CN110713517B - Hyaluronan oligopeptide and preparation and application methods thereof - Google Patents
Hyaluronan oligopeptide and preparation and application methods thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/91—Injection
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to a hyaluronan oligopeptide, and a preparation method and an application method thereof, and solves the technical problems that in the prior art, hyaluronan and oligopeptide substances are unstable in structure, hyaluronan and oligopeptide in functional cosmetics need to be added respectively, and are easy to be subjected to enzymolysis, poor in chemical stability, easy to be inactivated by environmental influence and short in action time. The invention provides a preparation method of a wrinkle-removing and anti-aging hyaluronan oligopeptide, which comprises the following steps: connecting hyaluronan and oligopeptide through a covalent bond form: epsilon-NH of Lys side chains in oligopeptide sequence2Amide bond is generated through amide condensation reaction with-COOH of the hyaluronan for connection, so as to generate hyaluronan oligopeptide; also provides an application method thereof. The invention is widely applied to the technical field of functional cosmetics and shaping and beautifying products.
Description
Technical Field
The invention belongs to the technical field of functional cosmetics, and particularly relates to a hyaluronan oligopeptide and a preparation and application method thereof.
Background
The development of functional cosmetics has now been the subject of the development of the cosmetic industry today, where the added bioactive ingredients have important regulatory effects on the growth and metabolism of cells, such as: polypeptide, polysaccharide, etc. can effectively treat skin injury and promote skin repair. The polypeptide has special biological activity, plays an important role in regulating the growth, development and metabolism of organisms, plays an important role in regulating and controlling the special physiological activity in the skin aging and nursing processes, and is widely applied to beauty skin care products. The beauty polypeptide is mainly a small molecular oligopeptide consisting of two to ten amino acids, can promote cell growth, differentiation, reconstruction and repair, fundamentally improve and repair skin injury, has obvious effects of removing wrinkles, resisting aging, whitening, removing freckles and the like, is an important component of functional cosmetics, and has extremely high use safety, for example: tripeptide-1 can stimulate the production of collagen, elastin, fibronectin and laminin, restore the elasticity and firmness of the skin, has the functions of anti-inflammation, antioxidation, wrinkle removal and anti-aging, and can be used for the production of hair and skin care products; the SIKVAV oligopeptide and IKVAV oligopeptide can promote cell adhesion, improve skin flexibility, prevent skin aging, etc. Hyaluronic Acid (HA) is a glycosaminoglycan, HAs a special water retention effect, is a substance which is found to have the best moisture retention in nature at present, can retain 1000 times of water of the self weight, can form gel after being dissolved in 1 percent of solution, prevents the water of the skin from losing through the epidermis, is called as an ideal natural moisture retention factor, and is also a very good transdermal absorption promoter. The unique molecular structure and physicochemical properties of hyaluronic acid have a variety of important physiological functions in the body, including prevention, repair and wound healing, and the like, and are widely used for the production of beauty skin care products. Wherein the small molecular hyaluronic acid can penetrate into dermis, remove oxygen free radicals, improve skin nutrition metabolism, promote proliferation and differentiation of epidermal cells, increase skin elasticity, remove wrinkle and prevent aging; the macromolecular hyaluronic acid can form a layer of protective film on the surface layer of the skin, can lock water in the skin and can prevent the skin from being damaged by external adverse environmental factors. AcHA is a derivative of hyaluronic acid modified by acetylation, is a novel and efficient skin softening factor, has hydrophilicity and lipophilicity, can highly soften cuticle, has strong affinity to skin, has water retention capacity 2 times higher than that of hyaluronic acid, and has a skin care effect superior to that of hyaluronic acid. The functional cosmetic containing the oligopeptide or the hyaluronic acid has the advantages of safety, stability, easy absorption and good effect, and is favored by consumers. Meanwhile, the functional cosmetics have high added value and large market space, the profits of cosmetic manufacturers are greatly increased, and more enterprises and research institutions are invested in the research and development of the functional cosmetics.
Patent CN106109296B discloses a moisturizing essence containing sodium hyaluronate and sodium acetylhyaluronate; CN104740643A discloses a stable hyaluronic acid solution loaded with bioactive protein or polypeptide, which needs to add hyaluronic acid and bioactive protein or polypeptide respectively; CN10
9157471A discloses a skin care composition with repairing and moisturizing functions, which is added with hyaluronic acid and oligopeptide respectively; CN108904317A discloses a polypeptide combination method for removing wrinkles. However, these patents disclose the addition of hyaluronic acid and polypeptides, either alone or in combination, to products and do not mention hyaluronic acid oligopeptides that function as both active ingredients, and their development and use in cosmetic compositions. A single active ingredient has limited effect on cells, and better effects can be obtained by combining two active ingredients or using a component having the functions of both active ingredients. However, hyaluronidase and protease of human body can hydrolyze hyaluronic acid and oligopeptide, so hyaluronic acid and oligopeptide absorbed by skin penetration and on skin surface are easily subjected to enzymolysis inactivation, and meanwhile, due to unstable structure of the polypeptide substance and easy denaturation and inactivation caused by external adverse environmental factors, the active action time of the polypeptide substance is short, so that proper dosage forms and protective agents need to be selected according to component characteristics to improve stability and prolong the efficacy of the polypeptide substance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a hyaluronan oligopeptide which has the functional activity of hyaluronan and oligopeptide, stable chemical property, difficult environmental influence, obvious wrinkle-removing and anti-aging effects and long action time, and a preparation method and an application method thereof aiming at the defect of unstable structure of hyaluronan and oligopeptide substances.
