A composition with protective function against chemical liver damage and its preparation method

Abstract

The present invention belongs to the field of traditional Chinese medicine for treating liver diseases, and is particularly related to a composition having a protective function against chemical liver damage and a preparation method thereof. The raw materials of the composition are composed of the following components in parts by weight: 8-15 parts of kudzu root, 3-10 parts of hovenia dulcis, 3-12 parts of schisandra chinensis, 2-9 parts of ginseng and 2-10 parts of shiitake mushroom. The preparation method comprises: first extracting volatile oil from ginseng, shiitake mushroom and schisandra chinensis, enzymolyzing the filtrate, mixing the medicinal residues with kudzu root and hovenia dulcis to extract weak polar active ingredients, then extracting the active ingredients with water through a macroporous resin, and finally concentrating and drying the extracts to obtain a dry paste powder. The preparation method of the present invention has a high utilization rate of medicinal materials, and the obtained composition has a precise protective effect on chemical liver damage, is safe to take, and has controllable quality.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine for treating liver diseases, and particularly relates to a composition having a protective function against chemical liver damage and a preparation method thereof. Background Art

[0002] Chemical liver injury is caused by chemical hepatotoxic substances. Its clinical manifestations include loss of appetite, indigestion, pain in the liver area, nausea and vomiting. Chemical liver injury is generally chronic and often occurs in people with sub-health conditions. Treatment focuses on repairing damaged liver cells and protecting the liver.

[0003] Chemical liver injury causes usually manifest in the following aspects:

[0004] (1) Long-term or intermittent drinking of large amounts of alcohol can cause liver damage. Alcohol directly poisons liver cells, affecting liver function and metabolic disorders, thus making it impossible to utilize or store nutrients. 80% to 90% of the alcohol that enters the human body is metabolized in the liver. When drinking large amounts of alcohol for a long time, alcohol metabolites accumulate in the liver, which has a direct toxic effect on liver cells. The mechanism of acute alcoholic liver damage is that after the body ingests a large amount of ethanol, it undergoes a large amount of dehydrogenation under the catalysis of alcohol dehydrogenase, which causes the tricarboxylic acid cycle to be impaired and fatty acid oxidation to be weakened, thus affecting fat metabolism. Ethanol can increase α-glycerophosphate and promote triglyceride synthesis, resulting in fat deposition in liver cells; at the same time, ethanol can activate oxygen molecules, generating oxygen free radicals, leading to lipid peroxidation of liver cell membranes and depletion of reduced glutathione in the body.

[0005] (2) Some hepatotoxic substances can increase their toxicity by combining with non- or slightly toxic chemicals. For example, fatty alcohols (methanol, ethanol, isopropanol, etc.) can enhance the toxicity of halogenated hydrocarbons (carbon tetrachloride, chloroform, etc.). Chemically harmful substances can enter the liver through the gastrointestinal tract and blood circulation for transformation. Therefore, the liver is easily damaged by these toxic substances, causing chemical liver damage.

[0006] (3) The liver is the main site of drug metabolism. Long-term use of drugs can cause liver cell damage and easily induce liver disease.

[0007] (4) Improper diet will also increase the burden on the liver. Diseases enter the body through the mouth. Spoiled food and contaminated water can cause certain damage to the liver. For example, long-term consumption of food containing nitrosamines, moldy food (food contaminated by aflatoxin), food lacking trace elements, and drinking water contaminated by algae can cause damage to the liver and even lead to liver cancer.

[0008] Currently, there are many drugs and health products on the market that have protective functions against chemical liver damage. Chemical drugs have obvious adverse reactions due to long-term use and may damage organs or cause drug dependence and drug resistance. Pure natural Chinese herbal medicines that have protective effects against chemical liver damage have relatively fewer adverse reactions to the human body and can regulate multiple targets, making them suitable for long-term use.

[0009] Chinese invention patent CN106975069B discloses a liver-protecting composition and its preparation method. The composition comprises the following components: 1-5 parts of Hovenia dulcis fruit, 0.5-4 parts of Pueraria root, 0.5-4 parts of Lycium barbarum fruit, 0.1-3 parts of Poria cocos, 0.1-3 parts of corn oligopeptide powder, 0.5-1 parts of mussel polysaccharide, and 0.05-0.4 parts of Chrysanthemum morifolium. The composition prepared by this method has a certain therapeutic effect on improving chemical liver damage, but its effects on lipid metabolism and anti-free radical properties remain to be studied.

[0010] Chinese invention patent CN110876772A discloses a method for preparing and using a traditional Chinese medicine preparation with hepatoprotective properties. The composition is made from the following Chinese medicinal ingredients by weight: 3-5 parts of Pueraria root, 3-5 parts of Lycium barbarum fruit, 2-3 parts of Hovenia dulcis fruit, 1-2 parts of Ginseng, and 1-2 parts of Schisandra chinensis. However, this invention utilizes the medicinal ingredients in a low efficiency, which is detrimental to the efficacy of the drug, and its therapeutic effect on various liver injuries needs further improvement. Summary of the Invention

[0011] In order to address the deficiencies in the prior art, the present invention provides a composition with protective function against chemical liver damage and a preparation method thereof, which has high medicinal material utilization rate, low production cost, definite protective effect against chemical liver damage, safe administration and controllable quality.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] The invention discloses a composition having a protective function against chemical liver damage. The raw materials of the composition are composed of the following components in parts by weight: 8-15 parts of kudzu root, 3-10 parts of hovenia dulcis fruit, 3-12 parts of schisandra chinensis, 2-9 parts of ginseng and 2-10 parts of shiitake mushroom.

