Application of ligularia virgaurea alcohol extract in preparation of medicine for preventing or treating medicine-induced liver injury

Abstract

The invention discloses an application of ligularia virgaurea alcohol extract in preparation of a medicine for preventing or treating drug-induced liver injury, relates to the technical field of biological medicines, and proves that the ligularia virgaurea alcohol extract has an obvious effect of improving acute drug-induced liver injury for the first time. The traditional Chinese medicine composition can obviously improve serum biochemical indexes, inflammatory factor levels and hepatic tissue pathological states of mice with acute drug-induced liver injury, improve the activity of superoxide dismutase (SOD) and glutathione (GSH) in hepatic tissues, and reduce the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Pharmacodynamic tests prove that the ligularia nobilis alcohol extract has a remarkable liver injury protection effect, and is low in effective dose and free of toxic and side effects. Therefore, the invention may become an alternative drug for preventing and treating drug-induced liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to the use of an ethanol extract of Ligularia angustifolia in preparing a medicament for preventing or treating drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) refers to a toxic reaction to the liver caused directly or indirectly by a drug or its metabolites, resulting in significant impairment of liver metabolism, detoxification, immune regulation, and digestive functions. DILI has become a major global public health issue, both a leading cause of acute liver failure (ALF) and a key factor in drug withdrawal from clinical trials. Epidemiological studies have shown that the annual incidence of DILI in my country is as high as 23.8 per 100,000 patients, significantly exceeding the 4.1–7.4 per 100,000 patients in Western countries (and continues to rise).

[0003] Acetaminophen (APAP), a commonly used antipyretic and analgesic, is a core component of hundreds of over-the-counter and combination medications. While safe at therapeutic doses, overdose can induce acute liver injury (AILI). APAP-induced acute liver failure accounts for only approximately 5% of DILI cases in my country and is not currently a major cause. However, with the widespread use of APAP-opioid combination analgesics and the public's irregular use of antipyretic and analgesic medications, the risk of APAP hepatotoxicity continues to expand.

[0004] The existing antidote, N-acetylcysteine ​​(NAC), eliminates the toxic metabolite N-acetylbenzoquinoneimine (NAPQI) by replenishing glutathione (GSH). However, its efficacy is strictly limited to a narrow therapeutic window of ≤8 hours and is significantly limited in interventions for advanced liver damage (e.g., extensive hepatocellular necrosis). Therefore, there is an urgent clinical need for the development of novel liver-protective drugs with multi-target regulatory capabilities and a wide therapeutic window.

[0005] Xiaobaoluwu ( Ligularia intermedia Nakai Aster tataricus is a perennial herbaceous plant of the genus Ligularia in the Asteraceae family, widely distributed in Yunnan, Sichuan, Guizhou, Hubei, Gansu, Shaanxi, Henan, and throughout North China and Northeast China. Its dried rhizome is gourd-shaped. In traditional Chinese medicine, the rootless rhizome is called "light aster" or "iron aster," while the uncut rhizome is called "hairy aster." Its rhizome has long been used as a folk medicine, known as mountain aster, for its lung-moistening, cough-relieving, expectorant, heat-clearing, detoxifying, and bile-stimulating properties. It is used to treat coughs, influenza, and ulcers.

[0006] Modern compositional research confirms that Ligularia angustifolia contains terpenes, sterols, flavonoids, and volatile oils, exhibiting pharmacological activities such as anti-tumor and antioxidant properties. It is noteworthy that, while traditional Chinese medicine texts describe its efficacy as "clearing heat and detoxifying, promoting bile secretion and alleviating jaundice," systematic modern pharmacodynamic research on its hepatoprotective effects has yet to be conducted. In particular, there is a lack of validation of its efficacy in preventing and treating drug-induced liver injury (DILI) and a standardized analysis of the underlying active substances. Summary of the Invention

[0007] In view of the current rising incidence of drug-induced liver injury in my country and the defects of existing chemotherapeutic drugs such as single target, limited target, and frequent adverse reactions, the present invention provides an application of an ethanol extract of Ligularia stenocarpa in the preparation of drugs for preventing or treating drug-induced liver injury, providing a basis for basic research on the pharmacological effects of Ligularia stenocarpa and its development and utilization.

[0008] To achieve the above object, the technical solution of the present invention is as follows: The invention discloses an application of an ethanol extract of Ligularia stenocarpa in preparing a medicine for preventing or treating drug-induced liver injury. The ethanol extract of Ligularia stenocarpa is an extract of Ligularia stenocarpa in ethanol with a volume concentration of 75-85%.

