CN108402415A - The peptide that detaches preparation in abalone processed side product is anti-oxidant, antihypertensive function and application - Google Patents
The peptide that detaches preparation in abalone processed side product is anti-oxidant, antihypertensive function and application Download PDFInfo
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- CN108402415A CN108402415A CN201810018438.0A CN201810018438A CN108402415A CN 108402415 A CN108402415 A CN 108402415A CN 201810018438 A CN201810018438 A CN 201810018438A CN 108402415 A CN108402415 A CN 108402415A
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- abalone
- babs
- oxidant
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- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 229940117953 phenylisothiocyanate Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000013849 propane Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- KOUKXHPPRFNWPP-UHFFFAOYSA-N pyrazine-2,5-dicarboxylic acid;hydrate Chemical compound O.OC(=O)C1=CN=C(C(O)=O)C=N1 KOUKXHPPRFNWPP-UHFFFAOYSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006950 reactive oxygen species formation Effects 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000013319 spin trapping Methods 0.000 description 1
- 235000000891 standard diet Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical class OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/50—Molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Epidemiology (AREA)
- Molecular Biology (AREA)
- Marine Sciences & Fisheries (AREA)
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Abstract
The present invention relates to the methods by obtaining peptide in abalone processed side product, in particular to by abalone byproduct separation prepare peptide anti-oxidant and angiotensin i-converting enzyme (ACE) inhibiting effect and its to the antihypertensive effect of spontaneous hypertensive rat.Purposes of the peptide in preparing the drug and health food for treating spontaneous hypertension.Method by obtaining peptide in abalone byproduct, the method includes abalone processed side product by electrodialysis desalination, is then classified and is concentrated by ultrafiltration membrane system.
Description
Technical field
The present invention relates to anti-oxidant, the antihypertensive effect of peptide in abalone processed side product and applications, specifically, relating to
And anti-oxidant and angiotensin i-converting enzyme (ACE) inhibiting effect of peptide that is prepared by the separation of abalone processed side product and its right
The antihypertensive effect of spontaneous hypertensive rat.
Background technology
Abalone is the ocean gastropod with high commercial value and potentiality all over the world.In South Korea, abalone is to pass
The kind of important high-valued cultivation.It is in the distribution of South Korea west and south coastal area and sea-farming mostly, is public in fishery
The Important Economic species recognized.Due to the production of a large amount of abalones, develops the converted products of various abalones and sold, manufactured
In journey product is manufactured using the abalone for cooking and steaming.However, the remaining boiling after many manufacturing works abalones boil process
Liquid is nearly all dropped.In byproduct from abalone boiling process factory, containing 20% salt and rich in high concentration protein,
Carbohydrate, minerals and lipid.Find that this method of effective protein is widely used in improving and rise from BABs
The functional protein with trophism of grade.
Active oxygen (ROS) playing a key effect in origin of life and biological evolution.In vivo, ROS is in such as energy
Positive effect is played in generation, phagocytosis, cell cycle regulation and cellular signal transduction.On the other hand, excess generation
ROS can damage various important large biological molecules because they can attack lipid, protein, enzyme, carbohydrate and
DNA so that unbalance between ROS and defence and reparation antioxidant system.Free radical and other ROS include superoxide anion
(O2-) hydroxy radicals (OH) and non-free radical such as singlet oxygen (1O2) and hydrogen peroxide (H2O2) etc..Aerobic micro- life
Object inevitably forms hydroxyl radical free radical and ultra-oxygen anion free radical in respiratory.Free radical rises in many diseases
To important role.The uncontrolled generation of free radical is considered and many health diseases such as diabetes, cancer, neurological
Property and inflammatory disease are related.Many naturals have been confirmed as free radical or active oxygen scavenger.Recently, in food or
Natural is found in medicinal material, to replace the synthetized oxidation preventive agent for being constantly subjected to limitation due to the side effect of such as carcinogenicity
Research interest increase considerably.
