A composition for improving skin color and use thereof

Abstract

The present application relates to a kind of compositions for improving skin color and its application, belong to the field of cosmetics.The present application provides a kind of composition, the composition includes recombinant humanization type IV collagen and lavender exosome, the amino acid sequence of the recombinant humanization type IV collagen is as shown in SEQ ID NO.1, both compound use, can increase the expression of heat shock protein, improve cell viability under heat stress, inhibit the expression of endothelin and the production of glycation product, to improve the effect of skin skin color, and both have synergistic effect.The composition of the present application reduces the amount of recombinant humanization type IV collagen under the premise of achieving similar effects by using lavender exosome, significantly reduces the application cost of recombinant humanization type IV collagen.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for improving skin color and its application, belonging to the technical field of cosmetics. BACKGROUND

[0002] Skin color health is the key to maintaining the young state of the skin. Skin color is divided into "constitutional skin color" and "selective skin color". The constitutional skin color refers to the skin color of non-exposed parts, which is mainly determined by genetics; while the selective skin color refers to the skin color of exposed parts, which is affected by various regulating factors such as ultraviolet rays, heat, toxic substances in the environment, and can be adjusted through artificial intervention.

[0003] Ultraviolet (UV) radiation is the main external factor that causes skin damage and leads to skin redness. UV can stimulate skin cells to release a large amount of ROS (reactive oxygen free radicals), causing oxidation of proteins and lipids, and can regulate various cytokines by initiating downstream signal cascades. Inflammatory mediators and chemokines act on capillaries, inducing endothelial cells to express a large amount of endothelin-1 (ET-1), increasing vascular permeability, causing tissue fluid extravasation, and producing symptoms such as erythema and edema. After cell damage, a series of damage-associated molecular patterns are released, such as heat shock proteins (Hsp), which protect cells from damage caused by various stress factors such as free radicals, heat, and toxic substances in the environment, promote the correct folding, assembly of proteins, and correct the structure of proteins, and promote the self-repair of damaged cells. In addition, Hsp also participates in the transduction and regulation of cell proliferation, differentiation, and apoptosis. Hsp27 is a small molecular weight Hsp protein, which rapidly increases in expression in the early stage of stress response for a short period of time, and repairs the cells, but as time goes on, the expression of Hsp27 decreases, causing an imbalance in the process of cell damage repair.

[0004] Non-enzymatic glycosylation refers to the process of reacting the aldehyde or ketone group of reducing sugar with the free amino group in macromolecules such as proteins to form irreversible products-advanced glycation end-products (AGEs) without enzyme catalysis. AGEs are brown or black macromolecular substances with fluorescence characteristics, which exist in the epidermis and dermis. They can cause skin to appear dull and yellow by directly depositing on the skin tissue, and can also indirectly cause skin pigmentation by binding to the receptor for advanced glycation end products (RAGE) and activating the pigment secretion-related signal pathway.

[0005] Collagen is a functional protein, accounting for 30% of all proteins in the body, mainly existing in tissues such as skin, cartilage, tendon, etc., and plays an important role in maintaining the normal physiological functions of cells, tissues and organs. Type IV collagen is the main component of the dermal-epidermal junction (DEJ) and is used to maintain the stability of the basement membrane structure and function. Due to the certain risks of animal collagen in biological safety and immunogenicity, in recent years, recombinant collagen with higher purity and lower immunogenicity has been continuously developed to replace animal collagen. The biosynthetic recombinant humanized type IV collagen has a high consistency with the amino acid sequence of the natural collagen in the human body, can not only provide a scaffold for cell growth, but also has multiple active sites, can recognize and interact with specific receptors on the cell surface, and then activate the intracellular signal transduction pathway, participate in important physiological processes such as cell adhesion, migration, growth, proliferation and differentiation, but the cost of the biosynthetic recombinant collagen is high, which limits its wide application.

[0006] Lavender is a fragrant plant, and its essential oil is often extracted for use as aromatherapy, massage oil, fragrance, and cosmetic raw materials. In addition to preparing essential oil, lavender exosome extract obtained by plant exosome extraction method can play a role in anti-skin aging, inhibiting skin inflammation and anti-glycation (CN117187156A).

