Use of a ligusticum porteri alcohol extract in the preparation of a medicament for preventing or treating drug-induced liver injury

Abstract

The application discloses application of Ligusticum sinense alcohol extract in preparation of a medicine for preventing or treating drug-induced liver injury, and relates to the technical field of biological medicine.The application proves for the first time that the Ligusticum sinense alcohol extract has an obvious effect of improving acute drug-induced liver injury, can obviously improve serum biochemical indexes, inflammatory factor levels, and liver tissue pathological states of mice with acute drug-induced liver injury, and improve superoxide dismutase (SOD) and glutathione (GSH) activities in liver tissue and reduce malondialdehyde (MDA) and reactive oxygen species (ROS) levels.The pharmacodynamics test proves that the Ligusticum sinense alcohol extract has a significant liver injury protection effect, and has a low effective dose and no toxic side effect.Therefore, the application can become an alternative medicine for preventing and treating drug-induced liver injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of the ethanol extract of *Ligustrum sinense* in the preparation of remedies for the prevention or treatment of drug-induced liver injury. Background Technology

[0002] Drug-induced liver injury (DILI) refers to liver toxicity caused directly or indirectly by drugs or their metabolites, resulting in significant impairment of liver metabolism, detoxification, immune regulation, and digestive functions. DILI has become a major global public health problem, being both the leading cause of acute liver failure (ALF) and a core factor in drug withdrawal from clinical practice. Epidemiological studies show that the annual incidence of DILI in my country is as high as 23.8 / 100,000, significantly exceeding that of Western countries (4.1–7.4 / 100,000), and continues to rise.

[0003] Acetaminophen (APAP), a commonly used antipyretic analgesic, is a core component of hundreds of over-the-counter and combination drugs. While it has a good safety profile at therapeutic doses, overdose can induce acute liver injury (APAP-induced liver injury, AILI). APAP-induced acute liver failure accounts for only about 5% of drug-induced liver injury (DILI) cases in my country and is not currently a major contributing factor. However, with the widespread use of APAP-opioid combination analgesics and the public's improper use of antipyretic analgesics, the population at risk of APAP-induced liver toxicity is continuously expanding.

[0004] The existing antidote, N-acetylcysteine ​​(NAC), clears the toxic metabolite N-acetylbenzoquinone imine (NAPQI) by supplementing glutathione (GSH). However, its efficacy is strictly limited to a narrow treatment window of ≤8 hours, and its intervention effect on advanced liver injury (such as extensive hepatocellular necrosis) is significantly limited. Therefore, there is an urgent clinical need to develop novel hepatoprotective drugs that combine multi-target regulatory capabilities with a wide treatment window.

[0005] Xiaobaoluwu ( Ligularia intermedia Nakai *Ligustrum lucidum* is a perennial herb belonging to the genus *Ligustrum* in the family Asteraceae. It is widely distributed in Yunnan, Sichuan, Guizhou, Hubei, Gansu, Shaanxi, Henan, and parts of North and Northeast my country. Its dried rhizome is gourd-shaped. In traditional medicine, the rhizome with the fibrous roots removed is called "Guang Zi Wan" or "Tie Zi Wan," while the rhizome with the fibrous roots intact is called "Mao Zi Wan." In folk medicine, its rhizome has long been used as *Aster tataricus*, possessing properties such as moistening the lungs and lowering qi, relieving cough and expectoration, clearing heat and detoxifying, and promoting bile secretion and reducing jaundice. It is used to treat coughs, influenza, and ulcers.

[0006] Modern component studies have confirmed that *Ligustrum angustifolium* contains terpenes, sterols, flavonoids, and volatile oils, possessing pharmacological activities such as antitumor and antioxidant effects. It is noteworthy that while traditional Chinese medicine texts record its effects of "clearing heat and detoxifying, promoting bile secretion and reducing jaundice," systematic research on the modern pharmacodynamic mechanisms of its hepatoprotective effects has not yet been conducted, particularly lacking verification of its efficacy in preventing and treating drug-induced liver injury (DILI) and a standardized analysis of its active substance basis. Summary of the Invention

[0007] This invention addresses the rising incidence of drug-induced liver injury in my country, coupled with the shortcomings of existing chemotherapeutic drugs, such as single or limited target action and frequent adverse reactions. It provides an application of the alcoholic extract of *Ligustrum angustifolium* in the preparation of drugs for the prevention or treatment of drug-induced liver injury, thus providing a basis for the research and development of the pharmacodynamic material basis of *Ligustrum angustifolium*.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] The use of the ethanol extract of Ligustrum angustifolium in the preparation of drugs for the prevention or treatment of drug-induced liver injury, wherein the ethanol extract of Ligustrum angustifolium is an extract of Ligustrum angustifolium in ethanol with a volume concentration of 75-85%.

