Composition and medicine for preventing and treating acute liver injury and protecting liver

Abstract

The invention provides a composition and a medicine for preventing and treating acute liver injury and protecting liver. The composition comprises sweet orange flavone and dauricine. According to the traditional Chinese medicine composition disclosed by the invention, through a large number of researches, the sweet orange flavone and the dauricine can be synergistically interacted when being used for preventing and treating the acute liver injury; the sweet orange flavone and dauricine combined treatment group can more effectively relieve liver dysfunction, tissue injury and inflammatory state of acute liver injury model mice. Therefore, the combination of the sweet orange flavone and the dauricine can more effectively prevent and treat the acute liver injury and related diseases of the acute liver injury, and has the effect of protecting the liver.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, especially the treatment of acute liver injury, and specifically relates to a composition and drug for the prevention and treatment of acute liver injury and for 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] Currently, the main drugs for treating acute liver injury include N-acetylcysteine ​​and silymarin. N-acetylcysteine ​​is effective in the early stages but becomes ineffective in the later stages. Traditional compound treatments have complex ingredients, with multiple medicinal materials and numerous compounds interacting with each other, making quality standardization difficult and batch-to-batch stability hard to control.

[0004] Therefore, there is a need in this field for drugs with clearly defined ingredients, controllable quality, and good efficacy for the treatment of acute liver failure. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a composition and medicine for preventing and treating acute liver injury and protecting the liver, the composition comprising sweet orange flavonoids and puerarin, which can synergistically enhance the effects of treating acute liver injury and protecting the liver.

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

[0007] In a first aspect, the present invention provides a composition for preventing and treating acute liver injury, the composition comprising sweet orange flavonoids and puerarin.

[0008] In some embodiments, the molar ratio of sweet orange flavonoids and baicalein is 1:(1~3), preferably 1:(1~2).

[0009] A second aspect of the invention provides the use of the composition described above in the preparation of a medicament for the prevention and treatment of acute liver injury.

[0010] A third aspect of the invention provides the use of the composition described above in the preparation of a medicament for the prevention and treatment of acute liver injury-related diseases.

[0011] In some implementations, the acute liver injury-related diseases include cirrhosis and liver failure.

[0012] A fourth aspect of the invention provides the use of the composition described above in the preparation of a hepatoprotective medicament. In some implementations, the application includes reducing ALT and / or AST levels.

[0013] In some implementations, the application includes alleviating liver tissue damage.

[0014] In some implementations, the application includes alleviating liver inflammation.

[0015] In a fifth aspect, the present invention provides a medicament for preventing or protecting acute liver injury, wherein the active ingredient of the medicament comprises the composition described above.

[0016] In some embodiments, the dosage form of the drug includes solutions, powders, pills, capsules, etc.

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

[0018] Extensive research has revealed that hesperidin and guaiacine can synergistically enhance the prevention and treatment of acute liver injury. Compared to treatments with hesperidin or guaiacine alone, the combined treatment group showed more effective relief of liver dysfunction, tissue damage, and inflammation in mice with acute liver injury. Therefore, the combination of hesperidin and guaiacine can more effectively prevent and treat acute liver injury and related diseases, while also providing hepatoprotective effects.

[0019] The composition of this invention, containing sweet orange flavonoids and puerarin, has a clearly defined composition and controllable quality when used to prevent and treat acute liver injury and related diseases, ensuring the stability and reproducibility of the therapeutic effect, meeting modern pharmaceutical standards, and showing good application prospects. Attached Figure Description

[0020] Figure 1 The appearance of the livers of mice in each group.

[0021] Figure 2 The results show the ALT and AST levels in each group of mice.

[0022] Figure 3 The results of H&E staining of liver tissue from mice in each group are shown.

[0023] Figure 4 The results show the mRNA expression levels of relevant inflammatory factors 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] Sinensetin, chemical name 5,6,7,3',4'-pentamethoxyflavone, CAS number 2306-27-6, molecular formula C 20 H 20 O7, with a molecular weight of 372.373, is a flavonoid compound found in the fruits, juices, and Orthosiphonspicatus of citrus plants. Its main sources are Rutaceae plants such as dried tangerine peel, citrus peel, and Guangchenpi.

[0028] Dauricine, CAS No. 524-17-4, molecular formula C 38 H 44 N2O6, with a molecular weight of 624.77, is the main active ingredient derived from the Chinese herbal medicine *Radix Sophorae Flavescentis* (which has the effects of clearing heat and detoxifying, dispelling wind and relieving pain).

[0029] The following description is based on specific implementation methods.

[0030] Example 1 This embodiment studies the synergistic effect of sweet orange flavonoids and baicalein.

[0031] 1. Experimental Methods LX-2 cells were treated with different concentrations of drugs and divided into a normal group (normal culture), a model group, and a treatment group (treatment concentrations are shown in Table 6): sweet orange flavonoid group, puerarin group, and combination treatment groups with different ratios. Each treatment group was treated with the drug for 24 h. Then, the model group and treatment group were used to induce inflammatory modeling with 15 μM Ccl4 for 6 h. Cells from each group were then collected, and the expression of inflammatory factors in different groups was detected. The effect level and the cooperation index (CI) were calculated.

