Materials and reagents
The VAC dry extract BNO 1095 (DER 7–11:1) is commercially available as Agnucaston® and was provided by Bionorica SE, Neumarkt, Germany. For in vitro studies, solubilization of the test item was obtained by resuspension in 50 % ethanol (v/v) at a concentration of 40 mg/ml and homogenized by 5 min of vortexing, followed by a 30 min incubation at room temperature (RT) in an ultrasonic bath. The suspension was centrifuged at 3000 g for 10 min at RT and the supernatant was used immediately for the experiments.
Unless otherwise specified, all reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany).
In vivo measurements of uterine contraction, pain, and locomotor sedation in a rat dysmenorrhea model
Nulliparous, non-pregnant female Sprague Dawley rats were purchased at the age of 12 to 14 weeks from Charles River (Sulzfeld, Germany) and housed under specific pathogen-free conditions, with sterilized water and food given ad libitum. 12 days prior to the start of the experiments, the animals were randomly allocated to approximately-equal control (saline), treatment (BNO 1095), or comparator (carprofen) groups (n = 12–14 per group) and acclimatized in groups of 4 animals to their new cages. On Day 1 to Day 12, all animals were i.p. injected once-daily with 10 mg/kg body weight estradiol-benzoate. On Day 6 to Day 12, animals assigned to the treatment groups were dosed once-daily with an oral suspension (in sterile water) of BNO 1095 via oral gavage at one of three doses: 2.1 mg/kg body weight (comparable to five times the recommended human equivalent dose, 5× HED), 10.3 mg/kg body weight (25× HED) or 20.7 mg/kg body weight (50× HED) in a volume of 10 ml/kg, while animals of the control group received an equal amount of water. On Day 12, animals from the control, treatment and comparator groups received the following (all volumes equal between groups, respectively): water p.o. and a subcutaneous (s.c) injection of saline; BNO 1095 extract p.o. 3 hours after the last application of estradiol-benzoate and an s.c. injection of saline, or p.o. water and a s.c. injection of 5 mg/kg body weight of the NSAID carprofen (Pfizer, Berlin, Germany).
On Day 12, 1 hour after the vehicle or carprofen s.c. injection, oxytocin (10 I.U./ml, Hexal AG, Holzkirchen, Germany) was applied i.p. at a dose of 2,000 mI.U./kg (3.33 μg/kg) body weight to the animals of all groups to induce uterine contractions. Latency time and number of abdominal convulsions, indicated by writhings, after administration of oxytocin were monitored to estimate the degree of myometrial stimulation. In addition, the pain reaction was quantified according to the grimace scale as described by Sotocinal et al. , a method chosen due to its ease of automation and its ability to differentiate between spontaneous and evoked (e.g., in response to abdominal prodding) pain. Briefly, changes in orbital tightening, nose/cheek flattening, ear change and whisker change were quantified in a scale of 0 (normal), 1 (moderate) or 2 (obvious). In addition to measurements of uterine contraction and pain, a beam walking test was performed, as described by Goldstein et al.  with modifications according to Flierl et al.,  to analyze gait and latency while crossing a wooden bar to measure any potential influence of BNO 1095 or carprofen administration on motor coordination (a subjective scale from 0 to 16, where a score of 7 = normal and 3 = motor disorder) or sedation (measured by the amount of time between placing the animal on the beam and moving forward to cross the bar).
Rat experiments were conducted in accordance with the regulations for the care and use of laboratory animals and approved by the institutional animal ethics committee: a) EU: Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the Protection of Animals Used for Scientific Purposes; b) Directive CETS No. 123 (ETS 123): European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes Council of Europe (1986), Appendix A (2006).