[0007] The inventors have found that the current research on the efficacy of recombinant type IV collagen is relatively single. Patent CN117720638A mainly studies the antioxidant and anti-inflammatory effects of modified recombinant type IV collagen, and patent CN117024572A prepares a recombinant type IV collagen with anti-wrinkle effect. However, there is no related research on the improvement of skin color by protecting cells from environmental stress damage, enhancing cell self-repairing ability, inhibiting the expression of endothelin and anti-glycation of recombinant type IV collagen. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a composition for improving skin color and its application. The composition of the present application comprises recombinant humanized type IV collagen and lavender exosome, which can increase the expression of heat shock proteins, improve cell viability under heat stress, inhibit the expression of endothelin and the production of glycation products, thereby improving the skin color.

[0009] The technical solution of the present application is as follows:

[0010] A composition for improving skin color, comprising recombinant humanized type IV collagen and lavender exosome.

[0011] According to the present application, the amino acid sequence of the recombinant humanized type IV collagen is shown in SEQ ID NO. 1.

[0012] Further preferably, the preparation method of the recombinant humanized type IV collagen is as follows: the coding gene of the recombinant humanized type IV collagen shown in SEQ ID NO. 1 is codon-optimized according to the codon bias of E. coli, the codon-optimized coding gene is inserted into an expression vector pET28a to construct a recombinant plasmid, the recombinant plasmid is transformed into E. coli BL21 (DE3) to construct a recombinant strain, and the recombinant strain is cultured to the logarithmic growth phase, and then the recombinant humanized type IV collagen is obtained after IPTG induction.

[0013] Further preferably, the nucleotide sequence of the codon-optimized coding gene is shown in SEQ ID NO. 2.

[0014] In the present application, the lavender exosome can be prepared according to the method described in Example 1 of patent application CN117187156A.

[0015] According to the present application, in the composition-containing system, the final concentration of the recombinant humanized type IV collagen is 0.001-0.5 wt%, and the final concentration of the lavender exosome is 0.0001-0.5 wt%.

[0016] In the present application, the above-mentioned composition can increase the expression of heat shock proteins, improve cell viability under heat stress, inhibit the expression of endothelin and the production of glycation products, thereby achieving the effect of improving skin color.

[0017] In the present application, in the composition-containing system, in addition to the recombinant humanized type IV collagen and the lavender exosome, other raw material components allowed to be added in the cosmetic field can also be included, including but not limited to emulsifiers, emollients, humectants and thickening agents, etc.

[0018] The above-mentioned composition is applied in the preparation of cosmetics or skin care products.

[0019] According to the present application, the cosmetics or skin care products can increase the expression of heat shock proteins, improve cell viability under heat stress, inhibit the expression of endothelin and the production of glycation products, thereby achieving the effect of improving skin color.

[0020] A cosmetic or skin care product containing the above-mentioned composition.

[0021] According to the present application, the cosmetic or skin care product can be prepared in different dosage forms, such as serum, essence, cream, mask or lyophilized powder, etc.

[0022] In the present application, the cosmetic or skin care product further contains other raw material components allowed to be added in the cosmetic field, including but not limited to emulsifiers, emollients, humectants and thickening agents, etc.

[0023] In the present application, the cosmetic or skin care product can be used on the skin surface to improve skin color by increasing the expression of heat shock proteins, improving cell viability under heat stress, inhibiting the expression of endothelin and the production of glycation products.

[0024] Beneficial effects:

[0025] 1. The present application provides a composition comprising recombinant humanized type IV collagen and lavender exosomes, the amino acid sequence of the recombinant humanized type IV collagen is shown as SEQ ID NO. 1, and the two are used in combination to increase the expression of heat shock proteins, improve cell viability under heat stress, inhibit the expression of endothelin and the production of glycation products, thereby improving the skin color of the skin, and the two have a synergistic effect.

