CN117919113A - Anti-aging composition and application thereof - Google Patents
Anti-aging composition and application thereof Download PDFInfo
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- CN117919113A CN117919113A CN202410165392.0A CN202410165392A CN117919113A CN 117919113 A CN117919113 A CN 117919113A CN 202410165392 A CN202410165392 A CN 202410165392A CN 117919113 A CN117919113 A CN 117919113A
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Abstract
The invention belongs to the technical field of cosmetics, and discloses an anti-aging composition and application thereof. The active polypeptide composition SA-LFP, SPP19, AOP and the vitronectin are combined to form the anti-aging composition, and the active polypeptide composition can promote the absorption of the vitronectin which is an active ingredient in the anti-aging composition by a human body and enhance the anti-aging effect; solves the problems of poor percutaneous absorption and low availability of active ingredients of the existing product. After the bioactive peptide composition is compounded with the vitriol, the invention generates remarkable synergistic effect in the aspect of antioxidation effect and delays skin aging. The anti-aging composition can reduce the addition amount of the vitreous color due to the synergistic effect, reduce the irritation and reduce the inflammation risk caused by the use of the vitreous color.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to an anti-aging composition and application thereof.
Background
The skin is the largest and most important organ of the human body, covers the whole surface of the human body, and consists of epidermis, dermis and subcutaneous tissue, wherein the epidermis layer and the dermis layer play an important role. However, over time, the expression levels of the epidermis and dermis of the skin change due to various factors such as aging, internal and external environments, etc., and the expression of molecules involved in cell adhesion to the basal layer decreases, leading to a decrease in skin barrier function; the composition of the extracellular matrix of the dermis layer is also changed in a degenerative way along with the aging process, collagen is greatly lost, the activity of fibroblasts is weakened, the synthesized collagen is gradually reduced, the area of collagen fibers is also reduced, thus leading to loose reticular structure of the dermis layer of the skin, reduced moisture and toughness of skin tissues, and the skin is immediately subjected to the problems of dryness, roughness, wrinkles, slackening, damage and the like, thereby accelerating the skin aging. Skin aging is a complex and diverse process that is affected by both genetic, environmental and internal and external factors. In addition to changes in appearance, skin aging also reflects the health status of the body interior. Slower epidermal turnover, impaired barrier and reduced wound healing quality all lead to an increased risk of developing chronic diseases of the elderly.
In recent years, more and more functional skin care products have been developed to delay skin aging. However, most of the commercially available anti-aging skin care products improve the skin aging state by simply adding exogenous active substances (such as moisturizers, free radical scavengers, sunscreens, etc.), and the actual anti-aging effect is not satisfactory. In addition, the components of the skin care product can only be absorbed by the surface layer of the skin to a great extent, and can not really enter the deep layer of the skin so as to achieve the purposes of permanently updating and supplementing collagen, thus the skin care product can not better play the role of delaying skin aging. Currently, many anti-aging products such as glass color factor, A alcohol and the like are popularized in the market. The vitronectin, i.e., hydroxypropyl tetraoxypyrantriol, is a natural antioxidant and is one of the three anti-aging agents. The vitronectin can promote secretion of collagen and GAGs (glycosaminoglycans), which are extracellular matrix comprising hyaluronic acid, chondroitin sulfate, heparin, etc. However, the glass color has some defects, such as larger molecular structure, relatively lower permeability and lower usage amount, and the glass color has high cost, and the common product is only added in a small amount, so that the effect is limited. In addition, the stability of the vitriol is poor, the vitriol is extremely easy to lose efficacy under air and illumination and is not suitable for allergic skin groups, allergic reaction is easy to occur in the using process, and uncomfortable symptoms .(Vassal-StermannE,Duranton A,Black AF,Azadiguian G,Demaude J,Lortat-Jacob H,Breton L,Vivès RR.A New C-Xyloside induces modifications of GAG expression,structure and functional properties.PLoS One.2012;7(10):e47933.)