Application of Rubescensine A in preparing products for reducing skin ultraviolet light damage and improving skin photoaging

Abstract

The present invention discloses the use of Rubescensine A in preparing products for reducing skin ultraviolet light damage and improving skin photoaging. Studies have shown that Rubescensine A can significantly improve the vitality and proliferation ability of human dermal fibroblasts, reduce the proportion of aging-related β-gal staining positive cells, reduce DNA damage, and protect the skin from ultraviolet damage and aging. It can be used to prepare skin care products, cosmetics or medicines with anti-skin light damage and photoaging.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of Rubescensine A in preparing products for reducing skin ultraviolet light damage and improving skin photoaging. Background Art

[0002] Skin is exposed on the surface of the body, is the largest organ of the human body, and is also the first line of defense against the external environment. Compared with other organs of the body, the skin process is not only affected by force majeure such as genetic background (endogenous aging), but also by harmful substances in the environment such as ultraviolet radiation, air pollutants (such as NO, NO2, etc.), smoke, and volatilization of chemical substances. Among them, ultraviolet radiation is the culprit that exacerbates the natural aging of the skin on the basis of genetic background. Compared with non-exposed parts such as the trunk, exposed parts such as the head, neck and limbs are extremely susceptible to damage from ultraviolet rays, resulting in changes such as rough and loose skin.

[0003] Preventing or reversing skin photodamage and aging caused by ultraviolet rays has become an important target for human skin reversal. Fibroblasts are the most important cells in the dermis of the skin. After ultraviolet irradiation, they can lead to a decrease in the synthesis of extracellular matrix. The abnormal elastic fibers and collagen fibers produced by them cannot be degraded in time, which is an important cause of skin photodamage and aging. In vitro, ultraviolet rays acting on fibroblasts can lead to decreased activity of fibroblasts, cell cycle arrest, cell cycle arrest, and cell aging. At the same time, the synthesis of extracellular matrix such as collagen fibers and elastic fibers is reduced, and related secretory phenotypes such as increased synthesis of matrix metalloproteinases such as MMP1 and MMP3, increased degradation of extracellular matrix, and increased expression of inflammatory factors IL-1β and IL-6 are manifested. This kind of ultraviolet-induced damaged and aged fibroblasts is the basis and cause of skin ultraviolet damage and ultraviolet aging, and is also an important phenotype of skin photodamage and photoaging. Therefore, in vitro ultraviolet irradiation of fibroblasts to induce ultraviolet photodamage and aging is a good model for studying skin photodamage and aging and ultraviolet protection.

[0004] There have been many reports on the use of small molecule compounds to intervene in mouse models or in vitro ultraviolet-treated fibroblast photodamage and aging models, including vitamin C, vitamin E, rapamycin, etc. However, the clinical effects of these drugs on the treatment of skin photodamage and aging and ultraviolet protection are not ideal.

[0005] Oridonin, also known as Oridonin in English, is a natural biologically active substance isolated from the plant of the genus Rabdosia in the family Lamiaceae. Its main component is a natural organic compound of tetracyclic diterpenoids of the kaurenane type (ent-kau-renediterpenoid), with a molecular weight of 364.433, and is a small molecule compound. Previous studies have shown that Oridonin plays an important role in anti-tumor, anti-bacterial and anti-infection, but there are no reports on the use of Oridonin in the treatment of skin photodamage and UV protection. Summary of the invention

[0006] The present invention provides the use of Rubescensine A in preparing products for preventing / treating skin photodamage.

[0007] In the above application, the light damage refers to the damage to the dermis and fibroblasts in the dermis caused by ultraviolet light irradiation.

[0008] In the above application, the light damage is acute or chronic solar skin damage.

[0009] In the above application, the product is a skin care product, a cosmetic or a medicine.

[0010] In the above application, the dosage form of the product is emulsion, cream, gel, spray, ointment, oral administration, and injection.

[0011] The present invention also provides the use of Rubescensine A in preparing products for preventing skin photoaging.

[0012] In the above application, the photoaging refers to the aging of the dermis and fibroblasts in the dermis caused by ultraviolet light irradiation.

[0013] In the above application, the photoaging is skin aging caused by ultraviolet radiation.

[0014] In the above application, the product is a skin care product or a cosmetic.

[0015] In the above application, the dosage form of the product is emulsion, cream, gel, spray, ointment.

[0016] In the above application, the concentration of Rubescensine A in the product is 0.5-1 μM.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The oridonin provided by the present invention can significantly prevent and repair skin photodamage and photoaging caused by ultraviolet irradiation by improving the activity of fibroblasts, improving the proliferation ability of fibroblasts, reducing the proportion of senescence-related β-galactosidase staining-positive cells in fiber cells, reducing the proportion of apoptotic cells in fiber cells, and reducing the proportion of γ-H2A.X-positive cells in fiber cells, thereby protecting the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a light microscopic photograph of fibroblasts after ultraviolet irradiation.