[0026] 2. The composition of the present application reduces the amount of recombinant humanized type IV collagen used under the premise of achieving similar effects, greatly reducing the application cost of recombinant humanized type IV collagen by using lavender exosomes. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Column chart of Hsp27 protein content of HaCaT cells under the action of the compositions of Examples 1-4 and Comparative Examples 1-4;

[0028] Figure 2 Column chart of HaCaT cell viability under the action of the compositions of Examples 1-4 and Comparative Examples 1-4;

[0029] Figure 3 Column chart of HUVEC cell ET-1 content under the action of the compositions of Examples 1-4 and Comparative Examples 1-4;

[0030] Figure 4 Column chart of HSF cell CML content under the action of the compositions of Examples 1-4 and Comparative Examples 1-4;

[0031] Among them, the significance compared with the blank group is represented by #, 0.01 DETAILED DESCRIPTION

[0032] In order to better understand the present application, further elaboration is made below in conjunction with examples, but the scope of protection of the present application is not limited to the following examples only, and the examples should not be considered as limiting the scope of protection of the present application. The reagents and materials involved in the examples, if not specifically stated, are all ordinary commercially available products.

[0033] The lavender exosome used in the examples and comparative examples can be prepared according to the method described in Example 1 of patent application No. CN117187156A.

[0034] The amino acid sequence of the recombinant humanized type IV collagen in the examples of the present application is shown in SEQ ID NO. 1, which is prepared according to the patent 2024102556491, i.e. the codon optimization of the coding gene is performed according to the codon bias of Escherichia coli, the nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO. 2, the coding gene is inserted into the expression vector pET28a to construct a recombinant plasmid, the recombinant plasmid is transformed into Escherichia coli BL21 (DE3) to construct a recombinant strain, the recombinant strain is cultured to the logarithmic growth phase, IPTG is used for induction expression, and the recombinant humanized type IV collagen is obtained after cell disruption and purification.

[0035] The recombinant humanized type III collagen in Comparative Example 3 is obtained according to the preparation method described in patent CN111087463A;

[0036] The recombinant humanized type IV collagen in Comparative Example 4 is different from the recombinant humanized type IV collagen in the examples in that the amino acid sequence is different, the amino acid sequence is shown in SEQ ID NO. 3, the nucleotide sequence of the coding gene after codon optimization according to the codon bias of Escherichia coli is shown in SEQ ID NO. 4, represented as recombinant humanized type IV collagen-2, which can be prepared according to the conventional technical method in the art, or according to the preparation method of the recombinant humanized type IV collagen in the examples.

[0037] Example:

[0038] Examples 1-4 provide a composition mainly comprising recombinant humanized type IV collagen and lavender exosome, and the ingredient ratio is shown in Table 1. As a comparison, Comparative Examples 1-4 are also provided in Table 1, which are different from the examples in that they only contain one substance in the composition or the source of one substance is different. Through cell level experiments, the effects of the above-mentioned compositions on the expression of heat shock proteins, cell viability under heat stress, the expression of endothelins and the production of glycation products are verified.

[0039] Table 1. Raw material ingredients and ratio of compositions of Examples 1-4 and Comparative Examples 1-4 Unit: wt%

[0040]

[0041]

[0042] A composition consisting of recombinant humanized type IV collagen and lavender exosome was added to a skin care serum base to make a skin care product, Example 5, and a placebo group, Comparative Example 5, was also set up. The difference between Comparative Example 5 and Example 5 is that Comparative Example 5 does not contain the composition consisting of recombinant humanized type IV collagen and lavender exosome. The specific raw material ingredients and proportions are shown in Table 2, and the effect of improving skin color was tested by human efficacy experiment.

[0043] Table 2. Raw material ingredients and proportions of skin care serum of Example 5 and Comparative Example 5

[0044]

[0045] The products of Example 5 and Comparative Example 5 were both prepared by conventional methods in the art.

[0046] Experimental Example:

[0047] The products of the above examples and comparative examples were tested for performance, as follows:

[0048] The human immortalized keratinocytes (HaCaT cells) and human skin fibroblasts (HSF cells) used in the following tests were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; human umbilical vein endothelial cells (HUVEC cells) were purchased from Beina Creational Link (Suzhou) Co., Ltd.; other reagents and materials were ordinary commercially available products unless otherwise specified.

