Stomach acid degradation resistant liver protection peptide oral liquid as well as preparation method and application thereof

Abstract

The invention discloses liver-protecting peptide oral liquid capable of resisting gastric acid degradation as well as a preparation method and application of the liver-protecting peptide oral liquid, in particular to a method for synthesizing polypeptide CLKARIC through structural optimization design and constructing the peptide oral liquid with purified water, and relates to the technical field of biomedical materials. The peptide oral liquid provided by the invention has the advantages of strong antioxidant activity, high biological safety, obvious anti-inflammatory effect and high gastric acid environment stability, and can maintain the integrity of the active structure under the physiological conditions of gastrointestinal tracts. The preparation method is simple, the preparation process is good in repeatability, and clinical transformation potential is shown in the field of liver injury repair.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and more specifically relates to a preparation method and application of an oral solution of liver-protecting peptide resistant to gastric acid degradation. Background Art

[0002] In modern society, with the accelerated pace of life, environmental pollution, long-term mental stress and irregular eating habits, the human body is facing multiple harmful stimuli. These stimuli not only affect the normal physiological functions of the human body, but may also cause a series of health problems. Among them, reactive oxygen species (ROS), as a byproduct produced during normal cell metabolism, have physiological functions such as cell signal transduction to a certain extent, but their excessive accumulation will bring serious health risks. ROS can damage biological macromolecules such as cell membranes, proteins, lipids and nucleic acids, induce oxidative stress and the ensuing inflammatory response, thereby leading to cell damage, functional decline, and even inducing a series of pathological changes, such as liver damage, neurodegenerative diseases (such as Alzheimer's disease), skin aging and inflammatory diseases. The liver is an important metabolic organ in the human body, involved in the detoxification of toxins in the body and the regulation of various physiological functions. However, the liver is also a high-risk site for ROS accumulation, especially under the influence of unhealthy lifestyles, long-term drinking, drug use and other factors, the liver is susceptible to oxidative stress damage. Excessive ROS can cause oxidative damage to liver cells, destroy their cell membranes, proteins and DNA structures, and eventually lead to apoptosis or necrosis of liver cells, which in turn leads to serious diseases such as liver fibrosis, cirrhosis and even liver cancer. The role of oxidative stress in liver damage cannot be ignored, especially in acute and chronic liver damage. The generation and accumulation of ROS can lead to further intensification of the inflammatory response, thereby aggravating liver damage. Therefore, alleviating oxidative stress and protecting the liver from excessive oxidative damage have become important strategies for the prevention and treatment of liver diseases.

[0003] At present, most antioxidants available on the market are chemical synthetics, such as butylated hydroxyanisole, butylated hydroxytoluene and tert-butylhydroquinone, etc. These chemical antioxidants can effectively inhibit oxidative stress reactions to a certain extent. However, long-term use of these synthetic antioxidants may bring some side effects, such as organ damage, carcinogenicity, cytotoxicity, etc., and may even cause endocrine disruption. In addition, compared with traditional antioxidants such as glutathione, it is easily restricted by the redox environment in the body, and there is a situation where the concentration in the body is too low. The antioxidant effect of antioxidant peptides is not only more durable than glutathione, but also has stronger stability. In addition, the role of antioxidant peptides in inhibiting cell damage caused by oxidative stress, regulating immune response, protecting the liver, etc. is more extensive than glutathione, especially in the prevention and treatment of chronic diseases and liver diseases, it has a more significant effect. Therefore, finding antioxidants from natural sources, especially those natural products with good biocompatibility and safety, has become a hot topic in current research.

[0004] In response to the above problems, this study developed a new type of antioxidant peptide by directional design of active side chain combination. Based on the structural characteristics of different amino acid side chains, the peptide has excellent antioxidant activity and breaks through the research limitations of traditional antioxidant peptides. By rationally designing the amino acid arrangement and structure in the peptide chain, the peptide chain can exert significant antioxidant effects at lower concentrations and has good stability. After optimizing the sequence arrangement, the DDPH clearance rate and OH clearance rate were increased to 1.3 times and 2.1 times that of traditional peptides, respectively, and its broad-spectrum antioxidant effect was verified based on animal models: in the mouse acute liver injury model, the peptide can downregulate ALT and AST levels by 50% and 51%. This design not only overcomes the safety defects of synthetic antioxidants and the efficiency bottleneck of natural ingredients, but also provides an innovative and practical solution for the development of antioxidant therapeutic preparations through structural rationalization and functional synergy. The designed antioxidant peptide not only showed excellent antioxidant capacity in in vitro experiments, but also showed good biological activity, excellent antioxidant and hepatoprotective effects in in vivo experiments.

