An in vitro cell test method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics

Abstract

The present invention relates to an in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics, comprising (1) sunscreen cosmetic sample processing; (2) cell culture and cell inoculation; (3) UVA irradiation determination; (4) quantitative detection of ROS by an enzyme marker. This method uses the cell oxidative damage caused by long-wave ultraviolet radiation as a theoretical basis, and uses cells cultured in vitro as materials to establish an in vitro biological evaluation system that is different from the traditional sunscreen cosmetic evaluation method. An in vitro biological detection device innovatively applied in the experiment is scientifically and reasonably designed, has many advantages such as fast and simple operation, low cost, time saving, avoidance of operation pollution, relatively good data repeatability, etc., is safe to use, accurate in detection, and effectively conducts a scientific, reasonable and correct evaluation of the efficacy of sunscreen cosmetics, providing technical guarantee for the human safety and efficacy evaluation of sunscreen cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics testing, and relates to a detection technology for the ultraviolet protection effect of sunscreen cosmetics, in particular to an in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics. Background Art

[0002] Ultraviolet radiation can cause skin burns, skin aging, and even skin cancer to human skin. In life, ultraviolet rays can be divided into three frequency bands, including short-wave ultraviolet rays (UVC), medium-wave ultraviolet rays (UVB), and long-wave ultraviolet rays (UVA). Among them, long-wave ultraviolet rays UVA have the longest wavelength and the strongest penetrating power. They can directly penetrate the skin and reach the dermis of the skin, so they are also one of the important causes of skin photoaging. It has been reported that ultraviolet radiation UVA can cause a large increase in reactive oxygen species (ROS) in cells. As the skin is exposed to ultraviolet rays for a long time, the amount of ROS increases dramatically, resulting in direct attacks on cell membranes to phosphorylate their receptors, thereby causing oxidative damage to cells. At the same time, it can stimulate and regulate cell signals to control the expression of genes such as elastin and matrix metalloproteinases, accelerate collagen decomposition, and accelerate skin damage.

[0003] As an important means of protecting against ultraviolet rays, sunscreen cosmetics are widely used. At present, the sunscreen ability of sunscreen cosmetics is mainly tested by instrumental ultraviolet spectroscopy and SPF meter method, and the sun protection factor value (SPF and PFA value) is expressed. With the rapid development of the cosmetics market in recent years and the gradual maturity of in vitro cell culture technology, it is urgent to establish the application of cell culture technology in the evaluation of the efficacy of sunscreen cosmetics.

[0004] In addition, the current evaluation of the sunscreen effect of sunscreen cosmetics is generally carried out by human body method, instrument method, UV spectrometry and instrument method. The human body method is to apply sunscreen cosmetics directly on human skin. Although its observation endpoints of erythema and tanning can reflect the physiological response of human skin, it has the disadvantages of high cost, high uncertainty of data repetition due to large individual differences among subjects, and most importantly, the subjects have health risks. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics. The method takes the cell oxidative damage caused by long-wave ultraviolet radiation UVA as a theoretical basis, uses in vitro cultured cells as materials, and uses ROS, a biological indicator of oxidative damage caused by UVA irradiation to cells, as a detection indicator to establish an in vitro biological detection method applied to the detection and evaluation of sunscreen cosmetics. At the same time, an in vitro biological detection device applied to the detection and evaluation of sunscreen cosmetics is provided, so as to provide an efficient and reliable test device for in vitro cell tests.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] An in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics comprises the following steps:

[0008] ⑴Sunscreen cosmetic sample processing

[0009] Weigh the sunscreen sample to be tested, mix with anhydrous ethanol, take the mixed solution and mix with PBS to prepare a 4 mg / ml solution, and dilute with PBS to different concentrations;

[0010] ⑵Cell culture, cell inoculation

[0011] ①Take out the cryotube from liquid nitrogen, thaw it, centrifuge it, discard the supernatant, and culture it in a culture bottle. Then, change the medium after 24 hours and observe the growth of the cells. Change the medium at intervals. When the cell density reaches 1×10 6 cell / ml before passage;

[0012] ② Cryopreserved in cryopreservation tubes of cryopreservation solution, cells in logarithmic growth phase were plated. Two 24-well plates need to be plated in parallel, with UVA-irradiated plates and light-protected non-irradiated plates as controls;

[0013] ③ Take cells in the logarithmic growth phase and digest them with 0.25% trypsin and 0.02% EDTA digestion solution to form a single cell suspension. Use DMEM complete medium to adjust the cell concentration to 4×10 5 cell / ml and inoculated into a culture dish and cultured in an incubator;

