2.1. Chemicals

Dimethylsulfoxide (DMSO), thiobarbituric acid (TBA), and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were provided by commercial suppliers: POCh (Gliwice, Poland), Chempur (Piekary Slaskie, Poland). ADP and collagen have been purchased from Chrono-Log, Havertown, PA, USA).

Leaves of six-month Paulownia trees were collected from a local plantation at Łęka, Lubelskie Voivodeship, Poland (21°54′ N, 51°27′ E). A voucher specimen (IUNG/PCIV112/2017/1) was deposited at the Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy, Poland.

Freshly picked Paulownia leaves were chopped, frozen, and lyophilized (Martin Christ Gamma 2-16 LSC, Germany). Next, the freeze-dried leaves were milled in a laboratory mill (ZM200, Retsch, Haan, Germany) and sieved through a 0.5 mm sieve. The extract of paulownia leaves was obtained through sequential extraction. The powdered plant material was extracted with 5% methanol (v/v) using ultrasonic bath, at room temperature for 30 min, and still macerated using a magnetic stirrer for 30 min at room temperature. The content was centrifuged at 4000 g for 10 min. The residue was then re-extracted with 30% methanol (v/v) under the same conditions as above, and centrifuged at 4000× g for 10 min. Finally, the residue was re-extracted with 70% methanol and centrifuged at 4000× g for 10 min. The supernatants were pooled and concentrated under reduced pressure and freeze-dried. The extraction yield was 41.7%.

The crude methanol extract was then purified in a stepwise manner by a range of chromatographic methods. First, the extract was applied to a preconditioned RP-C18 column (80 × 70 mm, 140 µm; Cosmosil C18-PREP; Nacalai Tesque, Kyoto, Japan), and polar constituents were then removed (1% methanol, v/v), while active metabolites were eluted with 80% methanol (v/v) to give fraction A. The yield of this stage was 44.1%.

Next, fraction A was further separated by flash chromatography on a reversed phase column (140 × 12 mm, 40 μm; Cosmosil C18-PREP; Nacalai Tesque, Kyoto, Japan) connected to a Gilson HPLC apparatus. A linear gradient of aqueous acetonitrile (2–30% v/v) containing 0.1% formic acid over 140 min, was used as a mobile phase at a flow rate of 8 mL min⁻¹ at ambient temperature. Afterward, fraction A afforded three subfractions B–D. Fraction B constituted 23.4%, fraction C 33.6%, and fraction D 36.3% of fraction A.

The composition of the extract and fractions was determined by UHPLC-DAD-ESI-MS. Samples were chromatographed using an Acquity UPLC system (Waters, Milford, MA, USA), coupled with an Acquity TQD (Waters) mass detector on an Acquity BEH C18 column (100 × 2.1 mm, 1.7 μm; Waters). The mobile phase was composed of mixtures of solvent A (0.1% formic acid (FA) in Milli-Q water) and solvent B (0.1% FA in acetonitrile). The constituents of the samples were identified on the basis of their MS and UV spectra (identification was supported by spectra and molecular formulas obtained during previously performed LC-HRMS/MS analysis (Q-TOF) of a similar extract from Paulownia Clone in Vitro 112 leaves; data not shown) and literature data. The following elution program was used for semiquantitation of phenolic compounds: 0–1 min: 5% B; 1–14.9 min: 5–40% B (a concave-shaped gradient); 15–17 min: 99% B; 17.10–20 min: 5% B. The column was maintained at 50 °C, the flow rate was 0.500 mL min⁻¹, and the injection volume was 2.5 µL. The mass spectrometer was operated in negative and positive ion scanning modes. The following settings were used in negative mode: capillary voltage 2.80 kV, cone voltage 55 V, source temperature 150 °C, desolvation temperature 450 °C, cone gas (N₂) flow 100 L h⁻¹, desolvation gas (N₂) flow 900 L h⁻¹. In positive ion mode, the capillary voltage was 3.10 kV and the cone voltage was 60 V. UV-DAD detection (λ = 330 nm) was used for semiquantitation of phenolic compounds. Levels of individual phenolics were determined using calibration curves of verbascoside (HWI Analytik, Rüelzheim, Germany) and rutin (PhytoLab, Vestenbergsgreuth, Germany). The content of the phenylethanoids, as well as other derivatives of phenolic acids and all minor and unidentified compounds were expressed as equivalent of verbascoside; the content of the major flavonoids was expressed as rutin equivalent.