[0009] Optionally, the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

[0010] Optionally, the preparation method of the alcohol extract of Ligularia stenophylla comprises the following steps: taking fresh Ligularia stenophylla, drying it in the sun, performing reflux extraction with ethanol having a volume concentration of 75-85% as the extraction solvent, filtering the obtained extract, concentrating and freeze-drying to obtain the alcohol extract of Ligularia stenophylla.

[0011] Optionally, the preparation method of the alcohol extract of Ligularia stenophylla includes the following steps: taking the whole herb of Ligularia stenophylla, drying and crushing it, and soaking it in ethanol with a volume concentration of 80%; wherein the soaking time is 12 hours; then the Ligularia stenophylla powder and ethanol with a volume concentration of 80% are refluxed at 60-70°C for 1-3 times at a solid-liquid ratio of 1:20, and each reflux extraction time is 2 hours. After combining the extracts, they are filtered, concentrated and freeze-dried to obtain the alcohol extract of Ligularia stenophylla.

[0012] Optionally, the drug is used in any one of the following (1) to (3): (1) Reduce the levels of serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase; (2) reducing the levels of inflammatory factors in cells; the inflammatory factors include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6); (3) Increase the activity of superoxide dismutase and glutathione in liver tissue and reduce the levels of malondialdehyde and reactive oxygen species.

[0013] The present invention also discloses a pharmaceutical composition for preventing and / or treating drug-induced liver injury, comprising the ethanol extract of Ligularia angustifolia.

[0014] Optionally, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0015] Optionally, the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

[0016] The present invention aims to address the current rising incidence of drug-induced liver injury in my country and the defects of existing chemotherapeutic drugs such as a single target, limited target, and frequent adverse reactions. The invention provides an application of an ethanol extract of Ligularia stenophylla in the preparation of a drug for preventing or treating drug-induced liver injury. The ethanol extract of Ligularia stenophylla of the present invention is a natural plant active component, and its multiple components exert synergistic effects to synchronously regulate core pathological links of drug-induced liver injury such as oxidative stress and inflammatory response.

[0017] Validated in an APAP-induced mouse model of acute drug-induced liver injury, this invention demonstrates for the first time that an ethanol extract of Ligularia stenophylla has a significant effect on ameliorating acute drug-induced liver injury. The extract significantly improves serum biochemical parameters, inflammatory factor levels, and liver tissue pathology in mice with acute drug-induced liver injury. It also increases superoxide dismutase (SOD) and glutathione (GSH) activity in liver tissue, and reduces malondialdehyde (MDA) and reactive oxygen species (ROS) levels. Pharmacodynamic studies have demonstrated that the ethanol extract of Ligularia stenophylla has a significant protective effect against liver injury, with a low effective dose and no toxic side effects. Therefore, this invention may become a potential candidate for the prevention and treatment of drug-induced liver injury.

[0018] The implementation of the present invention will have the following beneficial effects: The present invention uses Ligularia angustifolia from Changbai Mountain area of ​​Jilin Province as raw material, extracts it with 80% ethanol by volume, and analyzes its active ingredients using UHPLC-Q Exactive-Orbitrap-MS technology to establish the pharmacological substance basis for preventing and treating drug-induced liver injury (DILI).

[0019] The present invention adopts plant alcohol extract to treat drug-induced liver injury, which has the characteristics of significant efficacy, safety, reliability, small toxicity and side effects, etc.

[0020] The plant alcohol extract of the present invention intervenes in drug-induced liver injury from a holistic, multi-component, and multi-target perspective, significantly reducing the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), and inflammatory factors TNF-α, IL-1β, and IL-6 in serum, increasing the activities of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue, and reducing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The tissue biochemical test results are obvious, and the extract has significant anti-inflammatory and anti-oxidative stress therapeutic effects, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Total ion chromatograms of the chemical components of the ethanol extract of Ligularia stenocarpa analyzed by UHPLC-Q Exactive-Orbitrap-MS technology in positive and negative ion modes.

[0022] Figure 2 The figure shows the effect of the ethanol extract of Ligularia angustifolia on serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AKP) in mice with drug-induced liver injury, where ### indicates p<0.001 compared with the control group, * indicates p<0.05 compared with the model group, ** indicates p<0.01, and *** indicates p<0.001.

[0023] Figure 3 The figure shows the effect of the ethanol extract of Ligularia angustifolia on glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) in the liver tissue of mice with drug-induced liver injury, where # indicates p<0.05 compared with the control group, ### indicates p<0.001, * indicates p<0.05 compared with the model group, ** indicates p<0.01, and *** indicates p<0.001.

[0024] Figure 4 The figure shows the effect of the ethanol extract of Ligularia angustifolia on serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in mice with drug-induced liver injury, where ### indicates p<0.001 compared with the control group, ** indicates p<0.01 compared with the model group, and *** indicates p<0.001.

