CN106800588A

CN106800588A - A kind of peptide and its application

Info

Publication number: CN106800588A
Application number: CN201510847635.XA
Authority: CN
Inventors: 华洋林; 王子偲; 唐健; 顾怀宇
Original assignee: Infinitus China Co Ltd
Current assignee: Infinitus China Co Ltd
Priority date: 2015-11-26
Filing date: 2015-11-26
Publication date: 2017-06-06

Abstract

The present invention relates to albumen field, more particularly to a kind of peptide and its application.During the present invention by tripeptides Arg-Arg-Tyr by acting on HSF, CCK-8 proliferation experiments, Edu methods detection cell proliferation experiment, the experiment of flow cytomery cell cycle are carried out to HSF.It is experimentally confirmed that tripeptides Arg-Arg-Tyr can effectively facilitate proliferation of human dermal fibroblasts, increase S+G2M phase proportions, it is used for skin senescence prevention or treatment determined curative effect, Small side effects, therefore has important practical significance.

Description

A kind of peptide and its application

Technical field

The present invention relates to albumen field, more particularly to a kind of peptide and its application.

Background technology

Aging, is the process for referring to that each organ dysfunction of body is universal, gradually reducing.Aging has two kinds not Same situation, a kind of is the physiological aging for occurring under normal circumstances；Another kind is the pathology that disease causes Property aging.It is a kind of natural law.But, when people use good habits and customs and hygienic measures, Just can effectively anti-aging, improve the quality of living.Theory of traditional Chinese medical science thinks, the growth of human body, development, Aging is in close relations with the prosperity and decline of function and meridian qi and blood.When body insufficiency of vital energy and blood, QI of channels and collaterals fortune Capable not smooth, function goes down, negative and positive disequilibrium, can cause and accelerate aging, shows as spirit Depressed, forgetful, shape is cold, and limb is cold, few dormancys of difference of receiving, waist-leg weakness, hair take off tooth shake, shortness of breath and fatigue, very then Face edema etc..Over the past thousands of years, people are exploring the secret of good health and a long life always, full of residing permanently to the youth, The yearning promoted longevity.

The mechanism for exploring aging generation is both an ancient problem, is again a brand-new scientific research field, In the very long history development procedure of medical science, it is believed that proposing the hypothesis of hundreds of agings altogether.Ancestral State's medical science have accumulated rich experience in terms of anti-aging, it is proposed that " imbalance of yin and yang is said ", " internal organs void declines ", " energy loss theory " etc., is impregnated with the understanding to nature macroscopic view.External ancient medicine man and wise man Scholar also explains aging from different perspectives, proposes warm theory, entropy theory, abrasion theory, oneself is poisoned Theory etc., plays a positive role for people's understanding aging.But because of historical conditions and the limit of scientific level System, these theories have significant limitation.

Modern study shows, skin senescence is reflected in cellular level as cell ageing, and fibroblast It is the bulk composition in corium, its aging and the change of biological characteristics cause skin aging to occur Major reason.At present, be all mostly by cosmetics for external use add EGF (EGF) come Improve skin, it has certain effect.But because the reason such as technology, the production and supply of EGF raw materials It is difficult to meet increasing need, the price of EGF remains high always, not only seriously constrains EGF Clinical research and clinical practice, also limit further genralrlization of the EGF cosmetics in consumer groups. Therefore, a kind of efficient EGF-R ELISA (EGFR) activating molecules are developed not only with huge Economic worth, also provides most basic guarantee for EGF researchs and develops broader practice prospect.

The content of the invention

In view of this, the present invention provides a kind of peptide and its application.The peptide can effectively facilitate skin into fiber finer Born of the same parents breed, anti aging effect, at the same also have it is easy to use, raw material sources extensively, it is cheap, drop The toxicity of low cosmetics, has a safety feature.

In order to realize foregoing invention purpose, the present invention provides following technical scheme：

The invention provides a kind of peptide, including：

(I), the amino acid sequence of Arg-Arg-Tyr；Or

(II), (I) described amino acid sequence is substituted, lacks or adds one or more amino acid and obtains Amino acid sequence, and with the same or analogous amino acid sequence of amino acid sequence function shown in (I) Row；

(III) and (I) or (II) described sequence at least 80% homology amino acid sequence；

The amino acid number of the peptide is 3~5.

In some specific embodiments of the invention, the amino acid of the addition is selected from Trp or Phe.

In other specific embodiments of the invention, the amino acid sequence of the peptide is Arg-Arg-Tyr.

The answering in the cosmetics for being used for preventing and/or treat aging are prepared present invention also offers the peptide With.

In the present invention, the aging is embodied on the whole：Height declines, spinal curvature, and skin loses Elasticity is gone, face gauffer increases, local skin, particularly face, hand etc., it is seen that pigmentation are in The brown spot for differing in size, referred to as senile plaque expelling.Sweat gland, smegma are reduced makes dry skin, lacks Weary gloss.Beard and hair is greyish white, and the domain or half often occurs in alopecia even bald, ptosis, peripheral cornea Ring white herring bone, is called arcus senilis (or old bow), caused by being lipidosis.Loss of tooth, but time Vary with each individual sooner or later.In terms of behavior, the elderly is slow in reacting, and stride is slow, and facial expression is gradually stayed Stagnant, failure of memory notes not concentrating, and language often likes repetition.Hypopsia, tends to long sight.Hearing Also easily degenerate.

