CN105175507B - A kind of pentadecapeptide and its application - Google Patents
A kind of pentadecapeptide and its application Download PDFInfo
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Abstract
The invention discloses a kind of pentadecapeptide and its applications, the amino acid sequence of the synthesis pentadecapeptide is as follows: Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro- Lys, it is abbreviated as WVAGLGYFTKNGGPK, molecular weight 1594.6,95% or more purity.Polypeptide of the invention uses Peptide synthesizer, is synthesized using solid-phase synthesis.The object of the present invention is to provide a kind of with antioxidation activity in vitro, protection erythrocyte hemolysis and the synthesis pentadecapeptide for promoting human skin fibroblasts collagen to generate, and can be applied to the fields such as bio-pharmaceuticals and cosmetics.
Description
Technical field
The present invention relates to the fields such as bio-pharmaceuticals and cosmetics, and in particular to a kind of synthesis pentadecapeptide and its application.
Background technique
Biologically active peptide is that have a variety of body metabolisms and physiological regulation function, absorption easy to digest, have promote immunological regulation,
Antibacterial, blood pressure lowering, anticancer, it is anti-oxidant the effects of dipeptides to complicated linear, ring structure different peptides general name, by 25
A natural amino acid is made up of with different form with arrangement mode peptide bond.Active peptide safety is high, is current international food
The function factor of boundary most popular research topic and great development prospect.Modern nutriology shows that protein is made through digestive enzyme
It is not fully absorbed, is absorbed in the form of low peptide mostly, and low peptide specific ionization amino acid in the form of free amino acid after
With higher nutritive value and biological value.
The ability that sample removes free radical is the evaluation most common method of its antioxidant activity.The aging of free radical and the mankind
Closely related with certain pathologic processes, they all have azygous single electron, attack internal cell, destroy lipid, protein
And DNA structure, eventually lead to the generation of the missing and disease of body function.It is certain in prevention to remove excessive free radical in vivo
Play a significant role during disease.
ABTS, 2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts, are a kind of water-soluble substances, can quilt
MnO2It is oxidized to more stable blue-green ABTS free radical, this free radical has stable maximum absorption band in 736 nm, resists
It is colourless ABTS that oxidant, which is capable of providing hydrogen atom for radical reduction, can be become according to absorbance of the solution at 736 nm
Change to assess antioxidant for clearing ABTS+Ability.DPPH(1,1- diphenyl -2- hardship diazanyl) it is a kind of alcohol-soluble substance,
Stabilized radical centered on nitrogen-atoms and dark purple is generated in alcoholic solution, which has maximum at 522 nm
Absorption peak, after anti-oxidant sample is added, solution can fade, can be by the variation of its light absorption value come the anti-oxidant work of test sample
Property.FRAP method (Ferric reducing antioxidant potential Assay) is measurement substance oxidation resistance
A kind of method.Principle is that polyphenoils can restore Ferric-tripyridyltriazine (Fe under acid condition3+-
TPTZ the Fe of blue) is generated2+- TPTZ then can be obtained total antioxygen in sample in the Fe2+-TPTZ of 596 nm measurement blue
Change ability.Three of the above model manipulation is easy, quick, controllability is used widely well.
Building cell oxidative damage model be evaluate sample whether there is the common method of oxidation resistance, and red blood cell by
It is easy in abundance, materials, it can be used as a kind of metabolic model of simple out-phase medium, be normal in external biological experiment
Use material.Free radical casts the first stone lipid and protein on erythrocyte membrane, keeps cell membrane damaged, releases the blood in red blood cell
Lactoferrin, referred to as erythrocyte hemolysis.AAPH,H2O2It is the substance of common induction erythrocyte hemolysis with Hemin.
Atomic force microscope (AFM) is a kind of high-resolution novel imaging tool, has been used extensively on biomedical boundary
In cell morphology and its characterization of ultra microstructure, principle is mutual between generated atom when scanning sample by detection probe
Active force obtains sample topography structural information, handles and is imaged through computer software.Free radical attack cells film be allowed to structure and
Function is lost, and cell haemolysis occurs.This conclusion is by observing control group, damage group and protection with atomic force microscope (AFM)
Group red blood cell pattern is further verified.
