Pyrocatechol

Folin–Ciocalteu reagent, gallic acid (3,4,5-trihydroxybenzoic acid, abbreviated as GA), anhydrous sodium carbonate (Na₂CO₃), were obtained from Sigma-Aldrich (St. Louis, MO, USA). Reagents pyrocatechol, catechin, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, and quercetin were of analytical grade and supplied from Merck (Darmstadt, Germany). The plant species O. mutabilis was collected in Mersin, Turkey, and identified by Dr. Riza Binzet (Location: C5 Mersin, Mersin-Gözne, around Darısekisi, rocky slopes and scrub area, 36°58′10.91″ N 34°34′11.79″ E, altitude of 780 m). Ultrapure water (18 MΩ cm at 25 °C) was provided by a Millipore Milli-Q Advantage A10.

Tamoxifen was purchased from Nacalai Tesque (Tokyo, Japan). Caffeine, caffeic acid, chlorogenic acid, pyrocatechol, and trigonelline were purchased from Sigma-Aldrich (St. Louis, MO). LY294002 and U0126 were purchased from Tocris Bioscience (Bristol, UK). Anti-ERα, anti-phospho-MEK1/2 (S217/221), anti-MEK1/2, anti-phospho-ERK1/2 (T202/Y204), anti-ERK1/2, anti-phospho-Akt (S473), anti-Akt, and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p53, anti-cyclin D1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Peroxidase-conjugated rabbit anti-mouse, goat anti-rabbit, and swine anti-goat secondary antibodies were from Dako (Glostrup, Denmark).

The principal flavonoids and phenolic compounds were detected and identified using an Agilent 1260 series HPLC system with an Eclipse C18 column (4.6 mm × 250 mm i.d., 5 μm) and water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) as the mobile phase at a flow rate of 1 ml/min. The mobile phase elution linear gradient was used as follows: 0 min (82% A:18% B); 0–5 min (80% A:20% B); 5–8 min (60% A:40% B); 8–12 min (60% A:40% B); and 12–20 min (82% A:18% B). The multi-wavelength detector used was monitored at a wavelength of 280 nm. The injection volume for each sample was 5 μl and the column temperature was maintained at 40°C. Each sample’s phenolic and flavonoid composition was determined by comparing their retention times and spectral reference data with the external standard controls. All standards, namely gallic acid, chlorogenic acid, catechin, methyl gallate, caffeic acid, syringic acid, pyrocatechol, rutin, ellagic acid, coumaric acid, vanillin, ferulic acid, naringenin, querectin, cinnamic acid, kaempferol, and hesperetin were purchased from Sigma-Aldrich (Khalil et al., 2020 (link); Abdel-Aziz et al., 2021 (link)).

Gallic acid, (+)-catechin, pyrocatechol, chlorogenic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, syringic acid, vanillin, taxifolin, sinapic acid, p-coumaric acid, ferulic acid, rosmarinic acid, 2-hydroxycinnamic acid, pinoresinol, quercetin, luteolin, and apigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vanillic acid, 3-hydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, apigenin 7-glucoside, luteolin 7-glucoside, hesperidin, eriodictyol, and kaempferol were obtained from Fluka (St. Louis, MO, USA). Finally, verbascoside, protocatechuic acid, and hyperoside were purchased from HWI Analytik (Ruelzheim, Germany). Methanol and formic acid of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd.)
Ethyl acetate and methanol were obtained from Carlo Erba Reagents (Milan, Italy). Ultra-pure water was obtained using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA). All chemicals were of analytical grade.