Materials
Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), antibiotics, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), indomethacin (IND), diclofenac sodium (DICLO), polyvinylpyrrolidone (PVP), Griess reagent (GR), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Aldrich (St. Louis, MO, USA). The murine colorectal cancer cell line CT26 and the murine J774A.1 macrophages were purchased from ATCC (Manassas, VA, USA), whereas cisplatin (CDDP) was acquired from PISA Laboratory (PISA™ Pharmaceutics, Guadalajara, Mexico).
Plant material
Salvia ballotiflora Benth was collected in Las Comadres, Municipality of Guadalcazar within the state of San Luis Potosi, Mexico in August 2017. A voucher specimen was preserved in the Isidro Palacios Herbarium of the Autonomous University of San Luis Potosí (SLPM43014). A taxonomist (José García Pérez) identified the plant material. S. ballotiflora is not an endangered species, and for this reason, a collection permit is not required by the Secretariat of Ecology and Environmental Management of San Luis Potosi (SEGAM-SLP).
Extract preparation
The dried and ground aerial parts (500 g) of S. ballotiflora were extracted with 3 L of chloroform at the boiling point for 4 h. The mixture was filtered and the solvent was evaporated to dryness under reduced pressure. The solid material was then washed with warm hexane (ECL) and a 5.07% yield was obtained.
Isolation of DEOX, ICT, and DAM
The diterpenes were isolated from ECL by open column chromatography using a mixture of hexane-ethyl acetate as described earlier [6].
Analysis by GC-MS
A mixture of 5 mg ECL or 1 mg DEOX, 1 mL of isooctane and 100 μL of bis(trimethylsilyl) trifluoroacetamide with 10% of trimethylsilyl chloride was heated at 100 °C for 30 min.
The ECL analysis was performed with a gas chromatography/mass spectrometer (Agilent Technology, model 6890 N) connected to a mass detector model 5973 with a DB-5HT capillary column. The injector temperature was set at 200 °C. The initial oven temperature (250 °C) was held for 2 min, then the temperature was increased at a rate of 15 °C/min up to 325 °C and maintained for 3 min. The splitless injection was performed at a ratio of 1:100 and the injector temperature was 325 °C. The spectrum was determined at 70 eV. DEOX was identified in ECL and extrapolation of this diterpene was performed on the standard curve (500–31 ppm). We use DEOX for the extract standardization because this diterpene is the most abundant of the three diterpenes in ECL [6].
Animals
One hundred fifty-two Male CD1 and forty-eight Balb/c mice (20–25 g, 6–7 weeks of age) from the animal facility of the Autonomous Metropolitan-Xochimilco University, were used. The experimental protocol (140) was approved by the Research Bioethics Committee of the Autonomous Metropolitan-Xochimilco University. All experiments were performed in compliance with the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999). Mice were maintained with free access to food (Lab Diet 5001) and water ad libitum. Animals were housed at 24 °C ± 1 °C with 12 h light/dark cycles. The mice were acclimatized in the laboratory for 2 weeks prior to the experiments, which began at 8:00 am. After experiments, conscious animals were sacrificed in a CO2 chamber.
Acute toxicity test
The Lorke methodology [11] was followed to evaluate the acute toxicity of ECL. Briefly, ECL in PVP (1:4) from 400 to 5000 mg/kg, was orally administered to mice as a single dose (n = 10 per test group) and compared with the negative control group. Mice were monitored, under open-field conditions every 24 h, during 72 h. At the end of 72 h, the number of animal deaths was recorded and the rest were sacrificed conscious in a CO2 chamber. Then, biopsies were obtained to identify possible damage to the stomach, spleen, liver, and kidneys.
Acute anti-inflammatory activity: TPA-induced mouse ear edema
A TPA (2.5 μg) solution in acetone (20 μL) was topically administered to both, the inner and outer surfaces of the right ear of mice, and acetone (vehicle) was administered to both surfaces of the left ear of mice from the ECL, positive (IND), and negative study groups (n = 8 per group). After 30 min, 2.0 mg/ear ECL or IND dissolved in acetone, was topically administered to the right ear, whereas the vehicle was applied to the left ear. After 6 h, conscious animals were sacrificed in a CO2 chamber, and 6 mm portions of the central sections of both ears were obtained [12]. The weight of these tissue portions were recorded and the percent inhibition of ear edema was determined as follows:
$$ \%\mathrm{Inhibition}=\left(\frac{\left({W}_t-{W}_{nt}\right)\mathrm{control}-\left({W}_t-{W}_{nt}\right)\mathrm{treated}}{\left({W}_t-{W}_{nt}\right)\mathrm{control}}\right)100 $$
Wt: weight of treated ear, Wnt: weight of non-treated ear.
Chronic anti-inflammatory activity
ECL (12.5, 25, 50, or 100 mg/Kg), 8 mg/kg IND (positive control) or vehicle were orally administered to each group of 8 mice. Samples were dissolved in saline solution with PVP (1:4). After 30 min, a TPA (2.5 μg) solution in acetone (20 μL) was topically applied to the inner and outer surfaces of the right ear of mice, whereas acetone alone was applied to both surfaces of the left ear. The topical application of TPA was carried out in the 1st, 3rd, 5th, 7th, and 9th day of the experiment. On the last day, conscious animals were sacrificed in a CO2 chamber, and the weight of 6 mm plugs from the central portion of both ears was recorded. The percent inhibition of edema was determined as described above [13].
