A composition with the efficacy of repairing cell photo-damage and its application

Abstract

The present invention discloses a composition having the efficacy of repairing cell photo-damage and its application. The composition having the efficacy of repairing cell photo-damage is composed of retinol and ginkgo biloba extract, and the mass ratio of retinol to ginkgo biloba extract is 1:(0.5 - 10). The present invention discovers that the compounding of retinol and ginkgo biloba extract can synergistically play a role in repairing cell photo-damage, and the compound composition of the present invention has good application prospects in cosmetic raw materials having the function of repairing photo-damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and particularly relates to a composition with the efficacy of repairing cell photo-damage and its application. Background Art

[0002] Skin photo-damage is the damage caused by long-term exposure of the skin to sunlight. When the skin is exposed to ultraviolet rays, obvious changes will occur in the skin collagen tissue, etc., and a large amount of reactive oxygen species (ROS) will be generated, causing cell damage and inflammation, etc.

[0003] Some natural plant extracts have excellent effects in aspects such as antioxidant; retinol has the effects of anti-wrinkle and reducing fine lines. How to develop cosmetic raw materials with the efficacy of repairing cell photo-damage is a technical problem to be solved. Summary of the Invention

[0004] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the specification of this application, to avoid obscuring the purpose of this part, the abstract of the specification, and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] As one aspect of the present invention, the present invention provides a composition with the efficacy of repairing cell photo-damage, wherein: the composition with the efficacy of repairing cell photo-damage is composed of retinol and ginkgo biloba extract, and the mass ratio of retinol to ginkgo biloba extract is 1∶(0.5 - 10).

[0006] As a preferred scheme of the composition with the efficacy of repairing cell photo-damage according to the present invention: the mass ratio of retinol to ginkgo biloba extract is 1∶(1 - 5).

[0007] As a preferred scheme of the composition with the efficacy of repairing cell photo-damage according to the present invention: the mass ratio of retinol to ginkgo biloba extract is 1∶3.

[0008] As one aspect of the present invention, the present invention provides the application of the composition with the efficacy of repairing cell photo-damage in cosmetics.

[0009] As one implementation mode of the present invention, the ginkgo biloba extract of the present invention includes extraction with 60% ethanol as a solvent, and the extract is obtained by precipitation, filtration, concentration, and freeze-drying to obtain ginkgo extract.

[0010] Advantages of the present invention: The present invention discovers through research that ginkgo biloba extract plays a major role in the down-regulation of ROS level. Ginkgo biloba extract and retinol synergistically inhibit the activation of the P38-MAPK pathway, and retinol plays a key role in the down-regulation of MMP-1 expression. Since ginkgo biloba extract down-regulates ROS and inhibits P38-MARK, it has a synergistic effect on the inhibition of MMP-1 expression, thereby enhancing the synergistic effect of the composition and synergistically playing a role in repairing cellular photo-damage. The compound composition of the present invention has good application prospects among the cosmetic raw materials with the function of repairing photo-damage. Description of the Drawings

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0012] Figure 1 It is the cellular photo-damage rate of the UVA damage model in Example 1.

[0013] Figure 2 It is the test results of the cell viability of each experimental group.

[0014] Figure 3 It is the test results of the cell viability after the compounding of active components in different ratios.

[0015] Figure 4 It is the microscopic photograph of cells in the photo-damage experiment.

[0016] Figure 5 It is the photograph of cell β-galactosidase staining.

[0017] Figure 6 It is the statistics of the proportion of the β-galactosidase staining area.

[0018] Figure 7 It is the fluorescence microscope picture of ROS.

[0019] Figure 8 It is the statistics of the relative intensity of intracellular ROS.

[0020] Figure 9 It is the statistics of the content of MMP-1.

[0021] Figure 10 It is the statistics of the relative expression level of C-JUN protein.

[0022] Figure 11 It is the statistics of the relative expression level of P38 protein.

