CN118286107A - Multi-effect composition containing recombinant humanized III type collagen and application thereof - Google Patents
Multi-effect composition containing recombinant humanized III type collagen and application thereof Download PDFInfo
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Abstract
The invention relates to a multi-effect composition containing recombinant humanized III type collagen and application thereof, belonging to the technical field of cosmetics. The invention provides a multi-effect composition for improving skin photoaging and photodamage effects through basement membrane repair, dermis regeneration, barrier repair, relieving skin inflammation and reducing apoptosis pathways caused by endoplasmic reticulum stress, which comprises one of recombinant humanized III type collagen and sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex, has a synergistic effect, and can improve skin photoaging or photodamage problems by increasing contents of integrin alpha 6, elastin and keratin 10 and reducing contents of interleukin 6 and Caspase-12.
Description
Technical Field
The invention relates to a multi-effect composition containing recombinant humanized III type collagen and application thereof, belonging to the technical field of cosmetics.
Background
Ultraviolet (UV) is one of the main factors responsible for exogenous aging of the skin and impaired barrier, and the accumulation of UV radiation can affect the homeostasis of the intracellular environment to some extent. For example, UVA indirectly causes oxidative damage to DNA, proteins and lipids by inducing oxidative stress, and influences the content of key proteins such as integrins in basement membrane (Dermal-EPIDERMAL JUNCTION, DEJ) and the transmission of plasma membrane signals and the synthesis and degradation of extracellular matrix (Extracellular matrix, ECM) such as collagen and elastin in dermis by activating TGF-beta/Smad and MAPK/AP-1 signal pathways. UVB can induce Toll-like receptors 2 (TLR 2) and TLR4 in keratinocytes to be expressed in high, activate downstream NF- κB molecules, release inflammatory factors to cause skin inflammation, and induce DNA damage of cells, thereby changing the expression of skin barrier-related proteins, preventing the normal assembly of the barrier and inducing barrier damage.
Skin damage and aging caused by ultraviolet irradiation are associated with excessive apoptosis. Endoplasmic reticulum is used as a site for lipid metabolism and cell storage and processing, and is involved in protein synthesis, folding and oligomerization, while maintaining normal physiological functions of cells. It is an important organelle for maintaining physiological functions of cells, and has high sensitivity to changes in intracellular environments. When irradiated by ultraviolet light, the processing or transport functions of proteins in the lumen of the endoplasmic reticulum are impaired, and endoplasmic reticulum stress occurs. Endoplasmic reticulum stress, in turn, further increases the accumulation of misfolded proteins, thereby activating the endoplasmic reticulum-specific protein, cysteine proteinase-12 (CYSTEINE ASPARTATE-specific protease, caspase-12), initiating the apoptotic cascade of endoplasmic reticulum pathways.
Collagen is the most abundant protein in the body of mammals, contains 28 subtypes, and has different distribution positions and functions of different types of collagen, and is involved in the regulation of basic activities such as cell adhesion, proliferation, migration, differentiation and the like. The recombinant III type collagen obtained by utilizing the synthetic biology technology has high consistency with the amino acid sequence of the natural collagen of the human body, has a plurality of active sites, can interact with the receptor on the cell surface, and regulates the basic functions of the cells. The recombinant III type collagen has different functions due to the differences of amino acid sequences, engineering strains, production processes, purification processes and the like of different factories.
Hyaluronic Acid (HA), also known as Hyaluronic Acid, is a macromolecular glycosaminoglycan commonly found in connective tissue of organisms, is one of the main components of skin extracellular matrix, HAs good viscoelasticity, lubricity, water absorption, plasticity and biocompatibility, and is known as an ideal natural moisturizing factor. In order to improve the solubility and expand the applicability thereof, sodium salt forms of hyaluronic acid and derivatives thereof are widely used in the cosmetic field.
The series of characteristics of the collagen and the hyaluronic acid substances enable people to gradually begin to think about whether the two raw materials can be used in a compounding way, so that the effects of the two raw materials are improved, and a synergistic effect is achieved. However, hyaluronic acid substances and collagen are affected by factors such as different charge properties, isoelectric points, pH values, ionic strength and the like, precipitation can occur in a pure water system, transparent solution is difficult to form, and the application of the hyaluronic acid substances and collagen is greatly limited. In the prior art, chen Xu et al in the research on compatibility and interaction of collagen/hyaluronic acid blend systems, suggest that the collagen/hyaluronic acid blend in a pure water system can be compatible only at a ratio of 9/1 due to the strong electrostatic attraction between collagen and hyaluronic acid; collagen and hyaluronic acid can be compounded in the presence of high concentrations of salt ions, but the high concentration of ionic environment still limits its application. Patent document CN114699335A, CN102492033A, CN107189119A, CN110075006a and the like solve the problem of elution by adjusting pH, crosslinking, adding a buffer system or forming a formulation and the like, but the above method also limits the range of application thereof, increasing the difficulty of operation and the cost of application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a multi-effect composition containing recombinant humanized III type collagen and application thereof. The multi-effect composition of the invention comprises one of recombinant humanized type III collagen and sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex, and has the effects of improving skin photoaging and photodamage through basement membrane repair, dermis regeneration, barrier repair, relieving skin inflammation and reducing apoptosis pathways caused by endoplasmic reticulum stress.
