CN115670969B - Composition for stimulating elastin formation - Google Patents
Composition for stimulating elastin formation Download PDFInfo
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- CN115670969B CN115670969B CN202211391503.7A CN202211391503A CN115670969B CN 115670969 B CN115670969 B CN 115670969B CN 202211391503 A CN202211391503 A CN 202211391503A CN 115670969 B CN115670969 B CN 115670969B
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- hyaluronic acid
- carnosine
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a composition for stimulating elastin formation, and belongs to the field of aging resistance. The composition provided by the invention comprises: hyaluronic acid or a pharmaceutically acceptable salt thereof, carnosine, and amino acids and buffers. The composition provided by the invention not only has the function of supplementing extracellular matrix, but also can promote elastin formation, and treat, alleviate and/or improve symptoms, disorders or diseases of skin or mucous membranes.
Description
Technical Field
The invention belongs to the technical field of medical cosmetology, in particular to a composition for stimulating elastin formation, more particularly relates to a composition comprising hyaluronic acid, carnosine, amino acid and the like, which can be used for promoting elastin formation, treating, relieving and/or improving symptoms, disorders or diseases of skin or mucous membrane, resisting aging and the like, and belongs to the field of anti-aging preparations.
Background
Human skin is an organ covering the whole body, in which the dermis is mainly composed of fibroblasts, and mast cells or tissue macrophages (dermis also contains blood vessels and nerve fibers) are also present. The acellular portion of the dermis (i.e., the region between cells) is referred to as the extracellular matrix, which is composed of a variety of extracellular components including proteins, such as collagen fibers and elastin, and also glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, and the like, and also proteoglycans and a variety of glycoproteins, such as fibronectin, and the like. These extracellular components are synthesized by dermal fibroblasts, so dysfunction and changes in extracellular matrix components can interfere with cellular performance and tissue integrity. Dysfunction and changes in the extracellular matrix components of human skin, mucous membranes may lead to skin aging, atrophy, damage or other disorders and/or diseases of the skin and mucous membranes associated with changes in the extracellular matrix components.
In addition, aging may occur in organs or tissues such as the skin of the human body with age and/or environmental changes. Natural aging of the skin can reduce the content of collagen and sodium hyaluronate in the skin, elastic fiber and collagen fibrosis, thinning the skin and the like, and the damage can appear on the skin at all parts of the whole body; skin aging may exhibit phenomena or conditions such as dry skin, roughness, telangiectasia, reduced elasticity, irregular pigmentation, seborrheic keratosis, etc.
Thus, methods of subcutaneously supplementing collagen and/or sodium hyaluronate, or stimulating collagen, elastin formation, etc., to maintain or increase the level of extracellular matrix components are effective means or protocols for treating, alleviating and/or ameliorating symptoms, disorders or diseases of the skin or mucosa associated with changes in extracellular matrix components, improving the state of skin aging.
Hyaluronic acid is a natural polysaccharide, which is formed by connecting the following repeated units: N-acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid is capable of binding and retaining moisture, plays a supporting role in skin hydration, and has excellent viscoelasticity. In order to avoid rapid degradation of exogenous hyaluronic acid in organs/tissues such as skin, a cross-linking agent and hyaluronic acid are often used in products, but the cross-linking agent (such as 1, 4-butanediol diglycidyl ether (BDDE)) is a component in non-human tissues, and is easy to have uncrosslinked or uncrosslinked condition, and the cross-linking agent is used as an external component, so that the safety risk is greatly improved, and the difficulty in component determination and production process and quality control of related products is also greatly improved.
Clinically, the collagen injection filler for the human body comprises human collagen and non-human collagen, and the human collagen has no immune risk, but is time-consuming and labor-consuming to obtain and has high price; the non-human collagen has a certain immune risk, and more uncertain factors exist in use; and collagen is relatively expensive; direct injection of collagen bulking agents is therefore not an optimal solution.
Elastin is an important load-bearing tissue in the human body, plays an important role in skin elasticity (elastin plays an important role in arteries, lungs, elastic ligaments, elastic cartilage and the like), is mainly responsible for restoring the stretched tissue to the original shape, is very important for maintaining skin elasticity, compactness and smoothness, and can cause human body to be subjected to changes such as disorder, diseases or aging if elastin in the body is dysfunctional or changed.
At present, part of products on the market claim to supplement collagen or stimulate collagen increase and have anti-aging effect, but the effects are not verified, and cannot be determined whether the collagen increase can be promoted or not, and further whether the elastin formation and increase can be promoted or not.
Amino acid is a structural unit of protein, and certain amino acid can be supplemented to provide raw materials for synthesis of collagen, so that possibility is provided for improving skin aging state, and the amino acid has no safety risk and is convenient and easy to obtain. However, how to reasonably and efficiently mix hyaluronic acid, amino acid and other substances to obtain safe, efficient and easily available anti-aging preparations is not known at present.
Thus, it would be highly useful and necessary to provide a composition that helps to maintain or increase the level of extracellular matrix components for treating, alleviating and/or ameliorating a symptom, disorder or disease of the skin or mucosa associated with changes in extracellular matrix components, including ameliorating a skin aging state.
Disclosure of Invention
Based on the problems and needs that exist today, the present invention provides a composition that can be used to treat, alleviate and/or ameliorate symptoms, disorders or diseases of the skin or mucosa associated with changes in extracellular matrix components, including improving the state of skin aging. The composition provided by the invention not only has the function of supplementing extracellular matrix of the filling material, but also can stimulate and promote fibroblast proliferation, collagen and elastin formation, treat, alleviate and/or improve symptoms, disorders or diseases of skin or mucous membrane, improve and solve aging and ageing phenomena of skin, such as water deficiency, dryness, pigmentation, wrinkles and the like.
