Alcohol-detoxifying peptides

Abstract

This invention belongs to the field of biomedical technology, specifically providing a novel active hangover-relieving peptide, the sequence of which is shown in SEQ ID NO.1 or NO.2. This invention also provides a composition containing this peptide, which can be a pharmaceutical, health product, or food. The peptide of this invention has a small molecular weight, is easy to synthesize, is safe and has no toxic side effects, and exhibits good safety and practicality.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to alcohol-detoxifying active peptides and their applications. Background Technology

[0002] Drunkenness, a common term for alcohol poisoning, refers to an abnormal state of bodily function that occurs after a person consumes a large amount of alcohol (ethanol) at once. It is classified into acute and chronic drunkenness. As is well known, ethanol is mainly absorbed into tissues and organs through the mouth, esophagus, stomach, and intestinal mucosa. The liver is the primary organ for alcohol metabolism. In the liver, 90% of ethanol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), and acetaldehyde is oxidized to acetic acid by aldehyde dehydrogenase (ALDH), ultimately producing CO2 and H2O which are excreted from the body. Numerous studies have demonstrated that the accumulation of alcohol and its harmful metabolites in the body is the main cause of alcoholic liver damage. Therefore, accelerating alcohol metabolism and reducing the retention of harmful metabolites in the body is crucial for maintaining health. Currently, finding safe and effective active ingredients from natural products to improve alcohol metabolism and alleviate alcoholic liver damage is a hot topic.

[0003] Previous studies have identified bioactive peptides from enzymatic hydrolysates of plants, aquatic organisms, livestock, poultry, and dairy products that accelerate alcohol metabolism and alleviate alcoholic liver damage. Microorganisms are a vast treasure trove of bioactive resources, containing abundant intracellular bioactive proteins. Small peptides produced after gastrointestinal digestion of exogenous protein molecules may have stronger biological effects, but the role of lactic acid bacteria-derived bioactive peptides in promoting alcohol metabolism and alleviating alcoholic liver damage has not been reported. This invention, based on previous screening of two bioactive peptides that increase ADH from enzymatic hydrolysates of ethanol-induced differentially expressed lactic acid bacteria proteins, explores their applications in alcohol metabolism. Summary of the Invention

[0004] One objective of this invention is to provide two novel active hangover-relieving peptides, YRPI and YRPG, with amino acid sequences Tyr-Arg-Pro-Ile and Tyr-Arg-Pro-Gly, respectively.

[0005] Another object of the present invention is to further provide the application of the tetrapeptide for hangover relief;

[0006] Accordingly, the present invention also provides compositions containing the tetrapeptide, such as hangover relief products, which may be drugs, health products or food for relieving hangovers.

[0007] Furthermore, any of the aforementioned methods for relieving hangovers, including the prevention and treatment of acute and chronic alcohol poisoning and / or alcoholic liver damage, can produce at least one of the following effects:

[0008] (1) It can accelerate the metabolism of alcohol in the body;

[0009] (2) It can shorten the residence time of ethanol and harmful metabolites in the body;

[0010] (3) Increase the activity of ADH and ALDH enzymes in the liver;

[0011] (4) Increase the levels of T-AOC, GSH and / or CAT in the blood, and decrease the level of MDA in the blood;

[0012] (5) Increase the levels of T-AOC, GSH and / or CAT in the liver and decrease the level of MDA in the liver;

[0013] (6) Reduce the levels of AST and / or ALT enzymes in the blood;

[0014] Preferably, the composition is a hangover remedy, which may be in the form of granules, capsules, or oral liquid.

[0015] Furthermore, the aforementioned hangover remedies also contain pharmaceutical excipients known to those skilled in the art.

[0016] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0017] The tetrapeptide of this invention has a small molecular weight, is easy to synthesize, and is safe and free of toxic side effects, exhibiting good safety and practicality. Compared with existing hangover remedies and other drugs, the raw materials are simple and easy to prepare, and the tetrapeptide alone can achieve a good hangover relief effect, making it easier to apply in pharmaceuticals, food, and health products, and more readily applicable for widespread use in hangover relief. Attached Figure Description

[0018] Figure 1 This is a graph showing the activation rate of ADH by the tetrapeptide described in this invention.

[0019] Figure 2 This is a graph showing the effect of the tetrapeptide described in this invention on the concentration of ethanol in mice.

[0020] Figure 3 The results of the tetrapeptide described in this invention on ethanol-metabolizing enzymes in mouse liver are shown in the figure (A: ADH activity in liver, B: ALDH activity in liver);

[0021] Figure 4 The results of the tetrapeptide described in this invention on antioxidant enzymes in mouse serum are shown in the figure (A: T-AOC content in mouse serum, B: GSH content in mouse serum, C: CAT content in mouse serum, D: MDA content in mouse serum).

