Pentapeptide and application thereof in preparation of medicine for preventing and / or treating alcoholic liver injury

Abstract

The invention provides pentapeptide and application thereof in preparation of a medicine for preventing and / or treating alcoholic liver injury. The pentapeptide with the amino acid sequence shown as SEQ ID NO: 1 not only can remarkably remove DPPH free radicals and ABTS free radicals and play an anti-oxidation role, but also can activate ADH enzyme and ALDH enzyme and play a role in promoting ethanol metabolism, so that the alcoholic liver injury is effectively prevented and / or treated, and the pentapeptide is very suitable for preparing the medicine for preventing and / or treating the alcoholic liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceutical manufacturing. More particularly, it relates to a pentapeptide and its application in the preparation of a medicament for preventing and / or treating alcoholic liver injury. BACKGROUND

[0002] Alcoholic liver injury is a liver disease caused by long-term heavy drinking. In the early stage, it usually presents as fatty liver, and can further develop into alcoholic hepatitis, liver fibrosis and cirrhosis, the main clinical features of which are nausea, vomiting, jaundice, and can have liver enlargement and tenderness, and can be complicated with liver failure and upper gastrointestinal bleeding, etc. Severe alcoholism can induce extensive hepatocyte necrosis, and even liver failure.

[0003] At present, the methods for treating alcoholic liver injury in clinical practice mainly include alcohol abstinence, surgical treatment, drug treatment, etc. Alcohol abstinence is the key to treating alcoholic liver injury, but many patients usually have poor compliance due to psychological dependence and physiological response; surgical treatment can remove diseased tissue, but surgery itself has risks, and a period of recovery is needed after surgery; drug treatment can relieve symptoms, but the drugs currently used mainly include polyene phosphatidylcholine, Fuzheng Huayu capsules, and Shui Fei Bi capsules, etc., and long-term use of such drugs can easily produce drug resistance and side effects. Therefore, it is urgent to find a safe and side-effect-free drug for treating alcoholic liver injury.

[0004] Polypeptides are compounds formed by connecting multiple alpha-amino acids together in a peptide chain, and have various biological activities, safety and no side effects. However, there are still few polypeptides reported to have anti-alcoholic liver injury activity, which is not conducive to the development of clinical drugs. SUMMARY

[0005] The present application aims to provide a pentapeptide as a new raw material that is safe and has no side effects for preparing a medicament for preventing and / or treating alcoholic liver injury.

[0006] The first object of the present application is to provide a pentapeptide.

[0007] The second object of the present application is to provide the application of the above-mentioned pentapeptide or its related biological material in the preparation of a medicament for preventing and / or treating alcoholic liver injury.

[0008] The third object of the present application is to provide a medicament.

[0009] The above objects of the present application are achieved by the following technical solutions: This invention discovers that the pentapeptide with the amino acid sequence shown in SEQ ID NO:1 can not only significantly scavenge DPPH and ABTS free radicals and exert antioxidant effects, but also activate ADH and ALDH enzymes, thereby promoting ethanol metabolism and effectively preventing and / or treating alcoholic liver injury. Therefore, this invention provides a pentapeptide with the amino acid sequence shown in SEQ ID NO:1, and the application of the above-mentioned pentapeptide or related biological materials in the preparation of drugs for the prevention and / or treatment of alcoholic liver injury, wherein the related biological materials are nucleic acid molecules capable of expressing the pentapeptide, or expression constructs, recombinant vectors, genetically engineered bacteria, or transgenic cell lines containing the nucleic acid molecules.

[0010] Preferably, the prevention and / or treatment of alcoholic liver injury is an antioxidant.

[0011] More preferably, the antioxidant is the scavenging of DPPH free radicals.

[0012] More preferably, the antioxidant is the scavenger of ABTS free radicals.

[0013] Preferably, the prevention and / or treatment of alcoholic liver injury involves promoting ethanol metabolism.

[0014] More preferably, the promotion of ethanol metabolism is the activation of ADH enzyme.

[0015] More preferably, the promotion of ethanol metabolism involves activating the ALDH enzyme.

[0016] Based on this, the present invention also provides a drug containing the above-mentioned pentapeptide or related biological materials; wherein the related biological materials are nucleic acid molecules capable of expressing the pentapeptide, or expression constructs, recombinant vectors, genetically engineered bacteria or transgenic cell lines containing the nucleic acid molecules.