[0049] I. Effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on Hsp27 protein content

[0050] 1. Experimental method: HaCaT cells were cultured in high-sugar DMEM medium at 37°C in a 5% CO2 constant temperature incubator. Logarithmic growth phase cells were diluted to 1.5 x 10 5 cells / mL using high-sugar DMEM medium, and then inoculated into 96-well plates at 100 μL per well. The experiment set up a blank control group, a negative control group (13 mW / cm 2 UVB), and a sample group (13 mW / cm 2UVB+Example 1~4 / Comparative Example 1~4), 3 wells in each group. After inoculation of cells, the 96-well plate was first placed in a 37°C, 5% CO2 incubator for 24 h, then the culture solution was discarded, 200 μL high-sugar DMEM culture solution was added to each well of the blank control group and the negative control group, 200 μL high-sugar DMEM culture solution containing the test sample was added to each well of the sample group, and the plate was placed in the incubator for 4 h. Then the culture solution was discarded and replaced with PBS buffer. Except for the blank control group, UVB irradiation was performed using a medium wave ultraviolet treatment instrument (13 mW / cm 2 UVB) was fixed at a position 10 cm above the 96-well plate, and irradiation was performed for 4 min. Then the PBS buffer was discarded, 200 μL high-sugar DMEM culture solution was added to each well of the blank control group and the negative control group, 200 μL high-sugar DMEM culture solution containing the test sample was added to each well of the sample group, and the plate was placed in the incubator for continuous culture for 16 h. After the culture was completed, the culture solution was discarded, the cells were washed twice with PBS buffer, and the content of Hsp27 protein was detected using an immunofluorescence method. The photograph was observed and taken under a fluorescence microscope, the target signal in the picture was analyzed using Ipwin32 picture analysis software, and the average value was calculated. Taking the blank control group as the reference, the relative IOD average value was calculated to represent the relative content of Hsp27 protein.

[0051] Among them, the test concentration of the composition sample of Example 1~4 and Comparative Example 1~4 is shown in Table 1, and the solvent is high-sugar DMEM culture solution.

[0052] 2. Experimental results: as shown in Table 2, the relative IOD average value of the sample group was significantly higher than that of the blank control group and the negative control group, indicating that the sample had a significant effect on the expression of Hsp27 protein. Figure 1As shown in Table 3, after UVB stimulation, the Hsp27 protein content in the negative control group was significantly higher than that in the blank control group (P<0.0001), which proved that the photodamage model was successfully established. Compared with the negative control group, after the compositions of Examples 1-4 and Comparative Examples 1 / 4 were added to the cells, the Hsp27 protein content was significantly increased (P<0.01), and Comparative Examples 2 and 3 had no significant effect on the Hsp27 protein content. Among them, the increase rates of Hsp27 protein content of Examples 1-4 were 62.65%, 45.03%, 58.43%, and 53.41%, respectively, and the increase rates of Hsp27 protein content of Comparative Examples 1-4 were 25.97%, 7.38%, 11.13%, and 16.28%, respectively. As can be seen from Comparative Examples 1 and 2, the use of recombinant humanized type IV collagen alone has a certain effect on the increase of Hsp27 protein content, while the use of lavender exosomes alone has no significant effect on the increase of Hsp27 protein content. However, compared with the results of Examples 1-4, it can be seen that the use of the two in combination increases the effect. It is proved that the composition of recombinant humanized type IV collagen and lavender exosomes can synergistically increase the Hsp27 protein content and enhance the self-repairing ability of the skin. In addition, the effect of Example 2 is better than that of Comparative Example 1, which shows that by adding lavender exosomes and recombinant humanized type IV collagen, the amount of recombinant humanized type IV collagen can be greatly reduced, and the application cost can be reduced. As can be seen from Comparative Examples 3 and 4 and Example 1, the composition of recombinant humanized type IV collagen and lavender exosomes used in the examples of the present application has a better effect on increasing the Hsp27 protein content, while the composition of recombinant humanized type III collagen and lavender exosomes has no significant effect on increasing the Hsp27 protein content. The composition of recombinant humanized type IV collagen with different amino acid sequences and lavender exosomes has a poor effect on increasing the Hsp27 protein content.

