CN114272361A - Composition and application method of collagen molecular monomer skin coating liquid - Google Patents
Composition and application method of collagen molecular monomer skin coating liquid Download PDFInfo
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Abstract
The invention relates to a composition and an application method of a collagen molecular monomer skin coating liquid, belonging to the technical field of biomedical materials. The invention discloses a skin coating liquid, which is prepared by applying the biological function that collagen molecular monomers can better promote the regeneration of skin tissues than collagen fibers, collagen fragments and collagen small molecular peptides. The main components of the collagen peptide are collagen molecule monomers with complete triple helical structures dissolved in ascorbic acid solution, and scars and skin aging at the healing part of the skin caused by various reasons can be effectively improved and repaired under the action of the collagen molecule monomers and the ascorbic acid solution. The application method comprises skin care, skin treatment, recovery of skin healing parts after various operations and traumas, and skin care after treatment such as skin wrinkle removal, skin micro-needle treatment, laser treatment, skin abrasion, cutin removal, laser repair and the like.
Description
Technical Field
The invention relates to a composition and an application method of a collagen molecular monomer skin coating liquid dissolved in an ascorbic acid solution, which is applied to the recovery of skin after operation and the improvement of skin aging. The invention belongs to the technical field of biomedicine.
Background
Collagen is an extracellular matrix fibrin, which is a major component of most connective tissues in mammals. The collagen family consists of more than 20 known types, and mammalian dermis contains mainly type I and type III collagen, with type I collagen accounting for about 85% of the total collagen. The collagen type I and type III molecules are trimeric proteins consisting of three alpha chains and have a molecular weight of about 30 ten thousand (300 kD). Type I collagen is a heteromer containing 2 α 1(I) chains and 1 α 2(I) chain, while type I II collagen is a homomer containing 3 identical α 1(III) chains, each of which contains about 1000 amino acid residues. They all have the typical repeating Gly-X-Y sequence, proline usually occupying the X position and hydroxyproline occupying the Y position. Due to the high content of proline and hydroxyproline, each alpha chain can form a left-handed helical structure, and the alpha chains are further folded and intertwined to form a complex triple helical structure. Collagen molecules with a triple helix structure can be assembled and polymerized automatically to form collagen fibers under physiological conditions, and the collagen fibers in a human body can be degraded by specific metalloproteases to form collagen fragments.
Collagen helps to maintain the compactness, tone and plumpness of human skin, prevents skin aging, and is one of the main factors helping to maintain the health of human skin. As people age, the synthesis of new collagen decreases and the degradation increases, and the physiological aging process of the skin is adversely affected by ultraviolet radiation, environmental factors, unbalanced diet, pollution and hormonal factors, resulting in a significant change of the skin, and after the normal level of epidermal hydration is lost, a significant structural change of extracellular matrix and degradation of collagen occur, with the result that the dermis is severely structurally and functionally damaged, and skin sagging, volume reduction and deep wrinkles are inevitably generated.
The most important direction of research in the medical field for many years has been dermal bioremodelling, aimed at improving the quality of the dermis by specific treatments and internally combating the defects due to skin ageing. The aim is to find and determine the optimal composition of matter that will stimulate fibroblasts, provide them with collagen precursors and induce direct activity to produce nascent collagen. Studies have found that the administration of exogenous collagen in vitro and under the dermis has some of the above-mentioned effects, and various products have been developed using this principle. Products are known on the market today which contain a collagen component, wherein the collagen is all fibres of type I and type III collagen or fragments thereof.
Further scientific research shows that the collagen molecular monomer has better effect of promoting the growth of fibroblasts compared with collagen fibers and collagen degradation segments. Hong Xia, Richard Nho et al reported That Collagen Polymerized to form fibers, as compared to Collagen molecular monomers, inhibited fibroblast growth in THE literature, "Polymerized Collagen inhibitors fibrous sheets purification and purification OF THE Formation OF a 1 integer-Protein Phosphophatase 2A-Tuberous Sclerosis Complex 2 Complex coat precursors S6K1 Activity" (THE JOURNAL OF BIOGICAL CHEMISY TRVOL.283, NO.29, July 18, 2008). Hidenori Koyama, Elaine W.raines et al, in the publication of fibrous Collagen inactivation tissue biology Muscle promotion of Cdk2Inhibitors (Cell, Vol.87, December 13, 1996) describe a decrease in the ability of Collagen molecules to promote Cell growth after degradation into Collagen fragments.