The present invention thus provides a composition consisting essentially of: hyaluronic acid and/or a pharmaceutically acceptable salt thereof, carnosine, amino acids or a pharmaceutically acceptable salt thereof, and a buffer; wherein the amino acids include glycine, proline, lysine, valine, alanine and leucine.
In some embodiments, the present invention provides a composition consisting essentially of: hyaluronic acid and/or a pharmaceutically acceptable salt thereof, carnosine, amino acids or a pharmaceutically acceptable salt thereof, and a buffer; wherein the amino acids are glycine, proline, lysine, valine, alanine and leucine.
The pharmaceutically acceptable salt of hyaluronic acid may be sodium hyaluronate, zinc hyaluronate, calcium hyaluronate or a combination thereof; the buffer is substantially free of potassium elements such as potassium ions, and the buffer is not a potassium salt such as potassium hydrogen phosphate or potassium dihydrogen phosphate, etc.
The molecular weight of the hyaluronic acid or a pharmaceutically acceptable salt thereof is not too high, too low, difficult to dissolve and difficult to inject, too low, easy to be hydrolyzed by enzymes or to have a weak effect or not to achieve a desired effect; in the present invention, the hyaluronic acid or a pharmaceutically acceptable salt thereof may have a molecular weight of about 110 kilodaltons to about 180 kilodaltons. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof preferably has a molecular weight of about 120 kilodaltons to about 170 kilodaltons. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof preferably has a molecular weight of about 130 kilodaltons to about 160 kilodaltons. In some embodiments, the molecular weight of the hyaluronic acid or pharmaceutically acceptable salt thereof is preferably from about 140 kilodaltons to about 160 kilodaltons. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof preferably has a molecular weight of about 145 to about 155 kilodaltons. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof preferably has a molecular weight of about 148 ten thousand, about 150 ten thousand, or about 152 ten thousand daltons.
In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof is hyaluronic acid or a sodium salt thereof.
The hyaluronic acid, or pharmaceutically acceptable salt thereof, may be present in an amount of about 2.5mg/mL to about 10mg/mL (e.g., about 3mg/mL, about 4mg/mL, about 5mg/mL, about 6mg/mL, about 7mg/mL, about 8mg/mL, about 9mg/mL, or about 10 mg/mL) based on the volume of the composition. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof is preferably present in an amount of about 3mg/mL to about 8mg/mL, or preferably about 3mg/mL to about 7.5mg/mL, or preferably about 4mg/mL to about 6mg/mL, or even more preferably about 4.5mg/mL, about 5mg/mL, or about 5.5mg/mL, based on the volume of the composition.
The carnosine may be L-carnosine (chemical name: S) -2- (3-aminopropionamide) -3- (1H-imidazol-4-yl) propionic acid), or a derivative of L-carnosine.
The carnosine may be present in an amount of 0.1mg/mL to 5.0mg/mL (e.g., 0.5mg/mL,1.0mg/mL,1.5mg/mL,3.0 mg/mL), preferably 0.5mg/mL to 2.0mg/mL, based on the volume of the composition. In some embodiments, the carnosine is present in an amount of 1.5mg/mL based on the volume of the composition, which is more beneficial to the composition in stimulating, promoting the formation of collagen and elastin, and anti-aging effects.
The pharmaceutically acceptable salt of the amino acid can be hydrochloride, sulfate, acetate, citrate, sodium salt, magnesium salt or calcium salt of the amino acid. In some embodiments, the pharmaceutically acceptable salt of the amino acid is the hydrochloride or sodium salt of the amino acid.
The amino acid disclosed by the invention is contained in the composition, so that nutrients are provided for skin, the formation of collagen and elastin is stimulated and promoted, and the composition has better disease symptom improvement and anti-aging effects.
According to the embodiment of the invention, the inventor finds that hyaluronic acid and/or pharmaceutically acceptable salts thereof, carnosine and amino acid or pharmaceutically acceptable salts thereof are combined according to the scheme of the invention, and the composition has synergistic effect on promoting the formation of collagen and elastin of skin, soft tissues and the like and anti-aging effect, is more beneficial to stimulating and promoting the formation of collagen and elastin, and can better improve the disease symptoms and aging phenomena of skin and the like.
The amino acid or pharmaceutically acceptable salt thereof may independently be present in an amount of 0.01mg/mL-2.0mg/mL, preferably 0.05mg/mL-1.5mg/mL or 0.05mg/mL-1.0mg/mL, based on the volume of the composition. In some embodiments, the amino acid or pharmaceutically acceptable salt thereof is present in an amount of 0.1mg/mL or 1.0mg/mL, independently, based on the volume of the composition, which is advantageous in stimulating, promoting elastin and/or collagen formation.
According to embodiments of the invention, the pH of the composition may be from 6.5 to 8.0, preferably from 6.8 to 7.6. In some embodiments, the pH of the composition is preferably 6.9,7.0,7.1,7.2,7.3,7.4 or 7.5. The pH range aids in making the composition suitable for application to the skin and/or suitable osmotic pressure.
According to an embodiment of the present invention, the composition provided herein has a potassium ion content of no more than 0.008mg/mL, preferably no more than 0.007mg/mL or preferably no more than 0.005mg/mL, calculated on the volume of the composition. In some embodiments, the composition is free of potassium ions other than the impurity potassium ions introduced by the materials used. The inventors found that controlling the content of potassium ions in the composition within the range described in the present invention is advantageous for the composition to promote the formation of collagen and elastin of skin, soft tissues, etc., and the anti-aging effect. If the content of potassium ions in the composition is outside the range of the present invention, the resulting composition according to the present invention, which promotes the formation of collagen and elastin of skin, soft tissues, etc., and reduces the anti-aging effect, is reduced.
