CN106478770A - A kind of Fructus Perillae oxidation resistance dipeptide and preparation method and application - Google Patents
A kind of Fructus Perillae oxidation resistance dipeptide and preparation method and application Download PDFInfo
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- CN106478770A CN106478770A CN201610891732.3A CN201610891732A CN106478770A CN 106478770 A CN106478770 A CN 106478770A CN 201610891732 A CN201610891732 A CN 201610891732A CN 106478770 A CN106478770 A CN 106478770A
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- cell
- fructus perillae
- dipeptide
- oxidation resistance
- oxidation
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Links
- 108010016626 Dipeptides Proteins 0.000 title claims abstract description 24
- 230000003647 oxidation Effects 0.000 title claims abstract description 22
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 30
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 25
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- 238000000034 method Methods 0.000 claims abstract description 8
- FSXRLASFHBWESK-HOTGVXAUSA-N Phe-Tyr Chemical group C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 FSXRLASFHBWESK-HOTGVXAUSA-N 0.000 claims abstract description 5
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 claims abstract description 5
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- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 abstract description 11
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- LXEKPEMOWBOYRF-QDBORUFSSA-N AAPH Chemical compound Cl.Cl.NC(=N)C(C)(C)\N=N\C(C)(C)C(N)=N LXEKPEMOWBOYRF-QDBORUFSSA-N 0.000 description 1
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- QRYRORQUOLYVBU-VBKZILBWSA-N Carnosic acid Natural products CC([C@@H]1CC2)(C)CCC[C@]1(C(O)=O)C1=C2C=C(C(C)C)C(O)=C1O QRYRORQUOLYVBU-VBKZILBWSA-N 0.000 description 1
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- CQOVPNPJLQNMDC-UHFFFAOYSA-N N-beta-alanyl-L-histidine Natural products NCCC(=O)NC(C(O)=O)CC1=CN=CN1 CQOVPNPJLQNMDC-UHFFFAOYSA-N 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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- 239000007800 oxidant agent Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- 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
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of Fructus Perillae oxidation resistance dipeptide and preparation method and application.This oxidation resistance dipeptide sequence is Phe Tyr（FY）, experiment in vitro shows, this peptide can effectively be removed ABTS, hydroxyl radical free radical and superoxide anion and have stronger reactive oxygen free radical Scavenging activity（ORAC）Deng.Meanwhile, this peptide can effectively suppress linoleic acid and Hepar Mus lipid peroxidation.Prove through cell experiment, this peptide is safe to cell at low concentrations, and has significant protective effect to the oxidative damage of cell；When this dipeptides with high concentration processes HepG 2 cell, show obvious inhibition of cancer cell effect.It is simple and the features such as anti-oxidant vigor is strong that anti-oxidation peptide involved in the present invention has a structure, can have important value as the excellent replacement of existing synthetic antioxidant to novel oxidation-resistant health product and food additive exploitation and application aspect.
Description
Technical field
The present invention relates to a kind of Fructus Perillae natural anti-oxidation dipeptides and preparation method thereof and field of food and health product neck
The application in domain, belongs to technical field of food biotechnology.
Background technology
Fructus Perillae, also known as Fructus Perillae, is that Labiatae Perilla annual herb plant Folium Perillae (Perillafrutescens) is done
Dry mature fruit.Folium Perillae originates in China, all has wild species and cultivation in China North China, south China, Central China, southwest and Taiwan Province
Kind, it is one of 60 kinds of medicinal and edible plants of the first batch of license of China's Ministry of Public Health.Folium Perillae begins to be loaded in as China's Chinese medicine medical material
Ming Dynasty's holy doctor Li Shizhen (1518-1593 A.D.)《Compendium of Materia Medica》, there is the work(of " promoting the circulation of QI to alleviate the stagnation in middle-JIAO, clearing away phlegm profit lung, and blood, warming middle-JIAO, pain relieving, Dingchuan, antiabortive "
Effect.Fructus Perillae is rich in oils and fatss, and due to being subject to processing the restriction of technical conditions, current Fructus Perillae is mainly used in China's food industry
In extracting edible oil, and the nutrient substance such as its residue rich in proteins also can only be used as animal feed or fertilizer, so
Not only result in the waste of a large amount of protein resources it is also possible to environment is caused with a certain degree of pollution.
