CN117568430A - Antarctic krill peptide with uric acid reducing activity and application thereof - Google Patents
Antarctic krill peptide with uric acid reducing activity and application thereof Download PDFInfo
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- CN117568430A CN117568430A CN202311480398.9A CN202311480398A CN117568430A CN 117568430 A CN117568430 A CN 117568430A CN 202311480398 A CN202311480398 A CN 202311480398A CN 117568430 A CN117568430 A CN 117568430A
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- Prior art keywords
- uric acid
- peptide
- antarctic krill
- krill peptide
- protease
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Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 89
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 title claims abstract description 84
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 229940116269 uric acid Drugs 0.000 title claims abstract description 82
- 241000239366 Euphausiacea Species 0.000 title claims abstract description 59
- 230000001603 reducing effect Effects 0.000 title claims abstract description 35
- 230000000694 effects Effects 0.000 claims abstract description 40
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides antarctic krill peptide with uric acid reducing activity, which is prepared by adding water into antarctic krill meat emulsion to prepare homogenate, then adding protease for enzymolysis, adjusting the pH value of a system to be 6-9, and carrying out enzymolysis for 3-6 hours at 50-55 ℃ to obtain antarctic krill peptide enzymolysis liquid; concentrating and drying the enzymolysis liquid to obtain antarctic krill peptide; the invention also provides a purified product of the antarctic krill peptide. The uric acid reducing effect of the antarctic krill peptide prepared by the invention is obviously enhanced. Cell experiments and in-vitro activity experiments show that the uric acid reducing peptide prepared by the method has a low IC50 value and can obviously reduce the uric acid content of a high uric acid model; animal experiments show that the antarctic krill peptide can obviously reduce the blood uric acid level of mice, increase the urine and fecal uric acid excretion level of the mice, and has a certain protection effect on the kidney function of rats.
Description
Technical Field
The invention belongs to the technical field of deep processing of aquatic products, and particularly relates to antarctic krill peptide with uric acid reducing activity and application thereof.
Background
Hyperuricemia (HUA) is a metabolic disease caused by imbalance in Uric Acid (UA) production and excretion in the body. When uric acid exceeds the metabolic threshold of the body in the human body, uric acid accumulates in large amounts in the body, which easily causes urate crystals to deposit in joints, kidneys and other tissues, thereby causing gout. At the same time, it may be accompanied by various metabolic syndromes, such as kidney stones, diabetes, etc. A number of epidemiological results indicate that prevalence is on an increasing annual trend. Allopurinol and febuxostat have strong anti-hyperuricemia activity, but they cause side effects such as allergy. Therefore, it is urgent to select safe and effective natural substances to alleviate HUA.
Natural peptides are considered as potential substances for anti-hyperuricemia substances. Oyster peptide has good Xanthine Oxidase (XOD) inhibitory activity; the sea fish protein peptide can effectively reduce serum UA level of HUA rats and reduce kidney inflammation induced by HUA; rice derived peptide AAAAMAGPK-NH2 reduced UA levels and kidney injury in HUA mice and exhibited anti-inflammatory activity; bonito hydrolyzates have a pronounced anti-hyperuricemia effect on HUA rats, probably because the peptide readily enters the active site of XOD to act; the walnut protein hydrolysate has remarkable anti-hyperuricase activity on HUA rats. In addition, try-containing peptides showed better XOD inhibitory activity.
Euphausia superba is rich in high-quality proteins and rich in variety of unsaturated fatty acids. They contain all amino acids essential to the human body and are well suited for the manufacture of nutritional supplements. A wide variety of krill oil or extract products have been developed, but the finely processed products of antarctic krill, such as antarctic krill peptide products, are of a lesser variety.
Disclosure of Invention
The invention aims to provide antarctic krill peptide with uric acid reducing activity and application thereof, and the antarctic krill peptide is used for preparing a product for reducing uric acid.