[0005] Through this innovative work, we have provided new ideas and technical support for the development and application of natural antioxidant peptides, which has important scientific research value and practical application prospects. In the future, these antioxidant peptides are expected to become safe alternatives to chemically synthesized antioxidants, and provide new treatment options in the fields of antioxidant and anti-inflammatory in clinical applications. Summary of the invention

[0006] In order to deal with the above problems, the present invention provides an oral liquid of liver-protecting peptide resistant to gastric acid degradation and its preparation method and application. It can effectively solve the problem of liver damage caused by oxidative stress, and has the advantages of strong antioxidant activity, high biosafety, significant anti-inflammatory effect and high stability in gastric acid environment.

[0007] The invention also provides a method for preparing the gastric acid degradation-resistant liver-protecting peptide oral liquid.

[0008] To solve the above problems, the technical solution of the present invention is as follows:

[0009] A method for preparing an oral liquid of liver-protecting peptide resistant to gastric acid degradation, characterized in that it comprises the following steps:

[0010] S1. Using solid phase synthesis method, amino acids protected by N 9 fluorenylmethyl carbonyl (Fmoc) were used as raw materials, and Fmoc was removed with 20% piperidine. PyBoP, a peptide coupling agent, HoBT, an activator of amino acid carboxyl group, and DIEA were used as catalyst for the reaction to crosslink and condense to form peptide bonds. The polypeptide chain was extended from the C-terminus to the N-terminus to synthesize the dichlororesin sphere polypeptide polymer.

[0011] S2, cutting the polymer prepared in step S1 with a cutting agent, dropping it into ice ether to precipitate a crude polypeptide product, standing it for a period of time, vacuum drying to remove anhydrous ether, adding water and freeze drying to obtain a crude polypeptide product.

[0012] S3. Add purified water to the crude polypeptide product prepared in step S2, mix thoroughly and sterilize to obtain an oral solution of liver-protecting peptide resistant to gastric acid degradation.

[0013] The step S1 is to swell the dichlororesin with DMF for 90 minutes, add amino acids and DIEA, wash with DMF for 3 times, each time for 2 hours, then add a connector sealant (methanol and DIEA) to seal the unreacted connector. Then add amino acids (3 times the amount of dichlororesin ball grafting), PyBoP, HoBT and DIEA to react for 2 hours, and then remove the Fmoc protecting group with an Fmoc protecting group remover (20% piperidine + 80% DMF). The desired dichlororesin ball polypeptide polymer is synthesized cyclically.

[0014] The amount of PyBoP, HoBT and DIEA used is 3 equivalents of PyBoP, 3 ​​equivalents of HoBT and 6 equivalents of DIEA per mole of dichlororesin substituent.

[0015] Preferably, the step S2 is:

[0016] The dichlororesin polypeptide polymer obtained in step S1 was washed three times with DMF, and then cut with a cutting agent (TFA: water: triisopropylsilane = 95:2.5:2.5) for 2 hours to cut the peptide chain from the resin, and then dropped into ice ether to precipitate the crude polypeptide product. After standing for a period of time, vacuum drying was performed to remove anhydrous ether, and water was added and freeze-dried to obtain the crude polypeptide product.

[0017] The amount of the cutting agent and the dichlororesin ball polypeptide polymer is 20-25 mL / g.

[0018] Preferably, the step S3 is:

[0019] The gastric acid-resistant liver-protecting peptide obtained in step S2 is added to purified water, and the concentration of the peptide in the oral solution is 2-2.5 mg / mL.

[0020] The organic solvent used in the present invention is N,N-dimethylformamide, trifluoroacetic acid and ether.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention adopts solid phase synthesis method combined with DPPH liquid chromatography to prepare and screen the antioxidant liver-protecting peptide CLKARIC, which has the advantages of high efficiency, rapidity and accurate screening.