(Sok J,Pineau N,Dalko-Csiba M,Breton L,Bernerd F.Improvement of the dermal epidermal junction in human reconstructed skin by a new c-xylopyranoside derivative.Eur JDermatol.2008May-Jun;18(3):297-302.) such as dermatitis, erythema and the like are caused. A alcohol produces innumerable physiological effects by binding to 6a acid receptors on cells after conversion to a acid in vivo, including defense against inflammation, regulating growth and differentiation of epidermal cells, promoting collagen production, and regulating cell regeneration rate. The skin care composition has strong antioxidation, can resist the generation of skin cell free radicals, reduce the damage of the free radicals to the skin and delay the skin aging; however, the alcohol A has larger irritation and is not suitable for sensitive skin, the general population needs to establish a tolerance stage, strict requirements are imposed on the use times and dosage, and excessive use easily causes thinning of skin horny layer and causes uncomfortable symptoms such as burning sensation, erythema, swelling and the like; moreover, the A alcohol has poor stability and photosensitivity, and can generate phototoxicity under the irradiation of ultraviolet rays after the face is put on, so that the A alcohol is generally recommended to be used at night. From these results, it was found that the above-mentioned compounds do not exert an anti-aging effect with a long-lasting, safe and high-efficiency .(Zasada M,Budzisz E.Retinoids:active molecules influencing skin structure formation in cosmetic and dermatological treatments.Postepy Dermatol Alergol.2019Aug;36(4):392-397.)
The vitrine and the A alcohol are gold components for resisting aging, are widely favored by the technical field of skin care products, but the low permeability of the vitrine and the A alcohol is an important reason for influencing the use of the skin care products, and the vitrine and the A alcohol often bring stronger skin irritation, and when a product relieving system is not strong enough, the inflammation-related cytokines and the like can be activated, so that the skin barrier is damaged, and the phenomena of inflammatory reaction, redness, burning, stinging, desquamation and the like of the skin are caused. It is therefore necessary to develop a soothing system that reduces both the skin irritation of both the vitriol and the alcohol a.
Bioactive peptides are a generic term for peptides ranging from dipeptides to complex linear ring structures, which are composed of 20 natural amino acids contained in proteins in different compositions and arrangements, and are multifunctional compounds derived from proteins. The bioactive peptide participates in the growth and development, metabolism and aging of human, has the functions of enhancing immunity, resisting bacteria and viruses, resisting oxidation, delaying aging, reducing blood pressure and the like, has strong activity and high safety, has no side effect on human bodies, and has been widely applied to cosmetics. Meanwhile, with the progress of society, the development of science and technology and the improvement of living standard, people pursue increasingly more consciousness of green, health and safety, green and safe cosmetics and functional cosmetics are more popular, and the idea of scientific skin care becomes a market hotspot. Therefore, it is necessary to combine bioactive peptide with effective raw materials to develop an anti-aging composition with high transdermal efficiency, safety, green health and good stability, solve the problems of poor raw material permeability, low availability and the like, provide a combined formula to solve the skin aging problem, delay skin aging, and apply the combined formula to the technical field of cosmetics so as to meet the increasing demands of consumers for treating or caring skin.
Disclosure of Invention
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
The invention provides an active polypeptide composition, which comprises SA-LFP, SPP19 and AOP, and specifically comprises 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP by mass percent; preferably, it comprises 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
The invention also provides an anti-aging composition, which comprises the following components in percentage by mass: 0.01% -1% of vitrein, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP.
Preferably, it comprises, in mass percent: 0.1% of vitrein and 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; more preferably, the method comprises the following steps in percentage by mass: 0.1% vitrein and 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
Or preferably, it comprises, in mass percent: 0.01% of vitrein and 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; more preferably, the method comprises the following steps in percentage by mass: 0.01% vitrein and 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
The invention also provides application of the polypeptide composition or the anti-aging composition in preparing cosmetics.
The invention also provides a cosmetic comprising, in mass percent, a vitreous factor, SA-LFP, SPP19 and AOP, preferably: 0.01% -1% of vitrein, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP.