[0014] (3) UVA irradiation measurement

[0015] The cells were incubated for 24 hours, washed twice with PBS, and 0.5 mL of PBS was added to each well of cells at 3.0 mW / cm 2 The light intensity of irradiated cells for 44 minutes;

[0016] ⑷ Quantitative detection of ROS by microplate reader

[0017] Use a fluorescence microplate reader for quantitative detection: Place the 24-well plate into the fluorescence microplate reader, with an excitation wavelength of 490nm and an emission wavelength of 530nm. When the relative fluorescence unit (RFU) increases, it means that the ROS content is high;

[0018] ⑸ Use statistical methods to conduct data statistics.

[0019] Moreover, in step (1), the solution was diluted with PBS to different concentrations, namely 100ug / ml, 150ug / ml, and 300ug / ml.

[0020] In addition, the medium was changed every 2 days in step (2), and the culture conditions in the incubator were 37°C and 5% CO 2 .

[0021] Moreover, the mechanical work of UVA irradiation in step (3) is 8 J / cm 2 .

[0022] An in vitro biological detection device for sunscreen cosmetics detection and evaluation comprises an adjustable steering bracket, an adjustable UVA irradiation mechanism and a detachable movable sterile operating table, wherein the adjustable UVA irradiation mechanism and the detachable movable sterile operating table are respectively arranged on both sides of the adjustable steering bracket.

[0023] Moreover, the adjustable steering bracket includes a base plate and a support rod, the support rod is arranged on the base plate, and a plurality of unit rotating sleeves are coaxially mounted on the support rod, and a cryotube placement rack, a culture bottle placement rack and an adjustable 24-well plate bracket are arranged in sequence on the unit rotating sleeves.

[0024] Moreover, the 24-well plate bracket includes a telescopic rod, one end of which is arranged on the corresponding unit rotating sleeve, and the other end is installed with an adjustable bracket. A groove begins on the adjustable bracket, and a sliding limiting column is arranged in the groove for limiting the placement of the 24-well plate.

[0025] Moreover, the adjustable UVA irradiation mechanism is spliced ​​and inserted on one side of the adjustable steering bracket through a first plug-in plate, an electric control box is arranged on the first plug-in plate, a threaded telescopic rod is arranged on the electric control box, and an adjustable telescopic UVA irradiation lamp is arranged on the upper part of the threaded telescopic rod.

[0026] Furthermore, the detachable movable sterile operating table is spliced ​​and inserted on the other side of the adjustable steering bracket through the second plug-in plate.

[0027] Moreover, the detachable movable sterile operating table body is a sterile operating organic glass cover.

[0028] The advantages and positive effects of the present invention are:

[0029] The evaluation method of the present invention uses the cellular oxidative damage caused by long-wave ultraviolet radiation as the theoretical basis, and uses cells cultured in vitro as materials to establish an in vitro biological evaluation system that is different from the traditional sunscreen cosmetics evaluation method. In the experiment, we found that after UVA irradiation, the amount of ROS generated in the cells increased significantly. The results confirmed that UVA irradiation can cause cellular oxidative damage, thereby causing human skin aging. The three sunscreen cosmetics significantly inhibited the increase of ROS and were statistically significant. The above results suggest that ROS, as one of the indicators of UVA damage to cells, is sensitive and can be used as a biomarker for qualitative evaluation of the ultraviolet protection effect of sunscreen cosmetics.

[0030] The in vitro biological detection device for detecting and evaluating sunscreen cosmetics innovatively applied in the experiment by the present invention has a scientific and reasonable design, and is quick and easy to operate. By organically combining an adjustable steering bracket, an adjustable UVA irradiation mechanism and a detachable and movable sterile operating table, sterile operation and UVA are efficiently combined and applied. In addition, an adjustable cryotube placement rack, a culture bottle placement rack and an adjustable 24-well plate rack are used. The device has many advantages such as quick and simple operation, low cost, time saving, avoidance of operation pollution, relatively good data repeatability, etc. The device is safe to use, accurate in detection, and effectively conducts a scientific, reasonable and correct evaluation on the efficacy of sunscreen cosmetics.

