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10 protocols using rutoside

1

TLC Analysis of Osmunda regalis Extract

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As samples for TLC, O. regalis extract and standards were used. They were applied to Silica gel 60 F264 plates (Merckmillipore.com), according to standard procedures [11 ]. As solvents ethyl acetate - formic acid - acetic acid - water (100:11:11:27) for flavones or toluene - ethyl acetate - formic acid (50:40:10) for polyphenolcarboxylic acids were used. UV detection was performed at 365 nm.
The following standards were used: ferulic acid, apigenin (Extrasynthese, Genay, France), chlorogenic acid (Carl Roth, Rothenfels, Germany), rosmarinic acid and rutoside (Sigma Aldrich, St. Louis, Missouri, USA).
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2

Phytochemical Profiling and Antioxidant Activity

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Acetic acid, ethanol 96%, methanol, and sucrose were from Chempur (Piekary Śląskie, Poland). HPLC-grade methanol and acetonitrile were purchased in Merck (Darmstadt, Germany).
Plant culture media components, plant growth regulators BA (6-benzyladenine) and NAA (1-naphthaleneAcetic acid) and agar were purchased in Duchefa Biochemie (Haarlem, Netherlands). Cultures were grown in the plant tissue-dedicated glass containers (V8630, Sigma-Aldrich, Saint Louis, MI, USA).
Commercially available standards: chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid, hyperoside (quercetin 3-galactoside), isoquercitrin (quercetin 3-glucoside), isorhamnetin, kaempferol, guaijaverin (quercetin 3-arabinoside), quercetin, rutoside (quercetin 3-rutinoside), and trifolin (kaempferol-3-galactoside) of HPLC grade (≥95.0%) purity were acquired in Sigma-Aldrich Saint Louis, MI, USA. Ammonium acetate, CuCl2⋅2H2O, DPPH, FeCl3⋅6H2O, Folin-Ciocalteu reagent, hydrochloric acid (HCl), Na2CO3, 2,9-dimethyl-1,10-phenanthroline (neocuprine), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) were also provided by Sigma-Aldrich. Deionised water (>15 MΩ) was produced in house (PureLab OptionR, Elga, High Wycombe, UK).
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3

HPLC-DAD Analysis of Phenolic Compounds

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The analysis was performed with the HPLC-DAD method described previously [52 (link),53 (link)]. For these estimations, the methanolic extracts were used (prepared as described in Section 3.4). An HPLC-DAD system (Merck-Hitachi, Merck KGaA, Darmstadt, Germany) and a Purospher RP-18e analytical column (4 × 250 nm, 5 mL; Merck) were used. Elution was performed with a mobile phase A (methanol:0.5% acetic acid, 1:4 v/v) and a mobile phase B (methanol). The gradient program was used. The temperature was set at 25°C, the flow rate at 1 mL/min, the injection volume at 20 μL, and the detection wavelength at 254 nm (UV spectra were recorded in the 220–350 nm range). Quantitative analyses were carried for the following compounds identified previously using the UHPLC-DAD-ESI-MS method [24 (link)]: p-coumaric acid, ferulic acid, and rutoside (Sigma-Aldrich Co., St. Louis, MO, USA).
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4

HPLC-DAD Analysis of Phenolic Compounds

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The analysis was performed using the HPLC–DAD method described previously (Ellnain-Wojtaszek and Zgórka 1999 (link); Sułkowska-Ziaja et al. 2017 (link)). For the estimation, methanolic extracts were used (prepared as described in "Sample preparation"). An HPLC–DAD system (Merck-Hitachi, Merck KGaA, Darmstadt, Germany) and a Purospher RP-18e analytical column (4 × 250 nm, 5 mL; Merck) were used. Elution was done with a mobile phase A (methanol:0.5% acetic acid, 1:4 v/v) and a mobile phase B (methanol). The gradient program was set as follows: 0–20 min, 0% B; 20–35 min, 0–20% B; 35–45 min, 20–30% B; 45–55 min, 30–40% B; 55–60 min, 40–50% B; 60–65 min, 50–75% B; and 65–70 min, 75–100% B. The hold time was 15 min. The other parameters were as follows: temperature 25°C, flow rate 1 mL/min, injection volume 20 μL, and detection wavelength 254 nm. Quantitative analysis was carried out for the following compounds identified previously using the UHPLC-DAD-ESI–MS method (Klimek-Szczykutowicz et al. 2020a (link)): p-coumaric acid, ferulic acid, and rutoside (Sigma-Aldrich Co. St. Louis, MO, USA).
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5