The genetic predisposition's (also referred to as age aging) and people that skin aging is mainly people are to the physiological reaction of environmental stress
The result of (also referred to as light aging).Light aging is effect of the external environment to the biological answer-reply of skin.Skin is to light aging
Response is usually related with the shortage of normal aquation, skin sagging and lines and wrinkled appearance.UVB irradiation can make into
Fiber finer intracellular reactive oxygen species generation (reactive oxygen species, ROS) accumulation, be more than its Scavenging activity, break oxidation with
Response to oxidative stress occurs for anti-oxidant balance, adjusts a series of cell effect of sequencing, or even the relevant signal of regulation aging
Access promotes the generation of cell ageing.Some researches show that the accumulation of ROS in the cell is skin photoage occurrence and development process
In important link.Human skin fibroblasts be application on human skin produce collagen main place, quantity number and cell
The yield of collagen and skin aging state are closely bound up.Therefore, the yield of human skin fibroblasts collagen be by
A kind of index for the evaluation skin aging state approved is widely applied.
Summary of the invention
There is antioxidation activity in vitro, protection erythrocyte hemolysis and promotion fell the object of the present invention is to provide a kind of
The synthesis pentadecapeptide that skin Collagen of Fibroblasts albumen generates, can be applied to the fields such as bio-pharmaceuticals and cosmetics.
The present invention is molten with ABTS free radical, DPPH radicals scavenging experiment, FRAP experiment, the red blood cell of APPH induction
Blood experiment and ultraviolet damage human skin fibroblasts are active come the anti-oxidant and anti aging effect for evaluating polypeptide.
Synthesis pentadecapeptide of the present invention is abbreviated as WVAGLGYFTKNGGPK, molecular weight 1594.6, sequence are as follows: Trp-
Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-Lys.Wherein,
Trp indicates that English name is Tryptophan, and Chinese is the corresponding residue of the amino acid of tryptophan；
Val indicates that English name is Valine, and Chinese is the corresponding residue of the amino acid of valine；
Ala indicates that English name is Alanine, and Chinese is the corresponding residue of the amino acid of alanine；
Gly indicates that English name is Glycine, and Chinese is the corresponding residue of the amino acid of glycine；
Leu indicates that English name is Leucine, and Chinese is the corresponding residue of the amino acid of leucine；
Tyr indicates that English name is Tyrosine, and Chinese is the corresponding residue of the amino acid of tyrosine；
Phe indicates that English name is phenylalanine, and Chinese is the corresponding residue of the amino acid of phenylalanine；
Thr indicates that English name is Threonine, and Chinese is the corresponding residue of the amino acid of threonine；
Lys indicates that English name is lysine, and Chinese is the corresponding residue of the amino acid of lysine；
Asn indicates that English name is Asparagine, and Chinese is the corresponding residue of asparagine；
Pro indicates that English name is proline, and Chinese is the corresponding residue of the amino acid of proline.
Amino acid sequence of the present invention uses standard Fmoc scheme, and by the screening of resin, reasonable polypeptide is closed
At method.The C- carboxyl end group of target polypeptides is connected in the form of covalent bond with an insoluble macromolecule resin, then with this
The amino of a amino acid acts on forming peptide bond as starting point with the carboxyl of another molecule amino acid.This process is constantly repeated, i.e.,
Available target polypeptides product.After the completion of synthetic reaction, protecting group is removed, peptide chain is separated with resin and is produced to get to target
Object.Peptide systhesis is the process that amino acid is added in a repetition, and synthesis in solid state sequence is synthesized from C-terminal to the end N.
The present invention mixes the synthesis pentadecapeptide of final concentration of 1-100 μ g/mL and normal human red cell, is incubated for, warp
After AAPH damages 2 h, so that cell haemolysis inhibiting rate reaches 56.76% ~ 86.60%.At synthesis pentadecapeptide with 100 μ g/mL
After reason with only with AAPH damage red blood cell group compared with, pattern is more smooth than simple AAPH processing group, orderly, present pie
Structure.With 5 μ g/mL sample treatment red blood cells, SOD vigor rises to 5.1958 by 2.5976 mgprot/ml
Mgprot/mL, the results showed that, the SOD activity of red blood cell can be cured relative to improving a lot in biology after synthesis pentadecapeptide processing
Medicine and cosmetics etc. are applied in fields.
