Healthy female and male volunteers aged 18 to 30 years participated in this study. Both study protocols were approved by the ethical committee of the Comenius University's Faculty of Medicine, Bratislava, Slovak Republic, and all participants gave written informed consent.
Protocol of single intake of Pycnogenol
Eleven volunteers (five female and six male) participated in this study. After a 24 hour diet free of flavonoids (no vegetables, fruits and fruit juices or marmalades, tea, coffee, cocoa, wine and beer) a venous blood catheter was inserted into an antecubital vein and blood samples were drawn to obtain basal values (t = 0 h at 8:00 a.m.). Subsequently, the volunteers received a single dose of six 50 mg tablets (preparation of study medication by DKSH, Market intelligence, Tokyo, Japan) containing 300 mg standardized maritime pine bark extract (Pycnogenol®, Horphag Research Ltd., UK) with 200 mL tap water. At 8:15 a.m. the volunteers had a standardized breakfast (two white rolls (bread) and 0.3–0.5 L of milk (1.5 % fat)), at 12:15 lunch (1/4 of only salted and baked chicken with white bread) and at 6:15 p.m. dinner (100 g ham, 50 g cheese (eidam) and bread with butter or margarine). After each blood sampling mineral water was served. Blood samples were obtained at t = 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 14 h. Samples were centrifuged and plasma was aliquoted, shock frozen and stored at -80°C until further analysis.
Protocol of repeated intake of Pycnogenol
Five volunteers (four female and one male) participated in this study. After a 24 hour diet free of flavonoids blood samples were drawn to obtain basal values. Subsequently, the volunteers took four 50 mg tablets containing 200 mg Pycnogenol every morning for five days to reach steady state conditions of constituents and/or metabolites of Pycnogenol. It was assumed that steady state plasma concentrations were reached after 5 days.
Four hours after the last intake of Pycnogenol on day five a second blood sample was obtained from each volunteer. Again, a 24 hour period of a diet free of flavonoids preceded this blood sampling. Blood samples were centrifuged and plasma was aliquoted, shock frozen and stored at -80°C until further analysis.
Chemicals and reagents
A spray-dried extract from maritime pine bark (Pycnogenol®) was generously provided by Horphag Research Ltd. (Geneva, Switzerland). The metabolites M1 (δ-(3,4-Dihydroxy-phenyl)-γ-valerolactone) and M2 (δ-(3-Methoxy-4-hydroxy-phenyl)-γ-valerolactone) were synthesized by Groβe Düweler The monomeric compounds (+)-catechin, (-)-epicatechin, ferulic acid, gallic acid, 4-hydroxybenzoic acid, caffeic acid, protocatechuic acid, sinapic acid und (±)-taxifolin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from Sigma-Aldrich or Merck (Darmstadt, Germany), if not stated otherwise. All chemicals used were of highest purity available.
Krebs-Ringer-HEPES buffer (pH 7.4) consisted of 118 mM NaCl, 4.84 mM KCl, 1.2 mM KH2PO4, 2.43 mM MgSO4, 2.44 mM CaCl2 × 2 H2O and 10 mM HEPES.
Preparation of plasma samples
Plasma samples of 2.0 mL were prepared for analysis. For determination of total plasma concentration, to each sample 20 U β-glucuronidase (type H-3 from Helix pomatia; EC 220.127.116.11) and 20 U sulfatase (type H-1 from Helix pomatia; EC 18.104.22.168) were added in 50 mM sodium acetate buffer (pH 5.0) and incubated for 2 h at 37°C under gentle shaking. Samples for determination of free plasma concentrations were prepared without prior incubation with enzymes. Each 20 μL of p-hydroxybenzoic acid methylester (100 μg/mL; for HPLC UV detection) and hydrochinone (10 μg/mL; for HPLC electrochemical detection) were added as internal standards and samples were acidified with 100 μL 1 M-HCl. Samples were extracted twice with each 3 mL acetic acid methylester for 20 min, using a roller mixer, followed by centrifugation (20°C) 5 min. The organic phases of both extractions were separated, combined and evaporated to dryness under a gentle stream of nitrogen at 25°C. The resulting residue was reconstituted in 100 μL methanol and subjected to HPLC analysis.