[0010] Optionally, the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

[0011] Optionally, the preparation method of the ethanol extract of Ligustrum angustifolium includes the following steps: after drying fresh Ligustrum angustifolium, reflux extraction is performed using ethanol with a volume concentration of 75-85% as the extraction solvent. The resulting extract is then filtered, concentrated, and freeze-dried to obtain the ethanol extract of Ligustrum angustifolium.

[0012] Optionally, the preparation method of the ethanol extract of *Ligustrum angustifolium* includes the following steps: after drying and pulverizing the whole herb of *Ligustrum angustifolium*, it is soaked in ethanol with a volume concentration of 80% for 12 hours; then, the *Ligustrum angustifolium* powder and ethanol with a volume concentration of 80% are refluxed at a ratio of 1:20 at 60-70℃ for 1-3 times, with each reflux extraction time being 2 hours; the extracts are combined, filtered, concentrated, and freeze-dried to obtain the ethanol extract of *Ligustrum angustifolium*.

[0013] Optionally, the drug may be used in any of the following (1) to (3):

[0014] (1) Reduce serum levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase;

[0015] (2) Reduce the level of inflammatory factors in cells; the inflammatory factors include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6).

[0016] (3) Increase the activity of superoxide dismutase and glutathione in liver tissue and reduce the levels of malondialdehyde and reactive oxygen species.

[0017] The present invention also discloses a pharmaceutical composition for the prevention and / or treatment of drug-induced liver injury, comprising scutellaria baicalensis extract.

[0018] Optionally, the pharmaceutical composition may also contain pharmaceutically usable excipients.

[0019] Optionally, the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

[0020] This invention addresses the rising incidence of drug-induced liver injury in my country, coupled with the shortcomings of existing chemotherapeutic drugs such as single-target action, limited target action, and frequent adverse reactions. It provides an application of *Ligustrum sinense* extract in the preparation of drugs for the prevention or treatment of drug-induced liver injury. The *Ligustrum sinense* extract of this invention is a natural plant active component, and its multiple components work synergistically to simultaneously regulate core pathological processes of drug-induced liver injury, such as oxidative stress and inflammatory responses.

[0021] Validated for the first time in an APAP-induced mouse model of acute drug-induced liver injury, this invention demonstrates that the ethanol extract of *Ligustrum angustifolium* significantly improves acute drug-induced liver injury. It significantly improves serum biochemical indicators, inflammatory factor levels, and liver tissue pathological state in mice with acute drug-induced liver injury, and increases the activities of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue while decreasing malondialdehyde (MDA) and reactive oxygen species (ROS) levels. Pharmacodynamic studies have shown that the ethanol extract of *Ligustrum angustifolium* has a significant protective effect against liver injury, with a low effective dose and no toxic side effects. Therefore, this invention may be a potential candidate drug for the prevention and treatment of drug-induced liver injury.

[0022] Implementing the embodiments of the present invention will have the following beneficial effects:

[0023] This invention uses *Ligustrum lucidum* from the Changbai Mountains region 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 pharmacodynamic material basis for preventing drug-induced liver injury (DILI).

[0024] This invention utilizes plant alcohol extracts to treat drug-induced liver injury, which is characterized by significant efficacy, safety, reliability, and minimal toxic side effects.

[0025] This invention utilizes plant alcohol extracts to address drug-induced liver injury holistically, with multiple components and targets. It significantly reduces serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), and inflammatory factors TNF-α, IL-1β, and IL-6. It also increases the activity of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue, and reduces malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The biochemical detection results are significant, demonstrating substantial anti-inflammatory and antioxidant stress-relieving effects, and it shows promising clinical application prospects. Attached Figure Description

[0026] Figure 1 Total ion chromatograms in positive and negative ion modes for the analysis of chemical components of the alcohol extract of *Ligustrum sinense* using UHPLC-Q Exactive-Orbitrap-MS.

[0027] Figure 2 The figure shows the effect of the alcohol extract of Ligustrum angustifolium on serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP) in mice with drug-induced liver injury. In the figure, ### 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.