Iridoids were semiquantified using the following method of elution: 0–2.5 min: 2% B; 2.50–10.0 min: 2–60% B; 10.10–12.10 min: 99% B; 12.20–16 min: 2% B. The column was maintained at 35 °C, the flow rate was 0.400 mL min⁻¹, and the injection volume was 2.5 µL. A negative ion SIM method was used: capillary voltage 2.80 kV, cone voltage 30 V, and with the other settings as for phenolics. Two ions were monitored, at m/z 407 (FA adduct of catalpol; much higher intensities were observed, as compared to deprotonated ion) and m/z 391 (FA adduct of aucubin/7-hydroxytomentoside); dwell times were set automatically. The iridoid content was determined on the basis of a calibration curve of catalpol (Sigma, St. Louis, MO, USA), and expressed as equivalent of catalpol.

The elution program for the semiquantitation of triterpenoids was: 0–0.5 min: 7% B; 0.5–11.90 min: 7–80% B (linear gradient); 12–13 min: 99% B; 13.10–15 min: 7% B. The column was maintained at 50 °C, the flow rate was 0.500 mL min⁻¹, and the injection volume was 2.5 µL. A negative ion SIM method was applied: capillary voltage 2.80 kV, cone voltage 80, and other settings as for phenolics. Four ions were monitored (in sequence, within set time ranges), at m/z 503, m/z 487, m/z 471, and m/z 455 (chosen on the basis of our preliminary qualitative analyses); the dwell time was 200 ms. The triterpenoid content was determined on the basis of a calibration curve of maslinic acid (Sigma), and expressed as equivalent of maslinic acid.

Stock solutions of the extract and fractions A–D of Paulownia Clone in Vitro 112 leaves were prepared with 50% (v/v) aq. soln. DMSO, a universal solvent for many different plant substances. The final concentration of DMSO in the tested samples was below 0.05% (v/v). In addition, as repeatedly confirmed by our earlier studies [5], the addition of a low concentration of DMSO to human plasma has no effect on oxidative stress or coagulation parameters [6].

Human blood and plasma were obtained from non-smoking men and women who were regular donors of a blood bank (RCKiK in Lodz, Poland) and a Medical Center (L. Rydygier Medical Center, in Lodz, Poland). None of the donors had taken any medication, any addictive substances or any antioxidant supplementation. All participants gave informed consent before being inclusion in the study. The study was performed according to the principles given in the Helsinki Declaration. Consent for the study was given by the University of Lodz Bioethical Commission (11/KBBN-UŁ/I/2019).

Plasma was isolated by differential centrifugation as described earlier by Walkowiak et al. [7]. To measure the hemostasis parameters, the plasma was incubated at 37 °C for 30 min with the extract and the four tested fractions (concentration range 1–50 µg/mL). To measure the oxidative stress parameters the plasma was incubated at 37 °C for 30 min with the extract and the four tested fractions (concentration range 1–50 µg/mL) with the addition of 4.7 mM H₂O₂/3.8 mM Fe₂SO₄/2.5 mM EDTA. The negative control was plasma not treated with H₂O₂/Fe, whereas the positive control was plasma treated with H₂O₂/Fe.

The protein concentration was calculated according to the procedure devised by Whitaker and Granum [8], on the basis of absorbance measurements at 280 nm (in tested samples).