A liver-protecting composition for alleviating liver damage caused by staying up late

Abstract

This invention discloses a liver-protective composition for alleviating liver damage caused by staying up late, relating to the field of liver peptide composition technology. It includes a liver peptide with the amino acid sequence LLVW and a liver peptide with the amino acid sequence LLVF. When the liver peptides LLVW and LLVF with specific amino acid sequences are combined in a specific ratio, they can synergistically alleviate liver damage caused by staying up late, thereby achieving a liver-protective effect.

Description

Technical Field

[0001] This invention relates to the field of liver peptide composition technology, and more specifically to a liver-protecting composition that alleviates liver damage caused by staying up late. Background Technology

[0002] As a core metabolic and detoxification organ in the human body, the liver undertakes key physiological functions such as toxin removal, substance synthesis, and energy metabolism. The integrity of its function directly affects the body's health. With the accelerated pace of modern life, staying up late has become a common habit. Long-term or frequent late nights can cause continuous and multi-dimensional damage to the liver.

[0003] From a physiological perspective, the core pathways through which staying up late causes liver damage include three aspects: First, disruption of the biological clock rhythm. Staying up late disrupts the body's diurnal rhythm balance, leading to abnormal expression of core biological clock genes such as Clock, Bmal1, and Per2 in liver tissue. This, in turn, regulates the rhythmic synthesis and activity of downstream metabolic enzymes and antioxidant enzymes, disrupting the normal metabolic sequence of the liver. Second, oxidative stress overload. During the state of staying up late, the sympathetic nervous system remains excited, promoting the excessive generation of reactive oxygen species (ROS). At the same time, the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver decreases, while the consumption of non-enzymatic antioxidants such as reduced glutathione (GSH) increases, leading to lipid overload. Oxidation products such as malondialdehyde (MDA) and DNA oxidative damage markers like 8-hydroxydeoxyguanosine (8-OHdG) accumulate in hepatocytes, directly attacking the hepatocyte membrane, mitochondria, and genetic material, leading to hepatocyte damage. Thirdly, detoxification function declines. Staying up late significantly inhibits the normal activity of hepatic cytochrome P450 enzyme systems (such as CYP1A2 and CYP3A4), while simultaneously reducing the expression of detoxification-binding enzymes such as glutathione S-transferase (GST) and uridine diphosphate glucuronide transferase (UGT). This results in reduced metabolic efficiency of endogenous toxins (such as bilirubin and lactate) and exogenous toxins, further exacerbating hepatocyte damage and creating a vicious cycle of damage-metabolic disorder-further damage. Furthermore, staying up late activates the NF-κB inflammatory pathway, promoting the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), inducing hepatocyte apoptosis, inhibiting hepatocyte regeneration, and accelerating the liver damage process.

[0004] For liver damage caused by staying up late, liver protection products on the market can be mainly divided into three categories: First, chemically synthesized drugs, such as thioproline and silymarin compounds, can improve liver cell damage to a certain extent, but they often have side effects (such as gastrointestinal discomfort and allergic reactions), and their mechanisms of action are singular, focusing on a single link of antioxidation or detoxification. They are difficult to address the multi-mechanism synergistic damage characteristics of liver damage caused by staying up late, including circadian rhythm disorder, oxidative stress, and inflammation activation, and their long-term safety is limited. Second, traditional Chinese medicine and natural product extracts, such as wolfberry extract, salvia miltiorrhiza extract, and artemisia capillaris preparations, have complex components, unstable content of effective active ingredients, and unclear mechanisms of action. They also have the problems of slow onset of action and poor targeting, and cannot quickly relieve acute liver damage after staying up late. Third, ordinary health products, such as vitamin C, vitamin E, and glutathione supplements, are mostly single nutrient supplements that can only help enhance antioxidant capacity. They lack the regulatory effect on the core damage pathways such as circadian rhythm disorder and abnormal detoxification enzyme function caused by staying up late, and their liver protection effect is limited.

