CN110713517A - Hyaluronan oligopeptide and preparation and application methods thereof - Google Patents
Hyaluronan oligopeptide and preparation and application methods thereof Download PDFInfo
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
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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 preparation and application methods 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 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 and prevent skin aging. 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 wrinkles and prevent aging; the macromolecular hyaluronic acid can form a protective film on the surface layer of the skin, can lock moisture 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, greatly increase the profits of cosmetic manufacturers, and more enterprises and research institutions invest in the research and development of the functional cosmetics.
Patent CN106109296B discloses a moisturizing essence containing sodium hyaluronate and sodium acetyl hyaluronate; CN104740643A discloses a stable hyaluronic acid solution loaded with bioactive protein or polypeptide, which needs to be added with hyaluronic acid and bioactive protein or polypeptide respectively; CN109157471A discloses a skin care composition with repair and moisture retention 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 polypeptide, 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 inactivated by enzymolysis, and meanwhile, due to the fact that the structure of the polypeptide substance is unstable and easy to denature and inactivate 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, the stability is improved, and the efficacy is prolonged.
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:
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 solid phase synthesis method, in the presence of an activation system and DIEA, using CTC resin as a solid phase synthesis carrier, sequentially condensing activated Fmoc-amino acids from C end to N end according to the amino acid sequences of the oligopeptide structures of formula II, formula III and formula IV to synthesize peptide resins, wherein the three N-end amino groups of the oligopeptide structures of formula II, formula III and formula IV contain Fmoc protecting groups, the Fmoc-amino acid sequences of the peptide resins of formula II of the synthetic oligopeptide are Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Lys (Dde) -OH, Fmoc-Ile-OH and Fmoc-Ser-OH, the Fmoc-amino acid sequences of the peptide resins of formula III of the synthetic oligopeptide are Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Lys (Dde) -OH and Fmoc-Iloc-Ser-OH, the Fmoc-peptide resins of formula IV of the synthetic oligopeptide are Fmoc-Lys-OH, Fmoc-Gly-Iloc-Ser-OH, the Fmoc-Iloc-peptide resins of formula IV of the synthetic oligopeptide are Fmoc-Gly-Lys-;
② removal of Lys side chain ε -NH from the Fmoc protecting group containing peptide resin prepared in step ①2The 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 peptide containing Fmoc protecting group prepared in step ②, and lyophilizing to obtain three pure oligopeptides with Fmoc protecting group at N-terminal amino group of oligopeptide formulas II, III and IV;
(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 group of N-terminal amino group of the oligopeptide in the product prepared in the step (3) to obtain a product of hyaluronan TBA oligopeptide;
(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 500Da), 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 100 kDa.
Preferably, the activating 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, D is DIC, and the activating method of the hyaluronan-TBA in step (3) is that the hyaluronan-TBA reacts with HOSU in the presence of EDC for 24 h.
Preferably, the peptide resin containing Fmoc protecting group prepared in step ① is removed in step (2) ② to remove the Lys side chain ε -NH2The method for removing the protecting group Dde of (2) comprises adding a deprotection solution to the Fmoc protecting group peptide resin prepared in the step (2) ① to react for 3 hours, wherein the deprotection solution is hydroxylamine hydrochloride, imidazole, DCM, NMP 25:18:16:100 (w/w/w).
Preferably, the peptide resin is cleaved in step (2) ② by adding 10ml of cleavage solution per g of peptide resin, reacting at 10-30 deg.C for 2-5h, and cleaving, wherein the cleavage solution is 20% TFE/DCM (v/v).
Preferably, the step (2) ③ is a step of purifying the crude peptide of oligopeptide containing Fmoc protecting group prepared in the step ② by reversed-phase high performance liquid chromatography with the proviso that the mobile phase A is H2And O, 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) the hyaluronan oligopeptide and the preparation and application methods thereof have the advantages that the obtained hyaluronan oligopeptide has the functional activities 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 moisturizing and wrinkle-removing effects and the anti-aging effects which are obviously superior to the effects of the oligopeptide and the hyaluronan which are respectively used and jointly used, and simultaneously has the advantages of stable chemical properties, improved enzymolysis resistance, prolonged half life, no environmental influence, obvious moisturizing, wrinkle-removing and anti-aging effects and long action time.
