Brassica juncea or its extract, compositions thereof, and uses thereof

Abstract

The application discloses use of a bupleurum falcatum or extract thereof in preparation of a medicine for treating and / or preventing diseases caused by liver damage and use in preparation of a medicine for treating and / or preventing diseases and / or conditions related to diseases caused by liver damage. The diseases caused by liver damage can be acute liver damage, chronic liver damage, liver fibrosis, liver cirrhosis, liver cancer and the like. The bupleurum falcatum or extract thereof has a liver protection effect, and proves to have a protective effect on diseases caused by liver damage through liver function indexes, oxidative stress levels and liver pathological staining, and is expected to become a new therapeutic drug for liver damage diseases.

Description

Technical Field

[0001] This application relates to the fields of traditional Chinese medicine or medicinal chemistry, specifically to water spinach or its extracts, their compositions and uses. Background Technology

[0002] The liver is the largest metabolic organ in the human body, and also the largest gland in the digestive system. It plays a crucial role in maintaining metabolism, eliminating toxins, and the production and release of proteins. Liver damage results from the combined effects of various internal and external factors, leading to abnormalities in the liver's structure and function. These factors may include hepatitis viruses, medications, alcohol, fat deposits, and other diseases or conditions. If liver damage continues to worsen, it can lead to fatty liver, cirrhosis, liver fibrosis, and even liver cancer, seriously endangering health. Reports indicate that approximately 3.5% of global deaths are directly related to liver damage.

[0003] With the rapid development of medical technology, many drugs for treating liver injury have been researched and reported, mainly including traditional Chinese medicine, biological drugs, chemical drugs, and trace elements. However, some studies have shown that some anti-liver injury drugs can still inevitably produce biotoxicity. Therefore, the prevention and treatment of liver injury remains a serious challenge.

[0004] Traditional Chinese medicine (TCM) believes that acute liver injury is often related to improper diet, physical weakness and fatigue, emotional imbalance, and external pathogenic factors. Its pathogenesis is primarily due to dampness accumulating in the liver and spleen, leading to liver and gallbladder dysfunction, spleen and stomach disharmony, and qi stagnation and blood stasis. Chinese herbal medicine is a treasure of traditional Chinese medicine. Many Chinese herbs and herbal components with effects such as clearing heat and detoxifying, promoting diuresis and relieving jaundice, promoting blood circulation and removing blood stasis, and soothing the liver and regulating qi can directly treat liver injury without producing significant toxic side effects. Therefore, the research and development of these drugs has important practical significance for the clinical treatment of liver injury. Summary of the Invention

[0005] Currently, there is no publicly available information on the protective effect of watercress against liver damage. This application, through extensive testing, has found that watercress or its extracts can be used to prevent and treat diseases caused by liver damage.

[0006] Specifically, this application relates to the following aspects:

[0007] 1. Use of water spinach or its extracts in the preparation of medicaments for the treatment and / or prevention of diseases caused by liver injury.

[0008] 2. Use of watercress or its extracts in the preparation of medicaments for the treatment and / or prevention of diseases and / or conditions associated with liver injury.

[0009] 3. The use described in item 1 or 2, wherein the disease caused by liver injury is selected from one of acute liver injury, chronic liver injury, liver fibrosis, cirrhosis, and liver cancer.

[0010] 4. The use described in item 1 or 2, wherein the cause of liver injury is selected from one of trauma, drugs, alcohol, chemical toxins, viruses, or autoimmune diseases.

[0011] 5. The use described in item 1 or 2, wherein the drug is used in mammals.

[0012] 6. The use described in item 1 or 2, wherein a single dose of the drug is equivalent to 10g to 200g of raw water spinach.

[0013] 7. The use described in item 6, wherein a single dose of the drug is equivalent to 25g to 100g of raw water spinach.

[0014] 8. The use described in item 1 or 2, wherein the water spinach extract is a water spinach extract or a water spinach alcohol extract.

[0015] 9. The use described in item 8, wherein the water extract of water spinach is extracted by the following method:

[0016] Grind: Take watercress and grind it into coarse powder;

[0017] Water heating and reflux extraction: Add water to the coarse powder, reflux extract, filter, add water again, reflux extract again, filter and combine the two filtrates;

[0018] Filtrate concentration: The filtrate was concentrated under reduced pressure to obtain the water extract of the water purslane.