[0005] Peptides have gained increasing attention in the field of liver protection due to their advantages such as small molecular weight, high bioavailability, few side effects, and well-defined mechanisms of action. Compared to proteins, peptides can be directly absorbed and utilized by the body, and can precisely target specific sites to regulate related physiological processes. However, most currently available liver-protecting peptide products target chemical or immune-mediated liver damage, and their mechanisms of action are mainly designed around a single antioxidant or anti-inflammatory pathway. They do not specifically address the multi-mechanism synergistic damage caused by sleep deprivation, which involves "circadian rhythm disruption, oxidative stress, and decreased detoxification," thus failing to provide precise and comprehensive relief for liver damage caused by sleep deprivation. Furthermore, existing peptide-based liver-protecting products are mostly single-peptide components, lacking synergistic formulations of different functional peptides, making it difficult to fully leverage the advantages of "multi-target synergistic regulation," resulting in poor liver-protecting effects.

[0006] Therefore, developing a liver-protecting composition that can precisely improve liver damage caused by staying up late has become an urgent need in the research and development of liver-protecting products. Summary of the Invention

[0007] In view of this, the present invention provides a liver-protective composition for alleviating liver damage caused by staying up late. Peptide molecules have significant advantages in the field of liver protection due to their small molecular weight, good biocompatibility, easy absorption and utilization by the human body, and low side effects. When liver peptides LLVW and LLVF with specific amino acid sequences are combined in a specific ratio, they can alleviate liver damage caused by staying up late through a synergistic effect.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a liver-protecting composition for alleviating liver damage caused by staying up late, comprising a liver peptide with the amino acid sequence LLVW and a liver peptide with the amino acid sequence LLVF.

[0009] Furthermore, the mass ratio of the LLVW to the LLVF is 1:(2~4).

[0010] Furthermore, the hepateptide composition further includes one or more pharmaceutically acceptable excipients.

[0011] Furthermore, the composition may be delivered to the human body via oral, injection, mucosal, transdermal, or topical administration.

[0012] Furthermore, the dosage form of the composition is an oral dosage form, an injectable dosage form, a topical dosage form, or a mucosal dosage form.

[0013] Secondly, the present invention provides the application of the hepatic peptide composition in the preparation of products that alleviate liver damage caused by staying up late.

[0014] Furthermore, the products include food or pharmaceuticals.

[0015] Thirdly, the present invention provides a drug for relieving liver damage caused by staying up late, comprising the aforementioned liver peptide composition.

[0016] Fourthly, the present invention provides a food for alleviating liver damage caused by staying up late, comprising the aforementioned liver peptide composition.

[0017] As can be seen from the above technical solution, this invention independently purifies two novel liver peptides with amino acid sequences LLVW and LLVF from natural biological raw materials. After purification, the purity reaches over 97%, exhibiting both high activity and good biocompatibility, laying a solid foundation for subsequent effects. Furthermore, the study unexpectedly discovered that combining the two liver peptides in a mass ratio of 1:(2~4) does not result in a simple additive effect of individual peptides, but rather produces a significant synergistic effect, significantly alleviating liver damage caused by staying up late. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is the mass spectrum of the liver peptide LLVW obtained by protease digestion.

[0020] Figure 2 This is the mass spectrum of the liver peptide LLVF obtained by protease digestion. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 The amino acid sequences LLVW (Leu-Leu-Val-Trp) and LLVF (Leu-Leu-Val-Phe) in this invention are prepared as follows: I. Raw material pretreatment 1. Select healthy pig liver (which has passed quarantine, is free from disease and drug residues), remove the fascia, blood vessels and connective tissue, rinse 3 times with 4℃ pre-cooled physiological saline, drain the surface water, and cut into small pieces of about 1cm³. 2. Add liver tissue to pre-cooled physiological saline at a ratio of 1:2 (mass-volume ratio, g / mL) into a high-speed tissue homogenizer and homogenize for 2 minutes under ice bath conditions to prepare a uniform liver tissue homogenate. 3. Add EDTA (ethylenediaminetetraacetic acid) to the homogenate at a final concentration of 0.05 mol / L and PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 0.1 mmol / L, stir well, and let stand at 4°C for 30 min to inhibit protease activity and prevent liver peptide degradation.

[0023] II. Liver peptide extraction 1. Transfer the pretreated liver homogenate to a centrifuge tube, centrifuge at 4℃ and 8000r / min for 20min, collect the supernatant (crude extract), and discard the precipitate (tissue fragments, large molecular proteins, etc.). 2. Slowly add ammonium sulfate to the supernatant until the saturation is 60%, stir at 4°C for 3 hours to allow the impurities and proteins to precipitate fully; 3. Continue centrifuging at 4℃ and 10000r / min for 25min, collect the supernatant (containing the target hepatic peptide), and discard the impurity protein precipitate; 4. Use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter the supernatant at 4°C and 0.3 MPa pressure. Collect the filtrate (to remove impurities and polymers with a molecular weight greater than 10 kDa) and retain the filtrate containing small peptides.

[0024] III. Preliminary purification (gel filtration chromatography) 1. Sephadex G-15 gel chromatography was used. The column (2.6 cm × 100 cm) was equilibrated with 0.02 mol / L, pH 7.0 phosphate-buffered saline (PBS) at a volume of 3 times the column volume. 2. Load the ultrafiltration filtrate into the chromatography column, with a loading volume of 5% of the column volume; 3. Elute with the same PBS buffer at a flow rate of 1 mL / min. Monitor the elution peak at 220 nm using a UV detector and collect the eluent in the corresponding molecular weight range (300-500 Da) (the collection range is preset according to the molecular weight of LLVW and LLVF). 4. Combine the collected eluents and freeze-dry them initially using a freeze dryer to obtain crude liver peptide powder.

[0025] IV. Purification (Reversed-phase high-performance liquid chromatography, RP-HPLC) 1. Dissolve the crude hepatin powder in 0.1% trifluoroacetic acid aqueous solution to prepare a sample solution of 10 mg / mL, and filter it through a 0.22 μm microporous membrane; 2. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a column temperature of 30 °C; mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile; 3. Gradient elution program: 0-20 min, the volume fraction of mobile phase B increases from 10% to 30%; 20-35 min, the volume fraction of mobile phase B increases from 30% to 45%; 35-40 min, the volume fraction of mobile phase B returns to 10% to equilibrate the column. 4. Elution flow rate 1.0 mL / min, detection at 220 nm wavelength using a UV detector, and collection of the target elution peaks for LLVW and LLVF according to retention time (retention time determined by standard reference: LLVW approximately 29.7 min, LLVF approximately 25.3 min). 5. The collected target eluents were freeze-dried to obtain high-purity liver peptide powder.

[0026] V. Purity Identification and Sequence Confirmation 1. Purity detection: The purity of both LLVW and LLVF was ≥96% by repeated HPLC analysis, with no obvious impurity peaks. 2. Sequence confirmation: The molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the amino acid sequence was determined by Edman degradation method. The obtained products were confirmed to be LLVW (molecular weight 516.6 Da) and LLVF (molecular weight 472.6 Da), which were completely consistent with the preset sequence. 3. Impurity detection: The residue of heavy metals (lead, mercury, cadmium) is ≤0.1mg / kg, the microbial limit meets the requirements of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), and there are no toxic or harmful substances residues.

[0027] Example 2 A composition of LLVW and LLVF in a mass ratio of 1:2: LLVW liver peptide 2g, LLVF liver peptide 4g.

[0028] Example 3 A composition of LLVW and LLVF in a mass ratio of 1:3 LLVW liver peptide: 1.5g, LLVF liver peptide: 4.5g.

[0029] Example 4 A composition of LLVW and LLVF in a mass ratio of 1:4 LLVW liver peptide: 1.2g, LLVF liver peptide: 4.8g.

[0030] Example 5 A composition of LLVW and LLVF in a mass ratio of 1:2 (oral tablets) Formula composition (dosage for 1000 tablets) LLVW liver peptide 2g, LLVF liver peptide 4g.

[0031] Pharmaceutically acceptable excipients: lactose 20g, microcrystalline cellulose 15g, sodium carboxymethyl starch 2g, magnesium stearate 0.5g.

[0032] Preparation steps: LLVW liver peptide, LLVF liver peptide, lactose, and microcrystalline cellulose are mixed evenly, and a 5% (whole mass) aqueous solution of povidone K30 is added as a binder to prepare a soft material. Granulation is performed by passing the wet granules through a 20-mesh sieve, drying the wet granules in a 60℃ oven for 2 hours, and then granulating the dried granules by passing them through an 18-mesh sieve. Sodium carboxymethyl starch and magnesium stearate are added, mixed evenly, and then compressed into tablets to make oral tablets containing 5 mg of LLVW and 5 mg of LLVF per tablet.

[0033] Example 6 A composition of LLVW and LLVF in a mass ratio of 1:3 (lyophilized powder for injection). Formula composition (for 1000 vials): LLVW liver peptide: 1.5g LLVF Liver Peptide: 4.5g Pharmaceutically acceptable excipients: 18g mannitol, appropriate amount of sodium bicarbonate for injection (adjust pH to 7.0).

[0034] Preparation steps: Take 800 mL of water for injection, add mannitol and stir to dissolve, then add LLVW liver peptide and LLVF liver peptide and stir until completely dissolved; Adjust the pH of the solution to 6.5 with sodium bicarbonate for injection, add water for injection to 1000 mL, and filter with a 0.22 μm microporous membrane for sterilization; The filtrate was dispensed into 10 mL vials, 1 mL per vial, and placed in a freeze dryer: pre-frozen to -40℃ and maintained for 2 hours, then sublimated (vacuum 10 Pa, temperature gradient -20℃→0℃→25℃, total time 12 hours), stoppered and capped to prepare lyophilized powder for injection.

[0035] Performance testing Animal selection: 100 SPF-grade SD rats, half male and half female, aged 6-8 weeks and weighing 200±20g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Adaptation feeding: One week before the experiment, 84 rats were placed in an SPF-grade animal room to adapt to the environment. The feeding conditions were: temperature 22±2℃, relative humidity 55±5%, and a basic light cycle of 12h light (06:00-18:00) / 12h darkness (18:00-06:00). The rats were allowed free access to standard rat feed and sterile drinking water. The rats' mental state, diet, water intake and defecation were observed daily. After ensuring that there were no abnormalities, the modeling process began.

[0036] An internationally recognized chronic incomplete sleep deprivation device was used to avoid interference from physical stress in the experiment. Device parameters: Customized acrylic sleep deprivation box (50cm×50cm×40cm), with 12 circular platforms, each 10cm in diameter and 8cm in height, placed inside the box. The platforms are spaced 5cm apart. The box is filled with clean water, with the water level 2cm below the surface of the platforms (to ensure that the rats will not drown when standing on the platforms, but if their muscles relax after falling asleep, they will fall into the water due to instability. After waking up, they will climb back onto the platforms on their own, thus achieving fragmented sleep deprivation). Deprivation period and duration: The modeling period lasted for 2 weeks. The daily sleep deprivation time was from 23:00 to 07:00 the next day (corresponding to the peak time for humans to stay up late, with 8 hours of sleep deprivation). During the rest of the time (07:00-23:00), the rats were moved to normal breeding cages and given free access to food and water and normal light to avoid excessive stress. Blank control group treatment: The rats in the blank control group were housed in the same size "sham deprivation box" (the platform height in the box was 15cm, the water level was 8cm from the platform surface, and the rats could sleep freely on the platform without sleep deprivation pressure). All other housing conditions and light cycles were completely consistent with the model group to exclude environmental factors.

