Application of aloin in light protection

Abstract

The invention discloses an application of aloin in light protection. The invention discloses a remarkable effect of aloin in the aspect of preventing ultraviolet-induced apoptosis, and provides a new solution for the field of light protection. Researches show that aloin can significantly reduce generation of active oxygen caused by UVB ultraviolet rays and shows an obvious antioxidant characteristic, and the effect is enhanced along with increase of the concentration of aloin. Besides, cell survival rate test and fluorescence microscope observation show that aloin can effectively prevent cell apoptosis caused by UVB ultraviolet rays, so that a light protection effect is achieved. The discoveries not only prove the wide application potential of aloin in the fields of cosmetics, beauty and medicine, but also provide scientific basis for the development of related products.

Description

Technical Field

[0001] The invention relates to the application of aloin, in particular to the application of aloin in preventing cell apoptosis caused by ultraviolet rays. Background Art

[0002] Ultraviolet rays are an important component of solar radiation, and the UVB component with a wavelength of 280-320nm has a particularly significant effect on human skin damage. UVB rays can penetrate the epidermis and directly act on DNA, not only inducing DNA damage, but also may trigger a series of complex biological reactions, such as oxidative stress, inflammatory response, photoaging and increased risk of skin cancer. This is mainly because UVB radiation prompts cells to produce a large amount of reactive oxygen species (ROS), including superoxide anions (O2 - ) and hydroxyl radicals (•OH), these active molecules can damage DNA, proteins and lipids in cells, leading to cellular dysfunction. In addition, reactive oxygen species react with the lipid bilayer in skin cells, destroying the integrity of the cell membrane and causing gene mutations by oxidizing DNA. Over time, these oxidative damages accelerate the aging process of the skin, manifested as collagen degradation, loss of skin elasticity and increased wrinkles. Therefore, finding compounds with antioxidant effects has become the key to resisting UVB damage. Among the many antioxidants, compounds such as quercetin and arbutin have been shown to effectively reduce the generation of reactive oxygen species induced by UVB. They show strong UV protection capabilities by inhibiting the production of pro-inflammatory factors and reducing apoptosis and damage to skin cells. Summary of the invention

[0003] The present invention has found that aloin, as a monomer compound, also has an excellent UV protection effect. It can effectively prevent UVB from damaging cells and avoid the occurrence of cell apoptosis, thereby protecting the skin from damage caused by ultraviolet radiation. This discovery provides new ideas and broad prospects for the application of aloin in the fields of cosmetics, beauty and medicine.

[0004] The present invention adopts the following technical solution: Application of aloin in photoprotection.

[0005] Preferably, the application of aloin in photoprotection: aloin is used to prevent cell apoptosis caused by ultraviolet rays.

[0006] Preferably, the application of aloin in photoprotection: aloin is used in the preparation of antioxidant skin care products. Wherein, when the concentration of aloin in the antioxidant skin care products is 25-100 μg / mL, effective antioxidant and photoprotective effects can be achieved. Preferably, it is 25-75 μg / mL, and the optimal antioxidant concentration of aloin is 50 μg / mL.

[0007] Preferably, the application of aloin in photoprotection: aloin is used in the preparation of antioxidant cosmetics. Wherein, when the concentration of aloin in the antioxidant cosmetics is 25-100 μg / mL, effective antioxidant and photoprotective effects can be achieved. Preferably, it is 25-75 μg / mL, and the optimal antioxidant concentration of aloin is 50 μg / mL.

[0008] Compared with the prior art, the present invention has the following significant advantages: The present invention proves that aloin can effectively protect skin cells from UV-induced death at a concentration of 25-100 μg / mL. Unlike traditional physical sunscreen, aloin can not only directly absorb ultraviolet rays, but also effectively inhibit cell apoptosis by regulating cell apoptosis-related signaling pathways. In addition, cells treated with aloin can still maintain a high survival rate after 24 hours, showing a lasting protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The scavenging rates of different concentrations of aloesin on ABTS free radicals (A) and DPPH free radicals (B) are shown. The results show that aloesin has a significant scavenging effect on ABTS free radicals, and when the concentration reaches 50 μg / mL, its scavenging rate exceeds 90%. In contrast, the scavenging effect of aloesin on DPPH free radicals shows drug dependence, but its scavenging rate is lower than that of ABTS free radicals. When the concentration reaches 200 μg / mL, the scavenging rate of DPPH free radicals is only 20%.

