CN114470150B - Application of chicken-derived small molecular peptide in preparation of product for preventing and improving liver injury and secondary symptoms thereof and product - Google Patents
Application of chicken-derived small molecular peptide in preparation of product for preventing and improving liver injury and secondary symptoms thereof and product Download PDFInfo
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- CN114470150B CN114470150B CN202210076927.8A CN202210076927A CN114470150B CN 114470150 B CN114470150 B CN 114470150B CN 202210076927 A CN202210076927 A CN 202210076927A CN 114470150 B CN114470150 B CN 114470150B
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Abstract
The invention discloses application of chicken-derived small molecular peptide in preparing a product for preventing and improving liver injury and secondary symptoms thereof and the product, and relates to the field of food and medical health. The small molecular peptide is chicken-derived free active peptide extracted from nature, is particularly dipeptide Cyclo (L-Phe-L-Phe), shows strong antioxidant capacity in cells, can improve the oxidative stress state of the liver by increasing the activity of antioxidant enzyme GPx, reducing the level of oxidative stress product malondialdehyde MDA and the like, obviously improves liver pathological injuries such as liver bleeding, liver fibrosis and liver cell apoptosis and the like, has small cytotoxicity, wide medication window and high safety, and can be applied to products such as functional food, health-care food, medicaments, compositions and the like for preventing or improving liver injury and related secondary symptoms thereof.
Description
Technical Field
The invention relates to the field of food and medical health, in particular to application of chicken-derived small molecular peptide in preparing a product for preventing and improving liver injury and secondary symptoms thereof and the product.
Background
The liver, as a main metabolic organ of a human body, participates in the processes of digestion, metabolism, excretion, detoxification, immunity and the like in vivo, and the normal function of the liver is very important for the health of the human body. With the rapid development of socioeconomic and the change of living environment, the psychological pressure of people is continuously increased, and people often relieve the pressure by drinking, so that the number of patients with alcoholic liver injury is increased year by year. In addition, environmental pollution and the incidence of food and drug safety are high, and liver dysfunction and organic injury are also increasing, especially drug-induced liver injury.
Clinically, many drugs can cause liver injury of different degrees, and acute liver injury is the main morbidity form. According to incomplete statistics, more than 1000 drugs in the current clinic have potential hepatotoxicity, and excipients, traditional Chinese medicines and health products of the drugs also have the possibility of inducing drug-induced liver injury, and in recent years, researches show that the pathological changes of the liver present a trend of complication and difficult cure.
At present, the medicines clinically used for treating liver injury mainly adopt modes of antioxidation, anti-inflammation, improvement of liver enzyme activity and the like, however, the medicines have certain side effects, and even aggravate the liver injury after long-term administration. Therefore, the search for a natural active small molecule with high safety, low toxic and side effects and suitability for long-term administration for developing functional foods, health foods, pharmaceuticals and compositions thereof has a huge market demand, and the research and development thereof are also concerned by people.
Disclosure of Invention
The invention provides application of chicken-derived small molecular peptide in preparing a product for preventing and improving liver injury and secondary symptoms thereof and the product, and provides natural active small molecular peptide which is high in safety, small in toxic and side effects and suitable for long-term administration.
In order to solve the technical problems, the invention provides an application of chicken-derived small molecular peptide in preparing products for preventing and improving liver injury and secondary symptoms thereof, wherein the small molecular peptide is free active peptide naturally extracted from chicken.
Preferably, the small molecule peptide is dipeptide Cyclo (L-Phe-L-Phe).
Preferably, the dipeptide Cyclo (L-Phe-L-Phe) has the structural formula:
preferably, the dipeptide Cyclo (L-Phe-L-Phe) includes natural products of animal, vegetable or microbial origin.
Preferably, the dipeptide Cyclo (L-Phe-L-Phe) includes compounds of the same or similar structure synthesized by fermentation, eukaryotic expression systems or artificially.
Preferably, the liver damage includes liver damage and associated clinical symptoms caused by chemical agents, such as alcohol, drugs, or carbon tetrachloride.
