CN105669853A - Activator for Nrf2-ARE pathway in high fischer ratio oligo-peptide from tuna - Google Patents

Activator for Nrf2-ARE pathway in high fischer ratio oligo-peptide from tuna Download PDF

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CN105669853A
CN105669853A CN201610110512.2A CN201610110512A CN105669853A CN 105669853 A CN105669853 A CN 105669853A CN 201610110512 A CN201610110512 A CN 201610110512A CN 105669853 A CN105669853 A CN 105669853A
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nrf2
tuna
peptide
pathway
activator
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CN105669853B (en
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王斌
赵玉勤
孙坤来
罗红宇
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Zhejiang Ocean University ZJOU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses an activator for an Nrf2-ARE pathway in high fischer ratio oligo-peptide from tuna. The ground meat of the tuna is taken as raw material; enzymatic hydrolysate is obtained by bienzyme hydrolysis; the enzymatic hydrolysate is treated by ultrafiltration, absorbed by activated carbon and purified by macroporous resin, so that the high fischer ratio oligo-peptide from the tuna is obtained; after that, active peptide Ile-Glu-Leu-Val-Trp is obtained by gel column chromatography and reversed-phase high-performance liquid chromatography separation and purification; the molecular weight measured by electrospray ionization-mass spectrometry (ESI-MS) is 658.76 Da; the prepared high-activity peptide has a good scavenging effect for hydroxy free radicals and superoxide anion free radicals; furthermore, the activator is capable of promoting Nrf2 protein accumulation and activating the Nrf2-ARE pathway, and can be used as medicines and functional products which are capable of treating liver injury, skin photoage, senility and the like as well as diseases closely related to the Nrf2-ARE pathway.