[0028] Figure 3 The figure shows the effect of the alcohol extract of *Ligustrum angustifolium* on glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) in the liver tissue of mice with drug-induced liver injury. In the figure, # indicates p < 0.05 compared to the control group, ### indicates p < 0.001, * indicates p < 0.05 compared to the model group, ** indicates p < 0.01, and *** indicates p < 0.001.

[0029] Figure 4 The figure shows the effect of the ethanol extract of Ligusticum esculentum on serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in mice with drug-induced liver injury. In the figure, ### indicates p<0.001 compared with the control group, ** indicates p<0.01 compared with the model group, and *** indicates p<0.001.

[0030] Figure 5 This is a H&E staining image of liver tissue from mice with drug-induced liver injury, obtained from the alcohol extract of *Ligustrum stenoptera*. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] The materials and reagents used in this invention were sourced from: *Ligularia stenoptera* collected in the Changbai Mountains region of Jilin Province. ALT, AST, AKP, TNF-α, IL-1β, and IL-6 reagent kits were purchased from Nanjing Jiancheng. GSH, MDA, SOD, and ROS reagent kits were purchased from Servicebio.

[0035] Example 1: Preparation of ethanol extract of Ligustrum stenoptera.

[0036] The ethanol extract of *Ligustrum angustifolium* in this embodiment is an ethanol extract of *Ligustrum angustifolium*. The preparation method includes the following steps: drying and pulverizing the whole herb of *Ligustrum angustifolium*, and soaking it in 80% ethanol for 12 hours; then, refluxing the *Ligustrum angustifolium* powder with 80% ethanol at a material-to-liquid ratio of 1:20 at 60-70°C three times, with each refluxing extraction time being 2 hours; combining the extracts, filtering, concentrating, and freeze-drying to obtain the ethanol extract of *Ligustrum angustifolium*.

[0037] The component identification results of the ethanol extract of *Ligustrum sinense* obtained in this embodiment are as follows: Figure 1 As shown in Table 1.

[0038] Table 1. Chemical composition analysis of *Ligustrum sinense* extract based on UPLC-Q-TOF / MS technology.

[0039]

[0040] Example 2: Pharmacodynamic evaluation of the ethanol extract of Ligusticum esculentum (XBTW) in preventing APAP-induced acute drug-induced liver injury in mice.

[0041] Experimental animal grouping and drug preparation: 50 SPF-grade C57BL / 6 mice (weighing 18-22g) were weighed after 1 week of acclimatization feeding and randomly divided into 5 groups of 10 mice each.

[0042] (1) control group;

[0043] (2) Model group (APAP, dosage 300 mg / kg);

[0044] (3) Low-dose group of Ligustrum stenoptera extract (XBTW, dosage 50 mg / kg);

[0045] (4) High-dose group of Ligustrum stenoptera extract (XBTW, dosage 100 mg / kg).

[0046] (5) Positive control group (Silymarin, dosage 100 mg / kg).

[0047] The ethanol extract of *Ligustrum angustifolium* (Example 1) was prepared into solutions with concentrations of 2.5 mg / ml and 5 mg / ml using 0.5% CMC-Na, and administered to mice in the low- and high-dose groups, respectively. Silymarin was prepared with 0.5% CMC-Na at a concentration of 5 mg / ml and administered to mice in the positive control group. All mice received an equal volume (0.2 ml / 10 g) via gavage. APAP is insoluble in physiological saline at room temperature and was dissolved using physiological saline by heating in a 45°C water bath. The solution was prepared fresh and used immediately at a concentration of 15 mg / ml, and all mice received an equal volume (0.2 ml / 10 g) via intraperitoneal injection.

[0048] Preparation and administration of APAP-induced acute drug-induced liver injury model on days 1-7: Low-dose and high-dose groups of *Ligusticum striatum* extract and the positive control group were administered prophylactically by gavage, while the control group and the model group were administered 0.5% CMC-Na by gavage.

[0049] Day 7: After oral administration of the drug, mice were fasted for 12 hours but allowed free access to water. The model group, the low- and high-dose groups of *Ligustrum lucidum* extract, and the positive control group were intraperitoneally injected with APAP. Mice in the control group were intraperitoneally injected with saline. Mice were fasted for 12 hours after intraperitoneal injection but allowed free access to water.

[0050] Serum and tissue samples were collected from mouse eyeballs. After standing for 1 hour, the blood was centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and aliquoted into 1.5 ml tubes and stored at -80°C in the dark for later use. The livers of mice were dissected, blotted dry with filter paper, and weighed. After weighing, the livers were divided into two portions. One portion was fixed with 4% paraformaldehyde, and the other portion was stored at -80°C in the dark for later use.