The buffer may adjust the pH and/or osmotic pressure of the composition, may be in any suitable amount, may be in any suitable concentration in aqueous solution, to facilitate providing the composition with an osmotic pressure, fluidity and/or stability suitable for dermal use, allowing the composition to be used for injection into the skin or soft tissue of the human body, or allowing the composition to be used for topical application, such as by application, plaster, etc. The buffering agent may include sodium chloride, hydrochloric acid,sodium hydroxide, sodium dihydrogen phosphate or a hydrate thereof, disodium hydrogen phosphate or a hydrate thereof; or the buffer is an aqueous solution prepared from one or more of sodium chloride, hydrochloric acid, sodium dihydrogen phosphate or hydrate thereof, disodium hydrogen phosphate or hydrate thereof and sodium hydroxide. In some embodiments, the buffer comprises disodium hydrogen phosphate or a hydrate thereof. In some embodiments, the buffer comprises sodium dihydrogen phosphate or a hydrate thereof. In some embodiments, the buffer comprises disodium hydrogen phosphate or a hydrate thereof, and further comprises sodium dihydrogen phosphate or a hydrate thereof. In some embodiments, the buffer comprises or is sodium dihydrogen phosphate and sodium hydroxide. In some embodiments, the buffer is disodium hydrogen phosphate (Na 2 HPO 4 ) Or a hydrate thereof and sodium dihydrogen phosphate (NaH) 2 PO 4 ) Or a hydrate thereof. In some embodiments, the buffer pH is 6.8-7.6. In some embodiments, the buffer pH is 7.0,7.2, or 7.4.
In some embodiments, the aforementioned composition consists essentially of: hyaluronic acid and/or sodium hyaluronate, L-carnosine, glycine or a salt thereof, proline or a salt thereof, lysine or a salt thereof, valine or a salt thereof, alanine or a salt thereof, leucine or a salt thereof, and a buffer; the buffer comprises one or more of sodium chloride, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate or hydrate thereof, disodium hydrogen phosphate or hydrate thereof; the pH of the composition is from 6.8 to 7.6. In some embodiments, the aforementioned composition consists essentially of: hyaluronic acid and/or sodium hyaluronate, L-carnosine, glycine, proline, lysine, valine, alanine, leucine and buffering agents; the buffer comprises one or more of sodium chloride, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate or hydrate thereof, disodium hydrogen phosphate or hydrate thereof; the pH of the composition is 6.8-7.6; can better stimulate and promote the formation of elastin and collagen and the cell regeneration.
In some embodiments, the aforementioned composition consists essentially of: hyaluronic acid and/or sodium hyaluronate, L-carnosine, glycine, proline, lysine, valine, alanine, leucine and buffering agents; wherein the buffer comprises one or more of sodium chloride, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate or a hydrate thereof, and disodium hydrogen phosphate or a hydrate thereof; the pH of the composition is 6.8-7.6; the molecular weight of the hyaluronic acid or sodium hyaluronate is 130-160 kilodaltons; can better stimulate and promote the formation of elastin and collagen and the cell regeneration.
In some embodiments, the aforementioned composition is formulation 1 or formulation 2, consisting essentially of the following components in amounts, respectively, calculated on the volume of the composition:
the method comprises the steps of carrying out a first treatment on the surface of the Wherein, the molecular weight of the sodium hyaluronate is 130 ten thousand-160 ten thousand daltons; the potassium ion content is lower than 0.008mg/mL.
The composition provided by the invention can be an injection for injection on the skin, soft tissues and other parts of a human body, can also be an external preparation for external application, is applied on the skin of the human body or is stuck on the skin of the human body.
The invention also provides a method for preparing the composition. The method can simply obtain the composition of the invention, so that the components in the composition cannot be degraded and the like, which are not in accordance with the quality requirements.
A method of preparing the aforementioned composition comprising: hyaluronic acid and/or a pharmaceutically acceptable salt thereof, carnosine, amino acid or a pharmaceutically acceptable salt thereof, and a buffer are mixed, optionally with water to a sufficient amount, and then sterilized to obtain a composition.
In some embodiments, a method of preparing the aforementioned composition comprises: hyaluronic acid and/or a pharmaceutically acceptable salt thereof, carnosine, amino acid or a pharmaceutically acceptable salt thereof are mixed with water, and then mixed with a buffer, optionally water is added to a sufficient amount, and then sterilized to obtain a composition.
In some embodiments, a method of preparing the aforementioned composition comprises: or comprises: mixing buffer, carnosine and amino acid or pharmaceutically acceptable salt thereof, optionally sterilizing, mixing with hyaluronic acid or pharmaceutically acceptable salt thereof, and sterilizing to obtain the composition.
In some embodiments, a method of preparing the aforementioned composition comprises: adding hyaluronic acid or pharmaceutically acceptable salt thereof, carnosine, amino acid or pharmaceutically acceptable salt thereof into an aqueous buffer solution serving as a solvent under stirring, fully mixing, optionally adjusting pH or adding water to a sufficient amount, and then sterilizing to obtain the composition.
In some embodiments, a method of preparing the aforementioned composition comprises: sodium hyaluronate, L-carnosine, glycine, proline, lysine, valine, alanine and leucine are mixed with water, a buffer is added, the pH is adjusted to 6.8-7.6, optionally water is added to a sufficient amount, and then the composition is sterilized.
In some embodiments, a method of preparing the aforementioned composition comprises: sodium hyaluronate, L-carnosine, glycine, proline, lysine, valine, alanine and leucine are mixed with a buffer solution, the pH is controlled to 6.8-7.6, optionally water is added to a sufficient amount, and then sterilized to obtain a composition.
In some embodiments, a method of preparing the aforementioned composition comprises: mixing L-carnosine, glycine, proline, lysine, valine, alanine and leucine with buffer solution, controlling pH to 6.8-7.6, mixing with sodium hyaluronate, optionally adding water to sufficient amount, and sterilizing to obtain composition.
