CN104387451B - Clam enzymolysis oligopeptide with repairing effect on non-alcoholic fatty liver disease cell model and preparation method thereof - Google Patents
Clam enzymolysis oligopeptide with repairing effect on non-alcoholic fatty liver disease cell model and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a clam enzymolysis oligopeptide with a repairing effect on a non-alcoholic fatty liver disease cell model, which is characterized by comprising the following amino acid sequences: gln Leu Asn Trp Asp, the invention also relates to a preparation method of clam enzymolysis oligopeptide with repair effect on the non-alcoholic fatty liver disease cell model, compared with the prior art, the invention has the advantages that: the NAFLD cell model is established by applying palmitic acid induction, damaged liver cells in vivo are fully simulated, and the result shows that after 15 mu g/mL palmitic acid is induced for 48 hours, the TG content of the established NAFLD in-vitro cell model is obviously increased compared with normal liver cells, and the oil red O staining shows that the number of lipid drops in the model group cells is increased compared with the normal group cells, and the cell death rate is low, the repeatability is good, thereby showing that the modeling method is simple and easy to implement, and meanwhile, the clam enzymolysis oligopeptide with the obvious repair effect on the NAFLD cell model is obtained by separating from clams.
Description
Technical Field
The invention relates to an extract obtained from an animal and a preparation method thereof, in particular to a clam extract having a repairing effect on a non-alcoholic fatty liver disease cell model and a preparation method thereof.
Background
Meretrix Meretrix L belongs to the phylum mollusca, family veneridae and genus Meretrix, the shell of the Meretrix Meretrix L is thick, the size of the two shells is equal, the Meretrix Meretrix L likes to grow on fine sandy beaches near an inner gulf and a river mouth where fresh water is injected, and the Meretrix Meretrix L is one of four cultured shellfish in China. The clam meat has delicious taste and contains protein, fat, trace elements and the like which are necessary for human bodies. The history of using the hard clam to treat diseases in China is long, and the book Ben Cao gang mu records that the hard clam can treat diseases such as sore, furuncle, swelling and toxin, mass elimination, alcoholism relief and the like.
Recent research shows that the clam extract has the effects of resisting tumor, resisting oxidation, reducing blood sugar, reducing blood fat, reducing blood pressure and the like, and is a health food to be developed. Among them, NAFLD (nonalcoholic fatty liver disease) is a pathological syndrome characterized mainly by liver cell steatosis and fat accumulation, and fat metabolism is affected by hormones such as estrogen, growth hormone, cortisol, glucagon, insulin, etc., which can rapidly convert many other substances such as proteins, saccharides, etc. into lipid substances through metabolism in a short time and accumulate in the human body, while the liver cannot completely remove the redundant fat, thereby causing the formation of fatty liver. At present, conservative treatments such as weight-losing treatment, lipid-lowering treatment, angiotensin converting enzyme inhibitor and the like are mainly adopted in the treatment method, but a specific method is not available in the conservative treatment method at present, and the effect is poor. In recent years, researchers in our country have conducted a great deal of research on the protection of liver damage by extracting bioactive substances from marine mollusks. Cerebroside is extracted from starfish and sea cucumber by Zhang Bei and the like to research the influence of cerebroside on the liver lipid metabolism of fatty liver rats, and the result shows that the cerebroside can reduce liver lipid accumulation.
However, epidemiological data show that NAFLD is not a liver disease, but rather is a centralized embodiment of metabolic syndrome-related components such as obesity, hyperlipidemia, insulin resistance and the like in the liver, wherein insulin resistance and genetic susceptibility are closely related to the pathogenesis of NAFLD. In the experiment, the oligopeptide is obtained by carrying out enzymolysis on the clam through protease, and acts on a NAFLD in-vitro cell model established by inducing normal liver Chang lever cells, namely 'human Zhang' liver cells through palmitic acid so as to discuss the repair effect of the clam enzymolysis oligopeptide on the NAFLD cell model and provide experimental basis for developing clam functional food.
Disclosure of Invention
The invention aims to solve the technical problem of providing a clam enzymolysis oligopeptide with high non-alcoholic fatty liver disease cell model repairability aiming at the current situation of the prior art.
The invention aims to solve another technical problem of providing a preparation method of clam enzymolysis oligopeptide with high non-alcoholic fatty liver disease cell model repairability aiming at the current situation of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: the clam enzymolysis oligopeptide with the repairing effect on the non-alcoholic fatty liver disease cell model is characterized by comprising the following amino acid sequences: gln Leu Asn Trp Asp are provided.