[0014] Preferably, the raw materials of the composition are composed of the following components in parts by weight: 8-10 parts of Pueraria root, 5-10 parts of Hovenia dulcis fruit, 6-12 parts of Schisandra chinensis, 4-9 parts of ginseng and 5-10 parts of shiitake mushroom.

[0015] Preferably, the raw materials of the composition are composed of the following components in parts by weight: 8 parts of Pueraria root, 5 parts of Hovenia dulcis fruit, 6 parts of Schisandra chinensis, 4 parts of ginseng, and 5 parts of shiitake mushrooms.

[0016] Preferably, the composition of the present invention further comprises a pharmaceutically acceptable excipient;

[0017] Preferably, the auxiliary material is selected from one or more of fillers, glidants, lubricants, binders, disintegrants or flavoring agents.

[0018] Another object of the present invention is to provide a method for preparing the above-mentioned composition, characterized in that it comprises the following steps:

[0019] (1) Ginseng, shiitake mushrooms and schisandra chinensis are crushed, and water is added to perform steam distillation extraction to obtain a filtrate 1, a medicinal residue 1 and a volatile oil; the volatile oil is included in the inclusion compound with β-cyclodextrin; the filtrate 1 is enzymatically hydrolyzed, the enzyme is inactivated at 95-100° C., and the mixture is concentrated and dried to obtain a dry paste powder 1;

[0020] (2) Grinding Puerariae Root and Hovenia Dulcis Fruit, mixing with the medicinal residue 1, adding 70-85% ethanol, heating and refluxing for extraction, filtering to obtain filtrate 2 and medicinal residue 2, concentrating and drying the filtrate 2 to obtain dry paste powder 2, adding water to the medicinal residue 2, refluxing for extraction, filtering, and concentrating to obtain a concentrated solution;

[0021] (3) mixing the macroporous adsorption resin and the macroporous anion resin, wet-loading the column, adsorbing the concentrated solution onto the column, eluting with 6-8 column volumes of water, then gradient eluting with an organic solvent, combining the eluates, concentrating and drying to obtain a dry paste powder 3;

[0022] (4) Evenly mix the inclusion compound, dry paste powder 1, dry paste powder 2, dry paste powder 3 and auxiliary materials to obtain the product.

[0023] Preferably, in step (1), the ratio of the mass of water to the total mass of ginseng, shiitake mushrooms and schisandra chinensis is 5-8:1; and the extraction time is 5-7 hours.

[0024] Preferably, in step (1), the mass ratio of the volatile oil to β-cyclodextrin is 1:4.5-5.5, the inclusion time is 1.5-2.5 hours, and the inclusion temperature is 50-65°C.

[0025] Preferably, the enzyme used in the enzymatic hydrolysis in step (1) is an enzyme mixture consisting of β-glucosidase and ficin in a mass ratio of 5-10:1, the amount of the enzyme mixture added is 0.1-0.3% of the mass of the filtrate 1, the enzymatic hydrolysis temperature is 45-60° C., the enzymatic hydrolysis time is 0.5-1.5 h, and the enzymatic hydrolysis pH is 4.0-6.0.

[0026] Preferably, the ratio of the mass of the 70-85% ethanol in step (2) to the total mass of Pueraria lobata, Hovenia dulcis and the medicinal residue 1 is 6-10:1; the extraction time is 1-2 hours; the mass ratio of the water to the medicinal residue 2 is 5-10:1, the number of times the medicinal residue 2 is refluxed with water for extraction is 1-2 times, and the extraction time each time is 1-1.5 hours.

[0027] The above-mentioned medicinal residues are all wet materials after filtration.

[0028] Preferably, the oral preparation is prepared after uniform mixing in step (4).

[0029] Preferably, in step (3), the mass ratio of the macroporous adsorption resin to the macroporous anion resin is 3-6:1; the eluent flow rate is controlled to be 0.8-1.2 mL / min during elution with water; and the organic solvent gradient elution is sequentially eluted with 3-5 column volumes of a mixed solution of 60% ethanol and acetone and 6-8 column volumes of a mixed solution of 95% ethanol and n-butanol.

[0030] Preferably, the ratio of the mass of the concentrated solution to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 is 1:3-5;

[0031] Preferably, the volume ratio of the 60% ethanol to acetone is 1:0.5-0.8; the volume ratio of the 95% ethanol to n-butanol is 1:1-3; and the elution flow rate during the organic solvent gradient elution is controlled to be 0.5-0.7 mL / min.

[0032] Another object of the present invention is to provide the use of the above composition or preparation method in the preparation of drugs for treating chemical liver damage.