Under normal circumstances, the reactive oxygen free radical ROS that body produces can be by the Antioxidant Enzyme Systems of itself（Superoxides
Dismutase, catalase and glutathion peroxidase etc.）And endogenous antioxidant（VE, VC, carnosine, gluathione
Peptide etc.）Maintain relatively low level under effect, protect body not injured by free radical.However, working as endogenouss or exogenous thorn
Swash and promote organism metabolism abnormal and produce a large amount of reactive oxygen free radical, or with advancing age, the polyphenoils of body and oxygen
When balance between agent is not normal, may result in oxidative stress, serious situation can cause oxidative damage, destroy intracellular DNA,
Protein and cell membrane, inducing cell apoptosis, accelerate human senility.Meanwhile, free radical is excessive, also can induce a series of diseases
Disease, such as senile dementia, cerebral thrombosiss, atherosclerosiss and cancer etc..
It would therefore be desirable to seek exogenous antioxidant to remove free radical excessive in vivo, to maintain the strong of body
Health.The antioxidant of synthetic such as 2,6- bis- tert-hydroxyl paracresol（BHT）, Butylated hydroxyanisole（BHA）, tertiary butyl is to benzene
Diphenol（TBHQ）And gallic acid（PG）Although Deng having stronger antioxidation, because it has to human liver, spleen, lung
, and there are potential teratogenesis, carcinogenesis in evil, the national governments ADI to its mandatory provision one after another（Acceptable daily intake）Value,
Control it to be excessively added.Then people progressively turn to sight and extract the Natural antioxidant from various plant and animals tissue.
Antioxidant activity polypeptide due to low toxicity, efficient the features such as, the antioxidant as food and body it is considered to be synthetic resist
The preferable replacer of oxidant.
Anti-oxidation peptide can remove reactive oxygen free radical superfluous in vivo, protection cell and mitochondrial normal configuration effectively
And function, prevent the generation of lipid peroxidation, help body to resist disease.Simultaneously that anti-oxidation peptide is permissible as food additive
Prevent fat-containing food from aoxidizing, can be used for the exploitation of health food and cosmetics etc. as functional factor, to raising China agricultural production
The added value of product deep processing has important function.
Both at home and abroad the research of Fructus Perillae is concentrated in a large number with refinement and the biological activity aspect of oils and fatss at present, and to its by-product
The protein development and utilization research being rich in thing is less, and its active polypeptide or its pharmacological research are not almost reported both at home and abroad
Road.Therefore, it is necessary to make full use of the Folium Perillae of China's very abundant（Fructus Perillae）Plant resourceses, carry out to it going deep into system grinding
Study carefully, for realizing the Fructus Perillae modernization of Chinese medicine and developing new, efficient, safe health food offer scientific basis.
Content of the invention
It is an object of the invention to provide a kind of prepare Fructus Perillae oxidation resistance dipeptide simple, that antioxidant activity is strong, and can
This oxidation resistance dipeptide to be applied to development and the exploitation of health food.
A kind of oxidation resistance dipeptide of the present invention, its aminoacid sequence is Phe-Tyr, is expressed as FY with single-letter, that is, by benzene
2 amino acid residues of AY are constituted.
This oxidation resistance dipeptide can effectively remove ABTS, hydroxyl radical free radical, and ultra-oxygen anion free radical simultaneously has stronger
Oxygen radical removing ability, there is good lipid peroxidation inhibition simultaneously；
This oxidation resistance dipeptide does not have toxic and side effects to normal Chinese hamster ovary celI；Under low concentration, this dipeptides to HepG-2 cell is
Safety, and at this concentration, can effective protection HepG-2 cell from hydrogen peroxide-induced cell injury；
Under 0.1mg/mL concentration, this oxidation resistance dipeptide is inhibited to HepG-2 cell line.