[0031] In summary, the in vitro cell method implemented by the in vitro biological detection device used for the detection and evaluation of sunscreen cosmetics in the present invention is used to qualitatively evaluate the ultraviolet protection effect of sunscreen cosmetics, which has the advantages of simple operation, rapidity, economy, and short cycle, and provides technical guarantee for the human safety and efficacy evaluation of sunscreen cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention tests three concentrations of sunscreen samples at 8J / cm 2 The result diagram of ROS concentration changes before and after UVA irradiation;

[0033] Figure 2 The figure is a schematic diagram of the structure of an in vitro biological detection device used for detection and evaluation of sunscreen cosmetics in the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0035] 1 Materials and methods

[0036] 1.1 Materials and reagents

[0037] Human epidermal keratinocyte cell line Hacat cells (Procell, catalog number CM-H113); DMEM culture medium (Thermo Corporation, USA); pancreatin (GIBCO, USA)-ethylenediaminetetraacetic acid (EDTA) (Sigma Corporation, USA); PBS buffer (Sigma Corporation, USA); three sunscreen cosmetics were test samples from Tianjin Customs Industrial Products Center.

[0038] CO2 cell culture incubator (MCO-15AC, Sanyo, Japan);

[0039] Fluorescence inverted phase contrast microscope (Olympus CKX41-F32FL / PH model )

[0040] -80℃ ultra-low temperature refrigerator (Japan Sanyo MDF-U4086S model);

[0041] Microplate reader (Thermo MK3, USA);

[0042] Skin phototoxicity experimental device (Tianjin Hepu 8130B);

[0043] Fluorescence quantitative PCR instrument (ABI 7300, USA);

[0044] Ultraspeed refrigerated centrifuge (American sigma model sigma3K15)

[0045] 24-well plate (Corning, USA)

[0046] 1.2 Sunscreen cosmetic sample processing

[0047] Weigh 32 mg of the sunscreen sample to be tested, mix with 320 ul of anhydrous ethanol, take 0.160 ml and mix with PBS to prepare a 4 mg / ml solution, dilute with PBS to different concentrations of 100 ug / ml, 150 ug / ml, 300 ug / ml. The three sunscreen samples were divided into group A, group B, and group C, and a blank group of PBS was used as a control.

[0048] 1.3 Cell culture and cell seeding

[0049] Take out the cryopreservation tube from liquid nitrogen, thaw, centrifuge, discard the supernatant, culture in a culture flask, then change the medium after 24 hours and observe the cell growth, change the medium every 2 days. When the cell density reaches 1×106cell / ml, it can be subcultured. Cryopreservation in cryopreservation tubes of cryopreservation solution. Take cells in the logarithmic growth phase for plating, and two 24-well plates need to be laid in parallel (UVA irradiated plates and non-irradiated plates treated with light protection as controls).

[0050] Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and 0.02% EDTA digestion solution to form a single cell suspension, adjust the cell concentration to 4×105cell / ml with DMEM complete medium and inoculate them into a culture dish, and culture them in an incubator (37°C, 5% CO2)

[0051] 1.4 UVA irradiation measurement

[0052] The inoculated cells were cultured for 24 hours, washed twice with PBS, 0.5 mL of PBS was added to each well of cells, and the cells were irradiated with a light intensity of 3.0 mW / cm2 for 44 minutes (8 J / cm2).

[0053] 1.5 Quantitative detection of ROS by microplate reader

[0054] Use a fluorescence microplate reader for quantitative detection: Place the 24-well plate into the fluorescence microplate reader, with an excitation wavelength of 490nm and an emission wavelength of 530nm. When the relative fluorescence unit (RFU) increases, it indicates that the ROS content is high.

[0055] 1.6 Statistical methods

[0056] Data Indicates that the student t test method was used, P>0.05 indicates no statistical significance; P<0.05 indicates a significant difference and statistical significance, and P<0.01 indicates an extremely significant difference and statistical significance.

[0057] An in vitro biological detection device for sunscreen cosmetics testing and evaluation, such as Figure 2 As shown, it includes an adjustable steering bracket, an adjustable UVA irradiation mechanism and a detachable movable sterile operating table, and the adjustable UVA irradiation mechanism and the detachable movable sterile operating table are respectively arranged on both sides of the adjustable steering bracket.

[0058] The adjustable steering bracket includes a base plate 9 and a support rod 5. The support rod is arranged on the base plate, and a plurality of unit rotating sleeves 4 are coaxially mounted on the support rod. A cryotube placement rack 3, a culture bottle placement rack 7 and an adjustable 24-well plate bracket are arranged in sequence on the unit rotating sleeves.

[0059] The 24-well plate bracket includes a telescopic rod 10, one end of which is arranged on the corresponding unit rotating sleeve, and the other end is installed with an adjustable bracket. A groove 11 is provided on the adjustable bracket, and a sliding limit column 12 is arranged in the groove for limiting the placement of the 24-well plate.