In vitro organ bath contraction experiments using excised rat uterine tissue
For the analysis of anti-convulsive activity using isolated rat uterine strips, female nulliparous Sprague–Dawley rats with body weights in the range of 250–275 g at delivery were obtained from Charles River Laboratories (Saint Germain sur l’Arbresle, France). Rats were treated with diethylstilbestrol (0.25 mg/kg, i.p.) 18 h prior to experiment (to synchronize hormonal conditions). Diethylstilbestrol was dissolved at the concentration of 0.5 mg/ml in corn oil (batch MKBH4894V, Sigma-Aldrich) and a volume of 0.5 ml/kg was injected. On the day of the experiment, rats were sacrificed by CO2 asphyxiation. The uterus was excised, cleaned from connecting tissues and longitudinal strips of uterus were rapidly dissected from each horn (2 strips per horn) and placed in 5 ml organ baths containing oxygenated Krebs solution of the following composition (in mM): NaCl 114, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11.7 (pH 7.4, aerated with 95 % O2 and 5 % CO2 at 37 °C). Uterine tissues were allowed to equilibrate at 1.0 g resting tension for at least 60 min, during which time the Krebs solution was replaced every 15 min and the tension readjusted if necessary. Following this equilibration period, myometrial contractions were induced by adding 0.5 nM (0.5 ng/ml) oxytocin, 1 μM (354.5 ng/ml) PGF2α or 50 nM (54.2 ng/ml) vasopressin to the baths. After contractions stabilized in amplitude and frequency, cumulative concentration-response curves to 10–1000 μg/ml BNO 1095 (dry extract solubilized as described above), positive control ritodrine (Sigma-Aldrich, Saint-Quentin Fallavier, France) and the vehicle control (aqueous ethanol solution) were recorded. Ethanol concentration in organ baths were at most 0.5 % for the maximal drug (or extract) concentrations tested. Data are presented as percent of control (relaxation before addition of test substance or vehicle).
All experiments using rat uterine tissue were performed in accordance with French legislation concerning the protection of laboratory animals and in accordance with a currently valid license for experiments on vertebrate animals, issued by the French Ministry for Agriculture and Fisheries.
In vitro organ bath contraction experiments using excised human uterine tissue
Samples of human uterus, obtained with informed consent, were taken from non-pregnant pre-menopausal donors. All uterine strips (approximately 15 mm length), were suspended in 25 ml organ baths containing physiological salt solution (PSS, 119.0 mM NaCl, 4.7 mM KCl, 1.2 mM MgS04, 24.9 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2 and 11.1 mM glucose), aerated with 95 % O2 and 5 % CO2 and maintained at approximately 37 °C. One side of the tissue strip was attached to an isometric transducer that records changes in tension. The uterine strips were allowed to equilibrate for at least 30 min. Uterine strips were then normalized to a standard passive tension (20 mN, approximately 2 g) to reduce signal variability prior to pharmacological intervention. The strips were then allowed to equilibrate for at least 1 hour, with washes with PSS every 15 min, to allow the development of spontaneous contractions. Any uterine strips failing to produce spontaneous contractions after tension was applied in the organ bath were rejected.
0.2 μM (0.2 μg/ml) oxytocin was added to increase the size and frequency of the spontaneous contractions. After contractions stabilized in amplitude and frequency, cumulative concentration-response curves to 10–400 μg/ml BNO 1095, the dihydropyridine calcium-channel blocker isradipine (positive control) and the vehicle control (aqueous ethanol solution) were recorded. The concentration of ethanol in organ baths was within the range of 0.013 to 0.5 % for the maximal concentration tested. Data are presented as percent of control (relaxation before addition of test substance or vehicle).
Inhibition of purified 5-lipoxygenase (5-LO) and cellular leukotriene biosynthesis in isolated human monocytes
In the cell-free (purified enzyme) assay, human recombinant 5-lipoxygenase (5-LO) was expressed in E. coli Bl21 (DE3) cells that were transformed with pT3–5LO, and purified by ATP-agarose column . For determination of the enzymatic activity, the purified enzyme was added to 1 ml of a 5-LO assay mix (PBS, pH 7.4, 1 mM EDTA, 1 mM ATP). After incubation for 10 min at 4 °C with vehicle (0.5 % ethanol) or BNO 1095, samples were pre-warmed for 30 s at 37 °C in the presence of 2 mM CaCl2 followed by addition of 20 μM (6.1 μg/ml) arachidonic acid. The reaction was stopped after 10 min at 37 °C by addition of 1 ml ice-cold methanol, and 200 ng PGB1 were added. Formed metabolites were extracted and analyzed by HPLC as previously described . 5-LO products include LTB4 all-trans isomers and 5-H(p)ETE. Results are reported as percentage of vehicle control.