The enzymolysis preparation of Fructus Perillae anti-oxidation peptide：Fructus Perillae egg is obtained from the Fructus Perillae dregs of rice using alkali extraction and acid precipitation
In vain, using alkaline protease by it under optimum enzymolysis condition（PH 9.80, enzyme addition 3000U/g, concentration of substrate 3%（w/
v））Solution enzyme 5.00h, boiling water bath 10min inactivate, and are centrifuged 10min through 12000rpm, take supernatant lyophilization, as Fructus Perillae
Protein digestion product；
Described oxidation resistance dipeptide isolation and purification method：Fructus Perillae protein digestion product utilization Sephadex G-25 gel chromatography
Carry out separating, with deionized water as eluent, flow velocity 0.3 mL/min, the light absorption value at measurement elution fraction 214nm wavelength；Receive
Collection has optimal antioxidant activity part, using reversed-phase high-performance liquid chromatography（RP-HPLC）Separate further；RP-HPLC washes
De- gradient is 0 ~ 60min, 5% ~ 40%（V/V）Acetonitrile；Flow velocity is 2.0 mL/min, Detection wavelength 214 nm, collects retention time
Eluting peak for 35min, lyophilization obtains final product described oxidation resistance dipeptide.
It is an advantage of the current invention that：
Described oxidation resistance dipeptide has strong free radical scavenging activity, to hydroxyl radical free radical, superoxide anion and ABTS free radical with
And reactive oxygen free radical has stronger scavenging action, show that it has important value in terms of antioxidant activity development and application.
The IC to ABTS free radical and hydroxyl radical free radical clearance rate for the Fructus Perillae Natural Antioxidant Peptides FY50Value be respectively 7.23 μ g/mL and
0.41mg/mL;When concentration is for 1.0mg/mL, it is 40.36 ± 1.28% to the clearance rate of ultra-oxygen anion free radical；Natural
The ORAC value of anti-oxidation peptide FY is 3.62 ± 0.14 μm of ol TE/mg polypeptides.
Additionally, this Natural Antioxidant Peptides FY has to external linoleic acid lipid peroxidation and Hepar Mus lipid peroxidation very well
Inhibition.Under conditions of the anti-oxidation peptide FY for 1.0mg/mL for the concentration is mixed with linoleic acid and is placed on 40 DEG C, first four days
It illustrates that this Natural Antioxidant Peptides FY can alleviate lipid peroxidation effectively to the suppression ratio of linoleic acid peroxidation more than 40%
Speed, is prevented from corrupt containing fatty foods, increases its shelf life.This Natural Antioxidant Peptides FY is to mouse liver extract lipid
The IC of peroxidating suppression ratio50It is worth for 6.81 mg/mL, illustrate that certain density anti-oxidation peptide FY has good guarantor to hepatic tissue
Shield effect.
This Natural Antioxidant Peptides FY is at low concentrations to normal Chinese hamster ovary celI and the equal no cytotoxicity of HepG-2 cell, and has
Protect HepG-2 from hydrogen peroxide induced injury；High concentration FY there is certain cancer resistant effect.Chinese hamster ovary celI
After processing 24 hours through 0.1mg/mL FY, normal cell ratio 99.7% is obviously improved with respect to untreated fish group 96.7%, explanation
After processing through FY, more preferably, FY has protective effect to Chinese hamster ovary celI to Chinese hamster ovary celI state.HepG-2 cell is through 0.05mg/mL FY
After processing 24 hours, normal cell ratio 81.0% is increased slightly with respect to untreated fish group 79.1%, illustrates at this concentration, FY pair
HepG-2 cell does not have toxic action；But after processing HepG-2 cell with 0.1 mg/mL FY, the ratio of normal cell 61.9%
Substantially reduce, apoptotic cell ratio substantially increases, and illustrates that, under this concentration, FY can induce HepG-2 apoptosis, have certain
Antitumaous effect.HepG-2 cell is through 1mM H2O2After induced damage, normal cell ratio drops to 44.3%, and dead cell reaches
To 39.9%, if using 0.05mg/mL FY pretreatment HepG-2 cell before hydrogen peroxide-induced oxidative damage, can reduce
The apoptosis rate of cell（Normal cell 57.8%, dead cell 34.5%）, illustrate that this Natural Antioxidant Peptides FY can be effectively protected
HepG-2 cell is by hydrogen peroxide induced injury.
Brief description
Fig. 1：Fructus Perillae dregs of rice crude protein enzymatic hydrolysate Sephedex G-25 gel filtration chromatography figure.
Fig. 2：Gel chromatography eluting peak c peak reversed-phase high-performance liquid chromatography figure.
Fig. 3：The mass spectrum of anti-oxidation peptide.