[0053] Table 3. Effect of compositions of Examples 1-4 and Comparative Examples 1-4 on Hsp27 protein expression

[0054] Group Relative IOD average SD P-value Elevation rate (%) Blank control group 1.00 0.01 / / Negative control group 2.71 0.03 ＜0.0001 / Example 1 4.41 0.10 ＜0.0001 62.65 Example 2 3.93 0.17 0.0002 45.03 Example 3 4.29 0.08 ＜0.0001 58.43 Example 4 4.16 0.04 ＜0.0001 53.41 Comparative Example 1 3.41 0.15 0.0013 25.97 Comparative Example 2 2.91 0.14 0.0770 7.38 Comparative Example 3 3.01 0.20 0.0596 11.13 Comparative Example 4 3.15 0.08 0.0009 16.28

[0055] II. Effect of compositions of Examples 1-4 and Comparative Examples 1-4 on cell viability under heat stress

[0056] 1. Experimental method: Take HaCaT cells in logarithmic growth phase, dilute the cell density to 1×10 5The cells were inoculated at 1 x 104cells / mL, and then inoculated into 96-well plates at 100 μL per well, and incubated overnight in a 37°C, 5% CO2 constant-temperature incubator. The blank control group, negative control group (45°C), and sample group (45°C + Examples 1-4 / Comparative Examples 1-4) were set up in triplicate. After overnight incubation, the culture solution was discarded, 200 μL high-sugar DMEM culture solution was added to each well of the blank control group and negative control group, and 200 μL high-sugar DMEM culture solution containing the test sample was added to each well of the sample group, and the plates were incubated in an incubator (37°C, 5% CO2) for 24 h. After the incubation was completed, the blank control group was incubated in a 37°C incubator for 27.5 h, the negative control group and sample group were incubated in a 45°C incubator for 3.5 h, and then transferred to a 37°C incubator for 24 h, and then the cell viability was determined by the MTT method.

[0057] The test concentration of the composition samples of Examples 1-4 and Comparative Examples 1-4 is shown in Table 1, and the solvent is high-sugar DMEM culture solution.

[0058] 2. Experimental results: As shown in Table 2, the cell viability of the sample group was higher than that of the blank control group and negative control group, and the cell viability of the sample group was higher than that of the blank control group and negative control group. Figure 2As shown in Table 4, under the heat stress at 45℃, the cell viability of the negative control group was significantly lower than that of the blank control group (P<0.0001), which proved that the heat stress model was successfully established. Compared with the negative control group, after the compositions of Examples 1-4 and Comparative Examples 1 / 4 were added to the cells and then subjected to 45℃ heat stress induction, the cell viability was significantly improved (P<0.05), while the addition of Comparative Examples 2 and 3 had no significant effect on the cell viability. Among them, the cell viability improvement rates of Examples 1-4 were 56.29%, 34.11%, 50.08%, and 45.73%, respectively, and the cell viability improvement rates of Comparative Examples 1-4 were 18.86%, 1.12%, 2.16%, and 6.05%, respectively. As shown in Comparative Examples 1 and 2, the use of recombinant humanized type IV collagen alone had a certain effect on improving the cell viability under heat stress, while the use of lavender exosomes alone had no significant effect on improving the cell viability. However, compared with the results of Examples 1-4, the use of the combination of the two improved the effect, indicating that the composition of recombinant humanized type IV collagen and lavender exosomes could improve the cell viability under heat stress and had a certain protective effect on cells, and could have a synergistic effect. In addition, the effect of Example 2 was better than that of Comparative Example 1, indicating that the use of lavender exosomes and recombinant humanized type IV collagen could greatly reduce the amount of recombinant humanized type IV collagen and reduce the application cost. As shown in Comparative Examples 3 and 4 and Example 1, the composition of recombinant humanized type IV collagen and lavender exosomes used in the present application had a better effect on improving the cell viability, while the composition of recombinant humanized type III collagen and lavender exosomes had no significant effect on improving the cell viability. The composition of recombinant humanized type IV collagen with different amino acid sequences and lavender exosomes had a poor effect on improving the cell viability.