The invention firstly provides euphausia superba peptide with uric acid reducing activity, and the preparation method of the euphausia superba peptide comprises the steps of adding water into euphausia superba meat paste to prepare homogenate, adding protease to carry out enzymolysis, adjusting the pH value of a system to be 6-9, and carrying out enzymolysis for 3-6 hours at 50-55 ℃ to obtain euphausia superba peptide enzymolysis liquid; concentrating and drying the enzymolysis liquid to obtain antarctic krill peptide;
the homogenate is characterized in that the mass ratio of the antarctic krill minced meat to water is 1: 2-1: 5, a step of;
the addition amount of the protease is 0.5 to 4.0 percent
Further, the protease is flavourzyme, alkaline protease or trypsin; preferably, the antarctic krill peptide is prepared by adding 0.8% of alkaline protease for enzymolysis.
Furthermore, the enzymolysis liquid is purified, and the specific steps are as follows:
filtering Euphausia superba peptide with a microporous membrane of 0.22 μm, separating and purifying with SP-SephadexC25 cation exchange column, balancing with 0.02mol/L acetic acid buffer solution of pH4.0 for 90-120min, loading sample, linearly gradient eluting with 0.02mol/L acetic acid buffer solution of pH4.0 containing 0-0.25mol/L NaCl, and collecting the first main peak eluate with highest activity; then further separating and purifying the collected eluent by using a SephadexG15 gel column, wherein the eluent is ultrapure water with the flow rate of 0.4mL/min, and collecting a second main peak; finally, separating and purifying by reverse phase high performance liquid chromatography, wherein the eluent is acetonitrile and ultrapure water, the flow rate is 0.8mL/min, and the seventh main peak with highest activity is euphausia superba uric acid reducing peptide.
Further, the euphausia superba uric acid reducing peptide comprises a peptide segment with a sequence of DIFDPL, EFDGF, FDPLIQ, DLGGGTF, ADIFDPLIQ;
the antarctic krill peptide provided by the invention is used for preparing uric acid-reducing medicines or health-care products.
The invention also provides a uric acid reducing medicament or health-care product, which comprises the antarctic krill peptide.
The uric acid reducing medicine or health care product also comprises other components with uric acid reducing effect.
Compared with the prior art, the invention has the following advantages and effects:
(1) The antarctic krill peptide with uric acid reducing effect prepared by the enzymolysis technology simplifies the production process flow, reduces the production cost and has no pollution.
(2) The uric acid reducing effect of the target antarctic krill peptide prepared by the invention is obviously enhanced. Cell experiments and in-vitro activity experiments show that the uric acid reducing peptide prepared by the method has a low IC50 value and can obviously reduce the uric acid content of a high uric acid model; animal experiments show that the antarctic krill peptide can obviously reduce the blood uric acid level of mice, increase the urine and fecal uric acid excretion level of the mice, and has a certain protection effect on the kidney function of the mice.
(3) The antarctic krill peptide with uric acid reducing effect is extracted, fills up the gap in the market, and has good application and economic prospects.
Drawings
FIG. 1 is a graph of XOD inhibition activity of different euphausia superba lysates,
FIG. 2 is a graph showing the effect of different euphausia superba hydrolysates on uric acid content of the supernatant of HK-2 cells,
figure 3 is a graph of properties and activity of antarctic krill peptides,
FIG. 4 is a graph showing the effect of antarctic krill peptide on serum uric acid levels in mice,
figure 5 is a graph showing the effect of antarctic krill peptide on urine uric acid levels in mice,
FIG. 6 is a graph showing the effect of antarctic krill peptide on uric acid content in mouse feces,
figure 7 is a graph showing the protective effect of antarctic krill peptide on the kidneys of mice,
FIG. 8 is a chromatogram and an activity measurement chart of antarctic krill peptide subjected to cation exchange C25,
FIG. 9 is a chromatogram and an activity measurement chart of antarctic krill peptide through a gel column SephadexG15,
FIG. 10 is a chromatogram and activity assay of antarctic krill peptide by reversed phase high performance liquid phase separation.
