Application of plant extract sweet orange flavone in prevention and treatment of acute liver injury and protection of liver

Abstract

The invention provides application of plant extract sweet orange flavone in prevention and treatment of acute liver injury and protection of liver. Research finds that the sweet orange flavone can effectively reduce ALT and AST levels of acute liver injury model mice, inhibit liver cell inflammatory response and reduce liver cell injury. The invention further finds that the sweet orange flavone can improve the acute liver injury by inhibiting the expression of the p-NF-kappa B-p65 protein. Therefore, the sweet orange flavone can be used as a medicine for preventing and treating acute liver injury and acute liver injury related diseases, can protect the liver, has the advantages of definite components, controllable quality, good curative effect and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the treatment of acute liver injury, specifically the application of the plant extract sweet orange flavonoid in the prevention and treatment of acute liver injury and liver protection. Background Technology

[0002] Acute liver injury (ALI) is a syndrome of severe hepatocellular damage that occurs rapidly due to various etiologies. Its core features include extensive hepatocellular necrosis and apoptosis, severe inflammatory response, and a rapid deterioration of liver function. Clinical manifestations include jaundice, coagulation disorders, and significantly elevated transaminase levels. In severe cases, it can rapidly progress to acute liver failure (ALF), which has a high mortality rate.

[0003] Sinensetin, chemically known as 5,6,7,3',4'-pentamethoxyflavone, is a flavonoid compound found in the fruits, juices, and *Orthosiphon spicatus* of citrus plants, primarily from Rutaceae plants such as dried tangerine peel, citrus peel, and *Citrus reticulata* var. *sinense*. This compound possesses anti-allergic, antiviral, anticancer, analgesic, cardiovascular disease prevention, and antioxidant effects. It can also improve lung tissue damage caused by respiratory syncytial virus infection through the AMPK / mTOR signaling pathway.

[0004] Currently, traditional Chinese medicine-based compound treatments involve complex components, with numerous herbs and compounds interacting and posing significant challenges to quality standardization and batch-to-batch stability control. Therefore, there is a need in this field to explore more drugs with clearly defined components, controllable quality, and effective efficacy for the treatment of acute liver injury. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide the application of the plant extract sweet orange flavonoid in the prevention and treatment of acute liver injury and in the protection of the liver.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention is to provide the use of sweet orange flavonoids in the preparation of medicaments for the prevention and treatment of acute liver injury.

[0008] A second aspect of the present invention is to provide the use of sweet orange flavonoids in the preparation of medicaments for the prevention and treatment of diseases related to acute liver injury.

[0009] In some implementations, the acute liver injury-related diseases include cirrhosis and liver failure.

[0010] A third aspect of the invention is to provide the use of sweet orange flavonoids in the preparation of a hepatoprotective medicament.

[0011] In some implementations, the application includes reducing ALT and / or AST levels.

[0012] In some implementations, the application includes mitigating hepatocyte damage.

[0013] In some implementations, the application includes suppressing the inflammatory response of hepatocytes.

[0014] In some implementations, the application includes suppressing the expression of the COX-2 gene.

[0015] In some implementations, the application includes suppressing the expression of the IL-1β gene.

[0016] In some implementations, the application includes reducing the expression of the p-NF-κB-p65 protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] This invention has revealed that sweet orange flavonoids can effectively reduce ALT and AST levels in mice with acute liver injury, inhibit hepatocyte inflammatory responses, and reduce hepatocyte damage. Furthermore, it was found that sweet orange flavonoids improve acute liver injury by inhibiting the expression of p-NF-κB-p65 protein. Therefore, sweet orange flavonoids can be used as a drug for the prevention and treatment of acute liver injury and related diseases, and can protect the liver. It has advantages such as clearly defined components, controllable quality, and good efficacy, showing promising application prospects. Attached Figure Description

[0019] Figure 1 The liver phenotype of mice in each group is observed.

[0020] Figure 2 The results show the serum ALT and AST levels of mice in each group.

[0021] Figure 3 The results of H&E staining of liver tissue from mice in each group are shown.

