CN117304368A - Composition of sodium hyaluronate and collagen pentapeptide and application thereof - Google Patents
Composition of sodium hyaluronate and collagen pentapeptide and application thereof Download PDFInfo
- Publication number
- CN117304368A CN117304368A CN202311606423.3A CN202311606423A CN117304368A CN 117304368 A CN117304368 A CN 117304368A CN 202311606423 A CN202311606423 A CN 202311606423A CN 117304368 A CN117304368 A CN 117304368A
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- Prior art keywords
- sodium hyaluronate
- composition
- collagen
- derivative
- thousand
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- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical group [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 104
- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 69
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 69
- 102000008186 Collagen Human genes 0.000 title claims abstract description 49
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- 229920001436 collagen Polymers 0.000 title claims abstract description 49
- 239000000203 mixture Substances 0.000 title claims abstract description 43
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- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims description 2
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 2
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 34
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- NHGXDBSUJJNIRV-UHFFFAOYSA-M tetrabutylammonium chloride Chemical compound [Cl-].CCCC[N+](CCCC)(CCCC)CCCC NHGXDBSUJJNIRV-UHFFFAOYSA-M 0.000 description 10
- AGYUOJIYYGGHKV-UHFFFAOYSA-N 1,2-bis(2-chloroethoxy)ethane Chemical compound ClCCOCCOCCCl AGYUOJIYYGGHKV-UHFFFAOYSA-N 0.000 description 9
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- SJXHSFSHNKFRLN-UHFFFAOYSA-N 5-chlorosulfonyl-2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC(S(Cl)(=O)=O)=CC=C1O SJXHSFSHNKFRLN-UHFFFAOYSA-N 0.000 description 8
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Classifications
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- 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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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
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- A—HUMAN NECESSITIES
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- 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
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a composition of sodium hyaluronate and collagen pentapeptide and application thereof, and relates to the technical field of polypeptide medical cosmetology cosmetics. The composition comprises sodium hyaluronate or sodium hyaluronate derivatives; the sodium hyaluronate derivative comprises a sodium hyaluronate cross-linked derivative; the crosslinked derivative comprises a mixture crosslinked material of one or more of sodium hyaluronate with single-chain molecular weight of 1-8 ten thousand, 8-15 ten thousand, 15-30 ten thousand, 30-80 ten thousand and 80-200 ten thousand. A composition of sodium hyaluronate and collagen pentapeptide and its application, which combines sodium hyaluronate and collagen pentapeptide, has good ability of stimulating collagen regeneration and produces synergistic effect; and meanwhile, auxiliary mannitol or derivatives thereof are added, so that the collagen regeneration stimulating capability of the composition is further improved.
Description
Technical Field
The invention belongs to the technical field of polypeptide medical beauty cosmetics, and particularly relates to a composition of sodium hyaluronate and collagen pentapeptide and application thereof.
Background
Hyaluronic Acid (HA) consists of a repeating polymeric disaccharide of D-glucuronic acid and N-acetyl-D-glucosamine. Its structural stability depends on the stereochemistry of the disaccharide. The natural abundance, degradability and biocompatibility of HA in animals and humans makes it versatile in regulating various biological processes and maintaining homeostasis, such as skin repair, cancer diagnosis, wound healing, tissue regeneration, anti-inflammatory and immunomodulation. Hyaluronic acid has the ability to capture about 1000 times its weight of water, and its excellent ability to replenish water makes it one of the main ingredients of medical and cosmetic products. In addition, higher concentrations of hyaluronic acid molecules can become entangled, forming a continuous porous network. Such a network structure generates expansion pressure due to the increase of mutual repulsion between molecules and inside the molecules, in an effort to make the self structure relatively loose. When external pressure is applied to the hyaluronic acid network, the molecular network contracts, and when external pressure is withdrawn, the hyaluronic acid network will spring back to its original shape, or acquire a new shape, due to the internal expansion pressure. Because of its remarkable hydrodynamic properties, it can play an important role in the hydration and biomechanical integrity of the dermis when used for facial injection, providing elasticity and extensibility to facial skin.