Detailed Description
The preparation method of the antarctic krill peptide with uric acid reducing activity provided by the invention comprises the following steps:
(1) Pretreatment of antarctic krill: thawing Euphausia superba in running water, removing head and shell, and mincing with meat mincer.
(2) And (3) enzymolysis reaction: adding a certain mass of water into the antarctic krill meat emulsion to ensure that the feed liquid ratio is 1: 2-1: 5, adding 6 proteases with different amounts, adjusting the pH value of the system to 6-9, and preserving heat and hydrolyzing for 3-6 hours at 50-55 ℃; boiling and inactivating enzyme for 5-10 min to obtain euphausia superba enzymolysis liquid; concentrating and drying the enzymolysis liquid to obtain euphausia superba peptide dry powder;
(3) Screening of zymolyte: and evaluating uric acid reducing activities of different zymolytes through an XOD inhibitory activity and an HK-2 hyperuricemia cell model, and screening the antarctic krill peptide with optimal activity.
Taking the mass of euphausia superba meat as a calculation reference, the addition amount of protease accounts for 0.5-4.0%;
the pH value of the regulating system is regulated by using a NaOH solution or an HCl solution with the concentration of 1mol/L to be 8.5;
the protease in the step (2) comprises commercial flavourzyme, alkaline protease, pancreatin, neutral protease, composite protease and papain;
the enzyme adding amount in the step (2) is 0.1-4%;
as a preferable scheme, water with a certain mass is added into the minced euphausia superba, so that the feed-liquid ratio is 1:4, adding alkaline protease, wherein the enzyme addition amount is 0.8%, adjusting the pH value of the system to 8.5, and preserving the temperature at 55 ℃ for hydrolysis for 4 hours; and (5) boiling and inactivating enzyme for 10min to obtain euphausia superba enzymatic hydrolysate.
In order to enhance the effect of the prepared polypeptide, the enzymolysis liquid is purified, and the specific steps are as follows:
1) Filtering the antarctic krill peptide solution by a microporous filter membrane with the diameter of 0.22 mu m, and separating and purifying by an SP-SephadexC25 cation exchange column: firstly, balancing for 90min by using 0.02mol/L acetic acid buffer solution with pH of 4.0, loading samples, then linearly gradient eluting by using acetic acid buffer solution containing 0-0.25mol/L NaCl, and collecting the first main peak with highest activity.
2) Further separating and purifying by Sephadex G15 gel column, eluting with ultrapure water at flow rate of 0.4mL/min, and collecting second main peak.
3) The reverse-phase high performance liquid chromatography is utilized for further separation and purification, the eluent is acetonitrile and ultrapure water, the flow rate is 0.8mL/min, and the seventh main peak with the highest activity is the typical peptide fragment component of the euphausia superba uric acid reducing peptide.
Further, the component with the strongest activity comprises peptide fragments with the sequences of FDRLF (SEQ ID NO: 1) and FFDPDMGTK, FVKPPL, HLLPKF, LPDLR, SGPSLLH, VDDHFL, VDDHFLFR;
the euphausia superba peptide prepared by the method can be used for preparing uric acid reducing medicines or health products; when in use, the antarctic krill peptide can be compounded with Chinese herbal medicines with uric acid reducing effect.
In the following examples, the methods for determining the XO inhibitory activity using the colorimetry method are as follows:
(1) Solution configuration
0.2mol/L (pH 7.5) Phosphate Buffer (PBS): accurately weigh 30.0838gNa 2 HPO 4 ·12H 2 O and 2.4962g NaH 2 PO4·2H 2 O, dissolving with deionized water, and fixing the volume to 500mL.
Xanthine solution: 6.4mg of xanthine were weighed out accurately, dissolved in 1mL of 1M NaOH, and then 100mL of PBS was added, and the pH was adjusted to 7.5 with 1M HCl.
Xanthine oxidase: mu.L of the enzyme solution was diluted to 8mL with PBS.