[0022] Figure 4 The results show the mRNA expression levels of relevant inflammatory factors in each group of mice.

[0023] Figure 5 The results show the expression levels of p-NF-κB-p65 protein in each group of mice. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0026] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0027] The following description is based on specific implementation methods.

[0028] Example 1 This embodiment studies the therapeutic effect of sweet orange flavonoids on acute liver injury.

[0029] 1. Establishment and administration of an acute liver injury mouse model Model mice: SPF grade BALB / C mice, 6 weeks old, weighing 18-22g.

[0030] Modeling reagents: CCl4 (solvent is olive oil), CCl4 concentration is 5%.

[0031] Modeling method and dosage: Forty-two mice were randomly divided into six groups: normal control group (blank group), model group, positive control silymarin group (25 mg / kg), low-dose hesperidin group (25 mg / kg), and high-dose hesperidin group (50 mg / kg), with seven mice in each group. Three days before modeling, mice in the positive control silymarin group, low-dose hesperidin group, medium-dose hesperidin group, and high-dose hesperidin group were administered hesperidin or silymarin via gavage three times, with each administration 24 hours apart; the model group was given an equal amount of olive oil. Eighteen hours after the end of administration, mice in the model group, positive control silymarin group, low-dose hesperidin group, medium-dose hesperidin group, and high-dose hesperidin group were intraperitoneally injected with 200 μL of 5% CCl4 olive oil solution once; mice in the blank group were injected with an equal amount of olive oil.

[0032] Successful modeling indicators and time points: 18 hours after modeling, the serum ALT and AST levels in the model group were detected. They were significantly higher than those in the normal control group, indicating that the modeling was successful.

[0033] 2. Indicator Testing (1) Phenotypic view Eighteen hours after the model was created, the liver was harvested for photographing.

[0034] (2) Serum ALT and AST levels Eighteen hours after the last injection of CCl4 olive oil solution, serum was collected from each group of mice to detect ALT and AST levels. The method was as follows: blood from the mouse eyeballs was allowed to stand for 6 hours, then incubated at 12,000 rpm for 10 minutes. The supernatant was collected and the serum was tested using an instrument.

[0035] (3) H&E staining of liver tissue Eighteen hours after the last injection of CCl4-olized olive oil solution, liver tissue from each group of mice was collected for H&E staining, as follows: 1) Fix the tissue for 48 h, then rinse with running water for 4 h. Next, soak in 75%, 85%, and 95% ethanol for 1 h each. Then soak in anhydrous ethanol I for 40 min and anhydrous ethanol II for 40 min. Then transfer to xylene I and xylene II for 20 min each. Then transfer to wax I for 40 min and wax II for 40 min (the melting point of the wax used is 56℃). Finally, embed the tissue (when performing the wax I step, the embedding machine can be opened in advance to dissolve the wax block inside the embedding machine. The melting point of the wax block used in the embedding machine is 60℃). 2) Ensure the required materials are spread evenly to avoid any cracks during the fabrication process. Next, carefully drain off excess water and gently lift it out to maintain its original shape and structural integrity. 3) First, soak in xylene I and xylene II for 10 minutes each, then transfer to anhydrous ethanol for 5 minutes. After that, transfer to 95%, 85%, and 75% ethanol for 2 minutes each, and finally soak in pure water for 2 minutes. 4) Stain the cell nuclei with hematoxylin staining solution for about 10 minutes. During this time, observe the degree of staining of the cell nuclei with a microscope. After staining, transfer the sample to water to rinse in order to remove any impurities or unwanted dye components that may remain in the staining solution. 5) After adding eosin staining solution for about 10 seconds, observe the staining effect under a microscope. Stop the staining with pure water and wash until no staining solution remains. 6) Soak in 75%, 85%, and 95% ethanol for 20 seconds each, then in anhydrous ethanol for 1 minute, and finally in xylene I and xylene II for 10 minutes each. 7) Neutral resin sealing.

[0036] (4) Expression of related inflammatory factors Eighteen hours after the last injection of CCl4 olive oil solution, liver tissues of mice in each group were collected to detect the mRNA expression levels of COX2 and IL-1β.