Collagen pentapeptides are also known as pentapeptides-3 or pentapeptides-4, and the amino acid sequence is lysine-threonine-lysine-serine (KTTKS). Because of its ability to enhance skin remodeling by triggering cellular processes, it can inhibit collagenase activity and increase extracellular matrix (ECM) content, etc., are considered "signal peptides". KTTKS is a subfragment of type I collagen, and was first discovered by a group of researchers at the university of tennessee in 1993 to be the smallest sequence that can retain 80% of the original activity of the large number of parent peptides required to stimulate ECM synthesis. Stimulation with KTTKS is specific for type I collagen expression.
Polypeptides including collagen pentapeptides are generally unsuitable for systemic delivery because they are susceptible to extensive proteolytic degradation, and to extreme pH conditions (stomach). Topical application provides a more direct route to the site of action, and the metabolic processes of the route are greatly reduced, resulting in higher bioavailability. However, their passive penetration and penetration potential on the skin need to be considered when formulating collagen pentapeptides into topical compositions. First, KTTKS is extremely difficult to be absorbed and distributed by the stratum corneum due to its hydrophilic nature. Second, KTTKS is highly likely to interact significantly with proteolytic enzymes during slow diffusion, even if a small fraction diffuses through the stratum corneum. The two above-mentioned points add difficulty to their widespread use. Collagen pentapeptides are usually present in cosmetics in the form of lipophilic modification, and the skin transmittance is increased by modification of palmitoyl groups, but the effective utilization rate of the dermis layer can be only 3%, and the introduction of palmitoyl groups makes the collagen pentapeptides unavailable in conventional water-soluble skin products, and limits the expansion of the application thereof.
Disclosure of Invention
The invention aims to provide a composition of sodium hyaluronate and collagen pentapeptide and application thereof, wherein the composition combines the sodium hyaluronate and the collagen pentapeptide for application, has good collagen regeneration stimulating capability and generates synergistic enhancement effect; mannitol or its derivative is added simultaneously to further promote the collagen regeneration stimulating capacity of the composition.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a sodium hyaluronate derivative comprising a cross-linked sodium hyaluronate derivative; the crosslinked derivative comprises a mixture crosslinked material of one or more of sodium hyaluronate with single-chain molecular weight of 1-8 ten thousand, 8-15 ten thousand, 15-30 ten thousand, 30-80 ten thousand and 80-200 ten thousand.
Specifically, the crosslinking mode of the sodium hyaluronate crosslinking derivative can be selected from the physical crosslinking modes such as traditional thermal crosslinking, freeze thawing crosslinking, electron beam crosslinking, irradiation crosslinking and the like.
A composition comprising the non-crosslinked sodium hyaluronate or crosslinked sodium hyaluronate described above. The invention firstly uses the sodium hyaluronate and the collagen pentapeptide in a combined way to obtain a composition product, and exploits one of core components in the product: the novel application scene of the collagen pentapeptide provides a way that the bioavailability is far higher than that of the traditional external application, HA and KTTKS are combined and applied for systematic research, and the combination of the HA and the KTTKS proves that the HA and the KTTKS have good collagen regeneration stimulating capability and better effect when being combined for use, and the synergic enhancement effect is generated; the composition product can be applied to the fields of subcutaneous implantation, water light, microneedle introduction and the like; and further defines the ultimate biosafety concentration of the collagen pentapeptide, thereby providing guarantee for the safety and effectiveness of the product.
Specifically, the composition further comprises collagen pentapeptide (KTTKS).
Specifically, the composition also comprises auxiliary ingredients including one or more of mannitol, sorbitol and dextran; the use concentration of the auxiliary components is 0.5-10wt%. The invention provides a technical scheme for effectively proportioning sodium hyaluronate, polypeptide and auxiliary materials, which is favorable for skin rejuvenation and has excellent effect. On the premise of taking HA+KTTKS as a main body, mannitol and the like are added as auxiliary components, and the important point is that the auxiliary materials have anti-inflammatory and free radical adsorption capacities, so that an important auxiliary effect is achieved, the action effect of the composition is obviously improved, the expression of TGF-beta is better promoted, the generation of collagen is further promoted, the better MMP-1 inhibition effect is achieved, and the collagen regeneration stimulation capacity is obviously enhanced.