Description

A kind of Nrf2-ARE Pathway Activation agent in tuna high F value oligopeptide
Technical field
The present invention relates to a kind of animal high F value oligopeptide, be specifically related to the Nrf2-ARE Pathway Activation agent in a kind of tuna high F value oligopeptide.
Background technology
Oligo-peptide mixture be one by mixed small peptide (or the claim oligopeptide) system being made up of 3～7 residual amino acid residues of aminoacid. F value (Fischerratio) refers to the molal quantity ratio of branched-chain amino acid (BCAA:Val, Ile, Leu) and aromatic amino acid (AAA:Trp, Tyr, Phe) content. In the blood of normal person, F value is 3.0-3.5, and the F value suffering from hepatic disease patient only has 1.0 or lower, and the F value of high F value oligopeptide should be greater than 20, far above the ratio of this two amino acid in human body. Owing to it has aminoacid composition and the physiological function of uniqueness, have been subjected to showing great attention to of food and medicine circle.
Skipjack (Katsuwonuspelamis), belongs to perch shape catalogue, tuna suborder, Thunnidae, frigate mackerel subfamily, oceanic bonito genus, has another name called positive oceanic bonito, bomb fish, hard fish, pine fish, flying tiger fish. Skipjack is ocean property Important Economic fish. Skipjack distribution is relatively wide, in the Indian Ocean, the Pacific Ocean and the Atlantic Ocean water temperature waters higher than more than 15 degrees Celsius, has the trace of Skipjack and rich reserves, exploitation prospect optimism.
Summary of the invention
The technical problem to be solved is to provide the Nrf2-ARE Pathway Activation agent in a kind of tuna high F value oligopeptide.
The present invention solves that the technical scheme that above-mentioned technical problem is taked is: the Nrf2-ARE Pathway Activation agent in a kind of tuna high F value oligopeptide, it is characterized in that this activator is pentapeptide compound, aminoacid sequence is Ile-Glu-Leu-Val-Trp (IELVW), ESI-MS mensuration molecular weight is 658.76Da.
The preparation method of this activator is:
1) enzymolysis of tuna minced meat: depletion marlin minced meat homogenate, Gly-NaOH buffer (0.05mol/L is added by solid-to-liquid ratio 1g:10mL～15mL, pH9.5), pH to 9.0～10.0 is regulated with HCl or NaOH, solution temperature is modulated 55～65 DEG C, adds alkaline protease (enzyme activity >=1.95 × 10 according to the 1.0%～1.5% of tuna minced meat quality5U/g), after enzymolysis time 3.5h～4.0h, enzymolysis solution is incubated 10～15min in 90～95 DEG C, and enzyme denaturing is lived;Enzymolysis solution temperature is adjusted to 45～55 DEG C, regulates pH to 6.0～7.0, add compound protease (enzyme activity >=1 × 10 according to the 0.8%～1.0% of tuna minced meat quality3LAPU/g), enzymolysis time 2.5h～3.0h, enzymolysis solution is down to room temperature after being incubated 10～15min in 90～95 DEG C, in the centrifugal 15～20min of 8000～10000rpm, takes supernatant.
2) preparation of tuna minced meat high F value oligopeptide: take above-mentioned supernatant, adopts 1kDa ultrafilter membrane to carry out hyperfiltration treatment, collects molecular weight less than 3kDa part, obtains ultrafiltration enzymolysis solution; Adjust pH value to 2.5 ~ 3.5 ultrafiltration enzymolysis solution, it is that ultrafiltration enzymolysis solution is slowly added in the glass column being mounted with activated carbon granule with 0.3～0.5mL/min flow velocity by 10～15:1 according to material with activated carbon granule ratio, after completion of the sample, adopt 1mol/L ammonia and the dehydrated alcohol mixed solution elution chromatography post of the 1:1 ratio of 2～3 times of column volumes, collect eluent, dry, obtain absorbent charcoal fine purification polypeptide; Absorbent charcoal fine purification polypeptide is made into the solution of 10～20mg/mL, adjust pH value to 6.5～7.5, be slowly added into material and D101 macroporous resin than be 10～15:1 the glass column loading D101 macroporous resin in, impurity is removed with the distilled water eluting of 2～3 times of column volumes, again with 95% ethanol elution of 2～3 times of column volumes, collect 95% ethanol elution thing, spray drying, be tuna minced meat high F value oligopeptide.
3) in tuna minced meat high F value oligopeptide prepared by Nrf2-ARE Pathway Activation agent: by tuna minced meat high F value oligopeptide successively through gel filtration chromatography and reversed-phase high-performance liquid chromatography purification, obtain Nrf2-ARE Pathway Activation agent.
As preferably, the tuna in described step 1) is Skipjack (Katsuwonuspelamis).
As preferably, the gel filtration chromatography of described step 3) and the detailed process of reversed-phase high-performance liquid chromatography purification be:
Gel filtration chromatography: refining for above-mentioned macroporous resin polypeptide is dissolved in distilled water and is made into the solution that concentration is 20～30mg/mL, through polydextran gel SephadexG-15 column chromatography for separation, eluting is carried out with distilled water, flow velocity is 0.5～0.8mL/min, the every 3min of wash solution collects a pipe and detects in 220nm, merges solution in test tube by peak, relatively each peak impact on Nrf2 protein level, select the highest component lyophilizing of Nrf2 protein level, be gel chromatography zymolyte;
Reversed-phase high-performance liquid chromatography purification: above-mentioned gel chromatography zymolyte distilled water is made into the solution of 90～100 μ g/mL, reversed-phase high-performance liquid chromatography is utilized to be purified, according to Nrf2 protein level is affected to obtain 1 active polypeptide Ile-Glu-Leu-Val-Trp (IELVW), it is 658.76Da that ESI-MS measures molecular weight.
Further preferably, described reversed-phase high-performance liquid chromatography condition is: sample size 10～15 μ L; Chromatographic column KromasilC18(250mm × 4.6mm, 5 μm); Mobile phase: water-acetonihile gradient elution (0～40min acetonitrile concentration is at the uniform velocity risen to 50% by 0); Elution speed 0.8 ~ 1.0mL/min; Ultraviolet detection wavelength 220nm.
Compared with present technology, hydroxyl radical free radical and ultra-oxygen anion free radical are had good scavenging action by Nrf2-ARE Pathway Activation agent Ile-Glu-Leu-Val-Trp (IELVW) in a kind of tuna high F value oligopeptide provided by the present invention; Nrf2 protein accumulation can be promoted, activate Nrf2-ARE path, raise antioxidase and II phase detoxication enzyme removes nuisance, the damage of the active oxygen that protection body produces from oxidative stress;Can be used as the medicine of preparation hepatic injury, skin photoage, aging and other and the closely-related disease of Nrf2-ARE path or health food.
Accompanying drawing explanation
Fig. 1 is the polydextran gel SephadexG-15 tomographic map of the present invention.
