Application of hericium erinaceus composite extract in preparation of medicine for treating alcoholic liver injury

Abstract

The invention provides application of a hericium erinaceus composite extract in preparation of a medicine for treating alcoholic liver injury, and relates to the technical field of the medicine for treating the alcoholic liver injury. The hericium erinaceus composite extract is composed of a hericium erinaceus petroleum ether extract, a hericium erinaceus ethyl acetate extract and a hericium erinaceus water extract. The hericium erinaceus composite extract is prepared from hericium erinaceus as a raw material through the process steps of ethanol extraction, petroleum extraction, ethyl acetate extraction, filter residue enzymolysis fermentation, water extraction, drying, mixing and the like. Through multi-step extraction and enzymolysis fermentation processes, the purity and activity of the extract are improved, the effects of various active ingredients are integrated, the overall curative effect of the medicine is enhanced, and the obtained composite extract can be used for preparing the medicine for treating the alcoholic liver injury and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of alcoholic liver injury drugs, and in particular to an application of a Hericium erinaceus composite extract in the preparation of a drug for treating alcoholic liver injury. Background Art

[0002] Hericium erinaceus, also known as Hericium erinaceus, Hericium erinaceus, Hedgehog mushroom, etc., is a precious edible and medicinal fungus, named after its appearance that resembles a monkey's head. It is rich in protein, amino acids, polysaccharides, vitamins and minerals, and has the effects of promoting digestion, enhancing immunity and protecting nerves. In nature, Hericium erinaceus mainly grows in broad-leaved forests or mixed coniferous and broad-leaved forests in the northern temperate zone. Artificial cultivation requires specific environmental conditions, including suitable temperature, water, air and light.

[0003] At present, Hericium erinaceus is mainly used in the fields of health care products, medicines, foods and functional foods. Because it is rich in polysaccharides, proteins and various trace elements, it is widely used in the research and development of health care products, such as Hericium erinaceus nutritional powder and health drinks; in terms of medicine, Hericium erinaceus has anti-tumor, anti-oxidation, immunity enhancement and gastric mucosal protection effects, and is used to develop a variety of medicines, such as Hericium erinaceus tablets and Hericium erinaceus stomach-strengthening spirit; as a precious edible fungus, Hericium erinaceus is often used in the high-end catering market, made into Hericium erinaceus biscuits and canned foods; in addition, through innovative extraction technology, active ingredients such as polysaccharides of Hericium erinaceus are used in the research and development of functional foods to meet consumers' needs for health and health care.

[0004] The invention patent with publication number CN118556871A provides a composition for relieving gastrointestinal bloating, its preparation method and application. The composition is composed of pea protein powder, chickpea protein powder, hericium erinaceus powder and hawthorn powder. The composition combines plant-based protein bars with medicinal and edible ingredients, has a significant effect in relieving gastrointestinal bloating, and takes into account the easy absorption of nutrients and better flavor and taste, solving the problems of large side effects of Western medicine and unacceptable taste of traditional Chinese medicine.

[0005] The invention patent with publication number CN118388677A provides a method for extracting Hericium erinaceus polysaccharide and its application. The method adopts a combination of betaine alkalization of Hericium erinaceus polysaccharide and freezing treatment to improve the extraction rate of Hericium erinaceus polysaccharide. The prepared Hericium erinaceus polysaccharide has low protein content, high transparency and good stability, and can be used to prepare Hericium erinaceus polysaccharide biscuits.

[0006] It can be seen that the functional research on Hericium erinaceus in the prior art is mostly focused on the research and development of stomach-nourishing and stomach-protecting or functional foods. However, there are few studies and developments on other functions of Hericium erinaceus, which limits its application scope. Therefore, in-depth research on the various functional components of Hericium erinaceus and exploration of new extract application methods are of great significance to promote the development of related fields. Summary of the invention

[0007] In view of this, the present invention provides an application of a composite extract of Hericium erinaceus in the preparation of a drug for treating alcoholic liver injury. The present invention achieves the extraction of multiple different effective components of Hericium erinaceus by adjusting the extraction process, and utilizes the synergistic effect between different extraction components to obtain a composite extract with good therapeutic effect on alcoholic liver injury.