[0019] 10. The use described in item 8, wherein the purslane alcohol extract is extracted by the following method:

[0020] Grind: Take watercress and grind it into coarse powder;

[0021] Ethanol reflux extraction: Add ethanol to the crude powder, reflux and extract, filter, add ethanol again, reflux and extract again, filter and combine the two filtrates.

[0022] Filtrate concentration: The filtrate was concentrated under reduced pressure to obtain the purslane alcohol extract.

[0023] 11. Use of any one of items 1-10, wherein the medicament further comprises one or more other active substances.

[0024] 12. A composition for treating and / or preventing diseases caused by or related to liver injury, wherein the composition comprises watercress or an extract thereof, watercress or an extract thereof as the sole active ingredient in the composition.

[0025] 13. The composition of claim 12, wherein the composition further comprises one or more pharmaceutically acceptable carriers or excipients, or further comprises one or more other active substances.

[0026] 14. The composition of item 12 or 13, wherein the disease caused by liver injury is the disease covered by item 3 or 4.

[0027] 15. The composition of any one of items 12-14, wherein the watercress extract is the watercress extract of any one of items 8-10.

[0028] Water spinach or its extracts can improve liver function indicators, oxidative stress levels, and liver pathological damage, and have a protective effect against diseases caused by liver damage. It is expected to become a new therapeutic drug for liver damage diseases. Attached Figure Description

[0029] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:

[0030] Figure 1 Changes in serum alanine aminotransferase (ALT) in mice from different groups;

[0031] Figure 2 Changes in serum aspartate aminotransferase (AST) in mice from different groups;

[0032] Figure 3 Changes in reduced glutathione (GSH) in liver tissues of mice from different groups;

[0033] Figure 4 Changes in superoxide dismutase (SOD) in liver tissues of mice from different groups;

[0034] Figure 5 Images of H&E staining in the livers of mice from different groups. Detailed Implementation

[0035] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0036] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0037] This application provides the use of watercress or its extract in the preparation of a medicament for treating and / or preventing diseases caused by liver damage.

[0038] This application also provides the use of watercress or its extract in the preparation of a medicament for treating and / or preventing diseases and / or conditions related to liver damage.

[0039] Watercress (scientific name: *Rorippa indica* (L.) Hiern) is a plant belonging to the genus *Rorippa* in the family Brassicaceae. It is an annual herb with expectorant and antitussive properties. It is used for chronic bronchitis. It also has antihypertensive, diuretic, blood-cooling, and hemostatic effects. It is used for dizziness, hypertension, painful and difficult urination, hematuria, metrorrhagia, and leukorrhea. In addition to its medicinal value, it can also be eaten as a vegetable.

[0040] Liver injury primarily refers to damage to liver cells and abnormalities in liver structure and function. Liver injury stimulates the body to produce excessive amounts of highly reactive oxygen free radicals, inducing oxidative stress, triggering inflammatory responses, leading to liver cell damage and apoptosis, and the release of excessive transaminases into the bloodstream, thus affecting normal liver function. If not prevented and treated promptly, it may induce a series of complications or promote the development of other diseases.

[0041] The innovative discovery of this application is that watercress or its extracts have therapeutic or preventive effects on diseases caused by liver damage.

[0042] There are many causes of liver damage, mainly including drug poisoning, viral infection, excessive alcohol intake, immune response, exposure to toxic chemicals in the environment, and radiation damage. The *Nasturtium* or its extract described in this application has therapeutic or preventative effects on diseases caused by liver damage. This application does not limit the causes of liver damage; any liver damage involving liver cell damage, abnormal liver structure, or abnormal liver function falls within the scope of this application. In a preferred embodiment, the liver damage is caused by alcohol, viruses, drugs, and toxic chemicals.

[0043] The specific type of disease caused by liver injury is not limited in this application. Any disease caused by liver injury is within the scope of protection of this application, including but not limited to acute liver injury, chronic liver injury, liver fibrosis, cirrhosis, hepatitis, liver cancer, fatty liver, and liver failure. Examples include liver injury, fatty liver, hepatitis, cirrhosis, and liver failure caused by factors such as alcohol. In a preferred embodiment, the disease is either acute or chronic liver injury.

[0044] The diseases and / or conditions related to liver injury refer to other diseases or conditions caused by liver injury, which may occur due to liver injury or be relieved or disappear after treatment of liver injury.