The invention also provides a preparation method of the clam enzymolysis oligopeptide with the repairing effect on the nonalcoholic fatty liver disease cell model, which is characterized by comprising the following steps:
firstly, establishing a non-alcoholic fatty liver disease cell model, placing normal liver cells in DMEM culture solution at 37 ℃ and 5% CO2Culturing in the incubator, discarding culture solution after the cell adherence reaches 80%, digesting with 0.25% of trypsin, subculturing, selecting cells with good growth state, inducing normal hepatocytes with 15 mug/mL palmitic acid, collecting cells after treating for 12h, 24h, 48h and 72h, determining Triglyceride (TG) content, and selecting induction time for obviously increasing the TG content of the normal hepatocytes and enabling the cells to grow well to establish an in vitro non-alcoholic fatty liver disease cell model;
secondly, removing shells and viscera of the clams, putting the clams into distilled water, slightly stirring, washing off impurities, homogenizing, and storing at-20 ℃ or-80 ℃ for later use;
thirdly, performing enzymolysis on the clam homogenate prepared in the second step, respectively intercepting 3 different molecular segment enzymolysis solutions with the molecular weights of less than 5KDa, 5KDa-8KDa and more than 8KDa through an ultrafiltration membrane, performing G-25 sephadex chromatography, collecting each obtained peak, respectively acting the obtained product on the nonalcoholic fatty liver disease cell model cell in the first step, collecting enzymolysis solution with the largest content reduction rate of the nonalcoholic fatty liver disease cell model cell TG, then enabling the collected maximum peak enzymolysis solution to pass through a Zorbax SB-C18 chromatographic column at 280nm to obtain two elution peaks, collecting the large elution peak, performing high-efficiency liquid phase detection, and determining the purity of the large elution peak to show as a single component, and determining the amino acid sequence of the large elution peak to confirm to be Gln Leu Asn Trp Asp.
Further, the palmitic acid in the step (i) is 15 mug/mL, and normal liver cells are induced.
Furthermore, the degradation rate of the TG content of the nonalcoholic fatty liver disease cell model cell in the third step is maximally 5KDa molecular weight or less molecular fragment enzymatic hydrolysate, and the degradation rate of the TG content is 54.2%.
Further, in the third step, 5 proteases are respectively used for enzymolysis of the clam homogenate, wherein the 5 proteases are respectively neutral protease, trypsin, alkaline protease, pepsin and papain.
Further, in the third step, the enzyme which maximizes the reduction rate of TG content in the non-alcoholic fatty liver disease cell model cell is alkaline protease, the enzymolysis condition is 40 ℃, the pH is 9.5, the feed-liquid ratio is 1:2, the enzymolysis time is 8h, and the enzyme dosage is 1000U/g.
Compared with the prior art, the invention has the advantages that: the NAFLD cell model is established by using palmitic acid induction, damaged hepatic cells in vivo are fully simulated, and the result shows that after 15 mu g/mL palmitic acid is used for inducing for 48 hours, the TG content of the established NAFLD in-vitro cell model is obviously increased compared with that of normal liver cells, the fat drop quantity in the model group cells is found to be more than that of the normal group cells by oil red O dyeing, the cell death rate is low, the repeatability is good, the modeling method is simple and easy to implement, meanwhile, the invention separates the clam to obtain clam enzymolysis oligopeptide with obvious repair function to NAFLD cell model, which is characterized in that the lipid peroxidation degree in the cell is relieved, the oxygen free radical removing capability of the cell is enhanced, and the damage degree of the cell can be effectively reduced, further discussing the mechanism of the repair effect of clam enzymolysis oligopeptide on the NAFLD cell model, and carrying out animal in vivo experiments, thereby providing reliable experimental basis for developing liver protection functional food of clams.
Drawings
FIG. 1 is a G-25 sephadex chromatogram of an enzymolysis solution with a molecular segment less than 5 kDa;
FIG. 2 is an analysis chart showing the results of HPLC analysis and determination of the amino acid sequence of a target peptide;
FIG. 3-A is a graph showing the results of oil red O staining of Chang lever hepatocytes in the normal group;
FIG. 3-B is a graph showing the results of oil red O staining of Chang liver cells in the model group;
FIG. 3-C is a graph showing the results of oil red O staining of Chang lever liver cells in the drug group.