[0059] Table 4. Effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on cell viability under heat stress

[0060]

[0061]

[0062] III. Effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on ET-1 content

[0063] 1、Experimental method: HUVEC cells were cultured in ECM medium containing 5% FBS, 1% ECGS and 1% P / S in a constant temperature incubator at 37℃ and 5% CO2. Blank control group, negative control group (4 μg / mL of LPS), sample group (4 μg / mL of LPS + examples 1-4 / comparative examples 1-4) were set up, and each group was set up in triplicate. Logarithmic growth phase cells were taken, and the cell density was diluted to 1×10 5 Each well was added with 100 μL of the diluted cells, and the cells were cultured in a constant temperature incubator at 37℃ and 5% CO2 overnight. The culture medium was discarded, 200 μL of ECM culture medium was added to each well of the blank control group, 200 μL of ECM culture medium containing 4 μg / mL of LPS was added to each well of the negative control group, and 200 μL of ECM culture medium containing the test sample and 4 μg / mL of LPS was added to each well of the sample group, and the culture was continued for 24 h. After the culture was completed, the culture medium was collected, and the content of ET-1 was detected according to the instructions of the ELISA kit.

[0064] The test concentration of the composition sample of examples 1-4 and comparative examples 1-4 is shown in Table 1, and the solvent is ECM culture medium.

[0065] 2、Experimental results: As shown in Table 2, the ET-1 content of the sample group was significantly lower than that of the negative control group, and the difference was statistically significant (P <0.05). Figure 3As shown in Table 5, after LPS stimulation, the ET-1 content in the negative control group was significantly higher than that in the blank control group (P<0.0001), which proved that the stimulation model was successfully established. Compared with the negative control group, the ET-1 content in the sample group added with the compositions of Examples 1-4 and Comparative Examples 1 / 4 was significantly reduced (P<0.05), the sample group added with the compositions of Comparative Examples 2 / 3 had no significant inhibitory effect on the increase of ET-1 content, and even the sample group added with Comparative Example 2 had a significant promoting effect on the ET-1 content (P<0.05); among them, the inhibition rates of Examples 1-4 on the ET-1 content were 46.07%, 29.45%, 41.14% and 36.20% respectively, and the inhibition rates of Comparative Examples 1-4 on the ET-1 content were 19.28%, -9.38%, 4.84% and 11.59% respectively. From the results of Comparative Example 1 and Comparative Example 2, it can be seen that the recombinant humanized type IV collagen alone has an inhibitory effect on the increase of ET-1 content, but the lavender exosome alone can promote the increase of ET-1 content; compared with the results of Examples 1-4, it can be seen that the inhibitory effect is significantly improved when the two are used together; in addition, the effect of Example 2 is better than that of Comparative Example 1, which shows that by compounding the lavender exosome and the recombinant humanized type IV collagen, the amount of the recombinant humanized type IV collagen can be greatly reduced, and the application cost can be reduced. The above results show that the composition formed by the recombinant humanized type IV collagen and the lavender exosome can inhibit the permeability of blood vessels by inhibiting the ET-1 content, improve the skin color, and have a synergistic effect. Compared with the results of Example 1, it can be seen from Comparative Examples 3 and 4 that the composition of the recombinant humanized type IV collagen and the lavender exosome used in the examples of the present application has a better inhibitory effect on the ET-1 content, while the composition of the recombinant humanized type III collagen and the lavender exosome has no significant inhibitory effect on the expression of ET-1, and the composition of the recombinant humanized type IV collagen with different amino acid sequences and the lavender exosome has a poor inhibitory effect on the expression of ET-1.

[0066] Table 5. Effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on the ET-1 content

[0067] Group ET-1 average content (pg / mL) SD P-value Inhibition rate (%) Blank control group 100.22 3.57 / / Negative control group 219.90 9.35 ＜0.0001 / Example 1 118.59 5.36 0.0001 46.07 Example 2 155.13 10.05 0.0012 29.45 Example 3 129.44 3.31 0.0001 41.14 Example 4 140.29 14.84 0.0014 36.20 Comparative Example 1 177.50 8.16 0.0041 19.28 Comparative Example 2 240.52 4.66 0.0268 -9.38 Comparative Example 3 209.26 13.13 0.3166 4.84 Comparative Example 4 194.40 11.88 0.0432 11.59

[0068] Four, the effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on the sugar product CML content

