Sodium nitrite

Different chemical reagents used for analysis viz. ascorbic acid, calcium chloride, quercetin, aluminium chloride, sodium nitrite, sodium hydroxide pellets, gallic acid, folin-ciocalteu phenol reagent, anhydrous sodium carbonate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium bicarbonate, methanol, tri-chloro acetic acid, and acetone were of analytical grade and purchased from Sigma-Aldrich (St. Louis, USA) and Himedia (HiMedia Laboratories, Mumbai, India). Broccolis (cultivar: Pusa KTS-1) utilized in the study were obtained from the Farm section of ICAR-Central Institute of Agricultural Engineering in Bhopal, India (23.3156° N, 77.4037° E). The broccolis were harvested 125 days after sowing of seeds and 90 days of transplanting. Mass of individual heads were in the range of 350–450 g.

The O₂⁻ content was estimated spectrophotometrically by following the method given by Wu et al. [60 (link)]. Briefly, one gram of fresh plant sample was grounded in phosphate buffer (6 mL of 65 mM, pH 7.8) containing 2% polyvinylpyrrolidone, followed by centrifugation (at 5000× g for 15 min at 4 °C). The mixture was incubated at 25 °C for 30 min. After that, 0.5 mL of supernatant was mixed with 0.5 mL of phosphate buffer and 0.1 mL of hydroxylamine hydrochloride (10 mM, Sigma Alrdrich, Mumbai, India) and incubated again. Incubated mixture was than mixed with 3-amino benzene sulphonic acid and 1-naphthylamine (1 mL of 58 and 7 mM respectively, CDH, New Delhi) and kept at 25 °C for 20 min. The absorbance was recorded at 530 nm and the amount of O₂⁻ was calculated against the sodium nitrite (Himedia, Mumbai, India) as standard which was expressed as μmol g⁻¹ FW. The in-vivo visualisation of O₂⁻ was done by using the method mentioned by Frahry and Schopfer [61 (link)]. The leaves of B. juncea were first incubated in nitro blue tetrazolium (NBT, Himedia, Mumbai, India) prepared in potassium phosphate buffer with pH 6.4 and sodium azide (10 mM), followed by a process of decolouration (by immersing them in boiling ethanol), and photographed.

Fetal Bovine Serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), trichloroacetic acid (TCA), N-(1-naphthyl) ethylenediamine dihydrochloride, sulphanilamide, ascorbic acid, tannic acid, custom oligonucleotides were obtained from Sigma-Aldrich, MO, USA. Penicillin, streptomycin, potassium ferricyanide, H₂O₂, ferric chloride, ferrous sulphate (FeSO₄), and sodium nitrite were obtained from Himedia (Mumbai, India). Cellular Senescence Assay kits were purchased from Millipore Corp. (USA). Involucrin and filaggrin ELISA kits were purchased from Cloud-Clone Corp. (USA), Collagen-Type I kits were purchased from Blue Gene Biotech, China. All molecular biology reagents for PCR were obtained from Bio Rad, USA.

Bovine serum
albumin (BSA), 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS), nicotinamide adenine dinucleotide (NADH), nitroblue
tetrazolium (NBT), disodium hydrogen phosphate, n-butyl alcohol, 2-thiobarbituric acid (TBA), phenazine methosulfate
(PMS), glutathione reduced, potassium persulfate, gallic acid, quercetin
dihydrate, ferric chloride, sodium nitrite, methanol, and cumene hydroperoxide
were obtained from HiMedia Laboratories Pvt., Ltd. (Mumbai, India).
Glacial acetic acid, aluminum chloride, silver nitrate, hydrogen peroxide
(H₂O₂), and 5,5′-dithio-2-nitrobenzoic
acid (DTNB) were obtained from Merck Specialities Pvt., Ltd. (Mumbai,
India). Potassium ferricyanide and ferrous chlorides were obtained
from Loba Chemie Pvt., Ltd. (Mumbai, India). 1-Chloro-2,4-dinitrobenzene
(CDNB), cupric sulfate, trichloroacetic acid (TCA), Folin–Ciocalteu’s
reagent, sodium hydroxide, and ascorbic acid were obtained from SD
Finechem Ltd. (Mumbai, India). Doxorubicin (Getwell Oncology Pvt.,
Ltd., Haryana, India) was purchased from a local pharmacy.