Fig. 4：The scavenging action to free radical for the Natural Antioxidant Peptides FY.A：ABTS free radical scavenging activity；B：Hydroxyl free
Base scavenging capacity；C：Ultra-oxygen anion free radical scavenging capacity；D：Reactive oxygen free radical Scavenging activity (ORAC).
Fig. 5：The inhibitory action to linoleic acid lipid peroxidation (A) and Hepar Mus lipid peroxidation (B) for the Natural Antioxidant Peptides FY.
Fig. 6：Natural Antioxidant Peptides FY is to Chinese hamster ovary celI toxicity test.A：Normal Chinese hamster ovary celI；B：CHO+0.1mg/mL FY.
Fig. 7：Natural Antioxidant Peptides FY is to HepG-2 cytotoxicity experiment.A：HepG-2 cell；B：HepG-2+0.05mg/
mL FY；C：HepG-2+0.1mg/mL FY.
Fig. 8：The protective effect to hydrogen peroxide-induced HepG-2 cell oxidative damage for the Natural Antioxidant Peptides FY.A：HepG-
2 cells；B：HepG-2+1mM H2O2；C：HepG-2+1mM H2O2+0.05mg/mL FY；D：HepG-2+1mM H2O2+0.1mg/
mL FY.
Specific embodiment
The present embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and mistake
Journey, but protection scope of the present invention is not limited only to following embodiments.
Embodiment 1
The the isolating and purifying including Sephadex G-25 gel filtration chromatography and RP-HPLC of oxidation resistance dipeptide of the present invention
Chromatograph（RP-HPLC）Two steps.
The preparation of Folium Perillae seed crude protein enzymatic hydrolysate：First Fructus Perillae is carried out ungrease treatment with petroleum ether, then adopt alkali
Put forward the heavy method of acid and obtain Folium Perillae seed crude protein, digest Folium Perillae seed crude protein with alkaline protease, optimal at it using alkaline protease
Enzymatic hydrolysis condition：PH 9.80, enzyme addition 3000U/g, concentration of substrate 3%（w/v）Lower solution enzyme time 5.00h, boiling water bath 10min go out
Live, be centrifuged 10min through 12000rpm, take supernatant lyophilization, as Fructus Perillae protein digestion product；
Sephadex G-25 gel filtration chromatography：It is dissolved in Fructus Perillae enzymatic hydrolysate lyophilized powder in deionized water,
12000rpm is centrifuged 30min.Take 0.22 μm of aperture micro-filtrate membrane filtration of supernatant.Sephadex G-25 gel column（1.6cm×
100cm）Deionized water balances, by filtered sample upper prop.Deionized water eluting, flow velocity 0.3mL/min, in 214nm
Detect eluent light absorption value at wavelength, draw elution curve, as shown in Figure 1.Collect eluting c peak, lyophilization, -80 DEG C of low temperature
Save backup.
High performance liquid chromatography：Deionized water dissolving above-mentioned c peak lyophilized powder, is separated further using RP-HPLC.Liquid chromatograph
System is LC-20A, assembles Gemini 5 μ C18（250mm×10mm）Reversed-phase column（Phenomenex, UK）, with water and acetonitrile
（Containing 0.05%（V/V）Trifluoroacetic acid）The elution system constituting carries out gradient elution.Gradient：0 ~ 60min, 5% ~ 40%（V/V）
Acetonitrile；Elution flow rate 2.0 mL/min, Detection wavelength 214nm, elution curve is as shown in Figure 2.Collecting eluting stays the time to be
35.24min, lyophilization is oxidation resistance dipeptide of the present invention.
By the antioxidant composition collected lyophilization, using the compositional purity obtained by high performance liquid chromatography inspection.Warp
Detection, this anti-oxidation peptide compositional purity reaches 95%, can measure its aminoacid sequence.
It is used in conjunction with mass spectrum with liquid chromatograph（LC-MS/MS）Method measures aminoacid sequence（Fig. 3）, obtain this anti-oxidation peptide
Aminoacid sequence be Phe-Tyr (FY).