[0010] Figure 2 The cell viability of human immortalized keratinocytes (HaCaT) treated with different concentrations of aloesin for 1 hour (A), 12 hours (B), and 24 hours (C) is shown. The results show that aloesin treatment of cells for 1 hour had no significant effect on cell viability. After 12 hours of treatment, when the concentration was 100 μg / mL, the cell viability decreased to 89%, and after 24 hours, it further decreased to 78%. However, when the concentration was 50 μg / mL, the cell viability was not significantly affected even after 24 hours of treatment.

[0011] Figure 3 Shows the effects of different concentrations of aloesin on cell viability (A) and reactive oxygen species (ROS) generation (B) in human immortalized keratinocytes treated with UVB (225 mJ / cm²) for 1 hour. The results show that cell viability decreased slightly after 1 hour of UVB irradiation; however, after the addition of aloesin, cell viability increased significantly with increasing concentrations. In addition, UVB stimulated the generation of intracellular reactive oxygen species, but as the concentration of aloesin increased, the content of reactive oxygen species decreased significantly, showing a concentration-dependent effect.

[0012] Figure 4 Showing the changes in the content of reactive oxygen species under the microscope. The DCFH-DA fluorescent probe can react with intracellular reactive oxygen species to generate fluorescence. UVB irradiation significantly increased the level of intracellular reactive oxygen species, resulting in an increase in the fluorescence signal. However, as the concentration of aloesin increased, the fluorescence intensity showed a significant decreasing trend.

[0013] Figure 5 Shows the effect of different concentrations of aloin on cell survival after treating human immortalized keratinocytes for 12 hours (A) and 24 hours (B) after cells were irradiated with UVB (225 mJ / cm²). The results show that cell survival decreased significantly after 12 hours of UVB irradiation. However, after the addition of aloin, cell survival increased significantly with increasing concentration, and when the concentration was 50 μg / mL, the cell survival rate increased to 87%. After 24 hours of UVB irradiation, the cell survival rate without the addition of aloin further decreased to only 27%. In contrast, when aloin was added at a concentration of 50 μg / mL, the cell survival rate increased significantly to 78%.

[0014] Figure 6 The analysis of cell activity and cytotoxicity under the microscope was presented. Calcein AM (CalceinAcetoxymethyl Ester) is a reagent that can perform fluorescent staining on living cells. PI (PropidiumIodide, a red fluorescent dye for nucleic acids) cannot penetrate the cell membrane of living cells, so what is observed is dead cells with damaged cell membranes. The results showed that after UVB irradiation of cells for 12 hours, the Calcein fluorescence intensity increased with the increase of aloesin concentration, indicating that cell activity increased; on the contrary, the PI fluorescence intensity gradually decreased with the increase of aloesin concentration, indicating that cytotoxicity decreased.

[0015] Figure 7 The analysis of cell viability and cytotoxicity after 24 hours of aloin treatment under a microscope is shown. After UVB irradiation of cells for 24 hours, the fluorescence signal was sparse overall after staining with Calcein and PI fluorescent dyes due to the high cell death rate without the addition of drugs. With the increase of aloin concentration, the Calcein fluorescence signal gradually increased, indicating that the cell viability was improved. Under low concentration aloin treatment, the PI fluorescence signal was strong, reflecting a high proportion of dead cells; however, when the aloin concentration reached 100 μg / mL, the PI fluorescence signal was significantly weakened, indicating that the cytotoxicity was reduced.

[0016] Figure 8The cell apoptosis analysis under the microscope is shown. Hoechst staining reagent binds to apoptotic cells with chromatin condensation, showing white fluorescence signals under a fluorescence microscope, while the nuclei of normal cells show blue fluorescence. The results show that after 12 hours of UVB irradiation, as the concentration of aloesin increases, the number of apoptotic cells (white fluorescence) gradually decreases, while the number of normal cells (blue fluorescence) increases significantly.

