Polypeptide and application thereof
1. A polypeptide having the structure of formula (I):
Glu-Glu-Met-Gln-Arg-Arg-Ala（Ⅰ）
and derivatives, isomers, mixtures, cosmetically or pharmaceutically acceptable salts, hydrates or solvates thereof;
wherein R is at least one selected from Ser, Glu, Phe, Met, Arg, Asn, Glu and Leu.
2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
3. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
4. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
5. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
6. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
7. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is: Ser-Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala.
8. The polypeptide of any one of claims 1-7, wherein the polypeptide has one or more of an amino-terminal protecting group, a carboxy-terminal protecting group.
9. The polypeptide of any one of claims 8, wherein the amino-terminal protecting group is selected from optionally substituted C2-10Acyl or optionally substituted C2-10An amide group.
10. The polypeptide of any one of claims 8, wherein the carboxy-terminal protecting group is selected from optionally substituted C2-10An acyl group.
11. Use of a polypeptide according to any one of claims 1 to 10 as a muscle paralysis relaxant or as an anti-wrinkle agent in the skin.
Background
The skin is the protective barrier located on the outermost layer of the human body. As people age, the skin gradually becomes senescent. The phenomenon has various internal and external causes, and the internal cause is mainly because the self regeneration capacity of the human body is gradually weakened along with the increase of the age; the external inducers include ultraviolet and electromagnetic radiation, staying up overnight, etc. The superposition of these internal and external inducers can cause the facial skin to show the characteristics of increased wrinkles, loose skin, large loss of collagen, and the like.
In the last 90 s, foreign researchers discovered that botulinum toxin type A, which originally had the effect of treating muscle spasm, strabismus, and other diseases, had a good wrinkle-removing effect, and therefore began to apply this botulinum toxin type A to the fields of plastic surgery and cosmetology. Botulinum toxin is a neurotoxin protein produced by botulinum in a reproductive process, a 150kD polypeptide, which acts by inhibiting the release of peripheral motor nerve terminal presynaptic membrane acetylcholine, resulting in muscle relaxant paralysis and inhibition of muscle contraction. Botulinum toxin also faces a number of problems when applied as a wrinkle reduction product. First, botulinum toxin requires administration by injection and has a limited duration of effect after a single injection; secondly, botulinum toxin is expensive and relatively small in size for the audience; most importantly, some mechanisms are operated and used improperly, which can cause the user to have sequelae of facial stiffness, facial paralysis, etc. Consequently, scientists have begun to look for alternatives to the efficacy of botulinum toxin.
In recent years, with the continuous and intensive research on the structure and function of botulinum toxin, scientists try to design and synthesize a component with a remarkable botulinum-like effect, namely neuromuscular relaxing polypeptide. These polypeptides are superior to botulinum toxin in safety, and act in an anti-wrinkle manner primarily by inhibiting the release of pre-synaptic membrane acetylcholine from nerve terminals, blocking neurotransmission of muscle contraction messages; the second is the prevention of muscle contraction by inhibiting acetylcholine binding to and receptors on cell membranes. At present, scientists have developed and applied small molecular polypeptides in skin care products, which mainly include acetyl hexapeptide-8 (ayurrin), acetyl octapeptide-3, dipeptide diaminobutyrylbenzylamide diacetate and the like, and the polypeptides are added into the skin care products for smearing.
Acetyl hexapeptide-8 is most widely applied in the field of beauty treatment, is a bioactive polypeptide consisting of 6 amino acids through dehydration condensation, and the synthesis method mainly comprises a solid phase synthesis method and a liquid phase synthesis method, however, the anti-wrinkle effect of the polypeptide is not obvious, and the main reason of the unobvious effect is that the binding force of the polypeptide and a specific receptor SNARE protein combination for controlling and releasing acetylcholine is poor, and the acetyl hexapeptide-8 belongs to hydrophilic polypeptide and is difficult to penetrate through the epidermis layer to enter the dermis layer to play a role. Acetyl octapeptide-3 belongs to an extension peptide of acetyl hexapeptide-8, the binding rate of the acetyl octapeptide-3 and a specific receptor is improved compared with hexapeptide, but the transdermal property of the polypeptide is reduced along with the increase of the length of a peptide chain. Dipeptide diaminobutyrylbenzylamide diacetate is a synthetic tripeptide simulating snake venom serum, and plays an anti-wrinkle role by preventing acetylcholine nerve conduction.