Hypertension may lead to the generation of coronary heart disease, and angiocardiopathy and relevant can be reduced by being treated to it
The risk of stimulation.The adjusting of blood pressure is and renin-angiotensin system (RAS) (in controlling arterial pressure play a crucial role)
It is associated.Hypertension can lead to various angiocardiopathies, apoplexy and end-stage renal disease.Angiotensin i-converting enzyme (ACE I；
Dipeptidylcarboxypeptidase；EC 3.4.15.1) in adjusting blood pressure play important physiological function.It is by the C-terminal of various oligopeptides
Cut expeptidase obtained by dipeptides.The blood vessel dilatation that ACE passes through generation vasoconstrictor angiotensinⅡ and degradation
Agent, bradykinin has attempted synthesis Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe to be responsible for many researchs of to hypertension, such as captopril, Yi Napu
Profit, enalapril, alacepril and lisinopril, the above are now widely used for the essential hypertension and mental and physical efforts of the mankind
In the treatment of failure, synthesis Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe has been attempted in many researchs, such as captopril, enalapril, enalapril, A Lapu
Profit and lisinopril, the above are now widely used in the essential hypertension of the treatment mankind and the treatment of heart failure.
However, these synthetic drugs are considered the side effect for having certain, such as cough, taste interference and fash.It has been carried out recently perhaps
The enzymolysis product of more natural materials such as cheese whey, casein, maisin, tuna, sardine and maize yellow-powder
Although studying these substances be not as effective as those of synthesis；But they do not show known side effect yet.Other researchs
Show that Angiotensin II stimulates the formation of reactive oxygen species to expand oxidative stress under the conditions of hypertension.Therefore, in addition to control
Blood pressure processed, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe is also shown enhances animals and humans anti-oxidative defense system by inhibiting Angiotensin II formation
System.
Pass through ultrafiltration membrane system split pole and concentration (＜ in the abalone processed side product that production manufacturing works are generally discarded
1KDa).Measure the antioxidation in vitro and antiangiotensin I converting Enzymes of the most active peptide in abalone processed side product (BABs)
Activity determines the amino acid sequence of isolating active peptide, and is studied by the way that spontaneous hypertensive rat (SHR) is administered orally
The antihypertensive function of purified peptide.The BABs of the present invention can be reduced as a kind of synergist is related to radicals scavenging cell mechanism
Oxidation associated injury, and show ACE inhibiting effect.BABs has ACE inhibiting effect and anti-oxidant work in macrophage
Property, and the cytotoxic effect of BABs is not observed in macrophage RAW 264.7.BABs can be in antioxygen as a result,
Change, applied in antihypertensive therapy.Additionally, it is contemplated that will be helpful to develop interesting basic research and bioactive substance is potential
Using.
Invention content
The present invention provides the method by obtaining peptide in abalone processed side product, and the method includes by abalone processed side product
By electrodialysis desalination, then it is classified and concentrates by ultrafiltration membrane system.
Preferably the method further includes freeze-drying step at least once.
Preferably the method further includes by HPLC separating-purifyings.
The present invention provides a kind of biological peptide product, and the biology peptide product is made in aforementioned manners.
The biological peptide product of the freeze-drying, ingredient include 75.1% protein, 3.84% lipid, 21.06%
Carbohydrate.
The biological peptide product of the freeze-drying, ingredient include the amino acid of following content：19.1% His, 16.33%
Cys, 12.12% Asp.
The present invention provides the peptide obtained by abalone processed side product in application anti-oxidant, in anti-hypertension.
It is appreciated that the peptide of the present invention obtained by abalone processed side product can be applied or as prevention in terms of acology
Medicine application.
It is anti-oxidant, spontaneous hypertension for treating in production that the present invention provides the peptide obtained by abalone processed side product
Application in drug and health food.
Suitable daily dosage range is about 0.1mg/kg-100mg/kg.By convention, unit dose can give daily
Medicine one time 2 or primary or more, such as daily 2,3 or 4 are usually daily 1 or 2 time.
The present invention provides the pharmaceutical composition for the treatment of spontaneous hypertension, which includes the biological peptide of the present invention
Product and pharmaceutical acceptable carrier or excipient.
Including the convenient form of pharmaceutical composition of present invention biology peptide product can be tablet for oral administration, ball
Agent, capsule, syrup, pulvis or granule；For the sterile parenteral or subcutaneous solution, suspension of parenterai administration, institute
These are all well known in the art.