Detailed Description
The present invention is further illustrated by the following figures, sequence listing and examples.
Example 1
1. Establishment of NAFLD model
The normal liver Chang lever cells (called normal group cells) are placed in a DMEM culture solution, cultured at 37 ℃ in an incubator with 5% CO2, the culture solution is discarded after the cells are attached to the wall to reach 80%, digested by 0.25% trypsin, and subcultured. Selecting cells with good growth state, inducing the Chang lever cells by using palmitic acid of 15 mu g/mL, collecting the cells after treating for 12h, 24h, 48h and 72h, and determining the content of Triglyceride (TG), wherein the TG detection method is carried out according to the kit instructions. And selecting the induction time which obviously increases the TG content of the normal liver Chang lever cells and ensures that the cells grow well, and establishing an in vitro NAFLD cell model (hereinafter referred to as model group cells).
The result of the content of TG in the normal liver Chang lever cells after being induced by 15 mu g/mL palmitic acid shows that the content of TG in the palmitic acid is obviously increased after the action of the palmitic acid, but after the action of the palmitic acid for 48 hours, the content of TG in the cells is increased most, the content of TG in the cells is increased by about 300 percent compared with that in a normal group, and the growth state of the cells observed under a mirror is good, so that the palmitic acid induction time for establishing a NAFLD cell model is 48 hours (see table 1), and the number of lipid drops in the cells of the model group is found to be more than that in the cells of the normal group through oil red O staining, so that the cell death rate is low, the repeatability is good, and the.
TABLE 1 change in TG content after palmitic acid treatment of Chang lever cells
Note: p <0.05 compared to control.
2. Screening of optimal enzymolysis conditions of hard clam
Removing shell and viscera of Meretrix Linnaeus, adding into distilled water, stirring gently, washing off impurities, homogenizing, and storing at-20 deg.C. The clam homogenate is subjected to enzymolysis by using neutral protease, trypsin, alkaline protease, pepsin and papain, and the enzymolysis conditions are shown in table 2. The selection criteria for the best enzyme species are: and (3) treating the model group cells with the enzymolysis solution at the concentration of 10mg/mL for 24h, and screening the protease with the highest reduction rate of TG content in the model group cells. The orthogonal experimental method for designing the enzyme is designed from 4 levels of 5 factors including enzymolysis time, enzymolysis temperature, enzyme adding amount, feed-liquid ratio and pH, and the screening standard of the optimal enzymolysis condition is the same as the optimal enzyme species. The calculation method of the TG content reduction rate is as follows: the TG content reduction rate (%) (model group TG content-drug group TG content)/model group TG content × 100%.
TABLE 2 enzymatic conditions for different proteases
3. Screening result of clam optimum enzymolysis condition
The product was collected by enzymatic hydrolysis of clam homogenate using 5 proteases as in table 2 above, and the results are shown in table 3 by treating model group cells and measuring the intracellular TG content after 24 h. From Table 3, it can be understood that the alkaline protease substrate maximizes the reduction rate of TG content in the cells, and thus the alkaline protease is selected as the optimum enzyme species. An orthogonal experiment is carried out by applying 5-factor-4 levels of alkaline protease design, wherein the levels are respectively enzymolysis time (6h, 8h, 10h and 12h), enzymolysis temperature (40 ℃, 45 ℃, 50 ℃ and 55 ℃), material-liquid ratio (1:1, 1:2, 1:3 and 1:4), pH value (9, 9.5, 10 and 10.5) and enzyme adding amount (800U/g, 1000U/g, 1200U/g and 1400U/g), the alkaline protease enzymolysis product of the clam acts on model group cells, the experiment result is shown in table 4, B & gtD & gtC & gtA & gtE and the optimal condition combination is A1B2C2D2E2, the enzymolysis product obtained under the enzymolysis condition of 40 ℃, pH of 9.5, material-liquid ratio of 1:2, enzymolysis time of 8h and enzyme addition amount of 1000U/g has the best effect, and the TG content reduction rate can reach 54.2% (the specific results are shown in tables 3 and 4).
TABLE 3 variation of TG content in different protease enzymatic products on NAFLD cell models
Note: compared with the model group, P is less than 0.05.