Preferably, it comprises 0.1% of vitronectin and 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP in mass percent; more preferably, the method comprises the following steps in percentage by mass: 0.1% vitrein and 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
Or preferably, it comprises, in mass percent: 0.01% of vitrein and 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; more preferably, the method comprises the following steps in percentage by mass: 0.01% vitrein and 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
The key point of the invention is to provide an anti-aging composition with high skin permeability, which can be directly smeared on skin for use, and the research in CN117050164A shows that SPP19 has high skin permeability, so that the invention replaces the traditional chemical permeation promoters such as ethanol, propylene glycol and the like with the bioactive peptide SPP19 with high skin permeability, and the bioactive peptide SPP19 not only has high skin permeability, but also can promote permeation of other components; and the availability of the effective components can be increased, and the addition amount and cost of raw materials can be reduced. CN115517999a discloses an anti-aging composition and its application in beauty and skin care, the anti-aging composition comprises 0.1-5 parts of antioxidant, 0.0001-0.05 parts of compound polypeptide, 1-10 parts of panthenol, 0.3-2 parts of glycoside compound; the composite polypeptide used in the technical proposal has a plurality of categories, the polypeptide itself has the function of stimulating the growth of collagen, and the cosmetics prepared from the anti-aging composition need to be matched with a radio frequency beauty instrument for use so as to achieve the technical effect. The anti-aging composition has dependence on a radio frequency beauty instrument when in use, is inconvenient to operate, needs special technicians to operate, and severely limits the application of the anti-aging composition.
The composition disclosed by the invention is simple in use mode, can be directly smeared on skin for noninvasive use, and is safe and efficient. The micro-needles, injection and other modes have great skin infection risks, and can easily cause papules, pustules, folliculitis and even small pustules, and the skin care after the micro-needles are important and tedious, such as water prevention and bath prevention, outdoor activities reduction, sun protection, severe exercise prevention, sweat generation and the like.
The key of the invention is to provide a polypeptide composition which has excellent antioxidant, anti-aging, soothing and anti-inflammatory effects. Studies in CN117024518A have shown that SA-LFP has excellent oxidation resistance and repair effect; studies in CN116804052A have shown that AOP has excellent antioxidant effects. The invention utilizes the excellent oxidation resistance of SA-LFP and AOP, and can play a role in combination with the vitreous factor to cooperatively enhance the oxidation resistance and anti-aging effect.
The key point of the invention is to provide a safe and non-irritating anti-aging composition, and the bioactive peptide SA-LFP of the invention has the effects of relieving and resisting inflammation, is safe and non-irritating to skin, and further increases the sensitivity and stability of the composition. The A alcohol and the glass color used in the prior anti-aging products have large irritation to skin, are not suitable for sensitive skin, are extremely unstable and have high requirements on storage conditions.
Therefore, compared with the prior art, the invention has the following characteristics:
1) The anti-aging composition provided by the invention has high-efficiency skin permeability, can be directly smeared on skin, and the SPP19 bioactive peptide not only has high-efficiency skin permeability, but also can promote the absorption of active ingredients in the anti-aging composition by a human body, and reduces the addition amount and cost of raw materials.
2) The invention combines the active polypeptide components SA-LFP and AOP with antioxidant effect and the high-efficiency antioxidant component vitronectin for use, and mutually and synergistically plays roles;
3) The bioactive peptide SA-LFP has excellent antioxidant, anti-aging, relieving anti-inflammatory and repairing effects, is more stable and safe on the basis of having the antioxidant, repairing and anti-aging effects, reduces the irritation of products, can be applied to the field of cosmetics, solves the problem of application limitation of antioxidant raw materials such as vitrine, retinol, arbutin and the like caused by high-sensitivity irritation, and meets the increasing demands of consumers on treating or nursing skin.
