L ascorbic acid vitc

Mouse embryonic fibroblasts NIH3T3 (ATCC‐CRL‐1658) and PARP‐1 knock‐out (PARP1−/−) mouse embryonic fibroblasts (derived from PARP‐1 knock‐out mouse11) cell lines were cultured in high glucose Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), L‐glutamine and penicillin/streptomycin (all cell culture reagents were supplied by Biological Industries Israel, Beit Haemek Ltd.). Both cell lines were treated with either 1 mmol/L dimethyloxalylglycine (DMOG) (Frontier scientific, USA) for 24 hours, or with 10 µmol/L L‐ascorbic acid (VitC) (Sigma Aldrich, USA) for 48 hours. These concentrations correspond to the EC₅₀ for the two cell lines.

Zein powder (MW 20 kDa),
ethanol (≥99.7%), l-ascorbic acid (Vit C), phosphate-buffered
saline (PBS) 1×, sodium acetate, calcium chloride (CaCl₂), apramycin sulfate salt, trifluoroacetic acid (TFA), and ethanol/hexamethyldisilizane
(36%) were purchased from Sigma-Aldrich and used as received. Low
methoxy pectin powder was purchased from Silva Team. Lecithin (90%)
soybean was purchased from Alfa Aesar. The cell proliferation reagent
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] (MTS) and the CellTiter-Glo Luminescent viability
assay kit were obtained from Promega. HDFa, fibroblast basal medium
supplemented with Supplement Pack Fibroblast Growth Medium 2, 0.25%
trypsin–EDTA (1×), 2-(4-aminiodinophenyl)-6-idolecarbamide
dihydrochloride (DAPI), and Alexa Fluor 488 Phalloidin were purchased
from Thermo Fisher Scientific. HaCaT cells were purchased from the
Cell Line Service (Heidelberg, Germany).

The DPPH radical scavenging activity of the sample was examined as previously mentioned [17 (link)]. Briefly, a working DPPH• solution (6 μM) was freshly prepared by adjusting the absorbance of 2 mM DPPH• (Sigma-Aldrich, Singapore) stock solution to 0.80 ± 0.05 at 517 nm by using methanol. Then, 200 μL of sample at various concentrations or a serial dilution of L-ascorbic acid (VitC) (Sigma-Aldrich, Singapore) were reacted with 1.8 mL of working DPPH• solution for 30 min before measuring at 517 nm. The DPPH radical scavenging activity was calculated and compared with the activity with the Vit C standard curve. The result was expressed as mg Vit C equivalent/g sample.