The sterilization can be carried out by filtration sterilization using a microporous filter membrane, and other modes such as terminal sterilization, irradiation sterilization and the like can also be used. In some embodiments, the bacteria are filtered using a 0.22 μm filter.
In order to obtain the composition which can be used for injection and meet the sterile quality standard required by Chinese pharmacopoeia injection, the composition can also be prepared in a sterile environment or by adopting sterile production processes such as sterile operation and the like.
In some embodiments, a method of preparing the aforementioned composition comprises: adding L-carnosine, glycine, proline, lysine, valine, alanine and leucine into an aqueous solution of a buffering agent with pH of 6.8-7.6 serving as a solvent under stirring, fully mixing, adding sodium hyaluronate, fully mixing, preparing a solution containing 5mg/mL of sodium hyaluronate, 1.5mg/mL of L-carnosine, 0.1mg/mL of glycine, 0.1mg/mL of proline, 0.1mg/mL of lysine, 1mg/mL of valine, 1mg/mL of alanine and 1mg/mL of leucine, and filtering and sterilizing by a 0.22 mu m filter membrane to obtain the composition.
In some embodiments, a method of preparing the aforementioned composition comprises: adding L-carnosine, glycine, proline, lysine, valine, alanine and leucine into an aqueous solution of a buffering agent with pH of 6.8-7.6 serving as a solvent under stirring, fully mixing, then adding sodium hyaluronate, fully mixing, preparing into a solution containing 5mg/mL of sodium hyaluronate, 1.5mg/mL of L-carnosine, 0.3mg/mL of glycine, 0.1mg/mL of proline, 0.1mg/mL of lysine, 1mg/mL of valine, 0.5mg/mL of alanine and 1mg/mL of leucine, and then filtering and sterilizing by a 0.22 mu m filter membrane to obtain the composition.
The method for preparing the composition can prepare the composition into injection or external preparation.
The method for preparing the composition is simple, convenient and controllable, is easy to operate, can simply obtain the composition, and can prevent degradation, denaturation and other phenomena of non-compliance quality of the components in the composition.
Drawings
FIG. 1 shows the quantitative analysis of elastin secretion for C-series samples, where p <0.05;
FIG. 2 shows the effect of the extract of the HA series of samples on the elastin secretion of HFF-1 cells (24 h), representing p <0.05;
FIG. 3 shows the effect of a sample of the composition (extract) on HFF-1 cell proliferation (24 h, 48h, 72 h);
FIG. 4 shows the effect of a sample of the composition (extract) on HFF-1 cell proliferation (24 h, 48h, 72h, morphology);
FIG. 5 shows the effect of a sample of the composition (leach liquor) on HFF-1 cell migration (0 h, 12h, 24 h);
FIG. 6 shows the effect of a sample of the composition (extract) on HFF-1 cell collagen secretion (24 h);
FIG. 7 shows the effect of a sample of the composition (extract) on the secretion of elastin by HFF-1 cells (48 h), representing p <0.05;
FIG. 8 shows the effect of a composition sample (extract) on SOD activity of oxidative damage model cells, wherein a represents p <0.05 compared to control group, b represents p <0.05 compared to model group;
figure 9 shows the effect of a composition sample (leach) on MDA levels in oxidative damage model cells, where a represents comparison to control, p <0.05, b represents comparison to model, and p <0.05.
Detailed Description
The terms "comprising" or "including" are used in an open-ended fashion, i.e., including the teachings described herein, but not excluding additional aspects.
In the present invention, "consisting essentially of" means that the composition may further contain unavoidable impurities and water.
In the present invention, ppm means parts per million.
In the present invention, optional means may or may not be present.
In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "implementations," "specific examples," or "some examples" and the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In the present invention, room temperature is ambient temperature, at 20 ℃ to 30 ℃, or 22 ℃ to 28 ℃, or 25 ℃.
In the present invention, the molecular weight of hyaluronic acid or a pharmaceutically acceptable salt thereof is calculated on a weight average molecular weight basis.
In the invention, the content calculation method of the components comprises the following steps: the weight of the components in the composition per volume of the composition, e.g., sodium hyaluronate content of 5mg/mL, means that 5mg of sodium hyaluronate is contained per 1mL of the composition.
In the composition, the concentration of hyaluronic acid is calculated based on the total mass of hyaluronic acid or sodium hyaluronate, calculated as hyaluronic acid if hyaluronic acid is contained in the sample, calculated as sodium hyaluronate if sodium hyaluronate is contained in the sample, calculated as both amounts.
In the invention, the experimental methods of various test experiments of the sample to be tested are described below:
effect on HFF-1 cell proliferation: after HFF-1 cells were prepared into a cell suspension in DMEM high-sugar medium (containing 15% fetal bovine serum), the cells were inoculated into 96-well plates with an amount of 2X 10 cells per well 4 After the cells adhere to the wall, the original culture medium is discarded; adding each sample to be tested according to a grouping design, wherein each group is provided with 3 parallel holes; 37 ℃ and 5% CO 2 After culturing for 24 hours, 48 hours or 72 hours under the condition, the proliferation rate of the cells is detected by a CCK-8 method, and the cell morphology is observed by an inverted microscope.
Principle of CCK-8 method for cell proliferation assay: CCK-8, chemical name 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazolium monosodium salt, can be reduced to a water-soluble orange yellow formazan product by dehydrogenase in cell mitochondria in the presence of electron carrier PMS. For the same kind of cells cultured in parallel, the number of living cells is in direct proportion to the formazan generation amount, the faster the cell proliferation is, the better the state is, the more the formazan conversion amount is, the darker the color is, the light absorption value at 450nm is measured, the generated formazan amount is in direct proportion to the number of living cells, and the characteristics can be used for cell proliferation and toxicity detection analysis.
