The dry extract of the Dioscorea villosa (DV) root was obtained from Shaanxi Meih Biochemics Co., Ltd. (Lot number MH-06DI-090609), China (imported by Galena Química e Farmacêutica Ltda company, São Paulo, SP, Brazil).
Diosgenin (≥93%) was purchased from Sigma-Aldrich (St. Louis, MO, USA), LC-grade acetonitrile (JT Baker, Philipsburg, PA, USA) was used for LC analysis and methanol (Tedia, Fairfield, OH, USA) was used for sample preparation. Deionized water was purified by a Milli-Q system (Millipore, São Paulo, SP, Brazil). All the solvents were filtered through nylon 0.45 μm membranes (MFS) and degassed by ultrasonic bath before use.
The LC analyses were performed on a Shimadzu liquid chromatographic (Kyoto, Japan) Prominence system, equipped with a degasser DGU-20A5 Model, SIL-10A autosampler, two high pressure pumps LC-20AT, a SPD-M10Avp photodiode array detector (DAD) and a CBM 20A interface. Data collection was performed using LC Solution software. Analysis was carried out on the analytical RP-18A Synergi® C18 column (5 μm, 250 × 4.6 mm i.d., Phenomenex, Torrance, CA, USA) with a RP-18A C18 guard column (4 μm, 4 × 3 mm, Phenomenex, Torrance, CA, USA) using a gradient elution at 1.0 mL/min with a mobile phase consisting of water (A) and acetonitrile (B). Initially, an exploratory gradient from 5 to 100% (B) in 60 min was run as suggested by Snyder et al. (1997) . After optimization, the gradient elution condition used was: 30-40% (B) in 5 min, 40-68% (B) in 18 min, 68-100% (B) in 21 min, maintained in 100% (B) for 10 min. The return to initial chromatographic conditions (100-30% B) was doing in 10 min, followed by column conditioning for 10 min. The photodiode array detector was set at 205 nm for acquiring the chromatograms.
Samples of extract and diosgenin were dissolved in methanol:water (1:1 v/v) and methanol, respectively, at concentrations of 1.0 mg/mL each, and submitted to filtration through a cellulose membrane (pore diameter of 0.45 μm). Identification was based on co-injection of the standard compound and comparisons of absorption spectra.
Male and female Wistar rats (150-200 g) and male Swiss mice (24-30 g) were obtained from the Tiradentes University (Sergipe, Brazil). The animals were kept at conventional temperature conditions (20 ± 1°C) and lodged in polypropylene cages with food and water available ad libitum and a 12 h light:dark cycle (light from 6 am to 6 pm). Experimental protocols were approved by the Ethical Committee in Animal Care of the Tiradentes University (CEPA/UNIT #110310R) and all procedures were carried out in accordance with Animal Care. The experiments were performed between 7 am and 5 pm.
Acetic acid-induced writhing
This study was performed according to Broadbear et al. (1994)  with alterations [6, 20–22]. Mice (n = 6, per group) were injected intraperitoneally (i.p.) with 0.85% acetic acid at a dose of 10 ml/ kg. One hour before the acetic acid injection, the mice were pretreated orally (per os or p.o.) with DV (100, 200 and 400 mg/kg, per os or p.o.), morphine (MOR, 3 mg/kg, i.p.) or vehicle (0.9% saline with two drops of tween 80, the solvent for DV, Control group). Subsequently, writhing was counted for 15 min after a latency period of 5 min.
The procedure described by Hunskaar and Hole (1987)  was used with slight modifications [6, 20–22]. Nociception was induced by injecting 20 μl of 1% formalin in distilled water in the subplantar region of the right hind paw. Mice (n = 6 per group) were given DV (100, 200 and 400 mg/kg) and vehicle (saline + two drops of tween 80) (per os or p.o.) 1,0 h prior to formalin injection. The acetylsalicylic acid (ASA, 200 mg/kg) and morphine (MOR, 3 mg/kg) were administered i.p. 0.5 hr before formalin injection. These mice were individually placed in a transparent acrylic glass cage (25 cm × 15 cm × 15 cm) observation chamber. The amount of time spent licking the injected paw was indicative of pain. After injection of formalin, licking time was recorded from 0-5 min (first phase) and 15–30 min (second phase), representing neurogenic and inflammatory pain responses, respectively.
