Extraction method of cordyceps nigrum and medicinal application of extract of cordyceps nigrum

Abstract

The invention discloses an application of a cordyceps nigrum extract in preparation of a medicine for treating and / or preventing alcoholic liver diseases. The cordyceps nigrum extract is prepared by a method comprising the following steps: crushing a cordyceps nigrum raw material, performing ultrasonic extraction at 20-45 DEG C by taking water as a solvent, collecting an extracting solution, and concentrating and drying the extracting solution.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug component extraction, and particularly relates to an extraction method of Cordyceps nigar Wang et al. and the medicinal use of its extract. Background Art

[0002] With the change of people's lifestyle, the incidence of non-viral liver diseases is increasing year by year. Non-viral liver diseases mainly include alcoholic liver disease (ALD), alcoholic fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic liver disease, drug-induced liver disease, liver cirrhosis and liver cancer, etc., seriously affecting human health. ALD is the main representative of non-viral liver diseases and an important inducement for other liver diseases. Its initial stage usually presents as fatty liver, and then develops into alcoholic hepatitis, liver fibrosis and liver cirrhosis; in severe cases, clinical symptoms such as hepatocyte necrosis and liver failure will occur, seriously endangering physical health. ALD is usually caused by patients' long-term and excessive drinking. The treatment principles of alcoholic liver disease include abstinence from alcohol, nutritional support, reducing the severity of the disease, improving early secondary malnutrition, and symptomatic treatment of alcoholic liver cirrhosis and its complications. Since it is difficult for people with alcohol addiction to achieve complete abstinence from alcohol, the treatment effects of some glucocorticoids and other drugs are not satisfactory, and their drug effects vary from patient to patient. Liver transplantation may be an option for patients who do not respond well to drugs, but due to limited organ supply and possible post-transplant complications, it has great limitations in clinical treatment. In recent years, natural Chinese medicines have played an important role in the treatment of ALD due to their multi-targets, strong drug effects and low risk of side effects. Some Chinese medicine extracts and / or their combinations have been proven to be effective in preventing and treating ALD. For example, the decoction of Fructus Aurantii Immaturus and Rheum Palmatum can effectively improve chronic alcoholic liver injury in rats, and the formula for resolving turbidity and regulating lipid may reduce alcoholic liver injury, oxidative stress and inflammation by regulating the Nrf2 / Keap1 and MAPKs / NF-κB signaling pathways.

[0003] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0004] The purpose of the present invention is to provide an extraction method of Cordyceps nigar Wang et al. and the medicinal use of its extract, so as to overcome the defects in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the application of an extract of Cordyceps nigar Wang et al. in the preparation of a drug for treating and / or preventing alcoholic liver disease.

[0006] There is a type of cordyceps distributed in the southern part of Shanxi, and most previous literatures called it Cordyceps barnesii Thwaites ( CordycepsbarnesiiThwaitesIn 2016-2017, Professor Zhang Yongjie's team at Shanxi University collected a large number of samples of this cordyceps in Lishan, Shanxi Province over two years. Based on comprehensive morphological characteristics and multi-gene phylogenetic analysis, they ultimately determined its correct name to be *Cordyceps militaris*. Ophiocordycepsnigrella Currently, *Cordyceps militaris* has only been reported in Shanxi Province in China. *Cordyceps militaris* belongs to the kingdom Eukaryota, subkingdom Dikarya, phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Ophiocordycipitaceae, and its host is the larvae of scarab beetles (also known as grubs). A series of biochemical analyses show that, compared with *Cordyceps sinensis*, *Cordyceps militaris* has similar or even higher levels of amino acids, ergosterol and mannitol, mineral elements, cordycepic acid, and cordycepin. Furthermore, infrared spectroscopy analysis shows that the polysaccharide components in this *Cordyceps militaris* are consistent with those in *Cordyceps sinensis*. Existing research indicates that *Cordyceps militaris* has medicinal development value due to its relatively high safe dosage and rich content of cordycepin and other components; however, its extraction method and its therapeutic effect on alcoholic liver disease remain unclear.

[0007] This study first identified the optimal extraction method by comparing the differences in the content of major nutrients in Cordyceps militaris after different extraction methods. Then, an animal model of alcoholic liver injury was constructed to investigate the protective effect of Cordyceps militaris against alcoholic liver injury. Finally, based on this, the protective effects of different concentrations of Cordyceps militaris extract on alcoholic liver injury and the influence of drug toxicity were compared to screen the optimal drug concentration of Cordyceps militaris for the treatment of alcoholic liver disease, providing basic research data for subsequent drug development.

[0008] Preferably, in the above technical solution, the Cordyceps militaris extract is prepared by a method including the following steps: after pulverizing the Cordyceps militaris raw material, ultrasonic extraction is performed at 20℃-45℃ using water as a solvent, and the extract is collected, concentrated and dried.

[0009] The key components of Cordyceps militaris extract are: 1. Propranolol is the most abundant known small molecule among the metabolites extracted from Cordyceps militaris using the ultrasonic method. A search of the TCMSP database revealed its Chinese name as Propranolol L-, with the molecular formula C16H21NO2. It is an alkaloid, and Cordyceps is not found in related herbs. Searches for "Propranolol Cordyceps" or "Propranolol Cordyceps" in Chinese and English databases such as PubMed, Web of Science, and CNKI yielded no results. However, this substance is found to have hepatoprotective effects in these databases, leading us to believe it is a unique substance found only in Cordyceps militaris.