More preferably, mannitol derivatives may be used in the above composition instead of mannitol.
The mannitol derivatives include products of 5-chlorosulfonyl-2-hydroxybenzoic acid modified mannitol. The mannitol derivative prepared by adopting the 5-chlorosulfonyl-2-hydroxybenzoic acid modified mannitol shows better bioactivity, has stronger oxidation resistance and remarkably improves the adsorption removal capability of free radicals; the auxiliary components are added into the composition for compounding, so that better synergistic enhancement effect is achieved, TGF-beta expression is further promoted, generation of type I and type III collagen is further promoted, and MMP-1 inhibition effect is improved.
The preparation method of the mannitol derivative comprises the following steps: mixing 5-chlorosulfonyl-2-hydroxybenzoic acid with mannitol to react and obtain mannitol derivative.
Further specifically, the method for producing the mannitol derivative comprises:
and adding dry pyridine into 5-chlorosulfonyl-2-hydroxybenzoic acid, stirring for dissolving, slowly adding D-mannitol under ice-water bath, stirring for 2-4 hours, standing overnight, adding 1/3 volume of cold water, stirring for 2-4 hours at normal temperature, adding into ice water containing concentrated hydrochloric acid while stirring, carrying out vacuum filtration, washing with absolute ethanol to neutrality, dehydrating, recrystallizing with absolute ethanol, and carrying out vacuum drying to obtain mannitol derivatives.
Specifically, the solid-to-liquid ratio of 5-chlorosulfonyl-2-hydroxybenzoic acid to pyridine is 0.5-0.7 g:1mL; the molar ratio of the D-mannitol to the 5-chlorosulfonyl-2-hydroxybenzoic acid is 1:6-8; the volume ratio of the ice water to the pyridine is 2.5-3.5:1; the volume ratio of the concentrated hydrochloric acid to the ice water is 1:2-3.
More preferably, a sodium hyaluronate derivative comprises an esterified derivative, the chemical structure of which takes a sodium hyaluronate chain as a main chain structure, and the chemical grafting modification comprises chlorine-containing compounds;
the chlorine-containing compound comprises at least one of N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine and 1, 2-bis (2-chloroethoxy) ethane.
Specifically, the chlorine-containing compound is attached to the sodium hyaluronate chain structure by a chemical reaction of chlorine in its structure with carboxyl groups in the sodium hyaluronate structure.
Specifically, the method for producing a hyaluronic acid derivative comprises: mixing N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine and/or 1, 2-di (2-chloroethoxy) ethane with sodium hyaluronate, and reacting under the condition of tetrabutylammonium chloride to obtain the hyaluronic acid derivative.
Further specifically, the preparation method of the sodium hyaluronate derivative comprises the following steps:
and (3) taking sodium hyaluronate, N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine and 1, 2-bis (2-chloroethoxy) ethane, adding tetrabutylammonium chloride and N, N-dimethyl diamide, stirring and mixing uniformly, heating to 60-70 ℃, stirring and reacting for 2-3 days, recovering N, N-dimethyl diamide under reduced pressure, adding water, regulating the pH to 3-4, precipitating, filtering and vacuum drying to obtain sodium hyaluronate derivatives.
Specifically, the mass ratio of the sodium hyaluronate to the N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine is 1:0.1-0.2; the molar ratio of the N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine to the 1, 2-di (2-chloroethoxy) ethane is 1:0.4-0.6; the mass ratio of tetrabutyl ammonium chloride to sodium hyaluronate is 1:0.3-0.4.
Specifically, the solid-to-liquid ratio of sodium hyaluronate to N, N-dimethyl diamide is 0.2-0.35 g/1 mL; the volume ratio of water to N, N-dimethyl diamide is 1.4-1.8:1.
The invention also discloses application of the sodium hyaluronate derivative in preparing medical cosmetic filler, cosmetics or skin care products or medicaments.