Leukocyte migration to the peritoneal cavity: Leukocyte migration was induced by injection of carrageenan (500 μg/cavity, i.p., 500 μL) into the peritoneal cavity of mice 1 h after the administration of DV (100, 200 and 400 mg/kg, per os or p.o.), vehicle (saline 0.9% with two drops of tween 80) or dexamethasone (2 mg/kg, s.c., n = 6) using a modification of the technique previously described by Bastos et al. (2007) . Mice were euthanized by cervical dislocation 4 h after carrageenan injection. Shortly afterward, phosphate buffered saline (PBS) containing EDTA (1 mM, i.p., 10 mL) was injected. Fluid was collected immediately using a brief massage and centrifuged (2000 rpm, 5 min) at room temperature. The supernatant was removed and 1 mL of PBS was introduced to the precipitate. A 10 μL aliquot from this suspension was dissolved in 200 μL of Turk’s solution and the total number of cells was counted in a Neubauer chamber, under an optical microscope. The results were expressed as leukocyte number per mL. The percentage of leukocyte inhibition was calculated as (1 – T/C) × 100, where T represents the leukocyte count of the treated group and C represents the leukocyte count of the control group .
The following methods were performed according to the Organization for Economic Cooperation and Development test guidelines with slight modifications (OECD). The number of animals of this study was defined in compliance with Brazilian regulations for in vivo toxicological assays .
Subchronic toxicity study
Toxicological assays were performed with 20 male and 20 female rats distributed into 4 groups of 10 animals (experimental and control groups of male and female rats). For the subchronic study, the experimental groups received daily doses of DV (1 g/kg, per os or p.o.) dispersed in water (vehicle) over a period of 30 days. The product was stirred before being administered. The control groups received water only.
At the end of the period of administration, the animals were fasted for 12 h and then anesthetized with Ketamine (70 mg/kg) and Xylazine (12 mg/kg). Blood (3-5 ml) was collected by cardiac puncture. Subsequently, the animals were euthanized and a detailed study of the gross and microscopic features of the internal organs was performed, as well as haematological and biochemical analyses of the blood. The shape, size, texture, consistency and colour of the internal organs (lungs, heart, liver, stomach, pancreas, uterus/ovaries, brain and kidneys) were macroscopically observed for any sign of gross changes. The organs were then collected, weighed and preserved in 10% phosphate buffered formalin solution for subsequent histological procedures [4, 22, 26].
Acute toxicity study
Toxicological assays were performed with 12 male and 12 female rats, which were distributed into 4 groups of 6 animals (experimental and control groups of male and female rats). The animals received a maximum dose of 5 g/kg by oral administration (gavage). The experimental groups received DV (5 g/kg) dispersed in water (vehicle), whereas the control groups received water only. The product was stirred before being administered .
Specific behaviours (sedation, reduced ambulation, response to touch, analgesia and defecation) were observed and graded for 1, 2, 3 and 4 h after gavage. Finally, the animals were monitored daily for 14 days to observe the absence of lethality. At the end of the period of administration, the animals were fasted for 12 h, and then anesthetized with Ketamine (70 mg/kg) and Xylazine (12 mg/kg). Blood was collected by cardiac puncture. Subsequently, the animals were sacrificed and a detailed study of the gross and microscopic features of the internal organs was performed, as well as haematological and biochemical analyses of the blood. The position, shape, size, texture, consistency, and colour of the internal organs (lungs, heart, liver, stomach, pancreas, uterus/ovaries, brain and kidneys) were observed macroscopically, looking for any signs of gross changes. The organs were then collected, weighed and preserved in 10% phosphate buffered formalin solution for subsequent histological procedures [4, 22, 26].
Blood samples were collected into EDTA tubes. Measurements of erythrocytes, haemoglobin, haematocrit, leukocytes, neutrophils, lymphocytes, eosinophils, monocytes, basophils, VCM, HCM, CHCM, and platelets were determined using Sysmex Xs1000i automated equipment - Japan, according to the method described by Morris and Davey (1999) .
Serum was separated from non-heparinised blood and assayed for serum urea, creatinine, total protein, globulin, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBil), direct bilirubin (D.Bil), indirect bilirubin (Bil.IND), alkaline phosphatase (ALP), sodium (Na+), potassium (K+), cholesterol (CHOL), triglycerides (TRIG), glucose (GLUC) and uric acid. Biochemical parameters were determined by ARCHITECT C8000 automated equipment (Abbott, USA).
Formalin-fixed samples of the internal organs were dehydrated, diaphonized and embedded in paraffin according to protocols for routine histological procedures. Five micrometre thick sections of the paraffin-embedded tissues were obtained and stained with haematoxylin-eosin. Morphological analysis of the histological sections was performed by light microscopy following a closed numerical protocol so that the pathologist was not aware of what group was being evaluated until the end of the experiment.
Student’s t-test (GraphPad Prism 5.01 computer program) was employed for statistical analysis of the results. The data obtained from the antinociceptive study were evaluated by one-way analysis of variance ANOVA followed by Tukey’s tests. The percentage inhibition by antinociceptive agent was determined using the following equation: (Eq. 1) % Inhibition = 100 × (control-experiment)/control. Statistical evaluation of the consumption of food and water was carried out using the differentiation factor (f1) and similarity factor (f2) methods . All values were expressed as the back transformed mean ± S.D. or SEM. Differences below the probability level of 0.05 were considered statistically significant.