Proanthocyanidins

Phenolics in crude extracts and each fraction of lingonberries were identified and quantified by HPLC-PDA (Waters e2695 Alliance system, Waters, Milford, MA, USA) according to Raudone et al. with some modifications [27 (link)]. Chromatographic separation was carried out on an ACE C18 reversed-phase column (250 mm × 4.6 mm, particle size 3 µm; ACT, UK) with a gradient elution consisting of 0.1% trifluoroacetic acid in water (eluent A) and acetonitrile (eluent B) at a flow rate of 0.5 mL/min, injection volume of 10 µL, and column temperature maintained at 35 °C. The gradient pattern was 0 min, 10% B; 0–40 min, 30% B; 40–60 min, 70% B; 60–64 min, 90% B; 64–70 min, 10% B. Prior to HPLC-PDA analysis, all samples were dissolved in 70% ethanol until complete dissolution, obtaining a concentration of 1 mg/mL, and filtered through a pore size 0.2 µm PVDF syringe filters (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
The described modified method was validated following international guidelines [28 ]. The selectivity of peaks was evaluated and phenolic compounds were identified by comparing the retention times, UV spectra of the analytes with those of the reference compounds, and on the basis of previous reports on lingonberry phenolics. The PDA detector was set at a wavelength of 280 nm for proanthocyanidins and catechins, 360 nm for flavonols, 520 nm for anthocyanins, 330 for hydroxycinnamic acids, and 260 nm for hydroxybenzoic acids. All phenolics were quantified according to 5–7 points linear calibration curves of external standards, except well-known predominant compounds of lingonberry leaves—quercetin-3-O-(4”-(3-hydroxy-3-methylglutaryl)-rhamnoside (quercetin-HMG-rhamnoside), and 2-O-caffeoylarbutin, because of commercially unavailable standards. They were tentatively quantified using calibration curves of standard substances with similar chemical structures. Limits of detection (LOD) and of quantification (LOQ) were determined via the signal-to-noise ratio method. The trueness of the method was expressed as percent recoveries of phenolics at low, medium, and high concentrations of range, each analyzed in triplicate. To assess the repeatability and intermediate precision of the method, relative standard deviation percentages (% RSD) of peak areas of each quantified phenolic compound were calculated within (six times per day) and between days (three consecutive days), respectively, resulting in total repeatability of 18 replicates.

MALDI-MSI analysis was performed according to our previous study [24 (link)], with minor modifications. The flavan-3-ol standards and strawberry fruit sections were analyzed using a MALDI-TOF/TOF instrument (UltrafleXtreme, Bruker, Billerica, MA, USA) equipped with a 355 nm Nd:YAG laser, using a repetition rate of 1000 Hz. Data were acquired using a step size of 300 μm in negative-ion mode (reflector mode). The m/z values in the range of 240–1200 were measured. The laser diameter was set to the medium size. The instrument was calibrated externally using the exact m/z values of CHCA [M − H]⁻ ions (m/z 188.03532), bradykinin (1–7) [M − H]⁻ ions (m/z 755.38460), angiotensin II [M − H]⁻ ions (m/z 1044.52725), and angiotensin I [M − H]⁻ ions (m/z 1294.67025) as references. The spectra were acquired automatically using FlexImaging 4.1 software (Bruker). Normalization of spectra based on the total ion current was performed using the same software. The FlexImaging 4.1 software was also used to create two-dimensional ion-density maps and for peak analyses.
Matrix screening was performed according to our previous study [25 (link)]. Briefly, 1 µL of each standard (10 μg/mL) was mixed with equal volumes of CHCA (10 mg/mL in 70% aqueous methanol), 9AA (10 mg/mL in 70% aqueous methanol), and DAN (10 mg/mL in 80% aqueous methanol) on an ITO-coated glass slide; then, MALDI-MSI analysis was performed.
To analyze the strawberry fruit sections, frozen sections were took out from a freezer and dried in a vacuum desiccator for 30 min. Six milliliters of a DAN solution (10 mg/mL in 80% aqueous methanol) was sprayed uniformly over the section using a 0.18 mm nozzle caliber airbrush (Mr. Airbrush Custom Double Action; Mr. Hobby, Tokyo, Japan), after which MALDI-MSI analysis was performed. To investigate the spatial distribution of the identified proanthocyanidins, three different strawberry fruits were analyzed. The mass spectra and ion images of the identified flavan-3-ols in the three different strawberry fruits showed similar patterns (Supplementary Figure S3). The mass spectrum and ion images of one of the three different strawberry fruits are presented as representative data in Figure 2C–I.
To compare the intensities of the identified flavan-3-ols between the skin and vascular bundles, three sections of the same strawberry fruit were analyzed and their tissue-specific intensities were obtained using the region-of-interest function of FlexImaging 4.1 software. These data are shown in Figure 5.

The following materials were obtained from Sigma–Aldrich (St. Louis, MO, United States): guanosine 3′,5′-cyclic monophosphate sodium salt (cGMP, HPLC, 99%), zirconyl chloride octahydrate (ZrOCl₂, reagent grade, 98%), and bovine serum albumin (BSA, ≥98%). Guanosine monophosphate (GMP), sildenafil citrate (HPLC), and vardenafil HCl trihydrate (HPLC) were purchased from Aladdin (Shanghai, China). Deionized water was purified by a Milli-Q purification system (Millipore, Bedford, MA, United States). HPLC grade acetonitrile, HPLC formic acid, HPLC methanol, and HisPur™ Ni-NTA Resin were purchased from Thermo Fisher Scientific (Vilnius, Lithuania). Dimethyl sulfoxide (DMSO≥99%), ultrafiltration spin columns (0.5 mL, 10 kDa MWCO, PES, Sartorius), and 96-well black opaque plates were purchased from Beyotime Biotechnology (Shanghai, China). Proanthocyanidins and nine other standards were purchased from Baoji Herbest Bio-Tech Co., Ltd, and 2′-O-(6-[tetramethylrhodaminyl]aminopentylcarbamoylethylcarbonyl)guanosine-3′,5′-cyclic monophosphate trifluoroacetate salt (96%) was purchased from AAT Bioquest, Inc. (TAMRA-R-cGMP, Sunnyvale, CA, United States). All other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai China).

In order to determine procyanidin contents in the extracts, the vanillin-H₂SO₄ methodology was conducted with some modifications [17 (link)]. Two hundred and fifty µL of the extract was reacted with 250 µL of 1% vanillin (w/v, dissolved in methanol), followed by 250 µL of the 25% sulfuric acid solution (v/v, dissolved in methanol) and incubated at a temperature of 30 °C for 15 min. Absorbance was read at 500 nm using a Synergy HTX Multi-Mode microplate reader (Biotek, Rochester, VT, USA). Total tannin contents were estimated using a calibration curve with catechin (A₅₀₀ = 1.9461 (catechin) −0.0007, R² = 0.9990) using nine concentrations of catechin (10–100 µg/mL). Condensed tannin contents were expressed as milligrams of catechin equivalents per gram of dry extract (mg CE/g DE).

GSPE was kindly provided by Les Dérivés Résiniques et Terpéniques (Dax, France). According to the manufacturer, the GSPE composition used in this study contained monomers (21.3%), dimers (17.4%), trimers (16.3%), tetramers (13.3%), and oligomers (5–13 units; 31.7%) of proanthocyanidins. The exact phenolic composition of GSPE was determined by HPLC-MS/MS (TOF 6210, Agilent) [21 (link)], according to what was described by Quiñones et al. [22 (link)], and can be found in Supplementary Table S1.