The invention also discloses application of the composition in preparing medical cosmetic filler, cosmetics or skin care products or medicaments.
An aqueous product comprising the above composition.
Specifically, the concentration of sodium hyaluronate or sodium hyaluronate derivatives in the aqueous solution product is 0.2-1 wt%; preferably 0.5 to 1wt%.
Specifically, the concentration of the collagen pentapeptide in the aqueous solution product is 2-2000 ppm, preferably 20-500 ppm; more preferably 50 to 100ppm.
A powder product comprising the above composition.
Specifically, sodium hyaluronate or sodium hyaluronate crosslinked derivatives in the powder product are dissolved in water when in use, and the concentration is 0.2-1.5wt%.
Specifically, the concentration of the collagen pentapeptide in the powder product is 2-2000 ppm after the collagen pentapeptide is dissolved in water during use.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses the combination of the sodium hyaluronate and the collagen pentapeptide to obtain a composition product, has better effect when being used in combination, generates the synergistic enhancement effect, obviously enhances the collagen regeneration stimulation capability and increases the MMP-1 inhibition effect. Meanwhile, the invention provides a technical scheme of effective proportion of sodium hyaluronate, polypeptide and mannitol, which is favorable for skin rejuvenation and has excellent effect; the mannitol derivative is prepared by adopting the 5-chlorosulfonyl-2-hydroxybenzoic acid modified mannitol, the mannitol derivative has better antioxidation capability, and is added into the composition as an auxiliary component, and each component is compounded for use, so that better synergistic enhancement effect is achieved, the expression of TGF-beta is further promoted, the generation of collagen is further promoted, the MMP-1 inhibition effect is increased, and the collagen regeneration stimulation capability is obviously enhanced.
Therefore, the invention provides a composition of sodium hyaluronate and collagen pentapeptide and application thereof, wherein the sodium hyaluronate and the collagen pentapeptide are combined and applied, so that the composition has good collagen regeneration stimulating capability and synergistic enhancement effect; mannitol or its derivative is added simultaneously to further promote the collagen regeneration stimulating capacity of the composition.
Drawings
FIG. 1 shows the cytotoxicity test results in the examples of the present invention;
FIG. 2 is a graph showing the relative expression of the related anti-aging genes measured after the treatment of samples of collagen pentapeptide aqueous solution with different concentrations according to the embodiment of the present invention;
FIG. 3 is a graph showing the relative expression of the genes involved in determining the relative expression of the genes involved in the present invention after the treatment of aqueous HA solutions of different concentrations;
FIG. 4 is an infrared test result of sodium hyaluronate derivatives and sodium hyaluronate in an example of the present invention;
FIG. 5 shows the results of an infrared test of mannitol derivatives in the examples of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following describes in detail various embodiments of the present invention with reference to the embodiments. However, those of ordinary skill in the art will understand that in various embodiments of the present invention, numerous technical details have been set forth in order to provide a better understanding of the present application. However, the technical solutions claimed in the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments.
Example 1:
an aqueous product comprising sodium hyaluronate and collagen pentapeptide, formulated in a series of concentrations according to table 1:
TABLE 1 sample concentration ratios
Product cytotoxicity assays
The MTT method is used for testing cytotoxicity, HA is mixed with DMSO solvent containing bovine embryo serum to be used as a liquid culture medium, and the HA concentration in the culture medium simulates the HA concentration in a product.
The test results are shown in FIG. 1. From the analysis in the figure, three concentrations of 0.2%, 0.6% and 1.2% of sodium hyaluronate are selected and respectively added with KTTKS with different concentrations, and the obtained solution has no cytotoxicity reaction.
Example 2:
exploration of synergistic action mechanism of HA+KTTKS
The present study uses the QuantiGene Plex (QGP) method based on the Luminex200 suspension chip multiplex detection system, a detection method that incorporates branched DNA technology that directly measures RNA transcripts by signal amplification. The Luminex200 suspension chip multiplex detection system combines microsphere double fluorescent markers and liquid flow dispersion laser automatic detection technology, and can realize multichannel high-flux analysis of nucleic acid, antibodies and the like.