The technical scheme of the invention is as follows:
A multi-effect composition containing recombinant humanized III type collagen comprises the recombinant humanized III type collagen, sodium hyaluronate and derivatives thereof, wherein the sodium hyaluronate and the derivatives thereof are one of sodium hyaluronate, hydrolyzed sodium hyaluronate and dimethyl silanol hyaluronate complex.
According to the invention, the amino acid sequence of the recombinant humanized III type collagen is shown as SEQ ID NO. 1.
According to the present invention, it is preferable that the recombinant humanized type III collagen has a final concentration of 0.002 to 0.5wt% and the sodium hyaluronate and its derivatives have a final concentration of 0.001 to 0.5wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are sodium hyaluronate, the recombinant humanized type iii collagen has a final concentration of 0.002 to 0.5wt% and the sodium hyaluronate has a final concentration of 0.01 to 0.5wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are sodium hyaluronate, the recombinant humanized type iii collagen has a final concentration of 0.002 to 0.5wt% and the sodium hyaluronate has a final concentration of 0.01 to 0.45wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are hydrolyzed sodium hyaluronate, the recombinant humanized type iii collagen has a final concentration of 0.002 to 0.5wt% and the hydrolyzed sodium hyaluronate has a final concentration of 0.01 to 0.5wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are hydrolyzed sodium hyaluronate, the recombinant humanized type III collagen has a final concentration of 0.2 to 0.5wt% and the hydrolyzed sodium hyaluronate has a final concentration of 0.01 to 0.25wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are dimethyl silanol hyaluronate complexes, the final concentration of the recombinant humanized type III collagen is 0.002 to 0.5wt% and the final concentration of the dimethyl silanol hyaluronate complexes is 0.001 to 0.1wt% in a system containing a multi-effect composition.
According to the present invention, preferably, when the sodium hyaluronate and its derivatives are dimethyl silanol hyaluronate complexes, the recombinant humanized type III collagen has a final concentration of 0.01 to 0.5wt% and the dimethyl silanol hyaluronate complexes has a final concentration of 0.001 to 0.1wt% in a system containing a multi-effect composition.
In the present invention, the system comprising the multi-effect composition may comprise purified water, or other conventional cosmetic or skin care matrices, in addition to recombinant humanized type III collagen and sodium hyaluronate and derivatives thereof.
In the invention, the multi-effect composition can play a role in improving skin photoaging and photodamage through the ways of basement membrane repair, dermis regeneration, barrier repair, relieving skin inflammation, reducing apoptosis caused by endoplasmic reticulum stress and the like.
The application of the multi-effect composition in preparing cosmetics or skin care products.
Preferably, according to the present invention, the cosmetic or skin care product has an effect of improving photoaging and photodamage of skin.
A cosmetic or skin care product containing the above multi-effect composition is provided.
Preferably, according to the invention, the cosmetic or dermatological product is embodied in the form of a liquid, gel, cream, emulsion, mask or freeze-dried powder.
In the invention, the cosmetic or skin care product can be used on the surface of skin, and plays roles in improving skin photoaging and photodamage through the ways of basement membrane repair, dermis regeneration, barrier repair, skin inflammation relief, apoptosis reduction caused by endoplasmic reticulum stress and the like.
The beneficial effects are that:
1. The invention provides a multi-effect composition for improving skin photoaging and photodamage effects through basement membrane repair, dermis regeneration, barrier repair, relieving skin inflammation and reducing apoptosis pathways caused by endoplasmic reticulum stress, which comprises one of recombinant humanized III type collagen and sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex, and the two have synergistic effects, and can improve skin photoaging or photodamage problems by increasing the content of integrin alpha 6, elastin, keratin 10 (Keratins, KRT-10) and reducing the content of Interleukin 6 (Interleukin 6, IL-6) and Caspase-12.
2. The multi-effect composition of the invention reduces the consumption of the recombinant humanized III type collagen and greatly reduces the application cost of the recombinant humanized III type collagen by using the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex on the premise of achieving similar effects.
3. The invention surprisingly discovers that the recombinant humanized III type collagen used in the invention is compounded with 0.001-0.5 wt% of sodium hyaluronate and derivatives thereof in a pure water system within the concentration range of 0.002-0.5 wt%, so that stable and transparent solution can be formed, and the problem of the compounding stability of the recombinant humanized III type collagen, the sodium hyaluronate and derivatives thereof in the pure water system is solved.