Preferably, the liver damage includes liver damage caused by emotional stress, environmental pollution, pathogenic microorganisms or viruses, and related clinical symptoms.
Preferably, the product comprises a modifier, a composition, a functional food, a health food or a medical product synthesized by taking the small molecular peptide as a lead compound.
Preferably, the liver injury comprises altered liver function caused by oxidative stress.
Preferably, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken by using an organic reagent, wherein the organic reagent is one or more of acetic acid, diethyl ether, citric acid, formic acid, tartaric acid, malic acid, acetone, chloroform, ethyl acetate, dichloromethane, benzene and petroleum ether.
Preferably, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken by using acetic acid and diethyl ether as organic reagents.
As a preferable scheme, the extraction method of the dipeptide Cyclo (L-Phe-L-Phe) comprises the following steps:
and 4, dissolving the semi-finished product in a buffer solution, eluting through an ODS-HG-5 liquid chromatographic column, collecting fractions and drying to obtain dipeptide Cyclo (L-Phe-L-Phe) powder.
Preferably, the buffer solution is 80wt% acetonitrile aqueous solution.
In order to solve the technical problems, the invention also provides a product for preventing and improving liver injury and secondary symptoms thereof, which is prepared by adopting the small molecular peptide.
Preferably, the product is an oral preparation or an injection, and the oral preparation is a capsule, a tincture, a pill, a microemulsion, a tablet or a granule.
Preferably, the product is a beverage or food, the beverage is a carbonated beverage, a fruit juice beverage, a lactobacillus beverage, a sports beverage or a soybean milk beverage, and the food is a biscuit, a chocolate, a candy, a chewing gum, a snack, a jelly snack, bread, tofu, yogurt or meal replacement powder.
Compared with the prior art, the embodiment of the invention has the following beneficial effects:
the research of the application discovers that the chicken-derived dipeptide Cyclo (L-Phe-L-Phe) has strong oxidation resistance, the oxidation resistance is positively correlated with the concentration, the strong oxidation resistance is expressed in cells, the oxidative stress state of the liver is improved by increasing the activity of antioxidant enzyme GPx, reducing the level of oxidative stress product malondialdehyde MDA and the like, the liver volume is obviously improved to be reduced, the liver weight is reduced, liver fibrosis and liver cell apoptosis and other liver pathological injuries are obviously improved, the CPP has small cytotoxicity, wide medication window and high safety. Can be used for preparing functional food, health food, medicinal products, and composition for improving oxidative stress induced liver injury, or used as raw material for relieving and improving liver injury and related secondary symptoms.
Drawings
FIG. 1 shows the result of purity measurement of CPP in the first embodiment of the present invention;
FIG. 2 is the result of characterization and identification of CPP in the first embodiment of the present invention;
FIG. 3 is the result of the measurement of the in vitro antioxidant capacity of CPP in example II of the present invention;
FIG. 4 is the result of measurement of the effect of CPP on the activity of hepatocytes in example three of the present invention;
FIG. 5 is the result of detecting that CPP reduces ROS level in human hepatoma cells in the fourth embodiment of the present invention;
FIG. 6 is the overall morphology of chick embryo liver under the stereoscope and statistical results of liver weight and liver index in the fifth embodiment of the present invention;
FIG. 7-is the result of H & E staining of chick embryo liver in example five of the present invention;
FIG. 8 is the result of measuring the MDA level of the liver of the chick embryo treated with CPP to reduce AAPH in the sixth embodiment of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to find an active compound for preventing and ameliorating liver injury with good efficacy and high safety, the inventors first understood the pathological mechanism of liver injury. Alcohol, drugs, CCl4 and other substances generate a large amount of Reactive Oxygen Species (ROS) in the liver metabolic process, and the ROS damage biomacromolecules such as DNA, protein and lipid, damage signal pathways such as Nrf2 in cells and cause cell function damage, and induce cells to undergo apoptosis, scorching, iron death, necrosis and the like, and finally cause liver tissue dysfunction or organic damage.