[0017] Fig. 9 Shows cell apoptosis 24 hours after aloin treatment. After 24 hours of UVB irradiation, cells without aloin showed a higher mortality rate, and Hoechst staining results showed a significant decrease in normal cells (blue fluorescence) and an increase in the number of apoptotic cells (white fluorescence). However, similar to the results of 12 hours of irradiation, as the concentration of aloin increased, the number of apoptotic cells decreased and the number of normal cells increased significantly. DETAILED DESCRIPTION

[0018] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0019] Example 1 Analysis of free radical scavenging effect of aloesin The ABTS free radical scavenging ability was tested using the ABTS total antioxidant capacity detection kit (rapid method) of Biyuntian Biotechnology Co., Ltd. The ABTS free radical scavenging rate was calculated by comparing the absorbance of the OD734 control group with the absorbance of the extract samples of different concentrations. The calculation formula is: ABTS free radical scavenging rate (%) = ([OD734 control group absorbance - OD734 aloin absorbance] / OD734 control group absorbance) × 100. At the same time, DPPH was dissolved in anhydrous ethanol, and different concentrations of aloin samples were added, and the absorbance value of OD517 was measured using an enzyme marker. Based on the difference in absorbance between the OD517 control group and the aloin sample, the scavenging rate of DPPH free radicals was obtained. The calculation formula is: DPPH free radical scavenging rate (%) = ([OD517 control group absorbance - OD517 aloin absorbance] / OD517 control group absorbance) × 100.

[0020] Example 2 Analysis of the effect of aloesin on cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to detect the survival rate of human immortalized keratinocytes (HaCaT). Human immortalized keratinocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2 for 24 hours. Aloin was first dissolved in DMSO solvent and added to DMEM medium to prepare working solutions of different concentrations. The control group was added with an equal amount of solvent and DMEM medium to ensure that the solvent concentration was consistent with that of the treatment group. Subsequently, the cells were treated for 1 hour, 12 hours, and 24 hours, the medium was removed, and 90 μL of DMEM medium without FBS and 10 μL of 5 mg / mL MTT solution (prepared in PBS) were added to each well, and then incubated in a 37°C incubator for 4 hours. After the incubation, the supernatant was removed, and 150 μL DMSO was added to each well to dissolve the purple crystals, and the mixture was shaken on a shaker for 10 minutes to ensure complete dissolution. Finally, the optical density (OD) value of each well was measured at a wavelength of 490 nm using a microplate reader.

[0021] Example 3 Cellular Reactive Oxygen Species Analysis In order to investigate the effects of aloin on the survival rate and reactive oxygen species (ROS) levels of human immortalized keratinocytes (HaCaT cells) under UVB irradiation, first, the MTT method was used to analyze the survival rate of the cells. Subsequently, the reactive oxygen species detection kit provided by Bio-Sky Biotechnology Co., Ltd. was used to explore the level of intracellular reactive oxygen species. During the experiment, HaCaT cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then, the culture medium containing fetal bovine serum (FBS) was removed and replaced with DMEM culture medium without FBS. At this time, different concentrations of aloin were added and allowed to react for 1 hour. Afterwards, the cells were placed in a Bio-Sun UV irradiation system (Vilber Bio Imaging, France) at 225 mJ / cm 2The cells were irradiated with UVB at a dose of 1 μM. After UVB irradiation, the cells were placed back in the incubator and incubated for 1 hour. Thereafter, the cell viability was detected again using the MTT method. The steps of the reactive oxygen species detection kit were also analyzed immediately after the cells were incubated for 1 hour. In the reactive oxygen species analysis, the DCFH-DA probe was diluted in serum-free DMEM medium according to a certain ratio to reach a final concentration of 10 μM. After removing the original medium, an appropriate amount of DCFH-DA solution was added to each well and incubated in an incubator at 37°C for 30 minutes. After the incubation, the cells were carefully washed three times with serum-free medium to ensure that the probes that did not enter the cells were removed. Finally, a fluorescence microplate reader was used to measure the reactive oxygen species level, with the excitation wavelength and emission wavelength set to 488 nm and 525 nm, respectively. In addition, an inverted fluorescence microscope (Leica DMi8, Germany, equipped with an imaging system) was used to image the cells to capture the fluorescent images generated by reactive oxygen species. The fluorescence microplate reader data and microscope imaging results provide a comprehensive analysis basis for the changes in intracellular ROS levels.