Analysis of 5-LO inhibition in a cell-based assay was performed using human monocytes isolated from freshly withdrawn peripheral blood samples. Blood samples were obtained from consenting healthy donors (Institute of Transfusion Medicine, University Hospital Jena, Germany) who had declared that they had not taken any anti-inflammatory drugs within 10 days of providing the sample. The venous blood was centrifuged at 4,000 g for 20 min at 20 °C, PBMC were isolated by dextran sedimentation and centrifugation on Nycoprep cushions, and monocytes were collected by adherence as described above. Monocytes were finally resuspended in PGC buffer at the cell density of 2 × 106 cells/ml and incubated for 15 min at 37 °C with BNO 1095, vehicle or zileuton (3 μM or 0.71 μg/ml). Then, cells were stimulated at 37 °C with the Ca2+-ionophore A23187 (5 μM or 2.6 μg/ml). After 10 min, the reaction was stopped on ice and samples were centrifuged at 500 g for 10 min at 4 °C. Supernatants were collected and formed LTC4 was analyzed by ELISA (Enzo Life Sciences GmbH, Lörrach, Germany) according to manufacturer’s instructions. For analysis of LTB4, all trans LTB4 and 5-H(p)ETE using HPLC, 750 μL supernatant were mixed with 750 μL methanol and 22.5 μL of 1 N HCl, 150 ng PGB1, and 375 μL of PBS were added. Formed 5-LO products were extracted, analyzed by HPLC as described elsewhere  and presented as sum of all 5-LO products (i.e., LTB4, its all-trans isomers, and 5-H(p)ETE). Results are reported as normalized percentages vs. vehicle control (=100 %). Data represent three independent experiments, each collecting single data points.
Analysis of cytokine release from cultured human PBMCs
Cryopreserved PBMCs (Eurofins Panlabs Inc., Bothwell, WA, USA) were thawed and seeded into 96 well plates in culture media (RPMI 1640, 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-alanyl-L-glutamine) at a cell density of 5 × 104 cells/well. After incubation for 1 h at 37 °C, 10–300 μg/ml BNO 1095, vehicle control (0.3 % ethanol final concentration) or the positive control 100 nM (39.25 ng/ml) dexamethasone was added and incubated again for 1 h at 37 °C. After addition of 50 ng/ml LPS, PBMCs were incubated for 24 h at 37 °C. Collected supernatants were analyzed for cytokines using ProcartaPlex multiplex bead arrays for human IL-1β, IL-6, IL-8, MIP-1α and TNFα (Affymetrix, Santa Clara, CA, USA). Data represent two independent experiments in duplicate measurements.
Reactive oxygen species (ROS) release assay from isolated human macrophages
Human monocytes were isolated as described above. To obtain macrophages, the freshly isolated monocytes were incubated for 6 days with 20 ng/ml M-CSF at 37 °C in a 5 % CO2 atmosphere. For analysis of ROS formation, cells (human macrophages or neutrophils) were preincubated with the peroxide-sensitive fluorescence dye 2′,7′-dichlorofluorescein-diacetate (1 μg/ml) for 10 min at 37 °C. Then, 5–75 μg/ml (macrophages) or 1 – 100 μg/ml (neurophils) of the test compound or vehicle (containing 0.5 % ethanol) was added and after 10 min, cells were stimulated by 100 nM (61.7 ng/ml) (macrophages) or 1.62 μM (1 μg/ml) (neutrophils) phorbol 12-myristate 13-acetate (PMA). The fluorescence emission at 530 nm was measured after excitation at 485 nm in a thermally controlled (37 °C) 96-or 24-well plate in a spectrofluorometer. Diphenylene iodonium (DPI, 5 μM or 1.4 μg/ml) was used as control inhibitor. Data represent three independent experiments, each collecting single data points.
DPPH colorimetric radical scavenging (ROS-reduction) assay
The reaction of DPPH (2,2-diphenyl-1-picrylhydrazyl) with an antioxidant or reducing compound produces the corresponding hydrazine DPPH2, which is assessed by monitoring the photometric color change from purple to yellow. Briefly, 0.1–300 μg/ml BNO 1095 was added to 100 μl ethanol (blank controls) or to 100 μl of a solution of the stable free radical in ethanol buffered with acetate to pH 5.5 (50 μM, corresponding to 19.7 μg/ml), in a 96-well plate. Ascorbic acid (100 μL of 50 μM (8.8 μg/ml) solution ascorbic acid in ethanol, i.e. 5 nmol ascorbic acid) was used as reference compound. The absorbance was read at 520 nm after 30 min incubation under gentle shaking in the dark. Radical scavenging activity is expressed as % absorption of the subtracted blank control [A520 nm (DPPH, 50 μM + extract)-A520 nm (blank)] vs. [A520 nm (DPPH, 50 μM + vehicle) - A520 nm (blank)]. Data represent three independent experiments, each collecting single data points.
Unless otherwise stated, all values are presented as mean ± standard error of the mean (SEM) of at least two independent experiments analysed in triplicate measurements. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test and a p-value criteria of ≤ 0.05 was used to assess statistical significance. For in vitro experiments, IC50 values were calculated by non-linear regression using the equation (Y = 100/(1 + 10^((LogEC50-X)*HillSlope)), X = log of dose or concentration, Y = normalized response (0–100 %)). Curve fitting, IC50 value calculation, and statistical analyses were performed with GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego, CA).