In addition, in recent years, there has been much interest in the research and industrial application of small molecule bioactive peptides, particularly bioactive peptides of natural origin. The bioactive peptide has various sources, including plant, animal and microbial sources, and the bioactive peptide of natural source generally has the characteristic of high safety. The inventor finds that the chicken peptide has extensive biological activities such as oxidation resistance, inflammation resistance, immunoregulation and the like, and the chicken peptide mainly comprises oligomeric mixed peptide in the market at present, and the action mechanism and the material basis of the chicken peptide are not clear. The inventor finds that free small molecule dipeptide Cyclo (L-Phe-L-Phe) naturally existing in chicken origin, which is abbreviated as CPP, has good antioxidant effect, but the research on the small molecule is not deep at present, and the research on the small molecule in liver diseases is blank, so the small molecule forms the core content of the invention.
The application provides an application of small molecular peptide in preparing products for preventing and improving liver injury and secondary symptoms thereof, wherein the small molecular peptide is free active peptide naturally extracted from chicken sources, and specifically is dipeptide Cyclo (L-Phe-L-Phe).
In one embodiment, the dipeptide Cyclo (L-Phe-L-Phe) has the formula:
in one embodiment, the dipeptide Cyclo (L-Phe-L-Phe) may be a natural product derived from animals, plants, microorganisms, or the like, or a compound having the same structure or a similar structure synthesized by fermentation, eukaryotic expression system, artificial synthesis, or the like.
In one embodiment, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken meat by using an organic reagent, wherein the organic reagent is at least one of acetic acid, diethyl ether, citric acid, formic acid, tartaric acid, malic acid, acetone, chloroform, ethyl acetate, dichloromethane, benzene, petroleum ether, and the like.
In one embodiment, the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken meat using acetic acid and diethyl ether as organic reagents.
Since the CPP prepared is developed as a final product of functional foods, health foods, pharmaceuticals and compositions, it is preferable to select acetic acid and ether as the extraction solvent from the viewpoint of safety, but not limited thereto, and it is also within the contemplation of the present invention that the extract obtained by extraction using other organic solvents can remove organic residues by conventional means.
In one embodiment, the method for extracting the dipeptide Cyclo (L-Phe-L-Phe) specifically comprises the following steps:
and 4, dissolving the semi-finished product in a buffer solution, eluting through an ODS-HG-5 liquid chromatographic column, collecting fractions and drying to obtain dipeptide Cyclo (L-Phe-L-Phe) powder.
In one embodiment, the liver injury is liver injury and related clinical symptoms caused by chemical agents such as alcohol, drugs, carbon tetrachloride and the like, and also liver injury and related clinical symptoms caused by emotional stress, environmental pollution, pathogenic microorganisms, viruses and the like.
In one embodiment, the product can be a modified product synthesized by using the small molecule peptide as a lead compound, a mixture containing the unpurified small molecule peptide, a composition, a functional food, a health food, a medical product and the like.
In addition, the CPP may be combined with a carrier such as a conventional excipient, and may further contain an auxiliary material or additive such as a preservative, an antioxidant, a coloring agent, and a sweetener, if necessary.
In one embodiment, the product can be an oral preparation or an injection, and specifically can be a capsule, a tincture, a pill, an oral liquid, a tablet, a granule and the like.
The research of the application finds that the CPP has strong oxidation resistance, the index of the oxidative free Radical absorption Capacity (ORAC) is increased along with the increase of the concentration, and meanwhile, the CPP can obviously reduce the ROS level of a human hepatoma cell HepG2 and has strong oxidation resistance in the cell; the CPP can obviously improve the AAPH-induced liver volume reduction, the liver weight reduction and the pathological liver injury, and obviously reduce the AAPH-induced liver oxidative stress level, which is specifically shown in the reduction of the content of lipid peroxide Malondialdehyde (MDA), and shows that the small molecular peptide has obvious curative effect on preventing and improving the liver injury and the secondary symptoms thereof; the cytotoxicity of the CPP is measured by using MTT, and the CPP has small cytotoxicity, wide application window and high safety.