[0033] The four properties and five flavors of each medicinal material in the composition of the present invention and their efficacy:

[0034] Pueraria root: Sweet, pungent, and cooling. It enters the spleen, stomach, and lung meridians and has the effects of relieving muscle tension and reducing fever, promoting fluid production and quenching thirst, clearing rashes, promoting yang and stopping diarrhea, promoting blood circulation and activating collaterals, and detoxifying alcohol. It is used for exogenous fever and headache, stiff neck and back pain, thirst, polydipsia, measles that has not yet cleared up, dysentery, diarrhea, dizziness and headache, hemiplegia caused by stroke, chest pain and heartache, and alcohol toxicity. Pueraria root has the following benefits for the liver: 1. It improves the regenerative capacity of liver cells, restores normal liver function, promotes bile secretion, and prevents fat accumulation in the liver; 2. It promotes metabolism, strengthens the liver's detoxification function, and protects against alcohol damage; 3. It strengthens the autoimmune function of hepatobiliary cells and resists viral invasion. Puerarin contains saponins, which have protective effects on improving immunity, inducing apoptosis of activated hepatic stellate cells, effectively reversing chemical-induced liver fibrosis, and protecting against acute liver damage induced by carbon tetrachloride.

[0035] Hovenia dulcis (Houyi) Fruit: Sweet, neutral. It enters the Heart and Spleen meridians and has the effects of relieving coughs, relieving restlessness, clearing dampness and heat, and detoxifying alcohol. It is used to treat alcohol poisoning, thirst, vomiting, and constipation. Hovenia dulcis (Houyi) Fruit contains glucose, fructose, potassium nitrate, and peroxidase, which can significantly reduce blood ethanol concentrations, promote ethanol metabolism in the liver, and eliminate excess free radicals produced in the body after drinking. This reduces ethanol damage to liver tissue and prevents the metabolic abnormalities and liver diseases caused by alcohol poisoning.

[0036] Ginseng: Sweet, slightly bitter, and slightly warm. It enters the spleen, lung, heart, and kidney meridians, and has the effects of greatly replenishing vital energy, restoring the pulse and strengthening the body, tonifying the spleen and lungs, promoting the production of body fluids and nourishing blood, and calming the mind and improving intelligence. It treats symptoms such as physical weakness and collapse, cold limbs and weak pulse, poor appetite due to spleen deficiency, wheezing and coughing due to lung deficiency, thirst due to loss of body fluids, internal heat and thirst, qi and blood deficiency, chronic illness and weakness, palpitations and insomnia, impotence, and cold uterus. Ginseng is rich in nutrients such as ginsenosides, ginseng polysaccharides, ginsenols, ginseng polyacetylene compounds, maltol, essential amino acids, vitamins, and trace elements. It can enhance the activity of liver alcohol dehydrogenase and acetaldehyde dehydrogenase, promote the liver's metabolism of ethanol, and rapidly excrete the toxic substance acetaldehyde produced by ethanol metabolism, thereby effectively protecting the liver.

[0037] Schisandra chinensis: Sour, sweet, and warm. It enters the Lung, Heart, and Kidney meridians, and has astringent, strengthening Qi, promoting the production of body fluids, and tonifying the kidneys and calming the mind. It is used to treat chronic cough, weak asthma, nocturnal emission, spermatorrhea, enuresis, frequent urination, persistent diarrhea, spontaneous sweating, night sweats, thirst due to loss of body fluids, internal heat and thirst, palpitations, and insomnia. Schisandra chinensis protects the liver and promotes the regeneration of liver cells damaged by alcohol abuse, drug abuse, or hepatitis, thereby accelerating liver function recovery.

[0038] Shiitake mushrooms are sweet and neutral. They enter the liver and stomach meridians, stimulating appetite and strengthening the spleen. They are used to treat rickets, anemia, urinary incontinence, pox, measles, hypertension, and tonsillitis. Shiitake mushrooms are rich in polysaccharides and B vitamins. Shiitake polysaccharides protect the liver and enhance its detoxification function, while B vitamins act like a "fuel depot" in the body, accelerating metabolism and converting it into energy. This not only refuels the liver but also repairs damaged liver cells, prevents fatty liver degeneration, and enhances liver function.

[0039] Beneficial effects of the present invention:

[0040] (1) The prescription of the present invention is rationally combined based on the theory of the relationship between the liver and spleen in traditional Chinese medicine. The spleen is responsible for transportation and transformation and is the source of the biochemical transformation of qi and blood. The liver is responsible for storing blood, and the spleen is responsible for blood production and blood regulation. The spleen is healthy and functions well, and is intended to nourish the liver. The various medicinal ingredients in this prescription complement each other and have the effects of replenishing qi and nourishing blood, invigorating qi and producing body fluid, and strengthening the spleen and nourishing the liver.