[0008] The preparation method of the Hericium erinaceus composite extract of the present invention comprises the following steps:

[0009] S1. Dried Hericium erinaceus, crushed and sieved to obtain Hericium erinaceus dry powder;

[0010] S2, mixing the Hericium erinaceus dry powder with an ethanol solution for leaching, filtering after the leaching is completed, repeating 2 to 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0011] S3, mixing the extract and petroleum ether by oscillation for 10 minutes and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 2 to 3 times, combining the petroleum ether phase and the aqueous phase respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0012] S4, shaking and mixing the aqueous phase and ethyl acetate for 10 minutes, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 2 to 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining the ethyl acetate extract of Hericium erinaceus;

[0013] S5, enzymatically fermenting the filter residue, and then extracting it with water, repeating this process 2 to 3 times, combining the extracts, and obtaining a water extract of Hericium erinaceus;

[0014] S6. Drying and mixing the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus to obtain a composite extract of Hericium erinaceus.

[0015] Further, the drying temperature of the Hericium erinaceus is 30° C. to 40° C., and the water content of the Hericium erinaceus dry powder is 8% to 12%. Preferably, the drying temperature of the Hericium erinaceus is 35° C., and the water content of the Hericium erinaceus dry powder is 10%.

[0016] Further, the concentration of the ethanol solution is 70% to 80%, the extraction temperature is 30°C to 40°C, the extraction time is 1 to 3 hours, and the weight-volume ratio of Hericium erinaceus dry powder to the ethanol solution is 1g:(10-20)mL. Preferably, the concentration of the ethanol solution is 75%, the extraction temperature is 35°C, the extraction time is 2 hours, and the weight-volume ratio of Hericium erinaceus dry powder to the ethanol solution is 1g:15mL.

[0017] Furthermore, the volume ratio of the extract to petroleum ether is 1:(1-1.5). Preferably, the volume ratio of the extract to petroleum ether is 1:1.2.

[0018] Further, the volume ratio of the aqueous phase to ethyl acetate is 1:(1-1.5). Preferably, the volume ratio of the aqueous phase to ethyl acetate is 1:1.4.

[0019] Furthermore, the filter residue enzymatic hydrolysis and fermentation process is as follows: an enzyme preparation is added to the filter residue, and a starter is added for fermentation after enzymatic hydrolysis.

[0020] Furthermore, the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:(1-5), and the enzymatic activity of the cellulase is 2×10 4 ~4×10 4 U / g, the enzyme activity of the pectinase is 3×10 4 ~5×10 4 Preferably, the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2.5, and the enzymatic activity of the cellulase is 3×10 4 U / g, the enzyme activity of the pectinase is 4×10 4 U / g.

[0021] Furthermore, the enzymolysis temperature is 40°C to 50°C, and the enzymolysis time is 2 to 4 hours. Preferably, the enzymolysis temperature is 45°C, and the enzymolysis time is 3 hours.

[0022] Furthermore, the fermentation agent is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:(1-5), and the bacterial activity of Lactobacillus plantarum is 2×10 12 ~4×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 ~3×10 12 CFU / g. Preferably, the starter is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:3, and the bacterial activity of Lactobacillus plantarum is 3×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 CFU / g.

[0023] Furthermore, the fermentation temperature is 25°C to 30°C, and the fermentation time is 3 to 7 days. Preferably, the fermentation temperature is 30°C, and the fermentation time is 5 days.

[0024] Furthermore, the water extraction temperature is 40°C to 60°C, and the water extraction time is 1 to 2 hours. Preferably, the water extraction temperature is 50°C, and the water extraction time is 1.5 hours.

[0025] Furthermore, the weight-to-volume ratio of the filter residue to water is 1 g:(15-25) mL. Preferably, the weight-to-volume ratio of the filter residue to water is 1 g:20 mL.

[0026] Further, the mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is (3-7):(0.1-1):(5-10). Preferably, the mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is 5:0.5:7.

[0027] The present invention also provides a Hericium erinaceus composite extract prepared according to the method.

[0028] The Hericium erinaceus composite extract of the invention is used for preparing a medicine for treating alcoholic liver damage.

[0029] Compared with the prior art, the beneficial technical effects of the present invention are:

[0030] The invention extracts the effective components in Hericium erinaceus to the maximum extent through multi-step extraction and enzymatic fermentation processes, thereby improving the purity and activity of the extract.

[0031] The invention combines petroleum ether extract, ethyl acetate extract and water extract of Hericium erinaceus, integrates the effects of multiple active ingredients, and enhances the overall therapeutic effect of the medicine.

[0032] The extraction and fermentation process of the invention is carried out under relatively mild conditions, thus avoiding the destruction of active ingredients by high temperature and maintaining the biological activity of the extract.

[0033] The present invention ensures the high efficiency and repeatability of each step by optimizing the conditions of each step, such as temperature, time and ratio, thereby improving production efficiency and product quality.

[0034] The composite extract of the invention can be used to prepare medicines for treating alcoholic liver damage and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below in conjunction with the accompanying drawings.