[0023] (2) Compared with the prior art, the present invention has good biocompatibility and high stability, and can be absorbed orally to treat liver damage.

[0024] (3) The antioxidant liver-protecting peptide prepared by the present invention has the advantages of high efficiency in antioxidant activity and chelating metal ions, and can effectively cope with liver damage caused by oxidative stress.

[0025] (4) The antioxidant liver-protecting peptide prepared by the present invention is verified by using an acute liver injury model in mammals to verify its ability to treat and relieve liver injury in vivo. Oral administration in advance can improve the liver's ability to prevent oxidative stress, and can also further regulate the body's oxidative stress defense system and regulate enzyme activity, which is helpful for the treatment of liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0027] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 is the DPPH clearance and EC of CLKARIC prepared in Example 1 50 value

[0029] Figure 2 is the ABTS clearance rate and EC of CLKARIC prepared in Example 1 50 value

[0030] Figure 3 is the FRAP clearance and EC of CLKARIC prepared in Example 1 50 value

[0031] Figure 4 is the hydroxyl radical scavenging rate and EC of CLKARIC prepared in Example 1 20 value

[0032] Figure 5 Fe of CLKARIC prepared in Example 1 2+ Chelation and EC 50 value

[0033] Figure 6 is the hemolysis rate of mouse blood cells at different concentrations of CLKARIC;

[0034] Figure 7 Scanning electron microscopy images of mouse blood cells treated with different concentrations of CLKARIC;

[0035] Figure 8 Inverted microscope images of mouse blood cells treated with different concentrations of CLKARIC;

[0036] Fig. 9 To test the cytotoxicity of CLKARIC to cells;

[0037] Fig.10 Live / dead staining inverted fluorescence microscope images of L-929 cells treated with different concentrations of CLKARIC on days 1, 2, and 3;

[0038] Fig.11 The stability of CLKARIC at different concentrations in a simulated gastric fluid environment;

[0039] Fig.12 The stability of different concentrations of CLKARIC in a simulated digestion environment;

[0040] Fig.13 H&E staining of pathological sections of liver sections of different groups of liver damage;

[0041] Fig.14 H&E staining of pathological sections of heart, spleen, stomach and kidney in different groups of liver damage;

[0042] Fig.15 Figure 2 is the weight change of mice before and after liver injury;

[0043] Fig.16 Data graph of AST index in liver for preventing liver damage in mice;

[0044] Fig.17 Data graph of ALT index in liver for preventing liver damage in mice; DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with embodiments:

[0046] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0047] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0048] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] Example 1

[0050] This example provides a method for preparing the heptapeptide CLKARIC.

[0051] First, dichlorotrityl chloride resin was swollen with DMF, and amino acids and DIEA were added to react for 2 hours, and unreacted groups were blocked with methanol. The Fmoc protecting group was removed using a 20% piperidine solution, and then the corresponding amino acids, PyBoP, HoBt and DIEA were added in sequence. After reacting for 2 hours, the Fmoc protecting group was removed using a 20% piperidine solution, and the above steps were repeated until the last amino acid was connected. Finally, a cutting agent (TFA: water: triisopropylsilane = 95:2.5:2.5) was used for cutting, and the peptide was precipitated in ice ether, and finally freeze-dried in a freeze dryer for use.

[0052] S1. Using solid phase synthesis method, amino acids protected by N 9 fluorenylmethyl carbonyl (Fmoc) were used as raw materials, and Fmoc was removed with 20% piperidine. PyBoP, a peptide coupling agent, HoBT, an activator of amino acid carboxyl group, and DIEA were used as catalyst for the reaction to crosslink and condense to form peptide bonds. The polypeptide chain was extended from the C-terminus to the N-terminus to synthesize the dichlororesin sphere polypeptide polymer.

[0053] S2, cutting the polymer prepared in step S1 with a cutting agent, dropping it into ice ether to precipitate a crude polypeptide product, standing it for a period of time, vacuum drying to remove anhydrous ether, adding water and freeze drying to obtain a crude polypeptide product.