In the present invention, the aging is embodied in from tissue with organ level：Skeletal system --- bone group Knit with age aging it is calcareous decrescence, bone brittleness, easily fracture, wound healing is also than slow at an early age. Joint motion ability declines, and is susceptible to suffer from the fibrocartilage pad between arthritis, spinal vertebral due to cartilage atrophy It is thinning, cause backbone to shorten, this is that the elderly becomes a short reason.Skin --- old human dermis' breast Head step-down, makes epidermis be flattened with dermis interface, and epidermis is thinning, and corium reticular fibre is reduced, elastomer Elasticity and easy fracture are gradually lost, collagenous fibres update slack-off, and old fiber is in the majority, and collagen cross-linking increase makes The elasticity reduction of collagenous fiber network.Cutis laxa, no longer tightly invests sub-dermal structures, transparent in cytoplasm Matter acid is reduced and chondroitin sulfate increases relatively, makes the reduction of corium water content, and subcutaneous fat is reduced, sweat gland, , there is senile plaque expelling due to local melanocytic hyperplasia in sebaceous gland atrophy.Muscle --- the elderly's flesh weight with The ratio between body weight declines.Moisture, sodium and chloride outside myocyte have increase tendency, intracellular potassium content Then there is decline to be inclined to, additionally, muscle fibre quantity declines, diameter reduces, and whole muscle is seemed atrophy. This damped exponential model is different because function is different, and ST tends in different quick muscles or mixing flesh Extension, and ST tends to shorten in slow constrictor, this can influence the phase interaction of different motion unit With reduction muscle group coordinates the validity of mutual aid, it is likely that this is the hypodynamic reason of old man's flesh.When So, the Aging changes of motor unit are also not enough to explain all dyskinesia of the elderly, because nerveous system Complicated mechanism on system varying level can all produce influence to motion.Nervous system --- human brain weight at 90 years old Mitigate 10~20% during compared with 20 years old.The reason for causing loss of weight essentially consists in the forfeiture of nerve cell.This funeral Mistake has the specificity in region, and such as brain different zones Leukopenia degree is different.From birth to 10 years old god Breed to most through cell, no longer divided, cell has started to lose after 20 years old.But full brain cell radix Very big, part cell death will not cause the serious hindrance of function.Moreover people are solved to memory mechanism It is also few, therefore caused by hypomnesia is not necessarily cell loss.Meninx thickeies after the elderly, and gyrus reduces, Ditch, split wide and deep, ventricular cavity expands.Visible Nissl bodies are reduced in microstructure, and fat is brown Matter is deposited.Functionally then see that MNCV slows down, recent memory goes down seriously than remote memory, The physiologic sleep time shortens；The sensor capability such as sense of heat, tactile and seismesthesia all decline, and sense of taste threshold is raised, Audiovisual susceptibility declines.Respond is generally reduced, particularly in the feelings for requiring to be made decision by selection React more slow under condition.Cardiovascular system --- Aged Heart volume increases, there is presently no evidence table What harmful effect is bright lipofuscin deposition have to myocardial function.In the visible pacemaker cells of the conducting system of heart Quantity is reduced, and sinoatrial node increases with fibr tissue in internodal tract.In terms of artery, inner membrance also has different journeys The thickening of degree, therefore can cause parteriole luminal stenosis.Coronary arterial tree began to after 30 years old to be occurred Inner membrance is thickened, middle film fibrosis increasingly, some smooth muscles may necrosis, most prominent damped exponential model is Elastomer flaggy becomes.Arteriovascular degeneration, peripheral vascular resistance increases so that angiosthenia is raised.Breathing System --- the possible calcification of the elderly's costal cartilage in terms of form, bow-backed situation increased before causing thoracic cavity Footpath expands afterwards turns into " emphysematous chest ".Visible breathing expands with respiratory bronchiole under microscope, makes surrounding Alveolar is reduced.Digestive system --- generally the modal damped exponential model of digestive system is not notable, The tooth that falls is good no relevant with protection to tooth, may not be aging feature.Visible stomach secretes sour thin under microscope Born of the same parents are reduced with aging, and the cell number of hepatic tissue unit volume also declines, and Peyer's patches are at an early age Most obvious excretory system people and rat kidney are all weightless in old age, and up to 20~30%, glomerulus number is reduced, Normal glomerulus accounts for 95% at 40 years old, only remainings 63% at 90 years old, and proximal convoluted tubule length declines with volume, Basilar memebrane thickeies with the age, and interstitial tissue increases in medullary substance.Functionally glomerular filtration speed declines. The atrophy of internal system --- sexual gland is the most obvious damped exponential model of internal system.Such as women 45~50 Year or so ischomenia, estrogen secretion is remarkably decreased, and male gradually decreased from 50~90 years old androgen, Sexual hypofunction.Reproduction corresponding to this and gonophore produce various atrophic changes, and such as ovary lymph is thin The hormone that born of the same parents are formed, this results in immunity function decline.Molecular level --- device palace and the aging end of cell Return relevant with the aging of molecular level, first for extracellular molecule, be filled in the extracellular knot of whole body The basilar memebrane for forming tissue and epithelium has special damped exponential model.Connective tissue rich in collagen and Elastin laminin.Cross-bond is produced between age growth collagen molecules.30~50 years old rapid for crosslinking Increased period, the collagenous fibres water imbibition that increases with crosslinking declines, and loses toughness, tends to stiff, It is unfavorable for the activity of tissue.Elastin laminin is the main component of elastomer, can also be handed in aging Connection.Fibrous fracture, embrittlement, outward appearance yellow are deepened.Only know that it thickeies in aging as basilar memebrane, its Main component is also collagen, secondary for glycoprotein and carbohydrate.But how these molecules change is led Cause the thickening of film not clear.Additionally, also having blood, lymph certainly as extracellular material.These materials Often in running status, and constantly update, it is difficult to make the index of aging.

In some specific embodiments of the invention, the aging is skin senescence.

In the present invention, the cosmetics refer to, to smear, spray or other similar approach, to impose on human body Surface (such as epidermis, hair, nail, lip), plays cleaning, maintains, beautifies or eliminate bad gas The product of taste effect, the product using position to that can have abirritation.

(1) classify by application target.Cleaning cosmetics：To clearing skin, the cosmetics of hair. This kind of cosmetics such as cleansing cream, mildy wash, bath agent, hair-washing hair-care agent, shaving cream etc..Basic cosmetics： Before cosmetic, the based process of opposite size hair.For example various face creams of this kind of cosmetics, honey, toner, face Film, hair-cream, hair jelly etc. send out agent surely.Cosmetics：For face and the beautification articles for use of hair.It is this kind of Cosmetics refer to kermes, lipstick, eye shadow, the articles for use such as hair dyeing and perm, hair style treatment, fixed.Curative effect is made up Product：Daily chemical product between medicine and cosmetics.This kind of cosmetics such as freshener, deodorant, educate Hair agent, hair remover, hair dye, pest repellant etc..

(2) classify by using position.Skin applies some make up：Refer to face and skin cosmetic.This kind ofization For example various face creams of cosmetic, bath agent.Hair cosmetic：Finger sends out cosmetics special.This kind of cosmetics such as perfume Ripple, mousse, hair sprays etc..Cosmetics：Beautifying face product is referred mainly to, also including nail head The cosmetics of hair.Specific function cosmetics：Finger is added with the cosmetics of special role medicine.

(3) classify by formulation.Liquid make-up：Body lotion, shampoo, toner, perfume etc..Breast Liquid：Sweet class, milk.Cream kind：Profit face cream, vanishing cream, hair cream.Powder class：Face powder, talcum powder. It is block：Muffin, cosmetic container.It is bar-shaped：Lipstick, pomade.

(4) age-based classification.Baby applies some make up：Infant skin is tender and lovely, vulnerable.During preparation Low irritant raw material, essence should be selected will also select the excellent product of low stimulation.Teenager applies some make up：It is juvenile Skin is in the puberty, and skin condition is unstable, and acne easily long.Adjustment sebum secretion is can select to make Raw material, prepares weak oil-based cosmetic preparation.Men's cosmetics：The many rooms of male should be selected in lipid skin It is suitable to the raw material of lipid skin.Liquid is the special cosmetics of man after shaving cream, palpus.