[0036] Figure 1The changes of liver in rats with different treatments, where a: Con group; b: Mod group; c: L1 group;

[0037] Figure 2 H&E staining results of the livers of rats with different treatments, where a: Con group; b: Mod group; c: L1 group. DETAILED DESCRIPTION

[0038] The present invention provides an application of a Hericium erinaceus composite extract in preparing a drug for treating alcoholic liver damage. The preparation method of the Hericium erinaceus composite extract specifically comprises the following steps:

[0039] S1. Dried Hericium erinaceus, crushed and sieved to obtain Hericium erinaceus dry powder;

[0040] S2, mixing the Hericium erinaceus dry powder with an ethanol solution for leaching, filtering after the leaching is completed, repeating 2 to 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0041] S3, mixing the extract and petroleum ether by oscillation for 10 minutes and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 2 to 3 times, combining the petroleum ether phase and the aqueous phase respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0042] S4, shaking and mixing the aqueous phase and ethyl acetate for 10 minutes, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 2 to 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining the ethyl acetate extract of Hericium erinaceus;

[0043] S5, enzymatically fermenting the filter residue, and then extracting it with water, repeating this process 2 to 3 times, combining the extracts, and obtaining a water extract of Hericium erinaceus;

[0044] S6. Drying and mixing the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus to obtain a composite extract of Hericium erinaceus.

[0045] In one embodiment, the drying temperature of the Hericium erinaceus is 30° C. to 40° C., and the water content of the Hericium erinaceus dry powder is 8% to 12%.

[0046] In one embodiment, the drying temperature of the Hericium erinaceus is 35° C., and the water content of the Hericium erinaceus dry powder is 10%.

[0047] In principle, the present invention does not strictly limit the drying temperature of Hericium erinaceus, as long as the water content in the raw material of Hericium erinaceus can be reduced to the required range. However, the use of a drying temperature of 30°C to 40°C is more conducive to ensuring the quality and stability of the extract. The reason is that Hericium erinaceus contains a variety of active ingredients that are sensitive to temperature, and too high a drying temperature may cause them to degrade, thereby affecting the efficacy of the extract. Therefore, it is recommended to use low-temperature drying to process the raw materials.

[0048] Different from the drying temperature, the present invention strictly limits the water content of Hericium erinaceus dry powder to prevent the dry powder from absorbing moisture or agglomerating during storage, thereby affecting the extraction rate, and also avoiding the growth of microorganisms and affecting the quality of the extract.

[0049] In one embodiment, the concentration of the ethanol solution is 70% to 80%, the extraction temperature is 30° C. to 40° C., the extraction time is 1 to 3 hours, and the weight-to-volume ratio of Hericium erinaceus dry powder to the ethanol solution is 1 g: (10 to 20) mL.

[0050] In one embodiment, the concentration of the ethanol solution is 75%, the extraction temperature is 35° C., the extraction time is 2 h, and the weight-to-volume ratio of Hericium erinaceus dry powder to the ethanol solution is 1 g:15 mL.

[0051] The present invention utilizes 70% to 80% ethanol solution to effectively dissolve multiple active ingredients in Hericium erinaceus and has the best bactericidal effect; the extraction temperature of 30°C to 40°C can prevent the degradation of heat-sensitive components, while improving the permeability and solubility of the solvent; the extraction time of 1 to 3 hours can ensure that most of the active ingredients are extracted, while avoiding energy waste and cost increase caused by too long a time; the weight-to-volume ratio of 1g: (10 to 20) mL can ensure that the ethanol solution is in full contact with Hericium erinaceus dry powder, improve the extraction efficiency, and avoid using too much solvent. The setting of these parameters is based on single factor, orthogonal experiments and practical experience, aiming to maximize the extraction efficiency and the quality of the extract.

[0052] In one embodiment, the volume ratio of the extract to petroleum ether is 1:(1-1.5).

[0053] In one embodiment, the volume ratio of the extract to petroleum ether is 1:1.2.

[0054] In one embodiment, the volume ratio of the aqueous phase to ethyl acetate is 1:(1-1.5).

[0055] In one embodiment, the volume ratio of the aqueous phase to ethyl acetate is 1:1.4.

[0056] The present invention limits the volume ratio of the extract to petroleum ether and the volume ratio of the aqueous phase to ethyl acetate to optimize the efficiency and effect of the extraction process. Specifically, within a specific volume ratio range, it can ensure that the extractant is in full contact with the extract, thereby maximizing the extraction of the target component; at the same time, the appropriate volume ratio can avoid the use of too much extractant, reduce costs and reduce solvent residues; in addition, this ratio helps to form clear stratification during the extraction process, which is convenient for subsequent separation and collection. The setting of these parameters is based on single factor, orthogonal experiments and practical experience, and is intended to ensure the efficiency of the extraction process and the quality of the extract.

