Reagents
12-O-tetradecanoyl-phorbol-13-acetate (TPA), indomethacin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate (K2S2O8), Dulbecco’s Modified Eagle Medium (DMEM), L-glutamine, antibiotics (Penicillin-Streptomycin), dimethyl sulfoxide (DMSO), trypan blue, lipopolysaccharides from Escherichia coli serotype 0127:B8 (LPS), (N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400 W), rofecoxib, sodium nitrite (NaNO2), sodium nitroprusside (SNP), N-(1,1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, Phosphate Buffer Saline (PBS) tablets, O-Dianisidine, ethylenediaminetetraacetic acid (EDTA), hematoxylin and eosin were purchased from Sigma-Aldrich (St. Louis, MO, USA). fetal bovine serum (FBS) was obtained from GIBCO (Gaithersburg, MD, USA). Hydrogen peroxide (H2O2), hexadecyl-trimethylammonium bromide (HETAB), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) from Calbiochem® (San Diego, CA, USA). Organic solvents were analytical grade and obtained from Mallinckrodt Baker (San Diego, CA, USA).
Plant material
Leaves of Cryptostegia grandiflora (Apocynaceae) were collected at Pueblo Nuevo, Bolivar, Colombia (4° 10° 44’ 33” N; 75° 15’ 28” W; elevation 3 m.a.s.l.), in March 2011. The plant material was identified by Felipe A. Cardona N., and the voucher specimen has been deposited with the identification number HUA-175330 at the Herbarium University of Antioquia, Medellin, Colombia.
Preparation of ethanol extract and primary fractions
Dried and powdered leaves (689.9 g) were exhaustively extracted with ethanol by maceration at room temperature (25 ± 3°C). The extract was then filtered and concentrated in a rotary evaporator under controlled temperature (35–45°C) and reduced pressure. A portion of the concentrated total extract (10 g) was fractioned using liquid/liquid partition procedures with ether (1.4 g), dichloromethane (1.5 g), and methanol (3.8 g).
Experimental animals
Female ICR mice weighing 20–25 g were provided by Instituto Nacional de Salud, Colombia. Animals were allowed to acclimatize for 10 days before use and fed with standard rodent food and water ad libitum. They were housed in filtered-capped polycarbonate cages and kept in a controlled environment with temperature at 22 ± 3°C and relative humidity between 65 to 75%, under a cycle of 12 h light/darkness. All the procedures were performed in accordance with ethical guidelines on the care and use of animals in laboratory research, which were approved by the Ethics Committee of the University of Cartagena.
TPA-induced acute ear edema
The edema was induced by topical application of TPA (2.5 μg/ear) in acetone, according to the method described by De Young et al.[30]. Mice in groups (n = 6) were treated on the inner and outer surfaces of the right ear with an acetone solution containing test extracts/fractions from Cryptostegia grandiflora leaves (1 mg/ear in 20 μL of vehicle) or Indomethacin (0.5 mg/ear in 20 μL of vehicle), as reference drug. At the same time, the TPA solution was applied to both faces of the right ear. The left ear received only acetone, as a control. Four hours after the inflammation induction, animals were sacrificed by cervical dislocation and a disk (6 mm diameter) was removed from both ears (treated and untreated) and individually weighed on an Ohaus Pioneer™ (PA214) analytical balance. The degree of edema was indicated by the increase in the weight of the right ear disk over the left. The anti-inflammatory activity was expressed as percentage of the inhibition of edema in treated groups in comparison to control group (vehicle)[10].
Histopathologic analysis was performed according to standard protocols[31]. Ear samples were preserved in buffered formalin, embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin eosin. Subsequently, ear tissue was analyzed by a blinded pathologist using light microscopy, which assessed the presence of edema, epidermal hyperplasia/hypertrophy, hyperkeratosis, infiltration of mononuclear and polymorphonuclear cells, connective tissue disruption, and dermal fibrosis, using a scale of 0 to 4 (0, none; 1, mild; 2, mild; 3; moderate; 4, severe).
Myeloperoxidase (MPO) assay
MPO activity was measured according to the method by[32]. Ear tissue was weighed and homogenized in 10 vol of 50 mM PBS, pH 7.4. The homogenate was centrifuged at 4000 rpm for 30 min at 4°C. The pellet was again homogenized in 10 vol of 50 mM PBS, pH 6.0, containing 0.5% HETAB. The homogenate was subjected to cycles of freezing/thawing and brief periods of sonication (15 s) and centrifugation at 4000 rpm for 10 min at 4°C. Supernatants (20 μL) were mixed with 50 μL of o-dianisidine dihydrochloride (0.067%) and 50 μL of hydrogen peroxide (0.003%) in each well of a 96-well microplate, and incubated for 5 min. Enzyme activity was determined by measuring changes in the Optical Density at 450 nm (OD450), using a microplate reader (Multiscan EX Thermo®), and expressed as the inhibition percentage of MPO levels calculated against the absorbance presented by the control group.