[0017] Preferably, the drug further contains excipients.

[0018] Preferably, the dosage form of the drug is one of tablets, capsules, liquid preparations, or granules.

[0019] More preferably, the excipients are polyols (such as propylene glycol, glycerin, sorbitol, mannitol, and / or polyethylene glycol, used to improve drug stability, prevent drug dehydration or deterioration during storage, and also to improve drug taste and solubility), solid lubricants (such as stearic acid and / or magnesium stearate, used to reduce friction and ensure successful drug manufacturing), phosphate buffer (used to adjust the pH of the drug to meet human physiological requirements, thereby reducing irritation and adverse reactions), vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter, used as solvents or carriers to help drug components dissolve or disperse better), alginic acid (to maintain the physical stability of the drug and prevent drug particles from settling or agglomerating), starch (such as corn starch and / or potato starch, used to help tablets disintegrate rapidly after administration and release drug components, thereby improving drug bioavailability), stabilizers (to increase the physical stability of the drug and prevent drug deterioration or degradation during storage or transportation), and antioxidants (used to prevent drug...). The excipients include: (1) excipients that help extend the shelf life of a drug due to oxidation during storage or use; (2) flavoring agents (used to improve the taste of the drug and increase patient compliance); (3) tableting agents (used to compress loose granular substances into solid tablets for easy patient administration and carrying, and also to improve drug stability and bioavailability); (4) binders (such as gelatin, used to help drug components bind better); (5) wetting agents (such as sodium lauryl sulfate, used to help drug components disperse and dissolve better); (6) coloring agents (used to improve the appearance and color of the drug, making it easier to identify and distinguish); (7) isotonic salt solutions (used to adjust the osmotic pressure of the drug to meet human physiological requirements, thereby reducing irritation and adverse reactions); (8) sugars (such as lactose, glucose, and / or sucrose, used to help drug components disperse and dissolve better, while providing necessary energy support); and (9) cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and / or methyl cellulose, used to improve the compressibility and formability of the drug, and also used to control the drug release rate). The type of excipient can be selected according to the need to improve drug stability, activity, and / or bioavailability.

[0020] The present invention has the following beneficial effects: This invention provides a pentapeptide with an amino acid sequence as shown in SEQ ID NO:1, which can not only significantly scavenge DPPH and ABTS free radicals and exert antioxidant effects, but also activate ADH and ALDH enzymes to promote ethanol metabolism, thereby effectively preventing and / or treating alcoholic liver injury. It is very suitable for preparing drugs for the prevention and / or treatment of alcoholic liver injury. Attached Figure Description

[0021] Figure 1 The results are the purity analysis results for the pentapeptide.

[0022] Figure 2 This is the mass spectrometry identification result of the pentapeptide.

[0023] Figure 3 The graph shows the DPPH free radical scavenging rate of the pentapeptide.

[0024] Figure 4 The graph shows the ABTS free radical scavenging rate of the pentapeptide.

[0025] Figure 5 The graph shows the ADH activation rate of the pentapeptide.

[0026] Figure 6 The image shows the ALDH enzyme activity results after pentapeptide treatment. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] In this embodiment of the invention, each group of data was measured three times, and the results are expressed as mean ± standard deviation. One-way mean comparison (ANOVA) was performed using SPSS 27.0, and the LSD method was used for significance analysis between groups.

[0030] Example 1: Synthesis and Identification of Pentapeptides In this embodiment, a pentapeptide with the amino acid sequence shown in SEQ ID NO:1 was synthesized in a solid phase (with the assistance of Nanjing Peptide Valley Biotechnology Co., Ltd.). The purity of the synthesized pentapeptide was analyzed and identified by mass spectrometry. SEQ ID NO:1: WPGSP.

[0031] I. Purity Analysis of Pentapeptides The purity of the pentapeptide was analyzed using high performance liquid chromatography (HPLC). Analytical conditions: Column: Kromasil C18 (4.6 × 150 mm, 5 μm); Mobile phase A: acetonitrile (containing 0.1%... v / v Trifluoroacetic acid); Mobile phase B: Water (containing 0.1% ( v / v (trifluoroacetic acid); elution gradient: 0→0.01 min, 5% ( v / v A, 0.01 → 25 min, 50% ( v / v A, 25→30 min, 90% ( v / v )A; Flow rate: 1 mL / min; Detection wavelength: 214 nm; Sample loading volume: 20 μL.

