Application of beta-bisabolol in preparation of medicine for preventing or treating acute liver injury

Abstract

The invention discloses an application of beta-bisabolol in preparation of a medicine for preventing or treating acute liver injury, the beta-bisabolol can effectively improve liver blood stasis, edema and liver tissue lesion induced by LPS / D-GlaN, effectively reduce serum liver injury indexes such as glutamic-pyruvic transaminase and glutamic oxalacetic transaminase, inflammatory factors such as TNF-alpha, IL-6 and MCP-1, and improve liver injury effect. Meanwhile, the MDA level of liver tissues and liver cell apoptosis are reduced; in addition, the protective effect of 50 mg / kg of the compound beta-bisabolol on LPS / D-GalN induced acute liver injury of mice is superior to that of 300 mg / kg of a clinical first-line drug N-acetylcysteine. The beta-bisabolol has a remarkable protection effect on the acute liver injury induced by LPS / D-GlaN, has an important application value and a wide application prospect in prevention and treatment of the clinical acute liver injury, and opens up a new medicinal application of the sesquiterpene derivative beta-bisabolol.

Description

(I) Technical field

[0001] The invention belongs to the technical field of biomedicine and relates to an application of beta-bisabolene in preparing a medicine for preventing or treating acute liver injury. (II) Background technology

[0002] Acute liver injury (ALI) refers to liver cell necrosis or dysfunction caused by a variety of factors in a short period of time, manifested by a significant increase in liver function indicators (such as ALT, AST, TBIL, etc.). Its pathogenic factors mainly include: (1) drugs and toxins: drug-induced liver injury (DILI) is a common cause, such as acetaminophen overdose, anti-tuberculosis drugs, traditional Chinese medicine and dietary supplements; (2) viral hepatitis: hepatitis A, B, E virus, etc. can cause acute liver injury; (3) metabolic abnormalities: alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), etc.; (4) immune factors: autoimmune hepatitis or drug-induced immune response. About 99% of patients with acute liver injury can recover on their own, and only 1% may progress to acute liver failure (ALF), the latter of which has a mortality rate of more than 50%. In recent years, with the increase of drug abuse, especially traditional Chinese medicine and health products, and metabolic diseases, the incidence of DILI and NAFLD-related ALI has been on the rise. Currently, supportive therapy is the main treatment, including discontinuation of suspected drugs, nutritional support, artificial liver (such as plasma exchange), etc., but there is a lack of specific drugs. Severe patients require liver transplantation, but the shortage of donors and technical limitations lead to a poor prognosis.

[0003] The combined use of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) to induce acute liver injury in mice is currently a common model used to simulate clinical acute liver injury. D-GalN can inhibit hepatocyte uridylic acid metabolism, leading to UTP depletion, hindering RNA and protein synthesis, and inducing hepatocyte apoptosis. LPS can activate Kupffer cells to release inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), recruit mononuclear macrophages and neutrophils to infiltrate, amplify the inflammatory response, and aggravate hepatocyte necrosis. The pathological characteristics are characterized by large-area necrosis of hepatocytes, infiltration of inflammatory cells, and significant increase in liver function indicators, simulating the pathological process of viral or drug-induced liver injury in humans. The pathological mechanism of this model involves metabolic disorders and inflammatory responses, which are highly consistent with the pathogenesis of various clinical ALI. Through the study of this model, it has been found that multiple monomer components of traditional Chinese medicine have the potential to prevent and alleviate acute liver injury, such as baicalin, silymarin, and apigenin.

[0004] β-Bisabolene belongs to the monocyclic sesquiterpenoid compounds and is also a common bioactive compound in natural plant essential oils. It has a fragrance similar to sesame oil, so it can be used as a food flavoring. Modern pharmacological studies have shown that β-Bisabolene has a certain growth inhibitory effect on breast cancer cells and has potential anti-cancer activity; but other exact pharmacological activities are rarely reported in the literature. There is no application of β-Bisabolene in the treatment of liver damage-related diseases in the prior art, and there is no research report on β-Bisabolene in acute liver damage.

[0005] Therefore, the development of drugs with preventive and therapeutic effects is a top priority. (III) Summary of the invention

[0006] The present invention aims to provide an application of β-bisabolene in the preparation of drugs for preventing or treating acute liver injury, especially having a good alleviating effect on acute liver injury induced by LPS / D-GlaN, and having extremely strong liver-protecting activity. Natural sesquiterpene derivatives such as β-bisabolene show good development and utilization prospects in the preparation of drugs for preventing and treating acute liver injury, and provide support for solving the lack of specific drugs for the treatment of acute liver failure in clinical practice.