[0022] Figure 5The diagram shows the results of the tetrapeptide described in this invention on antioxidant enzymes in mouse liver (A: T-AOC content in liver, B: GSH content in liver, C: CAT content in liver, D: MDA content in liver).

[0023] Figure 6 The effect of the tetrapeptide described in this invention on mouse liver injury marker enzymes (A: AST activity in mouse serum, B: ALT activity in mouse serum); Detailed Implementation

[0024] The tetrapeptides Tyr-Arg-Pro-Gly (SEQ ID NO.1) and Tyr-Arg-Pro-Ile (SEQ ID NO.2) of this invention were chemically synthesized using a solid-phase method. The tetrapeptides were derived from differentially expressed lactic acid bacteria proteins induced by virtual enzymatic hydrolysis of ethanol in the previous stage. The technical solutions of this invention will be described more clearly and completely below through specific embodiments to facilitate the understanding of this invention by those skilled in the art, but this is not intended to limit the scope of protection of this invention.

[0025] Example 1

[0026] The effect of tetrapeptides (YRPI and YRPG) on ADH activity was determined in vitro. The method was as follows: Prepare two test tubes, and add 150 μL of buffer (including 75 μL of 0.1 mM sodium pyrophosphate buffer at pH 8.8, 25 μL of 11.5% (v / v) ethanol, and 50 μL of 27 mM NAD) to each tube. + Add YRPI and YRPG solutions (50 μL, 6 mmol / L) to each test tube, then incubate at 37°C for 5 min. Immediately after incubation, add 50 μL of 0.2 U / mL ADH enzyme solution to each test tube and mix thoroughly. Then, read the absorbance change of each solution at 340 nm wavelength, taking readings every 1 min for 5 consecutive minutes. Use the absorbance of the tube with added distilled water as the blank group. Calculate the relative enzyme activity. The results are shown below. Figure 1 As shown.

[0027] Figure 1 The results showed that the tetrapeptides YRPI and YRPG could increase the activation rate of ADH in vitro.

[0028] Example 2

[0029] This embodiment further demonstrates the effect of the tetrapeptide described in this invention on alcohol metabolism in mice through animal experiments. The specific experiments are as follows:

[0030] (1) Constructing an animal experimental mouse model

[0031] Eighty Kunming mice (20-22g) were acclimatized in the animal room for 7 days and then randomly divided into 5 groups: blank control group (Control), model group (Model), positive control group (Positive), YRPI group and YRPG group.

[0032] Control group: Mice were given physiological saline (30 mg / kg) by gavage daily according to their body weight, followed by a single gavage administration of 12 ml / kg of physiological saline.

[0033] Model group: Mice were given physiological saline (30 mg / kg) by gavage daily according to their body weight, followed by a single gavage administration of 12 ml / kg of 56-degree liquor.

[0034] Positive group: Mice were administered biphenyl diester emulsion (150 mg / kg) by gavage daily according to their body weight. After gavage administration of biphenyl diester emulsion, 12 ml / kg of 56-degree liquor was administered by gavage once.

[0035] YRPI group: mice were given tetrapeptide solution (30mg / Kg) by gavage daily according to their body weight. After gavage administration of YRPI active peptide, mice were given 12ml / Kg of 56-degree liquor by gavage once.

[0036] YRPG group: Mice were given tetrapeptide solution (30mg / Kg) by gavage daily according to their body weight. After gavage administration of YRPG active peptide, mice were given 12ml / Kg of 56-degree liquor by gavage once.

[0037] In the experimental group, blood was collected from the eyeballs 2 hours after gavage administration of baijiu (the blank group received physiological saline) on the first day. The blood was mixed with sodium citrate anticoagulant, incubated at 4°C, and then stored. The ethanol concentration in the blood was detected using gas chromatography with internal standard method. The results are as follows: Figure 2 As shown, alcohol intake caused a sharp increase in ethanol concentration in the model group mice to 4.69 mg / mL within 2 hours. Compared with the model group, gavage treatment with 30 mg / kg YRPI significantly reduced the ethanol content to 2.51 mg / mL, with a clearance rate of 46%; gavage treatment with 30 mg / kg YRPG significantly reduced the ethanol content to 2.32 mg / mL, with a clearance rate of 51%. These two tetrapeptides directly reduced the toxic effects of alcohol on mice.

[0038] Then, the gavage treatment was continued for 7 consecutive days using the above method. Two hours after the last gavage, serum and liver tissue were collected from the mice: blood samples were centrifuged at 4000 rpm and 4°C for 10 minutes to prepare serum; liver was prepared into a 10% liver homogenate using physiological saline, and then the homogenate sample was centrifuged at 3500 rpm and 4°C for 10 minutes, and the supernatant of the liver homogenate was collected. The serum and liver collected over 7 days of gavage were subjected to the following tests:

[0039] (2) Determine the activity of ethanol-metabolizing enzymes in mouse serum and liver.