[0019] Figure 2 The activity of fibroblasts after ultraviolet irradiation.

[0020] Figure 3 It is the proliferation capacity of fibroblasts after ultraviolet irradiation.

[0021] Figure 4 It is the proportion of cells positive for senescence-associated β-galactosidase staining in fibroblasts after ultraviolet irradiation.

[0022] Figure 5 is the proportion of apoptotic cells in fibroblasts after ultraviolet irradiation.

[0023] Figure 6 It is the proportion of γ-H2A.X positive cells in fibroblasts after ultraviolet irradiation. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0025] Example 1 This embodiment provides an emulsion for skin care products or cosmetics, and its formula, measured by mass fraction, is: oridonin (3%), carbomer (3%), polysorbate (15%), glycerol (3.5%), xanthan gum (1.5%), sodium hyaluronate (0.5%), and water (balance).

[0026] Example 2 The present embodiment provides a cream for skin care products or cosmetics, and its formula, measured by mass fraction, is: oridonin (5%), carbomer (2%), polysorbate (8%), glycerin (2%), xanthan gum (1%), sodium hyaluronate (0.8%), and water (balance).

[0027] Example 3 The present embodiment provides a gel for skin care products or cosmetics, and its formula, measured by mass fraction, is: oridonin (7.5%), carbomer (4%), polysorbate (12%), glycerol (3%), xanthan gum (2%), sodium hyaluronate (0.9%), and water (balance).

[0028] Effect verification test 1. Isolation and culture of normal human primary fibroblasts Preparation of single-cell suspension of normal human primary fibroblasts: Soak normal human skin tissue in 75% ethanol solution for 30 seconds, then wash with PBS 3-5 times until there is no blood. Remove subcutaneous tissue and blood vessels under sterile conditions, trim with scissors into 2mm×4mm strips, and transfer to 0.5% dispase at 4℃ for digestion overnight, about 16 hours. Serum digestion is terminated, and the epidermis and dermis are carefully separated with tweezers to remove the epidermis. The obtained dermis is placed in 0.25% trypsin-EDTA and incubated at 37℃ for 90 minutes, ground and filtered on a 200μm steel wire filter, and centrifuged at 1500 r / min for 10 minutes. Discard the supernatant, add 5ml of DMEM high-glucose medium containing 10% FBS, and press 5×10 5 The density of inoculation was 100 cm / bottle. 2 Culture in a culture dish at 37°C and 5% CO2, and change the medium once every 3 days.

[0029] Passaging and purification of fibroblasts: When the fibroblasts grow to nearly 70%-80% fusion (about 80% of the bottom area of ​​the bottle), discard the cell culture medium in the culture bottle, rinse twice with PBS solution, add 2ml of 0.25% trypsin-EDTA digestion solution, and incubate at 37℃ for 1-2min. Observe the morphological changes of fibroblasts under a phase contrast microscope. When most cells shrink, separate from each other, and become round and suspended, immediately add an equal amount of 10% FBS serum to terminate digestion. Transfer the cell suspension to a centrifuge tube and centrifuge (1000g, 5min), discard the supernatant, rinse twice with PBS solution, add new cell culture solution, blow evenly and count the cells, then move to a cell culture dish, culture at 37℃, 5% CO2, and change the solution once every 3 days. Generally, pure fibroblasts can be obtained after two passages.

[0030] The obtained light microscope pictures are as follows Figure 1 As shown, after ultraviolet irradiation, the fibroblasts became larger and flatter, and the number decreased; while after the addition of Rubescensine A, the number and morphology of fibroblasts were improved.

[0031] 2. CCK-8 cell viability assay and Ki-67 cell staining flow cytometry assay to detect fibroblast viability 1. CCK-8 method to detect cell activity: Fibroblasts from the UVB intervention group (UVB), UVB + Ori 0.5 μM, UVB + Ori 1 μM, and control group (control) were inoculated into 96-well plates, with 1×10 cells per well. 3 Each group has 3 duplicate wells. No cells are added to the blank wells, only culture medium is added. 5% CO2, 37℃ incubator for 48h, supernatant is discarded, and PBS is washed twice; CCK-8 10ul is added to each well, 5% CO2, 37℃ incubator is incubated for 4h; the absorbance value (A value) is measured by the microplate reader at a wavelength of 450nm. Cell proliferation rate = (A450 irradiation group - A450 blank group) / (A450 control group - A450 blank group) × 100%. Repeat 3 times.

[0032] The results are as follows Figure 2 As shown, the activity of fibroblasts decreased significantly after ultraviolet irradiation; after the addition of oridonin A, the activity increased significantly.

[0033] 2. Ki-67 cell staining flow cytometry detection of cell proliferation: Fibroblasts in the UVB intervention group (UVB), UVB + Ori 0.5 μM, UVB + Ori 1 μM, and control group (control) were cultured in a Ca-free medium at pH 7.4. 2+ and Mg 2+ The cells were washed twice with PBS, and after permeabilization, they were stained with Ki-67 fluorescent marker for 45 minutes, and then detected by flow cytometry to count the proportion of Ki-67 positive cells.