The technical scheme adopted by the invention is as follows:
a wrinkle-removing and anti-aging hyaluronan oligopeptide, wherein the structure of the hyaluronan oligopeptide is a glycopeptide having the structure of formula V, formula VI and formula VII:
a preparation method of a wrinkle-removing and anti-aging hyaluronan oligopeptide comprises the following steps: connecting hyaluronan and oligopeptide through a covalent bond form: epsilon-NH of Lys side chains in oligopeptide sequence2Amide bond is generated through amide condensation reaction with-COOH of the hyaluronan for connection, so as to generate hyaluronan oligopeptide;
the structure of hyaluronan is of formula I:
formula I;
the structure of the oligopeptide is respectively shown as formula II, formula III and formula IV:
preferably, the method comprises the following steps:
(1) Preparing hyaluronan into hyaluronan-TBA;
(2) Condensation reaction:
(1) by solid phase synthesis: under the existence of an activation system and DIEA, CTC resin is taken as a solid phase synthesis carrier, activated Fmoc-amino acids are sequentially condensed from C end to N end according to the amino acid sequences of the structures of the oligopeptides II, III and IV respectively, so as to synthesize peptide resin, and three kinds of peptide resin with Fmoc protecting groups contained in the N end amino groups of the structures of the oligopeptides II, III and IV respectively; the Fmoc-amino acid sequence of the synthetic oligopeptide condensed by the peptide resin with the structural formula II is as follows: fmoc-Val-OH, fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH, fmoc-Ile-OH, fmoc-Ser-OH; the Fmoc-amino acid sequence of the peptide resin condensed in sequence in the structural formula III of the synthetic oligopeptide is as follows: fmoc-Val-OH, fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH, fmoc-Ile-OH; the Fmoc-amino acid sequence of the synthetic oligopeptide, which is condensed in sequence by peptide resin with a structural formula IV, is as follows: fmoc-Lys (Dde) -OH, fmoc-His-OH, fmoc-Gly-OH;
(2) removing the Lys side chain epsilon-NH in the Fmoc protecting group-containing peptide resin prepared in the step (1)2The protecting group Dde and the cracked peptide resin respectively obtain three oligopeptide crude peptides of which N-terminal amino groups have Fmoc protecting groups, wherein the oligopeptide structures are shown in a formula II, a formula III and a formula IV;
(3) purifying the crude oligopeptide peptide containing the Fmoc protecting group prepared in the step (2), and freeze-drying to obtain three pure oligopeptides of which the N-terminal amino groups of the oligopeptides of the structures of the formulas II, III and IV contain the Fmoc protecting group;
(3) Activating the hyaluronan-TBA prepared in the step (1), and then carrying out condensation reaction with the pure oligopeptide prepared in the step (2); epsilon-NH of Lys side chains in the oligopeptide sequence2Amide bond is generated through amide condensation reaction with-COOH of the hyaluronan for connection, and the product hyaluronan TBA oligopeptide-Fmoc is generated;
(4) Removing Fmoc-protecting groups of N-terminal amino groups of the oligopeptides in the product prepared in the step (3) to obtain a product, namely hyaluronan TBA oligopeptides;
(5) Salt exchange: ultrafiltering the product of hyaluronan TBA oligopeptide prepared in the step (4) by using an ultrafiltration membrane (with the molecular weight cutoff of 500 Da), washing, and converting into Na by using a 732 type cation exchange column (resin)+And (3) freeze-drying the salt to obtain finished products of the hyaluronan oligopeptides with the structures of the formula V, the formula VI and the formula VII respectively.
Preferably, the molecular weight of the hyaluronan is 10kDa to 100kDa.
Preferably, in step (2) (1), the activation system is: a + D or A + B + C, wherein A is HOBT or HOAT, B is any one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC; the activating method of the hyaluronan-TBA in the step (3) comprises the following steps: hyaluronan-TBA was reacted with HOSU in the presence of EDC for 24h.
Preferably, the step (2) (2) removes the side chain ε -NH of Lys in the Fmoc-protecting group-containing peptide resin obtained in the step (1)2The method for removing the protecting group Dde comprises the following steps: to the peptide resin of Fmoc protecting group prepared in step (2) (1), deprotection solution was added at 100 (w/w/w/w) of hydroxylamine hydrochloride: imidazole: DCM: NMP = 25.
Preferably, the method for cleaving the peptide resin in step (2) (2) is: reacting for 2-5h at 10-30 ℃ according to the proportion of adding 10ml of lysis solution into each g of peptide resin, and cracking the peptide resin; lysate 20% TFE/DCM (v/v).
Preferably, the purification method in step (2) and (3) is to purify the crude peptide of oligopeptide containing Fmoc protecting group prepared in step (2) by reverse phase high performance liquid chromatography, and the conditions are as follows: mobile phase A is H2O, the mobile phase B is acetonitrile, gradient elution is adopted, the elution time is 60min, the flow rate is 80ml/min, the ultraviolet detection wavelength is 220nm, and the elution gradient B is 10-40% or 1-31%.
Any one, two or three of the three hyaluronan oligopeptides having the structures of formula V, formula VI and formula VII may be used as a cosmetic ingredient or added to a cosmetic.
Any one, any two or three of the three hyaluronan oligopeptides with the structures of formula V, formula VI and formula VII are used as skin fillers in facial injection cosmetic products.
The invention has the beneficial effects that:
(1) Aiming at the defect of unstable structure of hyaluronan and oligopeptide substances, the prepared hyaluronan oligopeptide has the functional activity of hyaluronan and oligopeptide, has the targeting performance of hyaluronan, effectively promotes the oligopeptide to penetrate a skin barrier, enhances the absorption and combination of skin on the oligopeptide, increases the using effect of the oligopeptide, has the moisture-preserving, wrinkle-removing and anti-aging effects which are obviously superior to the effects of separate use and combined use of the oligopeptide and the hyaluronan, has stable chemical properties, improves the anti-enzymolysis capacity, prolongs the half life, is not easy to be influenced by the environment, has obvious moisture-preserving, wrinkle-removing and anti-aging effects and has long acting time.
(2) The invention generates the hyaluronan oligopeptide by coupling the hyaluronan and the oligopeptide by adopting a covalent bond, radically endows the hyaluronan oligopeptide with more excellent characteristics by directly modifying and modifying chemically, has simple preparation and synthesis steps and simple and easy purification, and simultaneously provides an application method of the hyaluronan oligopeptide in cosmetics and cosmetic products, obviously enhances the effects of moisturizing, removing wrinkles and resisting aging when the functional cosmetics are used, and obviously improves the use satisfaction degree of users.
