Pentapeptide with anti-oxidation function and preparation method thereof
Technical field
The invention belongs to anti-oxidation peptide technical field, be specifically related to a kind of pentapeptide with anti-oxidation function and system thereof
Preparation Method.
Background technology
Numerous researchs show, body oxidativestress damage can cause Metabolic disorder, thus accelerates aging, cause
The diseases such as senile dementia, tumor and atherosclerosis.And the absorption by extraneous antioxidant can be one
Determine to remove in degree interior free yl, play slow down aging, prevent the effect of various disease.Nowadays great majority are anti-
Oxidant is all synthetic, in contrast, prepare from natural plants antioxidant have be prone to absorb,
The advantage that safety has no side effect.Meanwhile, China has profuse animal and vegetable protein resource, utilizes this advantage,
Exploitation Natural Antioxidant Peptides has great importance.
Changbaishan area, Jilin Province abounds with Semen coryli heterophyllae, although with a long history, but exploitation aspect is still at the beginning of comparison
In the stage of level, processing is only limitted to make cake, feedstuff and oil expression etc..Anti-with Changbai Mountain Semen coryli heterophyllae for raw material preparation
Oxidation peptide, will make full use of Changbai Mountain Semen coryli heterophyllae resource and the exploitation offer ginseng of Semen coryli heterophyllae polypeptide functional product
Examine.
Summary of the invention
An object of the present invention is to provide a kind of pentapeptide with anti-oxidation function and preparation method thereof, with long white
Mountain Semen coryli heterophyllae is raw material, uses enzyme to control technology for hydrolyzing and prepares anti-oxidation peptide and by gel permeation chromatography Sephadex
The technology such as G-25, Sephadex G-15 and RPHPLC (reversed-phase high-performance liquid chromatography) are isolated and purified, obtain many through the method
Peptide activity and purity are higher, and impurity is less.
The two of the purpose of the present invention are that Semen coryli heterophyllae anti-oxidation peptide carries out Structural Identification, use high performance liquid chromatography-electricity
Spraying flight time tandem mass spectrum (HPLC-ESI-TOF MS/MS) technology is the most square through de novo sequencing (de novo)
Method carries out interpretation of mass spectra, identifies two kinds of new anti-oxidation peptide AWDPE and DWDPK.
Pentapeptide with anti-oxidation function and preparation method thereof, described anti-oxidation peptide includes two peptides, is respectively
AWDPE (Ala-Trp-Asp-Pro-Glu) and DWDPK (Asp-Trp-Asp-Pro-Lys), its preparation process is as follows:
(1) preparation of Semen coryli heterophyllae anti-oxidation peptide
By 2~5g Semen coryli heterophyllae separated protein powders, (prepared by alkali extraction-acid precipitation, Functional Properties of the Protein
Isolate and Major Fractions of Pine Nut Proteins Prepared from the Changbai
Mountain in China, Dan Wu, Wei-hong Min) it is dissolved in 100mL distilled water and is configured to quality and divides
The solution of several 2%~5%, 90~100 DEG C of water-baths 10~20min make albuminous degeneration, after cooling regulate pH to 7~
10, add Alcalase 2.4L, maintain constant pH value by potentiometric titrimeter;After enzymolysis terminates, solution
At 90~100 DEG C, enzyme denaturing 10~20min, are cooled to room temperature, regulate pH to 7.0,5000~8000 with HCl
Rpm is centrifuged 10~20min, takes supernatant, will obtain thick Semen coryli heterophyllae anti-oxidation peptide after its lyophilization, measures anti-
Oxidation activity.
(2) Semen coryli heterophyllae anti-oxidation peptide is isolated and purified
Thick Semen coryli heterophyllae anti-oxidation peptide is added distilled water wiring solution-forming, and crosses the filter membrane of 0.22 μm;Then pass through
Sephadex G-25 sephadex column (1.6cm × 80cm) chromatography, with Ultraviolet Detector at 280nm
Detect under wavelength, and be collected component (1.5mL/ pipe) with automatic fraction collector, by the 3 of isolated
Plant Fraction collection, lyophilization, measure antioxidant activity;The strongest for activity component is carried out Sephadex G-15
Chromatography, Ultraviolet Detector detects at a wavelength of 280 nm, and collects (1.5mL/ with automatic fraction collector
Pipe), by the 2 of isolated kinds of Fraction collection, lyophilization, measure antioxidant activity;By the highest for activity group
Divide and carry out RPHPLC (reversed-phase high-performance liquid chromatography) separation, the 5 kinds of Fraction collection that will obtain, lyophilization, measure antioxidation
Activity, the highest component of activity be the pentapeptide AWDPE with anti-oxidation peptide function of the present invention and
DWDPK。
(3) Structural Identification of Semen coryli heterophyllae anti-oxidation peptide
Use ESI-TOF MS/MS to identify the aminoacid sequence of ace inhibitory peptide, use de novo method to carry out
Mass spectrometric data resolves.Structural Identification result is the pentapeptide AWDPE with anti-oxidation function of the present invention
And DWDPK.