Allopurinol: 1mg of allopurinol was weighed out accurately, and the volume was set to 100mL to prepare 10. Mu.g/mL of allopurinol.
(2) Sample pretreatment:
samples were prepared at different concentrations, 50. Mu.L of the sample to be tested (or water as a control) and 50. Mu.L of a xanthine oxidase solution with a concentration of 0.02U/mL were added to each well of a 96-well plate, the mixture was shaken for 30s, incubated at 25℃for 5min, 150. Mu.L of a 0.48mM xanthine solution was added, and after 30s shaking, incubated at 25℃for 25min, 80. Mu.L of 1M hydrochloric acid was added to determine the absorbance at 290 nm. Each sample was blank, i.e., 80. Mu.L of 1M hydrochloric acid was added while adding the enzyme.
(3) Calculation formula
Wherein:
a1-absorbance of the sample solution plus enzyme;
a2 absorbance of sample solution without enzyme
A3 absorbance of blank group in which buffer solution was used instead of sample solution
A4-absorbance without enzyme in blank.
In the following examples, the peptide samples were tested for their effect on HK-2 hyperuricase cells as follows:
HK-2 cell culture
(1) Cell resuscitation
Immediately after removal of the cells from the liquid nitrogen, the cells were heated in a 37℃water bath and gently shaken until completely thawed. After centrifugation at 1200r/min for 5min, the supernatant was discarded, 2mL of MEM complete medium (containing 10% fetal bovine serum FBS and 1% biantipenicillin-streptomycin mixture) was added, the cells were suspended by blowing, transferred to a T25 flask, then 3mL of medium was added, the flask was shaken in a "rice" shape to evenly distribute the cells, and the flask was placed in an incubator at 37℃with 5% CO2 for use, and fresh medium was replaced every 48 h.
(2) Cell passage
Observing the growth condition of HK-2 cells, and carrying out passage when the cell number reaches 80% -90% of the bottom plate of the culture flask. Sucking out the culture solution in the bottle, adding 3mL of PBS, washing twice, adding fresh culture medium, lightly blowing off the cells by using a pipetting gun, inoculating a proper amount of cells into a new T25 culture bottle, adding a proper amount of culture medium, and putting into an incubator for continuous culture to finish passage.
(3) Cell cryopreservation
Preparing a cell frozen stock solution: 60% MEM culture medium, 30% FBS and 10% dimethyl sulfoxide (DMSO), and sterilizing with 0.22 μm filter membrane. Centrifuging the cell suspension at 1200r/min for 5min, removing supernatant, adding cell freezing solution, blowing uniformly, and transferring to a freezing tube. And (3) placing the freezing tube into a gradient cooling freezing box, placing into a refrigerator at the temperature of-80 ℃, transferring to liquid nitrogen for freezing after 24 hours.
The operation steps are as follows:
(1) and (3) connecting plates: cells were counted after pancreatin digestion, seeded in 96-well plates at a density of 104 cells/well (diluted to 6.25 x 10 4 mu.L of complete medium for 24h.
(2) Pre-culturing: the control group and the model group were not treated, 100. Mu. Mol/L of purine alcohol was added to the positive group, 200. Mu. L of 1mg/mL or 5mg/mL of antarctic krill peptide was added to the positive group, AKP-L and AKP-H, respectively, and the positive group was pre-incubated for 24 hours.
(3) Induction: the culture medium was aspirated, each well was washed 3 times with PBS, 250. Mu.L of serum-free medium containing 3mmol/L of adenosine was added to the model group, the positive group and the sample group, and the control group was incubated for 24 hours with serum-free medium.
(4) Adding enzyme: 50 μl of 0.01U/mL XO (in PBS) was added to each well, the supernatant was collected 6h after treatment, and the uric acid content was determined by HPLC.
High performance liquid chromatography is used for measuring uric acid reducing activity of components:
the cell supernatant was filtered through a 0.22 μm filter, and the amount of uric acid produced in the liquid phase supernatant was used.