[0057] In one embodiment, the filter residue enzymatic hydrolysis and fermentation process is as follows: an enzyme preparation is added to the filter residue, and a starter is added for fermentation after enzymatic hydrolysis.

[0058] In one embodiment, the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:(1-5), and the enzymatic activity of the cellulase is 2×10 4 ~4×10 4 U / g, the enzyme activity of the pectinase is 3×10 4 ~5×10 4 U / g.

[0059] In one embodiment, the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2.5, and the enzymatic activity of the cellulase is 3×10 4 U / g, the enzyme activity of the pectinase is 4×10 4 U / g.

[0060] In one embodiment, the enzymolysis temperature is 40°C to 50°C, and the enzymolysis time is 2 to 4 hours.

[0061] In one embodiment, the enzymatic hydrolysis temperature is 45° C. and the enzymatic hydrolysis time is 3 h.

[0062] The present invention limits the key parameters involved in the enzyme preparation and the enzymatic hydrolysis process, which can ensure the synergistic effect of cellulase and pectinase under optimal conditions, effectively destroy the structure of the Hericium erinaceus cell wall, and promote the release of effective ingredients; at the same time, the appropriate enzyme activity and temperature range can prevent the inactivation of the enzyme, ensure the stability and high efficiency of the enzymatic hydrolysis process, and improve the extraction efficiency.

[0063] In one embodiment, the fermentation agent is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:(1-5), and the bacterial activity of Lactobacillus plantarum is 2×10 12 ~4×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 ~3×10 12 CFU / g.

[0064] In one embodiment, the fermentation agent is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:3, and the bacterial activity of Lactobacillus plantarum is 3×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 CFU / g.

[0065] In the present invention, Lactobacillus and Bacillus subtilis can work synergistically during the fermentation process. Lactobacillus can produce lactic acid, reduce the pH value of the environment, and thus inhibit the growth of harmful microorganisms; Bacillus subtilis can secrete a variety of enzymes to promote the decomposition of organic matter and improve fermentation efficiency. In addition, the appropriate bacterial activity and ratio can ensure that the two strains remain active and stable during the fermentation process, thereby improving the quality and biological activity of the final extract.

[0066] In one embodiment, the fermentation temperature is 25°C to 30°C, and the fermentation time is 3 to 7 days.

[0067] In one embodiment, the fermentation temperature is 30° C. and the fermentation time is 5 days.

[0068] In one embodiment, the water extraction temperature is 40° C. to 60° C., and the water extraction time is 1 to 2 hours.

[0069] In one embodiment, the water extraction temperature is 50° C. and the water extraction time is 1.5 h.

[0070] In one embodiment, the weight volume ratio of the filter residue to water is 1 g: (15-25) mL.

[0071] In one embodiment, the weight volume ratio of the filter residue and water is 1 g:20 mL.

[0072] The present invention first enzymatically hydrolyzes the Hericium erinaceus filter residue and then ferments it, and finally water extracts it to maximize the extraction of effective ingredients and improve the quality and functionality of the extract. Specifically, by adding cellulase and pectinase for enzymatic hydrolysis, the dense structure of the Hericium erinaceus cell wall is destroyed, and more effective ingredients, such as polysaccharides and proteins, are released; then fermentation is performed using Lactobacillus and Bacillus subtilis, which can further decompose complex organic matter and produce more biologically active substances, such as organic acids and enzymes, while inhibiting the growth of harmful microorganisms and improving the safety and stability of the extract; the final water extraction step can extract water-soluble components, such as polysaccharides and other water-soluble active substances, to ensure the comprehensiveness and diversity of the extract, and increase its functionality and application range. Through the combination of these three steps, the effective ingredients in the Hericium erinaceus residue can be maximized, and the quality and biological activity of the extract can be improved.

[0073] In one embodiment, the mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is (3-7):(0.1-1):(5-10).

[0074] In one embodiment, the mass ratio of the Hericium erinaceus petroleum ether extract, the Hericium erinaceus ethyl acetate extract and the Hericium erinaceus water extract is 5:0.5:7.

[0075] The present invention also provides a Hericium erinaceus composite extract prepared according to the method.

[0076] The Hericium erinaceus composite extract of the invention is used for preparing a medicine for treating alcoholic liver damage.

[0077] There are many forms of Hericium erinaceus extracts, each of which has its own unique ingredients and effects. The petroleum ether extract of Hericium erinaceus of the present invention mainly contains fat-soluble components such as fatty acids and sterols, the ethyl acetate extract of Hericium erinaceus is rich in polyphenols and flavonoids, the water extract of Hericium erinaceus mainly contains water-soluble components such as polysaccharides and oligosaccharides, and the alcohol extract includes a variety of polar components. The present invention uses the synergistic effect between the multiple extracts to make the composition have a good treatment and improvement effect on alcoholic liver injury.

[0078] The technical solution provided by the present invention is further described below in conjunction with embodiments.

[0079] Unless otherwise specified, all experiments were repeated three times. SPSS 21.0 was used for analysis of variance (ANOVA) and Duncan's multiple comparison analysis, and the results were expressed as mean ± SD.

[0080] Embodiment 1 A method for preparing a Hericium erinaceus composite extract, comprising the following steps:

[0081] S1, drying the Hericium erinaceus at 35°C until the moisture content is 10%, then crushing and sieving through a 100-mesh sieve to obtain Hericium erinaceus dry powder;

[0082] S2, mixing the Hericium erinaceus dry powder with 75% ethanol solution at a ratio of 1 g:15 mL, leaching at 35° C. for 10 h, filtering after the leaching, repeating 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0083] S3, mixing the extract and petroleum ether at a ratio of 1:1.2 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 3 times, combining the petroleum ether phase and the aqueous phase, respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0084] S4, mixing the aqueous phase and ethyl acetate at a ratio of 1:1.4 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining an ethyl acetate extract of Hericium erinaceus;

[0085] S5, adding the enzyme preparation to the filter residue, performing enzymolysis at 45° C. for 3 h, then adding the starter at 30° C. for 5 d, then adding water at a weight volume ratio of 1 g:20 mL of the filter residue to water, extracting at 50° C. for 1.5 h, repeating 3 times, combining the extracts, and obtaining an aqueous extract of Hericium erinaceus;

[0086] The enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2.5, and the enzymatic activity of the cellulase is 3×10 4 U / g, the enzyme activity of the pectinase is 4×10 4 U / g; the fermentation agent is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:3, and the bacterial activity of Lactobacillus plantarum is 3×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 CFU / g;

[0087] S6. Freeze-dry the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus in a mass ratio of 5:0.5:7 to obtain a composite extract of Hericium erinaceus.

[0088] Comparative Example 1

[0089] The same as Example 1, except that the filter residue is not fermented, and the specific steps are as follows:

[0090] S1, drying the Hericium erinaceus at 35°C until the moisture content is 10%, then crushing and sieving through a 100-mesh sieve to obtain Hericium erinaceus dry powder;

[0091] S2, mixing the Hericium erinaceus dry powder with 75% ethanol solution at a ratio of 1 g:15 mL, leaching at 35° C. for 10 h, filtering after the leaching, repeating 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0092] S3, mixing the extract and petroleum ether at a ratio of 1:1.2 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 3 times, combining the petroleum ether phase and the aqueous phase, respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0093] S4, mixing the aqueous phase and ethyl acetate at a ratio of 1:1.4 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining an ethyl acetate extract of Hericium erinaceus;

[0094] S5, adding the enzyme preparation to the filter residue, performing enzymolysis at 45° C. for 3 h, then adding water at a weight volume ratio of the filter residue to water of 1 g:20 mL, extracting at 50° C. for 1.5 h, repeating 3 times, combining the extracts, and obtaining an aqueous extract of Hericium erinaceus;

[0095] The enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2.5, and the enzymatic activity of the cellulase is 3×10 4 U / g, the enzyme activity of the pectinase is 4×10 4 U / g;

[0096] S6. Freeze-dry the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus in a mass ratio of 5:0.5:7 to obtain a composite extract of Hericium erinaceus.

[0097] Comparative Example 2

[0098] The same as Example 1, except that the filter residue was not subjected to enzymatic hydrolysis, and the specific steps are as follows:

[0099] S1, drying the Hericium erinaceus at 35°C until the moisture content is 10%, then crushing and sieving through a 100-mesh sieve to obtain Hericium erinaceus dry powder;

[0100] S2, mixing the Hericium erinaceus dry powder with 75% ethanol solution at a ratio of 1 g:15 mL, leaching at 35° C. for 10 h, filtering after the leaching, repeating 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0101] S3, mixing the extract and petroleum ether at a ratio of 1:1.2 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 3 times, combining the petroleum ether phase and the aqueous phase, respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0102] S4, mixing the aqueous phase and ethyl acetate at a ratio of 1:1.4 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining an ethyl acetate extract of Hericium erinaceus;

[0103] S5, adding the fermentation agent to the filter residue, fermenting at 30° C. for 5 days, then adding water at a weight volume ratio of 1 g:20 mL of the filter residue to water, extracting at 50° C. for 1.5 hours, repeating 3 times, combining the extracts, and obtaining an aqueous extract of Hericium erinaceus;

[0104] The fermentation agent is composed of lactobacillus plantarum and bacillus subtilis in a mass ratio of 1:3, and the bacterial activity of the plant lactobacillus plantarum is 3×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 CFU / g;

[0105] S6. Freeze-dry the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus in a mass ratio of 5:0.5:7 to obtain a composite extract of Hericium erinaceus.