[0041] (2) The present invention uses a specific complex enzyme for enzymatic hydrolysis, which significantly increases the concentration of effective ingredients such as polysaccharides and oligopeptides in ginseng, schisandra chinensis, and shiitake mushrooms, and has a significant therapeutic effect on chemical liver damage and acute liver damage caused by alcohol;

[0042] (3) The present invention establishes key quality attributes that affect efficacy during the preparation process and evaluates key process parameters. The results show that the amount of cyclodextrin used, the inclusion temperature, the ratio of macroporous adsorption resin to macroporous anionic resin, the type and polarity of the elution solvent, and the flow rate of the eluent significantly affect the extraction of the active ingredient. The present invention significantly improves the efficacy of the composition in treating liver damage through specific inclusion process parameters and purification processes. It has great clinical value and broad market prospects. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific embodiments.

[0044] Example 1

[0045] The composition of this example is composed of the following components in parts by weight: 8 parts of Pueraria root, 3 parts of Hovenia dulcis fruit, 3 parts of Schisandra chinensis, 2 parts of ginseng and 2 parts of Lentinus edodes.

[0046] The preparation method is as follows:

[0047] (1) Ginseng, shiitake mushrooms, and schisandra chinensis were crushed, and steam distilled for 5 h with 5 times the amount of water to obtain filtrate 1, medicinal residue 1, and volatile oil;

[0048] (2) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 rpm at a constant temperature of 65°C for 1.5 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex. The mass ratio of β-cyclodextrin to volatile oil was 4.5:1.

[0049] (3) 0.1% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 5:1) was added to the filtrate 1 and enzymatically hydrolyzed at 45°C for 0.5 h, during which the pH was continuously adjusted to 4.0. The enzyme was then inactivated at 100°C, concentrated, and dried to obtain a dry paste powder 1; β-glucosidase was purchased from Jiangsu Aofu Biotechnology Co., Ltd.; and ficin was purchased from Shaanxi Chenming Biotechnology Co., Ltd.

[0050] (4) Grind the Pueraria root and Hovenia dulcis fruit, mix with the medicinal residue 1, add 6 times the amount of 70% ethanol aqueous solution, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 5 times the amount of water to the filtered medicinal residue and reflux extract for 1.5 hours, filter, and concentrate to obtain a concentrate;

[0051] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed in a mass ratio of 3:1, the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:4, and the column was wet packed with a column diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 6 times the column volume of water, and the eluent flow rate was controlled to be 0.8 mL / min. The eluent was collected, and then eluted with 5 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.5) and 6 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:1) in sequence, and the eluent flow rate was controlled to be 0.5 mL / min. The eluents were collected, combined, concentrated, and dried under reduced pressure to obtain dry paste powder 3;

[0052] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0053] Example 2

[0054] The composition of this embodiment is composed of the following components in parts by weight: 15 parts of Pueraria root, 10 parts of Hovenia dulcis fruit, 12 parts of Schisandra chinensis, 9 parts of ginseng and 10 parts of Lentinus edodes.

[0055] The preparation method is as follows:

[0056] (4) Ginseng, shiitake mushrooms, and schisandra chinensis were crushed, and steam distilled for 7 h with 8 times the amount of water to obtain filtrate 1, medicinal residue 1, and volatile oil;

[0057] (5) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 r / min at a constant temperature of 50°C for 2.5 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex; wherein the mass ratio of β-cyclodextrin to volatile oil was 5.5:1.

[0058] (6) 0.3% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 10:1) was added to the filtrate 1 and enzymatically hydrolyzed at 60°C for 1.5 h, during which the pH was continuously adjusted to 6.0. The enzyme was then inactivated at 100°C, concentrated, and dried to obtain a dry paste powder 1; β-glucosidase was purchased from Jiangsu Aofu Biotechnology Co., Ltd.; and ficin was purchased from Shaanxi Chenming Biotechnology Co., Ltd.

[0059] (4) Grind the Pueraria root and Hovenia dulcis fruit, mix with the medicinal residue 1, add 10 times the amount of 85% ethanol aqueous solution, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 10 times the amount of water to the filtered medicinal residue and reflux extract twice, each time for 1 hour, filter, and concentrate to obtain a concentrated solution;

[0060] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed in a mass ratio of 6:1, and the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:3. The column was wet packed with a diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 8 times the column volume of water, and the eluent flow rate was controlled to be 1.2 mL / min. The eluent was collected, and then eluted with 3 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.8) and 8 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:3) in sequence, and the eluent flow rate was controlled to be 0.7 mL / min. The eluents were collected, combined, concentrated, and dried under reduced pressure to obtain dry paste powder 3;

[0061] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0062] Example 3

[0063] The composition of this example is composed of the following components in parts by weight: 8 parts of Pueraria root, 5 parts of Hovenia dulcis fruit, 6 parts of Schisandra chinensis, 4 parts of ginseng and 5 parts of Lentinus edodes.

[0064] The preparation method is as follows:

[0065] (1) Ginseng, shiitake mushrooms, and schisandra chinensis were crushed, and steam distilled for 6 h with 6 times the amount of water to obtain filtrate 1, medicinal residue 1, and volatile oil;

[0066] (2) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 rpm at a constant temperature of 55°C for 2 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex; wherein the mass ratio of β-cyclodextrin to volatile oil was 5:1.