[0045] As used herein, “treatment” means any improvement in any consequence of a disease, disorder, or condition, such as prolonged survival, lower morbidity, and / or reduction of side effects caused by alternative treatment modalities. In some embodiments, treatment comprises delaying or improving a disease, disorder, or condition (i.e., slowing or halting or reducing the development of the disease or at least one of its clinical symptoms). In some embodiments, treatment comprises delaying, alleviating, or improving at least one bodily parameter of a disease, disorder, or condition, including bodily parameters that the patient may not be able to discern. In some embodiments, treatment comprises physical (e.g., stabilizing discernible symptoms), physiological (e.g., stabilizing bodily parameters), or both, modulation of the disease, disorder, or condition.

[0046] As used in this article, the term “prevention” refers to preventive treatment of a disease or condition; or delaying the onset or progression of such a disease, disorder or condition.

[0047] The drug can be used in patients or other animals receiving the drug of this application to treat, prevent, alleviate, and / or relieve the diseases or conditions described in this application. In a preferred embodiment, the drug is used in mammals, including but not limited to humans, cattle, horses, sheep, pigs, goats, rabbits, cats, dogs, rats, and any other mammals with livers that may be damaged.

[0048] In a preferred embodiment, the drug is used on a human.

[0049] The dosage of the drug described in this application depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, and individual response of the patient or animal, the route of administration, and the frequency of administration. The dosage can be administered as a single dose or in several doses, such as two, three, or four doses. The dosage level must be selected based on the specific route of administration, the severity of the condition being treated, and the patient's condition and medical history.

[0050] However, it should be recognized that the total daily dosage of the drug in this application must be determined by the attending physician within the bounds of reliable medical judgment. For any given patient, the specific effective therapeutic dose level must be determined based on a variety of factors, including the disorder being treated and its severity; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate; the duration of treatment; any drugs used in combination or concurrently; and similar factors known in the medical field. For example, it is practiced in the art to start with a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0051] In a further preferred embodiment, the drug is administered in a single dose, i.e., a single-dose form. In one specific embodiment, the single dose is equivalent to 10g to 200g of raw water spinach, for example, 10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g, 50g, 55g, 60g, 65g, 70g, 75g, 80g, 85g, 90g, 95g, 100g, 110g, 120g, 130g, 140g, 150g, 160g, 170g, 180g, 190g, 200g, and any value between these values. In one specific embodiment, the single dose is equivalent to 25g to 100g of raw water spinach. In another specific embodiment, the single dose is equivalent to 25g to 50g of raw water spinach. Those skilled in the art should understand that, generally speaking, a person's weight is measured in 60 kg. If the person's weight is not 60 kg, this standard can be used for conversion.

[0052] Those skilled in the art will understand that the frequency and number of administrations can be adjusted based on the specific circumstances of the subject or the severity of liver injury.

[0053] In one specific embodiment, a single dose of the drug is administered to a person 1-3 times daily. In another specific embodiment, a single dose of the drug is administered to a person 1-3 times daily for 3 days to 1 month, for example, 3 days, 5 days, 7 days, 10 days, 14 days, 20 days, 1 month, etc.

[0054] The drug described in this application can be administered in unit dose form via enteral or non-enteric routes, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum, or rectum. Dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections. It can be a conventional formulation, a sustained-release formulation, a controlled-release formulation, or various microparticle delivery systems.

[0055] The water spinach extract can be a water extract or an alcohol extract of water spinach, or a mixture of the water extract and the alcohol extract.

[0056] Water spinach extract can be prepared using any part of the whole plant, such as roots, stems, leaves, and flowers.

[0057] The extraction method for the water extract of *Gnaphalium affine* can be any water extraction method available in the art. In a preferred embodiment, the water extract of *Gnaphalium affine* is prepared using the following method:

[0058] Grind: Take watercress and grind it into coarse powder;

[0059] Water heating and reflux extraction: Add water to the coarse powder, reflux extract, filter, add water again, reflux extract again, filter and combine the two filtrates;

[0060] Filtrate concentration: The filtrate was concentrated under reduced pressure to obtain the water extract of the water purslane.

[0061] The water can be any form of water in the art, such as purified water, deionized water, recycled water, etc.

[0062] The number of times the reflux extraction is performed is not limited in this application; it can be once, twice, three times, four times, or more.