(5) seven classes can be divided into by production process combination product feature.Emulsion class：Refer to various cream honey. Powder class：Various face powders, talcum powder.Beauty class：Refer to lipstick, eye shadow, mascara, nail polish etc..It is fragrant Water class：Perfume, cologne, floral water.Shampoo class：Refer to shampoo, body lotion, hair conditioner.Hairdressing class： Refer to hair dyeing, hair-waving, determine hair care product.Curative effect class：Add the cosmetics of medicine.

In some specific embodiments of the invention, the formulation of the cosmetics is aqua, mellite, milk Agent, paste, creme, pulvis, bulk, it is bar-shaped in one or more.

In some specific embodiments of the invention, the concentration of the peptide is 0.01-100 μM.

In other specific embodiments of the invention, the effective dose of described peptide is 0.1-10 μM.

In other specific embodiments of the invention, the effective dose of described peptide is 0.01,0.1, 10 or 100 μM.

This experiment uses molecular database.The database mainly includes Chinese herbal medicine monomer molecule, small molecule Compound, the compound structure extracted from natural products and the virtual compound produced with computer approach, In Computer-Aided Drug Design, it is mainly used in based on molecular property, pharmacophoric group and molecular docking Database search.On the one hand it can provide natural products three-dimensional structure information, on the other hand, database The important informations such as plant origin, traditional function, association area scientific research progress can also be provided.This Mesh passes through molecular docking technology, it is intended to search out the natural product for having specific bond with EGF-R ELISA Thing, so as to develop the natural products medicine of efficient, high selectivity, while natural products can also be studied The mechanism of action in vivo.With the accumulation of the natural products data in Chinese herbal medicine, this project experimental result With further research and development and value, be conducive to illustrating and probing into for small molecule mechanism of action.

During the present invention by tripeptides Arg-Arg-Tyr by acting on HSF, to application on human skin into Fibrocyte carries out CCK-8 proliferation experiments, Edu methods detection cell proliferation experiment, flow cytomery Cell cycle tests.It is experimentally confirmed that tripeptides Arg-Arg-Tyr can effectively facilitate HSF's increasing Grow, increase S+G2M phase proportions, it is used for skin senescence prevention or treatment determined curative effect, side effect It is small, therefore have important practical significance.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to reality The accompanying drawing to be used needed for example or description of the prior art is applied to be briefly described.

Fig. 1 shows EGFR molecular models：Shown in front view such as Fig. 1 (A), top view such as Fig. 1 (B) It is shown；

Fig. 2 shows influences of the Arg-Arg-Tyr to proliferation of human dermal fibroblasts；

Fig. 3 shows the influence that Arg-Arg-Tyr breeds to HSF；Wherein, Fig. 3 (A) shows fluorescence microscope Take pictures figure；Fig. 3 (B) shows fluorescence picture software analysis figure；

Fig. 4 shows cell cycle analysis figure；

Fig. 5 shows cell cycle statistic histogram；

Fig. 6 shows the visual results of control group in embodiment 3；

Fig. 7 shows the visual results of small peptide group in embodiment 3；

Control group HE dyes × 100 results during Fig. 8 shows embodiment 3；

Small peptide group HE dyes × 100 results during Fig. 9 shows embodiment 3.

Specific embodiment

The invention discloses a kind of peptide and its application, those skilled in the art can use for reference present disclosure, fit When modified technique parameter is realized.In particular, all similar replacements and change are to this area It is it will be apparent that they are considered as being included in the present invention for technical staff.The method of the present invention and Using being described by preferred embodiment, related personnel substantially can not depart from present invention, Realized to method described herein and using being modified or suitably changing with combining in spirit and scope With application the technology of the present invention.

In order to overcome prior art deficiency problem, it is an object of the invention to provide a kind of tripeptides --- The application of Arg-Arg-Tyr, it has significant curative effect in the cosmetics for preventing and/or treating skin senescence.

In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of tripeptides --- Applications of the Arg-Arg-Tyr in preparing for preventing and/or treating skin senescence cosmetics.

Further, described tripeptides --- Arg-Arg-Tyr is used to prevent and delaying skin protective agent.

The concentration of the tripeptides Arg-Arg-Tyr is 0.01-100 μM.

The present invention has following beneficial effect：SF can be effectively facilitated to breed, anti aging effect, Simultaneously also have it is easy to use, raw material sources extensively, it is cheap, reduce cosmetics toxicity, safety Performance is good.Specifically, during the present invention by tripeptides Arg-Arg-Tyr by acting on HSF, CCK-8 proliferation experiments, Edu methods detection cell proliferation experiment, streaming are carried out to HSF Cell instrument detection cell cycle experiment.It is experimentally confirmed that tripeptides Arg-Arg-Tyr can effectively facilitate application on human skin into Fibrocyte proliferation, increases S+G2M phase proportions, and it is used for skin senescence prevention or treatment curative effect is true Cut, Small side effects, therefore have important practical significance.

Raw materials used and reagent can be bought by market in a kind of peptide of present invention offer and its application.

With reference to embodiment, the present invention is expanded on further：

Embodiment 1：Computer molecular simulation screening function small peptide is tested

1.1 experiment materials

It is equipped with the supercomputer of 192AMD Opteron processor groups

1.2 experimental techniques：

The principle of high flux computer molecular simulation triage techniques is as follows：(1) the preliminary sieve of pharmacophore and target spot Choosing.Existing Chinese medicinal herb monomer database is utilized, analysis comprehensively is carried out to acceptors such as protein. The interaction of simulation Chinese herbal medicine monomer molecule and target recipient protein molecular, and obtain this several material With reference to score and action site.(2) molecular docking.It is small by continuing to optimize using semi-flexible docking calculation The position (orientation) of molecular compound and the dihedral angle (conformation) of intramolecule flexible bond, find small point The best conformation that sub- compound is acted on target macromolecule, it is allowed to which the conformation of small molecule changes, then Molecule in storehouse is docked with target molecule one by one, calculations incorporated free energy.Using Conjugated free energy as The foundation of docking result is evaluated, according to this score rank, the optimal molecule combined with target molecule is found out.(3) divide Subdynamics is simulated.Molecular dynamics is carried out to receptor-ligand complex in the case of virtual with computer Simulation, simulates the compound most stable of form in its natural state, and make data analysis.Can be very Good simulated in vivo environment, by fine calculating, can accurately represent the journey of acceptor and ligand binding Degree.Fine screening process uses the high-performance computer system of the series of dawn 5000, using multi-core parallel concurrent Calculation, greatly improves the progress of experiment.

