P hydroxybenzoic acid

Acetone HPLC purity (Sigma-Aldrich, Warsaw, Poland); carotenoids standards (99.9% purity): Lutein, zeaxanthin, α-carotene, β-carotene (Sigma-Aldrich, Warsaw, Poland); magnesium carbonate (Chempur, Warsaw, Poland); methanol HPLC/MC purity (Sigma-Aldrich, Warsaw, Poland), ortho-phosphoric acid 85% (Chempur, Warsaw, Poland); polyphenols standards: Gallic acid, chlorogenic acid, caffeic acid, benzoic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, kaempferol, kaempferol-3-O-glucoside, myricetin, quercetin, quercetin-3-O-rutinoside (Merck, Warsaw, Poland); ultrapure water (Sigma-Aldrich, Warsaw, Poland).

Folin-Ciocalteu’s phenol reagent, sodium carbonate (99%), catechin (98%), protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, p-coumaric acid, sinapic acid, ferulic acid, sodium nitrite (97%), aluminium chloride (99.9%), and vanillin (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Procyanidin B2, rutin, epicatechin, kaempferol, quercetin dehydrate, daidzein and genistein were purchased from Extrasynthese (Genay, France). Sulphuric acid (95–97%) and gallic acid (98%) were purchased from Fluka (Seelze, Germany). Sodium hydroxide (98%) was purchased from Merck (Darmstadt, Germany). Absolute ethanol (99.9%) and methanol (99.9%) were purchased from Carlo Erba Reagents (Rodano, Italy). Milli-Q^® water (18.2 MΩ.cm) was obtained in a Millipore–Direct Q3 UV System equipment (Molsheim, France).

Spelt (Triticum spelta L. cv. Ostro) seeds were obtained from a local mill in the Dolenjska region of Slovenia. The seeds were stored in the dark at 1 °C until analysis. The preparation of the germinated spelt seeds was reported in detail by Mencin et al. [4 (link)].
Methanol (99.9%), formic acid, sodium hydroxide and sodium carbonate were from Merck (Darmstadt, Germany). 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reagent, the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) reagent, Folin–Ciocalteu reagent, Trolox, sodium dihydrogen phosphate dehydrate, p-hydroxybenzoic acid, trans-ferulic acid, p-coumaric acid and caffeic acid were from Sigma-Aldrich (Steinheim, Germany). All of the chemicals and reagents used for the present study were of analytical quality. Water (Milli-Q; Millipore, Billerica, MA, USA) was used to prepare the working solutions.

All reagents, specifically 2,2´-diphenyl-1-picrylhydrazyl (DPPH), 2,2´-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), were purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade phenolic standards (protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, (−)-epicatechin, caffeic acid, p-coumaric acid, ferulic acid, rutin, quercetin, quercetin-3-rhamnoside, cAMP, and cGMP) were also purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC grade sugar standards (glucose, fructose, and sucrose), ascorbic acid (AA), phloridzin and kaempferol were purchased from Yuanye Biotechnology Ltd. (Shanghai, China). Methanol, acetonitrile and formic acid were purchased from Tedia Company, Inc. (Fairfield, CT, USA). Folin-Ciocalteu reagent and all other chemicals were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China).

Bee bread was purchased in Iasi County, Romania. The sample was harvested in the spring of 2020. The sample was stored at −20 °C prior to any analysis. The bee bread was defatted using n-hexane (the oil was used for fatty acids characterization, while the defatted bee bread was used for bioactive compounds analysis). Methanol, Folin-Ciocalteau reagent, ethanol, AlCl₃, sodium carbonate, ethyl ether, rosmarinic acid, p-coumaric acid, chlorogenic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, protocatechiuc acid, gallic acid, kaempferol, quercetin luteolin and myricetin, fructose, glucose, sucrose, melesitose and raffinose were purchased from Sigma–Aldrich (Taufkirchen, Germany). FAMEs mix was purchased from Restek (Lisses, France).

Crude pollen was purchased from a local beekeeper from Suceava County, Romania. The crude pollen was harvested in the summer of 2019 and was polyfloral. The samples were packed and stored at −20 °C until further analysis. The extraction of a lipid fraction from the pollen sample was performed by the Soxhlet method using pure hexane: 10 g of pollen was extracted with 100 mL of hexane into a Soxhlet apparatus, and the solvent was recirculated 5 times. Five batches were extracted as 10 g of crude pollen. The mixture of solvent and oil was separated using a rotary evaporator under vacuum at 65 °C. The defatted pollen was used for the extraction of polyphenols, while the lipid fraction was analyzed in terms of fatty acids composition.
Methanol, AlCl_3, Folin-Ciocalteau reagent, sodium carbonate, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, vanillic acid, chlorogenic acid, p-coumaric acid, rosmarinic acid, myricetin, luteolin, quercetin and kaempferol were purchased from Sigma–Aldrich (Taufkirchen, Germany).

Folin-Ciocalteu reagent, potassium acetate, sodium acetate, 2,2-diphenyl-1-Picrylhydrazyl (DPPH), 2,2′-Azino-di-(3-Ethylbenzthiazoline Sulfonic acid) (ABTS), Potassium hexacyanoferrate, thiobarbituric acid, xylenol orange, quinine hemisulfate and chloramine-T were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid (glacial) and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Phytochemical standards, including quercetin, gallic acid, catechin and p-hydroxybenzoic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA).