Sturgeon roe peptide for preventing alcoholic liver injury as well as preparation method and application of sturgeon roe peptide

Abstract

The invention discloses sturgeon roe peptide for preventing alcoholic liver injury as well as a preparation method and application of the sturgeon roe peptide. The peptide sequence is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL. The preparation method specifically comprises the following steps: (1) adding deionized water into the sturgeon roes, and homogenizing; (2) adjusting the pH value, adding alkaline protease, carrying out enzymolysis, carrying out enzyme inactivation, and cooling; and (3) performing centrifugal treatment, and taking supernate to obtain the traditional Chinese medicine composition. The sturgeon roe peptide disclosed by the invention has the efficacy of preventing or treating alcoholic liver injury, and is remarkable in hangover alleviating effect, simple to prepare and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and more particularly to a sturgeon roe peptide for preventing alcoholic liver damage, and a preparation method and application thereof. Background Art

[0002] Alcoholic Liver Disease (ALD) is a disease caused by long-term excessive drinking. During alcohol metabolism, ethanol is oxidized to acetaldehyde, which causes protein deformation and DNA damage, thereby inducing oxidative stress. The pathogenesis of alcoholic liver damage involves a variety of biological processes including oxidative stress, inflammatory response, lipid metabolism disorders, and hepatocyte apoptosis. Therefore, finding natural antioxidants that can reduce or prevent alcoholic liver damage has become one of the important directions of current liver disease research.

[0003] Sturgeon is an ancient bony fish, known as a "living fossil" because of its long history. According to the 2023 China Fisheries Statistical Yearbook, the output of China's sturgeon farming industry reached 130,951 tons in 2022, showing China's significant growth and potential in the field of sturgeon farming. Sturgeon products mainly include two categories: caviar and fish meat. Sturgeon is not only rich in nutritional value, but also has tender meat and no intramuscular spines, which makes it an ideal choice for cooking and eating. In recent years, many scholars have begun to pay attention to the bioactive components in sturgeon by-products, especially antioxidant peptides, antimicrobial peptides and chondroitin sulfate, and explored their application potential in different fields. For example, studies have found that the active peptides of sturgeon skin can significantly increase the activity of the antioxidant enzyme system in pork tissue, confirming that the active peptides of sturgeon skin have significant antioxidant effects. Studies have also shown that sturgeon head bone peptides have good antioxidant activity, and their scavenging rate in scavenging hydroxyl radicals and superoxide anions can reach 70%-95% at 5mg / mL. Other studies have further confirmed that the ability of sturgeon fish intestinal antioxidant peptides prepared by pepsin to scavenge DPPH at a concentration of 1.5 mg / mL is equivalent to 83.64% of vitamin C (Vc), and the scavenging rate of hydroxyl free radicals is as high as 78.06%. In addition, some scholars have successfully extracted extracts rich in antioxidant ingredients from sturgeon muscles and fish roe. These extracts not only showed a strong free radical scavenging effect in in vitro experiments, but also showed excellent activity in cell and mouse models, and the effects of these extracts were comparable to those of vitamin C and ginseng extracts.

[0004] In the production and processing of sturgeon roe sauce, strict screening and grading are usually carried out to ensure the quality and appearance of the final product. In this process, some broken and sticky roe will inevitably appear, which are often regarded as waste and discarded. But in fact, they are also rich in nutrients, including protein, unsaturated fatty acids, essential amino acids, etc. At present, the research on the role of sturgeon roe in preventing alcoholic liver damage is not sufficient, especially in terms of its sobering effect and its potential protective mechanism.

[0005] Therefore, how to develop a sturgeon roe product that can prevent alcoholic liver damage and sober up is a problem that technicians in this field urgently need to solve. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a sturgeon roe peptide for preventing alcoholic liver damage and a preparation method and application thereof, so as to solve the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A sturgeon roe peptide for preventing alcoholic liver damage, wherein the peptide sequence is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

[0009] A method for preparing the above-mentioned sturgeon roe peptide for preventing alcoholic liver damage, wherein the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL, specifically comprises the following steps:

[0010] (1) adding deionized water to sturgeon roe and homogenizing to obtain solution A;

[0011] (2) adjusting the pH value of solution A, adding alkaline protease, performing enzymolysis, deactivating the enzyme, and cooling to obtain solution B;

[0012] (3) Solution B is centrifuged and the supernatant is taken to obtain the sturgeon roe peptide for preventing alcoholic liver damage.

[0013] Furthermore, in the above step (1), the mass ratio of sturgeon roe to deionized water is 1:6.

[0014] Furthermore, in the above step (1), the homogenization speed is 8000 rpm and the time is 1 min.

[0015] Furthermore, in the above step (2), the amount of alkaline protease added is 1%.

[0016] Furthermore, in the above step (2), the pH value of the enzymatic hydrolysis is 8.0, the temperature is 55° C., and the time is 8 h.

[0017] Furthermore, in the above step (2), the enzyme is inactivated at a temperature of 90-100°C for 10-15 minutes; and cooled to room temperature.

[0018] Furthermore, in the above step (3), the centrifugal treatment temperature is 4°C, the rotation speed is 8000 rpm, and the time is 15 min.

[0019] The present invention also claims to protect the use of the above-mentioned sturgeon roe peptide or the sturgeon roe peptide prepared by the above-mentioned preparation method in the preparation of a drug for preventing or treating alcoholic liver injury, and the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

[0020] The present invention also claims to protect the use of the above-mentioned sturgeon roe peptide or the sturgeon roe peptide prepared by the above-mentioned preparation method in the preparation of hangover-relieving drugs or hangover-relieving foods, and the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

[0021] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The present invention adopts appropriate enzymolysis technology to extract sturgeon roe enzymatic hydrolysate (EHSR) from broken fish roe, and compares it with commercially available corn peptide for alcohol relief (Corn Oligopeptide, COP), evaluates the in vitro antioxidant capacity of the two in multiple biochemical indexes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and apoptosis level, improves the comprehensive utilization rate and economic value of sturgeon roe, and provides a scientific basis for developing novel natural antioxidants and liver protection drugs.

[0023] 2. The sturgeon roe peptide of the present invention has the effect of preventing or treating alcoholic liver damage, has a significant effect of sobering up, is simple to prepare and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of different alcohol concentrations on the viability of HepG2 cells;

[0025] Figure 2 The effects of sturgeon roe hydrolysate and corn peptide on the survival rate of HepG2 cells;

[0026] Figure 3 To study the effects of sturgeon roe hydrolysate and corn peptide on the survival rate of HepG2 cells damaged by alcohol;

[0027] Figure 4 To study the effects of sturgeon roe hydrolysate and corn peptide on AST, ALT and AST / ALT in HepG2 cells damaged by alcohol;

[0028] Figure 5 To study the effects of sturgeon roe hydrolysate and corn peptide on the content of MDA, SOD and T-AOC in HepG2 cells damaged by alcohol;

[0029] Figure 6 To study the effects of sturgeon roe hydrolysate and corn peptide on alcohol-induced cell apoptosis;

[0030] Figure 7 It is a bubble diagram (Note: the circle area represents the relative peak area). DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] The preparation method of sturgeon roe peptide for preventing alcoholic liver damage, wherein the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL, specifically comprises the following steps:

[0034] (1) Add 60 mL of deionized water to 10 g of sturgeon roe and homogenize at 8000 rpm for 1 min to obtain solution A;

[0035] (2) adjusting the pH value of solution A to 8.0, adding 1% by weight of alkaline protease, placing in a constant temperature oscillator at 55° C. for enzymatic hydrolysis for 8 h, then placing in a boiling water bath at 90° C. for enzyme inactivation for 15 min, and cooling to room temperature to obtain solution B;

[0036] (3) Solution B was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was collected to obtain sturgeon roe peptide for preventing alcoholic liver damage.

[0037] Example 2

[0038] The preparation method of sturgeon roe peptide for preventing alcoholic liver damage, wherein the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL, specifically comprises the following steps:

[0039] (1) Add 60 mL of deionized water to 10 g of sturgeon roe and homogenize at 8000 rpm for 1 min to obtain solution A;

[0040] (2) adjusting the pH value of solution A to 8.0, adding 1% by weight of alkaline protease, placing in a constant temperature oscillator at 55° C. for enzymatic hydrolysis for 8 h, then placing in a boiling water bath at 95° C. for enzyme inactivation for 12 min, and cooling to room temperature to obtain solution B;

[0041] (3) Solution B was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was collected to obtain sturgeon roe peptide for preventing alcoholic liver damage.

[0042] Example 3

[0043] The preparation method of sturgeon roe peptide for preventing alcoholic liver damage, wherein the peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL, specifically comprises the following steps:

[0044] (1) Add 60 mL of deionized water to 10 g of sturgeon roe and homogenize at 8000 rpm for 1 min to obtain solution A;

[0045] (2) adjusting the pH value of solution A to 8.0, adding 1% by weight of alkaline protease, placing in a constant temperature oscillator at 55° C. for 8 h, then placing in a boiling water bath at 100° C. for 10 min for enzyme inactivation, and cooling to room temperature to obtain solution B;

[0046] (3) Solution B was centrifuged at 4° C. and 8000 rpm for 15 min, and the supernatant was collected to obtain sturgeon roe peptide for preventing alcoholic liver damage.

[0047] Performance Testing

[0048] Study on the protective effect of sturgeon roe enzymatic hydrolysate on alcohol-induced oxidative damage in HepG2 cells

[0049] AbstractThis study aimed to compare the in vitro antioxidant capacity of enzymatic hydrolysate of sturgeon roe (EHSR) and commercially available corn peptide (COP) in terms of alcohol sobering effect. An alcohol-induced HepG2 cell oxidative damage model was established to study the protective effects of EHSR and COP at different protein concentrations (0.2, 0.5, and 1.0 mg / mL) on liver damaged cells. The antioxidant capacity of the two was compared and evaluated by measuring multiple biochemical indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and cell apoptosis levels. The results showed that the cell viability was 51.29% after 800 mM alcohol-induced injury for 24 hours, and the model was successfully established. At the same protein concentration, the cell viability of the EHSR treatment group was higher than that of the COP treatment group. In addition, compared with the COP group, the EHSR group showed more significant advantages in reducing AST, ALT and MDA levels, and enhancing SOD and T-AOC activities. In addition, 7 peptides with potential liver protection effects were obtained from EHSR through peptide identification and virtual peptide screening technology. In summary, EHSR showed a potential that was significantly better than COP in the in vitro hangover effect, providing a scientific basis for its development as a functional hangover product.

[0050] Keywords: sturgeon roe; HepG2; liver injury; antioxidant

[0051] 1 Materials and methods

[0052] 1.1 Experimental Materials

[0053] 1.1.1 Raw materials and reagents:

[0054] Fresh sturgeon roe, Guizhou Qianxun Biotechnology Co., Ltd.; corn oligopeptides, purchased from JD.com; alkaline protease (200U / mg) and total antioxidant capacity (T-AOC) kits, Shanghai Yuanye Biotechnology Co., Ltd.; HepG2 cells, Shanghai Anwei Biotechnology Co., Ltd.; DMEM culture medium, phosphate buffered saline (PBS), trypsin, and double antibody, Gibco, USA; fetal bovine serum (FBS), Hyclone; cell viability assay (CCK-8), superoxide dismutase (SOD), Beyotime Biotechnology Co., Ltd.; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits, Nanjing Jiancheng Bioengineering Institute; malondialdehyde (MDA), Beijing Solebold Technology Co., Ltd.; Annexvin V APC / PI double staining kit, Wuhan Elerite Biotechnology Co., Ltd.

[0055] 1.1.2 Instruments:

[0056] T25 homogenizer, IKA, Germany; constant temperature oscillator SHA-B, Shanghai Lichen Instrument Technology Co., Ltd.; 3110 Series carbon dioxide incubator, Thermo Corporation, USA; InfiniteM 200Pro microplate reader, TECAN, Austria; D2060RNovocyte flow cytometer, Agilent Technologies Co., Ltd.

[0057] 1.2 Experimental methods

[0058] 1.2.1 Preparation of sturgeon roe enzymatic hydrolysate (EHSR)

[0059] Referring to the preparation method of sturgeon roe peptide for preventing alcoholic liver damage in Example 1, the final product, sturgeon roe peptide, is EHSR.

[0060] 1.2.2 HepG2 cell culture

[0061] HepG2 cells were cultured in complete medium prepared with high-glucose DMEM, 10% FBS and 1% double antibody at 37°C and 5% CO. 2 When the cell density reaches about 90%, subculture at a ratio of 1:4. The remaining cells are frozen at -80℃ or in liquid nitrogen.

[0062] 1.2.3 Detection of cell viability using CCK-8 method

[0063] CCK-8 assay is a method commonly used to detect cell proliferation and cytotoxicity. By detecting the absorbance at a wavelength of 450nm, the number of living cells can be reflected. Therefore, CCK-8 can be used to evaluate cell proliferation, cytotoxicity or the cellular effects of drugs.

[0064] 1.2.4 Effects of different alcohol concentrations on HepG2 cell viability

[0065] HepG2 cells in the logarithmic growth phase were diluted to 2×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and 100 μL of cell suspension was added to each well. After inoculation, the microplate was placed in a 5% CO 2Pre-culture in an incubator. After 48 hours, discard the culture medium, add 100 μL of culture medium containing different concentrations of alcohol (0, 100, 200, 400, 500, 600, 800, 1000, 1200, 1500 mmol / L) to each well, and injure for 24 hours. Add 10 μL of CCK-8 solution to each well, and then use an enzyme reader to measure the absorbance at a wavelength of 450 nm to calculate the relative viability of HepG2 cells. Make 6 duplicate wells for each group, and repeat three times. Among them, relative cell viability = OD value of experimental group / OD value of control group × 100%.

[0066] 1.2.5 Effects of EHSR and COP on the survival rate of HepG2 cells

[0067] When HepG2 cells grew to the logarithmic growth phase, 2×10 4 The cells were inoculated into 96-well plates at a density of 100 μL and pre-cultured for 24 h. After the cells were fully attached, the culture medium was discarded and the cells were divided into a control group and a drug-treated group. The drug-treated groups were added with 100 μL of complete culture medium containing EHSR (protein concentration of 0.1, 0.2, 0.5, 1.0 mg / mL) and COP (protein concentration of 0.1, 0.2, 0.5, 1.0 mg / mL), and the control group was given an equal volume of complete culture medium. After continuing to culture for 24 h, the absorbance was measured according to the method in 1.2.4 to calculate the relative cell viability.

[0068] 1.2.6 Effects of EHSR and COP on the survival rate of HepG2 cells damaged by alcohol

[0069] According to the method in 1.2.5, the cells were cultured for 24 hours and divided into control group, model group, positive group and drug administration group. The control group and model group were added with 100 μL complete medium, the positive group was treated with complete medium containing COP with different protein concentrations (0.2, 0.5, 1.0 mg / mL), and the drug administration group was treated with EHSR with different protein concentrations (0.2, 0.5, 1.0 mg / mL). After incubation for 24 hours, the supernatant was discarded. Except for the normal control group, 800 mM ethanol solution was added to the remaining wells to induce damage, and CCK-8 was added 24 hours later to detect cell viability.