Example one
The CPP extraction method comprises the following steps:
1) Preparing chicken powder: cutting fresh chicken, drying, pulverizing at 10000rpm/min with a pulverizer, pausing for one minute every 2 minutes, pulverizing cumulatively for 5 minutes to obtain dry powder, and sieving with a 40-mesh sieve to obtain chicken powder.
2) Extracting small molecule peptides: weighing 135g of the chicken powder, adding 1 mol/L (mol/L) acetic acid solution to prepare 200mg/mL chicken acetic acid solution, immediately oscillating to fully extract after adding equal volume of ether solution, standing for 10 minutes, collecting ether layer solution, and drying by using a rotary evaporator to obtain dry powder, namely the crude powder of the small molecular peptide.
3) Preparing an active site containing CPP: dissolving the obtained dry powder in 80% acetonitrile/water solution, balancing by using the 80% acetonitrile/water solution as a buffer solution, loading a sample on a Sephadex hh-20 chromatographic column (6 multiplied by 18 cm), carrying out isothermal elution, collecting eluted fractions every 80mL of volume, identifying fractions containing CPP by using mass spectrometry, and drying the fractions containing the CPP to prepare the small-molecule peptide semi-finished product powder.
4) Preparing CPP: dissolving the dried powder prepared in the step 3) in 8% acetonitrile/water, balancing by using 8% acetonitrile/water solution as a buffer solution, then loading the sample on an ODS-HG-5 liquid chromatographic column, balancing by using the same buffer solution, collecting an elution fraction at a corresponding retention time of the CPP compared with a CPP standard, and then drying the CPP to prepare the CPP powder.
5) And (3) identifying CPP powder: dissolving the CPP powder prepared in the step 4) in dimethyl sulfoxide (DMSO), and detecting the purity of the CPP powder by using a high performance liquid chromatography-ultraviolet detector in series, wherein the chromatographic conditions are as follows: the liquid phase is separated by a reverse phase system, 0.1% formic acid water is used as an elution phase A, and acetonitrile is used as an elution phase B. Column model (HSS T3.8 μm, C18, 100X 2.1mm, waters Acquity). The elution procedure was as follows: 0min:25% by weight of B; 0.25min; 1.5min; 3 min; 5.5min; 9min; 9.5min; 15min. The flow rate was 0.25mL/min. The column temperature was 40 ℃. The sample size was 2. Mu.L. The detection wavelength of the ultraviolet detector is 254nm. And then, performing characterization verification on the CPP powder by using a high performance liquid chromatography tandem mass spectrum, wherein the conditions of the chromatography and the mass spectrum are as follows: LC-MS/MS analysis was performed by double ternary high performance liquid chromatography (DGLC) tandem quadrupole orbitrap high resolution mass spectrometer (Q-active) in Sermer fly (Thermo Fisher Scientific). The liquid phase is separated by a reverse phase system, 0.1% formic acid water is used as an elution phase A, and acetonitrile is used as an elution phase B. Column type (HSS T3.8 μm, C18, 100X 2.1mm, waters Acquity). The elution procedure was as follows: 0min:25% by weight of B; 0.25min; 1.5min; 3min; 5.5min; 9 min; 9.5min; 15min, 25%. The flow rate was 0.25mL/min. The column temperature was 40 ℃. The sample size was 2. Mu.L. The mass spectrum scanning mode is positive and negative ion switching full scan. The mass range of the primary mass spectrum scanning is 100-1000m/z, the resolution is 35,000, and the maximum injection time is 35ms; the MS2 scan resolution was 17,500, maximum injection time 50MS, collision energy 35eV, and scan isolation window set to 0.8m/z. The spraying voltage of the positive and negative ion capillary tubes is set to be 3.0kV, the temperature of the capillary tube is 350 ℃, and the heating temperature of the auxiliary device is 320 ℃. S-lens Rf was set to 60.
As shown in FIG. 1, the purity of the CPP powder prepared in this example was as high as 95% or more. As shown in FIG. 2, which is the result of mass spectrometric identification of CPP powder, the product obtained in this example was indeed CPP.