[0067] (3) 0.2% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 8:1) was added to the filtrate 1 and enzymatically hydrolyzed at 50°C for 1.0 h, during which the pH was continuously adjusted to 5.5. The enzyme was then inactivated at 100°C, concentrated, and dried to obtain a dry paste powder 1; β-glucosidase was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ficin was purchased from Shenzhen Love Biotechnology Co., Ltd.;

[0068] (4) Grind the Pueraria root and Hovenia dulcis fruit, mix with the medicinal residue 1, add 8 times the amount of 80% ethanol aqueous solution, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 8 times the amount of water to the filtered medicinal residue and reflux extract for 1 hour, filter, and concentrate to obtain a concentrated solution;

[0069] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed at a mass ratio of 5.5:1, and the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:5. The column was wet packed with a diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 8 times the column volume of water, and the eluent flow rate was controlled to be 1.0 mL / min. The eluent was collected, and then eluted with 5 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.6) and 6 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:2) in sequence, and the eluent flow rate was controlled to be 0.6 mL / min. The eluents were collected, combined, concentrated, and dried under reduced pressure to obtain dry paste powder 3;

[0070] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0071] Comparative Example 1

[0072] The difference between this comparative example and Example 3 is that 5 parts of the raw material shiitake mushrooms in the composition are adjusted to 5 parts of wolfberries, and the rest are consistent with Example 3.

[0073] The composition of this comparative example is composed of the following components in parts by weight: 8 parts of Pueraria root, 5 parts of Hovenia dulcis fruit, 6 parts of Schisandra chinensis, 4 parts of ginseng and 5 parts of wolfberry fruit.

[0074] The preparation method is as follows:

[0075] (1) Ginseng, wolfberry, and schisandra chinensis were crushed, and steam distilled for 6 h with 6 times the amount of water to obtain filtrate 1, medicinal residue 1, and volatile oil;

[0076] (2) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 rpm at a constant temperature of 55°C for 2 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex; wherein the mass ratio of β-cyclodextrin to volatile oil was 5:1.

[0077] (3) 0.2% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 8:1) was added to the filtrate 1 and enzymatically hydrolyzed at 50°C for 1.0 h, during which the pH was continuously adjusted to 5.5. The enzyme was then inactivated at 100°C, concentrated, and dried to obtain a dry paste powder 1; β-glucosidase was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ficin was purchased from Shenzhen Love Biotechnology Co., Ltd.;

[0078] (4) Grind the Pueraria root and Hovenia dulcis fruit, mix with the medicinal residue 1, add 8 times the amount of 80% ethanol aqueous solution, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 8 times the amount of water to the filtered medicinal residue and reflux extract for 1 hour, filter, and concentrate to obtain a concentrated solution;

[0079] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed at a mass ratio of 5.5:1, and the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:5. The column was wet packed with a diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 8 times the column volume of water, and the eluent flow rate was controlled to be 1.0 mL / min. The eluent was collected, and then eluted with 5 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.6) and 6 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:2) in sequence, and the eluent flow rate was controlled to be 0.6 mL / min. The eluents were collected, combined, concentrated, and dried under reduced pressure to obtain dry paste powder 3;

[0080] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0081] Comparative Example 2

[0082] The difference between this comparative example and Example 3 is that 5 parts of shiitake mushrooms are removed, and the other medicinal materials and their proportions are: 8 parts of Pueraria root, 5 parts of Hovenia dulcis fruit, 6 parts of Schisandra chinensis and 4 parts of ginseng; the rest are the same as Example 3.

[0083] The preparation method is as follows:

[0084] (1) Ginseng and Schisandra chinensis were crushed, and steam distilled for 6 h with 6 times the amount of water to obtain filtrate 1, residue 1, and volatile oil;

[0085] (2) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 rpm at a constant temperature of 55°C for 2 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex; wherein the mass ratio of β-cyclodextrin to volatile oil was 5:1.

[0086] (3) 0.2% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 8:1) was added to the filtrate 1 and enzymatically hydrolyzed at 50°C for 1.0 h, during which the pH was continuously adjusted to 5.5. The enzyme was then inactivated at 100°C, concentrated, and dried to obtain a dry paste powder 1; β-glucosidase was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ficin was purchased from Shenzhen Love Biotechnology Co., Ltd.;

[0087] (4) Grind the Pueraria root and Hovenia dulcis fruit, mix with the medicinal residue 1, add 8 times the amount of 80% ethanol aqueous solution, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 8 times the amount of water to the filtered medicinal residue and reflux extract for 1 hour, filter, and concentrate to obtain a concentrated solution;

[0088] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed at a mass ratio of 5.5:1, and the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:5. The column was wet packed with a diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 8 times the column volume of water, and the eluent flow rate was controlled to be 1.0 mL / min. The eluent was collected, and then eluted with 5 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.6) and 6 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:2) in sequence, and the eluent flow rate was controlled to be 0.6 mL / min. The eluents were collected, combined, concentrated, and dried under reduced pressure to obtain dry paste powder 3;

[0089] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0090] Comparative Example 3

[0091] The raw material of this comparative example composition is only 28 parts of shiitake mushrooms, and the preparation method is as follows:

[0092] (1) Crush the shiitake mushrooms, add 6 times the amount of water and perform steam distillation extraction to obtain a filtrate 1, a medicinal residue 1 and volatile oil;

[0093] (2) A 0.1 g / mL β-cyclodextrin aqueous solution was prepared in β-cyclodextrin, and the volatile oil was slowly added to the β-cyclodextrin solution. The solution was magnetically stirred at 250 rpm at a constant temperature of 55°C for 2 h, cooled, filtered, and the precipitate was washed with 1 times the amount of water and dried to obtain an inclusion complex; wherein the mass ratio of β-cyclodextrin to volatile oil was 5:1.