The scientific research results show that: the collagen molecular monomer has better skin regeneration promoting effect than collagen fiber or its segment, and the collagen molecular monomer has complete triple helical structure.
Collagen products commercially available at home and abroad for application to the skin field, and representative examples thereof include bovine Collagen fiber injection gel developed from cow leather by Collagen Corporation of the United states
And
the product "NITHYA" produced by Euroresearch, italy, using collagen type I fragments of horse hide. Also, the product "Shuangmei No. 1" of Taiwan Shuangmei science and technology Limited and the product "Fumeida" of Changchun Boratai medicine are LimeidaInjection glue developed with collagen fibers developed from cow hide. The products of the Shanxi brocade organism, namely 'Wei Mei', 'Chongyuan' and the products of the Shaanxi Juzi organism, namely 'Koijin' and 'Koxike', are skin protection products developed by utilizing the recombinant human collagen fragments.
The products containing the collagen components are all products in different dosage forms constructed in the form that collagen fibers formed by polymerizing collagen or collagen molecules are degraded to form collagen fragments, do not have the biological function of collagen molecule monomers, and can not effectively activate and induce the generation of new human autologous collagen, so the effects of repairing and caring the skin are limited.
Ascorbic acid, also known as vitamin C or L-ascorbic acid, is a six-carbon lactone produced by plant and some animal species, a polyhydroxy compound whose two adjacent enol-type hydroxyl groups at positions 2 and 3 in the molecule are readily dissociated to release H +, and thus has an acid property. Many important mammalian biosynthetic processes require ascorbic acid to be involved, including antibody and collagen formation, tissue repair, fat and protein synthesis. Its main physiological functions include: promoting the biosynthesis of ossein and being beneficial to the faster healing of tissue wounds. As a water-soluble antioxidant, it is mainly effective in strengthening connective tissue in vivo aqueous solution and promoting collagen synthesis.
Scientific research results for many years show that ascorbic acid has an important effect on skin aging resistance and repair, and the effect of ascorbic acid has been reviewed in "research progress on the skin health promoting effect of vitamin C" (vol. 36, 19, 10/2020/10/2020 in modern medicine and health). The effect of vitamin C to protect skin has been widely used in the fields of medical instruments, cosmetics, health products and foods.
The invention aims to develop a skin coating agent with skin care functions of collagen molecular monomers and ascorbic acid.
Disclosure of Invention
In the present invention, the "collagen molecule monomer having an intact triple helix structure" is defined as: tissue-extracted or recombinant xenocollagen, which is type I and type III collagen and retains its native triple helical structure, molecular weight and polypeptide chain sequence, and human allocollagen molecules. The meaning is well known and understood by those skilled in the art and is not intended to be ambiguous.
In the present invention, "ascorbic acid" is defined as: the meaning of vitamin C is well known and understood by those skilled in the art and is not intended to be ambiguous.
The instruments and reagents used to practice the invention are well known and understood by those skilled in the art and are available from commercial disclosures, unless otherwise specified. The collagen used in the embodiment of the invention is a self-made collagen molecular monomer.
The invention creatively applies the biological function of a collagen molecular monomer and aims to develop a skin coating liquid which promotes the growth of new collagen fibroblasts by using the collagen molecular monomer and promotes the synthesis of collagen by combining with ascorbic acid.
At present, a plurality of products for skin protection are available on the market, wherein the collagen is in the form of collagen fibers, collagen fragments or collagen polypeptides, and is not a collagen molecular monomer. Scientific research proves that the collagen molecular monomer has better promotion effect on the growth of fibroblasts than collagen fibers, collagen fragments or collagen polypeptides. Therefore, the invention discloses a skin coating liquid with the functions of promoting fibroblast growth by collagen molecular monomers and promoting collagen synthesis by ascorbic acid, and collagen in the components of the coating liquid maintains the form of the molecular monomers. The intact collagen molecular monomers are obtained from animal tissues using proprietary preparative techniques or by recombinant methods.