[0023] Figure 12Schematic diagram of the mechanism of the synergistic effect of retinol and ginkgo biloba extract.

[0024] Figure 13 Fluorescence staining picture of cellular collagen content. Detailed implementation manners

[0025] To make the above objects, features and advantages of the present invention more obvious and understandable, the following specific embodiments are used to describe the detailed implementation manners of the present invention in detail.

[0026] Example 1:

[0027] Establish a UVA damage model: Take well-conditioned HFF-1 cells (human skin fibroblasts), digest them with trypsin and count them by hemocytometry, and prepare a cell suspension of 1×10 4 cells / mL. Inoculate 100 μL per well into a 96-well plate. After the 96-well plate adheres to the wall in an incubator at 37 °C, 5% CO2 and saturated humidity for 24 h, discard the DMEM complete medium in the wells and carefully rinse once with PBS. Cover the cells with 100 μL of PBS per well and induce them under a UVA lamp. The irradiation doses of different reaction wells are 6.5, 8.7, 10.9 J / cm 2 . Use unirradiated HFF-1 cells as the control group, use the wells with only dimethyl sulfoxide as the blank group, and use UVA-irradiated HFF-1 cells as the experimental group. After irradiation, culture in serum-free DMEM medium for 24 h, then aspirate the liquid in the wells, wash each well 2 times with 1 mL of PBS, add 100 μL of freshly prepared 0.5 mg / mL MTT culture solution to each well, and culture in an incubator for 4 h. Aspirate the culture solution in the wells, add 100 mL of dimethyl sulfoxide (DMSO) to each well, place it on a shaker and shake at low speed for 5 min to fully dissolve the formazan crystals, and measure the absorbance of each well at OD 490 nm with an enzyme-linked immunosorbent assay detector. Calculate the survival rate of HFF-1 cells according to the following formula.

[0028]

[0029] In the formula: A1 is the absorbance value of the experimental group; A2 is the absorbance value of the control group; A0 is the absorbance value of the blank group. The experimental results are as Figure 1 shown. At an irradiation dose of 8.7 J / cm 2 , the light damage rate of HFF-1 cells reaches 22%.

[0030] Example 2:

[0031] Cell proliferation experiment:

[0032] Raw material 1: Ginkgo biloba extract (INCI name, commercially available product, preparation method in accordance with industry standards, referring to the Chinese Pharmacopoeia); Raw material 2: Retinol (INCI name, commercially available product).

[0033] Retinol ①, ginkgo biloba extract ②, and retinol∶ginkgo biloba extract = 1∶3 (mass ratio) ③ were used as samples for each experimental group, and samples of each experimental group with different final concentrations were prepared according to Table 1 (using serum-free DMEM as the solvent) for incubating HFF-1 cells.

[0034] HFF-1 cells in good condition were digested with trypsin and counted by hemocytometry, and cell suspensions were prepared at 1×10 4 cells / mL. 100 μL of the cell suspension was seeded into each well of a 96-well plate. The 96-well plate was placed in an incubator at 37 °C, 5% CO 2 , with saturated humidity, for 24 h for cell attachment. After attachment, the complete DMEM medium in the wells was discarded, and the cells were carefully rinsed once with PBS. Each well was covered with 100 μL of PBS and induced under a UVA lamp tube with an irradiation dose of 8.7 J / cm 2 . Untreated HFF-1 cells were used as the control group (cells were incubated with serum-free DMEM), and wells with only dimethyl sulfoxide were used as the blank group. The group with an irradiation dose of 8.7 J / cm 2 in Example 1 was used as the UVA light damage model group. After irradiation, HFF-1 cells were cultured in serum-free DMEM medium for 24 h. In the experimental groups, after UVA irradiation at 8.7 J / cm 2 , HFF-1 cells were cultured for 24 h in serum-free DMEM medium supplemented with samples of each experimental group at different final concentrations according to Table 1. Then, the liquid in the wells was aspirated, and each well was washed twice with 1 mL of PBS. 100 μL of freshly prepared 0.5 mg / mL MTT culture solution was added to each well, and the cells were cultured in the incubator for 4 h. The culture solution in the wells was aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was placed on a shaker and shaken at low speed for 5 min to fully dissolve the formazan crystals. The absorbance of each well was measured at 490 nm using an enzyme-linked immunosorbent assay reader, and the viability of HFF-1 cells in each experimental group was calculated according to the following formula.