Effect on HFF-1 cell collagen secretion: with reference to GB/T16886.5-2017 standard, the effect of samples on HFF-1 cell collagen secretion was evaluated by an enzyme-linked immunosorbent assay (ELISA). Inoculating HFF-1 cells on a 6-hole plate, discarding an original culture medium after the cells adhere to the wall, adding each sample to be tested according to a grouping design, and arranging 3 parallel holes in each group; 37 ℃ and 5% CO 2 Culturing for 24h or 48h, performing pancreatin digestion to collect cells, performing ultrasonic disruption, centrifuging for 20 min at 3000 rpm, collecting supernatant, performing subsequent operation according to the instruction of the type I collagen ELISA detection kit, and calculating the type I collagen level of each group based on the collagen level of the control group.
Effect on HFF-1 cell elastin secretion: with reference to GB/T16886.5-2017 standard, the effect of samples on HFF-1 cell elastin secretion was evaluated by an enzyme-linked immunosorbent assay (ELISA). Inoculating HFF-1 cells on a 6-hole plate, discarding an original culture medium after the cells adhere to the wall, adding each sample to be tested according to a grouping design, and arranging 3 parallel holes in each group; 37 ℃ and 5% CO 2 Culturing for 24 hours or 48 hours under the condition; pancreatin digestion and cell collection, ultrasonic disruption, centrifugation at 3000 rpm for 20 minutes, and supernatant collection; the elastin concentration of each group was determined using an elastin ELISA assay kit, and the elastin levels of each group were calculated based on the control group values.
Effects on HFF-1 cell antagonism of oxidative damage function: with reference to GB/T16886.5-2017 standard, the functional activity of the sample in antagonizing oxidative damage is evaluated by adopting two common oxidative damage biochemical indexes of SOD (superoxide dismutase) and MDA (malondialdehyde). Hydrogen peroxide (H) is established according to classical methods 2 O 2 ) Oxidative damage model using 800. Mu. Mmol/L H 2 O 2 And (5) molding. HFF-1 cells were seeded in 6-well plates at a cell density of 5X 10 5 After cells are attached to each other, the original culture medium is discarded, and each sample to be tested is added according to the grouping design, wherein the temperature is 37 ℃ and the CO content is 5 percent 2 Culturing for 24 hours under the condition; pancreatin digestion and collectionCells were sonicated, centrifuged at 2500 rpm for 10 min, and the supernatant was collected; and detecting the levels of SOD and MDA in each group according to the operation requirements of the SOD kit and the MDA kit.
Effect on HFF-1 cell migration: with reference to the GB/T16886.5-2017 standard, the effect of the sample on HFF-1 cell migration was evaluated using a cell scratch assay. Taking HFF-1 cells in logarithmic growth phase according to 2×10 5 Cell count/well was seeded in 6-well plates at 37℃with 5% CO 2 Culturing for 24h under the condition, streaking in a culture plate hole by using a 200 mu L pipette tip, and washing for 3 times by using PBS; adding each sample to be tested according to a grouping design, wherein each group is provided with 3 parallel holes; 37 ℃ and 5% CO 2 Culturing for 12 hours or 24 hours under the condition, and observing the migration condition of the cells under a microscope.
In order to better understand the technical solution of the present invention, the following further discloses some non-limiting examples, which are further described in detail.
In the examples below, reference is made to the following description of the preparation of the reagents/materials in part.
The preparation method of the complete culture medium comprises the following steps: DMEM high sugar medium +15% FBS (fetal bovine serum) +1% PS (penicillin 100U/mL and streptomycin 100 μg/mL).
PBS: phosphate buffer (NaH used) 2 PO 4 And Na (Na) 2 HPO 4 Formulated), 0.01mol/L, pH 7.0.
H 2 O 2 The preparation method of (hydrogen peroxide) solution comprises the following steps: diluting 30% hydrogen peroxide with PBS (pH 7.2-7.4) to 3% H 2 O 2 Filtering, sterilizing and packaging the solution, and preserving at 4 ℃;800 mu mol/L H 2 O 2 : 10 μl of 3% H was taken 2 O 2 Diluting the solution to 100 μl, and adding 2mL of complete culture medium into 16.5 μl; 400 mu mol/L H 2 O 2 : 1mL of 800. Mu. Mol/L H was taken 2 O 2 Diluting with complete culture medium at equal ratio to obtain the final product; 200 mu mol/L H 2 O 2 : 1mL of 400. Mu. Mol/L H was taken 2 O 2 Diluting with complete culture medium at equal ratio.
The preparation method of the leaching liquor of the sample comprises the following steps: leaching solution preparation is carried out by referring to a sample preparation method in GB/T16886.12-2017 medical instrument biological evaluation sample preparation and reference material: taking 0.1g of sample, adding 1mL of complete culture medium, shaking and mixing uniformly, and incubating in an incubator at 37 ℃ for 24 hours, and preparing at present.
A first part: the effect of each of the various components on various functions/characteristics of HFF-1 cells was tested separately, and the sample groupings, numbers, components, concentrations and formulation methods involved are described below.
The samples were grouped in the following table.
Grouping of samples
| Sample of | Sample information |
| Control group | Complete medium |
| A series | L-carnosine |
| B series | Glycine, proline and lysine in a mass ratio of 1:1:1 |
| C series | Valine, alanine and leucine in a mass ratio of 1:1:1 |
| HA series | Sodium hyaluronate with high, medium and low molecular weight |
The A-C series samples were formulated and their concentrations are shown in the tables below.