Molecular docking is the main experimental methods of this phase experiments.Given birth to by studying smaller ligand and acceptor The interaction of thing macromolecular, the binding pattern and affinity of the predictable compound of computer, are current medicines One of most important methods in field such as thing design.Molecular docking is smaller ligand and large biological molecule acceptor The process being mutually distinguishable by geometric match and energy match.The mistake of drug effect reaction is produced in drug molecule Cheng Zhong, drug molecule be combined with each other with target, first has to two molecules and is substantial access to, and takes and suitably takes To, both is mutually agreed with necessary position, interact, continue and adjusted by appropriate conformation It is whole, obtain a compound conformation for stabilization.Two molecules are correct in determining compound by molecular docking Relative position and orientation, research two conformations of molecule, the particularly conformation of substrate formed compound During change, be to determine mechanism of drug action, design new drug basis.The following is main several points Sub- docking calculation, refers to table 1：

Several main molecular docking methods of table 1

It is of the invention mainly to realize that molecular docking is calculated using Autodock.

Molecular docking is calculated：Ligand molecular is placed on the position in receptor active site, it is then mutual according to geometry The quality of the principle Real-Time Evaluation parts such as benefit, chemical environment complementation, energy complement and acceptor interaction, And find optimal binding pattern between two molecules.

Evaluation method：It is estimated by scoring functions.The arithmetic of each docking is in computer molecular sieve Select and can all carry out free energy function marking in platform, the process need to balance following two factors：It is ageing and Accuracy.This evaluation at present mainly includes following three part：1. the method for the regression parameter of experience is based on, Contribution of the various physical parameters to Conjugated free energy, such as FlexX programs are fitted with the method for multiple regression Number, hydrogen bond, the ionic bond of middle use part rotation key, the pi accumulation of hydrophobic and aromatic rings are acted on, with And hydrophilic interaction.This method quickly can directly estimate Conjugated free energy；2. the side of molecular force field is based on Method, the method only considers contribution of the heat content to energy, and the influence of entropy is not considered, generally, uses The non-binding effect in the standard field of force can serve as scoring functions, such as DOCK such as vacuum static electricity and model ylid bloom action Using the energy function of AMBER in program：

3. Knowledge based engineering scoring functions.It is initially applied to protein structure prediction, scoring functions statistical mechanics Method obtain the composite structure of protein-ligand, Conjugated free energy function is flat for intermolecular distance Can plus and to calculate.

1.3 experimental procedures：

1.3.1 clear and definite simulated object：

The simulated object of this term computer molecular simulation experiment is part to be calculated and EGFR albumen knots Structure (Fig. 1), wherein, Fig. 1 (A) is front view, and Fig. 1 (B) is top view.All EGFR albumen Structure comes from Protein Data Bank, and predominantly resolution isEGFR extracellular domain crystal structures (PDB is numbered：1NQL), and resolution isThe allosteric for combining EGF after EGFR (PDB is numbered extracellular domain crystal structure：1IVO).

The preparation of protein structure is the essential step of virtual screening.The structure of the protein target of virtual screening Can be from PDB storehouses (http://www.rcsb.org/pdb/index.htmL) in directly download and use；Can also lead to Cross and family in the sequence of homologous protein, structural information compare, Blast search and obtain.Removal acceptor molecule In all hydrones and metal ion.The description of binding site is followed by, suitable ligand binding is selected Pocket.This experiment is the accuracy of guarantee experimental result, and we such as combine according to biological function, are mutated Experiment information, manually selects binding site.

1.3.2 modeling scheme is formulated：

First by the PDB files that software SYBYL1.1 modifications are obtained, redundancy molecule and unrelated is deleted Group, to obtain pure protein structures；

Secondly software Autodock4.0 installation space coordinates are used, by Lamarckian genetic algorithm (Lamarckian genetic algorithm), it is stipulated that the binding site of drug molecule, selected avtive spot It is the binding site of part (referred to herein as EGF).Now initial setting up is finished.

1.3.3 molecular simulation is calculated:

Including docking and giving a mark, this step is the core procedure of virtual screening.Docking operation is that each is small Molecule is put into the ligand binding site of receptor protein, optimizes part conformation and position, is allowed to have most with acceptor Good combination；The marking of best combination conformation is given, all compounds are sorted according to marking, Ran Houcong Choose marking highest small molecule in compound library.

Using the AutoGrid modules in Autodock, the Conjugated free energy between part and acceptor is calculated. It is now preliminary screening, it is already possible to obtain the affine information of a part of molecule and EGFR；

Simulating external member using AMBER 11 carries out the simulation and data analysis of molecular dynamics.In EGFR Simulation in use the field of force be AMBER ff03, this field of force to the alpha-helix and β of secondary protein structure- The stress force distribution of folding is more balanced, therefore more suitable for the simulation of EGFR.Simulated environment is used TIP3P water box, i.e. water environment, because main component is water in human body, therefore test mesh according to this project , it is rational as the experiment condition of molecular simulation to choose water environment, can fully simulate the water in human body Environmental effect；

The geometry for carrying out ligand molecular using Gaussian03 programs is optimized, and is obtained most stable of quiet Energy of position；

Use remaining force field parameter of AMBER GAFF procedure stipulation parts；

The force field parameter that part is lacked is set using the antechamber external members of the softwares of AMBER 11.

, using the AutoGrid modules in Autodock, the combination between calculating part and acceptor is certainly for this phase experiments By energy.

1.3.4 the post processing of compound is hit:

The post processing for hitting compound (is absorbed by calculating the quasi-medicated property matter ADME/T of molecule Absorption, in vivo organ distribution distribution, metabolism metabolism, excretion excretion and poison Property toxicity) property estimation, exclude those molecules without quasi-medicated property matter.

Because EGFR is a relatively large molecular system, need to expend sizable in molecular simulation Amount of calculation, therefore we employ the simulated time of 5ns in medicine preliminary screening.Length is away from electrostatic force Calculated using Particle-Mesh Ewald methods, in calculating process, we with the addition of sodium ion, in The negative electrical charge produced with EGFR.

Finally, the Conjugated free energy of EGFR and part can be calculated, such that it is able to ligand activation The ability of EGFR makes a computer evaluation.Four steps are processed more than, and most of molecule is from compound Rejected in storehouse, form a compound library for size reasonable, the only compound to these suitable patent medicine or purchase Buy or synthesize or be isolated, actual biological test is then carried out again.

1.4 experimental results and analysis

Forgive having set up the albumen database of 8000 kinds of small peptide three-dimensional structures, by the sieve to the small peptide storehouse Choosing, because the spatial conformation of short peptide molecules is complicated, in actual molecular simulation screening, score absolute value It is more than 8 points " there is the target molecule for preferably combining effect ".Therefore, this experiment calculates knot according to final Really, obtain that there is the small peptide for preferably combining effect with EGFR, as a result as shown in formula I：

Arg-Arg-Tyr (Arg-Arg-tyrosine), simulates score twice：-9.5；-9.7

Embodiment 2：Influences of the Arg-Arg-Tyr to proliferation of human dermal fibroblasts

1 material

1.1 cell lines

HSF's strain (HSF), is purchased from Fudan University's IBS cell resource centers.