In this experiment, a total of 5 samples were designed to act on aged human fibroblasts (HFF 50) of 50 years old, as a control group, and the specific list is shown in Table 2 below:
TABLE 2 grouping of samples
Meanwhile, 6 year old human fibroblasts (HFF 6) were provided as an additional verification to approximately determine the regulatory range of the anti-aging gene expression.
Analysis of test results:
FIG. 2 shows the relative expression of the relevant anti-aging gene (TGF-. Beta.as transforming factor-. Beta.; COL1α1 is type I collagen gene) as determined by the QuantiGene Plex (QGP) assay after treatment of 50 year old human fibroblasts with samples of collagen pentapeptides at various concentrations. From the analysis in the figure, the stimulation effect of KTTKS on type I collagen is mainly related to a biosynthesis pathway, and the synthesis of type I collagen is further promoted by promoting the up-regulation of the expression of transforming growth factor beta (TGF-beta); while maintaining mRNA stability during the relevant process.
FIG. 3 shows the relative expression of the relevant anti-aging genes (TGF-. Beta.as the growth factor. Beta.; MMP1 as matrix metalloproteinase 1; COL1α1 as collagen type I gene; COL3α1 as collagen type III gene) as determined by the QuantiGene Plex (QGP) assay after treatment of 50 year old human fibroblasts with different HA samples. From the analysis in the figure, the expression of the growth conversion factor beta (TGF-beta) increases with the increase of the concentration of sodium hyaluronate, and the dose-dependent relationship is shown, while the introduction of KTTKS (100 ppm) has a strong promotion effect on the expression of TGF-beta, and the gain effect of the TGF-beta is not the superposition of the simple two effects. Therefore, HA and KTTKS jointly promote the expression of TGF-beta, further promote the generation of type I and type III collagen, and have synergistic promotion effect between HA and KTTKS.
Meanwhile, KTTKS HAs no obvious effect of inhibiting matrix metalloproteinase (MMP-1), and HAs excellent MMP-1 inhibition effect when combined with HA, thus proving that the KTTKS and the HA have synergistic effect. One target for exogenous aging factors is the dermal extracellular matrix produced by dermal fibroblasts. Ultraviolet light (UVB) activates proteolytic cleavage of dermal collagen fibrils by up-regulating the base MMP-1. As a result of collagen degradation, ECM networks are disrupted despite de novo collagen synthesis and repair, small defects remain in the ECM and accumulate over time. Therefore, the inhibition of MMP-1 can slow down the degradation rate of extracellular matrix and further promote the regeneration of collagen
In conclusion, the HA and the KTTKS are combined and applied for the first time and are subjected to systematic research, so that the HA and the KTTKS have good collagen regeneration stimulation capability, and the effect is better when the HA and the KTTKS are combined.
Example 3:
preparation of sodium hyaluronate crosslinked derivatives
Dissolving sodium hyaluronate (commercially available) with molecular weight of 8-15 ten thousand Da and sodium hyaluronate (commercially available) with molecular weight of 80-200 ten thousand Da in water, stirring and mixing uniformly at normal temperature, heating to 80 ℃, stirring for 1h, adding citric acid to adjust pH to 4.5, transferring into a high-pressure reaction kettle, reacting at 180 ℃ for 2h, cooling to 80 ℃, stirring for 10min, transferring into the high-pressure reaction kettle again, staying at 120 ℃ for 6h, cooling to normal temperature, and forming the partially physically crosslinked sodium hyaluronate 1. In the specific preparation process, the molar ratio of HA with the molecular weight of 8-15 ten thousand Da to HA with the molecular weight of 80-200 ten thousand Da is 3:1, the total concentration of sodium hyaluronate is 1.5wt%.
The preparation of the sodium hyaluronate crosslinked material 2 differs from the sodium hyaluronate crosslinked material 1 in that: in the preparation process, the molecular weight of 80-200 ten thousand Da is changed into 15-80 ten thousand Da (purchased in market);
the preparation of sodium hyaluronate crosslinker 3 differs from that of sodium hyaluronate crosslinker 1 in that: in the preparation process, the molecular weight of 80-200 ten thousand Da is changed into 1-8 ten thousand Da (commercially available).