[0051] Serum biochemical markers were detected using an enzyme-linked immunosorbent assay (ELISA) reader according to the kit instructions, measuring the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP).

[0052] Test results as follows Figure 2As shown, compared with the model group, the levels of AST, ALT, and AKP in the low- and high-dose groups of Ligustrum lucidum and the positive control group were significantly reduced (p<0.05, p<0.01, p<0.001), indicating that the ethanol extract of Ligustrum lucidum and silymarin can effectively protect against drug-induced liver injury in mice, with the low-dose group of Ligustrum lucidum ethanol extract showing better results than the high-dose group.

[0053] Liver tissue biochemical indicators were detected according to the kit instructions, including glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS).

[0054] The results are as follows Figure 3 As shown in the figure, compared with the model group, the activities of GSH and SOD in the low-dose and high-dose groups of *Ligustrum lucidum* and the positive control group were significantly increased (p<0.01, p<0.001), while the contents of MDA and ROS were significantly decreased (p<0.05, p<0.001). The results indicate that the ethanol extract of *Ligustrum lucidum* and silymarin can enhance the antioxidant capacity of mice with drug-induced liver injury and reduce the damage to the liver caused by oxidative stress.

[0055] Inflammatory factor markers were detected using an enzyme-linked immunosorbent assay (ELISA) reader according to the kit instructions, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

[0056] The results are as follows Figure 4 As shown in the figure. Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in the low- and high-dose groups of *Ligustrum lucidum* and the positive control group were significantly reduced (p<0.01, p<0.001). The results indicate that *Ligustrum lucidum* ethanol extract and silymarin can treat liver injury by reducing the levels of inflammatory factors in mice with drug-induced liver injury, with the low-dose group of *Ligustrum lucidum* ethanol extract showing better efficacy than the high-dose group.

[0057] Histopathological examination, such as Figure 5 The H&E staining results showed that, compared with the control group mice, the liver tissue of the model group mice exhibited significant hepatocyte necrosis, accompanied by nuclear dissolution or fragmentation, and the cytoplasm showed eosinophilic red staining, with hemorrhage areas also visible. In contrast, the liver tissue of mice treated with low- and high-dose Ligustrum lucidum and the positive control drug showed significantly reduced hepatocyte necrosis and hemorrhage.

[0058] The pharmacodynamic studies demonstrated that the ethanol extract of *Callicarpa angustifolia* exhibits significant hepatoprotective effects, reversing serum biochemical markers, liver tissue oxidative stress markers, and inflammatory factor markers in APAP-induced acute drug-induced liver injury in mice, with low effective doses and no toxic side effects. Therefore, the ethanol extract of *Callicarpa angustifolia* may be a potential candidate drug for the prevention and treatment of drug-induced liver injury.

[0059] Example 3: Pharmaceutical composition containing sclerotium truncatum extract

[0060] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of drug-induced liver injury, comprising a sclerotium thunbergii extract and pharmaceutically available excipients, which can be adjusted as needed by a technician.

[0061] The prepared drug composition can be administered via conventional routes.

[0062] The pharmaceutical compositions of the present invention contain a safe and effective amount of *Ligustrum lucidum* extract and pharmaceutically usable excipients. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into oral or injectable forms.

[0063] This drug can be used to prevent or treat drug-induced liver injury induced by acetaminophen.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of *Ligustrum sinense* ethanol extract in the preparation of drugs for the prevention or treatment of drug-induced liver injury, characterized in that, The *Ligustrum angustifolium* ethanol extract is an extract of *Ligustrum angustifolium* in 80% ethanol (v / v). The preparation method of the *Ligustrum angustifolium* ethanol extract includes the following steps: *Ligustrum angustifolium* whole herb is dried, pulverized, and then soaked in 80% ethanol (v / v) for 12 hours; subsequently, *Ligustrum angustifolium* powder and 80% ethanol (v / v) are refluxed at a ratio of 1:20 at 60-70℃ for 1-3 times, with each reflux extraction lasting 2 hours. The combined extracts are then filtered, concentrated, and freeze-dried to obtain the *Ligustrum angustifolium* ethanol extract.

2. The application according to claim 1, characterized in that, The drug-induced liver injury mentioned refers to acetaminophen-induced drug-induced liver injury.

3. The application according to claim 1, characterized in that, The drug is used in any of the following (1) to (3): (1) Reduce serum levels of 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.

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