[0032] The purity analysis results of the pentapeptide are as follows: Figure 1 As shown. By Figure 1 It is known that in the pentapeptide of the present invention, the main peak accounts for the majority, with only a few impurity peaks appearing, and the purity is as high as 99.15%.

[0033] II. Mass Spectrometry Identification of Pentapeptides The pentapeptide was identified using ESI-MS mass spectrometry. The molecular weight on the mass spectrum was compared with the theoretical value to determine if it was the target product. Mass spectrometry conditions: Mobile phase A: water (containing 0.1%... v / v Formic acid); Mobile phase B: Acetonitrile (containing 0.1% ( v / v (Formic acid); Flow rate: 0.2 mL / min; Run time: 1 min; Positive ion mode; Scan range: 0–2000 Da.

[0034] Mass spectrometry identification results of pentapeptides are as follows Figure 2 As shown. By Figure 2 It can be seen that the quasi-molecular ion peak (M+H)+ of the pentapeptide of the present invention is 543.43, carrying one charge, which is consistent with its theoretical molecular weight value.

[0035] In summary, the pentapeptide synthesized in this embodiment is the pentapeptide with the amino acid sequence shown in SEQ ID NO:1, and its purity meets the requirements of subsequent experiments.

[0036] Example 2: Antioxidant Activity Test of Pentapeptides I. DPPH free radical scavenging assay of pentapeptides (1) Solution preparation Preparation of pentapeptide solutions: The pentapeptide obtained in Example 1 was dissolved in ultrapure water to obtain pentapeptide solutions of 0.005, 0.01, 0.1, 0.5 and 1 mg / mL, respectively, which were then stored at -20 °C for later use.

[0037] Preparation of DPPH solution: Dissolve 4 mg of DPPH (2,2-biphenyl-1-picrylhydrazyl) solid in 100 mL of anhydrous ethanol and store at 4 °C in the dark for later use.

[0038] (2) Measurement method Sample group: In a 96-well plate, 150 μL of DPPH solution and 50 μL of pentapeptide solution were mixed and incubated in the dark for 40 min. The absorbance A1 was then measured in a microplate reader at a wavelength of 517 nm.

[0039] Control group: In a 96-well plate, 150 μL of anhydrous ethanol and 50 μL of pentapeptide solution were mixed and incubated in the dark for 40 min. The absorbance A2 was then measured in a microplate reader at a wavelength of 517 nm.

[0040] Blank group: In a 96-well plate, 150 μL of DPPH solution was mixed with 50 μL of ultrapure water, incubated in the dark for 40 min, and the absorbance A0 was measured in a microplate reader at a wavelength of 517 nm.

[0041] Four parallel samples were set up in each group, and the DPPH free radical scavenging rate of the pentapeptide solution was calculated according to the formula "DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0]×100%".

[0042] (3) Measurement results The results are as follows Figure 3 As shown, the DPPH free radical scavenging rate of the pentapeptide solution of 0.005 to 1 mg / mL reached (36.34% ± 3.47%) to (70.30% ± 9.89%), indicating that the pentapeptide of the present invention can significantly scavenge DPPH free radicals, exert antioxidant effects, and thus effectively prevent and / or treat alcoholic liver injury, making it very suitable for preparing drugs for the prevention and / or treatment of alcoholic liver injury.

[0043] II. ABTS Free Radical Scavenging Assay of Pentapeptide (1) Solution preparation Preparation of pentapeptide solutions: The pentapeptide obtained in Example 1 was dissolved in ultrapure water to obtain pentapeptide solutions of 0.005, 0.01, 0.1, 0.5 and 1 mg / mL, respectively, which were then stored at -20 °C for later use.

[0044] Preparation of ABTS stock solution: Dissolve 0.096 g of ABTS (2,2-aminodiphenyl-1-picrylhydrazine) solid in 25 mL of ultrapure water.

[0045] Preparation of potassium persulfate solution: Dissolve 0.017 g of potassium persulfate (K2S2O8) solid in 25 mL of ultrapure water.