Detailed Description
The present invention will be further described with reference to specific examples to assist understanding of the invention. The method used in the invention is a conventional production method if no special provisions are made; the starting materials used, unless otherwise specified, are conventional commercial products.
The specific meanings of the abbreviations used in the present invention are listed in the following table:
example 1
Preparation of hyaluronan-TBA:
(1) 300g of hyaluronan with a molecular weight of 50kDa was dissolved in 10L of deionized water and then passed through 732 (H)+) Type cation exchange column for separating Na therefrom+Conversion to H+To obtainAn eluent containing hyaluronan;
(2) Adding 200g of TBAOH into the eluent containing the hyaluronan in the step (1), stirring for 2h, and neutralizing to obtain a hyaluronan-TBA reaction solution;
(3) Performing ultrafiltration, washing and concentration on the hyaluronan-TBA reaction solution obtained in the step (2) by using an ultrafiltration membrane (with the molecular weight cutoff of 500D), and removing excessive TBAOH to obtain a hyaluronan-TBA product;
(4) And (4) freeze-drying the hyaluronan-TBA product obtained in the step (3) to obtain hyaluronan-TBA solid powder.
Example 2
Preparation of crude Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide:
(1) Swelling of the resin: taking 25g of CTC resin with the substitution degree of 1.2mmol/g, adding 200ml of DCM to swell the resin for 0.5h, draining the solvent, washing the resin twice by DMF, and draining the solvent;
(2) Preparation of Fmoc-Val-CTC resin: a) Mixing Fmoc-Val-OH, DIEA and the swelled CTC resin in the step (1) according to the molar mass ratio of 3; b) Adding a mixed solution of MeOH, DMF and DIEA into resin, reacting for 30min at 30 ℃, sealing the resin, washing the resin twice with DMF, and draining the solvent to obtain sealed Fmoc-Val-CTC resin;
(3) And (3) removing the Fmoc protecting group: adding a pip-DMF solution of 20% by volume fraction to the blocked Fmoc-Val-CTC resin obtained in the step (2), performing Fmoc deprotection twice under the condition of 10-30 ℃: after Fmoc protection removal for the first time and Fmoc protection removal for the second time, washing the resin with DMF until the pH is 7; the Fmoc protection removing time for the first time is 5min, and the Fmoc protection removing time for the second time is 10min, so as to obtain the resin for removing the Fmoc protection group;
(4) Amino acid activation: respectively dissolving 90mmol of Fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH, fmoc-Ile-OH and Fmoc-Ser-OH and 90mmol of HOBT in a proper amount of DMF at room temperature, adding 90mmol of DIC, and carrying out activation reaction for 5min to obtain activated amino acid;
(5) Amino acid condensation: sequentially adding the activated Fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH, fmoc-Ile-OH and Fmoc-Ser-OH obtained in the step (4) into the resin without the Fmoc protecting group obtained in the step (3), carrying out amino acid condensation reaction for 2h at the temperature of 25 ℃, and monitoring the reaction process by ninhydrin color reaction to obtain Fmoc-Ser-Ile-Lys (Dde) -Val-Ala-Val-CTC resin;
(6) And (3) removing the Dde protecting group: adding deprotection solution hydroxylamine hydrochloride, imidazole, DCM, NMP =25 (w/w/w) to the Fmoc-Ser-Ile-Lys (Dde) -Val-Ala-Val-CTC resin obtained in the step (5), reacting for 3h, washing the peptide resin with 300ml DMF and 300ml MeOH alternately for three times for 5 min/time, shrinking the peptide resin, draining the solvent, and drying in vacuum to obtain the Fmoc-Ser-Ile-Lys-Val-Ala-Val-CTC resin;
(7) Cracking: and (3) adding 10ml of lysis solution per g of peptide resin according to the proportion of 20% TFE/DCM (v/v) into a round-bottom flask, cracking the Fmoc-Ser-Ile-Lys-Val-Ala-Val-CTC resin obtained in the step (6), reacting for 2 hours at the temperature of 25 ℃, performing suction filtration, distilling the filtrate under reduced pressure, and evaporating the solvent to obtain the crude Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH.
Example 3
Preparation of crude Fmoc-Ile-Lys-Val-Ala-Val-COOH peptide:
(1) Swelling of the resin: taking 25g of CTC resin with the substitution degree of 1.2mmol/g, adding 200ml of DCM to swell the resin for 0.5h, draining the solvent, washing the resin twice by DMF, and draining the solvent;
(2) Preparation of Fmoc-Val-CTC resin: a) Mixing Fmoc-Val-OH, DIEA and resin according to the molar mass ratio of 3; b) Adding a mixed solution of MeOH, DMF and DIEA into resin, reacting for 30min at 10-30 ℃, sealing the resin, washing the resin twice with DMF, and draining the solvent to obtain sealed Fmoc-Val-CTC resin;
(3) Removing Fmoc protecting groups: adding a pip-DMF solution of 20% by volume fraction to the blocked Fmoc-Val-CTC resin obtained in the step (2), performing Fmoc deprotection twice under the condition of 10-30 ℃: after Fmoc protection removal for the first time and Fmoc protection removal for the second time, washing the resin with DMF until the pH is 7; the Fmoc removal protection time for the first time is 5min, and the Fmoc removal protection time for the second time is 10min, so that the resin for removing the Fmoc protection group is obtained;
(4) Amino acid activation: respectively dissolving 90mmol of Fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH and Fmoc-Ile-OH and 90mmol of HOBT in a proper amount of DMF, then adding 90mmol of DIC, and reacting for 5min at room temperature to obtain activated amino acid;
(5) Amino acid condensation: sequentially adding the Fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH and Fmoc-Ile-OH activated in the step (4) into the resin with the Fmoc protecting group removed, carrying out condensation reaction of amino acid for 2h at 25 ℃, monitoring the reaction process through ninhydrin color reaction, and finally obtaining Fmoc-Ile-Lys (Dde) -Val-Ala-Val-CTC resin;
(6) And (3) removing the Dde protecting group: adding deprotection solution hydroxylamine hydrochloride, imidazole, DCM: NMP =25, to the Fmoc-Ile-Lys (Dde) -Val-Ala-Val-CTC resin obtained in the step (5), reacting for 3h, washing the peptide resin with 300ml DMF and 300ml MeOH alternately for three times, wherein the washing time is 5 min/time, shrinking the peptide resin, draining the solvent, and drying in vacuum to obtain the Fmoc-Ile-Lys-Val-Ala-Val-CTC resin;
(7) Cracking: cleaving the Fmoc-Ile-Lys-Val-Ala-Val-CTC resin obtained in step (6) at a ratio of 20% TFE/DCM (v/v) per g of the peptide resin in a round-bottomed flask, reacting at 25 ℃ for 2 hours, suction-filtering, distilling the filtrate under reduced pressure, and evaporating the solvent to obtain crude Fmoc-Ile-Lys-Val-Ala-Val-COOH.