[0040] The activities of ADH and ALDH in the liver were detected using a kit (a method known to those skilled in the art), and the results are as follows: Figure 3 As shown, after mice ingested ethanol continuously for 7 days, the activities of ADH and ALDH in the liver of the model group were significantly reduced.

[0041] The test results demonstrated that continuous gavage administration of the YRPI alcohol-metabolizing peptide described in this invention for 7 days significantly increased the activity of hepatic ADH (p < 0.001) and ALDH (p < 0.05) enzymes, thereby improving the alcohol metabolism capacity of mice; YRPG alcohol-metabolizing peptide also significantly increased the activity of hepatic ADH (p < 0.001) and ALDH (p < 0.05) enzymes. YRPI and TRPG active peptides accelerate ethanol metabolism and reduce the damage to the liver caused by ethanol metabolites and ROS by enhancing the activity of ethanol-metabolizing enzymes.

[0042] (3) Determine the activity of antioxidant enzymes and the level of propylene glycol (MDA) in mouse serum.

[0043] Based on the collected mouse serum, the levels of total antioxidant (T-AOC), glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) in mouse serum were measured using a kit. Results Figure 4 As shown; Figure 4 The results showed that after 7 days of continuous gavage, YRPI active peptides significantly increased the levels of T-AOC (p<0.05), GSH (p<0.05), and CAT (p<0.05) antioxidant enzymes in mouse serum and significantly reduced the level of MDA (p<0.05) in serum. Figure 4 The results also showed that continuous gavage administration of YRPG peptides for 7 days significantly enhanced the levels of T-AOC (p<0.05) and GSH (p<0.05) in the liver of mice, reduced the content of MDA (p<0.05) in the liver, and reduced the oxidative stress damage caused by continuous heavy drinking in mice.

[0044] (4) Determine the activity of antioxidant enzymes and MDA levels in mouse liver.

[0045] Based on the collected mouse livers, the levels of total antioxidant (T-AOC), glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) in the mouse liver supernatant were measured using a kit. Results Figure 5 As shown; Figure 5The results showed that after 7 days of continuous gavage, YRPI active peptides significantly enhanced the levels of T-AOC (p<0.001), GSH (p<0.01), and CAT (p<0.01) antioxidant enzymes in the liver of mice, and significantly reduced the level of MDA (p<0.01) in the serum. Figure 5 The results also showed that continuous gavage administration of YRPG peptides for 7 days significantly enhanced the activities of T-AOC (p<0.01), GSH (p<0.001) and CAT (p<0.05) enzymes in mouse liver, reduced the content of MDA (p<0.01) in liver, and reduced the oxidative stress damage to mouse liver caused by continuous heavy drinking.

[0046] (5) Determine the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse serum.

[0047] Based on the collected mouse serum, the activities of AST and ALT in the mouse serum were measured using a kit (a method known to those skilled in the art). The results are as follows: Figure 6 As shown. By Figure 6 The results showed that, compared with the model group, continuous gavage administration of tetrapeptide YRPI for 7 days significantly reduced serum ALT levels (p < 0.05); continuous gavage administration of tetrapeptide YRPG for 7 days significantly reduced serum AST (p < 0.05) and ALT (p < 0.05). Therefore, overall, YRPI and YRPG have a protective effect against alcohol-induced hepatocellular damage.

Claims

1. The application of the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 in the preparation of hangover relief compositions.

2. The application as described in claim 1, characterized in that, This hangover remedy is used to prevent and treat acute and chronic drunkenness and / or alcoholic liver damage.

3. The application as described in claim 1, characterized in that, The hangover relief composition is used to produce at least one of the following effects: 1) Accelerates the metabolism of alcohol in the body; 3) Reduce the levels of malondialdehyde, alanine aminotransferase (ALT), and / or aspartate aminotransferase (AST) in the blood; 4) Reduces malondialdehyde levels in the liver; 5) Increase the levels of T-AOC, GSH, and / or CAT in the blood; 6) Increase the levels of T-AOC, GSH, and / or CAT in the liver; 7) Increase the activity of ADH and / or ALDH enzymes in the liver.

4. The application as described in claim 1, characterized in that, The hangover relief composition is a medicine used to relieve hangovers.

5. A hangover remedy composition containing the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. The hangover relief composition as described in claim 5, characterized in that, The hangover remedy composition is a medicine.

7. The polypeptide shown in SEQ ID NO. 2.

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