Fig. 2 polydextran gel SephadexG-15 prepares the reversed-phase high-performance liquid chromatography chromatogram of zymolyte.
Fig. 3 Ile-Glu-Leu-Val-Trp (IELVW) impact on Nrf2 protein expression level.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment:
The preparation method of the Nrf2-ARE Pathway Activation agent in a kind of tuna high F value oligopeptide, preparation technology flow process is as follows: tuna minced meat " double; two enzyme enzymolysis " zymolyte " ultrafiltration " activated carbon adsorption " purification by macroporous resin " tuna high F value oligopeptide " gel permeation chromatography " high performance liquid chromatography prepares " Nrf2-ARE Pathway Activation agent.
1) enzymolysis of tuna minced meat: take Skipjack minced meat homogenate, Gly-NaOH buffer (0.05mol/L is added by solid-to-liquid ratio 1g:12mL, pH9.5), pH to 9.3 is regulated with HCl or NaOH, solution temperature is modulated 60 DEG C, adds alkaline protease (enzyme activity >=1.95 × 10 according to the 1.2% of tuna minced meat quality5U/g), after enzymolysis time 3.5h, enzymolysis solution is incubated 12min in 95 DEG C, and enzyme denaturing is lived; Enzymolysis solution temperature is adjusted to 50 DEG C, regulates pH to 7.0, add compound protease (enzyme activity >=1 × 10 according to the 0.9% of tuna minced meat quality3LAPU/g), enzymolysis time 3.0h, enzymolysis solution is down to room temperature after being incubated 15min in 95 DEG C, in the centrifugal 15min of 9000rpm, takes supernatant.
2) preparation of tuna minced meat high F value oligopeptide: take above-mentioned supernatant, adopts 1kDa ultrafilter membrane to carry out hyperfiltration treatment, collects molecular weight less than 3kDa part, obtains ultrafiltration enzymolysis solution; Adjust pH value to 3.0 ultrafiltration enzymolysis solution, than for 12:1, ultrafiltration enzymolysis solution is slowly added into 0.3mL/min flow velocity in the glass column loading activated carbon granule with activated carbon granule according to material, after completion of the sample, adopt 1mol/L ammonia and the dehydrated alcohol mixed solution elution chromatography post of the 1:1 ratio of 3 times of column volumes, collect eluent, dry, obtain absorbent charcoal fine purification polypeptide; Absorbent charcoal fine purification polypeptide is made into the solution of 15mg/mL, adjust pH value to 7.0, it is slowly added into material with D101 macroporous resin ratio in the glass column loading D101 macroporous resin of 15:1, impurity is removed with the distilled water eluting of 3 times of column volumes, again with 95% ethanol elution of 3 column volumes, collect 95% ethanol elution thing, spray drying, be tuna minced meat high F value oligopeptide.
3) in tuna minced meat high F value oligopeptide prepared by Nrf2-ARE Pathway Activation agent: by tuna minced meat high F value oligopeptide successively through gel filtration chromatography and reversed-phase high-performance liquid chromatography purification, obtain Nrf2-ARE Pathway Activation agent.
As preferably, the gel filtration chromatography of described step 3) and the detailed process of reversed-phase high-performance liquid chromatography purification be:
1. gel filtration chromatography: refining for above-mentioned macroporous resin polypeptide is dissolved in distilled water and is made into the solution that concentration is 20mg/mL, through polydextran gel SephadexG-15 column chromatography for separation, eluting is carried out with distilled water, flow velocity is 0.6mL/min, the every 3min of wash solution collects a pipe and detects in 220nm, merges solution in test tube by peak, relatively each peak impact on Nrf2 protein level, select the highest component lyophilizing of Nrf2 protein level, be gel chromatography zymolyte Fr.3(Fig. 1).
2. reversed-phase high-performance liquid chromatography purification: above-mentioned gel chromatography zymolyte distilled water is made into the solution of 100 μ g/mL, (condition is: sample size 15 μ L to utilize reversed-phase high-performance liquid chromatography to be purified; Chromatographic column KromasilC18(250mm × 4.6mm, 5 μm); Mobile phase: water-acetonihile gradient elution (0～40min acetonitrile concentration is at the uniform velocity risen to 50% by 0); Elution speed 0.8mL/min; Ultraviolet detection wavelength 220nm. ), according to Nrf2 protein level affected to obtain 1 active polypeptide (Fig. 2).
3. structure detection: collect the polypeptide that the accumulative level of Nrf2 protein is the highest, the detection of inverted high performance liquid chromatography is simple spike, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Ile-Glu-Leu-Val-Trp (IELVW), and it is 658.76Da that ESI-MS measures molecular weight.
Free radical scavenging is tested: prepared tuna minced meat polypeptide Ile-Glu-Leu-Val-Trp (IELVW) is carried out free radical scavenging experiment. Test result indicate that: Ile-Glu-Leu-Val-Trp (IELVW) is to hydroxyl radical free radical (EC500.12mg/mL) with ultra-oxygen anion free radical (EC500.06mg/mL) there is good scavenging action.
The anti-oxidation peptide effect to activating Nrf2/ARE path: after the human embryonic kidney cell line HEK293 cell of trophophase of taking the logarithm is inoculated in 60mm ware 24 hours, it is separately added into enzymolysis solution and the Ile-Glu-Leu-Val-Trp (IELVW) of 10 μMs, continues to cultivate 4 hours. Supernatant discarded, cell RIPA buffer (the green skies Bioisystech Co., Ltd in Shanghai) cracks, and BCA method measures protein concentration. Each sample takes 20 μ g albumen and separates through SDS-PAGE, and then western blotting method detects the protein level of Nrf2 routinely.
Compared with present technology, hydroxyl radical free radical and ultra-oxygen anion free radical are had good scavenging action by Nrf2-ARE Pathway Activation agent Ile-Glu-Leu-Val-Trp (IELVW) in a kind of tuna high F value oligopeptide provided by the present invention; Nrf2 protein accumulation can be promoted, activate Nrf2-ARE path, raise antioxidase and II phase detoxication enzyme removes nuisance, the damage of the active oxygen that protection body produces from oxidative stress; Can be used as the medicine of preparation hepatic injury, skin photoage, aging and other and the closely-related disease of Nrf2-ARE path or health food.
Finally, in addition it is also necessary to be only the specific embodiment of the present invention it is noted that listed above. It is clear that the invention is not restricted to above example, it is also possible to there are many deformation. All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.
SEQUENCELISTING
<110>Oceanography Institute Of Zhejiang
<120>the Nrf2-ARE Pathway Activation agent in a kind of tuna high F value oligopeptide
<130>zjou-wb-201602-101
<160>1
<170>PatentInversion3.5
<210>1
<211>5
<212>PRT
<213>synthetic
<400>1
IleGluLeuValTrp
15