In step (1), utilize the pH value that 0.6mol/L NaOH tune pH value 7~10 is constant with maintenance, enzymolysis
Temperature is 40~70 DEG C, enzymolysis time is 3~7h, enzyme concentration 5000~10000U/g Semen coryli heterophyllae separation albumen.
Sephadex G-25 gel chromatography described in step (2) and Sephadex G-15 gel chromatography, on
Sample concentration 10~50mg/mL, loading volume 1~5mL, elution flow rate 0.1~3mL/min, eluent is pure
Water.
RPHPLC (reversed-phase high-performance liquid chromatography) described in step (2) uses C18 chromatographic column, and mobile phase A is containing matter
Amount mark 0.1%TFA distilled water, B is the acetonitrile containing mass fraction 0.1%TFA, sample concentration 0.1~
5mg/mL, loading volume 10~80 μ L, 100~60%B linear elutions 30~60min;Flow velocity 0.1~
1mL/min。
The present invention having the beneficial effect that compared to existing technology
Present invention firstly discloses pentapeptide with anti-oxidation function and preparation method thereof, use alkaline protease
Alcalase 2.4L enzymolysis Semen coryli heterophyllae separation albumen is prepared and Purification goes out two kinds of anti-oxidation peptides do not reported,
Be there is significant scavenging action, to Fe in DPPH, hydroxyl radical free radical, ABTS2+There is preferable chelate effect,
Possess stronger antioxidant activity, be a kind of good Natural antioxidant, have great researching value with wide
Wealthy market prospect.
Accompanying drawing explanation
Fig. 1 is the Semen coryli heterophyllae anti-oxidation peptide scavenging action curve chart to ABTS free radical;As shown in Figure 1, Semen coryli heterophyllae
When anti-oxidation peptide concentration reaches 3mg/mL, the clearance rate of ABTS free radical is reached 96.43%, and
Vc has reached 100% when 0.5mg/mL.In the range of experimental concentration, anti-oxidation peptide is to ABTS freely
Base clearance rate is maintained at 98%~100% after 3mg/mL, and now anti-oxidation peptide and Vc are to ABTS free radical
Clearance rate essentially identical, i.e. anti-oxidation peptide to ABTS free radical clearance rate is Vc 100%.Illustrate anti-
Oxidation peptide has stronger scavenging action to ABTS free radical, has stronger antioxidant activity.
Fig. 2 is the Semen coryli heterophyllae anti-oxidation peptide scavenging action curve chart to DPPH free radical;As in figure 2 it is shown, work as Vc
When concentration is 0.1mg/mL, clearance rate just reaches 91.22%.Semen coryli heterophyllae anti-oxidation peptide is clear to DPPH free radical
Except rate raises with the increase of concentration and gradually stable after 2mg/mL, reach 100% during 8mg/mL.
Fig. 3 is the Semen coryli heterophyllae anti-oxidation peptide scavenging action curve chart to hydroxyl radical free radical free radical;Vc is as one
Good antioxidant, when 0.5mg/mL, clearance rate just can reach 100%.Semen coryli heterophyllae anti-oxidation peptide is to hydroxyl
The scavenging action of free radical significantly improves with the increase of concentration, and when concentration is 8mg/mL, clearance rate reaches
More than 85%, and reach 100% when 24mg/mL.
Fig. 4 is the reducing power effect curves figure of Semen coryli heterophyllae anti-oxidation peptide;The sample having reducing power can make ferrum cyaniding
Fe in potassium3+It is reduced into Fe2+(potassium ferrocyanide), product and FeCl3Reaction generates at 700nm further
There is the Prussian blue (Fe at maximum extinction peak4[Fe(CN)6]3), therefore at mensuration 700nm, the height of light absorption value can
To reflect the size of sample reducing power, light absorption value is the biggest, then reducing power is the strongest.Result of the test such as 3.15
Shown in figure, along with the rising of concentration, the reducing power of Semen coryli heterophyllae anti-oxidation peptide gradually strengthens, during 10mg/mL,
A700 value is 1.055, it is possible to reach under same concentration the 38.55% of EDTA.