[0035]

[0036] In the formula: A1 is the absorbance value of the experimental group; A2 is the absorbance value of the control group; A0 is the absorbance value of the blank group.

[0037] Table 1

[0038]

[0039] To calculate the CI value, the above results were numerically normalized. According to Example 1, with an irradiation dose of 8.7 J / cm 2The cell damage rate of the UVA light damage model group at that time was 22%. After adding the samples of each experimental group, the cell light damage rate decreased, and the ratio of the cell damage rate of each experimental group to the cell damage rate of the light damage model group in Example 1 was calculated. For example, in Table 1, when 1 μg / mL of retinol was added, the light damage rate was: (1 - 0.8) * 100% = 20%. Therefore, the ratio of the cell damage rate of the experimental group to the cell damage rate of the light damage model group in Example 1 was: 0.2 / 0.22 = 0.909. The experimental data after normalization are shown in Table 2 and Figure 2 。

[0040] Table 2

[0041]

[0042]

[0043] When using CompuSyn software to calculate, when retinol + Ginkgo biloba extract (w:w = 1:3) was used in combination (total concentration was 20 μg / mL), CI = 0.383 (CI < 0.7), that is, it had a relatively strong synergistic effect.

[0044] Figure 2 Among them, the experimental conditions were: 5 μg / mL of retinol, 15 μg / mL of Ginkgo biloba extract, and the total concentration of the retinol + Ginkgo biloba extract composition (1:3) was 20 μg / mL.

[0045] The mass ratio of retinol to Ginkgo biloba extract was adjusted to 1:1, 1:2, 1:3, 1:5 respectively, and the total concentration of the composition was 20 μg / mL. The above experiment was repeated, and the effect of the combined use of retinol and Ginkgo biloba extract with different weight ratios in repairing cell light damage was calculated. The experimental results are shown in Figure 3 。Retinol and Ginkgo biloba extract both had the effect of synergistically repairing cell light damage in the range of mass ratio from 1:1 to 5. Among them, the preferred mass ratio was 1:3.

[0046] In Example 1, the irradiation dose was 8.7 J / cm 2 Cell micrographs of the light damage experiment of the UVA light damage model group, the retinol group with an added concentration of 5 μg / mL, the Ginkgo biloba extract group with an added concentration of 15 μg / mL, and the retinol + Ginkgo biloba extract group with a total concentration of 20 μg / mL and a mass ratio of 1:3 are shown in Figure 4 as shown.

[0047] Example 3:

[0048] Raw material 1: Ginkgo biloba extract (INCI name); Raw material 2: Retinol (INCI name, commercially available product, the preparation method is the industry standard, referring to the Chinese Pharmacopoeia).

[0049] Retinol ①, ginkgo biloba extract ②, and retinol∶ginkgo biloba extract = 1∶3 (mass ratio) ③ were used as samples for each experimental group, and samples of each experimental group with different final concentrations were prepared according to Table 1 (using serum-free DMEM as the solvent) for incubating HFF-1 cells.