[0034] The results are as follows Figure 3 As shown, the proliferation ability of fibroblasts decreased significantly after ultraviolet irradiation; after the addition of oridonin A treatment, the proliferation ability increased significantly.

[0035] 3. Identification of senescence-related β-galactosidase staining Senescence-associated β-galactosidase staining is a method for cell detection based on the upregulation of senescence β-galactosidase staining activity levels. Fibroblasts from the UVB intervention group (UVB), UVB+Russiconazole A treatment group (UVB+Ori 0.5μM, UVB+Ori 1μM), and control group (control) were treated in 12-well plates, washed twice with PBS, added with staining fixative, fixed at room temperature for 20 minutes, and then the fixative was discarded, washed twice with PBS, and 600μl of staining solution was added to each well. Cells stained blue-green under a microscope for about 36 hours are senescent cells, and the proportion of cells positive for senescence-associated β-galactosidase staining in the three groups of cells was statistically analyzed.

[0036] The results are as follows Figure 4 As shown in the figure, the proportion of senescence-associated β-galactosidase-positive cells in fibroblasts increased significantly after ultraviolet irradiation; after treatment with Rubescensine A, the proportion of senescence-associated β-galactosidase-positive cells decreased.

[0037] 5. Flow cytometry to detect cell apoptosis level (Annexin-V / 7-AAD) When apoptosis occurs, phosphatidylserine is exposed outside the cell membrane. Annexin-V can bind to phosphatidylserine (PS) and mark early apoptotic cells. 7-AAD can enter mid- to late-stage apoptotic cells. Co-staining of Annexin-V and 7-AAD can mark apoptotic cells.

[0038] 1. Collect the cells by digestion with 0.25% trypsin, add them into a 1.5ml centrifuge tube, add an equal amount of serum medium to neutralize, centrifuge and discard the supernatant, and wash with Annexin-V binding buffer at 7000rpm for 3min.

[0039] 2. Add Annexin-V (1:30) and 7-AAD (1:40) and incubate at room temperature in the dark for 15 minutes.

[0040] 3. Streaming on-machine testing.

[0041] The results are as follows Figure 5 As shown in the figure, the proportion of apoptotic cells in fibroblasts increased significantly after ultraviolet irradiation; after treatment with Rubescensine A, the proportion of apoptotic cells decreased.

[0042] 6. Immunofluorescence detection of cell DNA damage 1. Cell slides: After high-temperature disinfection of cell slides, place them at the bottom of a 24-well plate and inoculate the cells. After overnight, UVB irradiation and Rubescensine A treatment can be performed.

[0043] 2. Cells treated with UVB and Rubescensine A were fixed with 4% paraformaldehyde.

[0044] 3. Wash with PBS for 5 min three times, and then block in 5% BSA blocking buffer containing 0.3% Triton™X-100 for 60 min.

[0045] 4. Add mouse γ-H2A.X-antibody prepared in blocking buffer and incubate overnight at 4°C.

[0046] 5. Rinse with PBS. Incubate the specimen with the diluted fluorescent secondary antibody corresponding to mouse origin at room temperature for 1 hour in the dark.

[0047] 6. Wash with PBS, add DAPI diluted in blocking buffer, stain for 5 minutes, and rinse three times with PBS.

[0048] 7. Add antifade agent, observe the results under a fluorescence microscope, and then store in a 4 degree refrigerator.

[0049] The results are as follows Figure 6 As shown, the proportion of γ-H2A.X positive cells in fibroblasts increased significantly after ultraviolet irradiation; after treatment with oridonin, the proportion of γ-H2A.X positive cells decreased.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of Rubescensine A in the preparation of products for preventing / repairing skin photodamage.

2. The use according to claim 1, characterized in that: The photodamage is the damage to the dermis and fibroblasts in the dermis caused by ultraviolet light irradiation.

3. The use according to claim 1, characterized in that: The photodamage is acute or chronic solar skin damage.

4. The use according to claim 1, characterized in that: The product is a skin care product, a cosmetic or a medicine.

5. The use according to claim 1, characterized in that: The dosage forms of the product are emulsion, cream, gel, spray, ointment, oral administration and injection.

6. Application of Rubescensine A in the preparation of products for preventing skin photoaging.

7. The use according to claim 6, characterized in that: Photoaging is the aging of the dermis and fibroblasts in the dermis caused by ultraviolet light exposure.

8. The use according to claim 6, characterized in that: The photoaging is skin aging caused by ultraviolet radiation.

9. The use according to claim 6, characterized in that: The products are skin care products and cosmetics.

10. The use according to claim 6, characterized in that: The dosage form of the product is emulsion, cream, gel, spray and ointment.

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