2.1 Chemical and reagents
Keratinocyte culture medium (4th Military Medical University Tissue Engineering Center, Xi’An, China), PBS (Boster,Hubei, China), MTT (Sigma, Darmstadt, Germany), DMSO (Sigma, Darmstadt, Germany), RNA extraction kit (TaKaRa Bio, Tokyo, Japan), reverse transcription kit (TaKaRa Bio, Tokyo, Japan), fluorescent dye (TaKaRa Bio, Tokyo, Japan), AQP3 antibody (Santa, CA, USA), fluorescent secondary antibody (Santa, CA, USA), blocked sheep serum (Zsbio, Beijing, China), and antifluorescence quenching and sealing liquid (Beyotime, Shanghai, China) were used.
A CO2 incubator (Themo, MA, USA), an ultraclean workbench, an inverted microscope (Olympus, Tokyo, Japan), a micro-oscillator (Qilinbeier, Jiangsu, China), a microplate reader (BioTek, VT, USA), a real-time PCR instrument (Bio-Rad, CA, USA), and a laser confocal microscope (Olympus, Tokyo, Japan) were also used.
2.2 Plant Material Collection And Extraction Process Optimization
In this study, the whole grass of E. purpurea, the stems of D. nobile, the leaves of aloe, the roots of S. flavescens, and the fruits of Chinese wolfberry were smashed through a 6 mesh sieve. The effects of extraction temperature, solid-liquid ratio and extraction time on polysaccharide content were investigated by 3-factor and 3-level orthogonal tests.
2.3 Preliminary Qualitative Phytochemical Analysis Of Plant Extracts
A large number of reports have stated that plant polysaccharides have good moisturizing and skin care effects. To determine whether polysaccharides were present in the plant extracts, aqueous plant extracts were prepared for testing.
2.4 Test For Polysaccharide
Take 1 g of sample, add 1 ml 15% TCA solution, add a little 5% TCA solution, pour the supernatant into a 10 ml centrifuge tube, add a little 5% TCA solution to grind, pour the supernatant, and repeat 3 times. Centrifuge at 3,000 rpm for three times. After adding 2 ml of 6 mol / L hydrochloric acid to the colorimetric tube, shake well, and then water-bath in a 96 ° C water bath for 2 hours. After the water bath, cool with running water and add 2 ml of 6 mol / L sodium hydroxide to shake. Make up to a 25 ml volumetric flask. Pipette 0.2 ml of the sample solution, make up to 2.0 ml by distillation, then add 1.0 ml of 6% phenol and 5.0 ml of concentrated sulfuric acid, shake and cool for 20 minutes at room temperature and measure the optical density at 490 nm. Take two samples for each measurement. The polysaccharide content was calculated using a standard curve. The total polysaccharide content was detected[10–13].
2.5 Stability Investigation
The cosmetic plant composition was required to be strictly stable. The various conditions for testing the stability of a stock solution included room temperature (placed on the laboratory table top), protected from light (placed in a 28 ± 1 °C incubator cassette), illumination (in a 28 ± 1 °C constant temperature incubator placed under light), heat (placed in an oven at 45 ± 1 °C), alternating hot and cold (that is, a 28 ± 1 °C and 4 ± 1 °C alternating hot and cold incubator), refrigeration (placed in a 4 ± 1 °C refrigerator fresh-keeping layer), and freezing (placed in a -20 ± 1 °C freezer layer)[14]..
2.6 Cell Culture Of Keratinocytes
Keratinocytes were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37 °C. The medium was replaced every two days.
2.7 MTT Detection Of Keratinocytes
This test uses the MTT cell activity assay to screen for the maximum safe dose of the sample in keratinocytes. To ensure the validity of the MTT test results, 4% DMSO was used as a positive control (PC). The sample was set up with a gradient of 8 concentrations, and 3 replicate wells were set for each concentration. A solvent control (SC) and a zero well (cell culture) were set up[15].
2.8 Detection Of FLG And CLDN-1 Cell Immunofluorescence
Cells were seeded in 6-well plates and incubated overnight at 37 °C in a CO2 incubator. The sample was diluted with DMSO to prepare a plant moisturizing composition at 125 ppm, 250 ppm, and 500 ppm. When the plating rate of the 6-well plate reached 40%-50%, group administration was performed, and each group was provided in 3 duplicate wells. A blank control group (1‰ DMSO) was added to the keratinocyte culture medium, and the sample group was added to keratinocyte culture medium containing the corresponding concentration of the test substance. Culture was continued for 24 h at 37 °C in a 5% CO2 incubator. The culture solution was discarded, 1 ml of TRIzol was added per well, the lysed cells were pipetted, and then a sample was taken. RNA was extracted and reverse transcribed into cDNA, and then quantitative PCR detection was performed.
