Flavonols

Berry skins were freeze-dried (Cold Trap 7385020, Labconco, Kansas City, MO, United States). Dried tissues were ground with a tissue lyser (MM400, Retsch, Germany). Fifty mg of the powder were extracted with methanol: water: 7 M hydrochloric acid (70:29:1, V:V:V) to determine flavonol concentration and profile. Extracts were filtered (0.45 μm, Thermo Fisher Scientific, San Jose, CA, United States) and analyzed using reversed-phase high performance liquid chromatography (HPLC) coupled to a diode array detector (DAD). The HPLC system was an Agilent 1260 series (Agilent, Santa Clara, CA, United States) with a reversed-phase C₁₈ column LiChrospher^® 100, 250 mm × 4 mm with a 5 μm particle size and a 4 mm guard column of the same material. Anthocyanins may interfere significantly with the quantification of flavonols. Anthocyanin removal through solid phase extraction using a cationic exchange resin (e.g., Dowex 50X4-400, Acros Organics, Fair Lawn, NJ, United States) has been proposed for the determination of flavonols (Hilbert et al., 2015 (link)). However, the determination of flavonols is also possible avoiding co-elution between anthocyanins and flavonols (Downey and Rochfort, 2008 (link)). As Downey and Rochfort (2008) (link) method was not possible to implement directly on our HPLC system, the method was fine-tunned for our instruments. Flow was set to 0.5 ml per minute and temperature was set to 25°C. Two mobile phases were designed to always maintain the following proportions (V/V) of acetonitrile, 0–8 min 8%, at 25 min 12.2%, at 35 min 16.9%, at 70 min 35.7%, 70–75 min 65%, and 80–90 min 8%. This acetonitrile gradient and different isocratic concentrations of formic acid (HCOOH) from 1.8 to 10% were tested by adjusting the gradients and concentrations of two mobile phases (aqueous HCOOH and HCOOH in acetonitrile) as in Supplementary Information 3. A concentration 5% of HCOOH was the only one, avoiding coelution and allowing the simultaneous quantification (Figure 2 and Supplementary Information 4). The remaining volume up to 100% was achieved with purified water. For our HPLC system and column, a 5% HCOOH helped to avoid co-elution, separation of individual flavonols and a high degree of peak sharpness in both anthocyanins and flavonols.
For the identification of flavonols, standards of myricetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and syringetin-3-O-glucoside (Extrasynthese, Genay, France) were used. Flavonols were quantified determining the peak area of the absorbance at 365 nm. Quercetin-3-O-glucoside was used as a quantitative standard for all the flavonols. It must be noted that each individual anthocyanin and flavonol have a different molar relative response factors (e.g., absorbance per M unit) and even though calculating a response factors for each flavonol would have been possible using commercial standards, this is not the standard practice in the literature and would make comparisons of flavonol profiles harder.

The analysis was previously described by Sokół-Łętowska et al. [51 (link)]. The HPLC-PDA analysis was performed using a Dionex (Germering, Germany) system equipped with the diode array detector model Ultimate 3000, quaternary pump LPG-3400A, autosampler EWPS-3000SI, thermostated column compartment TCC-3000SD, and controlled by Chromeleon v.6.8 software (Thermo Scientific Dionex, Sunnyvale, CA, USA). The Cadenza Imtakt column C5-C18 (75 × 4.6 mm, 5 μm) was used. The mobile phase was composed of solvent C (4.5% aq. formic acid, v/v) and solvent D (100% acetonitrile). The elution system was as follows: 0–1 min 5% D in C, 20 min 25% D in C, 21 min 100% D, 26 min 100% D, 27 min 5% D in C. The flow rate of the mobile phase was 1.0 mL/min and the injection volume was 20 μL. The column was operated at 30 °C. Iridoids were detected at 245 nm, flavan-3-ols at 280 nm, phenolic acids and their derivatives at 320 nm, flavonols, flavanonols, flavones and flavanones at 280 and 360 nm, and anthocyanins at 520 nm.
Loganic acid and its derivatives were expressed as mg of loganic acid equivalents (LAE) per 100 g fresh weight (fw), loganin, sweroside and their derivatives as loganin equivalents (LoE) per 100 g fw, anthocyanins as cyanidin 3-O-glucoside equivalents (CygE) per 100 g fw, derivatives of quercetin and taxifolin as quercetin 3-O-glucoside equivalents (QgE) per 100 g fw, luteolin -O-dihexoside-hexoside as luteolin 7-O-glucoside equivalents (LgE) per 100 g fw, caffeoylquinic acids as mg of 5-O-caffeoylquinic (chlorogenic) acid equivalents (ChAE) per 100 g fw. Solutions of standards (1 mg/ml) were dissolved in 1 mL of methanol. The appropriate amounts of stock solutions were diluted with 50% aqueous methanol (v/v) acidified with 1% HCl in order to obtain standard solutions. Analytical characteristics for determination of phenolic compounds and iridoids are shown in Table S3.

Ethanol (≥ 99.9%, LiChrosolv^®), tetrahydrofuran (THF, ≥ 99.9%, LiChroSolv^®), n-hexane (SupraSolv^®), dichloromethane (< 99.8%, SupraSolv^®), chlorophyll a and b (analytical standards) and norflurazone (Pestanal^®, analytical standard) were obtained from Merck KGaA (Darmstadt, Germany). Tert-butyl methyl ether (≥ 99.9%, Rotisolv^®), 2-propanol (≥ 99.9%, Rotisolv^®), ammonium acetate (≥ 98%) and acetic acid (100%, Supra Quality) were purchased from Carl Roth GmbH (Karlsruhe, Germany). MEthanol (Chemsolute^®) and acetonitrile (Chemsolute^®) were purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany). Carotenoid standards were obtained from CaroteNature GmbH (Münsingen, Switzerland) and flavonol glycosides from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Abscisic acid (ABA) standard was purchased from Sigma Aldrich Chemie GmbH (Taufkirchen, Germany) and (+)-abscisic acid-d6, ≥ 98%) from Toronto Research Chemicals (North York, Canada). All solvents were of LC-MS quality and the water was of ultra-pure quality.

Intakes of total flavonoids and each flavonoid subclass in our study were downloaded from Flavonoid Values for US Department of Agriculture Survey Foods and Beverages (16 (link)), which are derived from (i) Database of Flavonoid Values for Food Codes; (ii) Flavonoid Intake Data Files from What We Eat in America and NHANES. The flavonoid database included data on the 6 main flavonoid subclasses: (i) flavones (apigenin and luteolin); (ii) anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin); (iii) flavanones (eriodictyol, hesperetin, and naringenin), (iv) flavonols (isorhamnetin, kaempferol, myricetin, and quercetin); (v) flavan-3-ols [catechins including: (−)-epicatechin, (−)-epicatechin 3-gallate, (−)-epigallocatechin, (−)-epigallocatechin 3-gallate, (+)-catechin, (+)-gallocatechin; theaflavin, theaflavin-3,3′-digallate, theaflavin-3′-gallate, theaflavin-3-gallate, and thearubigins]; (vi) isoflavones (daidzein, genistein, and glycitein), total flavones, total anthocyanidins, total flavanones, total flavan-3-ols, total flavonols, total isoflavones, subtotal catechins, and total flavonoids Daily flavonoid intake per participant was determined on the first and second days, and the mean of the two-day flavonoid intake was used in subsequent analyses.