[0106] Comparative Example 3

[0107] The same as Example 1, except that the filter residue was not subjected to enzymatic hydrolysis and fermentation, and the specific steps are as follows:

[0108] S1, drying the Hericium erinaceus at 35°C until the moisture content is 10%, then crushing and sieving through a 100-mesh sieve to obtain Hericium erinaceus dry powder;

[0109] S2, mixing the Hericium erinaceus dry powder with 75% ethanol solution at a ratio of 1 g:15 mL, leaching at 35° C. for 10 h, filtering after the leaching, repeating 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract;

[0110] S3, mixing the extract and petroleum ether at a ratio of 1:1.2 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 3 times, combining the petroleum ether phase and the aqueous phase, respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus;

[0111] S4, mixing the aqueous phase and ethyl acetate at a ratio of 1:1.4 (v / v) by oscillation for 10 min, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining an ethyl acetate extract of Hericium erinaceus;

[0112] S5, adding the fermentation agent to the filter residue, fermenting at 30° C. for 5 days, then adding water at a weight volume ratio of 1 g:20 mL of the filter residue to water, extracting at 50° C. for 1.5 hours, repeating 3 times, combining the extracts, and obtaining an aqueous extract of Hericium erinaceus;

[0113] The fermentation agent is composed of lactobacillus plantarum and bacillus subtilis in a mass ratio of 1:3, and the bacterial activity of the plant lactobacillus plantarum is 3×10 12CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 CFU / g;

[0114] S6. Freeze-dry the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus in a mass ratio of 5:0.5:7 to obtain a composite extract of Hericium erinaceus.

[0115] Comparative Example 4

[0116] Same as Example 1, except that the petroleum ether extract of Hericium erinaceus was not used.

[0117] Comparative Example 5

[0118] Same as Example 1, except that the Hericium erinaceus ethyl acetate extract was not used.

[0119] Comparative Example 6

[0120] Same as Example 1, except that: no Hericium erinaceus water extract was used.

[0121] Test Example 1 Effect of Hericium erinaceus compound extract on alcoholic liver injury

[0122] Establishment of rat alcoholic liver injury model:

[0123] After one week of adaptive feeding, SPF male SD rats weighing 180-220 g (6-8 weeks old) were randomly divided into 8 groups, each with 7 rats, namely: blank control group (Con), model control group (Mod), Example 1 group (L1), and comparative example 1-5 groups (D1, D2, D3, D4, D5). By referring to the fixed alcohol gavage modeling mode, the blank control group was gavaged with physiological saline every day to eliminate the stress difference, and the remaining groups were gavaged with 50% alcohol (10 mL / kg) by volume for three weeks.

[0124] Dosage:

[0125] During the modeling period, the blank group and the model group were gavaged with 0.5% CMC-Na (10 mL / kg) every afternoon; the Example and Comparative Example groups were gavaged with the Hericium erinaceus composite extract prepared in each group at a standard of 31.5 mg / kg / d. The gavage was performed at a fixed time every day, and the samples were collected after three consecutive weeks of gavage.

[0126] Rat organ index:

[0127] After the rats were anesthetized, blood was collected from the abdominal aorta and the liver, stomach, and spleen tissues were removed. They were immediately rinsed in pre-cooled saline. After the surface moisture of the organs was absorbed with absorbent paper, they were accurately weighed with an analytical balance and recorded. The organ index of the rats was calculated using the following formula:

[0128] Organ index (%) = [organ weight (g) / rat weight before sacrifice (g)] × 100%

[0129] The results of rat organ index detection are shown in Table 1:

[0130] Table 1 Results of rat organ index detection

[0131] Group Liver index Spleen index Renal Index Con 2.05±0.03 0.16±0.02 0.37±0.01 Mod <![CDATA[2.43±0.04 ** ]]> 0.17±0.02 0.37±0.01 L1 <![CDATA[2.15±0.04 ## ]]> 0.15±0.01 0.37±0.02 D1 <![CDATA[2.28±0.03 ## ]]> 0.16±0.03 0.36±0.03 D2 <![CDATA[2.25±0.02 ## ]]> 0.16±0.04 0.36±0.04 D3 <![CDATA[2.30±0.02 # ]]> 0.17±0.05 0.37±0.03 D4 2.32±0.05 0.15±0.04 0.38±0.05 D5 <![CDATA[2.31±0.03 # ]]> 0.16±0.03 0.36±0.02 D6 2.33±0.03 0.16±0.02 0.37±0.04