[0032] Cells from each group were collected, and the mRNA expression levels of IL-6, IL-10, CD206, and COX2 were detected using the following methods: (1) RNA extraction 1) Collect cells from each group and centrifuge to obtain the cell pellet; 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).

[0033] (2) 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 1.

[0034] Table 1 3) The reverse transcription procedure is shown in Table 2.

[0035] Table 2 (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 detection primers for each gene are shown in Table 3 below, and the specific preparation system is shown in Table 4.

[0036] Table 3 2) Table 5 lists the setup procedure for qRT-PCR; 3) Data usage The method is used to process and analyze the data.

[0037] Table 4 Table 5 The effect size and effect level were calculated based on the extent to which the expression levels of inflammatory factors decreased in the model group.

[0038] Generally, the effect size represents the percentage of the treatment group relative to the model group, expressed as a percentage (%).

[0039] The effect level is the normalized (decimal) form of the effect size, referring to the suppressed portion (Fraction Affected, Fa). For example, correspond This indicates that the concentration can inhibit the expression of inflammatory factors by 50%.

[0040] Given that the detection indicators include pro-inflammatory and anti-inflammatory factors with opposite trends, this embodiment uses relative effect value (fa, Fraction affected) to normalize the data.

[0041] For pro-inflammatory factors (the lower the index value, the better the drug effect), fa = (model group - treatment group) / (model group - normal group).

[0042] For anti-inflammatory factors (the higher the index value, the better the efficacy), fa = (treatment group - model group) / (normal group - model group).

[0043] After calculating the average fa value for each group, the Combination Index (CI) was calculated based on the Chou-Talalay intermediate-effect principle. When CI < 1, the drug combination was considered to have a synergistic effect.

[0044] The formula for calculating the synergistic index (CI) is as follows: For a given effect level, the formula for calculating the synergistic index (CI) is: .

[0045] Among them, D A and D B In combination therapy, the actual concentrations of drug A and drug B used to achieve effect Fa; (D) x ) A and (D) x ) B Based on the dose-response curves of individual drugs, calculate the concentrations required for each drug to achieve the same effect Fa.

[0046] Judgment criteria: CI < 1: synergistic; CI = 1: additive; CI > 1: antagonistic. The smaller the CI value, the stronger the synergistic effect.

[0047] The effect size calculation results are shown in Table 6, and the synergy index calculation results are shown in Table 7.

[0048] Table 6 Note: The combined treatment groups were: 2.5µM sweet orange flavonoids + 2.5µM puerarin, 5µM sweet orange flavonoids + 5µM puerarin, 7.5µM sweet orange flavonoids + 7.5µM puerarin, and 10µM sweet orange flavonoids + 10µM puerarin.

[0049] Table 7 The results showed that all CI values ​​were less than 1, indicating that the combined use of hesperidin and puerarin exhibited a clear synergistic effect at three different effect levels (ED50, ED75, and ED90). The strength of the synergistic effect increased with increasing effect level: at ED50 (moderate inhibition), CI = 0.85, indicating a mild synergistic effect; at ED90 (strong inhibition), CI = 0.40, indicating a very significant synergistic effect. This suggests that the combined use of hesperidin and puerarin can synergistically enhance the therapeutic effect.

[0050] Example 2 This embodiment studies the therapeutic effect of sweet orange flavonoids combined with puerarin on acute liver injury.

[0051] I. Experimental Methods 1. Establishment and administration of an acute liver injury mouse model Model mice: SPF grade BALB / C mice, 6 weeks old, weighing 18-22g.

[0052] Modeling reagents: CCl4 (solvent is olive oil), CCl4 concentration is 5%.

[0053] Modeling method and dosage: Forty mice were randomly divided into five groups: blank control group, model group, sweet orange flavonoid treatment group, puerarin treatment group, and sweet orange flavonoid and puerarin combined treatment group, with eight mice in each group. Three days before modeling, mice in the sweet orange flavonoid treatment group, puerarin treatment group, and sweet orange flavonoid and puerarin combined treatment group were administered sweet orange flavonoid, puerarin, or a combination of sweet orange flavonoid and puerarin (molar ratio of sweet orange flavonoid to puerarin 1:2) by gavage, respectively, three times, at a dose of 50 mg / kg each time, with each administration 24 hours apart. The model group was given an equal volume of PBS buffer. Eighteen hours after the end of administration, mice in the model group, sweet orange flavonoid treatment group, puerarin treatment group, and sweet orange flavonoid and puerarin combined treatment group were injected intraperitoneally with 200 μL of 5% CCl4 olive oil solution, once per mouse. Mice in the blank control group were injected with an equal volume of olive oil.

[0054] 2. Indicator Testing (1) 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 was collected from the mouse eyeballs, left to stand for 6 hours, centrifuged to collect the supernatant, and sent to the testing center for testing.