Example 4
Preparation of Fmoc-Gly-His-Lys-COOH crude peptide:
(1) Swelling of the resin: taking 37.5g of CTC resin with the substitution degree of 1.2mmol/g, adding 300ml of DCM to swell the resin for 0.5h, draining the solvent, washing the resin twice by DMF, and draining the solvent;
(2) Preparation of Fmoc-Lys (Dde) -CTC resin: a) Mixing Fmoc-Lys (Dde) -OH, DIEA and resin according to the molar mass ratio of 3; b) Adding a mixed solution of MeOH, DMF and DIEA into resin, reacting for 30min at 10-30 ℃, sealing the resin, washing the resin twice with DMF, and draining the solvent to obtain the sealed Fmoc-Lys (Dde) -CTC resin.
(3) Removing Fmoc protecting groups: adding a volume fraction of 20% PIP-DMF solution to the blocked Fmoc-Lys (Dde) -CTC resin obtained in step (2), and performing Fmoc deprotection twice at 10-30 ℃: after Fmoc protection removal for the first time and Fmoc protection removal for the second time, washing the resin with DMF until the pH is 7; the first Fmoc removal protection time is 5min, and the second Fmoc removal protection time is 10min, so that the resin with the Fmoc removal protection group is obtained;
(4) Amino acid activation: dissolving 135mmol of Fmoc-His-OH and 135mmol of Fmoc-Gly-OH and 135mmol of HOBT respectively in a proper amount of DMF, adding 135mmol of DIC, and reacting at room temperature for 5min to obtain activated amino acid;
(5) Amino acid condensation: sequentially adding the activated Fmoc-His-OH and Fmoc-Gly-OH in the step (4) into the resin with the Fmoc protecting group removed, carrying out condensation reaction of amino acid for 2h at 25 ℃, and carrying out ninhydrin color reaction to monitor the reaction process, thereby finally obtaining Fmoc-Gly-His-Lys-CTC resin;
(6) And (3) removing the Dde protecting group: adding 300ml deprotection solution hydroxylamine hydrochloride: imidazole: DCM: NMP =25 to the Fmoc-Gly-His-Lys-CTC resin obtained in the step (5) and then reacting for 3h, washing the peptide resin three times by using 300ml DMF and 300ml MeOH alternately, wherein the washing time is 5 min/time, shrinking the peptide resin, draining the solvent, and drying in vacuum to obtain the Fmoc-Gly-His-Lys-CTC resin;
(7) Cracking: and (3) adding 10ml of lysis solution 20% per g of peptide resin into a round-bottom flask, cracking the Fmoc-Gly-His-Lys-CTC resin obtained in the step (6) according to the proportion of TFE/DCM (v/v), reacting for 2 hours at 25 ℃, performing suction filtration, performing reduced pressure distillation on the filtrate, and evaporating the solvent to obtain the crude Fmoc-Gly-His-Lys-COOH.
Example 5
And (3) purification:
the crude Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide, the crude Fmoc-Ile-Lys-Val-Ala-Val-COOH peptide and the crude Fmoc-Gly-His-Lys-COOH peptide prepared in examples 2 to 4 were purified respectively;
using reverse phase high efficiencyPurifying three crude peptides by one step by liquid phase chromatography, wherein the mobile phase A is H2And O, taking acetonitrile as a mobile phase B, and adopting gradient elution: c18 preparation of the column (50X 250mm,10 μm) with an elution time of 60min: the elution gradient for the oligopeptides obtained in example 2 and example 3 was: phase B is 10-40%; the elution gradient for the oligopeptides obtained in example 4 was: phase B is 1% -31%; the flow rate is 80ml/min, and the ultraviolet detection wavelength is 220nm. And finally, obtaining the Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide, the Fmoc-Ile-Lys-Val-Ala-Val-COOH peptide and the Fmoc-Gly-His-Lys-COOH peptide through concentration and freeze-drying, wherein the purity of the three oligopeptides is 98.2%, 98.0% and 98.3% respectively.
Example 6
Preparing a finished product of hyaluronan oligopeptide:
(1) Activated hyaluronan: dissolving 50g of the hyaluronan-TBA prepared in example 1 in DMF, adding 17mmol of HOSU and 34mmol of EDC, reacting at 30 ℃ for 24h, pouring the reaction solution into ethyl acetate with the volume 4 times that of the reaction solution, stirring for 0.5h, standing overnight, and settling; filtering the precipitate, and washing the precipitate twice with ethyl acetate to obtain activated hyaluronan;
(2) Dissolving the activated hyaluronan obtained in the step (1) in DMF, adding 17mmol of any one of three pure oligopeptide products of Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide, fmoc-Ile-Lys-Val-Ala-Val-COOH peptide and Fmoc-Gly-His-Lys-COOH peptide prepared in the embodiment 5, adding 20.4mmol of DIEA, stirring for reaction, and monitoring the reaction process by adopting ninhydrin color reaction until no amino component in the reaction solution enables the reaction to be complete, thereby obtaining the hyaluronan oligopeptide protected by Fmoc;
(3) Adding diethylamine with the volume ratio of 10% into the reaction system of the Fmoc-protected hyaluronan oligopeptide obtained in the step (2), removing the Fmoc protecting group at the N end of the peptide chain, and then carrying out reduced pressure distillation on the reaction solution to evaporate the solvent to obtain a semi-finished hyaluronan oligopeptide;
(4) Dissolving the hyaluronic acid oligopeptide semi-finished product solid prepared in the step (3), performing ultrafiltration through an ultrafiltration membrane (with the molecular weight cutoff of 500D), washing, and converting TBA in the solution into Na through a 732 type cation exchange column+And respectively obtaining three hyaluronan oligopeptide finished products with structures of a formula V, a formula VI and a formula VII by freeze-drying.