[0063] The extraction method for the *Gynostemma pentaphyllum* alcohol extract can be any alcohol extraction method in the art. In a preferred embodiment, the *Gynostemma pentaphyllum* alcohol extract is prepared by the following method:

[0064] Grind: Take watercress and grind it into coarse powder;

[0065] Ethanol reflux extraction: Add ethanol to the crude powder, reflux and extract, filter, add ethanol again, reflux and extract again, filter and combine the two filtrates.

[0066] Filtrate concentration: The filtrate was concentrated under reduced pressure to obtain the purslane alcohol extract.

[0067] Ethanol can be any other common alcohol in the field, such as methanol, propanol, butanol, etc.

[0068] The number of times the reflux extraction is performed is not limited in this application; it can be once, twice, three times, four times, or more.

[0069] In a preferred embodiment, the medicament optionally also contains a pharmaceutically acceptable carrier or excipient. These include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gel, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above-mentioned components, the medicament of this application may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0070] In a preferred embodiment, the medicament of this application may further include one or more other active substances. These other active substances refer to substances other than Artemisia capillaris or its extracts that are active and produce certain functions. These active substances may be substances that treat or prevent diseases caused by liver damage or related to diseases caused by liver damage; they may also be substances that assist in the treatment or prevention of diseases caused by liver damage or related to diseases caused by liver damage; or they may not treat diseases caused by liver damage or related to diseases caused by liver damage, but may have a positive effect or function in other aspects. Examples of these active substances include one or more of Artemisia capillaris, Rehmannia glutinosa, Paeonia suffruticosa, Coptis chinensis, Rehmannia glutinosa, Bupleurum chinense, Prunella vulgaris, Achyranthes bidentata, and Scutellaria baicalensis. These other active substances may be the original medicinal material or its extracts (e.g., water extracts, alcohol extracts, or extracts of water extracts and alcohol extracts).

[0071] This application further provides a composition for treating and / or preventing diseases caused by liver injury or diseases and / or symptoms related to diseases caused by liver injury, wherein the composition comprises watercress or an extract thereof, with watercress or an extract thereof as the sole active ingredient in the composition. That is, watercress or an extract thereof is the sole active ingredient for treating and / or preventing diseases caused by liver injury or diseases and / or symptoms related to diseases caused by liver injury.

[0072] The term "composition" means a product comprising specified amounts of each specified ingredient, and any product derived directly or indirectly from a combination of specified amounts of each specified ingredient.

[0073] In a preferred embodiment, the composition further comprises one or more pharmaceutically acceptable carriers or excipients. The one or more pharmaceutically acceptable carriers or excipients are any of the carriers or excipients described above.

[0074] In a preferred embodiment, the composition further comprises one or more other active substances. The one or more other active substances are any of the active substances described above.

[0075] The diseases caused by liver damage are any of the diseases caused by liver damage described above.

[0076] The water spinach extract is any of the water spinach extracts described above.

[0077] The composition described in any one of the present application may be an injection, oral liquid, capsule, tablet, granule, or extract remixed dosage form.

[0078] The compositions according to any one of the claims of this application can be administered in a single dose via the enteric or non-enteric route, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum, or rectum. Dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. They can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

[0079] The dosage of the composition according to any one of the claims can be converted by referring to the dosage of watercress or its extract as described above.

[0080] In this application, the terms "alanine aminotransferase (ALT)" and "aspartate aminotransferase (AST)" refer to enzymes whose blood levels increase when the liver is damaged; these are enzymes whose properties are utilized as indicators of liver function. The terms "reduced glutathione (GSH)" and "superoxide dismutase (SOD)" are important components of the antioxidant enzyme system in biological systems; these are enzymes whose properties are utilized as indicators of oxidative stress levels.

[0081] The water spinach or its extract involved in this application has the efficacy of treating and / or preventing diseases caused by liver damage. This application has verified the effects of water spinach or its extract on serum AST, ALT, liver oxidative stress level and liver pathological structure through mouse model experiments.

[0082] Specifically, biochemical tests revealed that 16 hours after intraperitoneal injection of CC14, the serum AST and ALT levels in the model group mice significantly increased. The AST and ALT levels in the model group were significantly different from those in the control group (P<0.001). Regarding ALT levels, the silymarin-treated group, the high, medium, and low dose groups of water spinach water extract, and the medium and low dose groups of alcohol extract all showed improvement compared to the model group, with the low dose group of water spinach water extract showing the most significant improvement (P<0.001), superior to the positive control group. Similarly, the silymarin-treated group, the high, medium, and low dose groups of water spinach water extract, and the high, medium, and low dose groups of alcohol extract all showed improvement compared to the model group, with the low dose groups of water spinach water and alcohol extract showing the most significant improvement (P<0.001), all superior to the silymarin-treated positive control group.

