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conditions, the absorbance was recorded by a microplate reader (Molecular Devices E09090; San Francisco, CA, USA) at the wavelength of 520 nm. AgNPs synthesized from different plant extracts were tested at the different concen- tration from 100, 500 and 1000 μg mL−1 Arbutin was intro- duced as a positive control. Cell culture, UVB irradiation and sample treatment HaCaT cells originated from human epidermal keratinocytes (Sciencell, Carlsbad, CA, USA) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% peni- cillin–streptomycin at 37 °C in an atmosphere containing 5% CO2. When the cells reached more than 80% confluence, the cells sub-cultured in 35 mm culture dishes (1.0 × 105 cells) and were rinsed twice with phosphate-buffered saline (PBS). Then, the cells were irradiated with UVB (144 mJ cm−2) using UVB radiation machine (Bio-Link BLX-312; Vilber Lourmat GmbH, France). After UVB irradiation, the experi- mental group cells treated with AgNPs (1, 10 and 100 μg mL−1). The normal cells were treated with the same dose of AgNPs without UVB irradiation. The controls were without the treatment of AgNPs. Determination of cell viability To test the effect of AgNPs on the viability of HaCaT cells, MTT assay was carried out. MTT assay was performed as described previously [20]. Briefly, after 24 h of treatment, MTT at a final concentration of 0.1 mg mL−1 was added and further incubated for 2 h. The supernatants were removed and 1 mL dimethyl sulfoxide (DMSO) was added to dis- solve the formazan crystals. Absorbance was determined on a microplate reader (Molecular Devices Filter Max F5; Sun- nyvale, CA, USA) at a wavelength of 570 nm. Measurement of MMP‐1 and IL‐6 production The level of MMP-1 and IL-6 in the medium were deter- mined after 24 h of incubation with AgNPs using ELISA kits according to the manufacturer’s instructions. Each experi- ment was analyzed in triplicate. Reverse transcription (RT)‐PCR After 24 h of treatment, total RNA from HaCaT cells was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA). mRNA expression was determined by real-time PCR using SYBR green master mix in a BioRad CFX Connect Real- Time PCR Detection System (BioRad, Hercules, CA). The primer pairs as follows: MMP-1, sense 5′-TGGGAGGCA AGTTGAAAAGC-3′, antisense 5′-CATCTGGGCTGCTTC ATCAC-3′; PIP sense 5′-CACAGACAGCTATGACGTG-C- 3′ and antisense 5′-TCAGCAGAGAAGACCACCTG-3′. The PCR condition was carried out with an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denatura- tion at 95 °C for 5 s. The GAPDH gene was used for internal normalization (GAPDH sense 5′-CCAAGGAGTAAGACC CCTGG-3′ and antisense 5′-AGGGGAGATTCAGTGTGG TG-3′). PCR products were separated by 2% agarose gel electrophoresis with ethidium bromide staining. Statistical analysis The data are presented as mean ± standard deviation values of three independent experiments. Statistical analysis was performed using one-way ANOVA test. Statistical signifi- cance was set at P < 0.05. Results and discussion Synthesis and characterization of AgNPs UV–visible spectroscopy is a valuable technique used to detect the characteristic SPR pattern of metal nanoparticles. First, the AgNP synthesis was confirmed by visual obser- vation with the appearance of color change in the reaction mixture. The color of S. officinale extract was changed from pale yellow to light brown in 10 min, which resem- bles the synthesis of AgNPs, as the particles cause surface plasmon resonance (SPR) due to which light brown color appears in the supernatant. Thus, the appearance of a light brown color in the reaction mixture indicated the forma- tion of AgNPs [22]. In the UV–Vis absorption spectrum, a strong peak appeared at 468 nm for AgNPs synthesized from S. officinale (S-AgNPs), which is due to the SPR band of AgNPs (Fig. 1a). There is also a small peak appeared at 350 nm. The appearance of two peaks shows that the parti- cles are of different sizes. The peak at 350 nm is for smaller nanoparticles and on the other hand, the 468 nm peak is a result of larger size nanoparticles. FE-TEM was used for surface morphology and shapes of S-AgNPs, and the images were presented in Fig. 1b. The TEM analysis revealed the irregular shape of synthesized S-AgNPs. The synthesized S-AgNPs ranged in size of 20–94 nm. This size difference can be due to various factors such as temperature, pH, seed concentration or the reducing agent used. The elemental mapping results of the S-AgNPs indicate the maximum distribution of silver element, suggested that silver was the predominant element in the respective nanoproduct (Fig. 2a, b). The EDX analysis showed the highest peak at 3 keV for S-AgNPs (Fig. 2c). Some additional peak for carbon and copper appeared in the EDX spectrum which is due to the TEM grid used for the study. The number and percentage of Journal of Nanostructure in Chemistry 13PDF Image | green silver nanoparticles from leaf used as UVb protection
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