The active polypeptide composition SA-LFP, SPP19, AOP and the vitronectin are combined to form the anti-aging composition, and the active polypeptide composition can promote the absorption of the vitronectin which is an active ingredient in the anti-aging composition by a human body and enhance the anti-aging effect; solves the problems of poor percutaneous absorption and low availability of active ingredients of the existing product. After the bioactive peptide composition SA-LFP, SPP19 and AOP are compounded with the vitriol, the invention generates remarkable synergistic effect in the aspect of antioxidation effect and delays skin aging. The anti-aging composition can reduce the addition amount of the vitreous color due to the synergistic effect, reduce the irritation and reduce the inflammation risk caused by the use of the vitreous color.
Drawings
FIG. 1 shows the effect of different samples on fibroblast activity.
FIG. 2 shows the effect of different samples on collagen content.
FIG. 3 is a graph showing the effect of vitronectin and compositions on ROS content.
FIG. 4 shows the effect of different samples on inflammatory factors.
Detailed Description
Example 1: the raw materials of this example are as follows:
1. SA-LFP (CLAGRRRRSV, SEQ ID NO: 1), SPP19 (ANLDGSKRRR, SEQ ID NO: 2), AOP (ALFGKNGKNCP, SEQ ID NO: 3): the synthesis was performed by solid phase chemical method, by the company of Kinsrui Biotech Co.
2. The glass color factor is as follows: from Source leaf Biotechnology Co.
3. The present example provides an anti-aging composition comprising the formula: the composition of the composition comprises the following components in percentage by mass:
| Composition of the components | In mass percent (%) |
| SA-LFP | 0.001％-1％ |
| SPP19 | 0.001％-1％ |
| AOP | 0.001％-1％ |
| Glass color factor | 0.01％-1％ |
Example 2: preparation of a composition permeation detection related reagent:
1. Skin model
Back skin of Bama pig, 30 days old.
2. Receiving liquid
A0.9% aqueous sodium chloride solution was prepared using sterilized distilled water as a solvent.
3. Sample to be measured
And preparing a glass color factor penetration test sample by using sterilized distilled water as a solvent. Sample 1:0.1% glass color factor; sample 2: composition (0.1% vitrine+0.01% SA-LFP+0.01% SPP19+0.01% AOP); sample 3: composition (0.1% vitrine+0.01% TD-1).
4. Experimental method
In vitro skin permeation assays were performed using Franz transdermal diffusion cells. The specific operation is as follows: bama pigs were 30 days old and after sacrifice were given complete skin on their backs with scissors. Wiping dermis of skin with cotton ball stained with physiological saline, removing adhered subcutaneous tissue, washing skin with physiological saline, wiping, wrapping with tinfoil paper, and storing in refrigerator at-20deg.C. The prepared skin was fixed between the supply chamber and the receiving chamber of the Franz diffusion cell with the top layer of the skin facing the supply chamber, and the effective permeation area of the diffusion cell was 1.327cm 2. To the receiving chamber was added a volume of 3.8mL of 0.9% aqueous sodium chloride solution so that the liquid surface could be brought into close contact with the skin. 1mL of the glass color factor penetration test sample is added into the supply chamber, and the glass color factor mass percentage concentration in the glass color factor penetration test sample is ensured to be 0.1%. During the experiment, water bath heating is adopted, the temperature is kept at 32 ℃, and stirring is carried out at the rotating speed of 350 r/min. And quantifying the permeation effect at 8 hours.
5. Experimental results
TD-1 was the first transdermal enhancing peptide discovered by phage display technology, and studies have shown that TD-1 can promote transdermal administration. The 0.1% of the glassy factor is respectively mixed with 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP and TD-1, then different compositions are smeared on pigskin, and the content of the glassy factor in pool liquid is quantitatively received through liquid phase, and as shown in the result of the table 1, the permeability of the glassy factor alone is found to be only 4.07%, and after the glassy factor is compounded with 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP, the permeability of the glassy factor reaches 21.68 percent (P < 0.001), the permeability of the glassy factor is improved by 5.3 times, and after the glassy factor is compounded with TD-1, the permeability of the glassy factor is only 9.04 percent (P < 0.001). The method shows that the permeation of the glass color factor can be obviously promoted after the glass color factor is mixed with 0.01 percent of SA-LFP+0.01 percent of SPP19+0.01 percent of AOP, the permeation promoting effect is better than that of TD-1, the availability of the glass color factor can be effectively increased by combining the glass color factor with the polypeptide provided by the invention, the consumption of the glass color factor is reduced, and the inflammation promoting risk caused by using the glass color factor in a large amount is reduced.