[0132] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0133] Test Example 2 Determination of GSH, T-AOC, and MPO Activities in Liver Tissue

[0134] The activities of GSH, T-AOC and MPO in rat liver tissue were determined by conventional methods. The test results are shown in Table 2:

[0135] Table 2 Activity detection results of GSH, T-AOC and MPO in rat liver tissue

[0136] Group GSH (μmol / g.prot) T-AOC (μmol / g.prot) MPO(U / g) Con 66.12±3.11 1.60±0.05 4.02±0.18 Mod <![CDATA[27.01±1.35 ** ]]> <![CDATA[0.78±0.06 ** ]]> <![CDATA[5.93±0.26 ** ]]> L1 <![CDATA[55.34±2.26 ## ]]> <![CDATA[1.46±0.07 ## ]]> <![CDATA[4.47±0.22 ## ]]> D1 <![CDATA[50.47±3.148 ## ]]> <![CDATA[1.25±0.03 ## ]]> <![CDATA[4.83±0.14 ## ]]> D2 <![CDATA[47.79±2.63 ## ]]> <![CDATA[1.17±0.08 ## ]]> <![CDATA[4.98±0.36 ## ]]> D3 <![CDATA[53.42±2.42 ## ]]> <![CDATA[1.28±0.05 ## ]]> <![CDATA[4.67±0.25 ## ]]> D4 <![CDATA[55.38±3.57 ## ]]> <![CDATA[1.33±0.04 ## ]]> <![CDATA[4.58±0.21 ## ]]> D5 <![CDATA[54.42±3.06 ## ]]> <![CDATA[1.30±0.09 ## ]]> <![CDATA[4.62±0.17 ## <!-- 8 -->]]> D6 <![CDATA[56.25±2.75 ## ]]> <![CDATA[1.35±0.06 ## ]]> <![CDATA[4.54±0.13 ## ]]>

[0137] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0138] Test Example 3 Observation of general behavior status of rats:

[0139] Starting from gavage, the rats' hair, water intake, diet, feces, mental state and weight were observed and recorded daily. It was found that the rats in the blank group had a good mental state throughout the experiment, were lively and active, had pure white hair and luster, and had dry and oval feces; the group given alcohol gavage was in poor condition, with symptoms such as slow movement, lack of energy, and drowsiness, accompanied by loose stools, and rough and dark hair. After gavage with the extract, the state of rats in each group improved to varying degrees. Compared with the model group, the rats had more movements and less drowsiness. Among them, there was no significant difference in the mental state of the rats in Example 1 and the blank group.

[0140] During the experiment, the weight of rats showed a steady upward trend. The blank group rats grew rapidly in the early stage, and the growth trend began to slow down after 15 days. The model group rats grew slowly in the early stage after alcohol gavage, and the weight began to decline continuously after 11 days. The weight of rats in the experimental group showed an upward trend in the early stage, which was slower than that of the blank control group. Alcohol gavage can cause certain damage to the stomach of rats, and the rats' food intake decreases. However, after the intervention of the extract, the weight increased slightly, and the overall weight of the experimental group was higher than that of the model group.

[0141] Example 2

[0142] Same as Example 1, except that the mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is 3:0.1:5.

[0143] Example 3

[0144] Same as Example 1, except that the mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is 7:1:10.

[0145] Example 4

[0146] Same as Example 1, except that: the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:1, and the enzymatic activity of the cellulase is 2×10 4 U / g, the enzyme activity of the pectinase is 5×10 4 U / g.

[0147] Example 5

[0148] Same as Example 1, except that: the enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:5, and the enzymatic activity of the cellulase is 4×10 4 U / g, the enzyme activity of the pectinase is 3×10 4 U / g.

[0149] Example 6

[0150] Same as Example 1, except that the enzymolysis temperature is 40° C. and the enzymolysis time is 4 h.

[0151] Example 7

[0152] Same as Example 1, except that the enzymolysis temperature is 50° C. and the enzymolysis time is 2 h.

[0153] Example 8

[0154] Same as Example 1, except that the starter is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:1, and the bacterial activity of Lactobacillus plantarum is 2×10 12 The bacterial activity of the Bacillus subtilis is 3×10 12 CFU / g.