Embodiment 2
Natural anti-oxidation two peptide activity obtaining in embodiment 1 is studied：
ABTS free radical scavenging acts on：Prepare the ABTS of 7 mmol/L with distilled water+Solution and the potassium peroxydisulfate of 2.45 mmol/L
Solution（First 16 h must be placed using front at room temperature）, respectively by ABTS+Press 1: 1 volume ratio mixing with potassium persulfate solution,
Use the phosphate buffered solution of pH 7.4,5 mmol/L that mixed liquor is diluted to light absorption value A before use734For 0.70 ± 0.02.
The sample of 0.5 mL variable concentrations and ABTS free-atom aqueous solution mixing standing 10 min of 0.5 mL are taken to survey it under 734 nm
Light absorption value.Deionized water and reduced glutathion replace sample to make blank and positive control respectively.ABTS free radical
Removing vigor presses following equation（1）Calculate：
（1）
In formula, A0：Blank control group light absorption value；As：Sample sets light absorption value.
Ultra-oxygen anion free radical scavenging action：Take the Tris-HCl buffering that 0.4 mL sample solution adds 50 mmol/L
Solution（pH 8.3）0.4 mL, replaces sample as blank pipe with distilled water.Concussion mixes, and is incubated in 25 DEG C of water-baths
The pyrogallol hydrochloric acid solution 0.1mL of 1.5 mmol/L is added after 10min（25 DEG C of water-bath preheatings）, rapid mixing reaction 5 min,
Every 30s at 320 nm mensuration absorbance value A320.Deionized water and reduced glutathion replace sample to make sky respectively
White comparison and positive control.Make the time dependent regression equation of light absorption value, the slope of curve is mouse thymus cells speed, sample
Product press following equation to the removing vigor of superoxide anion（2）Calculate：
（2）
In formula, Δ A0/min：The blank control group light absorption value slope of curve；ΔAs/min：The sample sets light absorption value slope of curve.
Hydroxyl radical free radical scavenging capacity：Take 100uL sample and 100uL 6.0mmol/L ferrous sulfate, 100uL
After the mixing of 6.0mmol/L hydrogen peroxide, lucifuge is incubated 10min at room temperature, adds the salicylism reaction of 10uL 6.0mmol/L
30min, measures the light absorption value of reactant under 510nm wavelength.Do blank with distilled water.The scavenging capacity of hydroxyl radical free radical
By following equation（3）Calculate：
（3）
In formula,A s：The light absorption value of experimental group；A 0：The light absorption value of the experimental group without hydrogen peroxide；A b：The extinction of blank control group
Value
Reactive oxygen free radical Scavenging activity（ORAC）：50 μ L sample solution are blended in 100 μ L 70nM Fluresses
In opaque 96 orifice plates, and hatch 15min at 37 DEG C, then, add 50 μ L 200mM AAPH molten in each hole rapidly
Liquid, puts into rapidly fluorescence microplate reader after vibration 30s and carries out fluorescence measurement.Excitation wavelength 485nm, launch wavelength 530nm, every
1min measurement is once until fluorescence intensity no longer changes.In test, all reagent must be with 75 mM pH 7.0 phosphate buffer
Configuration.With phosphate buffer and GSH solution respectively as blank and positive control.Configuration 0.625 μM, 1.25 μM, 2.5 μ
M, 5 μM, 10 μM, 20 μM, 40 μM of watermiscible vitamin Es（Trolox）Solution is made antioxidation standard and is defined non-oxidizability.Fluorescence declines
Subtract area under the curve (AUC) to calculate by following equation (4)：
(4)
In formula：f0Represent initial fluorescence intensity, fiFluorescence intensity when representing i-th minute.Net fluorescence decay curve area
(net-AUC) press following equation (5) to calculate：
(5)
According to oxidation resistance and net-AUC linearly related determination Trolox standard curve, final ORAC value is expressed as μM
Trolox equivalent (TE)/mg polypeptide.
Linoleic acid peroxidation inhibitory activity：By 1.0 mg/ml sample 1.0 mL, 2.5%（V/V）Linoleic acid dehydrated alcohol is molten
Liquid 1.0 mL, 50 mmol/L phosphate（pH 7.0）2.0 mL and deionized water 1.0 mL are placed in tool plug test tube, keep away at 40 DEG C
Light constant temperature is cultivated 7 days.Measure the degree that thiocyanation ferrum value represents linoleic acid peroxidation every 24 h.Take the above-mentioned reactant liquor of 0.1 mL
With 4.7 mL 75%（V/V）Ethanol, 0.1 mL 30%（W/V）Ammonium thiocyanate solution, 0.1 mL 20 mmol/L protochloride iron salt
Acid（3.5%, W/V）Solution mixes, and measures absorbance value, light absorption value A500nm and linoleic acid oxygen after reaction 3 min under 500 nm
Change degree is proportionate.Deionized water makees negative control, makees positive control with GSH and BHA.