[0070] 1.2.7 Effects of EHSR and COP on AST and ALT in HepG2 cells damaged by alcohol

[0071] The cells were plated at 4.5 × 10 5 / mL concentration was inoculated in a 6-well plate, 2mL per well. After culturing the cells for 24 hours until they were fully attached, the old culture medium was removed and the six-well plates were divided into groups: control group, model group, positive group and drug group. The control group and model group were treated with 2mL complete culture medium, the positive group was treated with COP culture medium with a protein concentration of 1.0mg / mL, and the drug group was treated with EHSR culture medium with a protein concentration of 1.0mg / mL. After 24 hours of drug pre-protection, the cells were damaged with 800mM ethanol solution for 24 hours. After the damage was completed, the culture supernatant was aspirated and discarded, and 2mL PBS was added to each well for rinse. The cells were collected in an ice water bath environment for ultrasonic treatment. The parameters were set to 200W power, and each disruption lasted for 4s, with an interval of 30s. The disruption process was repeated 20 times for each group to obtain a fully lysed cell homogenate. The content of AST and ALT in the cells was detected according to the instructions. Six duplicate wells were made in each group, and repeated three times.

[0072] 1.2.8 Effects of EHSR and COP on the contents of MDA, SOD and T-AOC in alcohol-damaged HepG2 cells

[0073] The culture treatment was performed according to the method in 1.2.7. After the injury was completed, the culture supernatant was discarded, and 2 mL of PBS was added to each well for rinsing. Then, the cells were collected according to the instructions of each kit, and the contents of MDA, SOD and T-AOC in the cells were detected respectively. Each group had 6 replicate wells, and the process was repeated three times.

[0074] 1.2.9 HepG2 cell apoptosis detection

[0075] HepG2 cells were collected from the control group, model group, positive group and drug-treated group according to the method in 1.2.7. After adding each reagent, the apoptosis of HepG2 was detected by D2060RNovocyte flow cytometer (Agilent) according to the instructions of AnnexinVAPC / PI double staining kit (elabscience).

[0076] 1.2.10 Identification and virtual screening of bioactive peptide sequences in enzymatic hydrolysis products

[0077] Take an appropriate amount of sample and pass it through a C18 desalting column for desalting. The sample was analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. A total of 3 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 2% of phase B, and increased to 35% at 47 min with a nonlinear gradient, and increased to 100% within 1 min, and maintained for 12 min.

[0078] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200-2000; resolution: 70,000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28%; dynamic exclusion time: 30 s.

[0079] 1.3 Data processing

[0080] All experiments were repeated three times. Statistical significance analysis was performed using SPSS software version 27.0, and the significance level of the results was set at P < 0.05. Experimental data were plotted using GraphPadPrism software version 9.5, and all data results are presented in the form of mean ± standard deviation.

[0081] 2 Results and analysis

[0082] 2.1 Effects of different alcohol concentrations on HepG2 cell viability

[0083] Cell survival rate is one of the intuitive indicators to measure the degree of cell damage. The results of the effects of different concentrations of alcohol on the survival rate of HepG2 cells are as follows: Figure 1 shown.

[0084] Depend on Figure 1It can be seen that with the increase of alcohol concentration, the relative viability of cells showed a downward trend. When the alcohol concentration exceeded 200mM, the proliferation ability of cells would be significantly inhibited. The proliferation inhibition rate under 800mM alcohol treatment reached 51.29±0.008%, 1000mM alcohol inhibition rate reached 47.82±0.03%, and 1200mM alcohol inhibition rate was as high as 41.11±0.04%. To construct a cell oxidative stress model, a concentration with cell viability between 50% and 70% should be selected as the modeling concentration. Therefore, this experiment selected 800mM alcohol for 24h to induce alcoholic oxidative damage in HepG2 cells.

[0085] 2.2 Effects of EHSR and COP on the survival rate of HepG2 cells

[0086] The experiment used CCK-8 method to determine the effects of different concentrations of EHSR and COP on the survival rate of HepG2 cells, so as to analyze the toxic effects of these two substances on cells. Figure 2 shown.

[0087] Depend on Figure 2 It can be seen that after HepG2 cells were co-cultured with 0.1-1.0mmol / L EHSR and COP for 24 hours, the cell survival rate was higher than 95%. This result shows that within the concentration range of 0.1-1.0mmol / L, EHSR and COP have no obvious toxic effects on HepG2 cells. Taking into account comprehensive considerations, EHSR and COP with protein concentrations of 0.2, 0.5, and 1.0mg / mL were selected as low, medium, and high concentration groups for subsequent experiments to further explore their effects on HepG2 cells.