Example 4:
synergistic effect of series of sodium hyaluronate crosslinked derivatives and KTTKS
The specific test method is the same as in example 2. The test sample M1 was: sodium hyaluronate crosslinker 1 (prepared in example 3) at a concentration of 1.5% + KTTKS100ppm; test sample M2 was: sodium hyaluronate crosslinker 2 (prepared in example 3) at a concentration of 1.5% + KTTKS100ppm; test sample M3 (prepared in example 3) was: sodium hyaluronate cross-linked 3 concentration 1.5% + KTTKS100ppm; test sample M4 was: sodium hyaluronate crosslinker 1 concentration 1.5%; test sample M5 was: sodium hyaluronate crosslinker 2 concentration 1.5%; test sample M6 was: the concentration of the sodium hyaluronate cross-linked material 3 was 1.5%.
The test results are shown in table 3:
TABLE 3 results of relative Gene expression level test
From the analysis of the data in Table 3, it was found that the composition of crosslinked sodium hyaluronate and KTTKS prepared in this example, in which the composition of low crosslinking degree, medium crosslinking degree and high crosslinking degree was obtained according to the change in molecular weight, and that the composition of sodium hyaluronate M4 of high crosslinking degree and the composition of sodium hyaluronate M1 and KTTKS showed the highest relative expression of TGF-. Beta., COL1α1 and COL3α1 genes, showed that sodium hyaluronate for filling effect, which produced appropriate foreign body reaction, was able to stimulate collagen regeneration through TGF-. Beta.signaling pathway. Under the same group of conditions, the introduction of KTTKS can further improve the forward gain effect. The relative expression of MMP1 genes was less different between the same groups, but the expression of MMP1 was reduced and the degree of aging was reduced compared to crosslinked HA alone and compositions containing KTTKS.
Product cytotoxicity assays
The test method was the same as in example 1. From the analysis of the test results, the test sample solution obtained in this example showed no cytotoxic reaction.
Example 5:
synergistic effect of sodium hyaluronate derivative and KTTKS
The specific test method is the same as in example 2. The test sample M1 was: the concentration of the sodium hyaluronate derivative 1 prepared in the example is 0.6% + KTTKS100ppm; test sample M2 was: the concentration of the sodium hyaluronate derivative 2 prepared in the example is 0.6% + KTTKS100ppm; test sample M3 was: the concentration of the sodium hyaluronate derivative 3 prepared in this example was 0.6% + KTTKS100ppm.
Preparation of sodium hyaluronate derivative 1:
sodium hyaluronate (20-40 ten thousand molecular weight, commercially available), N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine and 1, 2-di (2-chloroethoxy) ethane are taken, tetrabutylammonium chloride and N, N-dimethyl diamide are added, stirred and mixed uniformly, the temperature is raised to 68 ℃, the stirring reaction is carried out for 2.5 days, the N, N-dimethyl diamide is recovered under reduced pressure, water is added, the pH is regulated to 3.6, precipitation is separated out, and the sodium hyaluronate derivative 1 is obtained through filtration and vacuum drying. In the specific preparation process, the mass ratio of the sodium hyaluronate to the N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine is 1:0.16; the molar ratio of N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine to 1, 2-bis (2-chloroethoxy) ethane is 1:0.51; the mass ratio of tetrabutyl ammonium chloride to sodium hyaluronate is 1:0.35; the solid-to-liquid ratio of sodium hyaluronate to N, N-dimethylformamide was 0.28g:1mL; the volume ratio of water to N, N-dimethylformamide was 1.6:1.
The preparation of sodium hyaluronate derivative 2 differs from sodium hyaluronate derivative 1 in that: in the preparation process, 1, 2-bis (2-chloroethoxy) ethane is replaced by N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine with equal molar weight.
The preparation of sodium hyaluronate derivative 3 differs from sodium hyaluronate derivative 1 in that: in the preparation process, 1, 2-bis (2-chloroethoxy) ethane with equal molar weight is adopted to replace N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine.