[0054] The step S1 is to swell the dichlororesin with DMF for 90 minutes, add amino acids and DIEA, wash with DMF for 3 times, each time for 2 hours, then add a connector sealant (methanol and DIEA) to seal the unreacted connector. Then add amino acids (3 times the amount of dichlororesin ball grafting), PyBoP, HoBT and DIEA to react for 2 hours, and then remove the Fmoc protecting group with an Fmoc protecting group remover (20% piperidine + 80% DMF). The desired dichlororesin ball polypeptide polymer is synthesized cyclically.

[0055] The amount of PyBoP, HoBT and DIEA used is 3 equivalents of PyBoP, 3 ​​equivalents of HoBT and 6 equivalents of DIEA per mole of dichlororesin substituent.

[0056] The step S2 is:

[0057] The dichlororesin polypeptide polymer obtained in step S1 was washed three times with DMF, and then cut with a cutting agent (TFA: water: triisopropylsilane = 95:2.5:2.5) for 2 hours to cut the peptide chain from the resin, and then dropped into ice ether to precipitate the crude polypeptide product. After standing for a period of time, vacuum drying was performed to remove anhydrous ether, and water was added and freeze-dried to obtain the crude polypeptide product.

[0058] Example 2

[0059] This embodiment provides an oral solution of liver-protecting peptide resistant to gastric acid degradation and a preparation method thereof.

[0060] With reference to Example 1, the heptapeptide of this example was prepared, and the obtained heptapeptide was added into purified water, mixed thoroughly and sterilized to prepare an oral solution of liver-protecting peptide resistant to gastric acid degradation, wherein the concentration of the peptide in the oral solution was 2-2.5 mg / mL.

[0061] Performance Test 1

[0062] The heptapeptide obtained in Example 1 was tested for its antioxidant properties.

[0063] DPPH free radical scavenging ability determination:

[0064] The scavenging ability of CLKARIC prepared by the present invention on DPPH free radicals is evaluated to support its application in the field of antioxidants. First, a 0.1 mM DPPH solution is prepared, dissolved in anhydrous ethanol, and stored in the dark. Then, antioxidant peptide solutions of different concentrations (50-1000 μg / mL) are prepared, and a standard antioxidant control group (GSH) is set. In a 96-well plate, 100 μL of the antioxidant peptide solution is mixed with 100 μL of the DPPH solution and allowed to stand at room temperature in the dark for 30 minutes. The absorbance value of each well is measured by a spectrophotometer at a wavelength of 517 nm, and the scavenging rate of DPPH free radicals is calculated using the following formula: DPPH scavenging rate (%) = [(A 空白 -A 样品 ) / A 空白 ]×100, where A 空白 is the absorbance of the blank group, A 样品 is the absorbance of the antioxidant peptide group. The results show that the short peptide CLKARIC prepared by the present invention has good antioxidant activity. The peptide has a significantly higher DPPH scavenging capacity than GSH. 50 The value was 88.45±48.66μg / mL, Figure 1 DPPH clearance and EC after treatment with different concentrations of CLKARIC and GSH 50 .

[0065] ABTS + Free radical scavenging ability determination:

[0066] This experiment aims to evaluate the scavenging ability of CLKARIC on ABTS free radicals. First, a 7mM ABTS solution was prepared and mixed with a 2.45mM ammonium persulfate solution in a 1:1 ratio and allowed to stand for 24 hours to form an ABTS free radical solution. Then, antioxidant peptide solutions of different concentrations (50-1000μg / mL) and a standard antioxidant control group (GSH) were prepared. In a 96-well plate, 100μL of the antioxidant peptide solution was mixed with 100μL of the ABTS free radical solution and reacted for 8 minutes. The absorbance of each well was measured at a wavelength of 734nm using a spectrophotometer. To calculate the ABTS free radical scavenging rate, the following formula was used: ABTS scavenging rate (%) = [(A 空白 -A 样品 ) / A 空白 ]×100, where A 空白 is the absorbance of the blank group, A 样品 is the absorbance of the antioxidant peptide group. The results show that the short peptide CLKARIC prepared by the present invention has good antioxidant properties. + The clearance was significantly higher than that of GSH and its EC 50 The value was 65.56±2.53μg / mL, Figure 2 ABTS treated with different concentrations of CLKARIC and GSH + Clearance and EC 50 .