[0010] 2. Schisandrin B is one of the known small molecule substances with relatively high content in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is schisandrin B, which belongs to the class of shikimic acid and phenylpropanoic acid. Its molecular formula is C23H28O6. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Schisandrin B" and "schisandrin B Cordyceps" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0011] 3. Glycyrrhizin is one of the known small molecule substances with relatively high content in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is glycyrrhizic acid, and its molecular formula is C42H62O16. It belongs to the class of terpenoids. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Glycyrrhizin" and "glycyrrhizic acid Cordyceps" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0012] 4. Octahydrocurcumin is one of the known small molecule substances with relatively high content in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is curcumin, and its molecular formula is C21H28O6. It belongs to the class of shikimic acid and phenylpropanoic acid substances. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Octahydrocurcumin" and "curcumin Cordyceps" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0013] 5. Hesperetin is one of the known small molecule substances with relatively high content in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is hesperetin. This compound belongs to the class of shikimic acid and phenylpropanoic acid. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Hesperetin" and "hesperetin Cordyceps" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0014] 6. Andrographolide is one of the known small molecules with relatively high contents in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is andrographolide. This compound belongs to terpenoids. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Andrographolide" and "Cordyceps andrographolide" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0015] 7. Rosmarinic acid is one of the known small molecules with relatively high contents in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is rosmarinic acid. This compound belongs to shikimic acid and phenylpropanoic acid. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Rosmarinic acid" and "Cordyceps rosmarinic acid" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0016] 8. Caffeic acid is one of the known small molecules with relatively high contents in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is caffeic acid. This compound belongs to shikimic acid and phenylpropanoic acid. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Caffeic acid" and "Cordyceps caffeic acid" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0017] 9. Ursolic acid is one of the known small molecules with relatively high contents in the metabolite data extracted from Cordyceps militaris by ultrasonic method. Its Chinese name is ursolic acid. This compound belongs to shikimic acid and phenylpropanoic acid. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when using keywords "Cordyceps Ursolic acid" and "Cordyceps ursolic acid" for retrieval, no directly relevant research papers were found. However, this substance can be found to have a hepatoprotective effect in the database.

[0018] 10. Corosolic acid is one of the known small molecules with relatively high content in the metabolite data extracted from Cordyceps pseudomilitaris by ultrasonic method. Its Chinese name is corosolic acid. This compound belongs to shikimic acid and phenylpropionic acid. In Chinese and English databases such as PubMed, Web of Science, and CNKI, when searching with keywords "Cordyceps Corosolic acid" and "Cordyceps corosolic acid", no directly relevant research papers were found. However, this substance can be found in the database to have a hepatoprotective effect.

[0019]

[0020] Preferably, in the above technical solution, the temperature of ultrasonic extraction is 20°C.

[0021] Preferably, in the above technical solution, the drug is used to reduce the increase of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), total cholesterol (TC) and / or triglyceride (TG) caused by alcoholic liver injury.

[0022] Preferably, in the above technical solution, the drug is used to increase the decrease of serum high-density lipoprotein cholesterol (HDL-C) caused by alcoholic liver injury.

[0023] Preferably, in the above technical solution, the drug is used to improve the histopathological changes of the liver caused by alcoholic liver injury, including reducing hepatic steatosis, inflammatory cell infiltration and hepatocyte necrosis.

[0024] Preferably, in the above technical solution, the administration dose of Cordyceps pseudomilitaris extract in the drug is 0.1 g / kg body weight to 0.3 g / kg body weight.

[0025] Preferably, in the above technical solution, the administration dose is 0.2 g / kg body weight.

[0026] A pharmaceutical composition for treating and / or preventing alcoholic liver disease, comprising a therapeutically effective amount of Cordyceps pseudomilitaris extract prepared by the method described in claim 2 or 3 and a pharmaceutically acceptable carrier.

[0027] Preferably, in the above technical solution, the content of Cordyceps pseudomilitaris extract in the pharmaceutical composition is such that its adult daily equivalent administration dose corresponds to 0.1 g / kg body weight to 0.3 g / kg body weight of the crude drug.

[0028] In summary, the water extract of Cordyceps pseudomilitaris is a traditional Chinese medicine that can scientifically and effectively treat alcoholic liver disease. By combining the intervention effects of water extracts of Cordyceps pseudomilitaris at different concentrations on different indexes in the serum of mice with alcoholic liver disease model and the toxic effects on the body weight of mice, the safe and effective administration concentration of the water extract of Cordyceps pseudomilitaris was finally screened out to be 0.2 g / kg. Attached Figure Description

[0029] Figure 1 Comparison of total sugar content obtained by three extraction methods. A: Standard curve; B: Content comparison.

[0030] Figure 2 PCA analysis of metabolites obtained by three extraction methods: red (CS method), blue (SY method), and green (JZ method).

[0031] Figure 3 Comparison of the types of small molecules of metabolites obtained by three extraction methods. A: SY method; B: CS method; C: JZ method.

[0032] Figure 4 Venn diagram comparing the types of small molecule metabolites obtained by three extraction methods.

[0033] Figure 5 Comparison of secondary mass spectra of seven target compounds in Cordyceps militaris extract with their corresponding standards. A: 6,7,4'-trihydroxyisoflavones; B: valine; C: DL-glutamic acid; D: L-uridine; E: inosine; F: L-tryptophan; G: vitamin B2. The horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the relative intensity. The spectra of the experimental sample (top) and the spectrum of the standard (bottom) are arranged in a mirror image for easy comparison.

[0034] Figure 6 Comparison of the contents of the above 20 main functional substances of Cordyceps militaris in the extracts of Cordyceps militaris obtained by water bath method and ultrasonic method.

[0035] Figure 7 Changes in mouse body weight after treatment with different concentrations of Cordyceps militaris ultrasonic extract (control group; pathological model group; 0.1 g / kg Cordyceps militaris ultrasonic extract treatment group; 0.4 g / kg Cordyceps militaris ultrasonic extract treatment group; 0.7 g / kg Cordyceps militaris ultrasonic extract treatment group; 1 g / kg Cordyceps militaris ultrasonic extract treatment group; 10 g / kg Cordyceps militaris ultrasonic extract treatment group; silymarin treatment group).

[0036] Figure 8 Changes in liver morphology (A), weight (B), and liver index (C) in mice after treatment with different concentrations of Cordyceps militaris ultrasonic extract.

[0037] Figure 9 Changes in body weight of mice after treatment with different concentrations of Cordyceps militaris ultrasonic extract (control group; pathological model group; 0.1 g / kg Cordyceps militaris ultrasonic extract treatment group; 0.2 g / kg Cordyceps militaris ultrasonic extract treatment group; 0.3 g / kg Cordyceps militaris ultrasonic extract treatment group; silymarin treatment group).

[0038] Figure 10 Changes in liver morphology, weight, and liver index in mice after treatment with different concentrations of Cordyceps militaris ultrasonic extract.

[0039] Figure 11 The results of biochemical detection in the serum of mice in each treatment group in the second animal experiment were as follows: (A) alanine aminotransferase, (B) aspartate aminotransferase, (C) alkaline phosphatase, (D) total cholesterol, (E) triglycerides, (F) low-density lipoprotein cholesterol, and (G) high-density lipoprotein cholesterol.