(2) The invention generates the hyaluronan oligopeptide by coupling the hyaluronan and the oligopeptide by adopting a covalent bond, fundamentally endows the hyaluronan oligopeptide with more excellent characteristics by directly carrying out chemical modification and modification, has simple preparation and synthesis steps and simple and easy purification, and simultaneously provides an application method of the hyaluronan oligopeptide in cosmetics and beauty products, obviously enhances the effects of moisturizing, wrinkle removing and anti-aging in the use of functional cosmetics, and obviously improves the use satisfaction 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)+) Form cation exchange column for separating Na from the solution+Conversion to H+Obtaining an eluent containing the 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 CTC resin swelled in the step (1) according to the molar mass ratio of 3:6:1, and reacting for 2h at 25 ℃ to obtain Fmoc-Val-CTC resin; 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) removing Fmoc protecting groups: adding a PIP-DMF solution with the volume fraction of 20% into the blocked Fmoc-Val-CTC resin obtained in the step (2), and carrying out Fmoc protection removal twice at the temperature 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, 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 development 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: 18:16: 100) (w/w/w) into 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 of DMF and 300ml of 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 lysate 20% TFE/DCM (v/v) into each g of peptide resin in 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 25 ℃, performing suction filtration, performing reduced pressure distillation on the filtrate, 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 3:6:1, and reacting for 2 hours at 25 ℃ to obtain Fmoc-Val-CTC resin; 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 with the volume fraction of 20% into the blocked Fmoc-Val-CTC resin obtained in the step (2), and carrying out Fmoc protection removal twice at the temperature 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 90mmol of HOBT in proper amount of DMF, adding 90mmol of DIC, and reacting at room temperature 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 and Fmoc-Ile-OH in the step (4) into the resin with the Fmoc protecting group removed, carrying out condensation reaction of amino acid for 2h at the temperature of 25 ℃, and carrying out ninhydrin color reaction to monitor the reaction process so as to finally obtain Fmoc-Ile-Lys (Dde) -Val-Ala-Val-CTC resin;
(6) and (3) removing the Dde protecting group: adding deprotection solution hydroxylamine hydrochloride, imidazole, DCM and NMP (25: 18:16: 100) (w/w/w) into the Fmoc-Ile-Lys (Dde) -Val-Ala-Val-CTC resin obtained in the step (5), reacting for 3h, washing the peptide resin three times by using 300ml of DMF and 300ml of MeOH alternately for 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: and (3) adding 10ml of 20% TFE/DCM (v/v) cracking Fmoc-Ile-Lys-Val-Ala-Val-CTC resin obtained in the step (6) into each g of peptide resin in a round-bottom flask, carrying out cracking for 2h at the temperature of 25 ℃, carrying out suction filtration, carrying out reduced pressure distillation on the filtrate, and evaporating the solvent to obtain the 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-Val-CTC resin: a) mixing Fmoc-Lys (Dde) -OH, DIEA and resin according to the molar mass ratio of 3:6:1, and reacting for 2 hours at 25 ℃ to obtain Fmoc-Val-CTC resin; 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-Val-CTC resin.
(3) Removing Fmoc protecting groups: adding a PIP-DMF solution with the volume fraction of 20% into the blocked Fmoc-Val-CTC resin obtained in the step (2), and carrying out Fmoc protection removal twice at the temperature 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 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 of deprotection solution hydroxylamine hydrochloride, imidazole, DCM, NMP, 25:18:16:100 (w/w/w), into the Fmoc-Gly-His-Lys-CTC resin obtained in the step (5), reacting for 3h, washing the peptide resin three times by using 300ml of DMF and 300ml of MeOH alternately, washing for 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 lysate 20% TFE/DCM (v/v) into each g of peptide resin in a round-bottom flask, cracking the Fmoc-Gly-His-Lys-CTC resin obtained in the step (6), reacting for 2 hours at 25 ℃, performing suction filtration, performing reduced pressure distillation on the filtrate, and evaporating the solvent to dryness to obtain the crude Fmoc-Gly-His-Lys-COOH peptide.
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;
three kinds of crude peptides are purified in one step by adopting a reversed-phase high-efficiency liquid-phase color boiling process, wherein a mobile phase A is H2And O, taking acetonitrile as a mobile phase B, and adopting gradient elution: c18 preparation of a column (50X 250mm, 10 μm) with an elution time of 60 min: 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 220 nm. 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 exists in the reaction solution so that the reaction is complete to obtain 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 finished products of the hyaluronan oligopeptides with structures of a formula V, a formula VI and a formula VII by freeze-drying.