[0094] (3) adding 0.2% enzyme mixture (a mixture of β-glucosidase and ficin at a mass ratio of 8:1) to the filtrate 1 and performing enzymolysis at 50°C for 1.0 h, during which the pH was continuously adjusted to 5.5, and then inactivating the enzyme at 100°C, concentrating, and drying to obtain a dry paste powder 1;

[0095] (4) Add 8 times the amount of 80% ethanol aqueous solution to the medicinal residue 1, heat and reflux extract for 1 hour, filter, concentrate and dry to obtain dry paste powder 2, add 8 times the amount of water to the filtered medicinal residue and reflux extract for 1 hour, filter, and concentrate to obtain a concentrated solution;

[0096] (5) The pretreated macroporous adsorption resin AB-8 and the macroporous anion resin D315 were mixed at a mass ratio of 5.5:1, and the ratio of the mass of the concentrate to the total mass of the macroporous adsorption resin AB-8 and the macroporous anion resin D315 was 1:5. The column was wet packed with a diameter-to-height ratio of 1:12. The concentrate was adsorbed on the column, and then eluted with 8 times the column volume of water, and the eluent flow rate was controlled to 1.0 mL / min. The eluent 1 was collected, and then eluted with 5 times the column volume of a mixed solution of 60% ethanol and acetone (volume ratio of 1:0.6) and 6 times the column volume of a mixed solution of 95% ethanol and n-butanol (volume ratio of 1:2) in sequence, and the eluent flow rate was controlled to 0.6 mL / min. The eluent 2 was collected, the eluents were combined, and concentrated and dried to obtain dry paste powder 3;

[0097] (6) The inclusion compound, dry paste powder 1, dry paste powder 2 and dry paste powder 3 are uniformly mixed to obtain a mixture, thereby obtaining a composition.

[0098] Comparative Example 4

[0099] The difference between this comparative example and Example 3 is that the enzyme used in step (1) is a mixture of β-glucosidase and papain, with a mass ratio of 8:1, and the rest is the same as Example 3. Papain was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0100] Comparative Example 5

[0101] The difference between this comparative example and Example 3 is that in step (3), the mass ratio of the macroporous adsorption resin AB-8 to the macroporous anion resin D315 is 2.0:1, and the rest is consistent with Example 3.

[0102] Each of the above compositions can be added with 10%-50% of the composition weight of a filler (such as dextrin, lactose or maltodextrin), and then 0.05-0.2% of magnesium stearate, or other excipients, and dry granulated or wet granulated to obtain granules, which can also be further prepared into oral preparations such as tablets and capsules.

[0103] Experimental Example 1 Therapeutic effect of the composition of the present invention on acute liver injury in mice induced by CCl4

[0104] 1.1 Experimental Animals

[0105] Clean-grade female Kunming mice, weighing 22-25 g, were housed in an animal room with a room temperature of 25°C, a humidity of 50%, and a 12-h dark and 12-h light cycle. They were raised in three-dimensional cages and given standard feed and free access to water. The experimental animals were provided by the Experimental Animal Center of Yanbian University.

[0106] 1.2 Experimental groups and drug administration

[0107] 110 Kunming mice were randomly divided into 11 groups, each with 10 mice: a blank group, a model group, groups of Examples 1 to 3, groups of Comparative Examples 1 to 5, and a positive control group of silymarin (100 mg / kg). The dose / kg administered to mice in each Example and Comparative Example group was equivalent to 1.5 g of the crude drug, where the crude drug amount represents the mass of the raw materials used to prepare the composition.

[0108] Experimental animals were orally gavaged daily. The blank control group and the model control group were given an equal amount of normal saline for a total of 7 days. Four hours after the last dose, mice in all groups except the blank group were gavaged with CCl4 (0.4% CCl4 in olive oil solution 0.1ml / 10g). The blank control group was given an olive oil solution without CCl4 0.1ml / 10g. The animals were fasted but not watered. 20 hours after CCl4 administration, blood was collected from the carotid artery, serum was separated, and ALT and AST were measured (ALT and AST were measured according to the kit instructions). The remaining blood was stored in liquid nitrogen or a low-temperature refrigerator (-80°C).

[0109] 1.3 Experimental results and statistical analysis

[0110] Experimental data were statistically analyzed using GraphPad Prism (USA) and expressed as mean ± standard deviation (SEM). All data were compared using one-way ANOVA and Tukey's multiple comparison test. The results are shown in Table 1.