[0022] Example 4 Cell activity and cytotoxicity analysis To explore the protective effect of aloin on human immortalized keratinocytes under UVB irradiation, especially its effect on cell survival and death, the Calcein AM / PI kit provided by Bio-Sky Biotechnology Co., Ltd. was used for cell fluorescence staining, and the fluorescence images were recorded using a fluorescence microscope. In the experiment, the cells were first irradiated with UVB at a dose of 225 mJ / cm² using the Bio-Sun UV irradiation system (Vilber Bio Imaging, France). Subsequently, the cells were returned to the incubator at 37°C and 5% CO2 and incubated for 12 hours or 24 hours, respectively. After the incubation, the cells were stained according to the instructions of the Calcein AM / PI kit. First, the original culture medium was carefully aspirated and the cells were gently washed once with PBS to remove any possible impurities. Then, the Calcein AM / PI detection working solution was prepared according to the recipe of the kit. According to the 96-well plate system, 100 μL of the detection working solution was added to each well. Subsequently, the cells were incubated at 37°C in the dark for 30 minutes to allow the dye to fully penetrate and react with the cells. During this process, Calcein AM dye enters the living cells and emits bright green fluorescence, while PI dye can penetrate the membrane of dead cells and emit red fluorescence. The fluorescence colors and excitation / emission wavelengths of these two dyes are different, providing a basis for distinguishing between living cells and dead cells. Finally, the fluorescent staining effect of the cells was observed and recorded under an inverted fluorescence microscope (Leica DMi8, Germany). Green Calcein AM fluorescence indicates living cells, while red PI fluorescence indicates dead cells.

[0023] Example 5 Cell apoptosis detection (Hoechst staining method) In order to study the protective effect of aloin on immortalized human keratinocytes under UVB irradiation, especially its effect on cell survival and apoptosis, the cell apoptosis-Hoechst staining kit provided by Bio-Sky Biotechnology Co., Ltd. was used to observe and record the state of cells with the help of fluorescence microscopy. During the experiment, the cells were first placed in the Bio-Sun UV irradiation system (Vilber Bio Imaging, France) at 225 mJ / cm 2 The cells were then placed back in an incubator at 37°C and 5% CO2 for 12 or 24 hours. After incubation, the cells were fixed and stained. First, the original culture medium was gently aspirated, and then the fixative was added to fix the cells. The fixation process lasted for 10 minutes, aiming to keep the cells in a stable morphology for subsequent staining observation. After fixation, the cells were washed twice with PBS for 3 minutes each time to remove excess fixative. Next, Hoechst 33258 staining solution was added to stain the cells. Hoechst dye can penetrate the cell membrane, bind to DNA and emit blue fluorescence, thereby marking the nuclear morphology of the cells. The staining process lasted for 5 minutes to allow the dye to fully bind to the cell nucleus. After staining, the cells were washed twice with PBS again to remove unbound dye. After washing, the nuclear staining of the cells was observed directly in the culture dish using a fluorescence microscope. The intensity and distribution of blue fluorescence reflect the activity and degree of apoptosis of the cells. The apoptosis of the cells was understood through image analysis under the microscope, and fluorescent images were taken for further analysis and comparison.

[0024] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of aloin in photoprotection.

2. The use of aloin in light protection according to claim 1, characterized in that Aloin is used to prevent cell apoptosis caused by ultraviolet rays.

3. The use of aloin in light protection according to claim 1, characterized in that Aloin is used in the preparation of antioxidant skin care products.

4. The use according to claim 3, characterized in that: The concentration of aloin in antioxidant skin care products is 25-100μg / mL.

5. The use according to claim 4, characterized in that: The concentration of aloin in antioxidant skin care products is 25-75μg / mL.

6. The use according to claim 4, characterized in that: The concentration of aloin in antioxidant skin care products is 50μg / mL.

7. The use of aloin in light protection according to claim 1, characterized in that: Aloin is used in the preparation of antioxidant cosmetics.

8. The use according to claim 7, characterized in that: The concentration of aloin in antioxidant cosmetics is 25-100 μg / mL.

9. The use according to claim 8, characterized in that: The concentration of aloin in antioxidant cosmetics is 25-75μg / mL.

10. The use according to claim 8, characterized in that: The concentration of aloin in antioxidant cosmetics is 50μg / mL.

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