[0050] HFF-1 cells in good condition were digested with trypsin and counted by hemocytometry, and cell suspensions of 30×10 4 cells / mL were prepared. 2 mL of the cell suspension was seeded into each well of a 6-well plate. The 6-well plate was placed in an incubator at 37 °C, 5% CO 2 , with saturated humidity, for 24 h for cell attachment. Then, the DMEM complete medium in the wells was discarded, and the cells were carefully rinsed once with PBS. Each well was covered with 2 mL of PBS and induced under a UVA lamp tube with an irradiation dose of 8.7 J / cm 2 . Untreated HFF-1 cells were used as the control group (incubating cells with serum-free DMEM), and wells with only dimethyl sulfoxide were used as the blank group. The group with an irradiation dose of 8.7 J / cm 2 in Example 1 was used as the UVA light damage model group. After irradiation, HFF-1 cells were cultured in serum-free DMEM medium for 24 h. In the experimental groups, after UVA irradiation at 8.7 J / cm 2 , samples of each experimental group with different final concentrations were added to the serum-free DMEM medium according to Table 1, and HFF-1 cells were cultured for 24 h. Then, the liquid in the wells was aspirated, and each well was washed twice with 1 mL of PBS. 100 μL of freshly prepared 0.5 mg / mL MTT culture solution was added to each well, and the cells were cultured in an incubator for 4 h. After that, the liquid in the wells was aspirated, washed once with PBS, and 1 mL of β-galactosidase staining fixative was added and fixed at room temperature for 15 minutes. The cell fixative was aspirated, and the cells were washed 3 times with PBS, 3 minutes each time. The PBS was aspirated, and 1 mL of staining working solution was added to each well. After sealing the plate with parafilm and incubating overnight at 37 °C, the samples were observed under a microscope, and the photos were processed with Image J software to obtain the percentage of the stained area. The experimental results are shown in Figure 5 and Figure 6 . Figure 6 In , the experimental conditions were: retinol 5 μg / mL, ginkgo biloba extract 15 μg / mL, and the total concentration of the retinol + ginkgo biloba extract composition (1∶3) was 20 μg / mL.

[0051] After UVA irradiation, intracellular cell damage markers were formed and stained quantitatively. From the change in the intracellular β-galactosidase content, it can be seen that both ginkgo biloba extract and retinol significantly reduced the expression of galactosidase, and the composition showed a synergistic effect in reducing cell photo-damage.

[0052] Example 4:

[0053] Take healthy HFF-1 cells, digest them with trypsin and count them using the hemocytometer technique. Prepare a cell suspension at a concentration of 30×10 4 cells / mL, and inoculate 2 mL per well into a 6-well plate. Incubate the 6-well plate in an incubator at 37°C, 5% CO 2 , with saturated humidity for 24 h until the cells adhere. Then discard the complete DMEM medium in the wells and carefully rinse once with PBS. Cover the cells with 2 mL of PBS per well and induce them under a UVA lamp tube with an irradiation dose of 8.7 J / cm 2 . Use non-UVA-irradiated HFF-1 cells as the blank control group, UVA-irradiated but untreated HFF-1 cells as the positive control group (UV group), and HFF-1 cells incubated with retinol, ginkgo biloba extract, and the composition (1:3) as the experimental group.

[0054] Dilute DCFH-DA 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. After irradiation, the UV group and the experimental group were cultured for 4 h in serum-free DMEM medium and serum-free DMEM medium supplemented with the experimental group samples, respectively. Then aspirate the liquid in the wells and add 1 mL of diluted DCFH-DA. Incubate in a 37°C cell incubator for 20 minutes. Wash the cells three times with serum-free DMEM medium to thoroughly remove the DCFH-DA that has not entered the cells. Observe and take pictures under a fluorescence microscope using an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and process the pictures with Image J software to obtain the relative fluorescence intensity.