The synthesis pentadecapeptide that concentration is 1-10 μ g/mL is added in human skin fibroblasts culture solution and is incubated for by the present invention,
Through ultraviolet (UVB) 80mJ/cm of medium wave2After damage, cell survival rate variation is little, so that intracellular collagen albumen yield improves
14.9-30.65%。
Compared with prior art, the invention has the advantages that and technical effect:
The present invention has synthesized the peptide for the first time, and uses iii vitro chemical anti-oxidation method and have detected the anti-of synthesis pentadecapeptide
Oxidation activity, the synthesis pentadecapeptide have very strong DPPH, ABTS+Free radical scavenging ability, and its electron supplying capacity compared with
By force, while the Red Blood Cells of Normal Persons applied to AAPH damage is protected, so that erythrocyte hemolysis rate significantly reduces, SOD activity
It is greatly improved, meanwhile, synthesis pentadecapeptide can promote the generation of the human skin fibroblasts collagen of ultraviolet damage.
Detailed description of the invention
Fig. 1 is synthesis pentadecapeptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-
The ESI-MS map of Pro-Lys, wherein abscissa is m/z (mass-to-charge ratio value), ordinate is that (signal is strong by Intensity
Degree).
Fig. 2 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-
The ABTS free radical scavenging activity comparison diagram of Lys.
Fig. 3 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-
The DPPH free radical scavenging activity comparison diagram of Lys.
Fig. 4 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-
The reducing power figure of Lys.
Fig. 5 is the Red Blood Cells of Normal Persons haemolysis inhibiting rate change curve that AAPH damage is carried out after synthesizing pentadecapeptide addition.
Fig. 6 is atomic force microscope (AFM) image of different disposal group red blood cell.
Specific embodiment
Below in conjunction with specific example, the invention will be further described, but implementation and protection scope of the invention is not limited to
This.For not specifically specified technological parameter, routine techniques progress can refer to.
Solid-phase synthesis peptides
It selects macromolecule resin (Chinese Peptide Co., Ltd.), according to amino acid sequence
The feature of Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro- Lys, first
The carboxyl of Lys is connected in the form of covalent bond with a resin, then the carboxyl of the amino of Lys and Pro, which shrink, reacts, processing
Afterwards, then Gly is added, the carboxyl reaction of the amino and Gly of Pro successively adds amino acid from right to left, added the last one Trp
After amino acid, then cuts off resin and obtain target polypeptides.It is purified using high performance liquid chromatography, column model is
kromasil C18Mobile phase A: -5, size 4.6*250mm contain the acetonitrile of 0.1% trifluoroacetic acid (TFA)；Mobile phase B: contain
The water of 0.1%TFA；A initial proportion is 20% during gradient elution, and in 0.01min to 25min, the ratio of A is risen to
45%, in 25min to 25.1min, the ratio of A rises to 100%, keeps 100% operation to 30min stopping, Detection wavelength
220nm.Polypeptide solution is collected, then liquid nitrogen quickly cooling is lyophilized.It obtains purity and is 95% or more product, and identified through ESI-MS
Structure (as shown in Fig. 1).
Synthesize the DPPH free radical scavenging activity experiment of pentadecapeptide
DPPH experiment: 180 μ L DPPH reagents (150 μM of DPPH, solvent are 80% ethyl alcohol, v/v) mixing, 20 μ L 1 ~
The sample of 100 μ g/mL, adds in 96 orifice plates.Blank group and model group are set simultaneously, and blank group is mixed for 180 μ L, 80% ethyl alcohol
20 μ L ultrapure waters are closed, model group is that 180 μ L DPPH reagents mix 20 μ L ultrapure waters.With microplate reader (infinite M200
Pro, TECAN, Switzerland) place 522 nm of measurement absorbance value, setting program shakes 30 s, every 2min survey one
It is secondary, 20 periods are surveyed altogether, it should be noted that, model group absorbance value is adjusted between 0.7-0.8 before experiment, in continuous mode
Room temperature is kept to be protected from light (result is shown in Fig. 2).