Example 7
Preparing a finished product of hyaluronan oligopeptide:
(1) dissolving 50g of the hyaluronan-TBA prepared in example 1 in DMF, adding 34mmol of HOSU and 68mmol 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 into 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 process by adopting ninhydrin color development 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 hyaluronan oligopeptide semi-finished product solid prepared in the step (3), and passing the solid through an ultrafiltration membrane(molecular weight cut-off 500D), ultrafiltering, washing, and separating with 732 type cation exchange column to convert TBA into Na+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 8
Preparing a finished product of hyaluronan oligopeptide:
(1) dissolving 50g of the hyaluronan-TBA prepared in example 1 in DMF, adding 51mmol of HOSU and 102mmol 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 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 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 solid prepared in the step (3), performing ultrafiltration and washing by an ultrafiltration membrane (with the molecular weight cut-off of 500D), and converting TBA in the solid into Na by a 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:
at room temperature, respectively adding butanediol with the mass fraction of 4% and PE9010 with the mass fraction of 0.8% as matrixes into 7 parts of sterile water with the same amount as that of the sterile water, respectively taking the butanediol and the PE9010 as an experimental group 1-an experimental group 6 and a blank group 7, respectively adding 0.5% of hyaluronan, oligopeptides, a composition of hyaluronan and oligopeptides, three hyaluronan oligopeptide finished products with the structures of formula V, formula VI and formula VII prepared in example 8 into the experimental group 1-the experimental group 6, respectively adding sterile water with the same amount as that of the blank group 7, uniformly stirring the mixture until the hyaluronan and the oligopeptides are completely dissolved, adjusting the pH value to 5-7, respectively obtaining 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 combined stock solution of hyaluronan and oligopeptides (three oligopeptides with the structures of formula II, formula III and formula IV respectively), An experiment group 4 of the hyaluronan oligopeptide stock solution with the structure shown in the formula V, an experiment group 5 of the hyaluronan oligopeptide stock solution with the structure shown in the formula VI, an experiment group 6 of the hyaluronan oligopeptide stock solution with the structure shown in the formula VII and a blank group 7.
The properties of the hyaluronan oligopeptides of the present invention are further illustrated by the experimental reports below.
(1) 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 types of oligopeptides having structures of formulae II, III, and IV, respectively), test group 3 of the combined stock solution of hyaluronan and oligopeptides (three types of oligopeptides having structures of formulae II, III, and IV, respectively), test group 4 of the hyaluronan oligopeptide stock solution of formula V, test group 5 of the hyaluronan oligopeptide stock solution of formula VI, test group 6 of the hyaluronan oligopeptide stock solution of formula VII, and blank group 7 are shown in tables 1 to 3.
(3) Determination of using effect of hyaluronan oligopeptide:
1. the moisturizing effect is as follows:
1.1 subject: selecting 110 volunteers, wherein the age is 35-40 years old, and performing an experiment 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 the test: 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 is divided into 7 test areas with interval of 1cm, and each test area is 3 × 3cm2The amount of the test sample was 0.2 g. 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. After the subject was allowed to sit still in a constant environment (measurement environment temperature of 20 ℃ C., relative humidity of 50%,) for 30min, a Corneometer CM 825 moisture tester was used to measure the blank value of the test site, and 5 points were fixed and measured in each area in a certain order 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.
Average moisture content growth rate%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 ℃ and relative humidity of 50%).
TABLE 1 Corneometer CM 825 moisture tester test empirical data
| Arms, hands, legs, etc | |
| The skin is drier | ＜35 |
| The skin is drier | 35-50 |
| The skin has sufficient moisture | ＞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 table 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 groups 4 to 6, the change of the MMV value of the skin tends to rise again in 120min, so that the effect of the hyaluronan oligopeptide is fully proved to be not limited on the surface of the skin, the hyaluronan oligopeptide also has a remarkable effect on the water replenishing and repairing of deep cells, and finally the slow reduction of the skin moisture is kept, so that 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 silicone gel of the skin wrinkles replicates the changes in the patch,analyzing by software to obtain the change of the wrinkle area of the facial 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, the skin wrinkle area reduction amount (%) was calculated as (skin wrinkle area before test-average skin wrinkle area per week)/skin wrinkle area before test × 100%0-St)/S0×100％。
TABLE 3 comparison of wrinkle-removing Effect of hyaluronan oligopeptide
The hyaluronic acid stock solution of experiment group 1, the oligopeptide stock solution of experiment group 2 (three oligopeptides with structures of formulas II, III and IV, respectively), the hyaluronan and oligopeptide (three oligopeptides with structures of formulas II, III and IV, respectively) combined stock solution of experiment group 3, the hyaluronan oligopeptide stock solution of formula V of experiment group 4, the hyaluronan oligopeptide stock solution of formula VI of experiment group 5, the hyaluronan oligopeptide stock solution of formula VII of experiment group 6, and blank group 7, wherein the wrinkle-removing effect test data results of the oligopeptides with structures of formulas II, III and IV in experiment groups 2 and 3 are similar, so the wrinkle-removing effect test data of the oligopeptide stock solution of experiment group 2 used in table 1 are the average value of the wrinkle-removing effect test data of the oligopeptides of three structures, and the wrinkle-removing effect test data of the hyaluronan and oligopeptide combined stock solution of experiment group 3 are also the average value of the wrinkle-removing effect test data of the mixed of three structures and hyaluronan Average value of (a).