Fig. 5 is the Semen coryli heterophyllae anti-oxidation peptide chelation curve chart to ferrous ion;0.5mg/mL EDTA can make Fe2+
Chelation percent reaches 100%, and when concentration is 10mg/mL, the chelation percent of anti-oxidation peptide is 90.25%, 12mg/mL
Time reach 99.30%.Visible Semen coryli heterophyllae anti-oxidation peptide is to Fe2+There is preferable sequestering power.
Fig. 6 is embodiment Sephadex G-25 sephadex chromatography collection of illustrative plates;By Sephadex G-25 gel
Semen coryli heterophyllae anti-oxidation peptide is separated into A1, tri-components of A2, A3 by chromatograph.According to gel chromatography principle, component A1
Should be for hydrolysis albumen the most thoroughly, molecular weight is more than 5000Da, and A2, A3 are the polypeptide that hydrolysis obtains,
Molecular weight is at below 5000Da.
Fig. 7 is embodiment Sephadex G-15 sephadex chromatography collection of illustrative plates;Sephadex G-15 glucosan coagulates
The molecular weight ranges that glue is suitable for is 100~1500, may be used for the exchange of buffer, desalination and separation molecular weight
Close little molecule.As it is shown in fig. 7, component A3 obtains two kinds of components of B1, B2 through G-15 is isolated and purified.
Fig. 8 is embodiment reversed-phase high-performance liquid chromatography separating spectrum;0~the 10min peak occurred is solvent peak, logical
In the case of Chang, solvent has certain uv absorption, forms solvent peak, the most all can be at 10 minutes
Occur before, have no effect on purification effect.Figure is composed peak and is concentrated mainly in 18~40min intervals, separates main
Obtain 5 kinds of components: C1, C2, C3, C4, C5.
Fig. 9 is two kinds of anti-oxidation peptide mass spectruies.
Detailed description of the invention
The present invention is explained further below in conjunction with embodiment, but the present invention is not limited in any way by embodiment.
(1) preparation of Semen coryli heterophyllae anti-oxidation peptide
Semen coryli heterophyllae separated protein powder and distilled water are configured to the solution of mass fraction 5%, water-bath 15min at 90 DEG C,
Thus destroy its protein structure.Utilize 0.6mol/L NaOH to adjust pH value to reach 9.5 after cooling, add Alcalase
2.4L (Novi of Denmark letter hydrolytic enzyme), addition 8000U/g separated protein powder, keep temperature 50 C and pass through
Potentiometric titrimeter adds the pH value that 0.6mol/L NaOH remains constant, and after enzymolysis 5h, solution goes out at 95 DEG C
Enzyme 15min, is cooled to room temperature, adds HCl and regulates pH to 7.0, and 5000r/min is centrifuged 10min, takes
Clear liquid, will obtain thick Semen coryli heterophyllae anti-oxidation peptide after its lyophilization.
(2) Semen coryli heterophyllae anti-oxidation peptide is isolated and purified
The thick Semen coryli heterophyllae anti-oxidation peptide of preparation, adds distilled water and is made into the solution of 30mg/mL, and cross 0.22 μm
Filter membrane.Separated by Sephadex G-25 sephadex column (1.6cm × 80cm), applied sample amount 2mL,
Eluent is distilled water, and elution flow rate 0.5mL/min detects at a wavelength of 280 nm with Ultraviolet Detector, and
It is collected (1.5mL/ pipe) with automatic fraction collector.Isolated A1, tri-components of A2, A3, point
Other lyophilization, measures antioxidant activity (the results are shown in Table 1), and component A3 has the strongest ABTS free radical
Scavenging activity and DPPH radical scavenging activity and reducing power;And the reducing power of component A2 and ABTS are certainly
Scavenging activity by base and DPPH free radical is the strongest;The total reducing power of A1 component and DPPH free radical are clear
Removing solid capacity is the most weak is below the most purified sample.To sum up, component A3 is selected to carry out next step isolated and purified.