Fig. 2 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-
The ABTS free radical scavenging activity comparison diagram of Lys.A is removing power of the sample to ABTS free radical of various concentration in 40min
Learn curve；B is the significance analysis of effect between sample various concentration when acting on 40min.Wherein the abscissa of a is Time in Fig. 2
(time), ordinate are ABTS scavenging rate (ABTS free radical scavenging activity).The abscissa of b is in Fig. 2
Concentration (concentration), ordinate are ABTS scavenging rate (ABTS free radical scavenging activity).The result shows that
The peptide is significant to the elimination effect of ABTS free radical, has basically reached maximum elimination effect in 2min after addition sample, has removed
The IC of ABTS free radical50Value is 5.32 μM, lower than 26.18 μM of positive control vitamin C.
Synthesize the ABTS free radical scavenging activity experiment of pentadecapeptide
ABTS experiment: 180 μ L ABTS reaction solutions mix the sample of 20 μ L, 1 ~ 100 μ g/mL, add in 96 orifice plates,
Concussion mixes.Blank group and model group are set simultaneously, and blank group is that 180 μ L, 80% ethyl alcohol mixes 20 μ L ultrapure waters, model group
20 μ L ultrapure waters are mixed for 180 μ L DPPH reagents.With microplate reader (infinite M200 pro, TECAN,
Switzerland the absorbance value at 736 nm) is measured.Setting program, shakes 30 s, and every 2min surveys primary, survey 20 altogether
Period, it should be noted that, model group absorbance value is adjusted between 0.7-0.8 before experiment, keeps room temperature to be protected from light in continuous mode,
As a result see Fig. 3.
Fig. 3 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-
The DPPH free radical scavenging activity of Lys.A is that the sample of various concentration in 40min is bent to the removing dynamics of DPPH free radical
Line；B is the significance analysis of effect between sample various concentration when acting on 40min.Wherein in Fig. 3 a abscissa be Time (when
Between), ordinate be DPPH scavenging rate (DPPH free radical scavenging activity).The abscissa of b is in Fig. 3
Concentration (concentration), ordinate are DPPH scavenging rate (DPPH free radical scavenging activity)
The result shows that the peptide is weaker to the elimination effect of DPPH free radical, in 40min, sample is to DPPH free radical
Concentration dependent is presented in elimination effect.In 24min, time and concentration-dependant is presented in elimination effect.After 24min
Elimination effect has the tendency that decrease.By Fig. 3 a it is found that sample has maximum elimination effect when action time is 24min.Therefore,
Significance analysis is carried out to the elimination effect of the sample of various concentration at this time, such as Fig. 3 b, the results showed that, it is dense in 10-100 μ g/mL
It spends in range, the sample trend significantly incremental with the increase presentation of concentration to the removing of DPPH free radical.But still it is much weak
In the effect of positive control VC.
Synthesize the FRAP experiment of pentadecapeptide
FRAP experiment: 180 μ L FRAP reaction solution (V300 mM acetate buffer solutions: V20 mM TPTZ: V20 mM iron chloride=10:1:1) it is mixed
The sample for closing 20 μ L, 1 ~ 100 μ g/mL, adds in 96 orifice plates, shakes, and mixes.Blank group and model group, blank are set simultaneously
Group is 200 μ L ultrapure waters, and model group is that 180 μ L FRAP reaction solutions mix 20 μ L ultrapure waters.With microplate reader (infinite
M200 pro, TECAN, Switzerland) measurement 596 nm place absorbance value, setting program, concussion 30 s, every 2min
It surveys once, surveys 20 periods altogether, as a result see Fig. 4.
Fig. 4 is synthetic peptide Trp-Val-Ala-Gly-Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro
The reducing power figure of-Lys.A is the sample of various concentration in 40min by TPTZ-Fe3+It is reduced to TPTZ-Fe2+'s
Kinetic curve；B is the significance analysis of effect between sample various concentration when acting on 40min.Wherein Fig. 4 a abscissa is Time
(time), ordinate are FRAP value (antioxidant activity).Fig. 2 b abscissa is Concentration (concentration), ordinate
For FRAP value (antioxidant activity).
The result shows that the synthetic peptide does not have reducing power substantially.