TABLE 1 penetration effect of different samples on the glass color factor
| Sample of | Glass color factor permeability (%) |
| 0.1% Glass color factor | 4.07±1.77 |
| 0.1% Vitrein+0.01% SA-LFP+0.01% SPP19+0.01% AOP | 21.68±1.59 |
| 0.1% Of vitrein+0.01% of TD-1 | 9.04±1.22 |
Example 3: performance test: skin fibroblast proliferation assay
1. Reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, sterile PBS, CCK8.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument, a CO 2 incubator, an ultra-clean bench and an inverted fluorescence microscope.
3. Cell strain
Skin Fibroblasts (HSF) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
4. Sample to be measured
Drug administration group: 0.01% glass color factor, 0.1% glass color factor, composition (0.01% SA-LFP+0.01% SPP19+0.01% AOP), composition (0.1% glass color factor+0.01% SA-LFP+0.01% SPP19+0.01% AOP).
Blank control group: PBS.
Model group: AAPH injury, PBS was added.
5. Experimental method
Taking HSF cells in logarithmic phase, adding 0.25% trypsin to digest and make adherent cells fall off, counting 1-4X 10 6 cells/mL, and preparing cell suspension; the cells were plated in 96-well plates at a density of 5X 10 4 cells/mL. After the cells are stabilized, the blank control group is added with the complete culture medium of the same amount of PBS, and the administration group is added with the complete culture medium containing samples to be tested with different concentrations for 24 hours. After the end of the incubation, the medium was discarded, and after incubation for 45min, the OD of each well was measured at 450nm, after which it was changed to basal medium containing 10% CCK 8.
6. Experimental results
Fibroblasts are the main cellular components of the skin, are distributed in the dermis of the skin, are the most common cells in loose connective tissue, can produce a large amount of collagen and elastic fibers, and play an important role in maintaining the structural stability and the skin elasticity of the skin. The viability and proliferation rate of fibroblasts determine the firmness of the skin and thus affect the condition of the skin. The viability of fibroblasts is reduced and the number is reduced, resulting in skin aging. As can be seen from fig. 1, the activity of the fibroblasts gradually decreased with increasing concentration of the vitriol, the 0.1% concentration of the vitriol showed only 76.5% of the activity of the fibroblasts, indicating that the vitriol had an effect on the active state of the fibroblasts, whereas the cell activity was not affected after the composition (0.01% sa-lfp+0.01% spp19+0.01% aop) acted on the cells, but the cell activity reached 113% after the vitriol and 0.01% sa-lfp+0.01% spp19+0.01% aop were compounded, significantly promoting proliferation of the fibroblasts, and the cell activity increased by 47.7% compared to the 0.1% vitriol (P < 0.001). The mixture of 0.01 percent of SA-LFP+0.01 percent of SPP19+0.01 percent of AOP and the vitriol can promote the proliferation of fibroblasts, repair the influence of the vitriol on the activity injury of the fibroblasts (P < 0.001) and strengthen the anti-aging effect of the fibroblasts.
Example 4: performance test: collagen content test
1. Reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, collagen I ELISA kit, BCA protein kit.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument, a CO 2 incubator and an ultra-clean workbench.
3. Cell strain
Human Skin Fibroblasts (HSF) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
4. Sample to be measured
Drug administration group: 0.01% glass color factor, 0.1% glass color factor, composition (0.01% SA-LFP+0.01% SPP19+0.01% AOP), composition (0.01% glass color factor+0.01% SA-LFP+0.01% SPP19+0.01% AOP).
Blank control group: PBS.