The inhibitory activity of Hepar Mus lipid peroxidation：Take murine liver tissue 1.0 g, after being rinsed with ice-cold normal saline,
Filter paper blots, and weighs.It is placed in 5 mL small beakers, measure the normal saline that 9 times of volume blocks amass, by 2/3 physiology
Saline is poured in beaker, shreds rapidly piece of tissue with eye scissorss, then the tissue shredding is poured into glass together with normal saline
In glass homogenizer, then with remaining normal saline flushing beaker and eye scissorss, and pour homogenizer into, be homogenized 6～8
Min, makes tissue homogenate, and whole process should be placed in ice bath and complete.By homogenate with 3000～4000 after homogenate completely
R/min is centrifuged 10～15 min, and supernatant is 10% liver tissue homogenate's liquid.
Quickly by 0.1ml anti-oxidation peptide sample（Concentration is 0.5 respectively, 1,2,4,8mg/ml）Even with 1ml 0.5% hepatic tissue
Serosity, 0.1ml 6mM FeSO4Solution, 0.1ml 60mM H2O2Solution mixes, and in 37 DEG C of water-bath 1h.It is subsequently added 1ml
15% trichloroacetic acid and 1ml 0.67% thiobarbituricacidα- solution, and heat 15min at 95 DEG C.It is cooled to 25 DEG C, then
3000rpm is centrifuged 10min.Absorbance under 532nm wavelength for the measurement.As a control group, distilled water is as blank group for GSH.Mus
Liver lipid peroxidation suppression ratio presses following equation（6）Calculate：
(6)
In formula, A0：Blank control group light absorption value；As：Sample sets light absorption value.
Measure through the present embodiment, Fructus Perillae Natural Antioxidant Peptides FY is to ABTS free radical and hydroxyl radical free radical clearance rate
IC50Value is 7.23 μ g/mL and 0.41mg/mL respectively（Fig. 4 A, 4B）;When concentration is for 1.0mg/mL, it is to superoxide anion certainly
It is 40.36 ± 1.28% by the clearance rate of base（Fig. 4 C）；The ORAC value of Natural Antioxidant Peptides FY is 3.62 ± 0.14 μm of ol TE/mg
Polypeptide（Fig. 4 D）.Under conditions of the anti-oxidation peptide FY for 1.0mg/mL for the concentration is mixed with linoleic acid and is placed on 40 DEG C, first four days
It is to the suppression ratio of linoleic acid peroxidation more than 40%（Fig. 5 A）, illustrate that FY can alleviate lipid peroxidation speed effectively, can
Prevent corrupt containing fatty foods, increase its shelf life.This Natural Antioxidant Peptides FY presses down to mouse liver extract lipid peroxidation
The IC of rate processed50It is worth for 6.81 mg/mL（Fig. 5 B）, illustrate that certain density anti-oxidation peptide FY has good protection to hepatic tissue
Effect.
Embodiment 3：
The natural anti-oxidation dipeptides obtaining in embodiment 1 is carried out cytotoxicity and antioxidation research：
Cytotoxicity experiment：Chinese hamster ovary cell through recovery（CHO）With hepatoma carcinoma cell (HepG-2) with containing 10% tire
The modified form RPMI-1640 culture medium of Ox blood serum, 2.0 mmol/L glutamate, Glus and 100 U/mL Pen .- Strep is moistening
, containing 5% carbon dioxide, temperature is adhere-wall culture 24 ~ 48 h in the environment of 37 DEG C.Add after being digested at 37 DEG C with pancreatin
RPMI-1640 culture medium terminates digestion, and adding appropriate RPMI-1640 culture medium to carry out piping and druming makes cell fully dispersed, inoculation
Continue culture until cell attachment area reaches more than 80% in 6 orifice plates.With final concentration of 0.05mg/mL's and 0.1mg/mL
FY is dyeed through flow cytomery every hole cell with double transfection reagent Annexin V-FITC and PI of cell after processing cell 24 h
Cell state.