Analysis of results:
the sodium hyaluronate derivative prepared by the embodiment is used together with KTTKS, the relative gene expression quantity of TGF-beta, COL1 alpha 1 and COL3 alpha 1 is obviously higher than that of sodium hyaluronate and KTTKS, the relative gene expression quantity of MMO1 is obviously lower than that of sodium hyaluronate and KTTKS, the effect of a test sample M1 is obviously better than that of a test sample M2 and a test sample M3, the sodium hyaluronate is subjected to chemical modification by using N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine to obtain the sodium hyaluronate derivative, and the sodium hyaluronate derivative is compounded with KTTKS, so that the compounding effect of the sodium hyaluronate derivative can be further enhanced, the expression of TGF-beta is better promoted, the generation of type I collagen and type III collagen is further promoted, and the MMP-1 inhibition effect is better; and under the condition that 1, 2-di (2-chloroethoxy) ethane and N1-gamma-chloropropyl-N4-beta-hydroxyethyl piperazine modified sodium hyaluronate are used simultaneously, the two have synergistic enhancement function, and the collagen regeneration stimulation capability is obviously enhanced.
Product cytotoxicity assays
The test method was the same as in example 1. From the analysis of the test results, the test sample solution obtained in this example showed no cytotoxic reaction.
Example 6:
investigation of the Co-use conditions of HA+KTTKS+mannitol or mannitol derivatives
The specific test method is the same as in example 2. Test sample N1 was: HA concentration 0.6% + KTTKS100ppm; +mannitol 1%; test sample N2 was: HA concentration 0.6% + KTTKS100ppm; +mannitol derivative 1%.
Preparation of mannitol derivatives:
adding dry pyridine into 5-chlorosulfonyl-2-hydroxybenzoic acid according to the solid-to-liquid ratio of 0.6g to 1mL, stirring for dissolving, slowly adding D-mannitol (the molar ratio of the D-mannitol to the 5-chlorosulfonyl-2-hydroxybenzoic acid is 1 to 7.2) under an ice water bath, stirring for 3 hours, standing overnight, adding 1/3 volume of cold water, stirring at normal temperature for 3 hours, adding into ice water containing concentrated hydrochloric acid (the volume ratio of the concentrated hydrochloric acid to the ice water is 1 to 2.5, the volume ratio of the ice water to the pyridine is 3 to 1) while stirring, carrying out vacuum suction filtration, washing with absolute ethanol to be neutral, dehydrating, recrystallizing with absolute ethanol, and carrying out vacuum drying to obtain the mannitol derivative.
The test results are shown in table 4:
TABLE 4 results of relative Gene expression level test
As shown by the data analysis in Table 4, the relative expression of the genes of TGF-beta, COL1 alpha 1 and COL3 alpha 1 of the test samples N1 and N2 is obviously higher than that of HA+KTTKS, the relative expression of the genes of MMO1 is obviously lower than that of sodium hyaluronate+KTTKS, and meanwhile, the effect of the test sample N2 is obviously better than that of the test sample N1, and the mannitol or the derivative thereof is compounded with HA and KTTKS to play a role in enhancing, so that the expression of TGF-beta is further promoted, the generation of I-type and III-type collagen is further promoted, and the MMP-1 inhibition effect is better; and the mannitol derivative is obtained by adopting the 5-chlorosulfonyl-2-hydroxybenzoic acid to chemically modify mannitol, so that the promotion and inhibition effects of the mixed sample can be obviously improved.
Product cytotoxicity assays
The test method was the same as in example 1. From the analysis of the test results, the test sample solution obtained in this example showed no cytotoxic reaction.
Example 6:
mannitol and derivative Performance characterization
Determination of free radical scavenging Effect
After DPPH is dissolved in a small amount of toluene, a solution of 120 mu mol/L is prepared by using 50% ethanol, 1.9mL of the solution is taken, 0.1mL of a test sample with the concentration of 2.0g/L is added, the solution is kept stand for 20min at room temperature, and then the absorbance at 525nm is measured. The clearance was calculated according to the following equation:
clearance/% = (A-A) 1 )/(A-A 0 )×100%
Wherein A is the absorbance of a control system, A 1 To test the absorbance of the sample, A 0 Absorbance was measured for the blank.