[0083] Oxidative stress level detection revealed that 16 hours after intraperitoneal injection of CC14, the levels of GSH and SOD in the liver of mice in the model group decreased significantly. The GSH and SOD levels in the model group were significantly different from those in the control group (P<0.05). As shown in the figure, high-dose, medium-dose, and low-dose groups of water extract and ethanol extract of water extract all increased the GSH content in liver tissue. In particular, the low-dose group of water extract and the medium-dose and low-dose groups of ethanol extract significantly increased the GSH content in mice, which was statistically significantly different from that in the model group (P<0.05) and much better than the positive control group treated with silymarin. High-dose, medium-dose, and low-dose groups of water extract and alcohol extract of purslane all increased the SOD content in liver tissue. In particular, the high-dose and medium-dose and low-dose groups of water extract and alcohol extract significantly increased the SOD content in mouse liver tissue, showing a statistically significant difference from the model group (P<0.05), and were far better than the positive control group treated with silymarin.

[0084] Further investigation through liver pathology and histology revealed that the model group showed extensive inflammatory cell infiltration and hepatocyte necrosis or nuclear condensation and dissolution in the liver tissue. Compared with the model group, the drug-treated mice showed significantly reduced hepatocyte damage, liver necrosis or nuclear condensation and dissolution, and inflammatory infiltration, with uniform cytoplasmic staining. This indicates that the water / alcohol extract of *Gynostemma pentaphyllum* can improve the structure of hepatocyte tissue, which is superior to the positive control group treated with silymarin.

[0085] Drugs commonly used in the prior art for treatment or prevention, such as silybin, the positive drug used in the present application, have low bioavailability, poor effect of being directly absorbed and utilized in the intestinal tract after administration, slow therapeutic effect, and relatively long treatment course. While other drugs such as bifendate can cause a significant and rapid decrease in transaminase, they do not produce obvious improvements in liver histology, and actually have poor anti-inflammatory and hepatocyte-protective effects. The Rorippa indica or its extract of the present application can significantly and rapidly reduce transaminase, protect the histological morphology of the liver, achieve comprehensive conditioning, and is mild and safe.

[0086] Examples

[0087] Experimental materials

[0088] Animals: SPF-grade male C57BL / 6 mice, weighing 22 (±2) g, 8 weeks old. Purchased from Zhuhai Beston Biotechnology Co., Ltd., Animal License No.: [SCXK (Yue) 2022-0051].

[0089] Test sample: Rorippa indica, purchased from Yulin Medicinal Material Market, Guangxi.

[0090] Reagents: 95% Ethanol (AR), Shanghai Macklin Biochemical Technology Co., Ltd.;

[0091] CCl4 (HPLC), Shanghai Macklin Biochemical Technology Co., Ltd.;

[0092] Olive oil (pharmaceutical grade), Shanghai Macklin Biochemical Technology Co., Ltd.;

[0093] Detection kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), reduced glutathione (GSH), and superoxide dismutase (SOD), Nanjing Jiancheng Bioengineering Institute Co., Ltd.

[0094] Silybin (SY), Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0095] Example 1 Preparation of Rorippa indica extract

[0096] In this example, alcohol extraction and water extraction are performed on Rorippa indica respectively, and the specific methods are as follows:

[0097] 1.1 Alcohol extraction

[0098] (1) Preliminary crushing: Take Rorippa indica and crush it into coarse drug powder.

[0099] (2) Ethanol heating reflux extraction: Take the dried coarse traditional Chinese medicine powder, add 8 times the mass of 95% ethanol, perform reflux extraction at 80°C for 1 hour, filter, then add 8 times the mass of 95% ethanol again, perform reflux extraction at 80°C for 1 hour, filter and combine the two filtrates.

[0100] (3) Filtrate concentration: The above filtrate was concentrated under reduced pressure at 60°C until the relative content of the original medicinal material was 1g / mL, to obtain the purslane alcohol extract.

[0101] 1.2 Water Extraction

[0102] (1) Preliminary crushing: Take water spinach and crush it into coarse powder.