TABLE 4 results of orthogonal experiments with alkaline protease enzymolysis
4. Determination result of clam enzymolysis polypeptide sequence
Performing ultrafiltration on the optimal enzyme species and the enzymolysis products obtained under the optimal enzymolysis conditions, respectively intercepting molecular segment products of less than 5kDa, 5kDa-8kDa and more than 8kDa, performing optimal molecular segment screening standard with the optimal enzyme species, performing ultrafiltration on the clam enzymolysis liquid obtained under the optimal enzymolysis conditions to obtain 3 different molecular segment products of less than 5kDa, 5kDa-8kDa and more than 8kDa, and acting on cells of a model group for 24 hours, wherein the detection result of TG content is shown in Table 5. As can be seen from Table 5, the < 5kDa molecular fragment maximizes the reduction in TG content in the cells of the model group from 36.12. mu. mol/G to 14.29. mu. mol/G, and therefore the enzymatic hydrolysate of the < 5kDa molecular fragment was selected for G-25 Sephadex chromatography. And then carrying out G-25 sephadex chromatography on the < 5kDa enzymolysis solution, collecting each obtained peak, acting the obtained product on cells of a model group to obtain a peak with the best effect, carrying out HPLC analysis, determining the amino acid sequence (the result is shown in figure 1 and figure 2), obtaining 4 peaks (namely a peak I, a peak II, a peak III and a peak IV) as shown in figure 1, and after collecting each peak to act on a NAFLD cell model group, leading the peak I component to reduce the content of TG in the cells most obviously. Performing high performance liquid chromatography elution on the peak I component to obtain 2 peaks as shown in figure 2, wherein the peak is detected to be oligopeptide consisting of 5 amino acids, and the amino acid sequence of the peak is as follows: Gln-Leu-Asn-Trp-Asp.
TABLE 5 variation of protease enzymatic products of different molecular fragments on the intracellular TG content in NAFLD model cells
Note: p <0.05 compared to model group.
Example 2 oligopeptide obtained by alkaline protease enzymolysis of Meretrix meretrix Linnaeus has obvious repairing effect on NAFLD cell model
Inoculating normal group cells and model group cells on a cover glass in a six-hole culture plate, abandoning nutrient solution after 24h to divide the nutrient solution into three groups of cells, namely normal group cells, model group cells and model group cells (hereinafter referred to as drug group) acting according to clam zymolyte prepared in example 1, culturing the normal group cells and the model group cells conventionally, treating the drug group cells at the drug concentration of 10mg/mL, and finishing the experiment after culturing for 24 h. Fixing cells with 10% neutral formaldehyde for 10min, adding oil red O staining solution, incubating for 15min, discarding staining solution, washing with PBS, staining with hematoxylin for 5min, washing with PBS, observing red granules in cytoplasm as positive staining of lipid drop under microscope, carefully distinguishing size and number of red granules, and taking pictures.
The results are shown in FIG. 3(A, B, C), in which the liver cells of the normal group, the model group and the drug group Chang lever liver cells were stained with oil red O, and the cells of the normal group were in an epithelial-like form, had protrusions around the cells, had good extensibility, and had a small amount of lipid droplets in the cytoplasm; the number of the model group cell processes is reduced, the morphology becomes round, and the number of lipid droplets in cytoplasm is obviously increased; the medicinal cells have protrusions, and the number of lipid drops in cytoplasm is obviously reduced, which indicates that the NAFLD cell model damage is relieved. Therefore, the oligopeptide obtained by enzymolysis of the clam with alkaline protease has obvious repairing effect on the NAFLD cell model.
Example 3 determination of indicators of hepatocyte function
Respectively measuring the contents of indexes such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Malondialdehyde (MDA), glutathione S transferase (GSH-ST), gamma-glutamyltranspeptidase (gamma-GT), superoxide dismutase (SOD) and the like in the Chang lever cells of the normal group, the model group and the drug group, and carrying out the detection method according to the kit instruction. The detection results of the liver cell function indexes are shown in Table 6, and it can be seen that the model group with the contents of MDA, GSH-ST, ALT, AST and gamma-GT is obviously higher than the normal group, the level of the drug group after the clam oligopeptide effect is obviously reduced, and the model group has statistical significance (P is less than 0.05) compared with the model group. The SOD content is reduced in the model group, the level of the drug group is increased, but the SOD content has no statistical significance compared with the model group. ALT is mainly present in liver cytoplasm and is the most sensitive detection index of liver function damage recommended by WHO; AST is mainly present in mitochondria, causing a significant increase in AST when severe liver destruction occurs. The MDA content can reflect the degree of lipid peroxidation and the severity of free radical attack on body cells, the SOD can specifically remove oxygen free radicals in vivo, and the SOD activity reflects the oxygen free radical removing capability of the body. GSH-ST is a group of enzymes involved in liver detoxification function, and when hepatocytes are damaged, the enzymes are rapidly released into the blood, resulting in an increase in GSH-ST activity. The experimental result shows that after the NAFLD cell model is subjected to oligopeptide enzymolysis for 24 hours, the SOD content is increased, the MDA and GSH-ST content are reduced, which indicates that the lipid peroxidation degree in the cell is relieved, and the oxygen free radical removing capability of the cell is enhanced; decreased levels of MDA and GSH-ST indicate decreased damage to the cells; meanwhile, the contents of ALT, AST and gamma-GT are obviously lower than those of model group cells, which shows that the damage degree of liver cells is reduced, and the results prove that clam enzymolysis oligopeptide has obvious repair effect on the NAFLD cell model.