[0067] Hydroxyl free radical scavenging ability assay:

[0068] This experiment aims to evaluate the ability of antioxidant peptides to scavenge hydroxyl radicals (·OH) by the salicylic acid method. First, a 10mM salicylic acid solution was prepared and the pH was adjusted to 6.0 using PBS buffer. Then, a reaction system for generating hydroxyl radicals was prepared, usually by reacting 10mM hydrogen peroxide with a solution containing ferrous ions to generate hydroxyl radicals. Next, antioxidant peptide solutions of different concentrations (50-1000μg / mL) were prepared, and a control group (GSH) was set up. In a 96-well plate, 100μL of antioxidant peptide solution was added to each well, and then 100μL of the hydroxyl radical generating system was added to mix with the salicylic acid solution and reacted for 30 minutes. The absorbance of each well was measured at a wavelength of 510nm using a spectrophotometer to calculate the hydroxyl radical scavenging ability. The scavenging rate calculation formula is: Hydroxyl radical scavenging rate (%) = [(A 空白 -A 样品 ) / A 空白 ]×100, where A blank is the absorbance of the blank group, and A sample is the absorbance of the antioxidant peptide group. The results show that the short peptide CLKARIC prepared by the present invention has good antioxidant activity, and the peptide has a significantly higher scavenging effect on hydroxyl free radicals than GSH. 20The value is 207.35±6425.10μg / mL, Figure 3 The hydroxyl radical scavenging rate and EC of patients treated with different concentrations of CLKARIC and GSH 20 .

[0069] Determination of the antioxidant capacity of iron ions:

[0070] This experiment aims to evaluate the reducing power of antioxidant peptides and determine their antioxidant capacity by the FRAP (Ferric Reducing Antioxidant Power) method. First, prepare the FRAP reagent solution, mix 10mM ferric chloride solution with 300mM sodium acetate buffer (pH 3.6) and 10mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in a ratio of 10:1:1 to form a FRAP reagent, and let it stand at room temperature. Then, prepare antioxidant peptide solutions of different concentrations (50-1000μg / mL) and set up a standard control group (GSH). In a 96-well plate, add 100μL of antioxidant peptide solution to each well, then add 100μL of FRAP reagent, mix gently, and let it stand for 30 minutes for reaction. Use a spectrophotometer to measure the absorbance at a wavelength of 593nm. By comparing with the standard curve, the reducing power of the antioxidant peptide is calculated, and the results are expressed as iron reducing equivalents (FE). The calculation formula of clearance rate is: FRAP value = (A 样品 -A 空白 ) / standard curve slope, where A 样品 is the sample absorbance, A 空白 is the absorbance of the blank group. The results show that the short peptide CLKARIC prepared by the present invention has good antioxidant activity. The peptide has a significantly higher FRAP clearance than GSH. 50 The value is 1723.99±407.35μg / mL, Figure 4 FRAP clearance and EC after treatment with different concentrations of CLKARIC and GSH 50 .

[0071] Fe 2+ Chelating capacity determination:

[0072] This study aimed to evaluate the effects of antioxidant peptides on Fe 2+ The chelation effect of FeCl2 was determined by the Ferrozine method. 2+ The source was added and FeCl2 was diluted to the desired concentration using 0.1 M phosphate buffer (pH 7.4). Next, antioxidant peptide solutions of different concentrations (50-1000 μg / mL) were prepared and a control group (GSH) was set up. The antioxidant peptide solution was added to the test tube, followed by the addition of Fe 2+The solution was mixed and allowed to stand for 10 minutes to ensure sufficient reaction. Then, Fe-L-Phenyl solution (0.1 mM) was added. 2+ A purple-red complex is formed. After the reaction is stable, the absorbance of the reaction system is measured at a wavelength of 562 nm using a spectrophotometer. 2+ Compare the absorbance of the solution and calculate the Fe of the antioxidant peptide 2+ Chelating capacity. 2+ The calculation formula of chelation rate is: Fe 2+ Chelation rate (%) = [(A 空白 -A 样品 ) / A 空白 ]×100, where A 空白 is the absorbance of the blank group, A 样品 is the absorbance of the antioxidant peptide group. The results show that the short peptide CLKARIC prepared by the present invention has good antioxidant properties. 2+ The chelating capacity is significantly higher than that of GSH. 50 The value is 1437.49±795.10μg / mL, Figure 5 Fe treated with different concentrations of CLKARIC and GSH 2+ Chelating capacity and EC 50 .