At present, no collagen molecular monomer is used as a skin care product. Because collagen molecular monomers have the capacity of self-polymerization assembly to form fibers and are insoluble in water, the existing preparation composition method of collagen fiber and collagen segment skin care products cannot be used for the collagen molecular monomers, and therefore, a new preparation composition method capable of maintaining the collagen molecules in a molecular monomer state instead of a fiber state or a segment state is innovatively invented.
The preparation and composition method of the coating liquid ensures that collagen molecules in the coating liquid are in a molecular monomer state and keeps the complete three-strand helical structure biological structure.
The invention provides an application method of a collagen molecular monomer coating liquid, which has wide application in the field of skin care and can be used in various ways of improving skin conditions. The product is suitable for cosmetic operation for skin recovery after skin healing, such as liposuction, abdominal plastic or human body sculpture. It is also suitable for skin care, such as wrinkle removal, laser treatment, skin abrasion, and desquamation.
The invention provides a collagen molecular monomer coating liquid composition, wherein ascorbic acid is used as an acidity regulator, and the synthesis of collagen needs the participation of the ascorbic acid, and if the ascorbic acid is deficient, the collagen cannot be normally synthesized. The effect of promoting collagen synthesis by using ascorbic acid is combined with a collagen molecular monomer, so that the effect of the coating liquid in the skin care process is improved.
The invention provides a collagen molecular monomer coating liquid, which is prepared by dissolving a collagen molecular monomer in an ascorbic acid solution, wherein the composition is in a solution state, and the pH value of the solution is between 2.0 and 4.5. Meanwhile, amino acids, nutritional trace elements, small molecule drugs, polypeptide molecular substances, hyaluronic acid and derivatives thereof and elastin can be added.
The invention relates to a coating liquid of a collagen molecular monomer with a complete triple helical structure dissolved in an ascorbic acid solution. In order to achieve the above objects, the present invention provides a composition of ascorbic acid solution containing collagen molecule monomer with complete triple helical structure.
The specific technical scheme of the invention is as follows:
the invention relates to a coating liquid formed by collagen molecular monomers dissolved in ascorbic acid solution, wherein the collagen is the collagen molecular monomers with a complete triple helical structure. In the following examples, it can be seen that the unique method and composition for preparing collagen monomers is employed in the present invention, so that collagen exists in the form of collagen monomers in the coating solution. The collagen molecular monomer is natural collagen molecular monomer prepared from animal related tissues through a complex and effective extraction, separation and purification process, and can also be recombinant collagen molecular monomer prepared through a biological engineering technology. The source of the animal tissue comprises relevant tissues of mammals and marine animals, and preferably, the source of the animal tissue is relevant tissues of cattle and pigs which are qualified for quarantine and relevant tissues of healthy people of legal origin. These tissues are rich in type I and III collagen, such as animal and human skin, tendons, blood vessels, pericardium, placenta, and the like. The collagen tissue extraction method is commonly performed by an alkaline method, an acid method, an enzymatic method and different combination methods of the above methods, and preferably, the method of combining the acid method and the enzymatic method is used in the present invention to prepare a high-purity collagen molecule monomer solution (see contents of examples 1 and 2 and fig. 1 and 2). The concentration of the collagen molecule monomer in the invention has a relevant action range of 0.01mg-5mg/ml, preferably 0.1-3.5 mg/ml. The ratio of the soluble monomer of the collagen molecule in the total amount of the collagen contained in the coating liquid is more than or equal to 50 percent, and preferably more than or equal to 55 percent.
In the present invention, through the study on the composition and preparation of the coating liquid, it was proved that the degree of collagen fiber formation by molecular polymerization of collagen molecular monomers is affected by the PH, and when the PH is lower than 4.0, no polymerization of collagen molecules occurs, and when the PH is higher than 5.0, polymerization of collagen molecules occurs, and the degree of collagen fiber formation by polymerization of collagen molecules increases with the increase of the PH (see the content of example 4). In order to keep the collagen molecules in a monomeric state in the coating liquid, the coating liquid is prepared to have a pH value of 2.0-4.5, preferably a pH value of 2.2-4.0. To ensure that the pH value of the coating solution is between 2.0 and 4.5, the invention dissolves the collagen molecular monomer in an ascorbic acid solution, the solution is acidic, and the ascorbic acid has the function of regulating the pH value. Wherein the concentration of the ascorbic acid is 0.1-15%, preferably 0.2-8%; the pH value of the ascorbic acid solution is between 2.0 and 4.0, and preferably between 2.2 and 4.0.