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 by 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 prolonged, and the hyaluronan oligopeptide has the targeting property of the hyaluronan and effectively promotes the penetration of the oligopeptide into the skin barrier, the absorption and combination of the skin to the oligopeptides are enhanced, the using effect of the oligopeptides is increased, and the wrinkle removing effect of the oligopeptides is obviously 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, the wrinkle-removing and anti-aging effects of the hyaluronan oligopeptide are obviously superior to the effects of separate use and combined use of the oligopeptide and hyaluronan, the effect is prolonged, the use effect of functional cosmetics is enhanced, the use satisfaction of users is improved, and the application range is wide.
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 the cosmetics comprises head washing, face washing, body washing and the like, and the application method of the hyaluronan oligopeptide can be realized.
The above are only examples of the present invention, and for example, the molecular weight of hyaluronan is 10kDa to 100kDa, the activation system in the condensation reaction step (2) ① is A + D or A + B + C, wherein A is HOBT or HOAT, B is HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC, and the method for cleaving peptide resin comprises reacting the peptide resin at 10ml of lysate per g of peptide resin at 10-30 ℃ for 2-5h, cleaving the peptide resin, and the like, thereby obtaining the hyaluronan oligopeptide and the preparation thereof.
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. An hyaluronan oligopeptide having the structure of a glycopeptide having the structure of formula V, formula VI and formula VII, respectively:
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 chain in the oligopeptide sequence2with-COOH of hyaluronan by amidePerforming condensation reaction to generate amido bond for connection to generate hyaluronan oligopeptide;
the structure of the hyaluronan is the structure of formula I:
the structure of the oligopeptide is respectively the structure of formula II, formula III and formula IV:
3. the method of claim 2, wherein the method comprises the steps of:
(1) preparing the hyaluronan into hyaluronan-TBA;
(2) condensation reaction:
① solid phase synthesis method, in the presence of an activation system and DIEA, using CTC resin as a solid phase synthesis carrier, sequentially condensing activated Fmoc-amino acids from C end to N end according to the amino acid sequences of the oligopeptide structures of formula II, formula III and formula IV to synthesize peptide resins, wherein the three N-end amino groups of the oligopeptide structures of formula II, formula III and formula IV contain Fmoc protecting groups, the Fmoc-amino acid sequences of the peptide resins of formula II of the synthetic oligopeptide are Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Lys (Dde) -OH, Fmoc-Ile-OH and Fmoc-Ser-OH, the Fmoc-amino acid sequences of the peptide resins of formula III of the synthetic oligopeptide are Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Lys (Dde) -OH and Fmoc-Iloc-Ser-OH, the Fmoc-peptide resins of formula IV of the synthetic oligopeptide are Fmoc-Lys-OH, Fmoc-Gly-Iloc-Ser-OH, the Fmoc-Iloc-peptide resins of formula IV of the synthetic oligopeptide are Fmoc-Gly-Lys-;
② removal of Lys side chain ε -NH from the Fmoc protecting group containing peptide resin prepared in step ①2The protective group Dde and the cracked peptide resin respectively obtain three N-terminal amino groups of oligopeptide structures shown in formula II, formula III and formula IV and contain Fmoc protectionOligopeptide crude peptide;
③ purifying the crude peptide containing Fmoc protecting group prepared in step ②, and lyophilizing to obtain three pure oligopeptides with Fmoc protecting group at N-terminal amino group of oligopeptide formulas II, III and IV;
(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 group of N-terminal amino group of the oligopeptide in the product prepared in the step (3) to obtain a product of hyaluronan TBA oligopeptide;
(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 500Da), 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.