Calibration curves of all known compounds of Pycnogenol were prepared by addition of the respective substances to 2.0 mL of Krebs-Ringer-HEPES buffer (pH 7.4). This was necessary because sufficient volumes of pooled blank plasma of blood donors who kept a 24 hour diet free of flavonoids was not available (total volume needed was about 800 mL for all experiments). Thus, buffer instead of plasma was used and treated analogously as plasma samples from volunteers. Since initial control experiments revealed that the incubation with β-glucuronidase and sulfatase did not produce interfering peaks in the HPLC chromatograms no enzymes were added to calibration curve samples.
Analysis of plasma samples by HPLC – UV/electrochemical dual detection
The HPLC system was a Waters HPLC (Milford, MA, USA) consisted of a 1525 binary pump, a 717plus autosampler, a 2487 dual wavelength absorbance detector set at the detection wavelength of 280 nm and an electrochemical detector CLC 100 (Chromsystems, Munich, Germany) set at an oxidation voltage of 0.5 V. The second detector was connected to the control system by a satellite interface (Waters). Data collection and integration were accomplished using Breeze™ software version 3.30. Analysis was performed on a Zorbax SB C8 column (150 × 4.6 mm I.D., 5 μm particle size, Agilent Technologies, Palo Alto, CA, USA).
Typically, 20 μL of sample were injected and separated at a flow rate of 1 mL/min. Isocratic elution was performed using water (containing 0.6 mM 1-octanesulfonic acid sodium salt, 0.27 mM ethylenediaminetetraacetic acid disodium salt, 0.04 M triethylamine; pH 2.95 adjusted with phosphoric acid) and acetonitrile (ACN, HPLC gradient quality, Fisher Scientific, Schwerte, Germany). Method A (for UV detection of ferulic acid, M2 (δ-(3-Methoxy-4-hydroxy-phenyl)-γ-valerolactone), U1, U2, U3, U4, U7, U8, and U9) used water/ACN at 85:15 (v/v). Method B (for electrochemical detection of catechin, caffeic acid, taxifolin, M1 (δ-(3,4-dihydroxy-phenyl)-γ-valerolactone), U1, U2, U4, U5, U6, U8, U9 and U10) used water/ACN at 88:12 (v/v).
The analytical method was validated according to ICH guidelines. The method fulfilled the quality criteria for selectivity, linearity, precision and accuracy. The calibration curves' working range was 0.5–20 ng/mL for caffeic acid, 1–50 ng/mL for M1, 10–100 ng/mL for catechin, taxifolin and ferulic acid, 50–200 ng/mL for M2. The recovery rates after extraction were 83.1 % (M2) to 98.0 % (taxifolin). The lower limits of quantitation were 0.5 ng/mL for caffeic acid, 1 ng/mL for M1, 10 ng/mL for catechin, taxifolin and ferulic acid, 50 ng/mL for M2.
Quantitation of pine bark extract constituents in tablets by HPLC – UV detection
Pycnogenol tablets prepared for this study (see above) were pounded and suspended in methanol to yield concentrations of 1 mg/mL, vortexted for 2 minutes, centrifuged and the supernatant collected. The procedure was repeated 4 times. The amount of taxifolin, ferulic acid, caffeic acid and catechin in the Pycnogenol tablets administered to the volunteers was calculated on the basis of calibration curves. They were constructed with five concentrations ranging from 5–20 μg/mL for taxifolin, from 1–10 μg/mL for ferulic acid, and from 0.1–8 mg/mL for caffeic acid. Analysis was performed on an Atlantis C-18d column (150 × 3.9 mm i.d, 3 μm particle size; Waters). The mobile phase consisted of water containing 1% acetic acid (A) and acetonitrile (B). The gradient elution started at 98 % eluent A increasing linearly to 84 % within 60 minutes and a flow rate of 1 mL/min. The UV detection wavelength was set to 280 nm.