With the improvement of living and economic levels of people, more and more people pay attention to skin care, and high importance is placed on anti-aging and anti-wrinkle. In recent years, various active polypeptides have been reported to be added into different skin care products, and the skin care products containing the polypeptides can relieve various skin problems such as dry lines, fine lines, roughness, color spots and the like of the skin. In addition, various polypeptide stock solutions emerge endlessly. The common polypeptides on the market at present comprise hexapeptide, pentapeptide, tripeptide, octapeptide and the like, and can be suitable for different parts of the face, the inner canthus, the forehead and the like according to the properties of different polypeptides.
However, there are still some technical problems with the polypeptide products currently on the market. First, the existing polypeptide products in the market have a problem of insufficient affinity with skin, and generally, the longer the peptide chain is, the more difficult it is to penetrate the skin barrier for the polypeptide product, so the existing polypeptide products in the market can improve the penetrability by reducing the length of the peptide chain, and at the same time, many effective parts on the peptide chain can be abandoned, resulting in the reduction of the polypeptide efficacy. The modified and modified pentadecapeptide has both penetrability and efficacy, and is innovative; in addition, the existing polypeptide products on the market have poor stability, and the polypeptide is easily identified and hydrolyzed by protease due to the special molecular structure of the polypeptide.
Disclosure of Invention
The polypeptide provided by the invention modifies and modifies the polypeptide structure to a certain degree through a special technical means, improves the stability of the peptide chain structure, and can exert the effect for a longer time.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, there is provided a polypeptide having the structure of formula (i):
Glu-Glu-Met-Gln-Arg-Arg-Ala（Ⅰ）
and derivatives, isomers, mixtures, cosmetically or pharmaceutically acceptable salts, hydrates or solvates thereof;
wherein R is at least one selected from Ser, Glu, Phe, Met, Arg, Asn, Gln and Leu.
Preferably, the amino acid sequence of the polypeptide is: Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: NELEEMQRRA, molecular weight: 1275.38.
preferably, the amino acid sequence of the polypeptide is: Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: RNELEEMQRRA, molecular weight: 1431.57.
preferably, the amino acid sequence of the polypeptide is: Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: MRNELEEMQRRA, molecular weight: 1562.77.
preferably, the amino acid sequence of the polypeptide is: Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: FMRNELEEMQRRA, molecular weight: 1709.94.
preferably, the amino acid sequence of the polypeptide is: Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: EFMRNELEEMQRRA, molecular weight: 1839.05.
preferably, the amino acid sequence of the polypeptide is: Ser-Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: SEFMRNELEEMQRRA, molecular weight: 1926.13.
preferably, the polypeptide comprises one or more of an amino-terminal protecting group, a carboxy-terminal protecting group.
Preferably, the amino-terminal protecting group is selected from optionally substituted C2-10Acyl or optionally substituted C2-10An amide group.
Preferably, the amino-terminal protecting group is selected from an acetyl or acetamide group.
Preferably, the carboxy terminal protecting group is selected from optionally substituted C2-10An acyl group.
Preferably, the carboxy terminal protecting group is selected from acetyl.
In a second aspect, there is provided the use of said polypeptide as a relaxant of muscle paralysis.
In a third aspect, the use of the polypeptide as an anti-wrinkle agent for skin is provided.
The amino acid sequence of the invention adopts a standard Fmoc scheme, and a reasonable polypeptide synthesis method is realized by screening resin. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymeric resin, and then the amino group of the amino acid is used as a starting point to react with the carboxyl group of another molecule of amino acid to form a peptide bond. The process is repeated continuously to obtain the target polypeptide product. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the solid phase synthesis sequence is synthesized from the C-terminus to the N-terminus.
Interpretation of terms:
ser represents the corresponding residue in the English name Serine and in the Chinese name Serine;
glu represents the corresponding residue of the English name Glutamic and the Chinese name Glutamic acid;
phe represents the corresponding residue in the English name phenylalanine and the Chinese name phenylalanine;
met represents the corresponding residue in the English name Methionine, and in the Chinese name Methionine (Methionine);
arg represents the corresponding residue in english name arginin, and in chinese name Arginine;
asn represents the corresponding residue in english under the name Asparagine and Asparagine in chinese;
leu represents the corresponding residue in England under the name Leucine and in Leucine;
ala represents the corresponding residue in the English name of Alanine and the Chinese name of Alanine;
gln represents the corresponding residue in the English name Glutamine and in the Chinese name Glutamine.
Compared with the prior art, the invention has the beneficial effects that:
the short peptide used by the existing daily chemical products is less than 10 peptides, and the stability, affinity and skin permeation barrier are not ideal. Type a botulinum toxin is limited by reversible dosage and action, and long-term injection may cause long-term, severe adverse reactions, such as dyspnea, muscle weakness, sarcoid granuloma, headache, flu-like symptoms, anaphylaxis, and the like. The A-type botulinum toxin simulated pentadecapeptide prepared by the invention has good stability, permeability and affinity, shows good biocompatibility, is used for relaxing facial expression muscle paralysis, and achieves the effects of improving facial contour and eliminating or reducing wrinkles.