[0025] Figure 5 This is the H&E staining of the liver tissue of mice with drug-induced liver injury by the ethanol extract of Ligularia angustifolia. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Sources of materials and reagents used in this study: Ligularia angustifolia was collected from the Changbai Mountain region of Jilin Province. ALT, AST, AKP, TNF-α, IL-1β, and IL-6 assays were purchased from Nanjing Jiancheng. GSH, MDA, SOD, and ROS assays were purchased from Servicebio.

[0030] Example 1: Preparation of Ligularia stenocarpa ethanol extract.

[0031] The alcohol extract of Ligularia stenophylla in this embodiment is an ethanol extract of Ligularia stenophylla, and the preparation method comprises the following steps: drying the whole herb of Ligularia stenophylla and crushing it, soaking it in ethanol with a volume concentration of 80% for 12 hours; then refluxing the Ligularia stenophylla powder and ethanol with a volume concentration of 80% at a solid-liquid ratio of 1:20 at 60-70°C for three times, each reflux extraction time being 2 hours, combining the extracts, filtering, concentrating and freeze-drying to obtain the alcohol extract of Ligularia stenophylla.

[0032] The results of component identification of the ethanol extract of Ligularia stenophylla prepared in this example are as follows: Figure 1 and as shown in Table 1.

[0033] Table 1 Analysis of chemical components of the ethanol extract of Ligularia stenophylla based on UPLC-Q-TOF / MS technology

[0034] Example 2: Pharmacodynamic evaluation of the ethanol extract of Ligularia stenophylla (XBTW) in preventing and treating APAP-induced acute drug-induced liver injury in mice.

[0035] Experimental Animal Grouping and Drug Allocation Fifty SPF-grade C57BL / 6 mice (weighing 18-22 g) were weighed after adaptive feeding for 1 week and randomly divided into 5 groups, with 10 mice in each group. (1) control group; (2) Model group (APAP, dosage 300 mg / kg); (3) low-dose group of Ligularia stenobractefolia ethanol extract (XBTW, dosage 50 mg / kg); (4) high-dose Ligularia stenobractefolia ethanol extract group (XBTW, dosage 100 mg / kg); (5) Positive control group (Silymarin, dosage 100 mg / kg).

[0036] The ethanol extract of Ligularia stenocarpa (Example 1) was prepared using 0.5% CMC-Na to a concentration of 2.5 mg / ml and 5 mg / ml, respectively. These solutions were administered to mice in the low- and high-dose Ligularia stenocarpa extract groups, respectively. Silymarin was prepared using 0.5% CMC-Na to a concentration of 5 mg / ml and administered to mice in the positive control group. All groups of mice received an equal volume (0.2 ml / 10 g) of the drug by oral gavage. APAP, which is insoluble in saline at room temperature, was dissolved in saline by heating in a 45°C water bath and prepared immediately before use at a concentration of 15 mg / ml. All groups of mice received an equal volume (0.2 ml / 10 g) of the drug by intraperitoneal injection.

[0037] Preparation and administration of APAP-induced acute drug-induced liver injury model. Days 1-7: The low-dose and high-dose groups of Ligularia angustifolia ethanol extract and the positive control group were given preventive oral administration, and the control group and model group were given 0.5% CMC-Na by oral administration.

[0038] Day 7: After oral administration, mice were fasted for 12 hours but not water. APAP was then injected intraperitoneally into the model group, the low- and high-dose Ligularia stenoclavata ethanol extract groups, and the positive control group. Normal saline was injected intraperitoneally into the control group. Mice were fasted for 12 hours after intraperitoneal injection.

[0039] Serum and tissue samples were collected from mouse eyeballs. After 1 hour of rest, the blood was centrifuged at 3000 rpm for 15 minutes. The supernatant was aliquoted into 1.5 ml tubes and stored at -80°C in the dark until use. The liver was dissected from the mouse, dried with filter paper, and weighed. After weighing, the liver was divided into two portions. One portion was fixed with 4% paraformaldehyde, and the other portion was stored at -80°C in the dark until use.

[0040] Serum biochemical index detection Serum biochemical index detection was performed using a microplate reader according to the kit instructions to detect the serum biochemical indicators aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AKP) levels.

[0041] Test results such as Figure 2As shown in the data, compared with the model group, the AST, ALT and AKP contents in the low-dose and high-dose Ligularia stenophylla groups and the positive control group were significantly reduced (p<0.05, p<0.01, p<0.001), indicating that the ethanol extract of Ligularia stenophylla and silymarin can effectively protect mice from drug-induced liver injury, among which the low-dose group of the ethanol extract of Ligularia stenophylla had better effect than the high-dose group.

