Anti oxi+ pollutant & dullness clarifying cleansing oil

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Preparation of samples

The dust PM1648a used in this study is urban particulate matter and was purchased from the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). It has an average particle size of 5.85 μm. NF and SN were purchased from Todipalm Korea (Korea) and Eco Plaza (Korea), respectively. CF and EC were provided in a mixture form by Kangs kosmetik (Korea). NF and SN were immersed in the distilled water at a sample : solvent ratio of 1:20 (w/v) for 24 h at 60℃. The mixtures were then homogenized at 60℃ for 4 h using a homogenizer (IKA, Staufen, Germany). The extracts were then filtered using the filter paper, concentrated at 60℃ using a rotary evaporator (IKA) and freeze-dried for 24 h. All freeze-dried extracts were stored at 4℃ prior to further experiments. All experiments were repeated in triplicate.

Reagents and instruments

Ascorbic acid, dimethyl sulfoxide (DMSO), methanol and 3-(4,5-dimethylthiaso-2-yl)-2,5-di Phenyl tetrazolium bromide (MTT) is Sigma-Aldrich, St. Louis (MI, USA). 1% antibiotic antifungal antibiotics, 10% fetal bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for cell culture. The CO2 incubator was purchased from Thermofisher Scientific (Waltham, MA, USA). Microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Was used in measures the absorbance of the Prostaglandin E2 (PGE2) ELISA assay kit (R&D system, Minneapolis, Minnesota, USA) and the non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA). To measure the concentration of free radicals in cells with dichlorofluorescin diacetate (DCFDA; Thermofisher Scientific), fluorescence intensity was measured using a Flourescence reader (Molecular Devices, San Joes, CA, USA).

Cell line and culture

Human keratinocytes (Human adult low calcium high temperature: HaCaT) cells used in this experiment were presold by the Korean Cell Line Bank, and DMEM medium containing 1% antibiotic antifungal and 10% FBS was used. Incubated in a CO2 incubator adjusted to 37℃, 5% CO2.

Cell viability measurement (MTT analysis)

MTT assay was performed to measure the effect of the sample on cell growth. Living cells with active metabolism reduce water-soluble yellow 3-(4,5-dimethylthiaso-2-yl)-2,5-diphenyltetrazolium bromide by the action of intracellular mitochondrial dehydrogenase, resulting in a purple insoluble form. HaCaT cells were placed in a 96-well plate of 1.0 × 104 cells/mL using DMEM medium supplemented with 10% FBS and cultured for 18 hours, and then the four mixtures were concentrated and cultured for 24 hours. Then, 50 μL of MTT solution was added and reacted for 4 hours. After the culture was completely removed and washed with PBS, 200 μL of DMSO was added to completely dissolve the precipitate, and the absorbance was measured at 540 nm using a microplate reader (Ha, 2014). The average absorbance value for each sample group was obtained, compared with the absorbance value of the control group to evaluate the cell growth rate, and then repeated three times.

Measurement of inflammation effect by fine particles

Inflammation expression effect by particulate PM1648a was measured by a PGE2 expression effect test (Kim et al., 2009; Park and Son, 2011). Raw264.7 cells were inoculated into a 48-well plate (SPL, Korea) at 1 × 105 cells/well, cultured for 18 hours, and then PM1648a were treated by concentration and cultured for 24 hours. Before collecting the culture medium, the control cells were treated with 1x lysis buffer (Promega) and incubated for 45 minutes, and then the culture medium was centrifuged at 3,000 rpm for 5 minutes. The obtained supernatant was measured for the amount of PGE2 expression using a PGE2 ELISA assay kit (R&D system), and the experiment was repeated three times. All samples were stored frozen below -20℃ until quantified.

Measurement of intracellular antioxidant effect (change in active oxygen concentration)

The change in the concentration of free radicals in cells was measured using the DCF-DA of the fluorescent probe. DCF-DA, a non-fluorescent substance, is oxidized by reactive oxygen species (ROS) when hydrogen peroxide-related peroxide is present in the cell, resulting in green fluorescence. Therefore, ROS can be measured via DCF-DA. 1 × 104 HaCaT cells were inoculated into a 96-well plate (SPL) and cultured for 18 hours. Four mixed concentrations and PM1648a were simultaneously treated and cultured for 24 hours. When incubation is complete, remove the medium and wash twice with PBS. After 10 μM DCF-DA was added to each well and incubated for 90 min in an incubator at 37℃ and 5% CO2, fluorescence intensities were measured at excitation 485 nm and emission 530 nm using a fluorescence reader.

Statistical processing

Measurement data was performed with a paired t-test, and it was confirmed that there was a statistically significant difference when the significance probability P<0.05 in the 95% confidence interval.