Example 7
Preparing a finished product of the hyaluronan oligopeptide:
(1) 50g of the hyaluronan-TBA prepared in example 1 is dissolved in DMF, 34mmol of HOSU and 68mmol of EDC are added, reaction is carried out at 30 ℃ for 24h, the reaction solution is poured into ethyl acetate with 4 times volume of the reaction solution, stirring is carried out for 0.5h, then standing overnight is carried out, and sedimentation is carried out; filtering the precipitate, and washing the precipitate twice with ethyl acetate to obtain activated hyaluronan;
(2) Dissolving the activated hyaluronan obtained in the step in DMF, adding 34mmol of any one of three pure oligopeptide products of Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide, fmoc-Ile-Lys-Val-Ala-Val-COOH peptide and Fmoc-Gly-His-Lys-COOH peptide prepared in the embodiment 5, adding 40.8mmol of DIEA, stirring for reaction, and monitoring the reaction progress by ninhydrin color reaction until no amino component exists in the reaction solution so as to ensure that the reaction is complete, thereby obtaining the hyaluronan oligopeptide protected by Fmoc;
(3) Adding diethylamine with the volume ratio of 10% into the reaction system of the Fmoc-protected hyaluronan oligopeptide obtained in the step (2), removing the Fmoc protecting group at the N end of the peptide chain, and then carrying out reduced pressure distillation on the reaction solution to evaporate the solvent to obtain a semi-finished hyaluronan oligopeptide;
(4) Dissolving the hyaluronic acid oligopeptide semi-finished product solid prepared in the step (3), performing ultrafiltration and washing through an ultrafiltration membrane (with the molecular weight cut-off of 500D), and converting TBA in the hyaluronic acid oligopeptide semi-finished product into Na through a 732 type cation exchange column+And respectively obtaining three hyaluronan oligopeptide finished products with structures of a formula V, a formula VI and a formula VII by freeze-drying.
Example 8
Preparing a finished product of hyaluronan oligopeptide:
(1) 50g of the hyaluronan-TBA prepared in example 1 is dissolved in DMF, 51mmol of HOSU and 102mmol of EDC are added, reaction is carried out for 24h at 30 ℃, the reaction solution is poured into ethyl acetate with 4 times volume of the reaction solution, stirring is carried out for 0.5h, then standing overnight is carried out, and sedimentation is carried out; filtering the precipitate, and washing the precipitate twice with ethyl acetate to obtain activated hyaluronan;
(2) Dissolving the activated hyaluronan obtained in the step (1) in DMF, adding 51mmol of any one of three pure oligopeptide products of Fmoc-Ser-Ile-Lys-Val-Ala-Val-COOH peptide, fmoc-Ile-Lys-Val-Ala-Val-COOH peptide and Fmoc-Gly-His-Lys-COOH peptide prepared in the embodiment 5, adding 61.2mmol of DIEA, stirring for reaction, and monitoring the reaction process by adopting ninhydrin color reaction until no amino component exists in the reaction solution so as to ensure that the reaction is complete, thereby obtaining the hyaluronan oligopeptide protected by Fmoc;
(3) Adding diethylamine with the volume ratio of 10% into the reaction system of the Fmoc-protected hyaluronic acid oligopeptide obtained in the step (2), removing the Fmoc protecting group at the N end of the peptide chain, and then carrying out reduced pressure distillation on the reaction solution to evaporate the solvent to obtain a semi-finished product of the hyaluronic acid oligopeptide;
(4) Dissolving the solid obtained in step (3), ultrafiltering with ultrafiltration membrane (molecular weight cut-off of 500D), washing, and converting TBA into Na with 732 type cation exchange column+And respectively obtaining three finished products of the hyaluronan oligopeptides with structures of a formula V, a formula VI and a formula VII by freeze-drying.
Example 9
Preparing a finished product of hyaluronan oligopeptide as a raw liquid of a moisturizing and anti-wrinkling cosmetic, wherein the raw liquid comprises the following components in percentage by weight:
at room temperature, in 7 parts of equivalent sterile water, 4% by mass of butanediol and 0.8% by mass of PE9010 are respectively added as substrates and respectively used as an experimental group 1-an experimental group 6 and a blank group 7, then, 0.5% by mass of hyaluronic acid, oligopeptides, a composition of hyaluronic acid and oligopeptides, three hyaluronic acid oligopeptide finished products with the structures of formula V, formula VI and formula VII prepared in example 8 are respectively added into the experimental group 1-the experimental group 6, after sterile water with equal mass is added into the blank group 7, the mixture is uniformly stirred until the mixture is completely dissolved, and after the pH value is adjusted to be 5-7, an experimental group 1 of a hyaluronic acid stock solution, an experimental group 2 of an oligopeptide stock solution (three oligopeptides with the structures of formula II, formula III and formula IV respectively), an experimental group 3 of a hyaluronic acid and oligopeptide of a hyaluronic acid stock solution (three oligopeptides with the structures of formula II, formula III and formula IV respectively), an experimental group 4 of an hyaluronic acid and a blank group 5 of an oligopeptide of an hyaluronic acid and a blank group 7 of an hyaluronic acid stock solution (three oligopeptides with the structures of formula II, formula III and formula IV) are respectively obtained.
The properties of the hyaluronan oligopeptides of the present invention are further illustrated by experimental reports below.