[0046] Preparation of ABTS free radical working solution: Mix ABTS stock solution and potassium persulfate solution at a volume ratio of 1:1, and let stand in the dark until the system turns blue-green.

[0047] (2) Measurement method Sample group: In a 96-well plate, 150 μL of ABTS free radical working solution and 50 μL of pentapeptide solution were mixed and incubated in the dark for 40 min. The absorbance A1 was then measured in a microplate reader at a wavelength of 734 nm.

[0048] Control group: In a 96-well plate, 150 μL of ultrapure water and 50 μL of pentapeptide solution were mixed and incubated in the dark for 40 min. The absorbance A2 was then measured in a microplate reader at a wavelength of 734 nm.

[0049] Blank group: In a 96-well plate, 150 μL of ABTS free radical working solution was mixed with 50 μL of ultrapure water, incubated in the dark for 40 min, and the absorbance A0 was measured in a microplate reader at a wavelength of 734 nm.

[0050] Four parallel samples were set up in each group, and the ABTS radical scavenging rate of the pentapeptide solution was calculated according to the formula "ABTS radical scavenging rate (%) = [1-(A1-A2) / A0]×100%".

[0051] (3) Measurement results The results are as follows Figure 4 As shown, the ABTS free radical scavenging rate of the pentapeptide solution of 0.005–1 mg / mL reached (54.79% ± 2.94%) to (91.37% ± 0.83%), indicating that the pentapeptide of the present invention can significantly scavenge ABTS free radicals, exert antioxidant effects, and thus effectively prevent and / or treat alcoholic liver injury, making it very suitable for preparing drugs for the prevention and / or treatment of alcoholic liver injury.

[0052] Example 3: Activity test of pentapeptide in promoting ethanol metabolism I. ADH enzyme activation assay of pentapeptides (1) Solution preparation Preparation of pentapeptide solutions: The pentapeptide obtained in Example 1 was dissolved in ultrapure water to obtain pentapeptide solutions of 0, 0.01, 0.1, 1, 2 and 4 mg / mL, respectively, which were then stored at -20 °C for later use.

[0053] Preparation of ADH enzyme solution: Dissolve ADH enzyme (360 U / mg) in ultrapure water to obtain a 0.2 U / mL ADH enzyme solution, and store it at -20 ℃ for later use.

[0054] Preparation of ADH enzyme working solution: Prepared according to the instructions of the alcohol dehydrogenase (ADH) kit.

[0055] (2) Measurement method Sample group: In a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of pentapeptide solution and incubated at 37 ℃ for 5 min. Then, 50 μL of ADH enzyme solution was added to initiate the reaction. Immediately after application, the absorbance was measured using a VIRIOSKAN LUX microplate reader at a wavelength of 340 nm. Scans were performed every 15 s for 10 min, for a total of 41 scans. The reaction kinetic curve was fitted, and the first derivative of the curve at 0 min was determined as the initial reaction rate V1.

[0056] Control group: In a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of ultrapure water and incubated at 37 °C for 5 min. Then, 50 μL of ADH enzyme solution was added to initiate the reaction. Immediately after plating, the absorbance was measured using a VIRIOSKAN LUX microplate reader at a wavelength of 340 nm. Scans were performed every 15 seconds for 10 min, for a total of 41 scans. The reaction kinetic curve was fitted, and the first derivative of the curve at 0 min was taken as the initial reaction rate V0.

[0057] The ADH activation rate of the pentapeptide solution was calculated using the formula “ADH activation rate (%) = [(V1-V0) / V0] × 100%”.

[0058] (3) Measurement results The results are as follows Figure 5 As shown, the ADH activation rate of the pentapeptide solution of 0.01–4 mg / mL reached (4.64% ± 4.03%) to (17.57% ± 8.75%), indicating that the pentapeptide of the present invention can effectively activate ADH enzyme and promote the metabolism of ethanol to acetaldehyde, thereby effectively preventing and / or treating alcoholic liver injury, and is very suitable for preparing drugs for the prevention and / or treatment of alcoholic liver injury.

[0059] II. ALDH enzyme activation assay of pentapeptides (1) Solution preparation Preparation of pentapeptide solutions: The pentapeptide obtained in Example 1 was dissolved in ultrapure water to obtain pentapeptide solutions of 0, 0.1, 1, 2 and 4 mg / mL, respectively, which were stored at -20 °C for later use.