[0103] (2) Water heating and reflux extraction: Take the dried crude powder of Chinese medicine, add 8 times the mass of water, reflux extract at 80℃ for 1 hour, filter, add 8 times the mass of water again, reflux extract at 80℃ for 1 hour, and combine the two filtrates after filtration.

[0104] (3) Filtrate concentration: The above filtrate was concentrated under reduced pressure at 60°C until the relative content of the original medicinal material was 1g / mL, to obtain water extract of water purslane.

[0105] Example 2: Experimental methods and results of the effect of purslane extract on carbon tetrachloride-induced acute liver injury in mice.

[0106] The aqueous extract and alcoholic extract of water spinach prepared in Example 1 were used in the following experiments.

[0107] 2.1 Experimental Methods

[0108] 2.1.1 Animal grouping and administration

[0109] Seventy mice were randomly divided into a control group (NC), a model group, a low-dose group treated with water extract of *Smilax china* (IRH-SL), a medium-dose group treated with water extract of *Smilax china* (IRH-SM), a high-dose group treated with water extract of *Smilax china* (IRH-SH), a low-dose group treated with alcohol extract of *Smilax china* (IRH-CL), a medium-dose group treated with alcohol extract of *Smilax china* (IRH-CM), a high-dose group treated with alcohol extract of *Smilax china* (IRH-CH), and a positive control group (SY). The positive control group was administered silymarin (SY) at a concentration of 150 mg / kg body weight / day. The low-dose group was administered silymarin at a concentration of 3.25 g / kg body weight / day, the medium-dose group at a concentration of 6.5 g / kg body weight / day, and the high-dose group at a concentration of 13 g / kg body weight / day, all by gavage once daily for 7 days.

[0110] 2.1.2 Establishment of an acute liver injury model using CCl4

[0111] Two hours after the last administration, all groups except the control group received a single intraperitoneal injection of 10% (v:v) concentration of CCl4 dissolved in olive oil (1 ml / kg) to induce an acute liver injury model; the control group received an equal volume of olive oil intraperitoneally.

[0112] 2.1.3 Sample Collection and Processing

[0113] Sixteen hours after CCl4 injection, mice were anesthetized with sodium pentobarbital, and blood was collected from the orbital cavity to euthanize them. The livers were then isolated for further analysis. Mouse whole blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and stored. The same portion of the mouse liver was used for histopathological examination, and the remaining liver tissue was stored at -80°C.

[0114] 2.1.4 Determination of Biochemical Indicators

[0115] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a kit method purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.

[0116] 2.1.5 Determination of Oxidative Stress Indicators

[0117] Reduced glutathione (GSH) and superoxide dismutase (SOD) were detected using a kit method purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.

[0118] 2.1.6 Processing of Pathological Sections

[0119] Liver tissues from the right lobe of the liver (with the largest cross-section, containing the portal vein area and the central vein area) of different mice were collected and fixed in 4% paraformaldehyde. Paraffin sections were prepared by washing, dehydration, clearing, paraffin embedding, sectioning and mounting, dewaxing, H&E staining, dehydration, clearing, and mounting. Histological lesions were observed under an optical microscope.

[0120] 2.1.7 Statistical Data Processing

[0121] All experimental data were statistically analyzed using GraphPad Prism 9.0 software. When the quantitative data met the normal distribution, one-way ANOVA was used with a sample size of N = 4 / 5. The p-values ​​were: #, P < 0.05; ##, P < 0.01; ###, P < 0.001; Compared with the model group (CCl4), *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0122] 2.2 Results

[0123] 2.2.1 Serum ALT and AST levels in each group of mice

[0124] exist Figure 1In the model group (CCl4), ALT activity was significantly higher than that of the control group (NC) (P<0.001), indicating successful modeling. Administration of the aqueous or alcoholic extracts of *Gnaphalium affine* prepared in Example 1 significantly reduced ALT activity. As shown in the figure, all groups (IRH-SH, IRH-SM, and IRH-SH) of the aqueous extract and the IRH-CM and IRH-CL of the alcoholic extract showed very good effects, with significant differences in ALT levels compared to the model group (P<0.001). In particular, the low-dose aqueous extract group (IRH-SL) was even more effective than the positive control group of silymarin (SY).