UV group: UV radiation, PBS was added.
5. Experimental method
Taking HSF cells in logarithmic growth phase, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, counting 1-4×10 6 cells/mL, and preparing cell suspension; the cells were seeded on 6-well plates at a density of 1X 10 5 cells/mL and a UV photoaging model was established when the cells were grown to about 80%. The blank control group is added with equal amount of PBS, the culture medium is supplemented to 2mL, and UV irradiation is not carried out; the UV group and the administration group were repeatedly washed to colorless by adding an appropriate amount of PBS, 200. Mu.L of PBS was added, and the mixture was irradiated under a UV lamp of 80mJ/cm 2 with a lamp-to-flask spacing of 15cm. After irradiation, PBS was discarded, the UV group was added to PBS solution and complete medium, and the dosing group was added with the double dilution drug and complete medium. The blank, UV, and dosing groups were incubated in a 5% CO 2 incubator at 37℃for a further 48h. After the end of the culture, the 1 st well cells were digested and counted, and diluted to 0.5X10 6 cells/mL, the cells of the remaining wells were scraped off with a cell scraper, after 500. Mu.L of the cells were resuspended, 50. Mu.L of ultrasound was taken for 30s from all wells, and the total protein was measured by BCA method, and the other wells were diluted according to the 1 st well protein concentration to give a total cell suspension concentration of 0.5X10 6 cells/mL. The cell suspension with the adjusted concentration was sonicated for 30s, centrifuged for 15min at 1500g, and the cell supernatant was collected to obtain a sample solution and operated according to the collagen I ELISA protocol. The OD of each well was measured sequentially with an microplate reader at 450nm over 15 min.
6. Experimental results
Collagen is the most abundant protein found in connective tissue, and collagenase is synthesized and secreted by fibroblasts, and can degrade collagen in skin to cause skin aging. Therefore, the method can inhibit the expression of collagenase in cells, and improve the content of collagen, and has important effects on preventing aging and increasing skin plumpness and compactness. In the ultraviolet overexposure environment, collagenase and elastase activities are greatly increased, elastin hydrolysis, and collagen synthesis is also inhibited. The experiment adopts a test sample to treat cells after ultraviolet radiation, and detects the content of collagen I in corresponding cells so as to determine whether the composition can promote the generation of collagen. The results showed (fig. 2) that the collagen content of the UV group was greatly reduced compared to the blank group, indicating that the photoaging model was successfully established. Compared with the UV group, 0.01% of the glass causes can not increase the collagen content in cells after ultraviolet irradiation, 0.1% of the glass causes, 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP can increase the collagen content by 26.6% (P < 0.001) and 35.2% (P < 0.001) respectively; and after 0.1% of the glass pigment is compounded with 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP, the content of collagen can be obviously improved, and compared with the content of collagen in a UV group, the content of collagen is increased by 48.9% (P < 0.001). The composition can promote collagen expression and has excellent collagen generation effect, and shows that the polypeptide mixed solution of 0.01 percent SA-LFP+0.01 percent SPP19+0.01 percent AOP can enhance the effect of low-concentration vitreogenesis, can reduce the addition amount of the vitreogenesis in the use process, and achieves the synergistic enhancement effect.
Example 5: performance test: antioxidant-ROS content assay
1. Reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, DCFH-DA probe, CCK8.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument, a CO 2 incubator, an ultra-clean bench and an inverted fluorescence microscope.
3. Cell strain
Human keratinocytes (HaCaT) were purchased from the national academy of sciences typical culture collection committee Shanghai cell bank.
4. Sample to be measured
Drug administration group: 0.01% vitriol, composition (0.01% SA-LFP+0.01% SPP19+0.01% AOP), composition (0.01% vitriol+0.01% SA-LFP+0.01% SPP19+0.01% AOP).
Blank control group: PBS.
Model group: AAPH injury, PBS was added.