Drawings
FIG. 1 is a photograph showing the results of a 3-hour hindlimb morphology photograph of a short peptide-treated mouse;
FIG. 2 is a graph showing photographs of hindlimb morphology at 1, 2, 3 and 4 hours after short peptide treatment of mice;
FIG. 3 is a graph showing the results of the grip of mice treated with the short peptide for 1, 2, 3 and 4 hours using a mouse grip tester;
FIG. 4 is a graph showing the results of measuring the change in endurance capacity of mice treated with the short peptide at 1, 2, 3 and 4 hours using a mouse fatigue tester.
Detailed Description
The present invention will be further illustrated with reference to specific examples.
Example 1:
this example provides a polypeptide having an amino acid sequence of: Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: NELEEMQRRA, molecular weight: 1275.38.
the polypeptide of the present example was prepared by the following method:
the amino acid sequence of the invention adopts a standard Fmoc scheme, and a reasonable polypeptide synthesis method is realized by screening resin. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymeric resin, and then the amino group of the amino acid is used as a starting point to react with the carboxyl group of another molecule of amino acid to form a peptide bond. The process is repeated continuously to obtain the target polypeptide product. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the solid phase synthesis sequence is synthesized from the C-terminus to the N-terminus.
First an amino acid protected with an Fmoc group to the alpha-amino group is attached to an insoluble support via a linker, finally the alpha-amino group is deprotected, the amino acid-linker-resin is washed with solution, and a second pre-activated alpha-amino protected amino acid is attached via a coupling reaction. Alternatively, instead of a single amino acid, a peptide fragment protected at the α -N terminus and side chain may be subjected to coupling reaction, followed by washing with a solution after completion of the condensation reaction, repeated deprotection and coupling until the desired peptide is obtained, and finally cleavage of the peptide-arm-resin.
(1) Binding the C-terminal amino acid to the resin;
(2) removing and washing Na-Fmoc;
(3) coupling and washing;
(4) repeating the step (2) to the step (3);
(5) and (4) removing the protecting group.
Example 2:
this example provides a polypeptide having an amino acid sequence of: Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: RNELEEMQRRA, molecular weight: 1431.57.
example 3:
this example provides a polypeptide having an amino acid sequence of: Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: MRNELEEMQRRA, molecular weight: 1562.77.
example 4:
this example provides a polypeptide having an amino acid sequence of: Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: FMRNELEEMQRRA, molecular weight: 1709.94.
example 5:
this example provides a polypeptide having an amino acid sequence of: Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: EFMRNELEEMQRRA, molecular weight: 1839.05.
example 6:
this example provides a polypeptide having an amino acid sequence of: Ser-Glu-Phe-Met-Arg-Asn-Glu-Leu-Glu-Glu-Met-Gln-Arg-Arg-Ala, abbreviated: SEFMRNELEEMQRRA, molecular weight: 1926.13.
example 7:
this example differs from examples 1-6 in that it adds an amino-terminal protecting group to the polypeptide sequence of any of examples 1-6;
the amino-terminal protecting group described in this example is selected from acetyl or acetamido;
in some embodiments, the amino-terminal protecting group is selected from propionyl or propionamido;
in some embodiments, the amino-terminal protecting group is selected from butyryl or butyramido;
in some embodiments, the amino-terminal protecting group is selected from isobutyryl or isobutyramido.
In some embodiments, the amino-terminal protecting group is selected from a secondary butyryl group or a secondary butyrylamino group.
In some embodiments, the amino-terminal protecting group is selected from a tertiary butyryl group or a tertiary butyrylamino group.
In some embodiments, the amino-terminal protecting group is selected from valeryl or valeramide.
Example 8:
this example differs from examples 1-6 in that it adds a carboxy-terminal protecting group to the polypeptide sequence of any of examples 1-6
The carboxy terminal protecting group in this example is selected from acetyl;
in some embodiments, the carboxy terminal protecting group is selected from propionyl or propionamido;
in some embodiments, the carboxy terminal protecting group is selected from butyryl or butyramido;
in some embodiments, the carboxy terminal protecting group is selected from a tertiary butyryl group or a tertiary butyrylamino group.
Unless otherwise specified, the polypeptides of examples 1-8 were synthesized by the well-known FMOC solid phase synthesis method.