[0040] Figure 12 Ultrastructural analysis of mouse livers in the second animal experiment: (A) Control group 100×, (B) Control group 200×, (C) Pathological model group 100×, (D) Pathological model group 200×, (E) 0.1g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (F) 0.1g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (G) 0.2g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (H) 0.2g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (I) 0.3g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (J) 0.3g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (K) Silymarin treatment group 100×, (L) Silymarin treatment group 200×.

[0041] Figure 13 The results of biochemical detection in the serum of mice in each treatment group in the first animal experiment were as follows: (A) alanine aminotransferase, (B) aspartate aminotransferase, (C) alkaline phosphatase, (D) total cholesterol, (D) triglycerides, (F) low-density lipoprotein cholesterol, and (G) high-density lipoprotein cholesterol.

[0042] Figure 14Ultrastructural analysis of mouse livers in the second animal experiment: (A) Control group 100×, (B) Control group 200×, (C) Pathological model group 100×, (D) Pathological model group 200×, (E) 0.1 g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (F) 0.1 g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (G) 0.4 g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (H) 0.4 g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (I) 0.7 g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (J) 0.7 g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (K) 1 g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (L) 1 g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (M) 10 g / kg Cordyceps militaris ultrasonic extract treatment group 100×, (N) 10 g / kg Cordyceps militaris ultrasonic extract treatment group 200×, (O) silymarin treatment group 100×, (P) silymarin treatment group 200×. Detailed Implementation

[0043] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0044] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0045] Conclusions and Analysis The complex chemical composition and diverse active ingredients of traditional Chinese medicine (TCM) pose significant challenges to its development. In contrast, TCM extracts possess advantages such as high content of active ingredients, good solubility, easy absorption, strong medicinal properties, high efficacy, and safety. Therefore, the extraction, separation, and purification of active ingredients in TCM are crucial for its development. This study selected water bath method, ultrasonic method, and decoction method as extraction methods for Cordyceps militaris. The content and types of polysaccharides and small molecule metabolites in the aqueous extracts obtained by the three methods were compared using the anthrone method and high-performance liquid chromatography (HPLC). The results showed that the ultrasonic method yielded a relatively higher total sugar content (…). Figure 1 B); the types of small molecule metabolites obtained by water bath and ultrasound methods are higher than those obtained by decoction (B); Figure 3 More importantly, further comparison of the contents of 20 major functional substances of Cordyceps militaris in small molecule metabolites obtained by water bath method and ultrasonic method revealed that the corresponding contents in the Cordyceps militaris water extract obtained by ultrasonic method were mostly higher than those obtained by water bath method. Figure 5Therefore, the ultrasonic method was chosen as the extraction method for Cordyceps militaris and used in subsequent animal experiments. In addition, solvent polarity, material-liquid ratio, extraction time and temperature have a very important influence on the extraction rate and retention rate of the effective components of traditional Chinese medicine. Previous research results showed that compared with ethanol, 50% ethanol, methanol and 50% methanol, water as the extraction solvent had a relatively high extraction rate [7]. Therefore, this study used the ultrasonic extract of Cordyceps militaris as the research object. At the same time, the rotary evaporation method was selected as the extraction method for Cordyceps militaris using the water bath method (Table 3), and the ultrasonic extraction method was set at 20°C (Table 4).

[0046] Experimental animal models play an important role in drug development, and can effectively test the efficacy, safety and effectiveness of drugs. The results of experimental animal models are affected by many factors. On the one hand, different types of experimental animals have different applicability due to their physiological, anatomical and genetic characteristics. Mice and rats are the most commonly used experimental animal models because they reproduce rapidly, are inexpensive and have clear genetic backgrounds, and are widely used in the preliminary screening of drugs, safety and efficacy assessments

[19] . For example, the C57BL / 6 mice used in this study are the most commonly used inbred experimental animals. Their advantages stem from their highly homozygous genetic background, stable reproductive performance and wide applicability, making them an important model animal for biomedical research and drug research. On the other hand, the experimental design of animal models is also an important part of drug research and testing. An ideal animal model of alcoholic liver disease can not only induce steatosis and increase serum ALT and other indicators, and cause liver inflammation, but is also easy to operate. At present, the main methods for constructing animal models of alcoholic liver disease include chronic alcohol diet, intragastric alcohol perfusion, alcohol gavage and adding alcohol to drinking water. This study employed an alcohol gavage method to establish a model, as this method effectively simulates alcohol consumption, allowing for the absorption of alcohol by the body and thus enabling the study of the systemic effects of alcohol on the entire organism (such as the brain and liver). Furthermore, the experimental method is simple to perform, and almost no mouse mortality occurred during the experiment. This study selected a four-week low-dose alcohol feeding model as the animal pathological experimental model. In addition, the biochemical and pathological results of this study showed that, compared with the control group, the serum ALT, AST, and AKP activities, TC and TG levels of the pathological model group mice were significantly increased, while HDL-C levels were significantly decreased. Figure 11 AE, Figure 11 G, Figure 13 AE, Figure 13 G), in the alcohol model group, hepatocytes were arranged disorderedly, and necrotic foci of hepatocytes were visible in the liver lobules. Under high magnification, the cytoplasm was loose, with a large number of lymphocytes aggregated and a large amount of inflammatory cell infiltration. Bile duct hyperplasia was frequently seen in the portal area, and mild fibrosis was also observed. Figure 12 CD Figure 14CD). Therefore, the experimental animal model constructed in this study met all the requirements for constructing an animal model of alcoholic liver disease. Furthermore, in our animal experiments, we found that compared with the control group, the liver weight and liver index of mice in the pathological model group were significantly increased (CD). Figure 8 BC and Figure 10 The liver coefficient (BC) is a commonly used indicator in toxicology experiments. Changes in its value can roughly reflect the type of liver disease. For example, an increased coefficient indicates that the liver may have fatty degeneration or hyperplasia and hypertrophy, while a decreased coefficient indicates liver atrophy or other degenerative changes. Based on this, we speculate that the increased liver coefficient in the pathological model group mice may be due to direct damage to hepatocytes by alcohol and its metabolites, leading to increased liver weight after liver inflammation, fatty degeneration, and fibrosis.