[0125] exist Figure 2 In the model group (CCl4), the AST activity was significantly higher than that of the control group (NC) (P<0.001), indicating successful modeling. Administration of the aqueous or alcoholic extracts of *Gnaphalium affine* prepared in Example 1 significantly reduced AST activity. As shown in the figure, the high-dose (IRH-SH), medium-dose (IRH-SM), and low-dose (IRH-SL) aqueous extract groups and the high-dose (IRH-CH), medium-dose (IRH-CM), and low-dose (IRH-CL) alcoholic extract groups of *Gnaphalium affine* all showed very good effects, with AST values ​​significantly different from the model group (P<0.05). In particular, both the aqueous and alcoholic extracts of *Gnaphalium affine* were more effective than the positive control group of silymarin (SY).

[0126] 2.2.2 Liver GSH and SOD levels in each group of mice

[0127] exist Figure 3 In the model group (CCl4), the GSH content in liver tissue was significantly lower than that in the control group (NC) (P<0.01). The Glehnia littoralis extract prepared in Example 1 could significantly increase the GSH content in liver tissue. As shown in the figure, the high-dose (IRH-SH), medium-dose (IRH-SM), and low-dose (IRH-SL) water extract and the high-dose (IRH-CH), medium-dose (IRH-CM), and low-dose (IRH-CL) alcohol extracts of Glehnia littoralis could all increase the GSH content in liver tissue. In particular, the low-dose (IRH-SL) water extract and the medium-dose (IRH-CM) and low-dose (IRH-CL) alcohol extracts of Glehnia littoralis could significantly increase the GSH content in mice, which was statistically significantly different from that in the model group (P<0.05) and much better than the positive control group treated with silymarin (SY).

[0128] exist Figure 4In the model group (CCl4), the SOD content in the liver tissue was significantly lower than that in the control group (NC) (P<0.05). Administration of the *Gnaphalium affine* extract prepared in Example 1 increased the SOD content in the liver tissue. As shown in the figure, the high-dose (IRH-SH), medium-dose (IRH-SM), and low-dose (IRH-SL) water extract groups and the high-dose (IRH-CH), medium-dose (IRH-CM), and low-dose (IRH-CL) alcohol extract groups of *Gnaphalium affine* all increased the SOD content in the liver tissue. In particular, the high-dose (IRH-SH) water extract group and the high-dose (IRH-CH), medium-dose (IRH-CM), and low-dose (IRH-CL) alcohol extract groups of *Gnaphalium affine* significantly increased the SOD content in the mouse liver tissue, showing a statistically significant difference from the model group (P<0.05), and were far superior to the positive control group treated with silymarin (SY).

[0129] 2.2.3 Liver pathological sections of mice in each group

[0130] Pathological sections were prepared from the liver tissues of mice in the normal group, model group, silymarin group, and high, medium, and low dose groups of water and alcohol extracts of *Gnaphalium affine*. For example... Figure 5 As shown, the normal liver control group (NC) exhibited clear lobular structure, no obvious hepatocyte lesions, abundant cytoplasm, and clear nuclear structure. In the model group (CCl4), hepatocyte degeneration, necrosis, or nuclear pyknosis and dissolution, along with inflammatory cell infiltration, were observed. The figures demonstrate that mice treated with high-dose (IRH-SH), medium-dose (IRH-SM), and low-dose (IRH-SL) water extracts and high-dose (IRH-CH), medium-dose (IRH-CM), and low-dose (IRH-CL) alcohol extracts of *Gynostemma pentaphyllum* showed clear liver lobular structure, significantly reduced hepatocyte damage, necrosis, nuclear pyknosis and dissolution, and inflammatory infiltration, with uniform cytoplasmic staining, all superior to the positive control group (SY) treated with silymarin.

Claims

1. The use of *Gnaphalium affine* extract in the preparation of a medicament for the prevention of acute liver injury caused by carbon tetrachloride, wherein the *Gnaphalium affine* extract is the sole active ingredient, and the *Gnaphalium affine* extract is a phytochemical extract; the phytochemical extract is extracted by the following method: Grind: Take watercress and grind it into coarse powder; Ethanol reflux extraction: Add ethanol to the crude powder, reflux and extract, filter, add ethanol again, reflux and extract again, filter and combine the two filtrates. Filtrate concentration: The filtrate was concentrated under reduced pressure to obtain the purslane alcohol extract.

2. The use as described in claim 1, wherein, The drug is used in mammals.

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