Phytochemical Analysis of Botanical Samples

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TLC was carried out on silica gel 60 F254 plates (Merck, Germany). Spots were visualized under UV light (254 and 365 nm) or by heating after spraying with 2% vanillin solution (in 96%H2SO4). Low resolution ESI-MS analyses were performed on a SCIEX API-3200 instrument (Applied Biosystems, Concord, Ontario, Canada) combined with a HTC-XT autosampler (CTC Analytics, Zwingen, Switzerland). The samples were introduced via autosampler and 2 µL loop injection. 1H, 13C NMR and 2D (COSY, HSQC, HMBC and NOESY) spectra were recorded on an Agilent DD2 400 NMR spectrometer and the chemical shifts were referenced to TMS or the solvent residual peak. UV spectra were measured with a Jasco V-560 UV/Vis spectrophotometer. CD spectra were acquired on a Jasco J-815 CD spectrophotometer and the specific rotation was measured with a Jasco P-2000 polarimeter. Infrared spectra (ATR) were recorded using a Thermo Nicolet 5700 FT-IR spectrometer. Rutoside, (+)-catechin and (−)-epicatechin analytical standards were obtained from Sigma Aldrich (Germany). Procyanidin B2 was used from the IPB-NWC in-house reference compound library (isolated from Bumelia sartorum Mart.) [48 (link)]. All the reagents and solvents used were of analytical and LC-MS grade.
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6

Trace Metal and Phenolic Acid Analysis

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Suprapur® nitric acid (65%) and Suprapur® hydrogen peroxide (30%) were purchased from Merck (Darmstadt, Germany). Quadruple-distilled water with a conductivity of less than 1 µS/cm was obtained using an S2–97A2 distillation apparatus (Chemland, Stargard Szczecinski, Poland). Standards of Zn(II), Fe(III), Mn(II), Mg(II), and Cu(II) at concentrations of 1 g/L were purchased from the District Office of Measures in Łodz, Poland.
The reagents used for the analysis of phenolic acids in the samples were as follows: MeOH and glacial acetic acid of analytical grade were purchased from Chempur, and MeOH of HPLC grade was purchased from Merck. The following standards were purchased: caffeic acid, chlorogenic acid, neochlorogenic acid, and rutoside (Sigma Aldrich, St. Louis, MI, USA); cryptochlorogenic acid, isochlorogenic acid, 4-feruloylquinic acid, and astragalin (ChromaDex, Los Angeles, CA, USA); and caffeine (Fluka Chemie AG, Buchs, Switzerland).
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7

Melanoma and Squamous Cell Carcinoma Assay

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A375 (ATCC CRL-1619) human malignant melanoma and human squamous cell carcinoma SCC-15 (ATCC CRL-1623) cell lines were purchased from LGC Standards (Łomianki, Poland). HaCaT immortalized human keratinocytes were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). Fetal bovine serum (FBS) was obtained from Pan-Biotech (Aidenbach, Germany). Dulbecco’s modified Eagle’s medium (DMEM)/high glucose, Dulbecco’s phosphate buffered saline (DPBS), mushroom tyrosinase from Agaricus bisporus, 3,4-Dihydroxy-l-phenylalanine (l-DOPA), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), neutral red solution (3.3 g/L), gallic acid, rutoside, quercetin, kojic acid, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of the reference compounds exceeded 95%. Acetonitrile, water, and formic acid for LC-MS analyses were purchased from Merck (Darmstadt, Germany). All other reagents were purchased from Honeywell (Warszawa, Poland). All solutions were prepared with ultrapure water (MilliQ Integral II, Merck, Darmstadt, Germany).
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8

Antioxidant Determination Protocol

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Reagents used to perform the antioxidant determinations—2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu reagent, the standards of gallic acid, chlorogenic acid, berberine, palmatine, jatrorrhizine, magnoflorine, quercetin, D-saccharic acid (glucaric acid), glucaric acid potassium salt and rutoside at purity exceeding 95% were purchased from Sigma-Aldrich (St. Louis, MO, USA). Spectroscopic grade solvents used for the LC-MS analyses—acetonitrile, water and formic acid, were manufactured by Merck (Darmstadt, Germany). The reagent grade chemicals used for the preparation of extracts and spectrophotometric determinations were purchased from Avantor Performance Materials (Gliwice, Poland).
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9

HPLC-DAD Analysis of Polyphenols

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This analysis was performed using the HPLC-DAD method described in previous works [52 (link),53 (link)]. Methanolic extract (prepared as described in Section 4.5) was used. A HPLC-DAD system (Merck-Hitachi, Merck KGaA, Darmstadt, Germany) and a Purospher RP-18e analytical column (4 × 250 nm, 5 mL; Merck) were used for the analysis. Elution was done with a mobile phase A (methanol:0.5% acetic acid, 1:4, v/v) and a mobile phase B (methanol). The gradient program was set as follows: 0–20 min, 0% B; 20–35 min, 0–20% B; 35–45 min, 20–30% B; 45–55 min, 30–40% B; 55–60 min, 40–50% B; 60–65 min, 50–75% B; and 65–70 min, 75–100% B. The hold time was 15 min. The other parameters were as follows: temperature, 25 °C; flow rate, 1 mL/min; injection volume, 10 μL; and detection wavelength, 254 nm. Quantitative analysis was carried for the following compounds detected using UHPLC-DAD-ESI-MS: p-coumaric acid, ferulic acid, gallic acid protocatechuic acid and rutoside (all compounds were purchased from Sigma-Aldrich Co. St. Louis, MO, USA).
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10

Cytotoxicity Assay Protocol

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Rutoside, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT), Tryphan blue solution 0.4%, Acridine orange, ethidium bromide,Hoechst, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS), phosphate buffer saline (PBS), and antibiotics were purchased from Gibco (Gibco BRL). Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Invitrogen (Invitrogen, Carlsbad, California, US). All the other reagents were bought from Merck.
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