[0055] The experimental results are shown in Figure 7 and Figure 8 . Figure 8 In 2 , the experimental conditions for each experimental group are as follows: retinol at 5 μg / mL, ginkgo biloba extract at 15 μg / mL, and the total concentration of the retinol + ginkgo biloba extract composition (1:3) at 20 μg / mL. After irradiation with UVA at 8.7 J / cm Figure 7 , strong ROS fluorescence intensity was generated in the cells, as shown in

[0056] Example 5:

[0057] Take healthy HFF-1 cells, digest them with trypsin and count them using the hemocytometer technique. Prepare a cell suspension at a concentration of 30×10 4 cells / mL, and inoculate 2 mL per well into a 6-well plate. Incubate the 6-well plate in an incubator at 37°C, 5% CO 2, after 24 hours of adhesion in a humidity-saturated incubator, discard the complete DMEM medium in the wells and carefully rinse once with PBS. Cover the cells with 2 mL of PBS per well and induce under a UVA lamp tube with an irradiation dose of 8.7 J / cm 2 . Use non-UVA-irradiated HFF-1 cells as the blank control group, UVA-irradiated but untreated HFF-1 cells as the positive control group (UV group), and HFF-1 cells incubated with retinol, ginkgo biloba extract, and the composition (1:3) as the experimental group.

[0058] After irradiation, the UV group and the experimental group were respectively cultured with serum-free DMEM medium and serum-free DMEM medium supplemented with the samples of the experimental group. Then, collect the cell culture medium and transfer it to a sterile centrifuge tube. Centrifuge at 1000×g for 10 min at 4°C. Take 100 μL of the culture medium supernatant and transfer it to the reaction wells. Seal the plate and incubate in an incubator at 37°C for 90 min. Add 100 μL of biotinylated antibody to the reaction wells. Seal the plate and incubate in an incubator at 37°C for 60 min. Add 100 μL of enzyme conjugate to the reaction wells. Seal the plate and incubate in an incubator at 37°C for 30 min. Add 100 μL of chromogenic substrate to the reaction wells. Seal the plate and develop color in the dark at 37°C for 15 min. Finally, add 50 μL of stop solution and immediately measure the OD value at wavelengths of 450 nm and 630 nm using an enzyme-linked immunosorbent assay reader. At the same time, extract the cell protein and detect the cell protein content by the BSA method at a wavelength of 562 nm. The data was normalized to the MMP-1 content per mg of cell protein.

[0059] The experimental results are shown in Figure 9 . The experimental conditions for each experimental group are as follows: retinol at 5 μg / mL, ginkgo biloba extract at 15 μg / mL, and the total concentration of the retinol + ginkgo biloba extract composition (1:3) at 20 μg / mL. UVA irradiation induced a significant increase in the content of matrix metalloproteinase MMP-1 in cells. From the change in the intracellular MMP-1 content, it can be seen that the down-regulation effect of ginkgo biloba extract alone on MMP-1 expression is not significant, while retinol alone has a significant effect on reducing MMP-1 expression, and the composition shows a more significant effect on down-regulating MMP-1 expression, indicating that retinol plays a key role in down-regulating MMP-1 expression, and the combined use of ginkgo biloba extract produces a synergistic effect, enhancing the effect of retinol.

[0060] Example 6:

[0061] Take well-conditioned HFF-1 cells, digest them with trypsin and count them by hemocytometry, and prepare a cell suspension at 30×10 4 cells / mL. Inoculate 2 mL per well into a 6-well plate. Incubate the 6-well plate at 37°C and 5% CO 2, after culturing in a humidity-saturated incubator for 24 hours and adhering to the wall, discard the complete DMEM medium in the wells and carefully rinse once with PBS. Cover the cells with 2 mL of PBS per well and induce under a UVA lamp tube with an irradiation dose of 8.7 J / cm 2 . Use HFF-1 cells without UVA irradiation as the blank control group, HFF-1 cells irradiated with UVA but not administered drugs as the positive control group (UV group), and HFF-1 cells incubated with retinol, ginkgo biloba extract, and the composition (1:3) as the experimental group.