Drawings
FIG. 1 is a graph showing the results of stability observation of examples 2/3/4 and comparative examples 7/8/9;
FIG. 2 is a bar graph of HaCaT cell IL-6 secretion by the compositions of examples 1-8 and comparative example 1/2/3/4/6/9;
FIG. 3 is a bar graph showing the levels of Caspase-12 in HaCaT cells under the influence of the compositions of examples 1-8 and comparative example 1/2/3/4/5/6/9;
FIG. 4 is a bar graph of KRT-10 content in a 3D epidermal skin model under the action of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9;
FIG. 5 is a bar graph of the elastin content of isolated skin tissue under the influence of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9;
FIG. 6 is a bar graph of integrin alpha 6 subunit content in ex vivo skin tissue under the influence of the compositions of examples 1-8 and comparative example 1/2/3/4/5/6/9;
In fig. 2 to 6, compared with the blank group, the significance is represented by #, 0.01 < P < 0.05 is represented by #,0.001 < P < 0.01 is represented by #,0.0001 < P < 0.001 is represented by # #, and P < 0.0001 is represented by # #; compared with the negative control group, the significance is expressed as the following, 0.01 < P < 0.05 is expressed as the following, 0.001 < P < 0.01 is expressed as the following, 0.0001 < P < 0.001 is expressed as the following, and P < 0.0001 is expressed as the following.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following examples, which are not intended to limit the scope of the invention.
Sodium hyaluronate and sodium hyaluronate hydrolyzed used in examples and comparative examples were all available from Fusarium furida pharmaceutical Co., ltd; the dimethyl silanol hyaluronate complex can be prepared as described in patent application number 201310317104.0. Other reagents and materials are common commercial products unless specified.
Examples:
Examples 1 to 8 provide a composition for improving photoaging and photodamage of skin by the effects of basement membrane repair, dermis regeneration, barrier repair, relieving skin inflammation, reducing apoptosis caused by endoplasmic reticulum stress, and the like, and the composition has the composition ratios shown in table 1. By way of comparison, comparative examples 1 to 9 are also provided in Table 1, which differ from the examples in that they contain only one substance in the composition/contain a composition of different origin or outside a specific range.
The amino acid sequence of the recombinant humanized III type collagen is shown as SEQ ID NO.1, and the recombinant humanized III type collagen is prepared according to the record of patent 202410290161.2; the recombinant humanized type III collagen of the comparative example is prepared according to the description of patent CN111087463A and is expressed as recombinant humanized type III collagen-2.
Table 1. Raw material components and ratio units of the compositions of examples 1 to 8 and comparative examples 1 to 9: wt%
The composition consisting of recombinant humanized type III collagen and sodium hyaluronate is added into a skin care emulsion matrix to prepare a skin care product with the effects of improving skin photoaging and photodamage, namely, example 9, and a placebo group, namely, comparative example 10 is arranged at the same time, the comparative example 10 is different from example 9 in that the composition consisting of recombinant humanized type III collagen and sodium hyaluronate is not contained in comparative example 10, the specific raw material components and the proportions are shown in table 2, and the effects of improving skin photoaging and photodamage are tested through a human body efficacy experiment.
TABLE 2 skin care emulsion raw materials and proportions of example 9 and comparative example 10
The products of example 9 and comparative example 10 were prepared by methods conventional in the art.
Experimental example:
the products of the above examples and comparative examples were subjected to performance testing as follows:
Preparing experimental materials:
3D epidermal skin model used in the following test An ex vivo skin tissue available from Guangdong Boxi biotechnology Co., ltd; other reagents and materials are common commercial products unless specified.
1. Stability observations of the compositions of examples 1 to 8 and comparative examples 7 to 9 in pure water systems
1. The experimental method comprises the following steps: adding the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex into purified water, stirring and mixing uniformly, and standing overnight to prepare mother solution. Adding the recombinant humanized III type collagen into purified water, stirring and mixing uniformly to prepare mother liquor. Adding purified water into the required amount of recombinant humanized III type collagen mother solution, uniformly mixing, dripping the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate composite mother solution into the recombinant humanized III type collagen solution until the final concentration of each component is the concentration shown in table 1, and observing the phenomenon.
2. Experimental results: as shown in table 3 and fig. 1 (showing the results of the part), examples 1 to 8 all formed stable colorless transparent solutions and were stable within 15 days at room temperature, comparative example 7 was colorless transparent liquid at the beginning, turned into white cloudy liquid after 15 days at room temperature, and comparative examples 8 and 9 had white floc precipitated, indicating that comparative examples 7 to 9 were unstable in pure water system. The stability phenomena of the comparative examples 7 and 8 show that when the addition amount of the recombinant humanized III type collagen exceeds a certain range, a white turbid liquid or precipitate is formed in a system consisting of the recombinant humanized III type collagen, sodium hyaluronate and derivatives thereof, and the stability is poor, so that the subsequent application of the composition is not facilitated; the recombinant humanized III type collagen used in the invention can be compounded with 0.001-0.5% of sodium hyaluronate and derivatives thereof in a pure water system in a concentration range of 0.002-0.5% by mass ratio in any proportion to form a stable transparent solution, so that the problem of stability of the recombinant humanized III type collagen and the sodium hyaluronate in the pure water system is solved, and the application range of the recombinant humanized III type collagen is widened. Meanwhile, the stability phenomenon of comparative example 9 indicates that not all recombinant humanized type III collagen can form a stable system within the above-mentioned ratio range.