The present invention achieved by the above technical route is a composition of collagen molecular monomer dissolved in ascorbic acid solution, which constitutes a collagen molecular monomer skin coating solution, and the composition includes, but is not limited to, collagen molecular monomer, amino acid, nutrient trace element, small molecule drug, polypeptide molecular substance, hyaluronic acid and its derivative, elastin and ascorbic acid, and can be a composition in various combination ratios. Preferably, the coating liquid component comprises collagen molecule monomer and ascorbic acid. The composition can be prepared into various dosage forms, and preferably, the dosage form of the composition is aqua, essence, emulsion, cream or facial mask. Preferably, the composition is in the form of an aqueous solution.
The application method of the coating agent comprises the step of applying the composition to the skin part required by a user.
The compositions of the present invention may be used in applications including skin care and post-operative skin healing applications. Due to the adoption of the technical route scheme, compared with the prior art, the invention has the following innovations and effects: the biological activity of collagen molecular monomer with complete triple helix structure is combined with the function of ascorbic acid for promoting collagen synthesis, thereby promoting fibroblast growth, and better promoting skin metabolism and skin care.
Drawings
FIG. 1 is a photograph of SDS-PAGE electrophoresis spectrum of the collagen molecular monomer solution prepared in example 1, in which electrophoresis bands are clearly visible, which are sequentially shown from left to right: marker; the prepared bovine achilles tendon collagen; the collagen band of each sample of the American bovine dermal collagen (Advanced Biomatrix) reference substance is a collagen molecular monomer alpha 2(I) chain, an alpha 1(I) chain, a dimer and a trimer thereof, which are sequentially in a band spectrum from bottom to top, and no hybrid proteins such as collagen fragments and the like.
FIG. 2 is a photograph of SDS-PAGE electrophoresis spectrum of the collagen molecular monomer solution prepared in example 2, in which the electrophoresis bands are clearly visible, from left to right: marker; preparing human placental collagen; the collagen band of each sample of the American bovine dermal collagen (Advanced Biomatrix) reference substance is a collagen molecular monomer alpha 2(I) chain, an alpha 1(I) chain, a dimer and a trimer thereof, which are sequentially in a band spectrum from bottom to top, and no hybrid proteins such as collagen fragments and the like.
FIG. 3 is a graph showing the results of example 3, wherein a sample of the human placental collagen molecular monomer solution prepared in example 2 was examined by Japanese JASCO J-810 circular dichroism spectroscopy using 10mM hydrochloric acid buffer solution at a sample concentration of 0.1mg/ml, and the spectral absorption curve was obtained by scanning at a wavelength ranging from 190 to 260nm, and the sample had a complete triple helix structure.
Detailed Description
The present invention is described in detail below with reference to specific embodiments. It will be appreciated by the public and those skilled in the art that the method of carrying out the invention in its various forms should not be limited by the examples set out below. These embodiments are provided so that this disclosure will be thorough and complete.
The invention provides a collagen molecular monomer coating solution dissolved in an ascorbic acid solution, which comprises the components of a collagen molecular monomer, ascorbic acid, water for injection and auxiliary materials.
In one embodiment, the coating solution consists of collagen molecule monomer, ascorbic acid and auxiliary materials.
In one embodiment, the collagen molecule monomer is obtained by extraction, separation and purification from healthy human placental tissue of legal origin. In another embodiment, the collagen molecule monomer is prepared from bovine achilles tendon.
In one embodiment, experiments have shown that collagen fibers may be formed by the monomeric form of collagen molecules in the coating solution, which is influenced by the pH of the system in which they are present. The proper pH value is adjusted by using ascorbic acid and phosphate buffer solution, so that collagen molecules in a coating solution system can be ensured to keep the monomer form.
In one embodiment, the coating composition further comprises adjuvants which are common and common adjuvants known and understood by those skilled in the art, for example, the adjuvants may be: phosphate buffer, antioxidants, and the like.