[0060] The adjustable UVA irradiation mechanism is spliced ​​and inserted on one side of the adjustable steering bracket through a first plug-in plate 14, an electric control box 13 is arranged on the first plug-in plate, a threaded telescopic rod 1 is arranged on the electric control box, and an adjustable telescopic UVA irradiation lamp 2 is arranged on the upper part of the threaded telescopic rod.

[0061] The detachable and movable sterile operating table is spliced ​​and inserted on the other side of the adjustable steering bracket through a second plug-in plate 8, and its body is a sterile operating organic glass cover 6.

[0062] When in use, the detachable and movable sterile operating table is spliced ​​and inserted on one side of the adjustable steering bracket through the second plug-in plate to form a sterile operating space, and the adjusting unit rotating sleeve is rotated into the sterile operating space to perform operations. After the operation is completed, the detachable and movable sterile operating table is removed, and the adjustable UVA irradiation mechanism is spliced ​​and inserted on the other side of the adjustable steering bracket through the first plug-in plate. At the same time, the adjusting unit rotating sleeve is rotated under the UVA irradiation lamp, and the threaded telescopic rod and the adjustable telescopic UVA irradiation lamp are adjusted according to actual needs to complete the UVA irradiation operation.

[0063] 2 Results and analysis

[0064]

[0065] Table 1 Cells were treated with three different concentrations of sunscreen samples at 8 J / cm 2 ROS content after UVA irradiation (Relative fluorescence unit RFU)

[0066] exist Figure 1 Before irradiation, there was no significant change in the comparison between groups A, B, and C and the control group (P>0.05). After UVA irradiation, the comparison between the control group and group B was P<0.05, and the comparison between group C and group B was P<0.05. After irradiation, the comparison between groups A, B, and C and the control group was P<0.01.

[0067] From Table 1 and Figure 1 It can be seen that before UVA irradiation, there was no statistical significance between the three different concentrations of sunscreen samples and the blank control group, and there was no significant difference in the amount of cellular ROS produced between different concentrations of the same sample. After the cells were irradiated with 8J / cm2 of UVA, the ROS in the control group cells increased significantly, and the ROS content in the cells treated with the three sunscreen samples increased slightly, P < 0.05, but had a significant inhibitory effect compared with the blank control group, P < 0.01. This shows that the three sunscreen cosmetics significantly inhibited the generation of ROS in cells under light, suggesting that the three sunscreen cosmetics have a significant protective effect against UVA-induced oxidative damage.

[0068] 3 Discussions

[0069] Sunscreen agents contained in sunscreen cosmetics can be roughly divided into UV absorbers and UV scatterers. UV absorbers are mainly organic substances, containing aminobenzoic acid esters, which absorb light in the UVB and UVA bands and are prone to phototoxic reactions. Sunscreen cosmetics are mostly applied to exposed parts and directly exposed to ultraviolet rays, so the efficacy evaluation of sunscreen cosmetics is very important for protecting consumer health.

[0070] At present, the human body method, instrumental method, ultraviolet spectrometry and instrument method are generally used internationally to evaluate the sunscreen effect of sunscreen cosmetics. The human body method is to apply sunscreen cosmetics directly on human skin. Although its observation endpoints of erythema and tanning can reflect the physiological response of human skin, it has disadvantages such as high cost and high uncertainty of data repetition due to large individual differences among subjects. The most important thing is that the subjects are at risk of human health. Although the in vitro instrumental method has the advantages of fast and simple operation, low cost, and relatively good data repetition, it is questioned because it is not a human experiment and has no clinical observation endpoint. Therefore, in order to launch safe and effective sunscreen cosmetics, it is necessary to adopt appropriate methods to scientifically, reasonably and correctly evaluate the sunscreen effect of cosmetics.

[0071] High concentrations of ROS have very active chemical and biological properties. ROS can undergo complex chemical reactions with cells in skin tissue, thereby producing a variety of biological effects and causing cell damage, leading to the decline of normal cell functions, photoaging of the skin, skin cancer, etc. In normal cells, there is a relatively complete defense system for removing ROS, including enzymatic and non-enzymatic systems. The components of the defense system play a synergistic role with each other. Under physiological conditions, a small amount of reactive oxygen species produced by cells can be removed by the defense system that removes reactive oxygen species in the cells. If a large amount of ROS induced by external stimuli cannot be removed in time by the antioxidant system in the skin, it will cause damage to the tissue.