[0037] The primers used for the detection are shown in Table 1.

[0038] Table 1 RNA extraction 1) Cut fresh liver tissue into small pieces and transfer them to homogenization tubes. Add 1 mL of Trizol and clean magnetic beads, and then homogenize by machine (homogenization conditions are the same as for protein tissue lysis). After homogenization, transfer the supernatant to an enzyme-free 1.5 mL EP tube, write the corresponding labels on the cap and tube body, and let it stand at room temperature for 5-10 minutes to allow for complete lysis. 2) Add 200 μl (1 / 5 Trizol) of chloroform, mix thoroughly by inverting, let stand at room temperature for 10 min, and then centrifuge in a low-temperature ultra-high speed centrifuge (12000 rpm, 15 min, 4℃). 3) When centrifugation is complete, carefully aspirate the clear liquid on top and transfer it to a new labeled 1.5 mL enzyme-free EP tube. Then add an equal volume of isopropanol and 1 μL of nucleic acid precipitation aid, invert and mix 10 times, and place in a 4°C refrigerator for 10 min. After centrifugation, centrifuge (12000 rpm, 10 min, 4°C). 4) Dilute anhydrous ethanol with DEPC water beforehand to prepare 75% ethanol. After centrifugation, discard the supernatant, add 1 mL of 75% ethanol to each tube, invert the tube to fully suspend the precipitate fragments in the 75% ethanol liquid, and continue centrifugation (12000 rpm, 5 min, 4℃). 5) After centrifugation, remove the supernatant, open the cap of the EP tube, and let it stand at room temperature for 5 minutes until the ethanol in the tube has completely evaporated. 6) Add 10 μL of DEPC water to each tube, mix it evenly, and determine its concentration using NanoDrop (1 μL of DEPC water is used as a blank control).

[0039] RNA reverse transcription 1) Calculate the total amount of RNA required for reverse transcription of 1 μg, add ddH2O to bring the volume to 16 μL, and then add 4 μL of 5×PrimeScript Buffer to each tube; 2) The total reverse transcription volume was 20 μL, as shown in Table 2.

[0040] Table 2 3) The reverse transcription procedure is shown in Table 3.

[0041] Table 3 Real-time quantitative PCR (qRT-PCR): 1) The total system of 20 μL = 0.4 μL rox + 10 μL mix + 0.44 μL cDNA + 8.36 μL LEPC water + 0.4 μL primer F + 0.4 μL primer R. The specific preparation system is shown in Table 4. 2) Table 5 lists the setup procedure for qRT-PCR; 3) Data usage The method is used to process and analyze the data.

[0042] Table 4 Table 5 (5) Network pharmacology analysis Analysis of the SwissTarget Prediction, PharmMapper, and Batman databases identified 159 targets related to puerarin. Further analysis of the Disgenet and GeneCards databases revealed 5012 target genes closely associated with acute liver injury. Venn diagrams were then used to merge drug-disease related targets, yielding 292 duplicate targets. These duplicate targets were imported into the String database to construct a PPI protein interaction network, which was then visualized using Cyctoscape software, creating a regulatory network of "drug-target-disease" to reveal the potential relationship between puerarin and acute liver injury.

[0043] II. Experimental Results 1. Liver phenotype like Figure 1 As shown, the livers of mice in the blank control group (NC) appeared smooth, reddish, and elastic. The livers of mice in the model group (Model) were noticeably rough, pale, soft, and fragile. Administration of sweet orange flavonoids improved CCl4-induced liver damage in mice. The liver lobes and edges of the model group (Model) showed significantly rougher textures compared to the blank control group (NC) (black arrows), and the color was duller. The low- and high-dose sweet orange flavonoid groups (Low: 25 mg / kg and High: 50 mg / kg, respectively) showed reduced roughness in the liver lobes and edges compared to the model group (red arrows), and the livers were redder in color.

[0044] 2. Serum ALT and AST levels like Figure 2As shown, compared with the control group (NC) mice, the serum ALT and AST levels in the model group (Model group) mice showed a significant increasing trend (p < 0.0001), indicating that intraperitoneal injection of 5% CCl4 induced acute liver injury in mice. Compared with the model group, the serum ALT and AST levels in mice treated with low-dose (25 mg / kg) and high-dose (50 mg / kg) sweet orange flavonoids were significantly decreased.