1.2 biochemical reagents

Pen .- Strep solution (100 ×), pancreatin cell dissociation buffer (containing phenol red), Phosphate-Buffered Saline (NaCl containing 135mM, 4.7mM KCl, 10mM Na₂HPO₄, 2mM NaH₂PO₄, pH It is worth 7.4), to be green skies Bioisystech Co., Ltd product；DMEM high glucose mediums, cell freeze Liquid storage, U.S.'s Gibco Products；Australia top grade hyclone, Gemini companies of the U.S.； TritonX-100, bovine serum albumin(BSA) (BSA) are the big bio tech ltd of Guangzhou Ztel；Sodium chloride, Absolute ethyl alcohol, paraformaldehyde, dimethyl sulfoxide (DMSO) (DMSO), are Guangzhou chemical reagent Co., Ltd product, Analysis is pure.

1.3 biochemical reagents boxes

CCK-8 kits, Japanese DOJINDO companies；The propagation detection examination of key Fluor488-EdU methods cell Agent box, cell cycle detection kit, are Nanjing Keygen Biotech's Products.

1.4 conventional biochemistry consumptive materials

6 holes, 24 holes, 96 porocyte culture plates, 25cm²Tissue Culture Flask, 0.22 μm of needle-based are sterile filtered Device, 15mL, 50mL sterile centrifugation tube is Jie Te biotech firms (JET) product；Cryopreservation tube, Sigma Products；Fluidic cell pipe, U.S. company BD product.

1.5 key instruments

EPICS XL-MCL type flow cytometers, U.S.'s Beckman Co μ Lter Products；Carbon dioxide Incubator, Biohazard Safety Equipment, are skill and think (ESCO) Science and Technology Ltd. high product；Inverted microscope, Japanese OLYMPUS Products；TW12 thermostat water baths, German J μ Labo Products；AL 104-IC electronic analytical balances, Switzerland's Mettler Products；The type ELIASAs of Model 680, the U.S. Bio-RAD Products；5810R table-type high-speed refrigerated centrifuges, German Eppendorf Products； Micropipettor, German Eppendorf companies；The intelligent inverted fluorescence microscopes of DMI4000B, Germany Leica Products；Cell counting count board, Shanghai refinement biochemical instrument company；Cell cryopreservation program temperature reduction box, U.S.'s Nalgene Products；Liquid nitrogen container, U.S.'s Thermo Products；80 DEG C of low temperature refrigerators of ﹣, U.S.'s Thermo Products；20 DEG C of low temperature refrigerators of ﹣, Qingdao Haier Products.

1.6 medicines

EGF, unlimited pole (China) Co., Ltd；Arg-Arg-Tyr (RRY) purity 99.33%, Shanghai is strong Credit bio tech ltd's product.

The preparation of 1.7 cell culture common experimental reagents

1.7.1 integral asepsis cell culture medium

DMEM high glucose mediums 90mL, hyclone 10mL are measured under aseptic condition, is mixed, plus Enter mycillin solution 1mL, packing after 0.22 μm of membrane filtration is degerming, be stored in 4 DEG C it is standby.

1.7.2EGF the preparation of working solution

(1) EGF is 1mg/ pipes, adds 10mL complete mediums, and concussion is mixed, and is made into 10mg/mL's Mother liquor, packing, 20 DEG C of ﹣ keeps in dark place；

(2) dilution of 10 μ L EGF mother liquors is taken before testing, finally configures other concentration respectively 100ng/mL's EGF working solutions.

1.7.3 small peptide Arg-Arg-Tyr working solutions are prepared

(1) small peptide Arg-Arg-Tyr is 10mg/ pipes, adds 20.125mL complete mediums, and concussion is mixed, 1000 μM of mother liquor is made into, is dispensed, 20 DEG C of ﹣ keeps in dark place；

(2) mother liquor dilution is taken before testing, other concentration is finally configured and is respectively 100,10,1,0.1,0.01 μM RRY working solutions.

1.7.4 4% paraformaldehyde solution

Weigh in the poly formic acid addition 900mI PBS of 40g, be sealed in wide-mouth bottle after being sufficiently stirred for, be placed in 60 DEG C of incubators heat dissolution, add PBS to be settled to 1000mL until completely dissolved, and 4 DEG C save backup.

1.7.5 3%BAS solution

Weigh in 3g BAS addition 100mL distilled waters, fully 4 DEG C of short-term preservations are standby after dissolving.

1.7.6 0.5%TritonX-100

Measure in 50 μ L TritonX-100 additions 9mLPBS, vibration is mixed, and is settled to 10mL, make it dense eventually Spend is 0.5%.

2 experimental techniques

2.1 cell culture processes

2.1.1 culture environment

HSF is incubated in the DMEM nutrient solutions containing 10% hyclone (pH7.2~ 7.4), in 5%CO₂, routine passage culture in 37 DEG C of saturated humidity incubator.

2.1.2 cell recovery

1) beaker of 37 DEG C of water-baths or 1000mL, built-in 2/3 glass 37 DEG C of warm water are prepared.

2) cryopreservation tube is taken out from liquid nitrogen, is immediately placed in warm water and is constantly stirred.Make to freeze thing in cryopreservation tube Melted within 1 minute.

3) with 75% alcohol wipe sterilize after, on clean bench open lid, with suction pipe suction out cell suspension, load from In heart pipe, then 10mL nutrient solutions are added, piping and druming makes cell suspend.

4) 1000rpm is centrifuged 10 minutes, abandoning supernatant.

5) precipitation plus 10mL nutrient solutions, piping and druming is uniform, then is centrifuged 10 minutes, abandons supernatant.

6) plus after appropriate culture medium cell is transferred in blake bottle, culture, second day in 37 DEG C of cell culture incubators Observation growing state.

2.1.3 the passage of cell

1) passed on when cell is paved with bottom of bottle.First original culture medium is outwelled, PBS is subsequently adding and is washed 3 It is secondary.

2) to the trypsase that 1mL 0.25% is added in bottle, blake bottle is shaken gently for, flows through digestive juice all Cell surface, then sop up or outwell after digestive juice again plus 1mL it is new tryptic digestive juice, gently Most of digestive juice is outwelled after shake again, is only stayed and is digested a little, basis of microscopic observation.

3) separated when discovery cell is connected to each other, when kytoplasm retraction is rounded but still end completely falls off, after discarding pancreatin 3mL DMEM culture mediums are added immediately, terminate digestion.