[0069] 1. Experimental method: human skin fibroblasts (HSF cells) were collected and prepared into a cell density of 1×10 4The cell suspension was inoculated in 24-well plates at 1 mL per well. The blank control group, the negative control group (methylglyoxal), and the sample group (methylglyoxal + Examples 1-4 / Comparative Examples 1-4) were set up in triplicate. After inoculation, the 24-well plates were incubated in a 37°C, 5% CO2 incubator for 24 h, and then the high-sugar DMEM culture medium containing 10% newborn calf serum was replaced and incubated in the incubator for 24 h. Then the culture medium was discarded, 2 mL of high-sugar DMEM culture medium was added to each well of the blank control group, 2 mL of high-sugar DMEM culture medium containing 0.3 mM methylglyoxal was added to each well of the negative control group, and 2 mL of high-sugar DMEM culture medium containing the test sample and 0.3 mM methylglyoxal was added to each well of the sample group, and the plates were incubated in the incubator for 48 h. After incubation, the cells were fixed with 4% paraformaldehyde, and the photographs were taken under a fluorescence microscope. Three field photographs were taken for each group, the target signal in the photographs was analyzed using Ipwin32 image analysis software, and the average value was calculated. The blank control group was used as a reference, and the relative IOD average value was calculated to represent the content of the glycation product CML.

[0070] The test concentration of the composition samples of Examples 1-4 and Comparative Examples 1-4 is shown in Table 1, and the solvent is high-sugar DMEM culture medium.

[0071] 2. Experimental results: Carboxymethyl lysine (CML) is a typical compound of AGEs, which can cause changes in skin color and make the skin yellow and dull. For example, Figure 4As shown in Table 6, after methylglyoxal stimulation, the CML content in the negative control group was significantly higher than that in the blank control group (P<0.0001), which proved that the modeling was successful. Compared with the negative control group, after the damaged cells were treated with the compositions of Examples 1-4 and Comparative Examples 2 / 3 / 4, the content of the glycation product CML was significantly reduced (P<0.0001), and Comparative Example 1 had no significant effect on the content of the glycation product CML; among them, the inhibition rates of Examples 1-4 on the content of the glycation product CML were 94.92%, 87.52%, 81.82%, and 82.80%, respectively, and the inhibition rates of Comparative Examples 1-4 on the content of the glycation product CML were 10.52%, 67.38%, 69.70%, and 71.66%, respectively. As can be seen from Comparative Example 1 and Comparative Example 2, the recombinant humanized type IV collagen alone has no significant inhibitory effect on the content of the glycation product CML, and the lavender exosome alone has a certain inhibitory effect on the content of the glycation product CML; but compared with the results of Examples 1-4, it can be seen that the inhibitory effect is enhanced when the two are used in combination; it is proved that the composition of recombinant humanized type IV collagen and lavender exosome can synergistically reduce the content of the glycation product CML and play an anti-glycation role. In addition, the effect of Example 2 is better than that of Comparative Example 1, which shows that by adding lavender exosome and recombinant humanized type IV collagen, the amount of recombinant humanized type IV collagen can be greatly reduced, and the application cost can be reduced. As can be seen from the results of Comparative Example 3 and Comparative Example 4 compared with Example 1, the composition of recombinant humanized type IV collagen and lavender exosome used in the present application has a better effect on reducing the content of the glycation product CML.

[0072] Table 6. Effect of the compositions of Examples 1-4 and Comparative Examples 1-4 on the content of the glycation product CML

[0073] Group Relative IOD average SD P-value Inhibition rate (%) Blank control group 1.00 0.12 / / Negative control group 3.74 0.12 ＜0.0001 / Example 1 0.19 0.03 ＜0.0001 94.92 Example 2 0.47 0.05 ＜0.0001 87.52 Example 3 0.68 0.03 ＜0.0001 81.82 Example 4 0.64 0.05 ＜0.0001 82.80 Comparative Example 1 3.35 0.22 0.0511 10.52 Comparative Example 2 1.22 0.05 ＜0.0001 67.38 Comparative Example 3 1.13 0.15 ＜0.0001 69.70 Comparative Example 4 1.06 0.07 ＜0.0001 71.66

[0074] V. Human efficacy test

[0075] 1. Selection and requirements of subjects

[0076] 60 subjects with dark and red facial skin (40 women and 20 men) aged 30-55 years were selected and divided into two groups (Example 5 group and Comparative Example 5 group), each group had 30 people (the same ratio of men and women), all subjects had no history of skin or systemic diseases, no abnormalities in the test site, and no application of any unrelated drugs or cosmetics during the test period.