| A series sample number | A1.0 | A1.5 | A2.0 |
| A series mother liquor volume (45 mg/mL, μL) | 33.3 | 50 | 67 |
| DMEM high sugar medium+15% fbs volume (mL) | 1.47 | 1.45 | 1.43 |
| Total volume (mL) | 1.5 | 1.5 | 1.5 |
| A series sample concentration (mg/mL) | 1.0 | 1.5 | 2.0 |
| Serial number of B series samples | B0.1 | B0.5 | B1.0 | B2.0 | B3.0 |
| B series mother liquor volume (15 mg/mL, μL) | 10 | 50 | 100 | 200 | 300 |
| DMEM high sugar medium+15% fbs volume (mL) | 1.49 | 1.45 | 1.4 | 1.3 | 1.2 |
| Total volume (mL) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| B series sample concentration (mg/mL) | 0.1 | 0.5 | 1.0 | 2.0 | 3.0 |
| C series sample numbering | C0.1 | C0.5 | C1.0 | C2.0 | C3.0 |
| C series mother liquor volume (15 mg/mL, μL) | 10 | 50 | 100 | 200 | 300 |
| DMEM high sugar medium+15% fbs volume (mL) | 1.49 | 1.45 | 1.4 | 1.3 | 1.2 |
| Total volume (mL) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| C series sample concentration (mg/mL) | 0.1 | 0.5 | 1.0 | 2.0 | 3.0 |
Wherein, the preparation method of A series mother liquor comprises the following steps: mixing L-carnosine 78.6mg with DMEM high-sugar culture medium 1.75mL to prepare 45mg/mL solution; the preparation method of the B series mother liquor comprises the following steps: 15mg of glycine, proline and lysine are respectively combined with 1mL of DMEM high-sugar culture medium to prepare 15mg/mL of solution; the preparation method of the C series mother solution comprises the following steps: valine, alanine and leucine were each mixed at 15mg with 1mL of DMEM high-sugar medium to prepare a 15mg/mL solution.
The preparation method of HA (sodium hyaluronate) series samples comprises the following steps: phosphate buffer (Na) at 0.01mol/L ph=7.0 2 HPO 4 /NaH 2 PO 4 ) Dissolving sodium hyaluronate to obtain a series of samples, wherein the molecular weight of the sodium hyaluronate in the high molecular weight group (H) is 150 ten thousand daltons, the molecular weight of the sodium hyaluronate in the medium molecular weight group (M) is 80-100 ten thousand daltons, and the molecular weight of the sodium hyaluronate in the low molecular weight group (L) is 30-50 ten thousand daltons, and the specific reference is given in the following table.
Example 1: testing the effect of A-series, B-series, C-series and HA-series samples (leaches) on HFF-1 cell proliferation
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control group, concentration groups of A series-C series, leaching liquor of HA series samples.
Experimental results:
1) B series and C series high concentration samples (3 mg/mL) have different degrees of inhibition on HFF-1 at different time points; the low concentration sample in the B series (less than or equal to 0.5 mg/mL) has no inhibition effect on HFF-1 cell proliferation basically, wherein, the B0.1 sample can obviously promote cell proliferation at 48h and 72h, and the B0.5 sample can obviously promote cell proliferation at 72h, but shows slight inhibition effect at 24h and 48h; the low-concentration samples (less than or equal to 2 mg/mL) in the C series have low inhibition on HFF-1 cell proliferation, and cytotoxicity is 0/I grade, so that the requirement of injection implantation is met;
2) The cytotoxicity of the A series samples (L-carnosine) at different concentrations and at different time points is 0 grade, wherein compared with the control group, the A1.0 and the A2.0 samples, the A1.5 samples can obviously promote the proliferation of HFF-1 cells at 72 h.
3) The inhibition of HFF-1 cell proliferation by the medium molecular weight group (M) in the HA sample (sodium hyaluronate) was higher than that of the high molecular weight (H) and low molecular weight groups (L), and H0.5 and H0.8 samples promoted cell proliferation at 72H.
From the results, it was found that different concentrations of amino acids have different effects on HFF-1 cell proliferation. Sodium hyaluronate of different molecular weights has different effects on HFF-1 cell proliferation.
Example 2: testing of the effects of B-series samples and HA-series samples (leaches) on the secretion of collagen by HFF-1 cells
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control group, B series concentration groups, leaching liquor of HA series samples.
Experimental results:
1) The B series can promote the collagen secretion of fibroblasts at medium and low concentrations, and inhibit the collagen secretion at higher concentrations (such as 3.0 mg/mL);
2) At 24h, the influence of the B series on collagen secretion is not obvious; at 48h, it showed significant stimulation of collagen secretion activity: the 0.1mg/mL-2.0mg/mL concentration samples of the B series can obviously stimulate the collagen secretion of cells, the 0.1mg/mL concentration sample has the best effect, the 0.5mg/mL concentration sample has a slightly inferior effect, and the high concentration sample of 3.0mg/mL has slightly inhibited collagen secretion, but the influence is not obvious.
3) In the HA sample (sodium hyaluronate), the high molecular weight group (H) HAs the strongest collagen secretion promotion effect, and the low molecular weight group (L) is slightly better than the medium molecular weight group (M); the effect of the samples with the same molecular weight and different concentrations on collagen secretion is basically unchanged.
Example 3: testing of the influence of C-series samples and HA-series samples (leaches) on the secretion of elastin by HFF-1 cells
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control group, concentration group of C series, leaching liquor of HA series sample.
Experimental results:
1) Culturing for 24 hours, wherein the C series has no obvious influence on the morphology of HFF-1 cells at different concentrations, and the phenomena of cell swelling, death and the like are not seen in a high-concentration group;
2) The C series can obviously promote the elastin secretion at different concentrations, and the promoting effect of the C series concentration on the elastin secretion is positively correlated with the concentration within the range of medium-low concentration gradient of 0.1-1.0mg/mL, and the promoting effect of the C series concentration on the elastin secretion is most obvious at the concentration of 1.0mg/mL (see figure 1).
3) At 24H, HA samples (sodium hyaluronate) generally tended to stimulate elastin secretion, with H0.5 samples in the high molecular weight group having very significant promotion of elastin secretion, other samples also significantly stimulated, promoted elastin secretion, with M0.6 > L0.5 > H0.8, M0.3 samples slightly inhibited elastin secretion in sequence, but not significantly (see fig. 2).