4) micropipettor is used, nutrient solution in bottle blowing is drawn, bottle wall cell dispersion is blown and beaten repeatedly.

5) after cell is uniformly dispersed, 1:3 passages.

2.1.4 cell freezes:

1) HSF of 80% or so degrees of fusion, method digestion as described above, centrifugation, abandoning supernatant will be grown to；

2) cells frozen storing liquid (4 DEG C of precoolings), piping and druming is added to be transferred to 2mL cell cryopreservation tubes after mixing in cell precipitation；

3) cryopreservation tube is put into cell cryopreservation program temperature reduction box, is placed in 80 DEG C of refrigerators of ﹣, and cryopreservation tube is taken out after 24 hours, Fast transfer to liquid nitrogen container is preserved.

2.2CCK-8 proliferation experiments

2.2.1 cell recovery and passage

Method is with 2.1.2 and 2.1.3.

2.2.2 cell suspension is prepared

Take the logarithm the cell in growth period, abandon old nutrient solution, with PBS washed cells 3~4 times, then with 0.25% Trypsin Induced, abandons trypsase and adds new culture medium and terminate digestion after digestion, piping and druming cell make its from Completely fallen off in bottle wall.Centrifuge tube centrifugation 1000r/min is conducted into, 5min is centrifuged.After centrifugation, abandon Fall supernatant, re-replace culture medium.

2.2.3 cell count

Tally and special cover glass are cleaned with 95% alcohol, is then dried at room temperature or is wiped dry with silk. A little cell suspension for preparing is taken, one end of cover glass is added dropwise the cell suspension of pettiness amount on tally, To prevent suspension from overflowing cover glass or overflowing into the glass guide channel of both sides during dropwise addition, sample-adding amount also should not it is very few or Band bubble.Observed under inverted microscope, under 10 × object lens, in the block plaid of four, count plate periphery Cell number.Result of calculation is substituted into following formula, cell density is drawn

Cell number/milliliter stoste=(4 cell number/4 of block plaid) × 10⁴

2.2.4 cell inoculation

The concentration results that reference cell tally is drawn, by 4X10³/ hole density, 200 μ L/ holes are inoculated in 96 orifice plates.The μ L of PBS liquid 200 are drawn with sample loading gun be injected separately into 96 orifice plate rims surroundings (resistance edge Effect), after the completion of bed board, close the lid, indicate the date.It is put into 5%CO₂, train in 37 DEG C of incubator 4~6h is supported, until cell adherent dosing completely.

2.2.5 pharmaceutical intervention is carried out

When cell is completely adherent, nutrient solution in each hole is suctioned out.If positive controls (EGF, 100ng/mL) ﹑ negative control groups (only adding single cell suspension to be not added with any medicine) and experimental group, experimental group is divided into dense Spend for 100,10,1,0.1,0.01 μM of every group of each concentration sets 5 multiple holes, each hole final volume is 200 μ L, Again 96 orifice plates are continued to be placed in 5%CO₂Cultivated in the saturated humidity incubator of 37 DEG C of ﹑, trained after dosing Foster 72h is measured.

2.2.6 colour developing and ELIASA detection：

Original fluid is removed under the 4h before cell culture terminates, aseptic condition, is added per hole 20 μ L10%CCK-8 solution, 37 DEG C of incubation 4h.Orifice plate is taken out, at ELIASA detection 450nm wavelength Light absorption value.Data output, record analysis.

2.3Edu methods detection cell propagation

2.3.1 cell recovery and passage

Method is with 2.1.2 and 2.1.3.

2.3.2 cell inoculation

Take the logarithm the HSF cells in growth period, after being rinsed with PBS, plus the digestion of pancreatin cell dissociation buffer, from The heart is collected, and abandons supernatant, adds fresh culture resuspended, is inoculated in containing thin according to the concentration in 10000/hole In 24 well culture plates of born of the same parents' creep plate, 37 DEG C, 5%CO are placed in₂12h is cultivated in incubator.

2.3.3 dosing and incubation

After cell growth is adherent, nutrient solution is suctioned out, add various concentrations, final concentration is respectively 100, 10th, negative control and Positive control wells are set up simultaneously and 3 multiple holes are respectively set up for 1,0.1,0.01 μM.Plus Continue culture 72h after medicine under the conditions of.

2.4.4 using the key Fluor488-EdU methods cell proliferation detecting kit detection of Nanjing Kai Ji companies production The influence of small peptide cell proliferation, to specifications step operation：

2.4.4.1EdU mark：

1) by cell culture complete medium and EdU solution according to 2000:The appropriate 50 μM of EdU dilutions of 1 proportions Liquid.

2) 50 μM of the μ L/ holes of EdU dilutions 200,37 DEG C, 5%CO are added₂Incubator is incubated 4h, abandons culture Base.

3) PBS cell, 5min/ times × 2 times, abandons PBS.

2.4.4.2 cell is fixed and promotees to ooze：

1) cell fixer (i.e. containing the PBS of 4% paraformaldehyde) 100 μ L/ holes are added, 30min is incubated at room temperature, is abandoned Cell fixer.

2) the PBS100 μ L/ holes containing 3%BSA, cleaning, 5min/ times × 2 times are added.

3) bleeding agent (PBS of 0.5%TritonX-100) 200 μ L/ holes are added, room temperature decolorization swinging table is incubated 20min, Abandon bleeding agent.PBS 1 time, 5min.

2.4.4.3EdU detect：

1) 1 × Click-iTEdU reaction buffers are prepared：Transfer 1 × Click-iTEdU reaction buffer reagent bottles In all of solution 4mL to 36mL deionized water in.

2) prepare a 10 × Click-iTEdU reaction additive liquid storages：Plus 1mL deionized waters to 10 × In Click-iTEdU reaction additive liquid storage test tubes, mix to whole dissolvings.

3) 1 × Click-iTEdU buffer solutions additive (being shown in Table lattice 2) are prepared：10 × liquid storage is diluted with deionized water To 1 ×, solution answers Fresh, and the same day is finished.

4) Click-iT reactant mixtures are prepared according to table 2.

The Click-iT reactant mixtures of table 2:

5) removal promotees sepage, is washed 2 times with the cleaning solution of the PBS of the 3%BSA of every hole 1mL, and removal is washed Wash liquid.

6) add 0.5mL Click-iT reactant mixtures to every creep plate, briefly rock culture plate to ensure reaction Mixture can be with uniform fold creep plate.

7) room temperature lucifuge is incubated 30min.

8) reactant mixture is removed, is washed 2 times with 1mL3%BSAin PBS per hole, remove cleaning solution.

2.4.4.4DNA dye:

1) every hole is washed 1 time with 1mLPBS, removes cleaning solution.

2) with PBS dilution Hoechst33342 liquid storages 1:2000 to 1 × Hoechst33342 solution, final concentration It is 5 μ g/mL.