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 activating system in step (2) ① is A + D or A + B + C, wherein A is HOBT or HOAT, B is HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, D is DIC, and the activating method of the hyaluronan-TBA in step (3) is reacting the hyaluronan-TBA with HOSU in the presence of EDC for 24 h.
6. The method of claim 3, wherein the peptide resin obtained by removing Fmoc protecting groups in step ① in step (2) ② has Lys side chain ε -NH2To ensureThe method for removing the protecting group Dde is to add a deprotection solution, hydroxylamine hydrochloride, imidazole, DCM and NMP (25: 18:16: 100) (w/w/w), to the peptide resin of the Fmoc protecting group prepared in the step (2) ① for reaction for 3 h.
7. The method of claim 3, wherein the step (2) ② comprises the step of cracking the peptide resin by adding 10ml of cracking solution per g of peptide resin at 10-30 deg.C for 2-5h, wherein the cracking solution is 20% TFE/DCM (v/v).
8. The method of claim 3, wherein the step (2) ③ is carried out by purifying crude peptide of Fmoc-protecting group prepared in step ② by reverse phase high performance liquid chromatography, with the proviso that phase A is H2And O, 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%.
9. The method of using a hyaluronan oligopeptide according to any one of claims 1-8, wherein any one, or any two, or three of the three hyaluronan oligopeptides having the structures of formula V, formula VI and formula VII are used as a cosmetic ingredient or added to a cosmetic.
10. The method of using a hyaluronan oligopeptide according to any one of claims 1-8, wherein any one, or any two, or three of the three hyaluronan oligopeptides having the structures of formula V, formula VI and formula VII are applied as dermal fillers to a cosmetic product for facial injection.
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| CN113402628A (en) * | 2021-06-07 | 2021-09-17 | 上海应用技术大学 | Amphiphilic high-bioactivity modified hyaluronic acid |
| CN115215948A (en) * | 2022-06-02 | 2022-10-21 | 润辉生物技术（威海）有限公司 | Low-molecular hyaluronan decarboxylated carnosine derivative, and preparation method and application thereof |
| CN117050146A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modified cosmetic peptide, preparation method and application thereof |
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| WO2018184516A1 (en) * | 2017-04-06 | 2018-10-11 | 湖南御家化妆品制造有限公司 | Moisturizing composition and application thereof in preparation of moisturizing cosmetic product |
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2019
- 2019-10-11 CN CN201910963468.3A patent/CN110713517B/en active Active
- 2019-10-11 CN CN202211211581.4A patent/CN115417914A/en active Pending
- 2019-10-11 CN CN202211211558.5A patent/CN115611967A/en active Pending
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| US20110195897A1 (en) * | 2008-06-17 | 2011-08-11 | Otsuka Chemical Co., Ltd. | Glycosylated glp-1 peptide |
| CN104995303A (en) * | 2012-12-18 | 2015-10-21 | 诺华股份有限公司 | Compositions and methods that utilize a peptide tag that binds to hyaluronan |
| 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 |
| WO2017216177A1 (en) * | 2016-06-14 | 2017-12-21 | Sederma | Peptide, composition comprising said peptide and uses thereof, in particular cosmetic uses |
| WO2018113802A1 (en) * | 2016-12-22 | 2018-06-28 | Contipro A.S. | Medical preparation with a carrier based on water insoluble hyaluronan conjugated to amino acids or peptides, method of preparation and use thereof |
| WO2018184516A1 (en) * | 2017-04-06 | 2018-10-11 | 湖南御家化妆品制造有限公司 | Moisturizing composition and application thereof in preparation of moisturizing cosmetic product |
| CN107261124A (en) * | 2017-06-09 | 2017-10-20 | 山东大学 | A kind of preparation method and the application of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113402628A (en) * | 2021-06-07 | 2021-09-17 | 上海应用技术大学 | Amphiphilic high-bioactivity modified hyaluronic acid |
| CN115215948A (en) * | 2022-06-02 | 2022-10-21 | 润辉生物技术（威海）有限公司 | Low-molecular hyaluronan decarboxylated carnosine derivative, and preparation method and application thereof |
| CN115215948B (en) * | 2022-06-02 | 2023-07-11 | 润辉生物技术（威海）有限公司 | Low-molecular hyaluronan decarboxylated carnosine derivative, preparation method and application |
| CN117050146A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modified cosmetic peptide, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110713517B (en) | 2022-11-01 |
| CN115611967A (en) | 2023-01-17 |
| CN115417914A (en) | 2022-12-02 |
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