For the use method of the present invention, the present invention is not limited at all, for example, the coating solution can be used in the aspects of anti-wrinkle, anti-fine line and anti-aging of skin, and the composition can be in the form of aqua, cream, lotion, essence, mask, etc.
The concentration of the collagen molecular monomer and ascorbic acid in the coating solution is not limited in the present invention, and may be adjusted as needed, for example, the concentration of the collagen molecular monomer in the coating solution may be 0.5mg/ml or 5mg/ml, and the concentration of ascorbic acid may be 0.02% or 12%
The invention provides a use method of a coating liquid, which comprises the step of applying any dosage form of the coating liquid to a skin part required by a user. For example, the coating liquid may be applied to the face, nose, abdomen, hands, etc. of the user, which may be determined according to the user's needs.
The invention provides application of the coating liquid to skin care and postoperative skin healing, and preferably the composition is used in aspects of anti-wrinkle and anti-fine line of skin.
The following is an example of the composition of a coating solution and its use according to the present invention, which is further elaborated on in the examples.
Example 1: preparation of bovine collagen solution
Taking fresh bovine achilles tendon qualified for quarantine, crushing and homogenizing, extracting according to the weight-volume ratio of homogenized tissue to extract (containing pepsin 3.5mg/ml) of 1: 8 under the acidic condition of PH2.0, centrifugally separating, carrying out salt multi-stage precipitation separation, dissolving the precipitate acid, and dialyzing by a dialysis membrane to obtain the natural bovine collagen molecular monomer solution. The obtained collagen was separated by SDS-PAGE, and the obtained collagen was prepared by using a DYCZ-25E electrophoresis apparatus and SDS-PAGE gel kit manufactured by Biyunstian Biotechnology Ltd, and the prepared separation gel had a concentration of 15%, the concentration of the concentrated gel was 10%, and the result was stained with Coomassie Brilliant blue, and is shown in FIG. 1. The band spectrum of alpha 1(I) chain and alpha 2(I) chain of collagen molecular monomer can be seen through SDS-PAGE electrophoresis detection and analysis, and hybrid proteins such as collagen fragments and the like are not contained.
Example 2: preparation of human collagen solution
Taking fresh and healthy human placenta, crushing and homogenizing, extracting and digesting the homogenate tissue and an extracting solution (containing 4.0mg/ml of pepsin) according to the weight-volume ratio of 1: 10 under the acidic condition of PH2.5, centrifugally separating, precipitating with salt, dissolving the precipitated acid, and performing ultrafiltration purification to obtain the natural human collagen molecular monomer solution. The obtained collagen was separated by SDS-PAGE, and the kit was prepared by DYCZ-25E electrophoresis and SDS-PAGE gel, which was manufactured by Biyuntian Biotechnology Ltd, and the prepared separation gel concentration was 10%, the concentration was 5%, and the result was shown in FIG. 2. The band spectrum of alpha 1(I) chain and alpha 2(I) chain of collagen molecular monomer can be seen through SDS-PAGE electrophoresis detection and analysis, and hybrid proteins such as collagen fragments and the like are not contained.
Example 3: circular dichroism chromatography detection of human collagen
Taking a collagen molecular monomer solution sample prepared in example 2, diluting to 0.1mg/ml, and carrying out CD spectral detection by using JASCO J-810 circular dichroism spectrometer: firstly, opening nitrogen, opening a circular dichroism instrument and software, adjusting zero by using buffer solution, and preserving as a substrate blank. And then taking the diluted 0.1mg/ml sample for CD atlas detection (wavelength range: 250-190 nm; scanning speed: 50 nm/min; frequency bandwidth: 1.0 nm; resolution: 0.1 nm). The structure of the collagen molecule monomer is detected by a circular dichroism chromatogram method, and the detection result is shown in figure 3. The results show that the sample has a complete triple helix structure.