[0088] 2.3 Effects of EHSR and COP on the survival rate of HepG2 cells damaged by alcohol

[0089] like Figure 3The results showed that in the alcohol-induced HepG2 cell injury model, the relative viability of the cells decreased to 50.54±0.03%, which was significantly different from the control group (P<0.05). Compared with the model group, the cell viability of the 0.2 mg / mL COP group did not change significantly (P>0.05), while the other pretreatment groups could significantly increase the cell viability (P<0.05) and showed a dose-dependent increase. Under the action of EHSR at protein concentrations of 0.2, 0.5, and 1.0 mg / mL, the survival rate of HepG2 cells increased by 25.73%, 51.11%, and 75.24%, respectively, showing a significant protective effect. Compared with COP, at the same protein concentration, the cell viability of the EHSR treatment group was higher than that of the COP treatment group (P<0.05). Specifically, the cell survival rate of the 1.0 mg / mL EHSR treatment group reached 88.57±0.03%, which was significantly higher than the 80.07±0.05% of the 1.0 mg / mL COP treatment group (P<0.05). The cell survival rate of the 0.5 mg / mL EHSR treatment group was 76.37±0.02%, which was not significantly different from the 1.0 mg / mL COP group (P>0.05), indicating that the two had similar protective effects on cells. The experimental results showed that EHSR could significantly improve the survival rate of HepG2 cells damaged by oxidative stress at three concentrations (P<0.05), and its protective effect was better than that of the COP treatment group to a certain extent.

[0090] 2.4 Effects of EHSR and COP on AST, ALT and their ratio in alcohol-damaged HepG2 cells

[0091] Transaminases include aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are released from the cells into the blood when liver cells are damaged, leading to increased levels of AST and ALT in the blood. Therefore, increased activity of these two enzymes has important diagnostic significance clinically.

[0092] Depend on Figure 4 It can be seen that compared with the control group, the AST and ALT levels in the model group were significantly increased (P<0.05), while EHSR and COP treatments could significantly reduce the AST and ALT levels in alcohol-damaged HepG2 cells (P<0.05), and the EHSR group was significantly lower than the COP group (P<0.05). This shows that EHSR has a significant effect on alleviating alcohol-induced liver cell damage, and its effect is better than COP.

[0093] In clinical practice, the severity of liver damage is often assessed by the AST / ALT ratio. Generally, AST / ALT < 1 indicates that the liver cells are in good condition and the liver damage is mild; when AST / ALT > 1, it indicates that the liver cell damage is aggravated, resulting in the release of a large amount of enzymes and a significant increase in AST.

[0094] Depend on Figure 4 It can be seen that the AST / ALT ratio of the control group was maintained within the normal range of less than 1, while after alcohol injury treatment, the AST / ALT ratio increased significantly to 3.60, indicating that alcohol caused significant damage to liver cells. Compared with the model group, after pre-protection treatment with EHSR and COP, the AST / ALT ratio of cells that were damaged by alcohol again was significantly reduced (P < 0.05), indicating that the two substances have a certain protective effect on alcohol-induced liver cell damage. In addition, the AST / ALT ratio of the EHSR treatment group decreased more significantly than that of the COP treatment group, indicating that EHSR has a more superior effect in protecting liver cells, which is consistent with the above 2.3 results.

[0095] 2.5 Effects of EHSR and COP on MDA, SOD and T-AOC contents in alcohol-damaged HepG2 cells

[0096] Malondialdehyde (MDA) is a product of lipid peroxidation, which can react with thiobarbituric acid (TBA) to produce chemical products. The absorbance value is measured by an enzyme marker. The MDA content can be used to detect the degree of oxidative damage to cells.