[0073] Performance Test 2

[0074] The heptapeptide obtained in Example 1 was subjected to blood compatibility and red blood cell morphology determination.

[0075] The hemolytic performance of CLKARIC prepared by the present invention is tested by mouse blood cell suspension. First, the freeze-dried CLKARIC powder is dissolved in sterile PBS, and a gradient concentration of 62.5-2000 μg / mL LKAHR solution is prepared. The blood cells washed with PBS are divided into sample tubes, and then the gradient concentration CLKARIC solutions are added to the blood cell sample tubes respectively, and the blood cells treated with sterile PBS are used as the negative control group, and the blood cells treated with Triton are used as the positive control group. All samples are incubated at 37 ° C for 2 hours. After the incubation, all samples are centrifuged and photographed, and the supernatant is taken for microplate reader test at 570nm. The above-treated blood cells are fixed with glutaraldehyde, dehydrated with gradient concentration ethanol, and then detected under SEM and inverted fluorescence microscope to observe the state of blood cells. The results show that the morphology of blood cells treated with LKAHR is intact, and there is no difference from the blood cells treated with the negative control group, indicating that CLKARIC prepared by the present invention is non-hemolytic. Figure 6 is the blood cell hemolysis rate of gradient concentration CLKARIC, Figure 7 and Figure 8These are the SEM images and inverted fluorescence microscope images of blood cells after CLKARIC treatment.

[0076] Performance Test 3

[0077] The heptapeptide obtained in Example 1 was subjected to cell compatibility test.

[0078] The cell compatibility of CLKARIC prepared by the present invention is tested by MTT toxicity and cell live / death experiments. L929 cells with good growth status are divided into 96-well plates (the number of cells per well is about 10,000) and cultured in a cell culture incubator for 24 hours. The CLKARIC solution prepared by gradient concentration cell culture fluid is added to a 96-well plate containing L929 cells and cultured for 24 hours. After cleaning, the pre-configured MTT reagent is added, and the MTT time is cleaned after incubation for 4 hours. After adding DMSO solution, the cell survival rate is tested under a microplate reader. The cell live / death experiment is to incubate L929 cells in a cell culture fluid containing LKAHR for 24, 48, and 72 hours, and then dyed with AM / PI dye, and placed under an inverted fluorescence microscope after 15 minutes to observe the cell growth state. The results show that the CLKARIC prepared by the present invention has no cytotoxicity and a high cell survival rate, indicating that it has good cell compatibility. Fig. 9 For the cytotoxicity test and cell viability of gradient concentrations of CLKARIC, Fig.10 Live / dead images of cells treated with different concentrations of CLKARIC.

[0079] Performance Test 4

[0080] The heptapeptide obtained in Example 1 was tested for stability.

[0081] This experiment aims to evaluate the stability of antioxidant peptides in artificial gastric juice and simulated digestive juice. First, an artificial gastric juice solution was prepared by mixing 0.1M hydrochloric acid (pH 1.5) with 0.2% pepsin solution to prepare a simulated gastric environment; at the same time, a simulated small intestinal juice solution was prepared by mixing 0.1M phosphate buffer (pH 6.8) with 1% trypsin solution to simulate the intestinal environment. Then, a certain amount of antioxidant peptide solution (1000μg / mL) was taken, mixed with artificial gastric juice or simulated small intestinal juice, and treated at 37°C for 10min, 20min and 30min. After the reaction, the insoluble matter was removed by a centrifuge, and the supernatant was taken for further analysis. The antioxidant activity of the antioxidant peptides at different time points was analyzed using an enzyme marker. The stability retention rate was calculated based on the ratio of the residual amount of the antioxidant peptide to the initial concentration, and its stability in the simulated digestive environment was evaluated based on the antioxidant activity. The results show that the CLKARIC prepared by the present invention does not change its structure due to environmental changes, indicating that it has high stability and can tolerate the digestive environment. Fig.11This is the drug stability diagram of CLKARIC after different artificial gastric juice tolerance times. Fig.12 This is the drug stability diagram of CLKARIC after digestion process.