[0047] The liver is the primary site of alcohol metabolism, and excessive alcohol intake can lead to liver tissue damage, increased hepatocyte membrane permeability, and the release of molecules such as AST, ALT, and AKP into the bloodstream. Therefore, serum ALT, AST, and AKP activities are commonly used clinically to reflect the degree of hepatocyte damage. This study found that, compared with the control group, the serum ALT, AST, and AKP activities of mice in the pathological model group were significantly increased, indicating that chronic alcohol gavage damages mouse hepatocytes and mitochondria. When different concentrations of *Cordyceps militaris* ultrasonic extract were administered, the serum ALT, AST, and AKP activities of mice in each group significantly decreased, indicating that the *Cordyceps militaris* ultrasonic extract has a hepatoprotective effect. Figure 11 AC, Figure 13 AC). Meanwhile, the liver is also a vital organ for lipid metabolism, playing a crucial role in the biosynthesis of lipids such as TC and TG, as well as in the transformation and excretion of lipids. Alcohol can impair hepatocyte function, affecting mitochondrial fatty acid β-oxidation, and disrupting the synthesis, transport, and metabolism of blood lipids, lipoproteins, and apolipoproteins, leading to fat deposition in the blood and hepatocytes, resulting in abnormal levels of TG, TC, LDH-C, and HDH-C in the blood. In this study, compared with the control group, the pathological model group mice showed significantly increased serum TG and TC levels and significantly decreased HDH-C levels. However, when different concentrations of *Cordyceps militaris* ultrasonic extract were administered, the serum TG and TC levels of mice in each group decreased, while the HDH-C level increased (…). Figure 11 DE and 11G; Figure 13 DE and 13G indicate that the ultrasonic extract of Cordyceps militaris has a protective function against lipid metabolism in hepatocytes. Furthermore, pathological section results showed that, compared with the pathological model group, the degree of hepatocyte pathological damage in mice treated with ultrasonic extracts of Cordyceps militaris at all concentrations was alleviated. Figure 12 and Figure 14 Therefore, extracts from Cordyceps militaris have a protective effect against alcoholic liver injury.

[0048] Silymarin is an active ingredient extracted from milk thistle fruit. It has functions such as scavenging reactive oxygen species, resisting lipid peroxidation, and promoting hepatocyte repair and regeneration

[24] . Clinically, it is often used to treat chronic hepatitis, cirrhosis and other diseases, and no obvious side effects have been observed in clinical use. Animal model experiments showed that slymarin has a significant protective effect on mice with acute liver injury caused by alcohol. Therefore, it was used as a positive control group in this study. Our experimental results showed that the serum ALT, AST, AKP activities, TC, TG and HDH-C contents and pathological section results of mice treated with different concentrations of Cordyceps militaris ultrasonic extract were significantly improved compared with those of the pathological model group. Figure 11-12 The therapeutic effects of the silymarin-treated group were similar, and in some cases, the therapeutic effects of certain indicators in the serum of mice in the 0.2 g / kg and 0.3 g / kg concentrations of Cordyceps militaris treatment groups were even better than those in the silymarin-treated group. Figure 11 (AB). Therefore, the ultrasonic extract of Cordyceps militaris has great potential for clinical application.

[0049] This study explored the protective effects of different concentrations of *Cordyceps militaris* ultrasonic extract on alcoholic liver injury. Biochemical and pathological results showed that 0.1-0.3 g / kg concentrations of *Cordyceps militaris* ultrasonic extract effectively alleviated alcohol-induced abnormal changes in serum ALT, AST, and other liver indicators, as well as liver inflammation, in a concentration-dependent manner. Figure 11-12 Furthermore, the ultrasonic extract of Cordyceps militaris at concentrations of 0.4 g / kg and / or 0.7 g / kg had a more significant effect in reducing the abnormal increases in ALT and AST levels induced by alcohol. Figure 13 (AB). However, concentrations of 0.3 g / kg and higher may cause a significant decrease in mouse body weight, and even treatment with 1 g / kg and 10 g / kg of Cordyceps militaris ultrasonic extract can cause a negative decrease in mouse body weight, affecting mouse health and normal development to varying degrees (Tables 6-7). Figure 7 , 9 Meanwhile, compared with the pathological model group, treatment with 0.2 g / kg of Cordyceps militaris ultrasonic extract effectively reduced liver weight, liver coefficient, and serum TG levels in mice. Figure 10 , Figure 11 E). Therefore, a concentration of 0.2 g / kg may be the optimal drug treatment concentration for Cordyceps militaris water extract.

[0050] 2. Instruments and Materials 2.1 Instruments Table 1. Instruments used in this study

[0051] 2.2 Reagents 0.52% ethanol solution was purchased from Beijing Hongxing Co., Ltd.; silymarin was purchased from Beijing Puxitang Biotechnology Co., Ltd.; sodium carboxymethyl cellulose was purchased from Henan Wanbang Chemical Technology Co., Ltd.; hematoxylin-eosin staining solution was purchased from Fuzhou Feijing Biotechnology Co., Ltd.; and the assay kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and alkaline phosphatase (AKP) were purchased from Nanjing Jiancheng Technology Co., Ltd.

[0052] 2.3 Experimental Samples Observational and experimental samples of Cordyceps militaris (collected in April 2025 from Lishan, Qinshui County, Shanxi Province).

[0053] 2.4 Experimental Animals Male SPF-grade C57BL / 6 mice, 6–8 weeks old, weighing (20±2) g, were purchased from Shanxi Sankeyue Biotechnology Co., Ltd., production license number: SCXK(Jin)2024-0003, and were housed in an environment with a temperature of (25±2)℃, humidity of (50±10)%, and a 12h light-dark cycle.

[0054] 3.1 Extraction method of Cordyceps militaris (1) Water bath extraction method (SY method) The Cordyceps militaris was ground into powder, and extracted with distilled water (solid-to-liquid ratio 1:25) in a water bath (temperature: 100°C; time: 60 min). The supernatant was collected by centrifugation at 4000 rpm for 10 min, and the supernatant was combined and filtered. The supernatant was concentrated to approximately one-third of its original volume using both rotary evaporation (temperature: 70-80°C; vacuum degree ≤ -0.1 MPa) and electric heating mantle concentration (temperature > 100°C), and then stored at -20°C for later use. When using, it should be dissolved in physiological saline and prepared fresh each time.

[0055] (2) Ultrasonic extraction method (CS method) The Cordyceps militaris was ground into powder, and then extracted with distilled water (solid-to-liquid ratio 1:25) using ultrasonic extraction (temperature: 20°C and 45°C; time: 30 min). The supernatant was collected by centrifugation at 4000 rpm for 10 min. The supernatants were combined and filtered, and then concentrated to approximately 1 / 3 of their original volume using a rotary evaporator (temperature: 70-80°C; vacuum degree ≤ -0.1 MPa). Finally, the concentrate was freeze-dried using a vacuum dryer to obtain the powder, which was then stored at -20°C for later use. When using, it should be dissolved in physiological saline and prepared fresh each time.