Synthesize the application that pentadecapeptide promotes the human skin fibroblasts collagen of UVB damage to generate
Human skin fibroblasts culture is in complete medium, and complete medium is mainly by the high sugar of basal medium
DMEM, 10% fetal calf serum (v/v) and 1% dual anti-(being made of penicillin and streptomysin, v/v) composition.Be placed in 37 DEG C,
CO2In the saturated humidity incubator that volume fraction is 5%.It is changed liquid 1 time every 2 d.90% left side of culture bottle is covered with to cell
The right side takes it to be inoculated in 96 well culture plates, every 100 μ L of hole, and concentration is 5 × 104cells/mL.After cell grows 24 h,
It inhales and abandons culture solution, washed 1 time with 200 μ L PBS, 200 μ L PBS are added, with 80 mJ/cm2UVB irradiation, inhales and abandons PBS, every hole adds
Enter 200 μ L samples (zero-dose is replaced with DMEM in high glucose, as normal control), continues to cultivate 72 h.Above-mentioned processing is taken to rent thin
Born of the same parents inhale and abandon culture solution, twice with sterile washing cell surface, ice-cold 70% ethyl alcohol, 200 μ L are added and is fixed, -80oC ice
Case places at least 10min, and after taking-up, twice with sterile washing cell surface, after air-dried moisture, it is full that 200 μ L 1% are added in every hole
With the scarlet dye liquor of picric acid-(m/v), in 4o(18-24 h) gently is shaken under the conditions of C, inhales and abandons dye liquor, is washed 2-4 times with sterile, until
Until not having dyeing liquor wash-off, 150 μ L 1M NaOH solutions are added, 150 r/min shake 15 min, and every hole is taken out 100 μ L and set
In 96 orifice plates, the absorbance value at 492nm is measured, the results are shown in Table 1.
Table 1
| UVB | Synthesize pentadecapeptide+UVB | |
| Human skin fibroblasts collagen yield (μ g/mL) | 11.9512±1.2067 | 15.6138±3.6714 |
The protection application that synthesis pentadecapeptide damages Red Blood Cells of Normal Persons AAPH
It is extracted health adult (30 years old with the anticoagulant tube (anti-coagulants sodium citrate: blood=1:9, v/v) containing sodium citrate
Blood is put in 4 DEG C of refrigerators and saves below), uses in one week.Face the used time, blood is placed in centrifuge tube in 1000 ~ 1500 g
It is centrifuged 8-15 min, removes the blood plasma on upper layer, red blood cell PBS(pH=7.4) it washes 2-3 times, until supernatant is colourless.
Finally 1000 ~ 1500 g are centrifuged 8-15 min in centrifuge, remove supernatant, obtain close hematocrit, use PBS
Be diluted to concentration be 20%(volume, v/v) red cell suspension.Red blood cell individually is handled with the synthesis pentadecapeptide of 100 μ g/mL,
It is incubated for 2 h jointly as blank control group using 0.1 mL, 20% red cell suspension and 0.3 mL PBS, determines that sample does not have
There is hemolytic.
Application Example 1
0.1 mL, 20% red cell suspension (is replaced, i.e., through the synthesis pentadecapeptide that 0.1 mL concentration is 0 μ g/mL with PBS
For model group) after 20 min of pretreatment, the AAPH solution of final concentration of 100 mM of 0.2 mL is added, microseism is protected from light
2 h are incubated for, the 50 μ L of reactant solution of each processing group is taken to be diluted to 1 mL, 1500 g centrifugation 12 with PBS buffer
Min takes supernatant in 96 orifice plates, its absorbance is surveyed at 540 nm with microplate reader, similarly, is diluted with distilled water anti-
It answers mixture to compare as full haemolysis, calculates hemolysis rate, as a result see Fig. 5；Fig. 5 is progress AAPH damage after synthesis pentadecapeptide addition
The Red Blood Cells of Normal Persons haemolysis inhibiting rate change curve of wound.Wherein abscissa is Concentration (concentration), ordinate is
Hemolysis inhibition (haemolysis inhibiting rate).The result shows that sample sets, relative to model group, haemolysis inhibiting rate significantly rises
Height, and concentration-dependant is presented.