[0077] 2. Test environment

[0078] The test site is constant temperature and humidity, the ambient temperature is 20-22℃, the relative humidity is 40-60%, and the subjects should be in stable state before the test. After the subjects' faces are washed with water at about 35℃, the test is started after they sit in the test environment for 30 min.

[0079] 3. Method of using the sample

[0080] The test sample is the product of Example 5 and the product of Comparative Example 5 (placebo), which is consistent with the matrix of Example 5, except that the composition composed of recombinant humanized type IV collagen and lavender exosome is not contained in Comparative Example 5. About 1 g of the sample is used each time, twice a day, after cleansing in the morning and evening, for 28 consecutive days.

[0081] 4. Test items

[0082] (1) Experimental method: The skin color of the two groups of subjects before using the product and after using the product for 28 days was detected using the skin color detector Colorimeter CL400, and statistical analysis was performed to calculate the change rate. The increase in the L* value of the skin color indicates that the skin color becomes lighter; the decrease in the a* value of the skin color indicates that the skin redness decreases; and the decrease in the b* value of the skin color indicates that the skin yellowing decreases.

[0083] (2) Experimental results: As shown in Table 7, after the subjects used the test sample for 28 days, the L* value change rate of Example 5 was 8.09%, and the L* value change rate of Comparative Example 5 was 1.85%, and there was a significant difference between them (P<0.0001); the a* value change rate of Example 5 was -16.77%, and the a* value change rate of Comparative Example 5 was -4.01%, and there was a significant difference between them (P<0.001); the b* value change rate of Example 5 was -13.30%, and the b* value change rate of Comparative Example 5 was -1.83%, and there was a significant difference between them (P<0.0001); indicating that the skin care product containing recombinant humanized type IV collagen and lavender exosome can improve the skin color.

[0084] Table 7. Test results of skin color index before and after using the product

[0085]

[0086]

[0087] Note: P<0.05 is considered to have a significant difference compared with Comparative Example 5.

[0088] The above results show that the composition comprising the recombinant humanized type IV collagen and lavender exosome can improve skin color by increasing the expression of heat shock proteins, improving cell viability under heat stress, inhibiting the expression of endothelin and the production of glycation products.

Claims

1. A composition for improving skin color, characterized by comprising: The composition comprises recombinant humanized collagen type IV and lavender exosome, and the amino acid sequence of the recombinant humanized collagen type IV is shown as SEQ ID NO.

1.

2. The composition of claim 1, wherein The preparation method of the recombinant humanized collagen type IV is as follows: the coding gene of the recombinant humanized collagen type IV shown as SEQ ID NO. 1 is codon-optimized according to the codon bias of E. coli, the codon-optimized coding gene is inserted into an expression vector pET28a to construct a recombinant plasmid, the recombinant plasmid is transformed into E. coli BL21 (DE3) to construct a recombinant strain, and the recombinant strain is cultured to logarithmic growth phase, and then the recombinant humanized collagen type IV is obtained after IPTG induction.

3. The composition of claim 2, wherein The nucleotide sequence of the codon-optimized coding gene is shown as SEQ ID NO.

2.

4. The composition of claim 1, wherein In the system containing the composition, the final concentration of the recombinant humanized collagen type IV is 0.001-0.5 wt%, and the final concentration of the lavender exosome is 0.0001-0.5 wt%.

5. Use of the composition of claim 1 in the preparation of a cosmetic.

6. Use according to claim 5, wherein The cosmetic can improve skin color by increasing the expression of heat shock proteins, improving cell viability under heat stress, inhibiting the expression of endothelin and the production of glycation products.

7. A cosmetic containing the composition of claim 1.

8. The cosmetic product according to claim 7, wherein The cosmetic can be prepared in different dosage forms, including serum, essence, cream, mask or lyophilized powder.

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