Cell culture conditions
J774A.1 murine macrophages and CT26 murine carcinoma cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 pg/mL streptomycin). Cell cultures were grown at 37 °C and 5% CO2.
Cell viability assay
The viability of the macrophages treated with ECL, DEOX, ICT, or DAM was determined. In a 96-well plate, 5000 cells/well were plated and the extract and the three diterpenes were applied at concentrations of 1 to 100 μg/mL for 24 h. Then, 10 μL of MTT (5 mg/mL) solution was placed in each well; 4 h later, the medium was removed and the formazan crystals formed in each well were dissolved with 100 μL of DMSO [14]. The absorbance was read at 540 nm in a BioRad spectrophotometer. The IC50 was calculated by linear regression.
Determination of Nitric Oxide (NO) and cytokine levels
In 6 well-plates, 5 × 105 macrophages J774A.1 were seeded per well. Macrophages were stimulated with 5 μg/mL LPS. After 2 h, cells were treated with ECL (10 μg/mL), DAM and ICT (25 μg/mL), DEOX (20 μg/mL), or reference drug (IND 17.1 μg/mL), and (culture medium with vehicle only). After 24 h, supernatants were collected for the quantification of NO and levels of IL-1β, IL-6, IL-10, and TNF-α, using commercial enzyme-linked immunosorbent assays (ELISA), following the manufacturer’s instructions (PROMEGA). The absorbance was recorded at 405 nm. For the quantitation of NO production, 100 μL of supernatant was mixed with 100 μL of Griess reagent in a 96-well plate. The reaction mixture was incubated at 37 °C for 30 min and the absorbance was measured at 540 nm with an ELISA reader (Bio-Rad). A 100% nitric oxide production was considered for the LPS group [15].
Membrane stabilization property
Blood samples were collected, maintaining aseptic conditions, from healthy human volunteers that did not consume non-steroidal anti-inflammatory drugs (NSAIDs), steroids, or oral contraceptives for 2 weeks prior to the experiment. Blood samples were washed with an equal volume of Alsever’s solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid, and 0.42% sodium chloride in water), and centrifuged at 3000 rpm for 10 min; then packed cells were washed three times with Alsever’s solution.
A 5% erythrocyte suspension was mixed with different concentrations (25–400 μg/mL) of ECL or DICLO prepared in PBS buffer. Distilled water and PBS buffer were used as negative controls. All samples were incubated at 37 °C for 30 min and centrifuged at 3500 rpm for 5 min [16]. The optical density was read at 450 nm. The percent protection was calculated with the following equation:
$$ \% Protection=100-\left(\frac{optical\ density\ of\ Test\ sample}{Optical\ density\ of\ Control}\ X\ 100\right) $$
In vitro anti-arthritic activity
The bovine serum protein (BSP) denaturation method [17] was used to evaluate in vitro anti-arthritic activity. A BSP solution (5% w/v aqueous solution) was prepared as follows: 0.45 mL of BSP solution and 0.05 mL of the drug treatment. The ECL concentrations were 25, 50, 100, 200, 500, 750, and 1000 μg/mL and the DICLO treatment included the same concentrations as ECL, and distilled water was used as a control. Samples were heated at 37 °C for 20 min and the temperature was increased to 57 °C for 3 min. After cooling, 2.5 mL PBS was added to the solutions. The absorbance was measured at 560 nm. The control represents 100% protein denaturation. The percentage of protein denaturation inhibition was estimated as follows:
$$ \%\mathrm{of}\ \mathrm{protein}\ \mathrm{denaturation}\ \mathrm{inhibition}=100-\left(\frac{\mathrm{Optical}\ \mathrm{density}\ \mathrm{of}\ \mathrm{Test}-\mathrm{Optical}\ \mathrm{density}\ \mathrm{of}\ \mathrm{Control}}{\mathrm{Optical}\ \mathrm{density}\ \mathrm{of}\ \mathrm{Test}}\ \mathrm{x}\ 100\right) $$
Apoptosis assay
CT26 cells were seeded in 100 mm2 culture plates (2 × 106 cells/plate). After 24 h, cells were incubated with CDDP (1.4 μg/mL), ECL (9.5 μg/mL), or DMSO at a final concentration of 0.01% (vehicle group). After treatment (48 h or 72 h), cells were detached with EDTA solution in 1X PBS, washed and centrifuged at 4500 rpm for 5 min. Cells were stained with the Annexin V/propidium iodide kit (Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry. Invitrogen) and read in a FacScan flow cytometer (BD Bioscience, USA). Data from 20,000 acquired events were analyzed using the Weasel cytometry analysis software v.2.6.1.
Antitumor assay
Balb/c mice were injected subcutaneously in their backs with CT26 cells (6.5 × 105) [18]. Eight days later, groups of 8 tumor-bearing mice (volume of 50 to 100 mm3) received oral doses of ECL (100, 200, and 300 mg/kg), dissolved in saline solution with PVP (1:4), 1 mg/kg CDDP (p.o. and i.p.) for 22 days, or 0.1 mL of saline solution. Tumors were measured using a Vernier, and size (in mm3) was calculated as follows:
$$ Tumor\ volume=\frac{length\ast width\ast height}{2} $$
At the end of the experiments, animals were sacrificed in a CO2 chamber, and tumors were excised and weighed.
Statistical analysis
Data are expressed as the mean ± S.E.M. When indicated, Student’s t-test and ANOVA followed by Dunnett’s test, were used. A P < 0.05 was considered significant. Results were processed using the SPSS software from IBM.