[0045] 3. H&E staining of liver tissue like Figure 3 As shown, in the control group (NC) mice, the hepatocyte nuclei were round with intact outlines, the sinusoidal structure was regular, and the hepatocyte cords were radially distributed from the central vein. In the model group (Model) mice, which were induced with only 5% CCl4, the hepatocyte cords were disordered (black arrows), the sinusoidal structure was incomplete and irregular, cell vacuoles were present, and the number of apoptotic and necrotic cells increased (red arrows). The hepatocyte cord arrangement trend tended to normalize and cell vacuoles decreased in the low-dose and high-dose hesperidin treatment groups. The results show that hesperidin treatment can alleviate liver inflammation and reduce hepatocyte damage in mice with acute liver injury.

[0046] 4. mRNA expression levels of relevant inflammatory factors like Figure 4 As shown, compared with the control group (NC), the COX2 transcription level in the model group (Model) was significantly increased, indicating the occurrence of inflammation in the body. Compared with the model group, the COX2 transcription level in mice treated with low-dose (25 mg / kg) and high-dose (50 mg / kg) sweet orange flavonoids was significantly decreased. Compared with the control group (NC), the IL-1β transcription level in the model group (Model) was significantly increased. Compared with the model group, the difference in IL-1β transcription level between the low-dose (25 mg / kg) and high-dose (50 mg / kg) sweet orange flavonoids groups was statistically significant. Compared with the model group, the reduction in IL-1β transcription level in the low-dose group was limited, while the reduction in IL-1β transcription level in the high-dose group was significant.

[0047] 5. Results of network pharmacology analysis Network pharmacology analysis revealed that the NF-κB signaling pathway may be involved in the therapeutic process of sweet orange flavonoids on acute liver injury (specific data omitted). The NF-κB family has a key core member, p65, which participates in many biological processes, such as inflammation, immunity, tumorigenesis, cell growth, and apoptosis. The expression level of phosphorylated p65 can also serve as an indicator of the severity of inflammation in the body. Therefore, the expression levels of p-NF-κB-p65 protein in each group were further examined. The results are as follows... Figure 5As shown, compared with the control group (NC), the expression level of p-NF-κB-p65 protein in the model group (Model) was significantly increased. Compared with the model group, the expression level of p-NF-κB-p65 protein was decreased in both the low-dose (Low) and high-dose (High) groups of sweet orange flavonoids. This indicates that sweet orange flavonoids can reduce the expression of p-NF-κB-p65 protein, and its effect is dose-dependent, suggesting that p-NF-κB-p65 protein may be involved in the treatment of acute liver injury by sweet orange flavonoids.

[0048] In summary, sweet orange flavonoids can be used to prepare drugs for the treatment of acute liver injury and related diseases.

[0049] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0050] 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 present invention. 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 modifications and improvements 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. Application of sweet orange flavonoids in the preparation of drugs for the prevention and treatment of acute liver injury.

2. Application of sweet orange flavonoids in the preparation of drugs for the prevention and treatment of acute liver injury-related diseases.

3. The application as described in claim 2, characterized in that, The acute liver injury-related diseases include cirrhosis and liver failure.

4. Application of sweet orange flavonoids in the preparation of drugs that protect the liver.

5. The application as described in any one of claims 1 to 4, characterized in that, The application includes reducing ALT and / or AST levels.

6. The application as described in any one of claims 1 to 4, characterized in that, The applications include reducing hepatocyte damage.

7. The application as described in any one of claims 1 to 4, characterized in that, The application includes suppressing the inflammatory response of hepatocytes.

8. The application as described in claim 7, characterized in that, The application includes suppressing the expression of the COX-2 gene.

9. The application as described in claim 7, characterized in that, The application includes suppressing the expression of the IL-1β gene.

10. The application as described in claim 7, characterized in that, The application includes reducing the expression of p-NF-κB-p65 protein.

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