[0055] (2) 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.

[0056] (3) Expression of related inflammatory factors Eighteen hours after the last injection of CCl4 olive oil solution, liver tissues from each group of mice were collected, and the mRNA expression levels of IL-6, IL-10, CD206, and COX2 were detected. The methods were the same as in Example 1, except for sample processing. The liver tissue processing method was as follows: 1) Fresh liver tissue was cut into small pieces and transferred to homogenization tubes. 1 mL of Trizol and clean magnetic beads were added, followed by machine homogenization (homogenization conditions were the same as for protein tissue lysis). After homogenization, the supernatant was transferred to enzyme-free 1.5 mL EP tubes, and appropriate labels were written on the caps and tube bodies. The tubes were allowed to stand at room temperature for 5-10 min to ensure complete lysis. 2) 200 μl (1 / 5 of Trizol) of chloroform was added, thoroughly mixed by inverting, and allowed to stand at room temperature for 10 min. The tubes were then centrifuged using a low-temperature ultracentrifuge (12000 rpm, 15 min, 4℃). Other steps were the same as in Example 1.

[0057] II. Experimental Results 1. Liver phenotype like Figure 1 As shown, the livers of mice in the blank control group (BL) were smooth, reddish, and elastic. The livers of mice in the model group (Model) were noticeably rough, pale, soft, and brittle. The sweet orange flavonoid group and the guaiac alkaloid group showed slight improvement compared to the model group, with a slightly smoother color and reduced granular texture, but still poor elasticity and brittleness. Compared to the sweet orange flavonoid (T) group and the guaiac alkaloid (D) group, the combined treatment group of sweet orange flavonoid and guaiac alkaloid showed a slightly smoother color, reduced granular texture, and better treatment effect.

[0058] 2. Serum ALT and AST levels like Figure 2As shown, compared with the blank control group (BL) mice, the serum ALT and AST levels in the model group (M) 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 the sweet orange flavonoid (Sin) and dau alkaloid (Dau) treatment groups were decreased, and the decrease was more significant in the combined treatment group.

[0059] 3. H&E staining of liver tissue like Figure 3 As shown, in the blank group (BL), the liver cell nuclei of mice were round with complete nuclear outlines, the sinusoidal structure was regular, and the hepatocyte cords were radially distributed from the central vein. In the model group (M), which was induced by 5% CCl4 alone, the hepatocyte cords were disordered, the sinusoidal structure was incomplete and irregular, cell vacuoles were present, and the number of apoptotic and necrotic cells increased. The hepatocyte cord arrangement trend tended to normalize and cell vacuoles were reduced in the groups treated with sweet orange flavonoids (T) and puerarin (D); the combined treatment group could more effectively reduce inflammation, reduce hepatocyte damage, and have fewer cell vacuoles.

[0060] 4. mRNA expression levels of relevant inflammatory factors like Figure 4 As shown, compared with the blank group (BL), the transcriptional level of the pro-inflammatory factor IL-6 in the model group (M) was significantly increased, while the transcriptional levels of the anti-inflammatory factors IL-10, CD206, and COX2 were significantly decreased, indicating the occurrence of inflammation in the body. Compared with the model group, the transcriptional levels of IL-6 in mice treated with sweet orange flavonoids (T) and puerarin (D) were significantly decreased, while the transcriptional levels of IL-10, CD206, and COX2 were significantly increased; the decrease was even more significant in the combined treatment group (G), indicating that the combined treatment group could more effectively improve the inflammatory state of the liver tissue in model mice.

[0061] In conclusion, the combined use of sweet orange flavonoids and puerarin in the treatment of acute liver injury can have a synergistic effect, effectively improving the treatment outcome.

[0062] 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.

[0063] 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. A composition for preventing and treating acute liver injury, characterized in that, The composition contains sweet orange flavonoids and piperine.

2. The composition according to claim 1, characterized in that, The molar ratio of sweet orange flavonoids to baicalein is 1:(1~3).

3. The use of the composition according to claim 1 or 2 in the preparation of a medicament for the prevention and treatment of acute liver injury.

4. The use of the composition according to claim 1 or 2 in the preparation of a medicament for the prevention and treatment of acute liver injury-related diseases.

5. The application as described in claim 4, characterized in that, The acute liver injury-related diseases include cirrhosis and liver failure.

6. Use of the composition according to claim 1 or 2 in the preparation of a hepatoprotective medicament.

7. The application as described in any one of claims 3 to 6, characterized in that, The application includes reducing ALT and / or AST levels.

8. The application as described in any one of claims 3 to 6, characterized in that, The applications include alleviating liver tissue damage and / or relieving liver inflammation.

9. A drug for preventing or protecting against acute liver injury, characterized in that, The active ingredient of the drug includes the composition as described in claim 1 or 2.

10. The medicament as claimed in claim 9, characterized in that, The dosage forms of the drug include solutions, powders, pills, and capsules.

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