Description of the drawings
Fig. 1 shows BABs to the cytotoxic effect of RAW 264.7；
Fig. 2 indicates that ESR spectrometers measure the free radical scavenging activity of various concentration BABs；
Fig. 3 a indicate BABs in 1,000 μ g/mL, 500 100 μ g/mL cell free radical scavenging activities of μ g/mL, and；
Fig. 3 b indicate BABs to DNA oxidation protections；
Fig. 4 indicates the ACE inhibitory activity of BABs；
Fig. 5 a indicate the RP-HPLC chromatograms of BABs；
Fig. 5 b indicate that the ESI MALDI-TOF-MS/MS of fraction 5 are measured；
Fig. 6 a indicate that Lineweaver-Burk Plot measure the ACE suppression modes of BABs；
Fig. 6 b indicate SBP variation diagrams after the administration of SHRs (n=6) single oral.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, in the following, by the specific implementation mode of the present invention
It is described in detail.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Based on the embodiments of the present invention, the institute that those of ordinary skill in the art are obtained without making creative work
There is other embodiment, shall fall within the protection scope of the present invention.
The purpose of the present invention is to provide the methods by obtaining peptide in abalone processed side product；By in abalone processed side product
The peptide of acquisition is in purposes anti-oxidant, in anti-hypertension.The peptide of the present invention is preparing the drug for treating spontaneous hypertension
With the purposes in health food.The present invention also provides the pharmaceutical composition for the treatment of spontaneous hypertension, the pharmaceutical composition packets
Containing the peptide and pharmaceutical acceptable carrier that are obtained by abalone processed side product or excipient.
In order to realize the present invention, " abalone processed side product " indicates abalone institute in steaming, boiling or similar processing technology
Remaining aqueous mixture.
Material and method
1, material
Abalone processing after remaining water cooking liquid, by Chengsan sea abalone Co., Ltds provide (Whan-Do,
Korea).ACE (EC 3.4.15.1) comes from rabbit lung, and hippuric acid-peptide substrate of the histidyl--leucine (HHL) as ACE comes from
Sigma Chemical companies.Free radical tests chemicals：Including 1,1- diphenyl -2- trinitrophenyl-hydrazines (DPPH), 5,5- bis-
Methyl-1-pyridine-N- oxygen (DMPO), FeSO4、H2O2, 2,2- azos-(2- aminopropyls amidine)-hydrochloride (AAPH) and a- (4-
Pyridyl group -1- oxygen)-N- tertiary butyls nitroketone (4-POBN), from Sigma Chemical companies purchase (St.Louis, MO,
USA).Other materials needed for cell culture medium and culture is obtained from Gibco Grand Island NY.All other reagent is
The commercially available superlative degree.
2, the preparation of boiling abalone byproduct (BABs)
Water cooking liquid passes through electrodialysis desalination (Micro Acilyzer model G3, Asahi Chemical Industry
Co., Tokyo, Japan), then by ultrafiltration membrane system (>It 1KDa) is classified and concentrates.BABs is lyophilized and is preserved at -20 DEG C,
Until using.
3, the general ingredient of BABs
Measure the general ingredients of BABs of freeze-drying.The crude fat of BABs, carbohydrate and albumen are measured according to AOAC methods
Matter content.The general composition of the freeze-drying BABs of mass production is as follows：75.1% (protein), 3.84% (lipid), 21.06%
(carbohydrate) is based on dry weight.
4, Analysis on amino acid components
The measurement of aminoacid ingredient, BABs (20mg) is under vacuum 110 in the 6N HCl containing 0.1% thioacetic acid
DEG C hydrolysis for 24 hours.With phenyl isothiocyanate derivatization reaction occurs for amino acid, uses automatic amino acid analyzer qualitative and quantitative analysis
(Biochrom 20；Pharmacia Biotech,Uppsala,Sweden).As shown in table 1, the aminoacid ingredient of BABs is by dredging
Aqueous and acidic amino acid such as His (19.10%w/w), Cys (16.33%), Asp/n (12.12%), Glu/n (7.85%),
Pro (5.12%), Ala (3.98%), Gly (3.69%) and Leu (3.41%) are formed, and explain their antioxidant activity.