Example 4: testing the effect of A-series samples and HA-series samples (leaches) on the function of antagonizing oxidative damage to HFF-1 cells
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control group, A series concentration groups, leaching liquor of HA series samples.
Experimental results:
1) The A series has remarkable antagonism on oxidative damage caused by hydrogen peroxide at different concentration gradients and different time points, and can increase the activity of endogenous antioxidant enzyme SOD (superoxide dismutase) and reduce the content of peroxidation product MDA (malondialdehyde); wherein, the effect of improving the activity of SOD by the combination of the concentration A of 1.5mg/mL is most remarkable;
2) In each concentration gradient, the oxidation antagonistic activity of the A1.5 sample is most prominent, which suggests that the antagonistic oxidative stress effect of the L-carnosine is optimal at the concentration of 1.5 mg/mL.
From the experimental results of the first part, it is known that the combination of amino acids and their concentration/content, the molecular weight of sodium hyaluronate and its concentration/content have a certain influence on various properties/characteristics of cells.
Based on the results of the first part of the experiment, the inventors devised samples of different combinations in order to obtain a composition advantageous for anti-aging, and conducted the second part of the experiment.
A second part: testing the effect of different combinations of samples on various functions/properties of HFF-1 cells, respectively, involving sample groupings, numbers, components, concentrations, and formulation methods, see the description below; wherein the control group (blank group) is complete medium.
Control group 1-control group 4 sample preparation method: by NaH 2 PO 4 And Na (Na) 2 HPO 4 The aqueous solution (0.01 mol/L, pH is 7.0, weigh each 0.1mol, constant volume to 1L, dilute to 0.01mol/L, get final product) of the mixture as solvent, add each amino acid and L-carnosine first, mix evenly, make the concentration of each component reach the design value, then filter and sterilize through the 0.22 μm filter membrane; then adding sodium hyaluronate, continuously stirring until completely dissolving, standing at 4deg.C for 12 hrAt this time, 0.22 μm filters were used for sterilization, and samples of each control group were obtained.
Pre-experiment example 1: effect of control samples (leaches) on HFF-1 cell proliferation
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, control 1-control 4 leaches.
Experimental results:
each group of samples showed little inhibition of proliferation of HFF-1 cells and no effect on cell morphology, demonstrating suitability for injection.
Pre-experiment example 2: effect of control samples (leaches) on HFF-1 cell collagen secretion
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, control 1-control 4 leaches.
Experimental results: the collagen levels of the control group 1-the control group 4 are higher than those of the control group, and the order of the collagen levels of the groups is as follows: control group < control group 2< control group 4< control group 3< control group 1.
Sample formulations of composition 1 and composition 2:
| composition sample number | Composition 1 | Composition 2 |
| Component (A) | Formula 1 (mg/mL) | Formula 2 (mg/mL) |
| Sodium hyaluronate | 5.0 | 5.0 |
| L-carnosine | 1.5 | 1.5 |
| Valine (valine) | 1.0 | 1.0 |
| Leucine (leucine) | 1.0 | 1.0 |
| Alanine (Ala) | 1.0 | 0.5 |
| Glycine (Gly) | 0.1 | 0.3 |
| Lysine | 0.1 | 0.1 |
| Proline (proline) | 0.1 | 0.1 |
| Buffering agents | NaH 2 PO 4 And Na (Na) 2 HPO 4 Is an aqueous solution of (a) | NaH 2 PO 4 And Na (Na) 2 HPO 4 Is an aqueous solution of (a) |
| pH | 6.8-7.6 | 6.8-7.6 |
Preparation method of composition 1 and composition 2: according to formula 1 and formula 2, respectively, with NaH 2 PO 4 And Na (Na) 2 HPO 4 The aqueous solution (0.01 mol/L, pH is 7.0, weigh each 0.1mol, constant volume to 1L, dilute to 0.01mol/L, get final product) of the mixture as solvent, add each amino acid and L-carnosine first, mix evenly, make the concentration of each component reach the formula requirement value, filter and sterilize through the 0.22 μm filter membrane; then sodium hyaluronate was added and stirred continuously until completely dissolved, and allowed to stand at 4℃for 12 hours, and then filtered through a 0.22 μm filter to be sterilized, to obtain composition 1 and composition 2, respectively.
The preparation method of the composition 3 comprises the following steps: according to the formulation and preparation method of composition 1, a small amount of potassium dihydrogen phosphate aqueous solution which was filtered and sterilized by a 0.22 μm filter was added to the obtained composition so that the potassium ion content in the composition was 0.008mg/mL, to obtain composition 3.
Example 5: effect of composition samples (leaches) on HFF-1 cell proliferation
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, composition 1 extract, composition 2 extract.
Experimental results:
composition 1 and composition 2 have little inhibition effect on proliferation of HFF-1 cells and no effect on cell morphology (see fig. 3 and 4), and the composition 1 and composition 2 provided by the invention have good cell compatibility and are suitable for injection.
Example 6: effect of composition sample (leaching solution) on HFF-1 cell migration
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, composition 1 extract, composition 2 extract.
Experimental results: compared with the control group, the compositions 1 and 2 can obviously promote the migration of HFF-1 to the scratch area (see figure 5), which shows that the compositions 1 and 2 provided by the invention can obviously improve the migration capacity of skin fibroblasts and promote the regeneration and repair of skin tissues.
Example 7: effect of composition samples (leaches) on HFF-1 cell collagen secretion
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, composition 1 extract, composition 2 extract.
Experimental results: the collagen levels of both composition 1 and composition 2 were significantly higher than the control (see fig. 6), indicating that composition 1 and composition 2 significantly promoted collagen secretion by skin fibroblasts.
Example 8: effect of composition samples (leaches) on HFF-1 cell elastin secretion
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control, control 1, composition 2, composition 3.
