Vitex agnus-castus dry extract BNO 1095 (Agnucaston^®) inhibits uterine hyper-contractions and inflammation in experimental models for primary dysmenorrhea

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).

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. [32], 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. [33] with modifications according to Flierl et al., [34] 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).

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) PGF_2α 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.

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).

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 [35]. 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 [36]. 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 [36] 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.

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.

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.

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 [A_{520 nm} (DPPH, 50 μM + extract)-A_{520 nm} (blank)] vs. [A_{520 nm} (DPPH, 50 μM + vehicle) - A_{520 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).