[0111] Table 1 Test results of biochemical indicators of mice in each group of Experimental Example 1

[0112] experimental group Number of mice ALT (IU / L) AST (IU / L) Blank group 10 <![CDATA[49.35±9.73 a ]]> <![CDATA[7.34±0.95 a ]]> Model Group 10 <![CDATA[245.01±3.17 b ]]> <![CDATA[61.93±5.03 b ]]> Positive group 10 <![CDATA[120.82±13.58 c ]]> <![CDATA[38.93±3.73 c ]]> Example 1 10 <![CDATA[79.01±16.69 d ]]> <![CDATA[29.36±6.95 d ]]> Example 2 10 <![CDATA[77.83±13.82 d ]]> <![CDATA[29.08±5.58 d ]]> Example 3 10 <![CDATA[76.95±12.87 d ]]> <![CDATA[28.89±2.97 d ]]> Comparative Example 1 10 <![CDATA[95.25±15.93 e ]]> <![CDATA[35.92±4.25 c ]]> Comparative Example 2 10 <![CDATA[101.53±17.15 e ]]> <![CDATA[40.15±7.68 c ]]> Comparative Example 3 10 <![CDATA[176.80±11.94 f ]]> <![CDATA[49.93±9.54 e ]]> Comparative Example 4 10 <![CDATA[83.04±16.81 e ]]> <![CDATA[33.26±8.41 c ]]> Comparative Example 5 10 <![CDATA[89.17±13.46 e ]]> <![CDATA[35.83±6.75 c ]]>

[0113] Note: Different letters in the same column indicate comparisons between corresponding groups, P < 0.05.

[0114] Results: The serum ALT and AST levels in the model group were significantly higher than those in the normal group (p < 0.001), indicating that the CCl4-induced acute liver injury model in mice was successfully established. The serum ALT and AST levels in the composition of the present invention were significantly lower than those in the model group and the control group (p < 0.05). The serum ALT and AST levels in the positive control group, silymarin, were also significantly lower than those in the model group. The combination of the various medicinal ingredients and the purification process had a significant, statistically significant impact on the efficacy results.

[0115] Experimental Example 2 Therapeutic Effect of the Composition of the Present Invention on Acute Liver Injury Induced by Ethanol in Mice

[0116] 2.1 Experimental animals

[0117] Clean-grade female Kunming mice, weighing 22-25 g, were housed in an animal room at 25°C, 50% humidity, 12 hours of darkness and 12 hours of light. They were raised in a three-dimensional cage and given standard feed and free access to water.

[0118] 2.2 Experimental groups and drug administration

[0119] The 110 Kunming mice were randomly divided into 11 groups, each with 10 mice: a blank group, a model group, groups of Examples 1 to 3, groups of Comparative Examples 1 to 5, and a positive control group of silymarin 100 mg / kg. The single dose of the raw drug was 1.5 g / kg for each Example and Comparative Example group.

[0120] Experimental animals were administered orally daily via gavage. The blank and model control groups received an equal volume of normal saline for a total of 7 days. Four hours after the last dose, mice in all groups except the blank group received a single gavage of 50% ethanol (12 ml / kg). The blank control group received distilled water and remained fasting. Twelve hours after ethanol administration, blood was collected from the carotid artery, serum was separated, and ALT was measured. Mice were sacrificed, and livers were harvested for analysis.

[0121] Determination of biochemical indicators of liver homogenate: Take part of the liver tissue, make 10% liver homogenate with 4°C normal saline, centrifuge at 3500 rpm for 15 minutes, take the supernatant for the determination of liver tissue TG, MDA, and GSH content, and store the remaining part in a low-temperature refrigerator (-80°C).

[0122] 2.3 Experimental results and statistical analysis

[0123] The experimental data were statistically analyzed using the GraphPad Prism program (GraphPad Software, Inc., San Diego, USA) and expressed as mean ± standard deviation (mean ± SEM). All data were compared using one-way ANOVA and Tukey's multiple comparison tests. The results are shown in Table 2.

[0124] Table 2 Test results of biochemical indicators of mice in each group of Experimental Example 2

[0125]

[0126] Note: Different letters in the same column indicate comparisons between corresponding groups, P < 0.05.

[0127] Result analysis: Lipid metabolism disorder is an early sign of alcoholic liver damage. Excessive alcohol intake can also lead to damaged mitochondrial function of hepatocytes, inhibit the oxidative utilization of liver lipids, and lead to hepatic triglyceride (TG) deposition.

[0128] The protective effect of the present composition on an ethanol-induced acute liver injury model in mice was evaluated by changes in TG, MDA, and GSH levels in liver homogenates and serum ALT. The changes in TG, MDA, GSH levels in liver homogenates and serum ALT in the model group were significantly higher than those in the normal group (p < 0.001). The TG, MDA, GSH levels in liver homogenates and ALT levels in the positive control group, the silymarin group, were significantly different from those in the model group. The TG and GSH levels in liver homogenates of the present invention group were significantly different from those in the positive control group. The decrease in MDA levels was comparable to that in the positive control group. These results demonstrate that the present composition significantly improves liver physiological function.