Experimental results: the elastin levels of each of composition 1, composition 2, and composition 3 were significantly higher than those of control and control 1, indicating that composition 1, composition 2, and composition 3 significantly promote elastin secretion from skin fibroblasts, wherein composition 3 was attenuated relative to composition 1 or composition 2 (see fig. 7).
Example 9: effect of composition samples (leaches) on HFF-1 cell antagonistic oxidative damage function
The experimental method and the sample preparation method are carried out according to the experimental method and the sample preparation method, wherein the experimental groups are as follows: control group, model group (800. Mu. Mmol/L H) 2 O 2 Complete medium of (C) 800. Mu. Mmol/L H) 2 O 2 The extract of composition 1 of (2) contained 800. Mu. Mmol/L H 2 O 2 The leaching solution of composition 2 of (2).
Experimental results: the SOD activity of the model group is obviously inhibited, the MDA level is obviously increased, which shows that in H 2 O 2 The risk of oxidative damage of HFF-1 cells under stress is significantly increased; composition 1 and composition 2 can obviously reduce SOD inhibition rate and MDA level, and resist oxidation injuryThe significantly enhanced ability of (see fig. 8, 9) indicates that composition 1, composition 2 significantly improved the ability of the cells to antagonize oxidative damage, reducing the risk of oxidative stress damage.
According to the results of the examples, the sodium hyaluronate composition solution with better anti-aging effect and stability can be obtained by adopting the sodium hyaluronate with the molecular weight and the amino acid with the content range.
While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications can be made to the embodiments or applications described herein, or in the appropriate combination, to practice and use the techniques of this invention, within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, implement and/or apply the techniques of the present invention with appropriate modifications of the conditions/parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.
Claims (7)
1. A composition for stimulating elastin formation, which consists of, based on the volume of the composition: hyaluronic acid and/or pharmaceutically acceptable salts thereof 5mg/ml, carnosine 1.5mg/ml, valine 1.0mg/ml, leucine 1.0mg/ml, lysine 0.1mg/ml, proline 0.1mg/ml, alanine 0.5-1 mg/ml, glycine 0.1-0.3 mg/ml, and buffering agents; the molecular weight of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 130-160 kilodaltons; the buffering agent is sodium dihydrogen phosphate or hydrate thereof, and disodium hydrogen phosphate or hydrate thereof; the pH of the composition is from 6.8 to 7.6.
2. The composition of claim 1, wherein the potassium ion content is no more than 0.008mg/mL, calculated on the volume of the composition.
3. The composition of claim 1, wherein the pharmaceutically acceptable salt of hyaluronic acid is sodium hyaluronate, zinc hyaluronate, calcium hyaluronate, or a combination thereof.
4. A composition according to any one of claims 1 to 3 wherein the hyaluronic acid or pharmaceutically acceptable salt thereof has a molecular weight of 145-155 kilodaltons.
5. A composition according to any one of claims 1 to 3, which consists of, based on the volume of the composition: sodium hyaluronate 5mg/ml, L-carnosine 1.5mg/ml, valine 1.0mg/ml, leucine 1.0mg/ml, lysine 0.1mg/ml, proline 0.1mg/ml, alanine 1.0mg/ml, glycine 0.1 mg/ml; or alternatively
The composition comprises the following components: sodium hyaluronate 5mg/ml, L-carnosine 1.5mg/ml, valine 1.0mg/ml, leucine 1.0mg/ml, lysine 0.1mg/ml, proline 0.1mg/ml, alanine 0.5mg/ml, glycine 0.3 mg/ml.
6. A process for preparing the composition of any one of claims 1-5, characterized by: the hyaluronic acid and/or pharmaceutically acceptable salts thereof, carnosine, various amino acids or pharmaceutically acceptable salts thereof and buffer are mixed, water is added to a sufficient amount, and then sterilization is carried out, so that the composition is obtained;
or: mixing hyaluronic acid and/or pharmaceutically acceptable salts thereof, carnosine, various amino acids or pharmaceutically acceptable salts thereof with water, then mixing with a buffer, adding water to a sufficient amount, and then sterilizing to obtain the composition;
or: mixing buffer, carnosine and various amino acids or pharmaceutically acceptable salts thereof, mixing with hyaluronic acid or pharmaceutically acceptable salts thereof, and sterilizing to obtain the composition.
7. The method of claim 6, wherein: the sterilization is microporous filter membrane sterilization, terminal sterilization or irradiation sterilization.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104189952A (en) * | 2014-08-05 | 2014-12-10 | 北京爱美客生物科技有限公司 | Injection for correcting skin wrinkles and preparation method thereof |
| CN106999390A (en) * | 2014-12-04 | 2017-08-01 | 专业营养股份公司 | The composition based on amino acid recovered for fibrous elasticity albumen in skin connective tissue |
| CN113260367A (en) * | 2020-09-02 | 2021-08-13 | 弹性实验室有限公司 | Composition for promoting elastin and collagen biosynthesis in connective tissue |
| CN114917409A (en) * | 2022-06-02 | 2022-08-19 | 瑞玞生物医学(深圳)有限公司 | Composition based on hyaluronic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104189952A (en) * | 2014-08-05 | 2014-12-10 | 北京爱美客生物科技有限公司 | Injection for correcting skin wrinkles and preparation method thereof |
| CN106999390A (en) * | 2014-12-04 | 2017-08-01 | 专业营养股份公司 | The composition based on amino acid recovered for fibrous elasticity albumen in skin connective tissue |
| CN113260367A (en) * | 2020-09-02 | 2021-08-13 | 弹性实验室有限公司 | Composition for promoting elastin and collagen biosynthesis in connective tissue |
| CN114917409A (en) * | 2022-06-02 | 2022-08-19 | 瑞玞生物医学(深圳)有限公司 | Composition based on hyaluronic acid |
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