Mobile phase: 20mM dipotassium hydrogen phosphate buffer solution, methanol=88:12;
chromatographic column: zorbax SB-C18 (3.5 μm, 4.6X100 mm);
sample injection volume: 10. Mu.L; flow rate: 0.3mL/min; column temperature: 30 ℃; detection wavelength: 290nm;
the running time is 22min.
In the following examples, the experimental methods of the effect of peptide samples on potassium oxazinate-induced hyperuricemia in mice are as follows:
1. adaptive feeding 14d
Standard feed, free drinking water. Serum was tested for uric acid, creatinine and urea nitrogen levels on day 7.
2. Molding for 30d, weighing the weight of the balance every other day, and collecting urine and feces
The normal group was intraperitoneally injected with 0.5% CMC-Na daily. The model group and other drug groups were fed with high purine feed, and 200mg/kg of potassium oxazinate was injected daily.
Note that: modeling was considered successful when serum uric acid levels reached 110. Mu. Mol/L.
3. Intervention is performed for 40d while modeling, the weight is weighed every other day, and urine and feces are collected
(1) Normal group: standard feed, daily intraperitoneal injection of 0.5% CMC-Na. After 1h, the stomach physiological saline is infused.
(2) Model group: high purine feed is injected with 200mg/kg of potassium oxazinate per day. After 1h, the stomach physiological saline is infused.
(3) Positive group: high purine feed is injected with 200mg/kg of potassium oxazinate per day. After 1h, the stomach was irrigated with 10mg/kg allopurinol.
(4) AKP-L group: high purine feed is injected with 200mg/kg of potassium oxazinate per day. 1h later, 200mg/kg of polypeptide is irrigated.
(5) AKP-H group: high purine feed is injected with 200mg/kg of potassium oxazinate per day. 1h later, 600mg/kg of polypeptide is irrigated.
4. Animal material
The mice were fasted and not watered for 8h after the last dose. The feces and urine are taken, the body mass is measured, the eyeball is taken for blood collection, and the serum is separated for standby. The liver, kidney and intestinal tract are rapidly stripped, weighed, and the kidney, cecum and colon are photographed. Each group was kept in liquid nitrogen for further use by taking 0.1 g. In addition, 0.5cm3 of liver, kidney and intestinal tract are respectively fixed in 10% neutral formaldehyde for standby.
The present invention will be described in detail with reference to specific embodiments and drawings.
Example 1: preparation of antarctic krill peptides with uric acid reducing activity
The preparation method of the antarctic krill peptide with uric acid reducing activity comprises the following steps:
1) Pretreatment of antarctic krill: thawing Euphausia superba in running water, removing head and shell, and mincing with meat mincer.
2) And (3) enzymolysis reaction: adding a certain mass of water into the antarctic krill meat emulsion to ensure that the feed liquid ratio is 1:4, adding protease, adjusting the pH value of the system to 8.5, and preserving the temperature at 55 ℃ for hydrolysis for 4 hours; boiling and inactivating enzyme for 10min to obtain Euphausia superba enzymolysis liquid; concentrating and drying the enzymolysis liquid to obtain euphausia superba peptide dry powder;
six proteases (flavourzyme, alkaline protease, trypsin, neutral protease, compound protease and papain) are selected for enzymolysis of euphausia superba, 5 enzyme adding gradients (0.2%, 0.4%,0.8%,1.6% and 3.2%) are respectively arranged, and the XOD inhibitory activity and uric acid content of a hyperuricic acid cell model are taken as screening indexes to perform process optimization on enzymolysis conditions. FIG. 1 shows that among 6 enzymes, flavourzyme, alkaline protease and trypsin have strong XOD inhibitory activity, and the activity is strongest when the amount of the flavourzyme added is 0.8% -3.2%.
The results of constructing a hyperuricemia cell model using adenosine-induced HK-2 cells are shown in FIG. 2, wherein most of the enzymatic hydrolysates showed uric acid lowering effects, and the alkaline proteolytic hydrolysates were strong in uric acid lowering. In combination with the XOD inhibitory activity results, euphausia superba peptide (AKP) with 0.8% alkaline protease added was finally screened for subsequent study.