The amino acid content (%) of table 1.BABs
5, cell culture and vitality test
RAW264.7 macrophages are cultivated, DMEM culture mediums include 10% fetal calf serum, 100 μ g/m l penicillin and 100 μ
G/m l streptomysins, 5%CO2, 37 DEG C of humid air environment.It is described using such as Hansen, Nielsen, and Berg. (1989)
Mtt assay measures cytotoxicity levels of the BABs to RAW264.7.Macrophage RAW264.7 is in 96 orifice plates with 4 × 103Cell/
The density in hole is inoculated with 24 hours, is washed out.The sample incubation of various concentration is used in 0.5mg/mL MTT solution.Three hours
Afterwards, supernatant is removed, microplate reader (Power Wave XS model are used；BioTek Instruments,Inc.,
Winooski, VT, USA) it measures to form formazans in 540nm.BABs at the concentration tested as shown in Figure 1 is to RAW264.7 cells
There is no cytotoxic effect (Fig 1.).The non-toxic concn (100-1,000 μ g/mL) of BABs is used for next experiment.
6, electron spin resonance spectrometer (ESR) measures free radical scavenging activity
According to following procedure, a variety of free radicals are generated, use JES-FAelectron spin resonance (ESR)
(JEOL Ltd., Tokyo, Japan) records spin adduct.Calculate the clearance rate of free radical with following formula, wherein H and
H0It is the opposite peak height for the Free Radical Signal that adduction is not added with sample respectively.
The scavenging effect of 6.1DPPH free radicals
It is free that DPPH is measured using the method by Ambigaipala, Al-Khalifa and Shahidi description in (2015)
Base scavenging capacity.30 μ L samples solution (ethyl alcohol is blank) are added in DPPH (60 μM) ethyl alcohol of 30 μ L.It is vigorously mixed 10s
Afterwards, 100 μ L quartz capillaries are then transferred the solution into, scavenging capacity is measured using ESR.Spin adduct ESR spectrometer essences
Really measure 2 minutes.Experiment condition is as follows：Magnetic field, 336.5 ± 5mT；Power, 5mW；Modulating frequency, 9.41GHz；Amplitude 1 ×
1000；Sweep time, 30s.
6.2. the scavenging capacity of hydroxyl radical free radical
The Fenton Haber-Weiss reactions that hydroxyl radical free radical is catalyzed by iron generate, the hydroxyl radical free radical and nitrone of generation
Spin trapping (DMPO) fast reaction (Chandrasekara and Shahidi, 2011).Obtained DMPO-OH adduct products
It is measured with ESR.Sample solution (20 μ L) and DMPO (0.3M, 20 μ L), FeSO4(10mM, 20 μ L), and H2O2(10mM, 20 μ L's)
Phosphate buffer solution (pH7.4) mixes, and solution is then transferred to 100 μ L quartz capillaries.After 2.5 minutes, ESR waves are used
Spectrometer records ESR wave spectrums.The experiment condition of use is as follows：Magnetic field, 336.5 ± 5mT；Power, 1mW；Modulating frequency, 9.41GHz；
Amplitude, 1 × 200；Sweep time, 4min.
6.3. ultra-oxygen anion free radical scavenging capacity
Ultra-oxygen anion free radical is generated by riboflavin/EDTA systems (Guo et al., 1999) of UV illumination.Reaction mixing
Object includes 0.3mM riboflavin, and 1.6mM EDTA, 800mM DMPO, sample is in UV lamp 365nm irradiations 1min.Reaction mixture turns
Move to 100 μ L quartz capillaries measurement.Experiment condition is as follows：Magnetic field, 336.5 ± 5mT；Power, 10mW；Modulating frequency,
9.41GHz；Amplitude, 1 × 1000；Sweep time, 1min.
6.4. Both peroxyl radical scavenging capacity
Both peroxyl radical is generated according to Hiramoto, Johkoh, Sako and Kikugawa, the method for (1993), letter
Yan Zhi, 2,2 '-azos (2- amidine propanes) dihydrochloride (AAPH) of 20 μ L 40mM and 20 μ L phosphate buffered saline solutions (PBS),
α-(4- pyridyl group -1- the oxygen)-N- tertiary butyls nitroketones (4-POBN) of 20 μ L40mM and the mixing of 20 μ L sample solution.Mixture exists
It is incubated 30min at 37 DEG C, is then transferred to 100 μ LESR quartz capillaries measurement.Experiment condition is as follows：Modulating frequency,
100KHz；Microwave power, 10mW；Microwave frequency, 9411MHz；Magnetic field, 336.5 ± 5mT；Sweep time, 30s.Fig. 2 shows four
The scavenging capacity of group separate sources free radical.BABs shows sizable DPPH, super oxygen, hydroxyl and mistake in 1,000 μ g/mL
The scavenging capacity of hydroperoxyl radical is aoxidized, clearance rate is respectively 87.4%, 88.2%, 78.2%, and 72.9%.In addition,
BABs shows four groups of free radical scavenging activities in a dose-dependent manner.