[0062] After irradiation, the UV group and the experimental group were respectively treated with serum-free DMEM medium and serum-free DMEM medium added with the samples of the experimental group. Then, the cells were digested with trypsin and the cell precipitate was collected. Take 1 ml of pre-cooled PBS and pipette it into a cell suspension. Centrifuge at 12000 g for 10 min at 4 °C. Aspirate the PBS and retain the cell precipitate. Add 150 μl of lysis buffer and lyse on ice for 30 minutes. Then centrifuge at 12000 g for 10 min at 4 °C. Transfer the supernatant to a new EP tube. Detect the protein concentration by the BSA method. Load an appropriate amount of protein sample into the loading wells of the SDS-PAGE gel. According to the electrophoresis of the pre-stained protein molecular weight standard, stop electrophoresis and start membrane transfer after it is expected that the target protein has been properly separated. After the membrane transfer is completed, immediately place the membrane into the pre-prepared Western washing solution, rinse for 1 - 2 minutes, aspirate the washing solution completely, and block on a shaker at 37 °C for more than 1 h. Refer to the instructions of the primary antibody and secondary antibody to incubate the primary antibody and secondary antibody. After incubation, perform protein detection. Use Image J software to quantitatively analyze each band.

[0063] The experimental results are shown in Figure 10 and Figure 11 . The experimental conditions for each experimental group: retinol 5 μg / mL, ginkgo biloba extract 15 μg / mL, and the total concentration of the retinol + ginkgo biloba extract composition (1:3) is 20 μg / mL.

[0064] UVA irradiation induces the production of ROS in cells, further activating the p38-MAPK signaling pathway. The activation and inhibition of the p38-MAPK signaling pathway were observed by the protein expression levels of C-JUN and P38. The results showed that both ginkgo biloba extract and retinol alone had the effect of inhibiting the activation of the p38-MAPK pathway, manifested as a decrease in the protein expression levels of C-JUN and P38. When the two were used in combination, a more significant effect was observed, indicating that ginkgo biloba extract and retinol synergistically inhibited the activation of the p38-MAPK pathway.

[0065] Figure 12Schematic diagram of the mechanism of the synergistic effect between retinol and ginkgo biloba extract discovered in the present invention. Ginkgo biloba extract plays a major role in downregulating the ROS level. Ginkgo biloba extract and retinol synergistically inhibit the activation of the P38-MAPK pathway. Retinol plays a key role in downregulating the expression of MMP-1. Since ginkgo biloba extract downregulates ROS and inhibits P38-MARK, it plays a synergistic role in inhibiting the expression of MMP-1, thus enhancing the synergistic effect of the composition and synergistically playing a role in repairing cell photo-damage.

[0066] Figure 13 It is a fluorescence staining picture of intracellular collagen after UVA irradiation of cells. After irradiation, the UV group and the experimental group were respectively cultured in serum-free DMEM medium and serum-free DMEM medium added with the sample of the experimental group. Then, they were incubated overnight at 4°C with collagen primary antibody, washed with PBS, added with FITC-labeled collagen secondary antibody and incubated for 1 h, stained with DAPI for the cell nucleus, and photographed under a fluorescence microscope after adding the anti-fluorescence quenching sealing solution (green is cytoplasmic collagen, blue is the cell nucleus). The pictures of different fluorescence colors were superimposed using Image J software. It can be seen from Figure 13 the figure that after UV irradiation, the green fluorescence of cytoplasmic collagen in cells was significantly weakened, and the composition (1:3) significantly increased the collagen content, indicated by the significant enhancement of green fluorescence.

[0067] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A composition with the efficacy of repairing photo-damage to cells, characterized in that: The composition with the efficacy of repairing photo-damage of cells is composed of retinol and ginkgo biloba extract, and the mass ratio of retinol to ginkgo biloba extract is 1:(1-5).

2. The composition with the efficacy of repairing photo-damage to cells according to claim 1, characterized in that: The mass ratio of retinol to ginkgo biloba extract is 1:

3.

3. Use of the composition with the efficacy of repairing photo-damage to cells according to claim 1 in the preparation of cosmetics.

4. The use according to claim 3, characterized in that: The composition is used for preparing cosmetics with the efficacy of repairing photo-damage of cells.

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