[0056] (3) Decoction extraction method (JZ method) Take a certain amount of Cordyceps militaris, crush it into coarse powder, boil it in distilled water (solid-to-liquid ratio 1:25) in an electric heating mantle for 30 minutes, centrifuge at 4000 rpm for 10 minutes and collect the supernatant. Filter out the residue, add distilled water again and boil for 30 minutes, collect the supernatant, filter out the residue, and repeat the above operation once more. Combine the supernatants from the three operations and collect them. Concentrate the supernatant to about 1 / 3 of its original volume using a rotary evaporator (temperature: 70-80°C; vacuum degree ≤-0.1Mpa), and store it in a 4°C refrigerator for later use.

[0057] 3.2 Determination of total soluble sugar content (anthrone method) (1) Preparation of standard products Prepare a 100 μg / mL glucose standard solution. Using this as the stock solution, dilute with distilled water to obtain a series of concentration gradients, as detailed below. Pipette 0.4 mL of the mixed sample into a new test tube and determine it simultaneously with the sample according to the assay method.

[0058] Table 2. Preparation of Glucose Standard Solution

[0059] (2) Sample pretreatment and measurement Accurately weigh an appropriate amount of sample, add 1 mL of distilled water, and extract by sonication for 10 min, followed by centrifugation at 12000 rpm for 10 min. Transfer 0.4 mL of the supernatant to a new test tube and dilute with distilled water to a specific volume. Then, in the test tube, mix the diluted sample solution with phenol and concentrated sulfuric acid, allow the reaction to proceed, and measure the absorbance at 490 nm.

[0060] (3) Sample measurement results The total sugar content in the sample can be calculated based on the standard curve: Total sugar content (mg / g) = C × V ÷ M, where C is the result calculated based on the standard curve; M is the actual weighed mass; and V is the total volume of the extract.

[0061] 3.3 NGM Detection in Traditional Chinese Medicine (1) Sample pretreatment Pretreatment of powdered drugs (SY group and CS group): Accurately weigh 50 mg of sample powder into a 2 mL EP tube using an analytical balance, add 2 homogenizing beads, and add 500 μL of extraction solution (methanol:acetonitrile:water (2:2:1, v / v / v)). The extraction solution contains an isotope-labeled internal standard. Vortex for 30 seconds; homogenize in a homogenizer (35 Hz, 240 s), then transfer to an ice-water bath and sonicate for 5 minutes; repeat the homogenization and sonication steps 3 times; place in a -40°C freezer for 30 minutes; centrifuge at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 minutes, and collect the supernatant; place the supernatant in a -40°C freezer for 10 minutes; centrifuge the supernatant at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 minutes; filter the supernatant through a 0.22 μm microporous membrane into a vial for analysis. Liquid reagent pretreatment (ZY group): After thawing, the samples were centrifuged at 12,000 rpm (centrifugal force 13,800 (×g), radius 8.6 cm) for 15 min at 4°C; 300 μL of supernatant was accurately pipetted and 1000 μL of extraction buffer (methanol:acetonitrile:water (2:2:1, v / v / v)) was added, the extraction buffer containing isotope-labeled internal standard; vortexed for 30 s, sonicated in an ice-water bath for 5 min; placed in a -20°C refrigerator and allowed to stand for 1 h; centrifuged at 12,000 rpm (centrifugal force 13,800 (×g), radius 8.6 cm) for 15 min at 4°C; the supernatant was filtered through a 0.22 μm microporous membrane into a vial for instrumental analysis.

[0062] 3.3.2 On-machine testing This project utilized a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UHPLC) with a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous containing 0.01% acetic acid, and Phase B was isopropanol:acetonitrile (1:1, v / v). The sample pan temperature was 4°C, and the injection volume was 2 μL. An Orbitrap Exploris 120 mass spectrometer was used for primary and secondary mass spectrometry data acquisition under the control of Xcalibur (version 4.4, Thermo) software. The detailed parameters for data acquisition are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320°C, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE20 / 30 / 40, Spray voltage: 3.8kV (positive) or -3.4kV (negative).

[0063] 3.3.3 Data Processing After the raw data was converted into mzXML format using ProteoWizard software, metabolite identification was performed using a collaboratively developed R package with BiotreeDB (V3.0) as the database. Then, visualization analysis was performed using a self-developed R package.

[0064] 3.4 Animal handling and grouping (1) Animal grouping for the first round of drug concentration screening: C57BL / 6 mice were randomly divided into a control group (Group A), a model group (Group B), mice treated with different doses of Cordyceps militaris (Group CG), and mice treated with silymarin (Group H), with 6 mice in each group. At 10:00 AM daily, mice in Group B and H were administered 30% alcohol (10 mL / kg) by gavage, while mice in Group A were administered an equal volume of physiological saline by gavage. At 4:00 PM daily, mice in Group CG were administered 0.1 g / kg, 0.4 g / kg, 0.7 g / kg, 1 g / kg, and 10 g / kg of Cordyceps militaris ultrasonic extract by gavage, respectively; mice in Group H were administered 0.05 g / kg of silymarin (diluted with 0.5% sodium carboxymethyl cellulose solution) by gavage; and mice in Groups A and B were administered an equal volume of physiological saline by gavage. All mice were administered the medication by gavage for 30 consecutive days, during which time coat color, body weight, and activity levels were observed. Mice were fasted and deprived of water for 12 hours prior to sampling. After measuring body weight, mice were euthanized by cervical dislocation, and blood was collected from the heart. Blood samples were centrifuged at 1500 x g for 15 minutes at 4°C, and the supernatant was further centrifuged at 12000 x g for 15 minutes at 4°C. Cell-free plasma samples were divided into equal portions in DNase / RNase-free tubes and stored at -80°C. Mouse liver tissue was dissected, and liver weight was measured. Liver index was calculated. Some liver tissue was directly fixed with 4% paraformaldehyde (pH 7.4) and used for paraffin embedding, while other liver tissue was directly stored at -80°C.

[0065] (2) Animal grouping for the second round of drug concentration screening: The results of the first round of animal experiments showed that, compared with the pathological model group, mice treated with 0.4 g / kg or higher concentrations of Cordyceps militaris ultrasonic extract had significantly lower body weight, while mice treated with 0.1 g / kg concentrations of Cordyceps militaris ultrasonic extract showed no significant change in body weight. Therefore, a second round of animal experiments was conducted.