[0097] Depend on Figure 5 It can be seen that compared with the control group, the MDA content in the alcohol model group increased significantly to 2.36±0.06nmol / mgprot (P<0.05). Under the pre-protective effect of 1.0mg / mL EHSR and COP, the MDA content decreased significantly by 44.35% and 35.63%, respectively. This result shows that EHSR and COP can significantly enhance the antioxidant capacity of cells and reduce alcohol-induced oxidative cell damage, further confirming the potential effectiveness of EHSR in protecting cells from oxidative stress.

[0098] Superoxide dismutase (SOD), as a key component of the antioxidant defense line inside cells, eliminates excess free radicals produced under oxidative stress and can effectively inhibit excessive oxidation reactions that may cause damage to cells.

[0099] Depend on Figure 5It can be seen that the superoxide dismutase (SOD) activity of the control group cells was higher, with a value of 16.74±0.46U / mgprot. After 24h of 800mM alcohol oxidative stress damage, SOD activity was significantly reduced to 0.70±0.04U / mgprot. Further results showed that pre-protecting cells with 1.0mg / mLEHSR could significantly increase SOD activity to 6.33±0.34U / mgprot (P<0.05), which was higher than 0.71±0.07U / mgprot in the COP treatment group. This result shows that compared with COP, EHSR shows a more significant effect in increasing SOD activity.

[0100] Total antioxidant capacity (T-AOC) refers to the overall content of various antioxidant active substances and enzyme systems in the body, and is an important indicator for measuring the body's ability to resist oxidative stress.

[0101] Depend on Figure 5 It can be seen that the total antioxidant capacity of HepG2 cells induced by alcohol decreased significantly (P<0.05). The total antioxidant capacity of cells pre-protected by EHSR and COP increased by 10.67% and 6.53%, respectively, indicating that both substances can enhance the antioxidant defense of cells, and the effect of EHSR is more significant (P<0.05). This result is consistent with the trend of changes in SOD activity, further confirming the positive role of EHSR in improving the antioxidant capacity of cells.

[0102] 2.6 HepG2 cell apoptosis detection

[0103] Depend on Figure 6 It can be seen that the apoptosis rate of the normal control group was 5.03%, and that of the alcohol model group was 54.61%, indicating that alcohol has a significant apoptosis-inducing effect on HepG2 cells. However, when the cells were pre-treated with 1.0 mg / mL of EHSR and COP, the alcohol-induced apoptosis rate decreased, among which the apoptosis rate of the COP pretreatment group was 43.37%, which was higher than the 21.32% of the EHSR group. These data show that both EHSR and COP can inhibit alcohol-induced apoptosis, but EHSR is more effective in reducing apoptosis, which may be related to its stronger antioxidant capacity.

[0104] 2.7 Virtual screening of bioactive peptides

[0105] PeptideRanker is used to predict the potential biological activity of peptides and rank them, and CPPpred is used to predict cell permeability and rank them, which can evaluate the cell penetration potential of peptides. The scores of both are between 0 and 1, and the higher the score, the greater the potential.

[0106] The peak area of ​​the peptide represents its content to a certain extent, so the peak area can be used as a filtering condition to improve the accuracy of the identified peptides and screen the main contributor peptides. Therefore, using PeptideRanker score>0.2, CPPpred score>0.2, and relative peak area>0.2% as filtering conditions, a total of 7 potential bioactive peptides were screened out, and the results are shown in Table 1.

[0107] Table 1 Biological information of 7 peptides

[0108]

[0109]

[0110] As shown in Table 1 , the hydrophobicity of the seven peptides ranges between +4.87 and +13.01 kcal / mol, the isoelectric point ranges between 5.40 and 6.74, the net charge is 0, and the molecular weight ranges between 358.22 and 1027.59 Da.

[0111] A bubble chart was created with the PeptideRanker score as the x-axis, the CPPpred score as the y-axis, and the peak area of ​​the peptide as the area of ​​the circle ( Figure 7 ), showing the relationship among the three.

[0112] There is a relationship between protein function, charge and hydrophobicity, and net charge and hydrophobicity can be used to describe the intermolecular forces of proteins. Studies have found that protein hydrophobicity has a significant correlation with emulsification properties and solubility. Therefore, PepDraw is used to measure the chemical properties of peptides: isoelectric point, net charge, and hydrophobicity.