Protection to hydrogen peroxide-induced hepatoma carcinoma cell oxidative damage：Hepatoma carcinoma cell (HepG-2) through recovery is pressed upper
State method culture, reach more than 80% in the adherent area of 6 orifice plate inner cells, the FY of final concentration of 0.05mg/mL and 0.1mg/mL
After using 1mmol/L hydrogen peroxide treatment cell 6h again after processing cell 18 h, with the double transfection reagent Annexin V-FITC of cell and
The cell state through flow cytomery every hole cell for the PI dyeing.
Measure through this embodiment, after Chinese hamster ovary celI is processed 24 hours through 0.1mg/mL FY, normal cell ratio 99.7%
(Fig. 6 B) is obviously improved with respect to untreated fish group 96.7% (Fig. 6 A), and after illustrating to process through FY, Chinese hamster ovary celI state can more preferably, FY pair
Chinese hamster ovary celI has protective effect.After HepG-2 cell is processed 24 hours through 0.05mg/mL FY, normal cell ratio 81.0%
(Fig. 7 B) is increased slightly with respect to untreated fish group 79.1% (Fig. 7 A), illustrates at this concentration, FY does not poison to HepG-2 cell
Effect；But after processing HepG-2 cell with 0.1 mg/mL FY, the ratio of normal cell 61.9% (Fig. 7 C) substantially reduces, and withers
Cell proportion of dying substantially increases, and illustrates that under this concentration, FY can induce HepG-2 apoptosis, has certain antitumaous effect.
HepG-2 cell is through 1mM H2O2After induced damage, normal cell ratio drops to 44.3%, and dead cell reaches 39.9% (figure
8B), if using 0.05mg/mL FY pretreatment HepG-2 cell before hydrogen peroxide-induced oxidative damage, cell can be reduced
Apoptosis rate（Normal cell 57.8%, dead cell 34.5%）(Fig. 8 C), illustrates that this Natural Antioxidant Peptides FY can effectively protect
Shield HepG-2 cell is by hydrogen peroxide induced injury.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of Fructus Perillae oxidation resistance dipeptide and preparation method and application
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2
<212> PRT
<213>Artificial sequence
<400> 1
Phe Tyr
1
Claims (3)
1. a kind of new Fructus Perillae oxidation resistance dipeptide it is characterised in that：Described anti-oxidation peptide aminoacid sequence is Phe-Tyr.
2. a kind of preparation method of Fructus Perillae oxidation resistance dipeptide as claimed in claim 1 it is characterised in that：Including as follows：
（1）The preparation of Fructus Perillae protein hydrolysate：First Fructus Perillae is carried out ungrease treatment with petroleum ether, then adopt alkali carries acid
Heavy method obtains Folium Perillae seed crude protein, digests Folium Perillae seed crude protein, its enzymatic hydrolysis condition with alkaline protease:PH 9.80, enzyme addition
3000U/g, concentration of substrate 3%w/v, solution enzyme time 5.00h, boiling water bath 10min inactivates, and is centrifuged 10min through 12000rpm, takes
Clear liquid lyophilization, as Fructus Perillae protein digestion product；
（2）The enzymatic hydrolysate of Fructus Perillae albumen carries out separating using Sephadex G-25 gel chromatography, with deionized water as eluting
Liquid, flow velocity 0.3 mL/min, collects elution fraction and measures light absorption value at 214nm wavelength, draw elution curve, collect eluting
Peak；To have the peak of antioxidant activity, be separated further using reversed-phase high-performance liquid chromatography RP-HPLC；The eluting ladder of RP-HPLC
Spend for 0 ~ 60min, 5% ~ 40%V/VAcetonitrile；Flow velocity is 2.0 mL/min, Detection wavelength 214 nm, and collection retention time is 35min
Eluting peak, lyophilization obtains final product described oxidation resistance dipeptide.
3. a kind of Fructus Perillae oxidation resistance dipeptide as claimed in claim 1 is in terms of antioxidant health-care product exploitation and food industry
Application.
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|---|---|
| CN106478770B (en) | 2019-12-17 |
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