TABLE 3 stability of the compositions of examples 1 to 8 and comparative examples 7 to 9 in pure water systems
| Name of the name | Phenomenon on day 0 | Phenomenon on day 15 |
| Example 1 | Colorless transparent solution | Colorless transparent solution |
| Example 2 | Colorless transparent solution | Colorless transparent solution |
| Example 3 | Colorless transparent solution | Colorless transparent solution |
| Example 4 | Colorless transparent solution | Colorless transparent solution |
| Example 5 | Colorless transparent solution | Colorless transparent solution |
| Example 6 | Colorless transparent solution | Colorless transparent solution |
| Example 7 | Colorless transparent solution | Colorless transparent solution |
| Example 8 | Colorless transparent solution | Colorless transparent solution |
| Comparative example 7 | Colorless transparent liquid | White turbid liquid |
| Comparative example 8 | White floc | White floc |
| Comparative example 9 | White floc | White floc |
2. Effect of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9 on the secretion of inflammatory factor IL-6
1. The experimental method comprises the following steps: human immortalized keratinocytes (HaCaT cells) were collected and cell suspensions were prepared with high sugar DMEM complete cell culture broth at a cell density of 1.3×10 5/mL, and 2mL of cell suspension was added to each well in a 6-well plate. The experiments set a blank control group, a negative control group (30 mJ/cm 2 UVB), a sample group (30 mJ/cm 2 UVB+examples 1-8/comparative example 1/2/3/4/6/9), and 3 duplicate wells were set for each group. The 6-hole plate is firstly placed in a cell incubator (5% CO 2, 37 ℃) for incubation for 24 hours, when the cell fusion rate reaches 50% -60%, the culture solution is discarded, 2mL PBS buffer solution is added, UVB irradiation is carried out except for a blank control group, and the irradiation dose is 30mJ/cm 2. After the irradiation, the PBS buffer solution is discarded, 2mL of high-sugar DMEM cell culture solution is added to each hole of the blank control group and the negative control group, 2mL of high-sugar DMEM cell culture solution containing the test sample is respectively added to the other groups, the mixture is placed in an incubator (37 ℃ C., 5% CO 2) for incubation and culture for 24 hours, and then cell culture supernatants are collected, and the secretion amount of IL-6 is detected according to the ELISA kit specification.
The test concentrations of the composition samples of examples 1 to 8 and comparative example 1/2/3/4/6/9 are shown in Table 1, and the solvent is a high-sugar DMEM cell culture solution.
2. Experimental results: IL-6 is a cytokine of the chemokine family, which induces recruitment and activation of inflammatory cells, promotes the release of inflammatory factors and the occurrence of inflammatory responses, and potentiates the actions of other inflammatory factors. As shown in fig. 2 and table 4, after UVB stimulation, the secretion amount of IL-6 in the negative control group was significantly increased (P < 0.0001) compared with the blank control group, demonstrating that the modeling of the photodamage model was successful; after UVB stimulation, the secretion of IL-6 was significantly reduced (P < 0.01) after addition of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9 to the injured cells, compared to the negative control. The inhibition ratios of IL-6 secretion in examples 1 to 8 were 58.88%, 53.42%, 54.45%, 46.71%, 42.74%, 44.28%, 41.10%, and 50.11%, respectively, and the inhibition ratios of IL-6 secretion in comparative example 12/3/4/6/9 were 9.61%, 12.53%, 13.94%, 11.59%, 8.06%, and 8.42%, respectively. The results of comparative examples 1 to 4 show that the recombinant humanized type III collagen or sodium hyaluronate and the derivatives thereof have a certain anti-inflammatory effect, but compared with the results of examples 1 to 8, the anti-inflammatory effect is obviously improved when the recombinant humanized type III collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate are compounded, and the composition formed by one of the recombinant humanized type III collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex can obviously reduce the skin inflammatory reaction induced by UVB through inhibiting the secretion of IL-6, plays roles of relieving skin inflammation and improving skin photodamage and can be synergistically enhanced. Meanwhile, as can be seen from comparison of the results of comparative example 9 with the results of example 1, the recombinant humanized type iii collagen used in the examples of the present invention has a better effect of the combination of sodium hyaluronate and its derivatives.