Meanwhile each processing group cell centrifuge is centrifuged 12 min in 1500 g, cell precipitation is collected, is washed 3 times with PBS
Centrifugation, discards supernatant liquid, is resuspended again.When film-making, takes 10 μ L red blood cell re-suspension liquids to be uniformly applied on clean mica sheet and (want
Ask that cell is non-overlapping, does not reunite), then use 2.5%(volume, v/v) the fixed cell of glutaraldehyde, after 5 min, rushed with ultrapure water
It washes silicon wafer 3 times, natural air drying silicon wafer is finally placed in atomic force microscope, the scanning cell morphology under tapping-mode, is used
NanoScope software analyzes experimental data, as a result sees Fig. 6.
Application Example 2
0.1 mL, 20% red cell suspension pre-processes 20 min through the synthesis pentadecapeptide that 0.1 mL concentration is 1 μ g/mL
Afterwards, the AAPH solution of final concentration of 100 mM of 0.2 mL is added, microseism is protected from light 2 h of incubation, takes the anti-of each processing group
50 μ L of object solution is answered to be diluted to 1 mL with PBS buffer, 1500 g are centrifuged 12 min, take supernatant in 96 orifice plates
In, its absorbance is surveyed at 540 nm with microplate reader, similarly, uses distilled water diluted reaction mixture as full haemolysis pair
According to as a result calculating hemolysis rate is shown in Fig. 5.
Application Example 3
0.1 mL, 20% red cell suspension pre-processes 20 min through the synthesis pentadecapeptide that 0.1 mL concentration is 5 μ g/mL
Afterwards, the AAPH solution of final concentration of 100 mM of 0.2 mL is added, microseism is protected from light 2 h of incubation, takes the anti-of each processing group
50 μ L of object solution is answered to be diluted to 1 mL with PBS buffer, 1500 g are centrifuged 12 min, take supernatant in 96 orifice plates
In, its absorbance is surveyed at 540 nm with microplate reader, similarly, uses distilled water diluted reaction mixture as full haemolysis pair
According to as a result calculating hemolysis rate is shown in Fig. 5.
Application Example 4
0.1 mL, 20% red cell suspension is through the synthesis pentadecapeptide pretreatment 20 that 0.1 mL concentration is 100 μ g/mL
After min, the AAPH solution of final concentration of 100 mM of 0.2 mL is added, microseism is protected from light 2 h of incubation, takes each processing group
50 μ L of reactant solution be diluted to 1 mL with PBS buffer, 1500 g are centrifuged 12 min, take supernatant in 96
In orifice plate, its absorbance is surveyed at 540 nm with microplate reader, similarly, uses distilled water diluted reaction mixture as full haemolysis
Control calculates hemolysis rate, as a result sees Fig. 5；Meanwhile each processing group cell centrifuge is centrifuged 12 min in 1500 g, it collects
Cell precipitation washes 3 centrifugations with PBS, discards supernatant liquid, be resuspended again.When film-making, 10 μ L red blood cell re-suspension liquids is taken uniformly to apply
In, (it is required that cell is non-overlapping, does not reunite), then fixing cell with 2.5% glutaraldehyde on clean mica sheet, after 5 min, use
Ultrapure water silicon wafer 3 times, natural air drying silicon wafer is finally placed in atomic force microscope, and scanning is thin under tapping-mode
Born of the same parents' pattern analyzes experimental data with NanoScope software, as a result sees Fig. 6.
Application Example 5
0.1 mL, 20% red cell suspension is that 0 μ g/mL(PBS replaces through 0.1 mL concentration, as normal control
Group) synthesis pentadecapeptide pre-process 20 min after, be added final concentration of 0 mM of 0.2 mL AAPH solution (use PBS
Replace, as normal control), microseism is protected from light 2 h of incubation, and the 50 μ L of reactant solution of each processing group is taken to be buffered with PBS
Liquid is diluted to 1 mL, and 1500 g are centrifuged 12 min, take supernatant in 96 orifice plates, surveyed at 540 nm with microplate reader
Its absorbance similarly uses distilled water diluted reaction mixture to compare as full haemolysis, calculates hemolysis rate, as a result sees Fig. 5.Together
When, each processing group cell centrifuge is centrifuged 12 min in 1500 g, collects cell precipitation, 3 centrifugations is washed with PBS, discards
Supernatant is resuspended again.When film-making, 10 μ L red blood cell re-suspension liquids is taken uniformly to be applied on clean mica sheet (it is required that cell is without weight
It is folded, do not reunite), then fix cell with 2.5% glutaraldehyde, after 5 min, with ultrapure water silicon wafer 3 times, natural air drying silicon
Piece is finally placed in atomic force microscope, the scanning cell morphology under tapping-mode, is analyzed with NanoScope software real
Data are tested, as a result see Fig. 6.