Example two
In-vitro antioxidant capacity determination of CPP: the in vitro antioxidant Capacity of CPP is measured by the Oxidative Radical Absorption Capacity (ORAC) test.
ORAC experimental process: adding 20 mu L of aqueous solution containing CPP with different concentrations into a 96-well plate to be set as an experimental group, wherein the CPP concentrations are respectively 0.4mmol/L, 0.8mmol/L and 1.5mmol/L, simultaneously setting a blank control group and a model group, adding 40 mu L of oxidative stress inducer AAPH into the experimental group and the model group, adding 20 mu L of phosphate buffer solution into all the groups, finally, quickly placing the 96-well plate into a fluorescence microplate reader with the set temperature of 37 ℃ after adding 20 mu L of fluorescein sodium into all the groups to start measurement, measuring one point every 2min, measuring 2h and ORAC values in total, and calculating the protection integral area corresponding to 1 mu mol/L Trolox on a fluorescence attenuation curve as a standard control.
The results are shown in FIG. 3, the in vitro antioxidant capacity of CPP is dose-dependent and increases with increasing dose, which indicates that the small molecule peptide has direct free radical scavenging activity, and can neutralize the oxidizing capacity of free radicals after entering cells or tissues, thereby reducing the damage to cell molecules.
EXAMPLE III
Determination of CPP effect on hepatocyte viability:
the cell viability is detected through a classical MTT experiment, MTT is yellow powder and is fully called thiazolyl blue, the detection principle is that MTT can penetrate through a cell membrane to enter cells, amber dehydrogenase in mitochondria of living cells is reduced into water-insoluble purple needle-shaped crystal formazan and is deposited in the cells, and dead cells do not have the function; crystals formed by living cells can be dissolved by dimethyl sulfoxide (DMSO), and the detected light absorption value can indirectly reflect the survival and growth conditions of the cells, so that the toxic and side effects of the medicine can be evaluated.
The specific experimental process is as follows:
1) The human hepatoma cell line HepG2 which is in a good state and is in a logarithmic growth phase and the growth density of which reaches about 80 percent in a culture dish is spread in a 96-well plate, 7000 cells are arranged in each well, and the cell is cultured in an incubator overnight and then subjected to a cell dosing experiment;
2) Discarding cell culture solution in a 96-well plate, adding 100 mu L of prepared CPP solution with serial concentrations into each well, wherein the concentration distribution of the CPP is 0.768nmol/L, 3.84nmol/L, 19.2nmol/L, 96nmol/L, 0.48 mu mol/L, 2.4 mu mol/L, 12 mu mol/L, 60 mu mol/L and 80 mu mol/L, simultaneously setting a blank control group, setting 6 multiple wells for each dose group, and placing the dose groups in an incubator for continuous culture for 24 hours;
3) After the drug effect is finished, adding 10uL of prepared 5mg/ml MTT solution into each hole, placing the MTT solution into an incubator to continue culturing for 3h, then discarding the stock solution, adding 200uL DMSO into each hole, shaking the MTT solution at room temperature for 10min, detecting the light absorption value (A) of each 570nm hole by using an enzyme-linked immunosorbent assay, and calculating according to the following formula:
cell survival (%) = (a test group-a blank)/(a control group-a blank) x 100%.
The results are shown in FIG. 4, the IC50 of CPP is about 60 μ M, which shows that CPP has less toxicity to liver cells, larger safety window and better safety.
Example four
Measurement of CPP to reduce ROS levels in human hepatoma cells:
oxidative stress often induces the production of large amounts of ROS that can impair cellular function and even induce cell death; therefore, this example investigates the effect of CPP on the increase in cellular ROS levels induced by the oxidative stress inducer AAPH by ROS probe and flow cytometry.