7, DCFH-DA measures cell activity oxygen
It is intracellular to assess for substrate using oxidation-sensitive dyestuff 2 ', 7 '-dichloro fluorescence acetoacetate (DCFH-DA)
Formation (Okimoto, Watanabe, Niki, Yamashita, and Noguchi, 2000) .RAW264.7 cells of ROS are in fluorescence
It is cultivated in micro 96 orifice plate, Hank ' the s balanced salt solutions (HBSS) of 20 μM of DCFH-DA is loaded in plate and dark place is incubated 20min.
Non-fluorescence DCFH-DA dyestuffs freely pass into intracellular and generate 2 ', 7 '-dichlorofluoresceins by intracellular esterase hydrolyzed
(DCFH).Then it handles cell with the Test extraction object of various concentration and is incubated 1h.Cells rinsed with PBS three times after, with HBSS
Dissolve 300 μM of H2O2And it is added into cell.DCFH aoxidizes to form DCF in the presence of ROS, and every 30 minutes at excitation wavelength (Ex)
It is measured using microplate reader under 485nm and launch wavelength (Em) 535nm.Draw the dose dependent and Time Dependent of BABs processing groups
Property effect and compared with the fluorescence intensity of control group and blank group.As shown in Figure 3a, by DCF after the DCFH oxidations that ROS is mediated
Emit fluorescence, time course increases to 150min.Being pre-processed with BABs reduces the dosage and Time Dependent of DCF fluorescence.100μg/
The BABs of mL generates sizable radicals scavenging effect after 30 minutes.More obvious, the BABs of 1,000 μ g/mL concentration can
Significantly to remove free radical in entire incubation time.The result shows that BABs can protect cells from ROS oxidative damages.
8, the separation of genomic DNA and DNA Oxidation Analysis
It using phenol/Proteinase K standardization program (Samnrook and Russell, 2001) and is improved, from RAW264.7
Cell extraction genome high-molecular-weight DNA.In short, cultivating cell in 10 cm dishes, washed twice, is scraped to 1 with PBS
In PBS of the milliliter containing 5mM EDTA.After centrifugation, cell dissolution is in RNase (0.5mg/mL), sodium acetate (0.2M), Proteinase K
In SDS (10%) solution of (10mg/mL).Mixture is incubated 1h respectively in 37 DEG C and 55 DEG C.After incubation, by phenol:Chloroform:It is different
Amylalcohol (25:24:1) with mixture with 1:1 ratio centrifuges 5min in 4 DEG C of 12,000 × g.Supernatant and 100% ice cold ethanol
With 1:1.5 ratio mixing simultaneously keeps 15min at -20 DEG C.With 12, after 000 × g centrifuges 5min, will be deposited in TE buffer solutions
Dissolving measures the DNA of purification with spectrophotometry in 260/280nm.The preparation of 100 μ LDNA reaction mixtures be pass through by
Predetermined concentration test sample (being compared with the distilled water of same volume) and final concentration of 200 μM of FeSO4And 2mM H2O2It presses
Same order is added in the genomic DNA of final concentration of 50 μ g/mL.The EDTA that final concentration of 10mM is added in mixture is incubated.
1% agarose gel electrophoresis separation, 100V are carried out to the aliquot (20 μ L) of the reaction mixture containing about 1 μ g DNA
30min.The ethidium bromide of 1mg/mL colours gel, usesGel image analysis software (Alpha
Innotech, CA, USA) UV lamp light visual inspection.As a result see Fig. 3 b, after reacting 30min, Fe (II)-is used only in control group
H2O2DNA almost all be degraded.However, when with BABs reaction mixtures, observe clearly dose-dependent
DNA antopxidations.The DNA damages of the DNA protection hydroxyl radical free radical inductions of the BABs processing of 250-1000 μ g/mL concentration ranges
Evil shows antioxidation.
