Ferulic acid

Ferulic acid (FA) and caffeic acid (CA) were purchased from ROTH company. Gallic acid (GA) was purchased from PolAura Company. Collagen (Coll) was isolated from the rat tail tendons. Collagen was freeze-dried and then dissolved at 1% concentration (in 0.1 M of acetic acid). The collagen solution was mixed with phenolic acid solutions such as Ferulic acid, Gallic acid, and caffeic acid. Each of the phenolic acids was dissolved separately in 0.1 M of acetic acid at 1% concentration. The phenolic acid solution was added to the collagen solution in the 10 w/w% ratios. The mixtures were mixed on the magnetic stirrer for 1 h at room temperature. Then they were frozen by using the lyophilizer (ALPHA 1–2 LDplus, CHRIST, − 20 °C, 100 Pa, 48 h). The 3D porous scaffolds were obtained and used for the biological assessment.

High purity chemicals: sodium carbonate, sodium acetate trihydrate, and anhydrous aluminum chloride were acquired from Sigma-Aldrich (Germany). Folin-Ciocâlteu reagent was purchased from Merck (Germany). The standard chemicals; chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, hyperoside, kaempferol, quercetol, myricetol, fisetin, gallic acid, aucubin, catalpol, and harpagoside were sourced from Sigma-Aldrich (Germany), ferulic acid, sinapic acid, gentisic acid, patuletin, luteolin from Roth (Germany), caftaric acid from Dalton (United States), harpagide, and 8-O-acetyl-harpagide from PhytoLab GmbH & Co. (Germany). HPLC grade solvents (methanol, acetonitrile, ammonium acetate, and silver nitrate) were purchased from Sigma-Aldrich. Distilled, deionised water was produced by a Direct Q-5 Millipore (Millipore SA, Molsheim, France) water system.

Methanol and ethanol were from VWR (Darmstadt, Germany) and were like all other solvents used of HPLC grade. Trolox, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), Folin-Ciocalteu reagent, quercetin, (−)-epicatechin, procyanidin B1, procyanidin B2, procyanidin C1, p-coumaric acid, trans-cinnamic acid and phloridzin dihydrate were from Sigma–Aldrich (Taufkirchen, Germany). Gallic acid, (+)-catechin, chlorogenic acid, ferulic acid, coffeic acid, rutin trihydrate and acetic acid were purchased from Carl Roth (Karlsruhe, Germany). Avicularin was used from Phytolab (Vestenbergsgreuth, Germany). Protocatechuic acid, hyperoside, isoquercitrin, quercitrin and procyanidin A2 were from Extrasynthese (Genay, France). Reynoutrin was purchased from Carbosynth Limited (Berkshire, UK). 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was from Fisher Scientific (Nidderau, Germany). HPLC grade water (18 MΩ) was prepared using a MicroPure purification system (Thermo Electron LED GmbH, Niederelbert, Germany). Sodium hydroxide, hydrochloric acid (Merck, Darmstadt, Germany), meta-phosphoric acid (Sigma–Aldrich) and all other chemicals were of analytical grade.

The reagents included in standard assay packets with colorimetric and kinetic methods were obtained from BioMaxima S.A., Lublin, Poland. Thiobarbituric acid, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), sulphanilamide, NEDD, VCl₃, methanol, diethyl ether, xylenol orange, [o-cresosulfonphtalein-3,3-bis(sodium methyliminodiacetate)], o-dianisidine dihydrochloride (3,3′-dimethoxybenzidine), ferrous ammonium sulphate, hydrogen peroxide, sulphuric acid, hydrochloride acid, glycerol, trichloroacetic acid (TCA), and trolox were obtained from several companies (Sigma, Fluka, Merck). Standards rutin, kaempferol, and chlorogenic acid were purchased from Sigma, Germany. Ferulic acid, gallic acid, luteolin, and quercetin were obtained from Roth, Germany. All other chemicals and solvents used in the study were of analytical grade.

Sinapinic and ferulic acid were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany); caffeic acid, chlorogenic acid, alpha-lactalbumin (bovine), and ammonium acetate were purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Methanol and acetonitrile were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). All chemicals were used in highest quality available. Water was purified before utilization via Direct-Q 3 UV-R system (Merck KGaA, Darmstadt, Germany).

For the phytochemical analysis by LC-MS/MS, a number of 25 polyphenols were used as standards. The following standards were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany): caftaric acid (≥97%), chlorogenic acid (≥95%), caffeic acid (≥98%), p-coumaric acid (≥98%), rutin (≥94%), apigenin (≥95%), kaempferol (≥97%), luteolin (≥98%), gentisic acid (≥98%), myricetol (≥96%), fisetin (≥98%), (+)-catechin (≥96%), (−)-epicatechin (≥90%), quercetin (≥95%), quercitrin (quercetin 3-rhamnoside) (≥78%), isoquercitrin (quercetin 3-d-glucoside) (≥98%), hyperoside (quercetin 3-d-galactoside) (≥97%), patuletin (≥98%), protocatechuic acid (3,4-dihydroxybenzoic acid) (≥97%), syringic acid (≥95%), vanillic acid (≥97%), rosmarinic acid (≥96%). Gallic acid (≥98%) was acquired from Merck (Darmstadt, Germany). Ferulic acid (≥99%) and sinapic acid (≥98%) were obtained from Roth (Karlsruhe, Germany). Solvents used for extraction and separation were of HPLC analytical-grade (methanol, ammonium acetate, acetonitrile) or analytical-grade (petroleum ether, chloroform, hydrochloric acid, acetic acid, potassium hydroxide). These solvents and Folin-Ciocâlteu reagent were purchased from Merck (Darmstadt, Germany). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Alfa-Aesar (Karlsruhe, Germany).

Ammoniac (NH₃), ammonium acetate, boric acid, ferulic acid, hydrochloric acid (HCl), kaempferol-3-O-glucoside, methanol, natrium hydroxide (NaOH), quercetin-3-O-glucoside, sinapic acid, and tert-butyl methyl ether were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Acetic acid, 2-propanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methylene chloride, potassium persulfate, and Trolox^® (6-hydroxyl-2,5,7,8-tetramethylchroman-2carbonic acid) were purchased from Merck KGaA (Darmstadt, Germany). 2,2′-Azinobis-(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) was purchased from Roche Diagnostic GmbH (Mannheim, Germany) and sulfuric acid from Grüssing GmbH (Filsum, Germany). Acetonitrile was obtained from J.T. Baker (Fisher Scientific GmbH, Grießheim, Germany) and tetrahydrofurane was from VWR International GmbH (Darmstadt, Germany). The standards of β-carotene, chlorophyll a, chlorophyll b, lutein, and zeaxanthin were purchased from CaroteNature^® GmbH (Münsingen, Switzerland) and pheophytin a from LGC Standards GmbH (Wesel, Germany).