The specific experimental process is as follows: the method comprises the following steps of (1) paving a human hepatoma cell line HepG2 which is in a good selection state and is in a logarithmic growth phase and has the growth density of about 80% in a culture dish in a 6-well plate, placing 5.5 x 104 cells in each well in an incubator for overnight culture, pre-protecting the HepG2 cells of other experimental groups for 2 hours by using CPP with different concentrations except a normal control group and a model group, wherein the CPP concentrations are 600nmol/L and 6000nmol/L, and then adding AAPH to samples of the model group and the experimental group for co-treatment for 1 hour; after the drug treatment, the cells were collected and incubated with ROS fluorescence probe DCFH-DA for 20 minutes, and the fluorescence intensity of the fluorescence probe was measured with a flow-type instrument to characterize intracellular ROS levels.
The result is shown in fig. 5, CPP can significantly reduce the ROS level of AAPH induced human hepatoma cells, which indicates that CPP has strong antioxidant capacity in cells.
EXAMPLE five
And (3) determining the effect of the CPP on relieving oxidative stress induced liver injury:
in addition to being used to study the physiological processes of embryonic development, chick embryo models are also widely used to evaluate the hepatotoxicity of drugs or chemical agents. For example, when sodium glutamate is studied in a chick embryo model, it was found that a high dose of sodium glutamate has potential hepatotoxicity, which inhibits hepatocyte differentiation, results in decreased hepatocyte chord density, widens tissue lacunae, and further induces portal vein hyperemia and vein wall fibrosis. Meanwhile, the experiments of chick embryos show that a large amount of edible salt damages an antioxidant signal pathway Nrf2-HO-1 and induces excessive active oxygen generation, and finally induces hepatocyte apoptosis and liver fibrosis. Therefore, the invention utilizes the chick embryo model to research the protective effect of the CPP on the oxidative stress induced liver injury.
The specific experimental process is as follows:
1) Randomly dividing chicken embryos into 5 groups of a control group (0.72% NaCl solution, bird physiological saline), an oxidative stress model group (0.75 mu mol/eggAAPH), a low concentration drug test group (AAPH +0.1 nmol/eggCPP), a medium concentration drug test group (AAPH +0.5 nmol/eggCPP), and a high concentration drug test group (AAPH +1 nmol/eggCPP);
2) AAPH and CPP of different concentrations were administered simultaneously every two days starting on day 2 of chick embryo incubation, and the control group was administered the same volume of 0.72% nacl solution for 17 days;
3) After the administration, the embryo is taken out by opening the shell, the weight of the chick embryo liver is respectively weighed, the liver shape is observed under a stereoscope, and then the chick embryo liver is fixed in paraformaldehyde for a week, embedded in paraffin and sliced in paraffin, and then H & E staining is carried out to observe the structure of the liver tissue.
The results are shown in fig. 6 and 7, AAPH inhibits chick embryo liver development, resulting in decreased chick embryo liver weight, and the results of staining pathological liver sections also indicate that AAPH causes severe liver damage, manifested as liver bleeding and inflammatory cell infiltration around portal veins; and the CPP can reverse the chick embryo development delay caused by AAPH, the chick embryo liver weight is obviously increased, and the liver injury is improved to some extent.
EXAMPLE six
CPP reduction of MDA levels assay of AAPH treated chick embryo livers:
malondialdehyde (MDA) is the most important lipid peroxidation product, and the relative content change of the Malondialdehyde (MDA) can reflect the degree of oxidative stress lipid peroxidation, and the content of the index can reflect the oxidative stress level of embryonic tissues.
The specific experimental process is as follows:
1) Weighing a part of each group of livers taken out in the fifth embodiment for detecting the MDA content, adding phosphate buffer solution PBS into each liver tissue to prepare liver homogenate, centrifuging at 4 ℃,12000rpm for 10 minutes, taking supernatant, and determining the protein concentration in the sample by using a BCA kit;
2) Then, lipid oxidation (MDA) kit provided by the bio-yunnan bio ltd was used to detect the MDA content of each group of liver tissues, and the following standards with concentration gradients were prepared according to the kit instructions: 1 mu mol/L, 2 mu mol/L, 5 mu mol/L, 10 mu mol/L, 20 mu mol/L and 50 mu mol/L, preparing MDA working solution, respectively adding 100uL of MDA working solution into 50uL of standard substance, blank lysate and sample to be detected, uniformly mixing, heating in boiling water for 15min, taking out, cooling to room temperature, and centrifuging at 3000rpm/min and 25 ℃ for 10min;
3) Transferring 100 mu L of centrifuged supernatant into a 96-well plate, detecting an OD value at a wavelength of 532nm by using an enzyme-labeling instrument, preparing a standard curve according to the data of the standard sample, calculating the MDA content of each sample to be detected according to the formula of the standard curve and the OD value corresponding to the sample, and standardizing by using the protein content of the sample.