TABLE 4 influence of examples 1-8 and comparative examples 1,2, 3, 4, 6, 9 on IL-6 secretion
3. Effect of the compositions of examples 1-8 and comparative example 1/2/3/4/5/6/9 on Caspase-12 content
1. The experimental method comprises the following steps: human immortalized keratinocytes (HaCaT cells) were collected and cell suspensions were prepared with high sugar DMEM complete cell culture broth at a cell density of 1.3×10 5/mL, and 2mL of cell suspension was added to each well in a 6-well plate. The experiments set a blank control group, a negative control group (30 mJ/cm 2 UVB), a sample group (30 mJ/cm 2 UVB+examples 1-8/comparative example 1/2/3/4/5/6/9), and 3 duplicate wells were set for each group. The 6-hole plate is firstly placed in a cell incubator (5% CO 2, 37 ℃) for incubation for 24 hours, when the cell fusion rate reaches 50% -60%, the culture solution is discarded, 2mL of PBS buffer solution is added, each group except for a blank control group is given with UVB irradiation with 30mJ/cm 2 dose, after the irradiation is finished, the PBS buffer solution is discarded, each hole of the blank control group and the negative control group is added with 2mL of high-sugar DMEM cell culture solution, and the sample groups are respectively added with 2mL of high-sugar DMEM cell culture solution containing a test sample. After incubation in an incubator (37 ℃ C., 5% CO 2) for 24 hours, cells were collected, diluted to 1X 10 6 cells/mL with PBS buffer, repeatedly frozen and thawed, cell lysate was collected, and Caspase-12 content was detected according to ELISA kit instructions.
The test concentrations of the composition samples of examples 1 to 8 and comparative example 1/2/3/4/5/6/9 are shown in Table 1, and the solvent is a high-sugar DMEM cell culture solution.
2. Experimental results: caspase-12 plays a role of an apoptosis promoter and effector in the endoplasmic reticulum stress IRE 1. Alpha. Pathway, and endoplasmic reticulum is subjected to endoplasmic reticulum stress under ultraviolet irradiation, so that Caspase-12 is activated to cause apoptosis. As shown in fig. 3 and table 5, after UVB stimulation, caspase-12 content in the negative control group was significantly increased (P < 0.0001) compared with the blank control group, demonstrating successful modeling of apoptosis model caused by endoplasmic reticulum stress under uv irradiation; after UVB stimulation, the Caspase-12 content was significantly reduced (P < 0.05) after the addition of the compositions of examples 1-8 and comparative examples 1-5 to the injured cells compared to the negative control, and comparative example 6 and comparative example 9 had no significant inhibitory effect on UVB-induced increase in Caspase-12 content. Wherein, the inhibition rates of examples 1 to 8 on Caspase-12 are 81.89%, 76.48%, 80.56%, 63.70%, 75.02%, 66.59%, 70.77% and 64.29%, respectively, and the inhibition rates of comparative examples 1 to 5 on Caspase-12 are 32.15%, 25.38%, 21.65%, 29.71% and 64.39%, respectively. Analysis shows that the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof used alone in comparative examples 1-4 have certain effect of inhibiting apoptosis, but compared with the results of examples 1-8, the inhibition effect is obviously improved when the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof are used in a compound way; in addition, the effect of comparative example 5 is equivalent to that of examples 4 and 8, and there is no significant difference, which means that on the premise of achieving similar effects, the use amount of recombinant humanized type iii collagen can be greatly reduced and the application cost can be reduced by adding one of the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complexes to be compounded with recombinant humanized type iii collagen. Taken together with the results, the composition formed by one of the recombinant humanized III type collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex can reduce the UVB-induced endoplasmic reticulum stress reaction, reduce apoptosis, play a role in improving skin photoaging and photodamage and can synergistically increase by inhibiting the generation of Caspase-12. In addition, it can be seen from comparison of the results of comparative examples 1 and 1 that comparative examples 6 and 9 show that the recombinant humanized type III collagen used in the examples of the present invention has a better effect of inhibiting apoptosis and also has a better effect of being compounded with sodium hyaluronate and its derivatives.
TABLE 5 influence of the compositions of examples 1-8 and comparative examples 1-6 and 9 on Caspase-12 content
4. Effect of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9 on KRT-10 content
1. The experimental method comprises the following steps: and (5) irradiating the 3D epidermis skin model with UVB to establish a skin photodamage model. The experiments set up a blank control group, a negative control group (600 mJ/cm 2 UVB) and a sample group (600 mJ/cm 2 UVB+examples 1-8/comparative example 1/2/3/4/6/9), 3 wells per group. The test solutions of different groups are respectively added on the surface of the model, the addition volume of the test solution is 25 mu L, and other groups except the blank control group are irradiated by UVB with dosage of 600mJ/cm 2, and the incubation is continued for 24 hours under the conditions of 37 ℃ and 5% CO 2. After incubation, the residual test liquid on the surface of the model is cleaned, the model for detection is circularly cut off, the fixation treatment is carried out by 4% paraformaldehyde, after the fixation is carried out for 24 hours, immunofluorescence detection is carried out on the barrier-related protein KRT-10, then, 3 visual field photos are acquired for each group, integrated optical density (Integral optical density, IOD) analysis is carried out on target signals in the pictures by adopting ipwin picture analysis software, an average IOD value is calculated, and relative IOD average value is calculated by taking a blank control group as a reference to characterize the relative expression quantity of the protein.