The synthesis principle of FMOC solid phase synthesis is as follows:
the C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymeric resin, and then the amino group of the amino acid is used as a starting point to react with the carboxyl group of another molecule of amino acid to form a peptide bond. The process is repeated continuously to obtain the target polypeptide product. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the solid phase synthesis sequence is synthesized from the C-terminus to the N-terminus.
Example 9:
this example provides the use of the polypeptide as a relaxant of muscle paralysis.
One experimental protocol:
the pentadecapeptide described in example 6 was used for experimental study of the relaxation of gastrocnemius paralysis in mice:
mice of the C57 strain aged 8-12 weeks were used;
the experimental design of the gastrocnemius injection of the mice is divided into a NaCl group (normal group), a hexapeptide group (control group) and a pentadecapeptide group (treatment group), and each group is not less than 3;
the control group of pentadecapeptide used for relaxing gastrocnemius paralysis of mice is hexapeptide imitating type A botulinum toxin;
dose screening of pentadecapeptide for relaxing gastrocnemius paralysis in mice:
the injection mode adopts a micro-injector, and the hind limb, the small leg and the gastrocnemius muscle are injected intramuscularly;
the administration mode adopts single injection and four-limb administration;
the administration dose is 10 mug, 20 mug and 30 mug respectively;
determining the optimal dosage 3h after injection by observing the shape and the relaxation degree of the hind limb and toe of the mouse;
pentadecapeptide for evaluation of the effect of the mouse on the relaxation of gastrocnemius paralysis:
administration dose: the dosage of the hexapeptide group is the same as that of the pentadecapeptide group which is the optimal screening;
injecting gastrocnemius of mice of different groups according to the scheme;
after injection, the effects of hexapeptide and pentadecapeptide on the paralysis and relaxation of gastrocnemius muscle of mice were evaluated in 1h, 2h, 3h and 4h respectively by using a mouse fatigue tester and a mouse grip tester.
II, experimental results:
1. dose screening of pentadecapeptide for relaxing gastrocnemius paralysis of mice
By observing the hindlimb morphology of mice injected with hexapeptide and pentadecapeptide in different dose groups, it is found that in 3 mice after injection, the muscle weakness of the hindlimb of the mice in the 20ug dose group appears most obviously, which is shown in the shrinkage and the weakness of the toes of the mice, and the pentadecapeptide group has better effect than the hexapeptide group (see figure 1). Figure 1, 6 represents an exemplary botulinum toxin type a hexapeptide; 15 represents the mimetic botulinum toxin type a pentadecapeptide.
2. Effect evaluation of pentadecapeptide on relaxing gastrocnemius paralysis of mice
Both hexapeptide and pentadecapeptide were administered as single, intramuscular injections into the extremities at a dose of 20 ug. Hind limb morphology observation and grip and endurance measurements were performed on mice at 1, 2, 3 and 4 hours post-injection, respectively.
By observing the shape of the hind limbs of mice at different time points of hexapeptide injection and pentadecapeptide injection, the hind limbs of mice of the more normal group hexapeptide group and pentadecapeptide group are found to have closed toes at 2 hours, and the phenomena still exist by 3 hours when the pentadecapeptide group has obvious crouch and muscle weakness compared with the hexapeptide group and the phenomena still exist by 4 hours (see figure 2). In FIG. 2, 6 represents the mimetic botulinum toxin type A hexapeptide; 15 represents the mimetic botulinum toxin type a pentadecapeptide.
Through the measurement of the holding power of mice injected with hexapeptide and pentadecapeptide at different time points, the experimental results show that the holding power of the mice among groups has no significant difference within 2 hours after injection, the holding power of the hexapeptide and the pentadecapeptide is obviously reduced compared with that of the normal group within 3 to 4 hours, and the holding power of the mice in the pentadecapeptide group is more significantly reduced (see figure 3). In FIG. 3, 6 represents the mimetic botulinum toxin type A hexapeptide; 15 represents the mimetic botulinum toxin type a pentadecapeptide.
Through the measurement of the endurance capacity of mice injected with hexapeptide and pentadecapeptide at different time points, the test result shows that 2 hours after the injection, the running time and the running distance of the mice of the hexapeptide and the pentadecapeptide are reduced compared with those of the mice of a normal group, and the endurance capacity of the mice is prompted to decline; the decline in endurance capacity was most significant in both groups of mice at 3 hours, and the pentadecapeptide group was more effective than the hexapeptide group (see fig. 4). In FIG. 4, 6 represents the mimetic botulinum toxin type A hexapeptide; 15 represents the mimetic botulinum toxin type a pentadecapeptide.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