[0082] Performance Test 5

[0083] The peptide oral solution obtained in Example 2 was tested for protection in a liver injury model.

[0084] This experiment was designed to evaluate the protective effect of antioxidant peptides on acute liver injury induced by carbon tetrachloride (CCl4) in mice. First, healthy male mice aged 6-8 weeks were selected and randomly divided into five groups: normal group (blank group), CCl4 model group (acute liver injury group), and antioxidant peptide group. All mice were fasted for 12 hours before the experiment. The dose of antioxidant peptide solution was set to 10μL / g, and normal saline was used as a control. Each group was gavaged at a dose of 10μL / g. The blank control group and the acute hepatitis model group were gavaged with normal saline every day, and the other groups were gavaged with the corresponding drug UP aqueous solution at a concentration of 4mg / mL for 7 consecutive days. Food and water were normally supplied during gavage. After gavage, all mice except the blank control group were intraperitoneally injected with 10μL / g 1.5% CCl4 / olive oil solution. After injection, water and food were cut off and the mice were killed at 16h. Blood samples and liver tissues were collected for analysis. By comparing the physiological indicators, liver function recovery and histological results of each group of mice, the protective effect of antioxidant peptides on CCl4-induced acute liver injury was comprehensively evaluated.

[0085] Performance Test 6

[0086] Toxicological analysis of the peptide oral solution obtained in Example 2 on the liver damage of mice

[0087] In order to more intuitively verify the protective and therapeutic effect of CLKARIC on liver injury, H&E staining sections were performed on the main organs of mice after treatment in each group for observation. Through histological examination, HE staining was used to observe the morphological changes of liver tissue and analyze the damage of liver cells. The protective effect of antioxidant peptides on CCl4-induced acute liver injury was evaluated by comparing the histological results of mice in each group. The results showed that compared with healthy mice, CCl4 could cause severe acute liver injury, which was manifested by a significantly higher rate of weight loss in mice than in the control group, obvious liver swelling and nodular granular lesions, leading to hepatomegaly, and may cause severe liver tissue damage. In contrast, the rate of weight loss in GSH-treated mice was significantly reduced, and the reduction in the CLKARIC group was more significant. The liver damage and inflammatory infiltration of mice in the GSH group were reduced. It is worth noting that the liver morphology of mice treated with CLKARIC tended to be normal. The results showed that CLKARIC can significantly reduce CCl4-induced liver oxidative stress damage. Fig.13 These are H&E stained sections of major organs in mice with liver injury.

[0088] In order to verify whether the peptide has any side effects on mouse organs, we took the heart, spleen, lung, and kidney of the mice treated in the above four groups and stained their organ slices to observe the effects of the peptide on the organs after entering the body's circulation. As shown in the figure, no pathological phenomena such as inflammation and local bleeding were found in the organs of the above four groups of mice. The results show that CLKARIC can be safely used in liver damage. Fig.14 H&E stained sections of the main organs in each group of mouse liver injury model.

[0089] Performance Test 7

[0090] Analysis of the anti-liver injury indexes of the peptide oral solution obtained in Example 2 in mice

[0091] To evaluate the extent of liver injury, a biochemical analyzer was used to measure the liver function indicators in serum, including enzyme activities such as ALT and AST. The protective effect of antioxidant peptides on CCl4-induced acute liver injury was evaluated by comparing the physiological indicators and liver function recovery of mice in each group, and the serum ALT and AST levels were measured. When exposed to CCl4, free radicals are produced in liver microsomes, leading to membrane lipid peroxidation, which leads to the release of ALT and AST in the cytoplasm of hepatocytes. Compared with the control group, the levels of these two transaminases were significantly increased in the CCl4 group. However, when these groups were treated with GSH and CLKARIC, the levels of the two transaminases were significantly reduced, indicating that GSH has a certain protective effect on CCl4-induced liver injury. Fig.16 and Fig.17 ALT and AST are liver function indicators of mouse liver injury model.