The test concentrations of the composition samples of examples 1 to 8 and comparative example 1/2/3/4/6/9 are shown in Table 1, the solvent is EpiGrowth culture solution, and the test solutions of the blank control group and the negative control group are EpiGrowth culture solution.
2. Experimental results: KRT-10 is a major structural protein of epidermis, and plays a very important role in the processes of cell proliferation and differentiation, inflammatory reaction, wound healing, etc., as a cytoskeleton to resist mechanical stress, maintain skin barrier function. As shown in fig. 4 and table 6, the KRT-10 content in the negative control group was significantly reduced (P < 0.001) compared with the blank control group after UVB stimulation, demonstrating that the skin photodamage model modeling was successful; after UVB stimulation, the KRT-10 content was significantly increased (P < 0.01) after addition of the compositions of examples 1-8 and comparative examples 1-4 to the damaged 3D epidermal skin model compared to the negative control, with no significant elevation of KRT-10 content by comparative examples 6 and 9. Wherein, the lifting rates of examples 1 to 8 on KRT-10 are respectively 186.46%, 154.62%, 159.89%, 132.47%, 126.31%, 119.07%, 122.88% and 135.65%, and the lifting rates of comparative examples 1 to 4 on KRT-10 are respectively 65.33%, 59.56%, 51.64% and 58.72%. Analysis shows that the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof in comparative examples 1-4 have a certain effect of improving the content of KRT-10, but compared with the results of examples 1-8, the improvement effect is obviously improved when the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof are compounded for use; it is demonstrated that the composition formed by one of the recombinant humanized III collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex can synergistically increase the content of KRT-10, improve the damaged barrier and play a role in improving the photodamage of skin. In addition, it can be seen from comparison of the results of comparative examples 1 and 1 that comparative examples 6 and 9 show that the recombinant humanized type III collagen used in the examples of the present invention has a better effect of increasing KRT-10 content and a better effect of compounding sodium hyaluronate and its derivatives.
TABLE 6 influence of the compositions of examples 1 to 8 and comparative examples 1,2, 3, 4, 6, 9 on the KRT-10 content
5. Effects of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9 on elastin content
1. The experimental method comprises the following steps: the skin photoaging model is established by the combination of UVA and UVB irradiation on the isolated skin tissue. The experiments set up a blank control group, a negative control group (30J/cm 2 UVA+50mJ/cm2 UVB) and a sample group (30J/cm 2 UVA+50mJ/cm2 UVB+ examples 1-8/comparative example 1/2/3/4/6/9), 3 duplicate wells per group. After the isolated skin tissue was first cultured for 2 days, the other groups, except for the blank group, were started to be irradiated and administered. The daily irradiation dose is UVA 30J/cm 2 and UVB 50mJ/cm 2, the culture medium is abandoned during irradiation, PBS buffer solution is replaced, UVA irradiation is firstly carried out, then UVB irradiation is carried out, the irradiation is carried out continuously for 4 days, fresh culture solution is replaced after each irradiation is finished, surface administration treatment is carried out, test solutions of different groups are respectively added to the surface of the isolated skin tissue, and the addition volume of the test solution is 10 mu L. After 4 days of continuous irradiation, the isolated skin tissue was cultured for 3 days, and only the administration treatment was performed without irradiation. After the culture is completed, the test liquid remained on the surface of the isolated skin tissue is washed, and the residual liquid is removed. Fixing by adopting 4% paraformaldehyde, then carrying out tissue embedding and tissue slicing, carrying out conventional immunohistochemical staining on elastin after roasting and dewaxing, photographing and observing under a microscope within 24 hours, collecting 3 pictures with different fields of view, carrying out relative IOD analysis on target signals in the pictures by using Ipwin picture analysis software, calculating an average value, and calculating the relative IOD average value to represent the relative expression quantity of the protein by taking a blank control group as a reference.
The test concentrations of the composition samples of examples 1 to 8 and comparative example 1/2/3/4/6/9 are shown in Table 1, the solvent is EpiGrowth culture solution, and the test solutions of the blank control group and the negative control group are EpiGrowth culture solution.
2. Experimental results: elastin plays an important role in skin elasticity, and its reduction or breakage is a major cause of skin aging leading to sagging, and wrinkles. As shown in fig. 5 and table 7, after ultraviolet stimulation, the content of elastin in the negative control group is significantly reduced (P < 0.01) compared with the blank control group, which proves that the skin photoaging model modeling is successful; after uv stimulation, the elastin content was significantly increased (P < 0.05) after addition of the compositions of examples 1-8 and comparative example 1/2/3/4/6/9 to the ex vivo skin tissue photoaging model, compared to the negative control. Wherein, the elastin-up rates of examples 1 to 8 were 163.66%, 156.25%, 143.27%, 91.13%, 111.59%, 135.52%, 127.85%, 105.89%, respectively, and the elastin-up rates of comparative example 12/3/4/6/9 were 66.28%, 50.73%, 41.76%, 61.82%, 35.54%, and 36.82%, respectively. Analysis shows that the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof in comparative examples 1-4 have certain effect of improving the content of elastin, but compared with the results of examples 1-8, the improvement effect is obviously improved when the recombinant humanized III type collagen or sodium hyaluronate and derivatives thereof are compounded for use; the composition formed by one of the recombinant humanized III type collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate compound can obviously increase the content of elastin, promote the regeneration of dermis, improve the skin elasticity, play a role in improving the photoaging of the skin and can be synergistic. In addition, it can be seen from comparison of the results of comparative examples 1 and 1 that comparative examples 6 and 9 show that the recombinant humanized type III collagen used in the examples of the present invention has a better effect of increasing the elastin content and a better effect of compounding sodium hyaluronate and its derivatives.
