic composite layered metal oxide; T2. Add cellulase and neutral protease to water, add NHS and EDC, stir to activate, then add silane coupling agent with amino group, stir to react, dialyze, freeze-dry, and obtain modified composite enzyme; T3. Add the modified composite enzyme and the magnetic composite layered metal oxide into ethanol, heat and stir to react, separate with a magnet, wash, and dry to obtain an immobilized enzyme.

9. The preparation method according to claim 8, characterized in that: The molar ratio of ferric nitrate, ferrous nitrate, cobalt nitrate and nickel nitrate in step T1 is 2:1:0.5-1.5:1-2, the pH value of the solution is adjusted to 9-10 by dropping ammonia water, the temperature of the heating crystallization is 85-95°C, the time is 4-6h, the temperature of the calcination is 400-500°C, and the time is 1-2h; the mass ratio of cellulase, neutral protease, NHS, EDC and silane coupling agent with amino group in step T2 is 10:3-5:4-6:3-5:2-4, and the silane coupling agent with amino group is selected from γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, At least one of N-β (aminoethyl) -γ-aminopropyl trimethoxysilane, N-β (aminoethyl) -γ-aminopropyl triethoxysilane, N-β (aminoethyl) -γ-aminopropyl methyl dimethoxysilane, N-β (aminoethyl) -γ-aminopropyl methyl diethoxysilane, and diethylenetriamine propyl trimethoxysilane, the stirring reaction time is 10-12h, and the dialysis bag pore size used for dialysis is 10-20KDa; the mass ratio of the modified composite enzyme and the magnetic composite layered metal oxide in step T3 is 3-5:10, the temperature of the heating and stirring reaction is 35-45°C, and the time is 1-3h.

10. A liver-protecting composition prepared by the preparation method according to any one of claims 1 to 9.

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