Fig. 6 is atomic force microscope (AFM) image of different disposal group red blood cell.Wherein a is normocyte, b
The red blood cell of 2 h is handled for the AAPH of 100 mM, c is the synthetic peptide Trp-Val-Ala-Gly- with 100 μ g/mL
Leu-Gly-Tyr-Phe-Thr-Lys-Asn-Gly-Gly-Pro-Lys adds 100 mM AAPH after pre-processing 20 min again
Cultivate the red blood cell of 2 h.
The result shows that normocyte has typical bi-concave structure, cell surface is smooth, surrounding height basic one
It causes.AAPH processing group cell surface becomes coarse, and cell seriously collapses, and highly decreases, in irregular shape.Synthesize pentadecapeptide
Pretreated protection group, cellular damage degree obviously weaken, and the smooth irregular pie structure in surface is presented.
Table 2
| AAPH | Synthesize pentadecapeptide+AAPH | |
| T-SOD activity (mgprot/mL) | 2.5976 | 5.1958 |
Application Example 6
0.1 mL, 20% red cell suspension is that 5 μ g/mL(PBS replace through 0.1 mL concentration, as normal control
Group) synthesis pentadecapeptide pre-process 20 min after, be added final concentration of 0 mM of 0.2 mL AAPH solution (use PBS
Replace, as normal control), microseism is protected from light 2 h of incubation, and each processing group cell centrifuge is centrifuged 12 in 1500 g
Min collects cell precipitation, washes 3 centrifugations with PBS, is added 4oThe ultrapure water of C pre-cooling, 150r/min shake 10min, 10000g
It is centrifuged 30min, collection supernatant is spare, is measured SOD activity therein, knot according to the specification of kit (Nanjing is built up)
Fruit is shown in Table 2.
Claims (4)
1. a kind of pentadecapeptide, it is characterized in that the amino acid sequence of the pentadecapeptide is Trp-Val-Ala-Gly-Leu-Gly-Tyr-
Phe-Thr-Lys-Asn-Gly-Gly-Pro-Lys；When the pentadecapeptide concentration is 1-100 μ g/mL, mixed with red blood cell,
Under the action of AAPH, after being incubated for 2 h, cell haemolysis inhibiting rate can be made to reach 56.76% ~ 86.60%, pattern is than simple AAPH
Processing group is smooth, orderly, pie structure is presented.
2. a kind of pentadecapeptide according to claim 1, it is characterised in that the pentadecapeptide can make cell haemolysis inhibiting rate high
Up to 86.60 ± 0.83%.
3. a kind of pentadecapeptide according to claim 1, it is characterised in that when the concentration of the pentadecapeptide is 5 μ g/mL, energy
Cell SOD activity is set to rise to 5.1958 mgprot/mL by 2.5976 mgprot/mL.
4. a kind of pentadecapeptide described in claim 1, which is characterized in that under the action of UVB, by the pentadecapeptide and fell
Skin is mixed at fiber, and after being incubated for 72 h, still, collagen yield improves 14.9-30.65%, and the concentration of the pentadecapeptide is
1-10 µg/mL。
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| CN107266530B (en) * | 2017-07-03 | 2020-08-14 | 大连理工大学 | Antioxidant ultrashort peptide, preparation method and application thereof |
| CN113512092A (en) * | 2021-06-28 | 2021-10-19 | 陕西未来多肽生物科技有限公司 | Polypeptide nano hybrid and application thereof |
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| 螺旋藻抗氧化肽的制备及其体外活性研究;欧赟等;《食品与生物技术学报》;20140115;第33卷(第1期);摘要 |
| 酶解动物蛋白制备抗氧化多肽研究进展;潘超等;《肉类工业》;20100425(第4期);27-29 |
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