TABLE 7 influence of the compositions of examples 1-8 and comparative examples 1,2, 3, 4, 6, 9 on the elastin content
6. Effect of the compositions of examples 1-8 and comparative example 1/2/3/4/5/6/9 on integrin alpha 6 subunit content
1. The experimental method comprises the following steps: the method comprises the steps of grouping and dosing the same elastin content test experimental method, taking isolated skin tissues for detection after treatment, fixing by adopting 4% paraformaldehyde, then embedding tissues and slicing tissues, performing conventional immunofluorescence staining on DEJ structure related protein integrin alpha 6 subunits after baking and dewaxing, photographing and observing under a fluorescence microscope within 24 hours, collecting 3 pictures with different fields of view, performing relative IOD analysis and area statistics on target signals in the pictures by using Ipwin picture analysis software, and calculating an average value. Relative IOD mean/area was calculated based on the blank to characterize protein relative expression.
The test concentrations of the composition samples of examples 1 to 8 and comparative example 1/2/3/4/5/6/9 are shown in Table 1, the solvent is EpiGrowth culture solution, and the test solutions of the blank control group and the negative control group are EpiGrowth culture solution.
2. Experimental results: the primary function of DEJ is to connect the epidermis and dermis, provide structural support for the epidermis layer, and act as a "conveyor" for the exchange of nutrients between the epidermis and dermis, controlling the structural integrity of the skin, and playing multiple roles in skin homeostasis and function. The integrin alpha 6 subunit is an important constituent of DEJ, constitutes the basic structure of DEJ, and is responsible for cell-to-matrix and cell-to-cell signaling. As shown in fig. 6 and table 8, after ultraviolet stimulation, the content of integrin alpha 6 subunit in the negative control group is obviously reduced (P < 0.001) compared with the blank control group, which proves that the skin photoaging model modeling is successful; after uv stimulation, the compositions of examples 1-8 and comparative example 1/2/5/6/9 were added to significantly increase the integrin α6 subunit content (P < 0.05) compared to the negative control group, and comparative example 3 and comparative example 4 did not significantly increase the integrin α6 subunit content after the addition of the compositions to the ex vivo skin tissue photoaging model. The rate of increase in the integrin α6 subunit in examples 1 to 8 was 179.26%, 168.70%, 164.39%, 132.28%, 155.74%, 146.20%, 153.48%, 134.02%, respectively, and the rate of increase in the integrin α6 subunit in comparative example 1/2/5/6/9 was 84.09%, 47.64%, 136.04%, 50.96%, 61.15%, respectively. Analysis shows that the recombinant humanized III collagen or sodium hyaluronate used alone in comparative examples 1-2 has a certain effect of improving the content of the integrin alpha 6 subunit, and the effect of the hydrolyzed sodium hyaluronate or dimethyl silanol hyaluronate complex used alone in comparative examples 3-4 on improving the content of the integrin alpha 6 subunit is not obvious, and compared with the results of examples 1-8, the recombinant humanized III collagen and sodium hyaluronate and derivatives thereof are compounded and used, especially when compounded and used with hydrolyzed sodium hyaluronate or dimethyl silanol hyaluronate complex, the more remarkable improving effect is shown; in addition, the effect of comparative example 5 is equivalent to that of examples 4 and 8, and there is no significant difference, which means that on the premise of achieving similar effects, the use amount of recombinant humanized type iii collagen can be greatly reduced and the application cost can be reduced by adding one of the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complexes to be compounded with recombinant humanized type iii collagen. The results show that the composition formed by one of the recombinant humanized III type collagen and the sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex can synergistically increase the content of an integrin alpha 6 subunit, promote the repair of a basement membrane and play a role in improving skin photoaging and photodamage. In addition, as can be seen from comparison of the results of comparative examples 1 and 1, comparative examples 6 and 9 show that the recombinant humanized type III collagen used in the examples of the present invention has a better effect of increasing the content of integrin alpha 6 subunit, and also has a better effect of compounding with sodium hyaluronate and its derivatives.
TABLE 8 influence of the compositions of examples 1-8 and comparative examples 1-6 and 9 on integrin alpha 6 subunit content
7. Human efficacy testing
1. Volunteer selection and request
60 Outdoor workers (40 women, 20 men) were screened, with facial skin redness, impaired barrier, loose skin and wrinkles, aged 35-55 years, divided into two groups (example 9 group and comparative example 10 group), 30 persons each (same ratio between men and women), all subjects had no history of skin or systemic illness, no abnormalities in the tested parts, and no application of any drugs or cosmetics unrelated to the experiment during the test.