[0042] Liver tissue biochemical indexes glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected according to the kit instructions.

[0043] The results are as follows Figure 3 Compared with the model group, the low- and high-dose Ligularia stenophylla groups and the positive control group showed significant increases in GSH and SOD activities (p<0.01, p<0.001), and significant decreases in MDA and ROS levels (p<0.05, p<0.001). These results suggest that Ligularia stenophylla ethanol extract and silymarin can enhance the antioxidant capacity of mice with drug-induced liver injury and reduce liver damage caused by oxidative stress.

[0044] Inflammatory factor index detection: The inflammatory factor indicators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected using an enzyme-labeled instrument according to the kit instructions.

[0045] The results are as follows Figure 4 Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in the low- and high-dose Ligularia stenophylla groups and the positive control group were significantly decreased (p < 0.01, p < 0.001). These results suggest that Ligularia stenophylla ethanol extract and silymarin can treat liver injury in mice with drug-induced liver injury by reducing the levels of inflammatory factors, with the low-dose Ligularia stenophylla ethanol extract group showing superior efficacy compared to the high-dose group.

[0046] Histopathological examination Figure 5 The H&E staining results shown in the figure show that compared with the control group, the liver tissue of the model group mice showed significant hepatocellular necrosis, accompanied by nuclear dissolution or fragmentation, eosinophilic red cytoplasm, and visible hemorrhage. In the liver tissue of mice treated with low and high doses of Ligularia stenophylla and positive drugs, the degree of hepatocellular necrosis and hemorrhage were significantly reduced.

[0047] The pharmacodynamic studies demonstrated that the ethanol extract of Ligularia stenophylla has a significant protective effect against liver injury. It reversed serum biochemical markers, liver tissue oxidative stress markers, and inflammatory cytokine levels in APAP-induced acute drug-induced liver injury in mice. The effective dose was low and there were no toxic side effects. Therefore, the ethanol extract of Ligularia stenophylla may become a potential drug candidate for the prevention and treatment of drug-induced liver injury.

[0048] Example 3: Pharmaceutical composition containing the ethanol extract of Ligularia stenophylla The present invention also provides a pharmaceutical composition for preventing and / or treating drug-induced liver injury, comprising an ethanol extract of Ligularia stenocarpa and pharmaceutically acceptable excipients, which can be adjusted by technicians as needed.

[0049] The prepared pharmaceutical composition can be administered through conventional routes.

[0050] The pharmaceutical composition of the present invention contains a safe and effective amount of the ethanol extract of Ligularia stenocarpa and pharmaceutically acceptable excipients. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared into an oral or injection form.

[0051] This drug can be used to prevent or treat acetaminophen-induced drug-induced liver injury.

[0052] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of an ethanol extract of Ligularia angustifolia in preparing a medicament for preventing or treating drug-induced liver injury, characterized in that: The Ligularia stenocarpa ethanol extract is an extract of Ligularia stenocarpa in ethanol with a volume concentration of 75-85%.

2. The use according to claim 1, characterized in that The drug-induced liver injury is acetaminophen-induced liver injury.

3. The use according to claim 1, characterized in that The preparation method of the Ligularia stenobracte ethanol extract comprises the following steps: Fresh Ligularia stenocarpa is sun-dried, and reflux extraction is performed using ethanol with a volume concentration of 75-85% as an extraction solvent. The obtained extract is filtered, concentrated and freeze-dried to obtain the Ligularia stenocarpa ethanol extract.

4. The use according to claim 3, characterized in that The preparation method of the Ligularia stenobracte ethanol extract comprises the following steps: The whole herb of Ligularia stenophylla is dried and crushed, and then soaked in 80% ethanol by volume for 12 hours. The Ligularia stenophylla powder and 80% ethanol by volume are then refluxed and extracted at 60-70°C for 1-3 times in a solid-liquid ratio of 1:20, with each reflux extraction time of 2 hours. The extracts are combined, filtered, concentrated and freeze-dried to obtain the Ligularia stenophylla alcohol extract.

5. The use according to claim 1, characterized in that Use of the drug in any one of the following (1) to (3): (1) Reduce the levels of serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase; (2) Reduce the level of inflammatory factors in cells; the inflammatory factors include TNF-α, IL-1β and IL-6; (3) Increase the activity of superoxide dismutase and glutathione in liver tissue and reduce the levels of malondialdehyde and reactive oxygen species.

6. A pharmaceutical composition for preventing and / or treating drug-induced liver injury, characterized in that: Including ethanol extract of Ligularia stenophylla.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 6, characterized in that The drug-induced liver injury is acetaminophen-induced liver injury.

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