TABLE 6 measurement results of hepatocyte function index
Note: p <0.05 compared to normal group; # compared to the model group, P < 0.05.
Sequence listing
<110> Zhejiang ocean academy
<120> clam enzymolysis oligopeptide with repairing function on non-alcoholic fatty liver disease cell model and preparation method thereof
<130> Not published yet
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 5
<212> PRT
<213> Meretrixmeretrix L.
<400> 1
Gln LeuAsnTrp Asp
1 5
QLNWD
- 1 -
Claims (2)
1. A clam enzymolysis oligopeptide with repairing effect on a non-alcoholic fatty liver disease cell model is characterized in that the amino acid sequence is as follows: gln Leu Asn Trp Asp are provided.
2. A method for preparing the clam enzymolytic oligopeptide with repairing effect on the nonalcoholic fatty liver disease cell model according to claim 1, which comprises the following steps:
firstly, establishing a non-alcoholic fatty liver disease cell model, placing normal liver cells in DMEM culture solution at 37 ℃ and 5% CO2Culturing in the incubator, discarding culture solution when the cell adherence reaches 80%, digesting with 0.25% trypsin, subculturing, selecting cells with good growth state, inducing normal liver cells with 15 μ g/mL palmitic acid, collecting cells after 48h treatment, and determining Triglyceride (TG) content to establish non-alcoholic lipid in vitroA fatty liver disease cell model;
secondly, removing shells and viscera of the clams, putting the clams into distilled water, slightly stirring, washing off impurities, homogenizing, and storing at-20 ℃ or-80 ℃ for later use;
thirdly, the clam homogenate prepared in the second step is subjected to enzymolysis by alkaline protease under the conditions of 40 ℃, pH 9.5, material-liquid ratio of 1:2, enzymolysis time of 8h and enzyme addition amount of 1000U/G, molecular segment enzymolysis liquid with molecular weight below 5KDa is intercepted by an ultrafiltration membrane, the reduction rate of TG content reaches 54.2 percent, G-25 glucan gel chromatography is used for collecting all obtained peaks, the obtained products respectively act on the nonalcoholic fatty liver disease cell model cells in the first step to screen enzymolysis liquid with the maximum reduction rate of TG content of the nonalcoholic fatty liver disease cell model cells, then the collected maximum peak enzymolysis liquid passes through a Zorbax SB-C18 chromatographic column under 280nm to obtain two elution peaks, the large elution peak is collected, the purity of the large elution peak is detected by a high-efficiency liquid phase to show as a single component, and the amino acid sequence of the large peak is determined, identified as Gln Leu Asn Trp Asp.
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| CN107880113A (en) * | 2017-10-09 | 2018-04-06 | 浙江海洋大学 | A kind of cod collagen peptide and purposes to nonalcoholic fatty liver model with repair |
| CN108277249A (en) * | 2018-01-25 | 2018-07-13 | 浙江海洋大学 | A kind of clam polypeptide production methods to nonalcoholic fatty liver model with repair |
| CN108315378A (en) * | 2018-04-23 | 2018-07-24 | 浙江海洋大学 | A kind of preparation method to Ruditapes philippinarum polypeptide of the hepatic injury with repair |
| CN108888752B (en) * | 2018-05-07 | 2023-02-17 | 广东海洋大学 | Application of isochrysis galbana polypeptide IZP-2 in preparation of medicine for preventing alcoholic liver injury |
| CN108743915B (en) * | 2018-05-07 | 2023-02-10 | 广东海洋大学深圳研究院 | Application of hippocampus polypeptide SP1 in preparation of medicine for preventing alcoholic liver injury |
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