2. Test environment
The test site is constant temperature and humidity, the ambient temperature is 20-22 ℃, the relative humidity is 40-60%, and the body of the subject is kept in a stable state before the test. After the face of the subject is cleaned by clear water at about 35 ℃, the test is started after sitting still for 30min in the test environment.
3. Sample application method
The test samples were the product of example 9 and the product of comparative example 10 (placebo), comparative example 10 was identical to the matrix of example 9, except that comparative example 10 did not contain a composition consisting of recombinant humanized type iii collagen and sodium hyaluronate. The preparation is applied 2 times daily, 1 time each after cleansing in the morning and evening, about 1g of sample each time, and 28 days continuously.
4. Test item
(1) The experimental method comprises the following steps: the subjects self-assessed the improvement in facial skin condition after use of the samples in the form of an answer questionnaire and the irritation of the samples. Questionnaire evaluations were performed on days 0 and 28. And carrying out statistical analysis on the test result.
Lifting/improvement after use of sample (rate of change) = (post-use data-pre-use data)/pre-use data×100%
Determination criteria: the scores before and after the use of the sample are obviously different, and the difference is a trend of good significance, and the sample is judged to have corresponding efficacy; the sample is considered to be mild and non-irritating when no adverse reaction occurs during use.
(2) Experimental results: as shown in table 9, subjects showed a significant 37.50% decrease in forehead wrinkle score, a significant 56.20% increase in skin elasticity score, a significant 50.88% increase in skin barrier ability score, a significant 31.61% decrease in Pi Fufan red score, and no adverse reaction during the test, demonstrating the mildness and no irritation of example 9 for the group of example 9 after 28 days of use of the test sample. Whereas the improvement was not evident in the comparative example 10 group. The skin care product containing the recombinant humanized III type collagen and the sodium hyaluronate composition can improve facial wrinkles, increase skin elasticity, reduce facial redness and improve skin barrier capability, thereby improving photoaging and photodamage.
TABLE 9 skin State scoring results before and after use
The above results indicate that a multi-effect composition comprising one of recombinant humanized type iii collagen and sodium hyaluronate/hydrolyzed sodium hyaluronate/dimethyl silanol hyaluronate complex can improve the effects of photoaging and photodamage of skin by basal membrane repair, dermal regeneration, barrier repair, relief of skin inflammation and reduction of apoptotic pathways caused by endoplasmic reticulum stress.
Claims (10)
1. A multi-effect composition containing recombinant humanized III type collagen is characterized by comprising the recombinant humanized III type collagen, sodium hyaluronate and derivatives thereof, wherein the sodium hyaluronate and the derivatives thereof are one of sodium hyaluronate, hydrolyzed sodium hyaluronate and dimethyl silanol hyaluronate complex.
2. The pleiotropic composition of claim 1, wherein the amino acid sequence of the recombinant humanized type iii collagen is shown in SEQ ID No. 1.
3. The pleiotropic composition of claim 1, wherein the final concentration of said recombinant humanized type iii collagen in the system comprising the pleiotropic composition is between 0.002 and 0.5wt% and the final concentration of said sodium hyaluronate and derivatives thereof is between 0.001 and 0.5wt%.
4. The multi-effect composition of claim 1, wherein when the sodium hyaluronate and derivatives thereof are sodium hyaluronate, the final concentration of the recombinant humanized type iii collagen in the system comprising the multi-effect composition is 0.002 to 0.5wt% and the final concentration of the sodium hyaluronate is 0.01 to 0.5wt%.
5. The multi-effect composition of claim 1, wherein when the sodium hyaluronate and derivatives thereof are hydrolyzed sodium hyaluronate, the recombinant humanized type iii collagen has a final concentration of 0.002 to 0.5wt% and the hydrolyzed sodium hyaluronate has a final concentration of 0.01 to 0.5wt% in a system comprising the multi-effect composition.
6. The multi-effect composition of claim 1, wherein when the sodium hyaluronate and derivatives thereof are dimethyl silanol hyaluronate complexes, the final concentration of the recombinant humanized type iii collagen in a system comprising the multi-effect composition is 0.002 to 0.5wt%, and the final concentration of the dimethyl silanol hyaluronate complexes is 0.001 to 0.1wt%.
7. Use of the multi-effect composition of claim 1 for the preparation of a cosmetic or skin care product.
8. The use according to claim 7, wherein the cosmetic or dermatological product has an effect of improving skin photoaging and photodamage.
9. A cosmetic or skin care product comprising the multi-effect composition of claim 1.
10. The cosmetic or dermatological product according to claim 9, characterized in that it is embodied in the form of a liquid, gel, cream, emulsion, mask or freeze-dried powder.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118286107A true CN118286107A (en) | 2024-07-05 |
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