[0066] C57BL / 6 mice were randomly divided into a control group (A'), a model group (B'), groups treated with different doses of *Cordyceps militaris* ultrasonic extract (C'-E'), and a silymarin treatment group (F'), with six mice in each group. At 10:00 AM daily, mice in groups B'-F' were administered 30% alcohol (10 mL / kg) by gavage, while mice in group A' were administered an equal volume of physiological saline by gavage. At 4:00 PM daily, groups C'-E were administered 0.1 g / kg, 0.2 g / kg, and 0.3 g / kg of *Cordyceps militaris* ultrasonic extract, respectively, while group F' was administered 0.05 g / kg of silymarin by gavage, and groups A'-B' were administered an equal volume of physiological saline by gavage. All mice were administered the extracts by gavage for 30 consecutive days, during which time coat color, body weight, and activity levels were observed. Mice were fasted and deprived of water for 12 hours prior to sampling. After measuring body weight, mice were euthanized by cervical dislocation, and blood was collected from the heart. Blood samples were centrifuged at 1500 x g for 15 minutes at 4°C, and the supernatant was further centrifuged at 12000 x g for 15 minutes at 4°C. Cell-free plasma samples were divided into equal portions in DNase / RNase-free tubes and stored at -80°C. Mouse liver tissue was dissected, and liver weight was measured. Liver index was calculated. Some liver tissue was directly fixed with 4% paraformaldehyde (pH 7.4) and used for paraffin embedding, while other liver tissue was directly stored at -80°C.

[0067] 3.5 Detection of related enzyme activities in mouse blood samples The relevant enzyme activities were measured according to the reagent instructions.

[0068] 3.6 Pathological examination of mouse liver tissue Mouse liver tissue was fixed with 4% paraformaldehyde (pH 7.4) solution for 48 h, dehydrated with ethanol gradient, embedded in paraffin, serially sectioned at 5 μm, dewaxed with xylene, dehydrated with ethanol gradient, stained with hematoxylin-eosin (HE) as usual, and observed under an optical microscope.

[0069] 3.7 Data Processing All data were analyzed using SPSS 27.0. The data are expressed as mean ± standard deviation. Differences in means among multiple samples were analyzed using one-way ANOVA and then multiple comparisons were performed. A p < 0.05 was considered statistically significant.

[0070] 4 Experimental Results 4.1 Comparison of yields of Cordyceps militaris using different water bath methods Water bath extraction is a common method for extracting traditional Chinese medicine, achieving a high extraction rate. Rotary evaporation and electrothermal extraction are two common water bath concentration methods. A yield comparison revealed that rotary evaporation yields a relatively higher rate (Table 3), therefore, the water bath method was specifically chosen as rotary evaporation.

[0071] Table 3. Comparison of dry powder yield of Cordyceps militaris using two water bath methods

[0072] 4.2 Effect of different temperatures on the extraction efficiency of Cordyceps militaris by ultrasonic extraction Ultrasonic extraction is a technique for extracting and separating components from traditional Chinese medicine. This technique utilizes the strong vibrations, high speeds, cavitation effects, thermal effects, and stirring properties of ultrasound to accelerate the entry of active ingredients into the solvent, thereby increasing the extraction rate, shortening the extraction time, and saving costs. Temperature is a key parameter in ultrasonic extraction of traditional Chinese medicine, significantly affecting extraction efficiency, component stability, and energy consumption. Analysis of the dry powder yield revealed that the ultrasonic extraction yield was relatively high at 20°C (Table 4). Therefore, the temperature for ultrasonic extraction was set at 20°C.

[0073] Table 4. Comparison of yields at different temperatures using ultrasonic methods

[0074] 4.3 Comparison of total sugar content using three methods Decoction is a traditional method for extracting active ingredients from medicinal herbs by boiling them in water, and it is one of the oldest extraction techniques in traditional Chinese medicine preparations. Based on this, we compared the total sugar content in *Cordyceps militaris* extract using the anthrone method, comparing the water bath method, the ultrasonic method, and the decoction method. The results showed that the ultrasonic method yielded a relatively higher total sugar content.

[0075] 4.4 Comparison of the types and quantities of small molecule metabolites using the three methods We used ultra-high performance liquid chromatography (UHPLC) to detect small molecule metabolites in *Cordyceps militaris* extracts obtained by three methods. Principal component analysis showed significant differences in the metabolic levels of the extracts obtained by the three methods, exhibiting a clear separation trend. Figure 2 ).

[0076] The number of small molecule metabolites obtained by the water bath method and the ultrasonic method were 2020 and 1976, respectively, which is far higher than the number of small molecule metabolites obtained by the decoction method (1067). Figure 3 AC). Meanwhile, the types of small molecule metabolites obtained by the three methods differed. Specifically, the water bath method and the ultrasonic method yielded 428 and 421 specific small molecule metabolites in the Cordyceps militaris extract, respectively, significantly higher than the 160 specific small molecule metabolites in the Cordyceps militaris preparation obtained by the decoction method. Figure 4 Therefore, compared with decoction, water bath and ultrasonic methods are more suitable as extraction methods for Cordyceps militaris.

[0077] 4.5 Qualitative analysis of metabolites in Cordyceps militaris extract obtained by water bath method and ultrasonic method To clarify the specific identities of metabolites and screen for superior extraction methods for active ingredients, we further conducted targeted identification and qualitative analysis of the main compounds. We compared the seven main compounds initially identified in the *Cordyceps militaris* extract with their corresponding standards. Under the same LC-MS / MS analytical conditions, we verified the results by comparing the retention times (with a deviation <0.1 min) and primary and secondary mass spectra of the sample and standard chromatograms. If the precise primary mass number of the sample matched that of the standard, and the mass-to-charge ratio (m / z) and relative intensity of the main fragment ions in its secondary mass spectrum highly matched those of the standard, then the sample was identified as the correct compound.

[0078] like Figure 5 As shown, the chromatographic peaks of the seven target compounds in the Cordyceps militaris extract all have a Dot Product Score (DP) higher than 0.96 compared to their corresponding standards. Among them, the DP value of valine (B) reaches 1. This means that the fragment ions produced in the experiment (as shown by the horizontal axis m / z in the figure) not only correspond completely with the ions in the reference spectrum in terms of species, but also their relative intensity ratios (as shown by the vertical axis in the figure) are almost completely consistent. This systematically confirms that the qualitative analysis of these seven components in the Cordyceps militaris extract is accurate and reliable.