(1) And (4) detecting items:
and detecting the moisturizing effect and wrinkle removing effect of the hyaluronan oligopeptide.
(2) And (3) testing a sample:
the moisturizing and wrinkle removing effects of test group 1 of the hyaluronic acid stock solution prepared in example 9, test group 2 of the oligopeptide stock solution (three oligopeptides having structures of formulae II, III, and IV, respectively), test group 3 of a combined stock solution of hyaluronic acid and oligopeptides (three oligopeptides having structures of formulae II, III, and IV, respectively), test group 4 of the hyaluronic acid oligopeptide stock solution having structure of formula V, test group 5 of the hyaluronic acid oligopeptide stock solution having structure of formula VI, test group 6 of the hyaluronic acid oligopeptide stock solution having structure of formula VII, and blank group 7 are shown in tables 1 to 3.
(3) Determination of using effect of hyaluronan oligopeptide:
1. moisture retention effect:
1.1 subject: selecting 110 volunteers, wherein the age is 35-40 years old, and performing experiments on the skin of the forearms of the volunteers;
1.2 test samples: stock solutions of experimental group 1 to experimental group 6 and blank group 7 prepared in example 9 were used as test substances;
1.3 before testing: the examinee needs to wash the inner sides of the forearms of both hands uniformly with clear water, and measurement marks are made on the inner sides of the forearms of both hands of the examinee after the washing. The left and right forearms in this experiment were each marked with two test areas.
1.4 in the test: the arm of each tester divides 7 test areas with the interval of 1cm, and each test area is 3 multiplied by 3cm2The amount of the test sample was 0.2g. The subjects applied one test substance per area, and applied stock solutions of experimental group 1 to experimental group 6 and blank group 7 prepared in example 9 to 7 test areas, respectively. Subject in a constant ringAfter sitting still for 30min in the environment (the measurement environment temperature is 20 ℃, the relative humidity is 50 percent), a Corneometer CM 825 moisture tester is used for measuring the blank value of the tested part, and 5 points are fixedly measured in each area according to a certain sequence to obtain an average value. Then the special person is responsible for smearing the sample, timing is started, and the MMV value change is measured at each time according to the design of the test. And subtracting a blank value from the average value detected in each test area every time to obtain the change of the MMV value in the time period, and removing the blank value to obtain the increase rate of the MMV value.
Mean moisture content increase rate% = (MMV)t-MMV0)/MMV0×100％，
In the formula: MMV0-the MMV of the skin before application,
MMVt-MMV of the skin t time after application.
The test experience reference data of the detection method is shown in the table 1: the following data are obtained under normal room temperature conditions (measured ambient temperature of 20 ℃ C., relative humidity of 50%).
TABLE 1Corneometer CM 825 moisture tester test empirical data
| Arms, hands, legs, etc | |
| The skin is drier | ＜35 |
| The skin is drier | 35-50 |
| The moisture of the skin is sufficient | ＞50 |
The Corneometer CM 825 moisture tester was used to test the skin of the forearms of 70 volunteers in stock solution 4h of experimental group 1-experimental group 6 and blank group 7 prepared using example 9, and the results of the variation of MMV value of the skin by each test object were averaged, and the data results are shown in table 2.
TABLE 2 comparison of moisturizing effect of hyaluronan oligopeptide
Hyaluronic acid stock solutions of experiment group 1, oligopeptide stock solutions of experiment group 2 (three kinds of oligopeptides with structures of formulas II, III and IV respectively), hyaluronan and oligopeptide combination stock solutions of experiment group 3 (three kinds of oligopeptides with structures of formulas II, III and IV respectively), hyaluronan oligopeptide stock solutions of experiment group 4 with structures of formula V, hyaluronan oligopeptide stock solutions of experiment group 5 with structures of formula VI, hyaluronan oligopeptide stock solutions of experiment group 6 with structures of formula VII, and blank group 7; the results of the moisturizing effect test data of the three oligopeptides with the structures of formula II, formula III and formula IV in the experimental group 2 and the experimental group 3 are similar, so the moisturizing effect test data of the oligopeptide stock solution of the experimental group 2 used in table 1 is the average value of the moisturizing effect test data of the three oligopeptides, and the moisturizing effect test data of the combined stock solution of the hyaluronan and the oligopeptides in the experimental group 3 is also the average value of the moisturizing effect test data of the mixture of the three oligopeptides with the hyaluronan.
As can be seen from the comparison of the moisturizing effect of the hyaluronan oligopeptide in table 1 and the data results in the table, the change of the MMV value of the skin is gradually reduced along with the increase of time, and the blank group 7 has the worst moisturizing effect, so that the skin of a human body cannot be effectively moisturized and locked when only a sterile water matrix is given when moisturizing substance components are not added to the skin; the moisturizing effect of the hyaluronan oligopeptide stock solution of the experimental group 4-the experimental group 6 is obviously higher than that of the hyaluronan stock solution of the independent experimental group 1 and the oligopeptide stock solution of the experimental group 2, and simultaneously is also obviously higher than that of the hyaluronan and oligopeptide combined stock solution of the experimental group 3, so that the substantial improvement of the moisturizing effect of the hyaluronan oligopeptide prepared by the invention is fully proved, the hyaluronan and oligopeptide are not generated by simply combining the hyaluronan and the oligopeptide, but the hyaluronan oligopeptide is generated by adopting a covalent bond coupling mode for the hyaluronan and the oligopeptide, the hyaluronan oligopeptide is fundamentally endowed with more excellent characteristics by directly carrying out chemical modification, the stability of the hyaluronan and the oligopeptide is improved, the enzymolysis resistance is improved, the half-life is prolonged, the action time is enhanced, and the moisturizing effect is remarkably improved. Particularly, in the experimental group 4-the experimental group 6, the change of the MMV value of the skin tends to rise back in 120min, so that the effect of the hyaluronan oligopeptide is fully proved to be not limited on the surface of the skin, the effect of the hyaluronan oligopeptide on water replenishing and repairing of deep cells is also remarkable, the slow reduction trend of the skin moisture is finally generated, and the long-term moisturizing effect is achieved.
