Flavones

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.

The 3 different T. comosus dried extracts (100 mg each one) were dissolved in 2 ml of the corresponding extraction solvents, namely water (TCI), ethanol 70% (TCT), and ethanol 50% (OpTC). After centrifugation (6,000 × g, 10 min, at 4°C), the supernatants were transferred to HPLC vials and supposed to untargeted phenolic profiling through high-resolution mass spectrometry (HRMS) using a Q-Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, United States) coupled to a Vanquish ultra-high-pressure liquid chromatography (UHPLC) pump, equipped with heated electrospray ionization (HESI)-II probe (Thermo Scientific, United States) (Babotă et al., 2022 (link); Nicolescu et al., 2022 (link)). The post-acquisition data filtering was accomplished using MS-DIAL software (version 4.70), while the annotation was achieved via spectral matching against FoodDB and Phenol-Explorer databases. Overall, the mass accuracy (setting a 5-ppm tolerance for m/z values), isotopic pattern, and spectral matching were used to calculate a total identification score, considering the most common HESI + adducts for the chromatographic conditions adopted, thus reaching a level 2 of confidence in annotation.
Semi-quantitative appreciation of each previously annotated phenolic class was made by analyzing representative pure standard compounds under the same conditions: ferulic acid (phenolic acids), quercetin (flavonols), catechin (flavanols), cyanidin (anthocyanins), luteolin (flavones and other flavonoids), resveratrol (stilbenes), and oleuropein (other remaining phenolics). A linear fitting (R² > 0.99) was built and used for quantification, results being expressed as mg equivalents (Eq.)/g lyophilized extract (n = 3).

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.

Flavone was purchased from Sigma-Aldrich chemicals
and used without
any purification. R2PI experiments have been performed employing a
molecular beam setup described previously.^{35 (link)} (see also Supporting Information SI1).
Briefly, flavone was heated up to 150 °C within a glass container
in order to obtain sufficient vapor pressure and expanded using neon
at 1.5 bar as a carrier gas through a General Valve pulsed nozzle
with a 0.5 mm orifice diameter, which was kept 5 °C higher than
the main body in order to avoid clogging. After being skimmed by a
2 mm skimmer, the molecular beam entered an ionization chamber where
ions or electrons were detected using either a reflection time-of-flight
(R.M. Jordan Co.) setup for mass-resolved ion detection or a custom-built
setup (R.M. Jordan Co.) for electron detection.
(1 + 1′)
R2PI excitation spectra were recorded using a frequency-doubled
Sirah Cobra-Stretch dye laser operating on DCM/Pyrromethene 597 pumped
by a Spectra-Physics Lab 190 Nd/YAG laser for excitation and a Neweks
PSX-501 ArF excimer laser (193 nm, 6.42 eV) for ionization. Typically,
pulse energies of 10–50 μJ and 1 mJ were used for excitation
and ionization, respectively. For recording ionization threshold spectra,
the same pulsed dye laser system has been used in combination with
another pulsed dye laser system consisting of a frequency-doubled
Sirah Precision Scan dye laser operating on DCM or Pyrromethene 597
and pumped by a Spectra Physics Lab 190 Nd/YAG laser. In these experiments,
typical pulse energies were employed of 1 mJ for the pump laser and
2–4 mJ for the probe laser.
In order to analyze the observed
electronic transitions and to
determine the ionization energy of flavone and its complexes, DFT
has been used to determine the equilibrium geometries and harmonic
force fields of flavone in the electronic ground state of the neutral
(S₀) and cation (D₀), while time-dependent DFT
(TD-DFT) was employed to optimize the geometry of the molecule in
the first three electronically excited singlet states of the neutral
and to determine the associated harmonic force fields. Such calculations
have been performed at both the B3LYP/TVZP and wB97XD/cc-pVDZ level.^{36 (link)−39 (link)} For comparison with the experimental results, the obtained equilibrium
geometries and force fields were used to obtain Franck–Condon
spectra at wB97XD/cc-pVDZ level for S_n ← S₀ transitions for which vibrational frequencies
were scaled using a scaling factor of 0.953.^{40 (link),41} All calculations have been performed with the Gaussian16, Rev.C.01
suite of programs.⁴²