[0079] 4.6 Comparison of the contents of 20 main substances in Cordyceps militaris extract obtained by water bath method and ultrasonic method Based on the above identification results, we further quantitatively compared the contents of 20 major active substances of Cordyceps in the extracts obtained by water bath method and ultrasonic method to determine the optimal extraction method.

[0080] Table 5. 20 main substances in Cordyceps sinensis based on literature review

[0081] As shown in Table 5, a literature review revealed that 2'-deoxyadenosine, mannitol, and 20 other substances are the 20 most common metabolites found in Cordyceps.

[0082] like Figure 6 As shown, comparing the contents of these 20 substances in *Cordyceps militaris* extracts obtained by water bath extraction and ultrasonic extraction revealed that the ultrasonic extraction method yielded extracts with significantly higher contents of most of the 20 main active substances compared to the water bath method. Specifically, tryptophan, glutamic acid, valine, uridine, and inosine were significantly higher in the ultrasonic extract. Therefore, ultrasonic extraction was chosen for subsequent functional experiments on *Cordyceps militaris*.

[0083] 4.7 Results of the first round of animal experiments (1) Changes in mouse body weight after treatment with different concentrations of Cordyceps militaris ultrasonic extract

[0084] Table 6. Changes in net body weight of mice in each treatment group over 30 days (Results of the first round of animal experiments) Changes in net body weight gain of mice in each group (n=7, x±s, *p<0.05 vs. control group); # (p<0.05 vs. pathological model group) like Figure 7 As shown in Table 6, the pathological model group resulted in a significant decrease in mouse body weight compared to the control group. Compared to the model group, the mouse body weight was significantly reduced in the treatment groups of 0.4 g / kg, 0.7 g / kg, 1 g / kg and 10 g / kg Cordyceps militaris ultrasonic extract, while there was no significant change in mouse body weight in the treatment groups of 0.1 g / kg and silymarin.

[0085] (2) Effects of different concentrations of Cordyceps militaris ultrasonic extract on liver weight and liver index in mice like Figure 8 As shown in BC, compared with the control group, the pathological model group significantly increased liver weight and liver index in mice. Compared with the pathological model group, the liver weight and liver index of mice treated with 1 g / kg, 10 g / kg concentrations of Cordyceps militaris ultrasonic extract and silymarin treatment groups were significantly decreased. The liver index of mice treated with 0.7 g / kg concentration of Cordyceps militaris ultrasonic extract was significantly decreased, but the liver weight did not change significantly. The liver weight and liver index of mice treated with other concentrations of Cordyceps militaris ultrasonic extract did not change significantly. In addition, there were no significant morphological changes in the livers of mice in any treatment group.

[0086] 4.9 Results of the second round of animal experiments The first round of animal experiments revealed that mice treated with 0.4 g / kg or higher concentrations of Cordyceps militaris ultrasonic extract showed a significant decrease in body weight, while the 0.1 g / kg concentration showed no significant change. Therefore, a second round of animal experiments was conducted, with concentrations of 0.1 g / kg, 0.2 g / kg, and 0.3 g / kg to be selected as the treatment concentrations for Cordyceps militaris ultrasonic extract.

[0087] (1) Changes in mouse body weight after treatment with different concentrations of Cordyceps militaris ultrasonic extract Table 7. Changes in net body weight of mice in each treatment group over 30 days (Results of the first round of animal experiments)

[0088] Changes in net body weight gain of mice in each group (n=7, x±s, *p<0.05 vs. control group); # (p<0.05 vs. pathological model group) like Figure 9As shown in Table 7, the pathological model group resulted in a significant decrease in mouse body weight compared to the control group. Compared to the model group, the mouse body weight was significantly reduced in the 0.3 g / kg Cordyceps militaris ultrasonic extract treatment group, while there were no significant changes in mouse body weight in the 0.1 g / kg, 0.2 g / kg Cordyceps militaris treatment groups and the silymarin treatment group.

[0089] (2) Effects of different concentrations of Cordyceps militaris ultrasonic extract on liver weight and liver index in mice like Figure 10 As shown in BC, compared with the control group, the pathological model group resulted in a significant increase in liver weight and liver index in mice. Compared with the pathological model group, the liver weight and liver index of mice treated with 0.2 g / kg concentration of Cordyceps militaris ultrasonic extract and silymarin treatment were significantly reduced, while the liver weight and liver index of mice treated with other concentrations of Cordyceps militaris ultrasonic extract showed no significant changes. Furthermore, there were no significant morphological changes in the livers of mice in any of the treatment groups.

[0090] 4.9 Liver function test results like Figure 11 As shown in the AD diagram, compared with the control group, the serum ALT, AST, and AKP activities and TC levels in the pathological model group were significantly increased. Compared with the pathological model group, all concentrations of Cordyceps militaris ultrasonic extract treatment groups reduced the serum ALT, AST, and AKP activities and TC levels to varying degrees, showing a concentration-dependent effect. The serum ALT, AST, and TC levels reached their lowest values ​​after treatment with 0.3 g / kg Cordyceps militaris ultrasonic extract. Furthermore, compared with the pathological model group, silymarin also reduced the serum ALT, AST, and AKP activities and TC levels in mice, but the reduction in ALT, AST, and TC levels was slightly lower than that in the 0.2 g / kg and / or 0.3 g / kg Cordyceps militaris ultrasonic extract treatment groups.

[0091] like Figure 11 As shown in E, compared with the control group, the serum TG content of mice in the pathological model group was significantly increased; compared with the pathological model group, the treatment groups with 0.2 g / kg Cordyceps militaris ultrasonic extract and silymarin significantly reduced the serum TG content of mice. Other concentrations of Cordyceps militaris ultrasonic extract treatment groups also led to a decrease in serum TG content, but there was no significant difference.

[0092] like Figure 11 As shown in G, compared with the control group, the HDL-C content in the serum of mice in the pathological model group was significantly reduced; compared with the pathological model group, the HDL-C content in the serum of mice treated with 0.2 g / kg and 0.3 g / kg concentrations of Cordyceps militaris ultrasonic extract and silymarin treatment groups was significantly increased.

[0093] Furthermore, there were no significant changes in LDL-C levels in any of the treatment groups. Figure 10 F).