Claims (1)

1. the Nrf2-ARE Pathway Activation agent in a tuna high F value oligopeptide, it is characterised in that: this activator is pentapeptide compound, and aminoacid sequence is Ile-Glu-Leu-Val-Trp, ESI-MS mensuration molecular weight is 658.76Da.
CN201610110512.2A 2016-02-29 2016-02-29 Nrf2-ARE pathway activator in tuna high F value oligopeptide Active CN105669853B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164440A (en) * 2017-05-11 2017-09-15 浙江海洋大学 High F value oligopeptide Coarse liquid separation purification process

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009091305A (en) * 2007-10-10 2009-04-30 Yaizu Suisankagaku Industry Co Ltd Novel peptide and method of manufacturing the same, and antioxidant
CN103073621A (en) * 2012-12-20 2013-05-01 浙江海洋学院 Minced tuna protein antioxidative peptide and its preparation method and use
CN103275181A (en) * 2013-05-23 2013-09-04 浙江海洋学院 Tuna ground meat polypeptide angiogenesis inhibiting factor as well as preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009091305A (en) * 2007-10-10 2009-04-30 Yaizu Suisankagaku Industry Co Ltd Novel peptide and method of manufacturing the same, and antioxidant
CN103073621A (en) * 2012-12-20 2013-05-01 浙江海洋学院 Minced tuna protein antioxidative peptide and its preparation method and use
CN103275181A (en) * 2013-05-23 2013-09-04 浙江海洋学院 Tuna ground meat polypeptide angiogenesis inhibiting factor as well as preparation method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164440A (en) * 2017-05-11 2017-09-15 浙江海洋大学 High F value oligopeptide Coarse liquid separation purification process

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