5. Experimental method
Taking HaCaT cells in logarithmic growth phase, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, counting 1-4×10 6 cells/mL, and preparing cell suspension; the cells are inoculated on a 96-well plate according to the density of 1X 10 5/mL, and an oxidative damage model is established when the cells grow to about 80 percent. Blank control was not subjected to AAPH injury and equal amount of PBS was added; model group and dosing group, adding the final concentration of 75mM AAPH complete medium effect 2h. After completion of the application, the complete medium and the multiple dilution were added to the dosing group, and the model group was added with an equal amount of PBS solution and complete culture. The blank, model, and dosing groups were incubated in a 5% CO 2 incubator at 37℃for 24h. After the end of the incubation, the cells were washed with PBS, added with basal medium and 10. Mu.M of DCFH-DA probe solution, incubated at 37℃for 20min, after washing 2 times with basal medium, the OD of each well was measured at excitation wavelength 488nm and emission wavelength 525nm, and after replacing with basal medium containing 10% CCK8, the OD of each well was measured at 450nm after 45min incubation.
6. Experimental results
ROS, reactive oxygen species, damage biological membranes and peroxidate cell membrane phospholipids, producing lipid peroxides, which can cause cellular DNA damage, leading to aging. And the active oxygen can destroy the integrity of cell membranes and an oxidation resistance system in cells, so that cell inflammation, cell apoptosis and tumor formation are caused, elastin, collagen and hyaluronic acid are degraded, and skin elasticity is reduced, roughness is caused, and wrinkles are formed. Therefore, the ROS content in the cells is reduced, and the ROS-containing antioxidant and antiaging agent has an important effect on resisting oxidization and preventing aging. After AAPH acts on cells, a large amount of ROS reactive oxygen species is generated, and the relative fluorescence intensity, relative fluorescence intensity=ros fluorescence intensity/cell activity, is obtained by measuring the fluorescence absorbance at a specific wavelength and the cell activity. As shown in FIG. 3, after 0.01% vitrine, 0.01% SA-LFP+0.01% SPP19+0.01% AOP act on the cells, the ROS content can be slightly reduced. Compared with the AAPH-acting group, the reduction rate of the ROS by the 0.01% glassy color factor group is only 12.8%; the reduction rate of ROS by the 0.01% SA-LFP+0.01% SPP19+0.01% AOP group was only 24.5%. However, after 0.01% of the glass pigment is compounded with 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP, namely the composition is acted, the ROS content can be remarkably reduced, the reduction rate reaches 52.7% (P < 0.001), and the effect is better than that of the single 0.01% of the glass pigment, 0.01% of SA-LFP+0.01% of SPP19+0.01% of AOP, and the ROS reduction rate is 2 times higher; the polypeptide mixed solution and the vitriol factor can synergistically enhance the antioxidation effect.
Example 6: anti-inflammatory test
1. Reagents and materials
Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, lipopolysaccharide (LPS), IL-6ELISA kit, and tnfa ELISA kit.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument, a CO 2 incubator and an ultra-clean workbench.
3. Cell strain
Mouse macrophages (raw 264.7) were purchased from the national academy of sciences of the classical culture collection committee (academy of sciences of china) Shanghai cells.
4. Sample to be measured
Drug administration group: 0.01% of a glassy cause, a composition (0.01% glassy cause +0.01% SA-LFP +0.01% SPP19+0.01% AOP).
Blank control group: PBS.
5. Experimental method
Taking Raw264.7 cells in the logarithmic growth phase, adding 0.25% trypsin digestive juice, and digesting to enable the adherent cells to fall off to prepare a cell suspension; the cells were seeded at a density of 5X 10 5 cells/mL in 96-well plates and allowed to grow to about 80%. Adding PBS into a blank control group; the lipopolysaccharide with the concentration of 1ug/mL is added into the administration group, 0.01% of vitriol plus 0.01% of SA-LFP plus 0.01% of SPP19 plus 0.01% of AOP are respectively added into the administration group to incubate cells together, and the incubation is continued for 24 hours in a culture box with 5% CO 2 at 37 ℃ after the addition. After the culture, the cell culture fluid was collected to obtain a sample fluid, and the sample fluid was subjected to the procedure according to the IL-6 and TNF alpha ELISA protocol.