As shown in FIG. 3, the present invention characterizes the enzymatic hydrolysate with an alkaline protease addition of 0.8%. Fig. 3A shows the molecular weight distribution of antarctic krill peptide, and the result shows that the molecular weight of antarctic krill peptide AKP added with 0.8% alkaline protease is more than 90% of that of antarctic krill peptide AKP with the molecular weight of less than 1KDa, which indicates that the molecular weight of AKP is smaller and is easy to be absorbed by the body. Studies have shown that small molecular weight (1 kDa) oligopeptides prepared by enzymatic methods are more readily absorbed and exhibit specific biological activities. FIG. 3B shows the XOD inhibitory Activity IC of AKP 50 The value is 3.232mg/mL, which is far smaller than 14.47mg/mL of bonito hydrolysate and 60.12mg/mL of oyster protein hydrolysate, which shows that the XOD inhibitory activity is higher and uric acid generation can be effectively inhibited. In addition, MTT experiments showed that AKP of 50-5000 μg/mL had no significant inhibition on proliferation of HK-2 cells (FIG. 3C), and AKP significantly reduced uric acid levels of the hyperuricemic cell model with increasing concentration (FIG. 3D).
Hyperuricemia is a metabolic disease caused by imbalance in uric acid production and excretion. Potassium oxazinate-induced hyperuricemia mice model were used, and mice were gavaged with 200 and 600mg/kg AKP. As shown in FIG. 4, the serum uric acid levels were significantly elevated (142.40.+ -. 26.25. Mu. Mol/L) (P < 0.01) in the model group compared to the normal group (82.57.+ -. 7.45. Mu. Mol/L), demonstrating that hyperuricemia has been successfully established. Positive group serum uric acid levels were significantly lower than those of HUA mice (62.30 ±4.05 μmol/L) (P < 0.01). Serum uric acid levels in AKP-L and AKP-H groups were significantly lower than in the model group, and were dose dependent, demonstrating that AKP could reduce uric acid levels in mice by reducing serum uric acid, indicating that AKP is a potential option for alleviating hyperuricemia.
Under normal physiological conditions, decreased excretion results in elevated serum uric acid levels, so excretion of uric acid in vivo is also an important way to decrease uric acid, usually by renal, intestinal reabsorption and urine excretion from the body. As shown in FIG. 5, the model group showed significantly reduced urine uric acid excretion (31.76.+ -. 3.84. Mu. Mol/L) compared to the normal group (92.10.+ -. 22.48. Mu. Mol/L). AKP can significantly increase urine uric acid excretion in mice and is concentration dependent (P < 0.01). In addition, high doses of AKP can also significantly increase the uric acid excretion from the mouse feces (fig. 6), indicating that AKP can reduce uric acid levels in vivo by increasing uric acid in urine and feces.
Hyperuricemia often leads to oxidative stress and renal dysfunction. Tissue section results of kidneys showed that normal groups of kidney tissues were clear and intact with closely ordered cell arrangement (fig. 7). The model group has the phenomena of irregular cell arrangement, vacuolation and expansion of cytoplasm, unclear boundary between adjacent proximal tubular cells and the like. The positive group had a reduced degree of tubular interstitial lesions compared to the model group, but there was still some degree of tubular interstitial fibrosis. Intervention of AKP reduced PO-induced pathological lesions in a concentration-dependent manner, suggesting a protective role in renal pathophysiology.
Example 2: purification and sequencing of AKP Polypeptides
The embodiment carries out refining purification and sequence identification on antarctic krill peptide with uric acid reducing activity, and comprises the following steps:
1) The euphausia superba peptide solution prepared in example 1 was filtered through a 0.22 μm microporous membrane and purified by separation using an SP-sephadex c25 cation exchange column: firstly, balancing for 90min by using 0.02mol/L acetic acid buffer solution with pH of 4.0, loading samples, then linearly gradient eluting by using acetic acid buffer solution containing 0-0.25mol/LNaCl, and collecting the first main peak with highest activity.