[0113] From Table 1 and Figure 7 It can be seen that the peptide sequence LGGL has the highest PeptideRanker score of 0.71; LAGL has the highest CPPpred score of 0.48; and ILKPTDAGTL has the largest relative peak area of ​​0.62%.

[0114] The above results indicate that the liver protective biological activity of EHSR in this study may be closely related to the seven peptides in Table 1 identified by virtual screening technology, which requires further experimental verification.

[0115] 3 Conclusion

[0116] In this study, we compared EHSR and COP in detail in terms of cell protection against liver injury, antioxidant capacity, inhibition of lipid peroxidation, and cell apoptosis. Under the action of 0.2, 0.5, and 1.0 mg / mL EHSR, the survival rate of HepG2 cells increased by 25.73%, 51.11%, and 75.24%, respectively. At the same protein concentration, the cell viability of the EHSR-treated group was higher than that of the COP-treated group. In terms of lipid peroxidation, we found that the MDA content levels pre-protected by 1.0 mg / mL EHSR and COP decreased by 44.35% and 35.63%, respectively. In addition, EHSR has a stronger effect in enhancing the activity of SOD and T-AOC. Studies on cell apoptosis also showed that both EHSR and COP can effectively reduce the proportion of apoptotic cells, but the former has a more significant effect. This further proves the important role of EHSR in maintaining cell survival and function. In order to further explore its active ingredients, we identified the peptides in the hydrolysate and used virtual peptide screening technology to screen out 7 peptides with potential liver-protecting effects. In view of the significant biological activity of these peptides and their application prospects in the medical field, this study proposes intellectual property protection for these peptides to ensure their legitimate rights and interests in future scientific research and commercialization. Overall, EHSR performed better than COP in various antioxidant indicators, suggesting that it has broad application potential in the field of hangover and health products. This study not only provides new ideas for the development of hangover drinks, but also lays the foundation for future related research.

[0117] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sturgeon roe peptide for preventing alcoholic liver damage, characterized in that: The peptide sequence is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

2. A method for preparing sturgeon roe peptide for preventing alcoholic liver damage as claimed in claim 1, characterized in that: The peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL, and specifically comprises the following steps: (1) adding deionized water to sturgeon roe and homogenizing to obtain solution A; (2) adjusting the pH value of solution A, adding alkaline protease, performing enzymolysis, deactivating the enzyme, and cooling to obtain solution B; (3) Centrifuging solution B and taking the supernatant to obtain the sturgeon roe peptide for preventing alcoholic liver damage.

3. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (1), the mass ratio of the sturgeon roe to deionized water is 1:

6.

4. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (1), the homogenization speed is 8000 rpm and the time is 1 min.

5. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (2), the amount of alkaline protease added is 1%.

6. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (2), the pH value of the enzymatic hydrolysis is 8.0, the temperature is 55° C., and the time is 8 hours.

7. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (2), the enzyme is inactivated at a temperature of 90-100°C for 10-15 minutes; and the step of cooling to room temperature is performed.

8. The method for preparing a sturgeon roe peptide for preventing alcoholic liver damage according to claim 2, characterized in that: In step (3), the centrifugal treatment temperature is 4°C, the rotation speed is 8000 rpm, and the time is 15 min.

9. Use of the sturgeon roe peptide according to claim 1 or the sturgeon roe peptide prepared by the preparation method according to any one of claims 2 to 9 in preparing a drug for preventing or treating alcoholic liver injury, characterized in that: The peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

10. Use of the sturgeon roe peptide according to claim 1 or the sturgeon roe peptide prepared by the preparation method according to any one of claims 2 to 9 in preparing a hangover-relief drug or hangover-relief food, characterized in that: The peptide sequence of the sturgeon roe peptide is ILKPTDAGTL, ALPLDPK, LGGL, VNRDL, LLGQ, VFVLN or LAGL.

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