2. Wrinkle removing effect:
2.1 subject: selecting 70 volunteers, wherein the age is 35-55 years old, dividing the volunteers into 7 groups, and carrying out skin experiments on 10 persons in each group;
2.2 test samples and methods of use thereof: 0.5g of each of the stock solutions of experimental group 1-experimental group 6 and blank group 7 prepared in example 9 was uniformly applied to facial skin of volunteers once a day for 4 weeks;
before the experiment, firstly, the facial skin wrinkle area S of the tested person before the experiment is measured by a Visoline VL 650 wrinkle tester0: the change of the silica gel copy membrane of the skin wrinkles is analyzed by software to obtain the change of the wrinkle area of the face skin;
in the test, the time point of 2 pm every day is respectively taken, the measurement environment temperature is 20 ℃, the relative humidity is 50%, and the facial skin wrinkle area S is measured by a Visiline VL 650 wrinkle testert: the change of the silica gel copy membrane of the skin wrinkles is analyzed by software to obtain the change of the wrinkle area of the face skin;
finally, skin wrinkle area reduction amount (%) = (skin wrinkle area before test-average skin wrinkle area per week)/skin before test was calculatedWrinkle area × 100% = (S)0-St)/S0×100％。
TABLE 3 comparison of wrinkle-removing effect of hyaluronan oligopeptide
The hyaluronic acid stock solution of experimental group 1, the oligopeptide stock solution of experimental group 2 (three oligopeptides with structures of formulas II, III and IV, respectively), the combined stock solution of hyaluronic acid and oligopeptides of experimental group 3 (three oligopeptides with structures of formulas II, III and IV, respectively), the hyaluronan oligopeptide stock solution of formula V of experimental group 4, the hyaluronan oligopeptide stock solution of formula VI of experimental group 5, the hyaluronan oligopeptide stock solution of formula VII of experimental group 6, and blank group 7, wherein the wrinkle-removing effect test data results of the oligopeptides of formulas II, III and IV of experimental group 2 and experimental group 3 are similar, so the wrinkle-removing effect test data of the oligopeptide stock solution of experimental group 2 used in table 1 are the average value of the wrinkle-removing effect test data of the oligopeptides of formula III and IV, and the wrinkle-removing effect test data of the combined stock solution of hyaluronic acid and oligopeptides of experimental group 3 are also the average value of the wrinkle-removing effect test data of the mixture of the oligopeptides of the three structures and hyaluronic acid.
As can be seen from table 2 comparing the wrinkle-removing effect of hyaluronan oligopeptide with the data in the table, the reduced amount of the skin wrinkles of the blank group 7 is almost unchanged, which proves that the wrinkles of the facial skin of a human body cannot disappear by themselves after being formed without adding a wrinkle-removing product; the wrinkle removing effect of the hyaluronan oligopeptide stock solution of the experimental group 4-the experimental group 6 is obviously higher than that of the hyaluronan stock solution of the independent experimental group 1 and the oligopeptide stock solution of the experimental group 2, and simultaneously is also obviously higher than that of the hyaluronan and oligopeptide combined stock solution of the experimental group 3, so that the significant improvement of the wrinkle removing effect of the hyaluronan oligopeptide prepared by the invention is fully proved, the hyaluronan oligopeptide is not generated by simply combining the hyaluronan and the oligopeptide, but the hyaluronan oligopeptide is generated by adopting a covalent bond coupling mode of the hyaluronan and the oligopeptide, the hyaluronan oligopeptide is fundamentally endowed with more excellent characteristics by directly carrying out chemical modification, the stability of the hyaluronan and the oligopeptide is improved, the enzymolysis resistance is improved, the half-life period is prolonged, the action time is prolonged, the hyaluronan oligopeptide has the targeting performance of the hyaluronan, the oligopeptide is effectively promoted to penetrate through a skin barrier, the absorption and the combination of the skin to the oligopeptide are enhanced, the use effect of the oligopeptide is increased, and the wrinkle removing effect is significantly improved. Preferably, the addition amount of the hyaluronan oligopeptide is 0.001-25%, so that the moisturizing and wrinkle-removing effects are most remarkable.
The chemical modification is adopted to connect the oligopeptide with the hyaluronan by a covalent bond connection mode, the obtained hyaluronan oligopeptide has the characteristics of compound activity, strong product efficacy and long efficacy duration, and the using effect of the method is superior to the effect of the hyaluronan or the oligopeptide when being used alone and the effect of the method is superior to the effect of the hyaluronan and the oligopeptide when being used together. Also provided are methods of use thereof in cosmetic and cosmetic products.
In conclusion, the invention aims at the defect that the structures of the hyaluronan and oligopeptide substances are unstable, and solves the technical problems that the hyaluronan and the oligopeptide in the functional cosmetics in the prior art need to be added respectively, are easy to be subjected to enzymolysis, have poor chemical stability, are easy to be subjected to inactivation caused by environmental influence and have short action time; the prepared hyaluronan oligopeptide has the functional activities of hyaluronan and oligopeptide, has the wrinkle-removing and anti-aging effects obviously superior to the effects of the oligopeptide and hyaluronan which are used respectively and jointly, prolongs the effect, enhances the using effect of functional cosmetics, improves the using satisfaction of users and has wide application range.
Adding amount of hyaluronan oligopeptide; the application range of the hyaluronan oligopeptide is as follows: can also be used for preparing cosmetics with sunscreen, moisture keeping, skin nutrition supplementing, antiinflammatory, antioxidant, wrinkle removing, antiaging and skin repairing effects; the cosmetic is in the form of aqueous solution, lotion, essence, gel, foundation, cream, and pack; the application range of cosmetics comprises head washing, face washing, body washing and the like, and the application method of the hyaluronan oligopeptide can be realized.