2) Further separating and purifying by Sephadex G15 gel column, eluting with ultrapure water at flow rate of 0.4mL/min, and collecting second main peak.
3) And (3) further separating and purifying by using reverse-phase high performance liquid chromatography, wherein the eluent is acetonitrile and ultrapure water, the flow rate is 0.8mL/min, and the seventh main peak with the highest activity is the peptide fragment component with the highest activity of the antarctic krill uric acid reducing peptide.
4) The column used for UHPLC was Agilent Advance-Bio Peptide Map C column (2.1X105 mm,2.7 μm). UHPLC parameters: mobile phase a:0.1% formic acid-acetonitrile, mobile phase B:0.1% formic acid-water, flow rate: 0.25mL/min, column temperature: gradient elution procedure at 40℃for 0-2 min (5%A), 2-27 min (5%A-20% A), 27-37 min (20% A-35% A), 37-39 min (35% A-80% A). Scanning range: 50-1500 m/z, electrospray mode: electrospray positive ions, electrospray voltage 5500v.
Separating and purifying antarctic krill uric acid reducing peptide by an SP-SephadexC25 cation exchange column to obtain 5 components, wherein the F1 component has the highest XOD inhibition activity (figure 8), and collecting the peak with the highest activity for further separation and purification. Further separation and purification were performed using a SephadexG15 gel column to obtain 4 fractions, and G2 having the highest activity was collected for further separation and purification (FIG. 9). Finally, further separating and purifying by reverse phase high performance liquid chromatography to obtain peak R7 with strongest activity, and IC thereof 50 The value was 1.679mg/mL (FIG. 10), which was far lower than the pre-purification fraction, indicating that the XOD inhibitory activity was far higher than the pre-purification fraction.
The most active R7 component was sequenced and its amino acid sequence is shown in Table 1.
Table 1: euphausia superba peptide sequence listing
The mass spectrum results are shown in table 1, in the peptide with the confidence coefficient of more than or equal to 95%, the number of amino acids is less than or equal to 10 amino acids, and the peptide contains more hydrophobic amino acids, so that AKP has stronger uric acid reducing activity. Amino acid residues near the XOD active center can form a hydrophobic pocket, an important domain that peptides containing a larger number of hydrophobic amino acids can easily access. After the polypeptides in table 1 are synthesized, experimental results show that the polypeptides have better uric acid reducing activity, and the amino acids can be combined with XOD through hydrophobic interaction and the like, so that the spatial structure of the enzyme can be changed to inhibit the activity of the enzyme.
In conclusion, the euphausia superba enzymolysis product has stronger in vitro and in vivo uric acid reducing activity, such as XOD inhibition, uric acid content reduction of a cell model, serum uric acid level reduction in a mouse body and the like. In addition, the H & E staining result shows that the euphausia superba enzymatic hydrolysate has a certain protection effect on the kidneys. Finally, the activity of the obtained components is far higher than that of the zymolyte after a series of refining and purification, so the uric acid reducing peptide prepared by the method has better application prospect.
Claims (10)
1. The euphausia superba peptide with uric acid reducing activity is characterized in that euphausia superba meat paste is added with water to prepare homogenate, protease is added for enzymolysis, the pH value of the system is regulated to be 6-9, and enzymolysis is carried out for 3-6 hours at 50-55 ℃ to obtain euphausia superba peptide enzymolysis liquid; and concentrating and drying the enzymolysis liquid to obtain the antarctic krill peptide.
2. The antarctic krill peptide of claim 1, wherein the homogenate has a mass ratio of antarctic krill meat emulsion to water of 1: 2-1: 5, a step of; the addition amount of the protease is 0.5-4.0%.