[0129] The embodiments of the present invention are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention. In addition, after reading the technical contents of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all such equivalent forms also fall within the scope of protection defined by the appended claims of the present application.

Claims

1. A composition having a protective function against chemical liver damage, characterized in that: The raw materials of the composition are composed of the following components in parts by weight: 8-15 parts of kudzu root, 3-10 parts of hovenia dulcis fruit, 3-12 parts of schisandra chinensis, 2-9 parts of ginseng and 2-10 parts of shiitake mushroom.

2. The composition according to claim 1, characterized in that The raw materials of the composition are composed of the following components in parts by weight: 8-10 parts of kudzu root, 5-10 parts of hovenia dulcis fruit, 6-12 parts of schisandra chinensis, 4-9 parts of ginseng and 5-10 parts of shiitake mushroom.

3. The composition according to claim 1, characterized in that The raw materials of the composition are composed of the following components in parts by weight: 8 parts of kudzu root, 5 parts of hovenia dulcis fruit, 6 parts of schisandra chinensis, 4 parts of ginseng and 5 parts of shiitake mushroom.

4. The composition according to claim 1, characterized in that The composition further includes pharmaceutically acceptable excipients.

5. The composition according to claim 4, characterized in that The auxiliary material is selected from one or more of fillers, glidants, lubricants, binders, disintegrants or flavoring agents.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that: The steps include: (1) Ginseng, shiitake mushrooms and schisandra chinensis are crushed, and water is added to perform steam distillation extraction to obtain a filtrate 1, a medicinal residue 1 and a volatile oil; the volatile oil is included in the inclusion compound with β-cyclodextrin; the filtrate 1 is enzymatically hydrolyzed, the enzyme is inactivated at 95-100°C, and the mixture is concentrated and dried to obtain a dry paste powder 1; (2) Grind the Pueraria root and Hovenia dulcis fruit, mix them with the medicinal residue 1, add 70-85% ethanol, heat and reflux to extract, filter to obtain filtrate 2 and medicinal residue 2, concentrate and dry the filtrate 2 to obtain dry paste powder 2, add water to the medicinal residue 2, reflux to extract, filter, and concentrate to obtain a concentrated solution; (3) Mix the macroporous adsorption resin AB-8 and the macroporous anion resin D315, wet-pack the column, adsorb the concentrated solution onto the column, and then elute with 6-8 times the column volume of water, then gradient elute with an organic solvent, combine the eluates, and concentrate under reduced pressure to obtain dry paste powder 3; (4) Evenly mix the inclusion compound, dry paste powder 1, dry paste powder 2, dry paste powder 3 and auxiliary materials to obtain; The enzyme used for enzymatic hydrolysis in step (1) is an enzyme mixture consisting of β-glucosidase and ficin in a mass ratio of 5-10:1; The organic solvent gradient elution in step (3) is eluted with a mixed solution of 60% ethanol and acetone for 3-5 times the column volume and a mixed solution of 95% ethanol and n-butanol for 6-8 times the column volume.

7. The preparation method according to claim 6, characterized in that The ratio of the mass of water to the total mass of ginseng, shiitake mushrooms and schisandra chinensis in step (1) is 5-8:1; and the extraction time is 5-7 hours.

8. The preparation method according to claim 6, characterized in that In step (1), the mass ratio of volatile oil to β-cyclodextrin is 1:4.5-5.5, the inclusion time is 1.5-2.5 hours, and the inclusion temperature is 50-65°C.

9. The preparation method according to claim 6, characterized in that The amount of the enzyme mixture added in step (1) is 0.1-0.3% of the mass of the filtrate 1, the temperature of the enzymatic hydrolysis is 45-60° C., the time of the enzymatic hydrolysis is 0.5-1.5 h, and the pH of the enzymatic hydrolysis is 4.0-6.

0.

10. The preparation method according to claim 6, characterized in that The mass ratio of the 70-85% ethanol in step (2) to the total mass of Pueraria root, Hovenia dulcis fruit and the medicinal residue 1 is 6-10:1; the extraction time is 1-2 hours; the mass ratio of water to the medicinal residue 2 is 5-10:1; the medicinal residue 2 is subjected to reflux extraction with water 1-2 times, and each extraction time is 1-1.5 hours; and the oral preparation is prepared after uniform mixing in step (4).

11. The preparation method according to claim 6, characterized in that The mass ratio of the macroporous adsorption resin to the macroporous anion resin in step (3) is 3-6:1; and the flow rate of the eluent is controlled to be 0.8-1.2 mL / min during elution with water.

12. The preparation method according to claim 6, characterized in that The volume ratio of 60% ethanol to acetone in step (3) is 1:0.5-0.8; the volume ratio of 95% ethanol to n-butanol is 1:1-3; and the flow rate of the eluent is controlled to be 0.5-0.7 mL / min during the organic solvent gradient elution.

13. Use of the composition according to any one of claims 1 to 5 or the composition prepared by the preparation method according to any one of claims 6 to 12 in preparing a drug for treating chemical liver injury.

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