Ferrous chloride

In addition, 100 µL of solution of each FPH at different concentrations was added to a microplate (Eon 2, BioTek, Winooski, VT, USA). Furthermore, 0.5 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) was used as positive control. To start the reaction, 20 µL of 0.5 mM ferrous chloride (Sigma-Aldrich, St. Louis, MO, USA) was added to the plate and mixed in a plate mixer for 3 min at 300–600 rpm. Subsequently, 20 µL of 2.5 mM ferrozine (Sigma-Aldrich, St. Louis, MO, USA) was added. Blanks consisted of only iron chloride and Ferrozin. The plate was mixed and left for 10 min in dark at room temperature. The absorbance at 562 nm was measured using a microtiterplate-reader (Eon 2, BioTek, Winooski, VT, USA). Ferrous chelating activity (FCA) was calculated as inhibition percentage (I_FCA) using mean absorbance as: $I_{FCA}(\%)=(\frac{A_{\mathrm{blank}}-\left(A_{\mathrm{sample}}-A_{\mathrm{blind}}\right)}{A_{\mathrm{blank}}})\times 100\%$
where A_blank is the absorbance of blank, A_sample is the absorbance of sample, and A_blind is the absorbance of blind (only FPH).
Based on the inhibition percentages for the different concentration for each FPHs, the MCA was determined as the EC₅₀ based on linear regression. Each sample concentration was measured in triplicate.

Organic solvents used for extraction were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Other chemicals and standards of analytical grade or higher were also sourced from Sigma Aldrich, such as 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), 3-(2-Pyridyl)-5,4,4-dimethoxybenzaldehyde,4-triazine-p,5,6-diphenyl-1,6-hydroxy-2,6-Tris(2-pyridyl)-s-triazine (TPTZ), 7,8-tetramethylchroman-2-carboxylic acid (Trolox), aluminium trichloride, anhydrous sodium carbonate, catechin hydrate, disodium ethylenediaminetetraacetic acid (EDTA-Na₂), ferrous chloride, Folin–Ciocalteu reagent, gallic acid monohydrate, phloroglucinol, potassium persulfate, p’-disulfonic acid monosodium salt hydrate (Ferrozine), sodium acetate, trisodium phosphate, and quercetin. HPLC grade standards including phloroglucinol, gallic acid, chlorogenic acid, syringic acid, synaptic acid, catechin, epicatechin, and epigallocatechin were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Vanillin was obtained from Chem-Supply Pty Let., Adelaide, SA, Australia. The Milli-Q water used was obtained from Millipore Milli-Q Gradient Water Purification System (Darmstadt, Germany).

Reagents: Folin-Ciocalteu, sodium persulfate, gallic acid (GA), BHT, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid, diammonium salt (ABTS), Trolox, ferrous chloride, and 2,2-diphenyl-1-picrylhydrazyl (DPPH), were purchased from Sigma-Aldrich (Athens, Greece); 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka Chemica (Athens, Greece). Dimethylsulfoxide (DMSO), (Merck KGaA, Darmstaadt, Germany) was used as a solvent. Water was obtained from a Milli-Q water purification system (TGI Pure Water Systems, Topway Global, Greenville, SC, USA). Lichrosolv hypergrade for LC-MS acetonitrile was supplied by Merck (Darmstadt, Germany). Water (18.2 MΩ) was from a Milli-Q water system (Millipore, Bedford, MA, USA). Acetic acid was from LGC Standards (Middlesex, UK). Eighteen standard phenolic compounds were used (gallic acid, protocatechuic acid, HydroxyTyrosol, 4-hydroxybenzoic acid, chlorogenic acid, gentisic acid, caffeic acid, vanillic acid, syringic acid, p-coumaric acid, rutin, ferulic acid, trans-m-hydroxycinnamic acid, o-coumaric acid, salicylic acid, luteolin, eriodictyol and cinnamic acid) purchased from Sigma Aldrich.

Analytical-grade reagents were used in this experiment [11 (link),12 (link)]. Phenolic contents and antioxidant activities of fruit extracts were measured using; Folin–Ciocalteu (F-C) phenol reagent, sodium carbonate, gallic acid, aluminum chloride, sodium acetate, quercetin, DPPH, ABTS, potassium persulfate, ferrous chloride, ferrozine, ethylenediaminetetraacetic acid (EDTA), iron (II) heptahydrate, L-ascorbic acid, and 3-hydroxybenzoic acid were purchased from Sigma Aldrich (St. Louis, MO, United States) while 30% hydrogen peroxide (H₂O₂) and 98% sulfuric acid (H₂SO₄) were purchased from Chem-Supply Pty Ltd. (Adelaide, SA, Australia) and RCI Labscan Ltd. (Bangkok, Thailand). Pure phenolic standards and LC-MS grade formic acid were also purchased from Sigma Aldrich (St. Louis, MI, USA). HPLC vials were purchased from Agilent Technologies (Santa Clara, CA, USA) while 96 well plates were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia).

Polycaprolactone diol (PCL-diol, M_n ~ 2000), tin(II) 2-ethylhexanoate (Sn-Oct), hexamethylene diisocyanate (HDI: >98%), 1,4-diaminobutane (putrescine: 99%), 2-propanol (IsOH, anhydrous: 99.5%), inulin from dahlia tubers (n ≈ 36, M_r ~ 5000), ferric chloride hexahydrate (FeCl₃·6H₂O, 97%), ferrous chloride (FeCl₂, 98%), and doxorubicin hydrochloride (DOXO, for HPLC: 98–102%) were purchased from Sigma Aldrich Co. Mexico; ammonium hydroxide (NH₄OH, ~29%), sodium citrate tribasic dihydrate (Na₃C₆H₅O₇·2H₂O, ~98%), 2-propanol were purchased from J.T. Baker. Ethylene dichloride (ACS reagent grade) was purchased from Macron Chemicals and Hartmann physiological solution was purchased from the local drugstore. All reactants were used as received.