[0072] The present invention uses the cell oxidative damage caused by long-wave ultraviolet radiation as the theoretical basis, and uses cells cultured in vitro as materials to establish an in vitro biological evaluation system that is different from the traditional sunscreen cosmetics evaluation method. In the experiment, we found that after UVA irradiation, the amount of ROS generated in the cells increased significantly. The results confirmed that UVA irradiation can cause oxidative damage to cells, thereby causing human skin aging. The three sunscreen cosmetics significantly inhibited the increase of ROS and were statistically significant. The above results suggest that ROS, as one of the indicators of UVA damage to cells, is sensitive and can be used as a biomarker for qualitative evaluation of the ultraviolet protection effect of sunscreen cosmetics.

[0073] In summary, the in vitro cell method of this study is used to qualitatively evaluate the ultraviolet protection effect of sunscreen cosmetics and has the advantages of simple operation, rapidity, economy, and short cycle, providing technical support for the human safety and efficacy evaluation of sunscreen cosmetics.

[0074] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics, characterized in that: The following steps are involved: ⑴Sunscreen cosmetic sample processing Weigh the sunscreen sample to be tested, mix with anhydrous ethanol, take the mixed solution and mix with PBS to prepare a 4 mg / ml solution, and dilute with PBS to different concentrations; ⑵Cell culture, cell inoculation ①Take out the cryotube from liquid nitrogen, thaw it, centrifuge it, discard the supernatant, and culture it in a culture bottle. Then, change the medium after 24 hours and observe the growth of the cells. Change the medium at intervals. When the cell density reaches 1×10 6 cell / ml before passage; ② Cryopreserved in cryopreservation tubes of cryopreservation solution, cells in logarithmic growth phase were plated. Two 24-well plates need to be plated in parallel, with UVA-irradiated plates and light-protected non-irradiated plates as controls; ③ Take cells in the logarithmic growth phase and digest them with 0.25% trypsin and 0.02% EDTA digestion solution to form a single cell suspension. Use DMEM complete medium to adjust the cell concentration to 4×10 5 cell / ml and inoculated into a culture dish and cultured in an incubator; (3) UVA irradiation measurement The cells were incubated for 24 hours, washed twice with PBS, and 0.5 mL of PBS was added to each well of cells at 3.0 mW / cm 2 The light intensity of irradiated cells for 44 minutes; ⑷ Quantitative detection of ROS by microplate reader Use a fluorescence microplate reader for quantitative detection: Place the 24-well plate into the fluorescence microplate reader, with an excitation wavelength of 490nm and an emission wavelength of 530nm. When the relative fluorescence unit (RFU) increases, it means that the ROS content is high; ⑸ Statistical methods are used to conduct data statistics, In step (1), the solution was diluted with PBS to different concentrations, namely 100ug / ml, 150ug / ml, 300ug / ml, In step (2), the medium was changed every 2 days. The culture conditions in the incubator were 37°C, 5% CO2, The mechanical work of UVA irradiation in step (3) is 8 J / cm 2 , The test evaluation method is completed by an in vitro biological detection device used for sunscreen cosmetics detection and evaluation, the device includes an adjustable steering bracket, an adjustable UVA irradiation mechanism and a detachable and movable sterile operating table, the adjustable UVA irradiation mechanism and the detachable and movable sterile operating table are respectively arranged on both sides of the adjustable steering bracket, the adjustable steering bracket includes a bottom plate and a support rod, the support rod is arranged on the bottom plate, a plurality of unit rotating sleeves are coaxially sleeved on the support rod, a cryopreservation tube placement rack, a culture bottle placement rack and an adjustable 24-well plate bracket are arranged in sequence on the unit rotating sleeve, the 24 The well plate bracket includes a telescopic rod, one end of which is arranged on the corresponding unit rotating sleeve, and the other end is installed with an adjustable bracket. A groove is provided on the adjustable bracket, and a sliding limit column for limiting the placement of a 24-well plate is provided in the groove. The adjustable UVA irradiation mechanism is spliced ​​and inserted on one side of the adjustable steering bracket through a first plug-in plate. An electrical control box is provided on the first plug-in plate rack, and a threaded telescopic rod is provided on the electrical control box. An adjustable telescopic UVA irradiation lamp is provided on the upper part of the threaded telescopic rod. The detachable and movable sterile operating table is spliced ​​and inserted on the other side of the adjustable steering bracket through a second plug-in plate.

2. The in vitro cell test evaluation method suitable for evaluating the ultraviolet protection effect of sunscreen cosmetics according to claim 1, characterized in that: The detachable and movable aseptic operating table body is a aseptic operating organic glass cover.

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