[0007] The technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a use of β-bisabolene in the preparation of a drug for preventing or treating acute liver injury.

[0009] The β-bisabolene is a sesquiterpene derivative, and its structural formula is as follows:

[0010]

[0011] Preferably, the drug is a drug that reduces serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), TNF-α, IL-6 and MCP-1 levels.

[0012] Preferably, the drug is a drug that inhibits hepatocyte apoptosis.

[0013] Preferably, the drug is a drug for preventing and treating LPS / D-GlaN-induced acute liver failure or acute-on-chronic liver failure, and the minimum effective concentration is 30 mg / kg.

[0014] In a second aspect, the present invention provides a drug for preventing and treating acute liver injury, wherein the drug contains β-bisabolene as an active ingredient.

[0015] Preferably, the drug further comprises other active pharmaceutical ingredients for preventing and treating acute liver injury, that is, the drug uses β-bisabolene as a single active ingredient or together with other drugs for preventing and treating acute liver injury as active ingredients.

[0016] In a third aspect, the present invention provides a pharmaceutical preparation, which comprises the drug for preventing and treating acute liver injury and a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the pharmaceutically acceptable carrier or excipient is selected from one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an ion exchanger, a flavoring agent or an antioxidant.

[0018] Preferably, the dosage form of the pharmaceutical preparation includes but is not limited to capsules, tablets, injections, granules, emulsions, ointments, patches, pills, syrups and the like.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention provides an application of β-bisabolene in the preparation of a drug for preventing or treating acute liver injury, in particular, the application of β-bisabolene in preventing and treating acute liver injury in mice induced by LPS / D-GlaN. Experimental studies have shown that β-bisabolene can effectively improve liver congestion, edema and liver tissue lesions induced by LPS / D-GlaN, effectively reduce serum liver injury indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), inflammatory factors TNF-α, IL-6 and MCP-1, and reduce hepatocyte apoptosis; in addition, the protective effect of the compound β-bisabolene at a dose of 50 mg / kg on acute liver injury in mice induced by LPS / D-GalN is better than that of the clinical first-line drug N-acetylcysteine ​​300 mg / kg.

[0020] The β-bisabolene of the present invention has a significant protective effect on acute liver injury induced by LPS / D-GlaN, has important application value and broad application prospects for the prevention and treatment of acute liver injury in clinic, and opens up a new pharmaceutical use of the sesquiterpene derivative β-bisabolene. (IV) Description of the drawings

[0021] Figure 1 , ALT and AST levels in serum of mice with acute liver injury by LPS / D-GalN in different groups; compared with the normal group, ## , p<0.01; compared with the model group, ** , p<0.01.

[0022] Figure 2 , levels of inflammatory factors TNF-α, IL-6 and MCP-1 in the serum of different groups of mice with acute liver injury by LPS / D-GalN; compared with the normal group, ##, p<0.01; compared with the model group, **, p<0.01; compared between the two groups, &&, p<0.01.

[0023] Figure 3, liver index of different groups of mice with acute liver injury by LPS / D-GalN (n=8); compared with the control group, ## P<0.01; compared with the model group, ** p<0.01.

[0024] Figure 4 , MDA levels in liver tissues of mice with acute liver injury by LPS / D-GalN in different groups; compared with the normal group, ## , p<0.01; compared with the model group, ** , p<0.01; comparison between the two groups, &, p<0.05.

[0025] Figure 5 , HE staining micrographs of liver tissues in different groups of mice with acute liver injury caused by LPS / D-GalN.

[0026] Figure 6 , Tunel staining micrographs of liver tissues in different groups of mice with acute liver injury using LPS / D-GalN. (V) Specific implementation methods

[0027] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0028] The male C57BL / 6 mice used in the examples of the present invention, weighing 18-22 g, were purchased from the Animal Center of Zhejiang Chinese Medical University.

[0029] Drugs and reagents: β-Bisabolene was purchased from Shanghai Xinbo Chemical Technology Co., Ltd., batch number: EALAFH; Tween-80 was purchased from Wuxi Yatai United Chemical Co., Ltd., batch number: 2020-01-01; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., batch number: 0000135218; D-galactosamine hydrochloride (D-GlaN) was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the one-step TUNEL in situ apoptosis detection kit was purchased from Elabscience, batch number: 5HNHLC9S4P; BD TM Cytometric Bead Array (CBA) Mouse inflammation Kit was purchased from BD, batch number: 1351786; N-acetylcysteine ​​was purchased from Bio-Tech, batch number: 030619190321; other reagents were of analytical grade. ALT, AST and MDA kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0030] Instruments: biological tissue paraffin embedding machine (Thermo Fisher Scientific); MagNA Lyser fully automatic tissue homogenizer (ROCHE, Germany); multifunctional microplate reader (Perkin Elmer); rotary slicer (Lecia); upright microscope (Motic).