By component A3, add distilled water and be made into the solution of 30mg/mL, and cross the filter membrane of 0.22 μm.With
Sephadex G-15 sephadex column (1.6cm × 80cm) separates, applied sample amount 2mL, and eluent is
Distilled water, elution flow rate 0.5mL/min, detects at a wavelength of 280 nm with Ultraviolet Detector, and uses automatic portion
Catcher is divided to be collected component (1.5mL/ pipe).Isolated B1, two kinds of components of B2, lyophilization,
Measuring antioxidant activity (the results are shown in Table 2), the antioxidant activity of component B2 is better than B1, so collecting component
B2, utilizes high performance liquid chromatography the most isolated and purified.
Use high performance liquid chromatography, component B2 is carried out isolated and purified.It is made into the solution of 1mg/mL, and
Cross the filter membrane of 0.22 μm.Chromatographic condition: instrument: Japan's Shimadzu chromatograph of liquid;Chromatographic column: Diamonsil
C18(250*4.6,5μm);Flowing phase (A: distilled water+mass fraction 0.1% trifluoroacetic acid;B: acetonitrile+quality
Mark 0.1% trifluoroacetic acid), applied sample amount 20 μ L, elution flow rate 0.5mL/min, detect wavelength 220nm,
Column temperature 30 DEG C.95~70%B linear elutions.5 kinds of components of isolated: C1, C2, C3, C4, C5, by
Less in the sample size of reversed-phase high-performance liquid chromatography isolated, so selection microplate reader mensuration hydroxyl radical free radical,
ABTS, three indexs of reducing power, concentration is 0.5mg/mL.Result is as shown in table 3, with other 4 kinds of groups
Split-phase ratio, C2 has the strongest reducing power and ABTS radical scavenging activity, and stronger DPPH is freely
Base Scavenging activity, so the antioxidant activity of C2 is the strongest, C4 takes second place, and other composition activities are more weak.Therefore I
Select C2 component is collected and Structural Identification.
(3) Structural Identification of Semen coryli heterophyllae anti-oxidation peptide
Use Agilent 1200 type sharp separation liquid chromatographic system and Agilent 6520Q-TOF mass spectrograph.Liquid
Phase condition: chromatographic column: Agilent Eclipse Plus C18 (2.1 × 150mm, 3.5 μm);Binary linear gradient is washed
De-: flowing phase: (A) 0.1% aqueous formic acid;(B) acetonitrile;Eluent gradient: 0~30min, 5%-30%B;
Column temperature 35 DEG C, flow velocity 0.4mL/min, sample size 5 μ L.
Mass Spectrometry Conditions: use electron spray cation scan pattern (ESI+);Mass scan range m/z 100~
2000;Dry gas stream speed (N2) is 9L/min, dryer temperature 300 DEG C, is atomized voltage 35psig, hair
Tubule voltage is 3.5kV, fragmentation voltage 175V, taper hole voltage 65V, eight grades of bar radio-frequency voltages 250V, two
Level MS/MS collision voltage 12-15eV.
Peptide section sequence resolves and uses Peaks 7.5 software (Bioinformatics Solutions Inc).Identified hazel
The structure of core anti-oxidation peptide is AWDPE and DWDPK respectively, and their molecular weight is 616.25 Hes respectively
659.29Da。
The above is only the preferred embodiments of the present invention, it is noted that before without departing from the principle of the invention
Put, other permutation and combination methods of the aminoacid sequence of two kinds of anti-oxidation peptides and some improvement that polypeptide is made and
Modify, also should be regarded as protection scope of the present invention.
Each component Antioxidant Indexes of table 1:G-25 isolated is than living
|
ABTS free radical scavenging activity |
DPPH free radical scavenging activity |
Reducing power |
A1 |
24.06 |
93.88 |
0.028 |
A2 |
137.25 |
142.62 |
0.074 |
A3 |
344.96 |
258.4 |
0.256 |
Thick peptide |
12.1 |
100.62 |
0.160 |
Each component Antioxidant Indexes of table 2:G-15 isolated is than living
|
ABTS free radical scavenging activity |
DPPH free radical scavenging activity |
Reducing power |
B1 |
277.19 |
150.33 |
0.021 |
B2 |
400.87 |
271.32 |
0.30 |
Table 3: each component Antioxidant Indexes of RP-HPLC isolated is than living
|
ABTS free radical scavenging activity |
DPPH free radical scavenging activity |
Reducing power |
C1 |
101.33 |
57.93 |
0.007 |
C2 |
1021.73 |
761.44 |
0.852 |
C3 |
565.22 |
439.07 |
0.344 |
C4 |
608.71 |
721.43 |
0.211 |
C5 |
37.95 |
22.37 |
0.002 |