C18 μ bondapak column

Optical rotations were measured on an Atago AP-300 digital polarimeter with a 1 dm microcell and a sodium lamp (589 nm). NMR data were recorded on a Bruker DRX-600 spectrometer at 300 K (Bruker BioSpinGmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI Cryoprobe at 300 K. All 2D NMR spectra were acquired from MeOH-d4 (99.95%, Sigma-Aldrich, Milano, Italy), and standard pulse-sequences and phase cycling were used for the DQF-COSY, HSQC, and HMBC spectra. The NMR data were processed using TOPSPIN 3.2 software (Bruker Biospin, Rheinstetten, Germany). HRESIMS data were obtained on a Q Exactive Plus mass spectrometer, Orbitrap-based FT-MS system, equipped with an ESI source (Thermo Fischer Scientific Inc., Bremen, Germany). Column chromatography was performed over Sephadex LH-20 (Pharmacia). RP-HPLC separations were carried out using a Shimadzu LC-8A series pumping-system equipped with a Shimadzu RID-10A refractive index detector and Shimadzu injector (Shimadzu Corporation, Kyoto, Japan) on a C 18 μ-Bondapak column (30 mm × 7.8 mm, 10 µm, Waters-Milford, Milford, MA, USA). TLC separations were conducted using silica gel 60 F 254 (0.20 mm thickness) plates (Merck, Germany) and Ce(SO₄)₂/H₂SO₄ as spray reagent (Sigma-Aldrich, Milano, Italy).

The HPLC-grade ethanol, acetonitrile, and formic acid were purchased from VWR International srl (Milano, Italy). The HPLC-grade water (18 mW) was prepared by a Milli-Q purification system (Millipore Co., Bedford, MA, USA). The quercetin reference standard was purchased from Sigma-Aldrich S.p.a. (Milano, Italy). The alpinetin, caffeic acid prenyl ester, luteolin, pinobanksin 5-methyl ether, and pinobanksin 3-O-acetate used as reference standards were isolated from the propolis extract by size exclusion column chromatography (Sephadex LH-20, 5 × 100 cm, flow rate 1.0 mL/min) eluting with methanol, followed by RP-HPLC using a C₁₈ μ-Bondapak column—30 cm × 7.8 mm, 10 μm (Waters, Milano, Italy)—at a flow rate of 2.0 mL/min using a mixture of MeOH−H₂O as the eluent.

Electrospray ionization mass spectrometry (ESI-MS) experiments were performed on an Applied Biosystem API 2000 triple-quadrupole mass spectrometer. Optical rotations were determined on a Jasko P-2000 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance NEO 400 spectrometer equipped with an RT-DR-BF/1H-5 mm-OZ SmartProbe (¹H at 400 MHz and ¹³C at 100 MHz), δ (ppm), J in Hz, using a solvent signal for the calibration (¹³CD₃OD at δ_C 49.0 and residual CD₂HOD at δ_H = 3.31). The heteronuclear single quantum coherence (HSQC) spectra were optimized for an average ¹J_CH of 140 Hz; the gradient-enhanced heteronuclear multiple bond correlation (HMBC) experiments were optimized for a ³J_CH of 8 Hz. Droplet counter-current chromatography (DCCC) fractionation was performed on DCC-A apparatus (Tokyo Rikakikai Co., Tokyo, Japan) equipped with 250 glass-columns. High-performance liquid chromatography (HPLC) was performed using a Waters 510 pump equipped with a Rheodyne 7125 injector and a Waters 401 differential refractometer as the detector on a C₁₈ μ-Bondapak column (30 cm × 3.9 mm i.d) (Waters, Milford, MA, USA).