9, ACE inhibitory activity measures
It is improved using Ambigaipala, Al-Khalifa and Shahidi (2015) method and inhibits to live to carry out ACE
Property analysis.Reaction mixture includes：5mM HHL are as substrate, the 50mM sodium borate buffer liquids (pH of 0.3MNaCl and 5mlACE
8.3).Sample (50 μ L) is added to above-mentioned reaction mixture (50 μ L) and is mixed with the 8.3mM HHL (150 μ L) containing 0.5MNaCl
It closes.It is incubated 30min at 37 DEG C, 0.1M HCl (250 μ L) are added and terminate further reaction.The extraction of 1.5mL ethyl acetate is added
Hippuric acid.After centrifuging (800 X g, 15min), 1mL upper liquids are transferred in teat glass and at room temperature under vacuum
Evaporate 2h.Hippuric acid is re-dissolved in 3.0mL distilled water, UV spectrophotometers (Model U-3210, Hitachi are used
Co., Tokyo, Japan) in 228nm measurement absorbances.It is handled with the BABs of 125-1,000 μ g/mL concentration ranges to measure ACE
Inhibitory activity.As shown in Fig.4, BABs shows apparent ACE inhibitory activity, all tests various concentration (125-1,000
μ g/mL), 1,000 μ g/mL concentration, which shows highest ACE inhibitory activity (inhibiting rate 81.8%) and measures IC50 values, is
0.62mg/mL。
10, the bioactivity of active peptide is oriented to separation
BABs is classified with ultrafiltration membrane system and concentrates (＜ 1KDa).Then the powder of freeze-drying passes through RP-HPLC color
Spectrometry (RP-HPLC) detaches (being subject to according to the method for Liu et al. people (2013) improved).Use 18 alkyl silica gel column
(5 μm of Inertsil ODS-3,10 × 250mm, particle size), methanol:Water (15:85) it is mobile phase, 1.0mL/min
Velocity separation.215nm monitors (Shimadzu RP-HPLC) (see Fig. 5 a.).The fraction of all collections is lyophilized and is surveyed again
Free radical scavenging activity and ACE inhibitory activity are determined, to showing that the fraction MALDI-TOF/TOF-MS of most highly active is analyzed to survey
Determine peptide sequence.Fraction 5 shows strongest radicals scavenging and ACE inhibitory activity in 1mg/mL.Fraction 5 is to DPPH, hydroxyl, super oxygen
Clearance rate with hydrogen peroxide is respectively 64.2%, 77.7%, 58.2% and 60.2%.Also show that high ACE inhibitory activity
(75.3%) and IC50 values are 0.39mg/mL (table 2.).
(1mg/mL) free radical scavenging activity of fraction 5 and ACE inhibiting effect of table 2.RP-HPLC separation
11, peptide sequencing
MALDI-TOF/MS measures the amino acid sequence of purified peptide.The fraction 5 detached by RP-HPLC, uses methanol:Water:
0.1%TFA (30:69:1) it dissolves.Using alpha-cyano -4- hydroxycinnamic acids (CHCA) as matrix, equipped with patent
The MALDI-TOF/MS of smartbeam laser is emitted in 355nm with 200Hz, and light is obtained with Bruker autoflex speed
Spectrum.Using laser induced breakdown technology (LIFTTM), by set laser intensity, by the fragment ion intensity of parent compound by
30% adjusts to 45%.Wave spectrum is recorded using cation reflective-mode and uses the BAS of trypsin digestion as TOF calibration standards
Solution.3.2 version softwares of BioTools (Medzihradszky and Chalkley, 2015) of RapiDeNovo module into
Row de novo methods are sequenced.Using Matrix Science Mascot softwares processing and with known protein sequence in database
Row compare.The structure determination of fraction 5 is ATPGDEG (MW 752kDa)
12, the suppression mode of ACE is measured
In order to illustrate inhibiting mechanisms of the BABs to ACE, the BABs of various concentration is added into each reaction mixture, according to
The method of Bush et al. (1984) is simultaneously improved.Adding or be not added with according to the substrate (HHL) of above procedure various concentration
ACE inhibitory activity (control, 50 μM, 100 μM) is measured when BABs applies Lineweaver-Burk Plot in the presence of the inhibitors
Measure ACE power.The result shows that the ACE suppression modes of purified peptide are Noncompetition inhibition (Fig. 6 a).