6. Experimental results
Lipopolysaccharide LPS binds to cell surface receptors, activates inflammatory cytokine gene expression and causes an inflammatory response, and then cells secrete a series of pro-inflammatory factors, also known as the senescence-associated secretion phenotype (SASPs), which promotes chronic inflammation and pushes normal cells into the senescence process; thus inhibiting inflammatory factor expression, reducing inflammatory response, and facilitating the prevention of cells from entering the aging process. The experiment adopts a test sample to treat lipopolysaccharide-induced inflammatory cell model, and detects the content of IL-6 and TNF alpha in corresponding cells so as to determine whether the composition can inhibit the expression of inflammatory factors IL-6 and TNF alpha. As shown in fig. 4, the concentrations of IL-6 and TNF alpha in the model group were significantly increased compared to the blank group, indicating that the inflammatory model was successfully induced, but the concentration of inflammatory factor was not decreased or even increased after 0.01% of the vitreofactor acted on the cells, and the concentration of TNF alpha was increased by 14.5% after 0.01% of the vitreofactor acted on the model group (P < 0.001), indicating that the vitreofactor aggravated the inflammatory response of the cells; compared with the single 0.01% vitriol effect group, the 0.01% vitriol+0.01% SA-LFP+0.01% SPP19+0.01% AOP composition significantly reduces the inflammatory factor content after the cells are acted, the IL-6 concentration is reduced by 16.2% (P < 0.001), and the TNF alpha concentration is reduced by 34.6% (P < 0.001). The composition has excellent anti-inflammatory effect, and the polypeptide mixture SA-LFP+SPP19+AOP and the vitronectin are compounded to reduce inflammatory reaction caused by the vitronectin and reduce the irritation of the vitronectin in the use process.
Claims (10)
1. A polypeptide composition comprising SA-LFP, SPP19 and AOP.
2. The active polypeptide composition of claim 1, comprising, in mass percent: 0.001% -1% SA-LFP, 0.001% -1% SPP19 and 0.001% -1% AOP.
3. The active polypeptide composition of claim 2, comprising 0.01% sa-LFP, 0.01% spp19, and 0.01% aop.
4. An anti-ageing composition comprising a vitronectin and a polypeptide composition as claimed in any one of claims 1 to 3.
5. The anti-aging composition of claim 4, comprising, in mass percent: 0.01% -1% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP.
6. The anti-aging composition of claim 5, comprising, in mass percent: 0.1% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; preferably, the method comprises the following steps in percentage by mass: 0.1% vitrine, 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
7. The anti-aging composition of claim 5, comprising, in mass percent: 0.01% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; preferably, the method comprises the following steps in percentage by mass: 0.01% vitrein, 0.01% SA-LFP, 0.01% SPP19, and 0.01% AOP.
8. Use of a polypeptide composition according to any one of claims 1 to 3 or an anti-ageing composition according to any one of claims 4 to 7 for the preparation of a cosmetic.
9. Cosmetic, characterized in that it comprises a vitreous factor and a polypeptide composition according to any of claims 1 to 3, preferably comprising, in mass percent: 0.01% -1% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP;
More preferably, the method comprises the following steps in percentage by mass: 0.1% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; preferably, the method comprises the following steps in percentage by mass: 0.1% vitrine, 0.01% SA-LFP, 0.01% SPP19 and 0.01% AOP.
10. The cosmetic as claimed in claim 9, comprising, in mass percent: 0.01% of vitrine, 0.001% -1% of SA-LFP, 0.001% -1% of SPP19 and 0.001% -1% of AOP; preferably, the method comprises the following steps in percentage by mass: 0.01% vitrein, 0.01% SA-LFP, 0.01% SPP19, and 0.01% AOP.
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| CN202410165392.0A CN117919113A (en) | 2024-02-05 | 2024-02-05 | Anti-aging composition and application thereof |
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