[0031] Example 1. Protective effect of β-bisabolene on acute liver injury induced by LPS / D-GlaN in C57BL / 6 mice

[0032] 1. Grouping and administration of drugs to mice:

[0033] Forty C57BL / 6 male mice were randomly divided into five groups, with eight mice in each group, namely, the control group, the model group, the positive drug group, the β-bisabolene low-dose group, and the β-bisabolene high-dose group.

[0034] The control group and the model group were intraperitoneally injected with 8.0 mL / kg of solvent (PBS solution containing 1% Tween 80 by volume); the positive drug group was intraperitoneally injected with 300 mg / kg of N-acetylcysteine ​​(NAC) (dissolved in the above solvent); the β-bisabolene low-dose group was intraperitoneally injected with 30 mg / kg (dissolved in the above solvent); the β-bisabolene high-dose group was intraperitoneally injected with 50 mg / kg (dissolved in the above solvent). The mice were injected intraperitoneally once a day for 3 consecutive days. One hour after the last administration, the control group was injected with PBS 8 mL / kg, and the other groups were injected with LPS (40 μg / kg, solvent PBS) and D-GlaN (400 mg / kg, solvent PBS) to establish the model. 6 hours after the model establishment, the mice were anesthetized, blood was collected from the abdominal aorta, and after the blood coagulated, it was centrifuged at 3500 rpm at 4°C for 10 min. The supernatant was cryopreserved at -80°C for the detection of indicators in serum. The liver was dissected and weighed, some tissues were fixed with 4% formaldehyde, and the rest of the tissues were quickly frozen in liquid nitrogen and cryopreserved at -80°C for the detection of indicators in liver tissue.

[0035] 2. Determination of ALT and AST content in serum:

[0036] According to the instructions of the ALT and AST kits, the ALT and AST levels in the serum of each group of mice were measured. The results are shown in Figure 1. β-Bisabolene can significantly reduce serum liver injury indicators: After LPS / D-GlaN modeling, the liver injury indicators ALT and AST in the serum increased significantly, reaching 43.401 times and 7.193 times that of the control group, respectively, indicating that the liver tissue of mice was severely damaged after modeling; similarly, each drug-treated group could effectively reduce the levels of ALT and AST, especially the ALT level of the high-dose β-bisabolene group was only 2.470 times that of the control group, which was significantly lower than the positive drug group (7.028 times that of the control group); the AST level of the high-dose β-bisabolene group was equivalent to that of the positive drug group; the above results showed that β-bisabolene has good hepatoprotective activity.

[0037] 3. Determination of TNF-α, IL-6 and MCP-1 content in serum:

[0038] According to BD TM Cytometric Bead Array (CBA) Mouse inflammation Kit instructions were used to detect the levels of inflammatory factors TNF-α, IL-6 and MCP-1 in serum. The results are shown in Figure 2 . β-Bisabolene can significantly reduce the level of inflammatory factors in serum: Excessive inflammatory response is one of the important causes of acute liver injury. From the perspective of serum inflammatory factor levels, the levels of TNF-α, IL-6 and MCP-1 in the model group were significantly increased, reaching 3.339 times, 224.885 times and 54.229 times that of the control group; with the increase of β-bisabolene treatment dose, the levels of each factor in each serum decreased significantly, especially the TNF-α level in the high-dose β-bisabolene group was almost equivalent to that in the control group; in addition, the levels of TNF-α, IL-6 and MCP-1 in the high-dose β-bisabolene group were also significantly lower than those in the positive drug group, showing good anti-inflammatory activity.

[0039] 4. Liver index determination:

[0040] Weigh the mouse body weight and liver weight to calculate the liver index (liver weight mg / mouse body weight g). The results are shown in Figure 3 β-Bisabolene can effectively reduce the liver index: the liver index after LPS / D-GlaN modeling reached (60.169±6.784) mg / g body weight, which was significantly increased compared with the control group (46.013±3.452) mg / g body weight, indicating that the liver tissue in the model group was severely damaged, and tissue fluid extravasation led to liver tissue edema and weight increase; and each drug-treated group could effectively reduce the liver index, indicating that β-Bisabolene can effectively alleviate LPS / D-GlaN-induced acute liver injury.