3. The antarctic krill peptide of claim 1, wherein the protease is a flavourzyme, an alkaline protease or trypsin.
4. The antarctic krill peptide of claim 1, wherein the protease is an alkaline protease.
5. The purified product of the antarctic krill peptide of claim 1, which is characterized in that the purified product is obtained by filtering the antarctic krill peptide of claim 1, separating and purifying by using an SP-Sephadex C25 cation exchange column, balancing for 90-120min by using 0.02mol/L acetic acid buffer with pH of 4.0, linearly gradient eluting by using 0.02mol/L acetic acid buffer with pH of 4.0 added with NaCl after loading, and collecting the first main peak eluent with highest activity; then further separating and purifying the collected eluent by using a SephadexG15 gel column, wherein the eluent is ultrapure water with the flow rate of 0.4mL/min, and collecting a second main peak; finally, separating and purifying by reverse phase high performance liquid chromatography, eluting with acetonitrile and ultrapure water at a flow rate of 0.8mL/min, collecting the seventh main peak, and drying to obtain purified product.
6. The purified product of claim 1, wherein the purified product comprises a peptide of sequence DIFDPL, EFDGF, FDPLIQ, DLGGGTF, ADIFDPLIQ.
7. Use of the antarctic krill peptide of claim 1 for the preparation of uric acid lowering preparations.
8. Use of the purified product of antarctic krill peptide of claim 5 or 6 for the preparation of uric acid lowering preparations.
9. A uric acid lowering product, comprising any one or more of the antarctic krill peptide of claim 1, the purified product of antarctic krill peptide of claim 5 or claim 6.
10. The uric acid lowering article of claim 9, further comprising other uric acid lowering components.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960699A (en) * | 2014-05-23 | 2014-08-06 | 中国海洋大学 | Euphausia superba peptide replenisher containing calcium, iron and zinc |
| CN110478373A (en) * | 2019-09-23 | 2019-11-22 | 中国水产科学研究院东海水产研究所 | Application of the antarctic krill oil in the drug or health food of preparation inhibiting hyperuricemia or antigout |
| CN113243477A (en) * | 2021-05-14 | 2021-08-13 | 中国海洋大学 | Composition for preventing browning of beverage and application thereof |
| CN114507702A (en) * | 2022-02-03 | 2022-05-17 | 中国海洋大学 | Ocean euphausia superba peptide and application thereof |
| CN116589533A (en) * | 2023-06-19 | 2023-08-15 | 中国海洋大学 | Euphausia superba small molecule active peptide, and preparation method and application thereof |
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960699A (en) * | 2014-05-23 | 2014-08-06 | 中国海洋大学 | Euphausia superba peptide replenisher containing calcium, iron and zinc |
| CN110478373A (en) * | 2019-09-23 | 2019-11-22 | 中国水产科学研究院东海水产研究所 | Application of the antarctic krill oil in the drug or health food of preparation inhibiting hyperuricemia or antigout |
| CN113243477A (en) * | 2021-05-14 | 2021-08-13 | 中国海洋大学 | Composition for preventing browning of beverage and application thereof |
| CN114507702A (en) * | 2022-02-03 | 2022-05-17 | 中国海洋大学 | Ocean euphausia superba peptide and application thereof |
| CN116589533A (en) * | 2023-06-19 | 2023-08-15 | 中国海洋大学 | Euphausia superba small molecule active peptide, and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| LORENZO SORI 等: "Computation meets experiment: Identification of highly efficient fibrillating peptides.", CRYSTENGCOMM., 4 July 2023 (2023-07-04), pages 1 * |
| 刘云娇 等: "响应面优化南极磷虾蛋白酶解工艺及蛋白肽组分分析", 现代食品科技, 28 November 2018 (2018-11-28), pages 144 - 151 * |
| 李明杰 等: "南极磷虾肽制备工艺优化及抗氧化测定", 食品工业科技, 1 February 2012 (2012-02-01), pages 279 - 282 * |
| 王庆慧 等: "南极磷虾肽对高尿酸血症小鼠脂代谢的改善及作用机制", 中国海洋大学学报, 5 September 2022 (2022-09-05), pages 54 - 60 * |
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