[0060] Preparation of ALDH enzyme solution: Dissolve ALDH enzyme (15 U / mg) in ultrapure water to obtain 0.2 U / mL ALDH enzyme solution, and store at -20 ℃ for later use.

[0061] Preparation of ALDH enzyme inactivation solution: Dissolve ALDH enzyme (15 U / mg) in ultrapure water to obtain 0.2 U / mL ALDH enzyme solution. Inactivate the enzyme at 100 ℃ for 5 min and then store at -20 ℃ for later use.

[0062] Preparation of ALDH enzyme working solution: Prepared according to the instructions of the acetaldehyde dehydrogenase (ALDH) kit.

[0063] (2) Construction of standard curve The acetaldehyde dehydrogenase kit contains the following reagents: standard (powder, 1 vial), reagent 2 (2.5 mL, 1 vial), and reagent 3 (70 mL, 1 vial). Dissolve the standard in 1.41 mL of distilled water to obtain a 1 nmol / μL standard solution. Then dilute this solution with distilled water to prepare six concentration gradients (0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol / μL). Take 20 μL of each concentration gradient, add 6.5 μL of reagent 2 and 174 μL of reagent 3, mix well, and incubate at 37 ℃ for 5 min. Read the absorbance at 450 nm. Plot a standard curve y = ax + b with standard concentration as the x-axis and absorbance as the y-axis.

[0064] (3) Measurement method Sample group: In a 96-well plate, mix 180 μL of ALDH enzyme working solution with 10 μL of pentapeptide solution, then add 10 μL of ALDH enzyme solution and mix well. After 30 s, measure the absorbance A1 in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, measure the absorbance A2 again. Calculate ΔA1 = A2 - A1.

[0065] Control group: In a 96-well plate, 180 μL of ALDH enzyme working solution was mixed with 10 μL of pentapeptide solution, and then 10 μL of ALDH enzyme inactivation solution was added and mixed. After 30 s, the absorbance A3 was measured in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, the absorbance A4 was measured again. The result was ΔA0 = A4 - A3.

[0066] The ALDH enzyme activity after treatment with the pentapeptide solution was calculated according to the formula “ALDH enzyme activity (nmol / min / mL) = (△Ab) / avt”, where △A = △A1 - △A0, v is the system volume (200 μL) and t is the reaction time (30 min).

[0067] (4) Measurement results The results are as follows Figure 6 As shown, the ALDH enzyme activity reached (2.56±0.24) to (3.46±0.38) nmol / min / mL after treatment with a pentapeptide solution of 0.1–4 mg / mL. This indicates that the pentapeptide of the present invention can effectively activate ALDH enzyme, promote the conversion of acetaldehyde, a metabolite of ethanol, to acetic acid, and thus effectively prevent and / or treat alcoholic liver injury, making it highly suitable for preparing drugs for the prevention and / or treatment of alcoholic liver injury.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A pentapeptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the pentapeptide or related biomaterials of claim 1 in the preparation of a medicament for the prevention and / or treatment of alcoholic liver injury, characterized in that, The relevant biological material is a nucleic acid molecule capable of expressing the pentapeptide, or an expression construct, recombinant vector, genetically engineered bacteria, or transgenic cell line containing the nucleic acid molecule.

3. The application according to claim 2, characterized in that, The prevention and / or treatment of alcoholic liver injury is based on antioxidants.

4. The application according to claim 3, characterized in that, The antioxidant is the scavenging of DPPH free radicals.

5. The application according to claim 3, characterized in that, The antioxidant is used to scavenge ABTS free radicals.

6. The application according to claim 2, characterized in that, The prevention and / or treatment of alcoholic liver injury involves promoting ethanol metabolism.

7. The application according to claim 6, characterized in that, The process of promoting ethanol metabolism involves activating the ADH enzyme.

8. The application according to claim 6, characterized in that, The process of promoting ethanol metabolism involves activating the ALDH enzyme.

9. A drug, characterized in that, The invention comprises the pentapeptide of claim 1 or related biological materials thereof; the related biological materials are nucleic acid molecules capable of expressing the pentapeptide, or expression constructs, recombinant vectors, genetically engineered bacteria or transgenic cell lines containing the nucleic acid molecules.

10. The drug according to claim 9, characterized in that, The drug also contains excipients.

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