[0041] 5. Determination of MDA in liver tissue:

[0042] Weigh the liver tissue samples of each group, add pre-cooled PBS solution at a volume ratio of 1:9, homogenize in an automatic tissue homogenizer, 6000rpm, 30s, centrifuge the homogenate at 2500rpm for 10min, and take the supernatant; according to the instructions of the MDA kit, determine the MDA content in the liver tissue. The results are shown in Figure 4 The MDA content in the liver tissue of the model group increased significantly, reaching (5.785±0.804) nmol / mg protein, which was 5.8 times higher than that in the control group (1.018±0.371) nmol / mg protein, indicating that lipid peroxidation in the liver tissue cells was more serious; β-bisabolene treatment could reduce the MDA level in the liver tissue in a dose-dependent manner, especially the MDA level in the 50 mg / kg high-dose group (1.318±0.424) nmol / mg protein, which was significantly lower than that in the positive drug group (2.283±0.456) nmol / mg protein, showing significant liver protective activity.

[0043] 6. HE staining of liver tissue:

[0044] Take routine paraffin sections of liver tissue samples from each group, perform HE staining to compare and analyze liver damage, and take pictures under an upright microscope. The results of HE sections are shown in Figure 5 The liver cells of the control group mice had clear outlines and normal morphological structure, while the model liver cells were wrinkled, the gaps were enlarged, the hepatic cords were disintegrated, and there was a lot of diffuse bleeding; both β-bisabolene and NAC drug treatment significantly improved liver tissue bleeding and inflammatory infiltration, but a small amount of red blood cell extravasation and aggregation could still be seen in the β-bisabolene 30mg / kg and NAC 300mg / kg treatment groups, and the β-bisabolene 50mg / kg group was almost equivalent to the control group; it can be seen that the effect of the high-dose β-bisabolene treatment group was significantly better than the positive drug NAC, indicating that β-bisabolene significantly improved liver tissue lesions.

[0045] 7. Tunel staining of liver tissue apoptosis:

[0046] According to the instructions of the kit, the Elabscience one-step TUNEL in situ apoptosis detection kit was used to stain the liver tissue sections for apoptosis. Finally, a sealing agent containing DAPI nuclear dye was added to seal the sections. The fluorescence microscope was used to record and analyze the results. Figure 6 .from Figure 6 The results showed that LPS / D-GlaN treatment led to massive apoptosis of hepatocytes, and low and high doses of β-bisabolene treatment significantly reduced hepatocyte apoptosis in a dose-related manner. In particular, the high-dose group was basically equivalent to the control group and was significantly better than the positive drug group, indicating that β-bisabolene can significantly reduce hepatocyte apoptosis.

[0047] 8. Conclusion

[0048] β-Bisabolene can effectively improve liver congestion, edema and liver tissue lesions induced by LPS / D-GlaN, effectively reduce serum liver injury indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), inflammatory factors TNF-α, IL-6 and MCP-1, and reduce liver tissue MDA level and hepatocyte apoptosis; in addition, the compound β-Bisabolene 50 mg / kg dose has a better protective effect on acute liver injury induced by LPS / D-GalN in mice than the clinical first-line drug N-acetylcysteine ​​300 mg / kg. The β-Bisabolene of the present invention has a significant protective effect on acute liver injury induced by LPS / D-GlaN.

Claims

1. A use of β-bisabolene in the preparation of a drug for preventing or treating acute liver injury.

2. The use according to claim 1, characterized in that The drug is a drug for reducing serum alanine aminotransferase, aspartate aminotransferase, TNF-α, IL-6 and MCP-1 levels.

3. The use according to claim 1, characterized in that The drug is a drug for inhibiting hepatocyte apoptosis.

4. The use according to claim 1, characterized in that The medicine is a medicine for preventing and treating acute liver failure or acute-on-chronic liver failure induced by LPS / D-GlaN.

5. A drug for preventing and treating acute liver injury, characterized in that: The drug contains the β-bisabolene described in claim 1 as an active ingredient.

6. The drug according to claim 5, characterized in that The medicine also includes other active pharmaceutical ingredients for preventing and treating acute liver injury.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the drug for preventing and treating acute liver injury according to claim 5 or 6 and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical preparation according to claim 7, characterized in that The pharmaceutically acceptable carrier or excipient is selected from one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an ion exchanger, a flavoring agent or an antioxidant.

9. The pharmaceutical preparation according to claim 7, characterized in that The dosage forms of the pharmaceutical preparation include, but are not limited to, capsules, tablets, injections, granules, emulsions, ointments, patches, pills, and syrups.

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