13, zoopery and systolic blood pressure measure
Spontaneous hypertensive rat (10 week old, male, SHR/Hos, SPF grades, weight 180-240g) tail systolic blood
Pressure is more than 180mmHg, comes from Korea Research Institute of Bioscience and Biotechnology
(DaeJeon,Korea).Spontaneous hypertension rat (SHRs) is individually placed in the steel cage in room, and room temperature is kept for 24 DEG C and given
Give 12h Dark-light cycles and laboratory standard diet.Tap water is provided without limitation.Active fraction salt water dissolution, to SHR with
The dosage of 10mg/kg weight (BW) is orally perfused using metal irrigation stomach device.By peptide to the reduction efficiency and Kato of systolic pressure (SBP)
Puli is compared.Captopril is perfused in method identical with bullfrog carnosine.With the salting liquid administration control mouse of same volume.
After sample oral medication, after SHR is heated 10min in 37 DEG C of rooms, using tail sleeve method Softron BP systems
(Softron BP-98A, Tokyo, Japan) measures the SBP behind oral medication 1,2,3,6, and 9h.The results show that peptide and Kato
After Puli's oral medication, SBP is significantly reduced, and observes the maximum reductions of SBP respectively at 3 and 6h (Fig 6b.).
Statistical analysis
Data are represented as the average value (n=3) of mean value ± standard error.Student t, which is examined, to be used for determining the level of signifiance (P<
0.05)。
Claims (9)
1. the method by obtaining peptide in abalone processed side product, the method includes taking off abalone processed side product by electrodialysis
Then salt is classified and is concentrated by ultrafiltration membrane system.
2. the method as described in claim 1 further includes freeze-drying step at least once.
3. method as claimed in claim 2 further includes by HPLC separating-purifyings.
4. a kind of biology peptide product, the biology peptide product are made with claims 1 to 3 any one of them method.
5. biology peptide product as claimed in claim 4, ingredient includes 75.1% protein, 3.84% lipid,
21.06% carbohydrate.
6. biology peptide product as claimed in claim 4, ingredient include the amino acid of following content：19.1% His,
16.33% Cys, 12.12% Asp.
7. by the biological peptide described in any one of claim 4 to 6 in application anti-oxidant, in anti-hypertension.
8. by the biological peptide described in any one of claim 4 to 6 in production for treating anti-oxidant, spontaneous hypertension medicine
Application in object and health food.
9. treating the pharmaceutical composition of spontaneous hypertension, which includes the biological peptide product described in claim 4
With pharmaceutical acceptable carrier or excipient.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105905A (en) * | 2018-09-25 | 2019-01-01 | 济南知尔医药科技有限公司 | A kind of health-care polypeptide product and preparation method thereof |
| CN110256529A (en) * | 2019-05-30 | 2019-09-20 | 集美大学 | A kind of anti-oxidation peptide and its application |
| CN110256530A (en) * | 2019-05-30 | 2019-09-20 | 集美大学 | A kind of anti-oxidation peptide and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104000246A (en) * | 2014-06-13 | 2014-08-27 | 福建省水产研究所 | Method for extracting abalone active elements from abalone cooking liquor |
| CN106616959A (en) * | 2015-11-10 | 2017-05-10 | 华仁药业股份有限公司 | Enteral nutrition preparation for patient in organ transplantation perioperative period and preparation method thereof |
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2018
- 2018-01-09 CN CN201810018438.0A patent/CN108402415A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104000246A (en) * | 2014-06-13 | 2014-08-27 | 福建省水产研究所 | Method for extracting abalone active elements from abalone cooking liquor |
| CN106616959A (en) * | 2015-11-10 | 2017-05-10 | 华仁药业股份有限公司 | Enteral nutrition preparation for patient in organ transplantation perioperative period and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 何东平等: "《多肽制备技术》", 30 June 2013, 中国轻工业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105905A (en) * | 2018-09-25 | 2019-01-01 | 济南知尔医药科技有限公司 | A kind of health-care polypeptide product and preparation method thereof |
| CN109105905B (en) * | 2018-09-25 | 2021-08-24 | 山东国和堂制药有限公司 | Polypeptide health product and preparation method thereof |
| CN110256529A (en) * | 2019-05-30 | 2019-09-20 | 集美大学 | A kind of anti-oxidation peptide and its application |
| CN110256530A (en) * | 2019-05-30 | 2019-09-20 | 集美大学 | A kind of anti-oxidation peptide and its application |
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