3) 1mL1 × Hoechst33342 solution is added per hole, room temperature lucifuge is incubated 30min.Remove Hoechst33342 Solution.

4) the every hole of 1mLPBS washings 2 times, removes cleaning solution.Seen under fluorescence microscope blueness and green fluorescence Examine, every cell climbing sheet randomly selects 5 visuals field and taken pictures, and image is entered using Image J softwares Row counts proliferating cell nuclear and total cell check figure mesh.

5) new proliferative cell excites lower nucleus to send the prominent light of green in blue light, all of cell (including new propagation and The cell of non-multiple fission) nucleus sends blue prominent light under burst of ultraviolel.By red proliferating cell nuclear Number and blue total cell nuclear phase ratio, be calculated EdU positive cell percentages, can detect cell Proliferative conditions.

2.4 flow cytomery cell cycles

2.4.1 cell recovery and passage

Method is with 2.1.2 and 2.1.3.

2.4.2 cell inoculation

Take the logarithm the HSF cells in growth period, after being rinsed with PBS, plus the digestion of pancreatin cell dissociation buffer, from The heart is collected, and abandons supernatant, adds fresh culture resuspended, and adjustment cell concentration is 1.0x10⁵/ mL, 2mL/ Hole is inoculated in 6 orifice plates, is placed in 37 DEG C, 5%CO₂12h is cultivated in incubator, treats that cell is completely adherent Afterwards, serum-free medium domestication 24h, makes cell cycle synchronization.

2.4.3 dosing and incubation

Nutrient solution is suctioned out, various concentrations are added, final concentration is respectively 100,10,1,0.1,0.01 μM Negative control and Positive control wells are set up simultaneously and respectively set up 4 multiple holes.Continue training after dosing under the conditions of Support 72h.

2.4.4 the shadow of small peptide cell cycle is detected using the cell cycle detection kit of Nanjing Kai Ji companies production Ring, to specifications step operation：

1) once (2000rpm, 5min is centrifuged) and collects and adjust cell concentration with PBS washed cells and be 1x10⁶/ mL, takes 1mL single cell suspensions；

2) after the single cell suspension centrifugation for preparing, supernatant is removed, it is 70% cold second that volume fraction is added in cell The μ L of alcohol 500 are fixed (2 hours to overnight), 4 DEG C of preservations, and fixer is washed away with PBS before dyeing；

3) 100 μ L RNase A37 DEG C water-baths 30min are added；

4) add 400 μ L PI dyeing to mix, 4 DEG C of lucifuge 30min；

5) machine testing on, red fluorescence at record excitation wavelength 488nm；

6) experiment is repeated 3 times；

7) using the software analysis datas of ModFit LT 4.1.Result is with proliferation index (proliferous index, PI) That is S and G₂M phase cell percentages represent, computing formula：PI=(S+G₂/M)/(G₀/G₁+S+ G₂/ M), the cell proportion size in the active proliferation phase is shown with this.Experiment is repeated 4 times.

2.5 statistical procedures

All experimental datas are added and subtracted standard deviation (x ± s) and are represented with mean, multigroup as a result with the software processings of SPSS 11.0 Between compare and use one-way analysis of variance, compare two-by-two using Dunnett-t inspections.P ＜ 0.05 are have statistics Learn meaning.

3 experimental results

3.1CCK-8 methods detect influences of the Arg-Arg-Tyr to HSF multiplication capacities

It is small with (0.01,0.1,1,10,100 μM) treatment HSF cells 72 of various concentrations Arg-Arg-Tyr When, cck-8 detect HSF cells propagation, compare with negative control group, Arg-Arg-Tyr 0.01,0.1, 1st, 10,100 μM for the treatment of groups and negative control group comparing difference significantly (p<0.05).Wherein 1 μM group with 0.01st, 0.1,10,100 μM of groups are compared, P<0.05, statistically significant, i.e. Arg-Arg-Tyr concentration For 1 μM when, the quantity of born of the same parents is most.But it is (positive right for 1 μM of Arg-Arg-Tyr and 100ng EGF According to group) difference between the effects of two groups are not statistically significant.Specific data are shown in Table 3, Fig. 2.

To sum up, various concentrations Arg-Arg-Tyr (0.01,0.1,1,10,100 μM) can cause HSF Propagation.We select 0.1,1,10 μM of Arg-Arg-Tyr to process cell in follow-up experiment.

The Arg-Arg-Tyr of table 3 intervenes lower each group cell OD values (x ± s)

Note：*P<0.05, pharmaceutical intervention group is compared with negative control group；#P<0.05,1 μM of Arg-Arg-Tyr group Compared with other concentration Arg-Arg-Tyr groups.

3.2EdU methods detect influences of the Arg-Arg-Tyr to HSF multiplication capacities

Cell 72h is processed with 0.1,1 and 10 μM of Arg-Arg-Tyr and 100ng EGF respectively, is used EdU detects the proliferation number of cell.As shown in Fig. 3 (A) and Fig. 3 (B)：Blueness figure (Hoechst) The all staining cell cores under the fluorescence microscope visual field are represented, green figure (EdU) is represented newly breeds in the visual field Nucleus, Merge is both fusion figures.Compared with Control groups, increasing occurs in 1 μM of cell of group Grow (P<0.05).Result shows that 1 μM of Arg-Arg-Tyr can cause cell to be bred.But for 1 μM The difference between the effects of two groups of Arg-Arg-Tyr and 100ngEGF are not statistically significant.

Influences of the 3.3RRY to the HSF cell cycles

As shown in Figure 4,5 apply stimulate 72h after, experimental group (1 μM of Arg-Arg-Tyr) with compare Group is compared, and DNA pre-synthesis phases (G1) cell percentage has been reduced, and reflects cell Substantially (P ＜ 0.05) is raised the PI proliferation indexs [i.e. (S+G2M)] of proliferation activity.Result shows 1 μM Arg-Arg-Tyr can cause cell breed (P<0.05).But for 1 μM of Arg-Arg-Tyr and The difference between the effects of two groups of 100ngEGF are not statistically significant.

The animal experiment of embodiment 3

1 materials and methods

1.1 experimental animals:SD mouse 18, female, the scholar 0.6g of body weight 18.0, purchased from Zhongshan University's zoopery Center.

1.2 experiment reagents：Arg-Arg-Tyr (RRY) purity 99.33%, Shanghai is shone by force the limited public affairs of biotechnology Department's synthesis；1 μM of solution is formulated as with distilled water.

1.3 instruments:

Leica paraffin slicing machines, German Leica Products；Shandon Paraffin Section Flotation Bath Stand piece machine, U.S.'s Thermo Fisher Scientific Products；The upright micro-imaging systems of DM 2500B System, German Leica Products.