[0155] Example 9

[0156] Same as Example 1, except that the starter is composed of Lactobacillus plantarum and Bacillus subtilis in a mass ratio of 1:5, and the bacterial activity of Lactobacillus plantarum is 4×10 12CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 .

[0157] Example 10

[0158] Same as Example 1, except that the fermentation temperature is 25° C. and the fermentation time is 7 days.

[0159] Embodiment 11

[0160] Same as Example 1, except that the fermentation temperature is 30° C. and the fermentation time is 3 days.

[0161] Example 12

[0162] Same as Example 1, except that the water extraction temperature is 40° C. and the water extraction time is 2 h.

[0163] Example 13

[0164] Same as Example 1, except that: the water extraction temperature is 60° C. and the water extraction time is 1 h.

[0165] After testing, the effect of the Hericium erinaceus composite extract obtained in Example 2-13 was not significantly different from that of the Hericium erinaceus composite extract in Example 1 (P>0.05).

[0166] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a Hericium erinaceus composite extract in the preparation of a drug for treating alcoholic liver damage, characterized in that: The Hericium erinaceus composite extract is composed of Hericium erinaceus petroleum ether extract, Hericium erinaceus ethyl acetate extract and Hericium erinaceus water extract in a mass ratio of (3-7): (0.1-1): (5-10); The preparation method of the Hericium erinaceus composite extract comprises the following steps: S1. After drying, the Hericium erinaceus is crushed and sieved to obtain Hericium erinaceus dry powder; S2, mixing the Hericium erinaceus dry powder with an ethanol solution for leaching, filtering after the leaching is completed, repeating 2 to 3 times, combining the filtrate to obtain a filtrate and a filter residue, and concentrating the filtrate to obtain an extract; S3, mixing the extract and petroleum ether by oscillation for 10 minutes and then standing for extraction, collecting the petroleum ether phase, repeating the extraction 2 to 3 times, combining the petroleum ether phase and the aqueous phase respectively, concentrating the petroleum ether phase under reduced pressure, removing the petroleum ether, and obtaining the petroleum ether extract of Hericium erinaceus; S4, shaking and mixing the aqueous phase and ethyl acetate for 10 minutes, and then standing for extraction, collecting the ethyl acetate phase, repeating the extraction 2 to 3 times, combining the ethyl acetate phase and the aqueous phase, respectively, concentrating the ethyl acetate phase under reduced pressure, removing ethyl acetate, and obtaining the ethyl acetate extract of Hericium erinaceus; S5, enzymatically fermenting the filter residue, and then extracting it with water, repeating this process 2 to 3 times, combining the extracts, and obtaining a water extract of Hericium erinaceus; S6. Drying and mixing the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus to obtain a composite extract of Hericium erinaceus.

2. The use according to claim 1, characterized in that: The concentration of the ethanol solution is 70% to 80%, the extraction temperature is 30° C. to 40° C., the extraction time is 1 to 3 hours, and the weight-to-volume ratio of Hericium erinaceus dry powder to the ethanol solution is 1 g: (10 to 20) mL.

3. The use according to claim 1, characterized in that: The filter residue enzymatic hydrolysis and fermentation process is as follows: an enzyme preparation is added to the filter residue, and a starter is added for fermentation after enzymatic hydrolysis.

4. The use according to claim 3, characterized in that: The enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:(1-5), and the enzymatic activity of the cellulase is 2×10 4 ~4×10 4 U / g, the enzyme activity of the pectinase is 3×10 4 ~5×10 4 U / g.

5. The use according to claim 3, characterized in that: The enzymolysis temperature is 40° C. to 50° C., and the enzymolysis time is 2 to 4 hours.

6. The use according to claim 3, characterized in that: The fermentation agent is composed of lactobacillus plantarum and bacillus subtilis in a mass ratio of 1:(1-5), and the bacterial activity of the plant lactobacillus plantarum is 2×10 12 ~4×10 12 CFU / g, the bacterial activity of Bacillus subtilis is 2×10 12 ~3×10 12 CFU / g.

7. The use according to claim 3, characterized in that: The fermentation temperature is 25°C to 30°C, and the fermentation time is 3 to 7 days.

8. The use according to claim 1, characterized in that: The water extraction temperature is 40° C. to 60° C., and the water extraction time is 1 to 2 hours.

9. The use according to claim 1, characterized in that: The weight volume ratio of the filter residue and water is 1g:(15-25)mL.

10. The use according to claim 1, characterized in that: The mass ratio of the petroleum ether extract of Hericium erinaceus, the ethyl acetate extract of Hericium erinaceus and the water extract of Hericium erinaceus is 5:0.5:7.

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