Mannitol and the mannitol derivatives prepared in example 4 were subjected to the above test, and the results are shown in table 5:
TABLE 5 free radical scavenging effect test results
From the data analysis in table 5, the clearance rate of the mannitol derivative to the DPPH free radical is obviously higher than that of mannitol, which indicates that the mannitol derivative obtained by chemically modifying mannitol with 5-chlorosulfonyl-2-hydroxybenzoic acid can obviously improve the free radical adsorption clearance capability of the mannitol derivative.
Test example 1:
infrared sign
The testing is carried out by adopting a Fourier infrared spectrometer, and the testing range is 500-4000 cm -1 。
The sodium hyaluronate crosslinked product prepared in example 3 was subjected to the above test, and the characteristic peaks in the infrared spectrum thereof were 3275 to 3390 (b), 1615(s), 1405 (m), 1150, 1077, 1045(s), 946 (m), 893 (w). The wave number error of the actually measured band of the fingerprint area is less than +/-5 cm -1 。
The sodium hyaluronate derivative 1 prepared in example 4 and sodium hyaluronate were subjected to the above-described test, and the results are shown in fig. 4. As can be seen from the analysis of the figure, compared with the infrared test result of sodium hyaluronate, in the infrared spectrum of the sodium hyaluronate derivative 1 prepared in example 3, the infrared spectrum is 3000-2800 cm -1 The characteristic absorption peak intensity of methylene in the range is obviously improved, and the peak intensity is 1350-1250cm -1 The characteristic absorption peak intensity of C-N bond in the range is enhanced, and the characteristic absorption peak intensity is 1100-1000 cm -1 The characteristic absorption peak intensity of the C-O bond in the range was enhanced, and the above results indicate that sodium hyaluronate derivative 1 in example 3 was successfully produced.
The mannitol derivatives prepared in example 5 were subjected to the above test, and the results are shown in fig. 5. From the analysis of the graph, 3000-2800 cm -1 Characteristic absorption peak of methyl and methylene in the range of 1500-1400 cm -1 Characteristic absorption peak of benzene ring appeared in the range of 1353cm -1 、1183cm -1 The characteristic absorption peak of s=o appearing nearby, the above result indicates successful preparation of mannitol derivative in example 3.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A sodium hyaluronate derivative comprising a cross-linked sodium hyaluronate derivative; the crosslinked derivative comprises a mixture crosslinked material of one or more of sodium hyaluronate with single-chain molecular weight of 1-8 ten thousand, 8-15 ten thousand, 15-30 ten thousand, 30-80 ten thousand and 80-200 ten thousand.
2. A composition comprising sodium hyaluronate or a sodium hyaluronate derivative of claim 1.
3. The composition of claim 2, wherein: the composition further comprises a collagen pentapeptide.
4. The composition of claim 2, wherein: the composition also comprises auxiliary components including one or more of mannitol, sorbitol and dextran.
5. Use of a sodium hyaluronate derivative according to claim 1 for the preparation of a medical cosmetic injection filler, a cosmetic or a skin care product or a medicament.
6. Use of the composition of claim 2 for the preparation of a medical cosmetic injection filler, a cosmetic or skin care product or medicament.
7. An aqueous product comprising the composition of any one of claims 2 to 4.
8. The aqueous product of claim 7, wherein: the concentration of the sodium hyaluronate or sodium hyaluronate derivative in the aqueous solution product is 0.2-1.5wt%; the concentration of the collagen pentapeptide is 2-2000 ppm.
9. A powder product comprising the composition of any one of claims 2-4.
10. A powder product as claimed in claim 9, wherein: sodium hyaluronate or sodium hyaluronate crosslinked derivatives in the product are dissolved in water when in use, and the concentration is 0.2-1.5wt%; the concentration of the collagen pentapeptide in the product is 2-2000 ppm after the collagen pentapeptide is dissolved in water during use.
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