2.9 Detection Of QP3 Cell Immunofluorescence
Cells were seeded in 24-well plates and incubated overnight at 37 °C in a CO2 incubator. The sample was diluted with DMSO to prepare a plant moisturizing composition at 125 ppm, 250 ppm, and 500 ppm. When the cell plating rate in the 24-well plate reached 40%-50%, group administration was performed, and three duplicate wells were set in groups. The blank control group was supplemented with keratinocyte culture medium containing 1‰ DMSO, the sample group was supplemented with keratinocyte culture medium containing the corresponding concentration of the test substance, and culture was continued for 24 h at 37 °C in a 5% CO2 incubator. After incubation, the medium in the 24-well plate was discarded, and the slides were washed three times with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stored in a refrigerator at 4 °C. Cells were permeabilized, blocked with goat serum blocking solution at 37 °C for 30 min, incubated with primary antibody solution at 4 °C overnight, and incubated with secondary antibody solution at 37 °C for 2 h before the cell nuclei were dyed with Hoechst dye. (The infected nuclear region can be excited by blue light to emit blue fluorescence.) A photo was taken under a fluorescence microscope. Green fluorescence of the target protein was excited by the blue light channel in the same field of view, and the blue fluorescence of the nuclear region was excited by ultraviolet light and photographed separately.
2.10 Erythrocyte Hemolysis Test
Stimulation of the red blood cell membrane broke the red blood cell membrane, causing a certain degree of hemolysis. The absorbance of the red blood cell suspension after the action of chemical substances was measured by spectrophotometry, and the hemolysis rate was calculated. Among the tested chemicals, 0.02% sodium dodecyl sulfate (SDS) is potent as a PC for this test[16].
2.11 Sample Preparation For The 3T3 Neutral Red Test
The plant moisturizing composition was formulated into solutions of 8 concentrations (7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL) with Hank’s balanced salt solution (HBSS). Chlorpromazine (CPZ) hydrochloride was dissolved in absolute ethanol to prepare the highest-concentration mother liquor (20.00 mg/mL), which was then diluted to different concentrations with 50% absolute ethanol. The concentration was 0.02 mg/mL, and each solution was diluted 100 times with HBSS before use and standby application.
2.12 Cell Inoculation For The 3T3 Neutral Red Test
Well-grown Balb/c3T3 cells were inoculated in the wells of a 96-well culture plate (except for the peripheral wells) according to the guidelines of OECD432 so that the number of cells per well is 1.0 × 104, and then the plate was placed at 37 ± 1 °C and 5 ± 1% CO2 for approximately 24 h to humidify the culture.
2.13 Dosing And Lighting For The 3T3 Neutral Red Test
After culturing of Balb/c 3T3 cells for 24 h, the culture solution was removed, and the cells were gently washed twice with 150 µL of prewarmed HBSS. Then, 100 µL of each test substance at different concentrations was added to each well. The control substance was HBSS, and there were 6 duplicate wells per concentration and 2 plates for each test substance and the PC, which were labeled “light plate” and “no light plate”, respectively. The culture plates were incubated at 37 ± 1 °C and 5 ± 1% CO2 for 1 h, and the light plates were irradiated for 45 min under UVA with a light intensity of 1.7 ± 0.2 mw/cm2 until the light dose reached 5.0 J/cm2. The no-light plate was wrapped with tin foil and incubated with the light plate for the same time in a dark room at room temperature. After the end of the light treatment, the HBSS containing the test substance/control substance was removed, and the cells were gently washed twice with 150 µL of preheated HBSS per well. Then, the HBSS was replaced with fresh 10% (v/v) FBS and DMEM containing 4 mM glutamine, 100 IU penicillin and 100 µg/mL streptomycin, and the cells were cultured at 37 ± 1 °C and 5 ± 1% CO2 for 18–22 h.
2.14 3T3 Neutral Red Test
Approximately 3 h before the end of culture, the medium of each well was discarded, the cells were gently washed once with 150 µL of prewarmed HBSS per well, and then 100 µL of 50.0 µg/mL neutral red was added serum-free per well. DMEM was cultured for 3 h at 37 ± 1 °C and 5 ± 1% CO2. After the completion of the culture, the medium containing neutral red was discarded, and the cells were washed with 150 µL of preheated HBSS. Then, 150 µL of neutral red eluate was added to each well (water:ethanol:acetic acid = 49:50:1, v/v/v), an oscillator rapidly shook the sample until a homogeneous solution was formed, and then the absorbance (OD540) of each well was measured with a spectrophotometer at 540 nm[17].
2.15 Statistical analysis
Based on the measured absorbance values (OD540), Phototox Version 2.0 software was used to calculate the photo irritation factor (PIF) and mean photo effect (MPE) of each test substance. Under light and no-light conditions, the OD540 was measured for each well. The IC50, PIF and MPE of the test samples and the PC under light and no-light conditions were calculated using Phototox software.