1.4 experiment packets and treatment:

Using random number method, 12 SD mouse are randomly divided into 2 groups, every group 6：Blank control group：Not Give any treatment；1 μM of Arg-Arg-Tyr solution small peptide group.Above-mentioned each group starts the previous day back of the body in experiment Portion's shaving (through in whole experiment process), scope is 4 × 4cm²Size, applies in shaving area skin and uses Above-mentioned relative medicine, 1 times/day, continuous 1 month.

1.5 skin surfaces are visually observed

The color and luster of mouse back skin is visually observed, smooth degree and wrinkle situation etc. judge that animal skin changes Situation, as the observation index of macroscopical figure and features.

1.6 Histomorphologicals

Concrete operations are as follows:

1.6.1. collection of specimens

30d is tested, all chloraldurates of animal intraperitoneal injection 10% are anaesthetized, cervical dislocation puts to death animal. Quick clip back of the body back about 4 × 4cm²The depilation skin of size, carefully removes subcutaneous superabundant fats, takes part Skin sample, carefully shakeouts, and 10% formaldehyde fixes more than 48 hours, and work done in the manner of a certain author is carried out for Skin slice Histomorphological.

1.6.2. FFPE

The skin histology that taking-up is fixed, is rinsed overnight with flowing water, and next day carries out the FFPE of following steps:70% Ethanol 1h → 80% ethanol 1h → 90% ethanol 1h → 95% ethanol (I) 1h → 95% ethanol (II) 1 H → 100% ethanol (I) 1h → 100% ethanol (II) 1h → n-butanol 1h → dimethylbenzene 30min → molten wax (I) 1.5h → molten wax (II) 2h → embedding.

1.6.3 microsection manufacture

Conventional dermal tissue paraffin section, thickness 6nm, will make section Yu room temperatures Xia Liao it is dry overnight, then 4 refrigerator sealing preserves are put into, it is standby.

1.6.4 skin histology HE dyeing

1) section dewaxing and rehydration

The ethanol 2 of dimethylbenzene (I) 2min → dimethylbenzene (II) 10min → 100% ethanol 2min → 95% Min → 80% ethanol 2min → 75% ethanol 2min → originally wash 2min.

2) dye：Haematine dye 3min → running water embathes and returns blue 3min → aquae destillata and embathe 3min；

3) it is dehydrated transparent：70% ethanol 2min → 80% ethanol 2min → 90% ethanol 2min → 1% eosin stains 3s → 95% ethanol 2min → 100% ethanol (I) 2min → 100% ethanol (II) 2min → dimethylbenzene (I) 6 Min → dimethylbenzene (II) 6min → neutral gum mounting, airing.

4) microscopy observation.

The depth for often examining under a microscope dyeing is taken in HE dyeing, as a result should be cell so as to regulation and control in time Core is dyed to blueness, and kernel is clearly demarcated, and cytoplasm is dyed red, blue nucleus and red by Yihong Endochylema is with distinct contrast.

2 results

2.1 each group mouse back skin gross morphologies are observed

Blank control group mouse skin is ruddy, smooth, without obvious wrinkle, full of elasticity；Experimental group (1 μM of Arg-Arg-Tyr solution small peptides group) ruddy gloss of skin, thickness is moderate, with good skin Elasticity, and compared with blank control group mouse dorsal part epidermis without pachyderma, furfur, color burn etc. no Good reaction.(see Fig. 6~7).

2.2 each group mouse back skin tissue morphologies are observed

HE results show that each Rotating fields of blank control group mouse skin are complete, and subcutaneous hair follicle, sebaceous glands are satisfied Full, epidermis rule, epidermis is clear with corium boundary line, and just, even dyeing is fine and close, collagen for skin corium thickness Fiber bundle-like is arranged, neatly fine and close；Experimental group (1 μM of Arg-Arg-Tyr solution small peptides group) mouse skin Skin epidermis structural integrity, cell layering is clear, and thickness is normal, cell component and moderate number, corium The visible wavy fibr tissue of layer, aligned orderly is evenly distributed, reasonable organization, and and blank control group Compared to indifference.(see Fig. 8~9).

3 conclusions

The present invention is visually observed by integrated use, HE colouring methods, in terms of tissue morphology biology Experiment, more comprehensively demonstrates small peptide Arg-Arg-Tyr and influence of the glabridin to SD mouse skins.Knot Fruit display small peptide Arg-Arg-Tyr has no toxic side effect to normal SD mouse skin histology, can for skin safe Lean on.

Trp or Phe is added on the basis of small peptide Arg-Arg-Tyr, can be reached same or similar Effect, with the effect of Arg-Arg-Tyr without significant difference.

The preparation of the cosmetics of embodiment 4

Acceptable auxiliary material is obtained cosmetic according to customary preparation methods in taking Arg-Arg-Tyr peptides addition cosmetics Product.The cosmetics refer to smear, spray or other similar approach, impose on human body surface (such as epidermis, Hair, nail, lip etc.), play cleaning, maintenance, beautification or eliminate the product of bad smell effect, The product using position to that can have abirritation.

The formulation of the cosmetics is liquid, mellite, milk agent, paste, creme, pulvis, bulk, rod One or more in shape.

The concentration of the peptide is 0.01-100 μM.

The inspection of the cosmetics of embodiment 5

Cosmetics prepared by embodiment 3 are tested according to cosmetics national standard, as a result such as table 4：

The cosmetics test stone of table 4 and result

Result above shows that the obtained cosmetics of the present invention meet the phase of National Standard of the People's Republic of China Close regulation.

The above is only the preferred embodiment of the present invention, it is noted that general for the art For logical technical staff, under the premise without departing from the principles of the invention, some improvement and profit can also be made Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims

1. a kind of peptide, it is characterised in that it includes：

(I), the amino acid sequence of Arg-Arg-Tyr；Or

The amino acid number of the peptide is 3~5.

2. peptide according to claim 1, it is characterised in that the amino acid of the addition is selected from Trp Or Phe.

3. peptide according to claim 1 and 2 is preparing the cosmetics for preventing and/or treating aging In application.

4. application according to claim 3, it is characterised in that the aging is skin senescence.

5. the application according to claim 3 or 4, it is characterised in that the effective dose of described peptide It is 0.01-100 μM.

6. the application according to any one of claim 3 to 5, it is characterised in that the cosmetics Formulation be aqua, mellite, milk agent, paste, creme, pulvis, bulk, it is bar-shaped in one or more.

7. a kind of cosmetics, it is characterised in that including peptide as claimed in claim 1 or 2.

8. cosmetics according to claim 7, it is characterised in that the effective dose of described peptide is 0.01-100μM。

9. cosmetics according to claim 7 or 8, it is characterised in that its formulation is aqua, honey Agent, milk agent, paste, creme, pulvis, bulk, it is bar-shaped in one or more.