Claims

1. An oral liquid of liver-protecting peptide resistant to gastric acid degradation, characterized in that: The amino acid sequence of the polypeptide chain is Cys-Ile-Arg-Ala-Lys-Leu-Cys (CLKARIC), which has stability, antioxidant properties and liver damage prevention properties, and can prevent oxidative stress liver damage through oral administration.

2. A method for preparing an oral liquid of liver-protecting peptide resistant to gastric acid degradation, characterized in that: The following steps are involved: S1 adopts solid phase synthesis method, using amino acid protected by N-9 fluorenemethyl carbonyl (Fmoc) as raw material, removing Fmoc with 20% piperidine, crosslinking and condensing under the conditions of peptide coupling agent PyBoP, amino acid carboxyl activator HoBT and reaction catalyst DIEA to form peptide bonds. The polypeptide chain extends from the C-terminus to the N-terminus to synthesize dichlororesin sphere polypeptide polymer. S2: Cut the polymer prepared in step S1 with a cutting agent, drop it into ice ether to precipitate a crude polypeptide product, let it stand for a period of time, vacuum dry it to remove anhydrous ether, add water and freeze-dry it to obtain a crude polypeptide product. S3: adding purified water to the crude polypeptide product prepared in step S2, and sterilizing after thorough mixing to obtain an oral solution of liver-protecting peptide resistant to gastric acid degradation.

3. The method for preparing the antioxidant liver-protecting peptide oral liquid resistant to gastric acid degradation according to claim 2, characterized in that: The specific steps of step 1 are: The dichlororesin was swelled with DMF for 90 minutes, and amino acids and DIEA were added. It was washed with DMF 3 times, 2 hours each time, and then the linker sealant (methanol and DIEA) was added to seal the unreacted linker. Then amino acids (3 times the grafting amount of dichlororesin balls), PyBoP, HoBT and DIEA were added to react for 2 hours, and then the Fmoc protecting group remover (20% piperidine + 80% DMF) was used to remove the Fmoc protecting group. The desired dichlororesin ball polypeptide polymer was synthesized cyclically. The amount of PyBoP, HoBT and DIEA used is 3 equivalents of PyBoP, 3 ​​equivalents of HoBT and 6 equivalents of DIEA per mole of dichlororesin substituent.

4. The method for preparing the antioxidant liver-protecting peptide oral liquid resistant to gastric acid degradation according to claim 2, characterized in that: The specific steps of step 2 are: The dichlororesin ball polypeptide polymer was washed three times with DMF, and then cut with a cutting agent (TFA: water: triisopropylsilane = 95:2.5:2.5) for 2 hours to cut the peptide chain from the resin, and then dropped into ice ether to precipitate the crude polypeptide product. After standing for a period of time, the anhydrous ether was removed by vacuum drying, and water was added and freeze-dried to obtain the crude polypeptide product. The amount of the cutting agent and the dichlororesin ball polypeptide polymer is 20-25 mL / g.

5. The preparation method according to claim 2, characterized in that: The concentration of the peptide in the gastric acid-resistant liver-protecting peptide oral solution is 2-2.5 mg / mL.

6. Use of the oral liquid of liver-protecting peptide resistant to gastric acid degradation according to claim 1, 2 or 5 in the preparation of antioxidants.

7. The hepatoprotective peptide resistant to gastric acid degradation according to claim 1, characterized in that: The peptide chain is one of a linear peptide chain, a circular peptide chain or a branched peptide chain.

8. An oral liquid of liver-protecting peptide resistant to gastric acid degradation, characterized in that: The invention comprises the gastric acid-resistant liver-protecting peptide or its derivative according to claim 1, and a pharmaceutically acceptable delivery carrier, wherein the delivery carrier is a liposome or a nanoparticle.

9. The oral liquid of liver-protecting peptide resistant to gastric acid degradation according to claim 1, characterized in that: The oral solution maintains stability in simulated gastric fluid (pH 1.2, containing 0.32% pepsin) and simulated intestinal fluid (pH 6.8, containing 0.1% pancreatin).

10. The antioxidant liver-protecting polypeptide chain resistant to gastric acid degradation according to claim 1, characterized in that: The peptide chain prevents alcohol or CCl4-induced oxidative stress liver damage through oral absorption.

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