The foregoing is merely exemplary of the invention and, for example; the molecular weight of the hyaluronan is 10 kDa-100 kDa; the activation system in the condensation reaction steps (2) and (1) is as follows: a + D or A + B + C, wherein A is HOBT or HOAT, B is any one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC; the method for cracking the peptide resin comprises the following steps: the hyaluronan oligopeptide and the preparation thereof can be realized by adding 10ml of lysate into each g of peptide resin, reacting for 2-5h at 10-30 ℃, cracking the lysate and the like.
However, the above description is only an embodiment of the present invention, and the scope of the present invention should not be limited by this, and all equivalent changes and modifications made in the claims of the present invention should be covered by the present invention.
Claims (10)
1. A hyaluronan oligopeptide having a structure that is a glycopeptide having the formula V:
2. the method of claim 1, wherein the method comprises the steps of: connecting hyaluronan and oligopeptide through a covalent bond form: epsilon-NH of Lys side chains in the oligopeptide sequence2Amide bond is generated through amide condensation reaction with-COOH of the hyaluronan for connection, so as to generate hyaluronan oligopeptide;
the structure of the hyaluronan is the structure of formula I:
the structure of the oligopeptide is shown as a formula II:
3. the method of claim 2, comprising the steps of:
(1) Preparing the hyaluronan into hyaluronan-TBA;
(2) Condensation reaction:
(1) by solid phase synthesis: in the presence of an activation system and DIEA, CTC resin is used as a carrier for solid phase synthesis, activated Fmoc-amino acids are sequentially condensed from C end to N end according to the amino acid sequence of the oligopeptide formula II structure, peptide resin synthesis is carried out, and the peptide resin of which the N-terminal amino group of the oligopeptide formula II structure contains Fmoc protecting group is synthesized; the Fmoc-amino acid sequence of the synthetic oligopeptide condensed by peptide resin with a structural formula II is as follows: fmoc-Val-OH, fmoc-Ala-OH, fmoc-Val-OH, fmoc-Lys (Dde) -OH, fmoc-Ile-OH, fmoc-Ser-OH;
(2) removing the Lys side chain epsilon-NH in the Fmoc protecting group-containing peptide resin prepared in the step (1)2The protecting group Dde is used for cracking peptide resin, and oligopeptide crude peptide with the structure of oligopeptide formula II and the Fmoc protecting group contained in N-terminal amino is obtained;
(3) purifying the crude oligopeptide peptide containing the Fmoc protecting group prepared in the step (2), and freeze-drying to obtain a pure product of the oligopeptide with the structure of the oligopeptide formula II, wherein the N-terminal amino group of the oligopeptide contains the Fmoc protecting group;
(3) Activating the hyaluronan-TBA prepared in the step (1), and then carrying out condensation reaction with the pure oligopeptide prepared in the step (2); epsilon-NH of Lys side chain in the oligopeptide sequence2Amide bond is generated through amide condensation reaction with-COOH of the hyaluronan for connection, and the product hyaluronan TBA oligopeptide-Fmoc is generated;
(4) Removing Fmoc-protecting groups of N-terminal amino groups of the oligopeptides in the product prepared in the step (3) to obtain a product, namely hyaluronan TBA oligopeptides;
(5) Salt exchange: ultrafiltering and washing the product of hyaluronan TBA oligopeptide prepared in the step (4) by an ultrafiltration membrane with the molecular weight cutoff of 500Da, and converting the product of hyaluronan TBA oligopeptide into Na by 732 type cation exchange column resin+And (3) freeze-drying the salt to obtain the finished product of the hyaluronan oligopeptide with the structure of the formula V.
4. The method according to claim 3, wherein the molecular weight of the hyaluronan oligopeptide is 10 kDa-100 kDa.
5. The method of claim 3, wherein the activation system in step (2) (1) is: a + D or A + B + C, wherein A is HOBT or HOAT, B is any one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC; the method for activating the hyaluronan-TBA in the step (3) comprises the following steps: hyaluronan-TBA was reacted with HOSU in the presence of EDC for 24h.
6. The method of claim 3, wherein the Fmoc protecting group of the peptide resin obtained in step (1) is removed in step (2) (2) to remove the side chain ε -NH Lys in the peptide resin2The method for removing the protecting group Dde comprises the following steps: adding deprotection solution to the peptide resin of the Fmoc protecting group prepared in the step (2) (1) for reaction for 3h, wherein the deprotection solution is composed of hydroxylamine hydrochloride, imidazole, DCM and NMP, and the mass ratio of the hydroxylamine hydrochloride to the imidazole to the DCM to NMP is 25.
7. The method of claim 3, wherein the step (2) (2) comprises the steps of: reacting for 2-5h at 10-30 ℃ according to the proportion of adding 10ml of lysis solution into each g of peptide resin, and cracking the peptide resin; the cracking solution consists of TFE and DCM, wherein the volume ratio of TFE to DCM is 2.
8. According to the rightThe method for preparing hyaluronan oligopeptide according to claim 3, wherein the purification method in steps (2) and (3) is to purify the crude peptide of Fmoc protecting group prepared in step (2) by reversed-phase high performance liquid chromatography, and the conditions are as follows: mobile phase A is H2O, taking acetonitrile as a mobile phase B, and adopting gradient elution, wherein the elution time is 60min, the flow rate is 80ml/min, the ultraviolet detection wavelength is 220nm, and the elution gradient B is 10-40%.
9. The method of any one of claims 1-8, wherein the hyaluronan oligopeptide having the structure of formula V is used as a cosmetic component or added to a cosmetic product.
10. The method for using hyaluronan oligopeptide according to any one of claims 1-8, wherein the hyaluronan oligopeptide having formula V is used as a dermal filler to prepare a cosmetic product for facial injection.
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| WO2015050943A1 (en) * | 2013-10-04 | 2015-04-09 | The Regents Of The University Of California | Hyaluronic acid and alginate hydrogel composition |
| AU2015367377A1 (en) * | 2014-12-18 | 2017-07-20 | Galderma S.A. | Grafting of cyclodextrin by amide bonds to an ether cross-linked hyaluronic acid and uses thereof |
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| CN110713517A (en) | 2020-01-21 |
| CN115611967A (en) | 2023-01-17 |
| CN115417914A (en) | 2022-12-02 |
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