As a result, AAPH significantly increased the level of MDA in liver tissue, while administration of CPP at various concentrations effectively reduced the MDA content, as shown in FIG. 8. The results show that CPP can reduce the level of lipid oxidation in tissues, reduce the accumulation of lipid oxidation products and damage to the addition modification of biological macromolecules such as proteins and genes.
The above-mentioned embodiments are provided to further explain the objects, technical solutions and advantages of the present invention in detail, and it should be understood that the above-mentioned embodiments are only examples of the present invention and are not intended to limit the scope of the present invention. It should be understood that any modifications, equivalents, improvements and the like, which come within the spirit and principle of the invention, may occur to those skilled in the art and are intended to be included within the scope of the invention.
Claims (7)
1. The application of the chicken-derived small molecular peptide in preparing the product for preventing and improving the liver injury and the secondary symptoms thereof is characterized in that the small molecular peptide is chicken-derived free active peptide which is extracted from nature, the small molecular peptide is dipeptide Cyclo (L-Phe-L-Phe), and the liver injury is the change of the liver function caused by oxidative stress.
2. The use of the chicken-derived small molecule peptide of claim 1 in the preparation of a product for preventing and improving liver injury and its secondary symptoms, wherein the liver injury is caused by chemical agents, emotional stress, environmental pollution, pathogenic microorganisms or viruses, and related clinical symptoms, and the chemical agents are alcohol, drugs or carbon tetrachloride.
3. The use of the chicken-derived small molecule peptide of claim 1 in the preparation of a product for preventing and ameliorating liver injury and its secondary symptoms, wherein the product comprises a medical product.
4. The use of the chicken-derived small molecule peptide of claim 1 in the preparation of a product for preventing and ameliorating liver damage and its secondary symptoms, wherein the dipeptide Cyclo (L-Phe-L-Phe) is extracted from chicken meat using acetic acid and diethyl ether as organic reagents.
5. The use of the chicken-derived small molecule peptide of any one of claims 1 to 4 in the preparation of a product for preventing and improving liver injury and secondary symptoms thereof, wherein the dipeptide Cyclo (L-Phe-L-Phe) is extracted by the following method:
step 1, drying and crushing chicken to obtain chicken powder;
step 2, adding an organic reagent into the chicken powder to perform small molecule peptide extraction, collecting the organic solvent and drying to obtain crude small molecule peptide powder, wherein the organic reagent is acetic acid and diethyl ether;
step 3, dissolving the powder in an acetonitrile water solution with the concentration of 80%, eluting the powder through a Sephadex hh-20 chromatographic column, collecting fractions and drying the fractions to obtain small molecular peptide semi-finished product powder;
and 4, dissolving the semi-finished product in 8% acetonitrile water solution, eluting by an ODS-HG-5 liquid chromatographic column, collecting fractions and drying to obtain dipeptide Cyclo (L-Phe-L-Phe) powder.
6. The application of dipeptide Cyclo (L-Phe-L-Phe) in preparing products for preventing and improving liver injury and secondary symptoms thereof is characterized in that the dipeptide Cyclo (L-Phe-L-Phe) is a natural product of animal, plant or microorganism sources or a compound which is artificially synthesized and has the same structure.
7. The use of the dipeptide Cyclo (L-Phe-L-Phe) according to claim 6 for the preparation of a product for preventing and ameliorating liver damage and its secondary symptoms, wherein the artificial synthesis comprises fermentative synthesis or eukaryotic expression system synthesis.
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