[0037] General condition monitoring: Record the weight, food intake, and water intake of each group of rats daily, observe their mental state (such as whether they are lethargic or have reduced activity), coat luster, and defecation. If a sudden drop in weight (more than 10% of the initial weight) or obvious disease symptoms occur, remove the rats in time. Stress level control: Change the water in the deprivation chamber once a week during the modeling period to keep the water clean; polish the platform surface to avoid scratching the rat's feet; after sleep deprivation each day, gently wipe the rat's body surface dry with a dry towel to prevent it from getting cold; Model stability verification (day 21 after modeling): After modeling, three rats in the model group were randomly selected, sacrificed, and samples were collected to verify the effectiveness of the model.

[0038] After successful modeling, the rats were divided into 7 groups, and 12 rats were selected as a blank control group. The experimental grouping and administration methods are as follows: Blank control group: normal saline (oral 20ml / kg·d) -1 ); Model control group: physiological saline (oral administration 20 mg / kg / day) -1 ); Positive control group: Oral administration of thiopronine tablets 20 mg / kg·d -1 ; Experimental group 1: Oral administration via gavage. Example 2: Raw material drug 20 mg / kg·d -1 ; Experimental Group 2: Oral administration via gavage. Example 5: Drug 20 mg / kg / day -1 ; Experimental Group 3: Oral administration via gavage. Example 3 drug 20 mg / kg·d -1 ; Experimental group 4: Intravenous injection of drug 15 mg / kg·d from Example 6 -1 ; Experimental group 5: Oral administration via gavage 20 mg / kg / day of the drug used in Example 4 -1 ; Dosage regimen: Start administering medication on day 7 of model establishment and continue for 2 weeks.

[0039] Sample collection and indicator detection Serum liver function indicators: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (ELISA detection kit); Liver detoxification enzyme activity: glutathione S-transferase (GST, a key detoxification enzyme) in the liver (ELISA kit).

[0040] The results are shown in Table 1.

[0041] Table 1. Results of in vivo experiments (SD rats) on liver detoxification-related indicators (x±s, n=12) As shown in Table 1, the liver peptide compositions in Examples 2-6 all have the effect of improving liver protection and alleviating liver damage, and the effect is significant. It is speculated that they can alleviate liver damage caused by staying up late.

[0042] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A liver-protecting composition for alleviating liver damage caused by staying up late, characterized by comprising, This includes liver peptides with the amino acid sequence LLVW and LLVF.

2. The liver-protective composition for alleviating liver damage caused by staying up late according to claim 1, characterized in that, The mass ratio of the LLVW to the LLVF is 1:(2~4).

3. The liver-protective composition for alleviating liver damage caused by staying up late according to claim 1, characterized in that, The hepateptide composition also includes one or more pharmaceutically acceptable excipients.

4. The liver-protective composition for alleviating liver damage caused by staying up late according to claim 3, characterized in that, The composition is intended for delivery to the human body via oral, injectable, mucosal, transdermal, or topical administration.

5. The liver-protective composition for alleviating liver damage caused by staying up late according to claim 4, characterized in that, The composition is available in oral, injectable, topical, or mucosal formulations.

6. The use of the liver peptide composition according to any one of claims 1-5 in the preparation of a product that alleviates liver damage caused by staying up late.

7. The application according to claim 6, characterized in that, The products include food or medicine.

8. A drug for relieving liver damage caused by staying up late, characterized in that, The liver peptide composition includes any one of claims 1-5.

9. A food product for alleviating liver damage caused by staying up late, characterized in that, The liver peptide composition includes any one of claims 1-5.

Images