Example 4: effect of solution pH on collagen molecule polymerization
Taking 9 collagen molecule monomer solutions of 5ml each, adjusting the collagen molecule monomer solutions to different pH values by using phosphate buffer solution and ascorbic acid 2% concentration solution, detecting the degree of collagen fibers in the collagen molecule monomer solutions under different pH values by using a 313nm wavelength of an ultraviolet spectrophotometer, and expressing the degree by using absorbance values, wherein the experimental results are as follows:
note: . + -. represents infinitesimal; + represents have; -represents none
The experimental result shows that the pH value of the collagen molecular monomer solution is within the range of 2.2-4.0, the solution is colorless and transparent, almost has no absorbance value, and no molecular monomer aggregation is generated to produce collagen fibers. After the pH value is more than 5.06, the absorbance value is obviously increased along with the increase of the pH value, and fine filiform precipitates are separated out in the solution, which indicates that collagen molecular monomers are aggregated to generate collagen fibers.
Example 5
50ml of coating solution was prepared from the collagen monomer solution extracted in example 1, and the protein concentration was measured at 562nm using a microplate reader BCA assay kit method (protein assay kit of Biyunshi Biotechnology, Inc.) and bovine collagen (Advanced Biomatrix) as a control. Adding collagen molecular monomer solution extracted from water for injection and example 1 into a 50ml volumetric flask to make the collagen concentration be 0.5mg/ml and the ascorbic acid concentration be 0.8%, stirring for 10 minutes at room temperature, adjusting the pH value of the solution to be 3.5, fixing the volume to 50ml, filtering by a 0.45 micron membrane, placing in a brown flask, and keeping away from light at 4 ℃.
Example 6
100ml of coating solution was prepared from the collagen monomer solution extracted in example 2, and the protein concentration was measured at 562nm using a microplate reader BCA assay kit method (protein assay kit from Bilun Biotechnology Ltd.) and bovine collagen (Advanced Biomatrix) as a control. Adding water for injection and the collagen molecular monomer solution extracted in example 2 into a 100ml volumetric flask to ensure that the collagen concentration is 1.5mg/ml and the ascorbic acid concentration is 2%, stirring for 10 minutes at room temperature until the collagen molecular monomer solution is completely dissolved, adjusting the pH value of the solution to be 3.0, fixing the volume to be 100ml, filtering by a 0.45 micron membrane, placing in a brown flask, and storing in a dark place at 4 ℃.
The embodiments of the present invention have been described in detail with reference to the specific examples, but the present invention is not limited to the above embodiments. The scope of the invention is to be determined by the following claims and their equivalents. Any person skilled in the art can make variations using the above-disclosed technical contents, but any modifications, equivalent variations and modifications of the present invention based on the technical essence of the present invention still fall within the protection scope of the present invention.
Claims (12)
1. Relates to a composition of a collagen molecular monomer skin coating liquid and an application method thereof, wherein the coating liquid mainly comprises a collagen molecular monomer with a complete triple helical structure dissolved in an acidic solution.
2. The collagen molecule monomer of claim 1, which is a type I or type III collagen molecule monomer.
3. The collagen molecule monomer according to claim 2, having an intact triple helix structure.
4. The collagen of claim 2, which is a collagen purified by tissue extraction or a recombinant collagen.
5. The collagen molecule monomer according to claim 2, having a concentration of 0.01mg/ml to 5 mg/ml.
6. The collagen molecule monomer according to claim 2, wherein the collagen molecule monomer is not less than 50% of the total amount of collagen contained in the coating solution.
7. The acidic solution of claim 1 having a pH of between 2.0 and 4.5.
8. The collagen molecule monomer according to claim 1, which is a collagen molecule monomer having an intact triple helix structure dissolved in an ascorbic acid solution.
9. The ascorbic acid solution of claim 8, having an ascorbic acid concentration of 0.01% to 15%.
10. The ascorbic acid solution of claim 8, having a pH of between 2.0 and 4.0.
11. The collagen molecule monomer skin coating liquid according to claim 1, which is a composition comprising amino acids, nutritional trace elements, small molecule drugs, polypeptide molecule substances, hyaluronic acid and its derivatives, and elastin in various combination ratios.
12. The method of using the collagen molecular monomer skin coating solution according to claim 1, which comprises skin care and coating of various post-operative skin healing sites and skin trauma healing sites, and can be used for wrinkle removal, skin microneedle treatment, laser treatment, dermabrasion and exfoliating.
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