[0094] 4.10 Histopathological changes in liver tissue like Figure 12 As shown, the control group had intact liver lobule structure, and the hepatocytes were arranged neatly radially around the central vein, with no obvious pathological changes observed. Figure 12 AB); In the alcohol model group, hepatocytes were arranged disorderedly, and necrotic foci of hepatocytes were visible in the liver lobules. Under high magnification, the cytoplasm was loose, with a large number of lymphocytes aggregated and a large amount of inflammatory cell infiltration. Bile duct hyperplasia was frequently seen in the portal area, and mild fibrosis was also observed. Figure 12 CD); the group treated with 0.1 g / kg concentration of Cordyceps militaris ultrasonic extract still showed portal duct hyperplasia and inflammatory cell infiltration, but no focal hepatocellular necrosis was observed. Figure 12 EF); In the 0.2 g / kg concentration treatment group, hepatocytes and hepatic cords were neatly arranged, with occasional hepatic congestion, and inflammatory cells were basically eliminated. Figure 12 In the 0.3 g / kg concentration treatment group, the liver lobule structure was close to normal, the hepatocytes and hepatic cords were neatly arranged, and no obvious abnormalities were observed in the portal areas between adjacent lobules. Figure 12 IJ); In the silymarin treatment group, the liver lobule structure was clear, the liver cells were neatly arranged, there was no fibrosis, and no inflammatory cell infiltration was observed. Figure 12 KL).

[0095] 4.11 Biochemical results and liver tissue pathological section results of the first batch of experimental animals (1) Biochemical experimental results like Figure 13 As shown in AC, compared with the control group, the serum ALT and AST activities and TC levels in the pathological model group were significantly increased. Compared with the pathological model group, all concentrations of Cordyceps militaris ultrasonic extract treatment groups reduced the serum ALT, AST activities, and TC levels to varying degrees in a concentration-dependent manner, with the lowest values ​​for ALT, AST activities, and TC levels observed after treatment with 0.7 g / kg (or 1 g / kg) concentration of Cordyceps militaris ultrasonic extract. Furthermore, compared with the pathological model group, silymarin also reduced the serum ALT, AST, and AKP activities and TC levels in mice, but the reduction in ALT and AST activities was significantly lower than that in the 0.4 g / kg, 0.7 g / kg, 1 g / kg, and 10 g / kg concentrations of Cordyceps militaris ultrasonic extract treatment groups.

[0096] like Figure 13As shown in E, compared with the control group, the serum TG content of mice in the pathological model group was significantly increased. Compared with the pathological model group, the treatment groups of 0.7 g / kg, 1 g / kg and 10 g / kg Cordyceps militaris ultrasonic extract and the silymarin treatment group could significantly reduce the serum TG content of mice. Other concentrations of Cordyceps militaris ultrasonic extract treatment groups could also lead to a decrease in serum TG content, but there was no significant difference.

[0097] like Figure 13 As shown in G, compared with the control group, the HDL-C content in the serum of mice in the pathological model group was significantly reduced; compared with the pathological model group, the HDL-C content in the serum of mice treated with 0.4 g / kg and 0.7 g / kg concentrations of Cordyceps militaris ultrasonic extract and silymarin treatment groups was significantly increased.

[0098] Furthermore, there were no significant changes in LDL-C levels in any of the treatment groups. Figure 13 F).

[0099] like Figure 14 As shown, the control group had intact liver lobule structure, and the hepatocytes were arranged neatly radially around the central vein, with no obvious pathological changes observed. Figure 14 AB); In the alcohol model group, hepatocytes were arranged disorderedly, and necrotic foci of hepatocytes were visible in the liver lobules. Under high magnification, the cytoplasm was loose, with a large number of lymphocytes aggregated and a large amount of inflammatory cell infiltration. Bile duct hyperplasia was frequently seen in the portal area, and mild fibrosis was also observed. Figure 14 CD); Inflammatory cell infiltration was observed around the portal area in the 0.1 g / kg dose group of Cordyceps militaris ultrasonic extract, but no focal hepatocyte necrosis was observed. Figure 14 EF); In the 0.4 g / kg and 0.7 g / kg dose groups, the liver lobule structure was close to normal, the hepatocytes and hepatic cords were neatly arranged, and no obvious abnormalities were observed in the portal areas between adjacent lobules. Figure 14 GJ); Hepatic sinusoidal congestion was observed in the 1 g / kg and 10 g / kg dose groups, with lymphocyte aggregation and occasional hepatic congestion. Figure 14 KN); In the silymarin group, the liver lobule structure was clear, the liver cells were neatly arranged, there was no fibrosis, and no inflammatory cell infiltration was observed. Figure 14 OP).

[0100] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The use of a Cordyceps militaris extract in the preparation of a medicament for the treatment and / or prevention of alcoholic liver disease.

2. The application as described in claim 1, characterized in that, The extract of Cordyceps militaris contains: propranolol L-, schisandrin B, glycyrrhizic acid, curcumin, hesperidin, andrographolide, rosmarinic acid, caffeic acid, ursolic acid, and corosolic acid.

3. The application as described in claim 1 or 2, characterized in that, The Cordyceps militaris extract is prepared by a method including the following steps: pulverizing Cordyceps militaris raw material, using water as a solvent, ultrasonically extracting it at 20-45°C, collecting the extract, and concentrating and drying it.

4. The application as described in any one of claims 1-3, characterized in that, The drug is used to reduce the elevation of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), total cholesterol (TC), and / or triglycerides (TG) caused by alcoholic liver injury.

5. The application as described in any one of claims 1-3, characterized in that, The drug is used to increase the reduction of serum high-density lipoprotein cholesterol (HDL-C) caused by alcoholic liver injury.

6. The application as described in any one of claims 1-3, characterized in that, The drug is used to improve the pathological changes in liver tissue caused by alcoholic liver injury, including reducing hepatocellular steatosis, inflammatory cell infiltration, and hepatocellular necrosis.

7. The application as described in any one of claims 1-3, characterized in that, The dosage of the Cordyceps militaris extract in the drug is from 0.1 g / kg body weight to 0.3 g / kg body weight.

8. The application as described in claim 7, characterized in that, The dosage is 0.2 g / kg body weight.

9. A pharmaceutical composition for treating and / or preventing alcoholic liver disease, characterized in that, The extract comprises a therapeutically effective amount of Cordyceps militaris extract prepared by the method of claim 2 or 3, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The amount of the Cordyceps militaris extract in the pharmaceutical composition is such that its equivalent daily adult dose corresponds to 0.1 g / kg body weight to 0.3 g / kg body weight of the raw drug.

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