Cell culture
The murine RAW 264.7 macrophage-like cell line was obtained from the American Type Culture Collection (TIB-71; Rockville, MD, USA) and routinely cultured in DMEM supplemented with 2 mM L-glutamine, antibiotics (100 IU/mL of penicillin-100 μg/mL streptomycin) and heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% CO2.
MTT assay
The mitochondrial-dependent reduction of MTT to formazan was used to assess the possible cytotoxic effects of Cryptostegia grandiflora leaves on RAW 264.7 cells[33, 34]. Macrophages (2×105 cells/mL) were cultured at 37°C and allowed to grow to confluence. Subsequently, the culture medium was replaced with various concentrations of test extracts/fractions or 1400 W for 30 min, followed by stimulation with of E. coli LPS (1 μg/mL). Control group was incubated with the same amount of DMSO. Triton X-100 (2%) was used as positive control. After 24 h, the medium was removed and cells incubated with MTT solution (3 mg/mL). Four hours later medium was carefully aspirated, formazan crystals were dissolved in DMSO, and OD550 was measured using a microplate reader (Multiscan EX Thermo®).
NO• and PGE2 production
The evaluation of the anti-inflammatory activity was performed in a manner similar to that described for cell viability. In brief, RAW 264.7 cells (2×105 cells/mL) were allowed to grow to confluence at 37°C. The adherent cells were treated for 30 min with various concentrations of fractions from Cryptostegia grandiflora leaves, 1400 W or Rofecoxib, and stimulated with LPS (1 μg/mL). Control cells were cultured under the same conditions but were not exposed to the effect of LPS. Twenty four hours later, culture supernatants were collected and stored at −20°C until use.
NO• release was determined spectrophotometrically by the accumulation of nitrite (NO2−), using the Griess reaction[35]. Briefly, 100 μL of cell culture medium was mixed with 100 μL of Griess reagent (1:1 mixture of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 1% sulfanilamide in 5% H3PO4), and incubated at room temperature for 5 min. The OD550 of the samples was measured using a microplate reader (Multiscan EX Thermo®) and compared with a standard curve prepared with NaNO2 (1–200 μM). Levels of PGE2 were determined using commercially available competitive ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Final results were expressed as pg/mL of supernatant.
NO radical scavenging effect
NO-scavenging effect of fractions of Cryptostegia grandiflora leaves was determined by the method of[36] with some modfications. Sodium nitroprusside (SNP) in aqueous solution spontaneously generates NO• which interacts with oxygen to produce NO2− ions that can be estimated using Griess reagent. Scavengers of NO• compete with oxygen, leading to reduced production of NO2−[37]. Briefly, SNP (5 mM) in PBS, pH 7.4, was mixed with test compounds and incubated at 25°C for 120 min. After this period, samples were incubated with an equal volume of Griess reagent for 5 min. The OD550 was measured using a microplate reader (Multiscan EX Thermo®) and compared with standard solutions of NaNO2 (1–200 μM).
DPPH radical scavenging activity
DPPH-scavenging effect was determined using the method described by[38], with some modifications. In brief, 75 μL of fractions from Cryptostegia grandiflora and ascorbic acid, at various concentrations, were plated into 96-well plates, mixed with 150 μL of DPPH (100 μg/mL; in methanol), and incubated at room temperature (25 ± 3°C). Vehicles were used as negative controls. After 30 min, the disappearance of DPPH radical absorption was determined at OD550 using a microplate reader (Multiscan EX Thermo®).
ABTS radical scavenging activity
ABTS-scavenging effect was determined using the method described by[39], whit modifications. Briefly, 10 μL of various concentrations of fractions from Cryptostegia grandiflora or ascorbic acid, used as positive controls, were mixed with 190 μL of ABTS (3.5 mM; in ethanol) and K2S2O8 (1.25 mM). The mixture was incubated at room temperature for 5 min. After this time, the disappearance of ABTS radical absorption was determined at OD405 using a microplate reader (Multiscan EX Thermo®).
Statistical analysis
Results from three independent assays were expressed as mean ± standard error of the mean (S.E.M) and analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc test, to determine the differences between groups. Values of p < 0.05 were considered significant. LC50 and IC50 values were calculated using non-linear regression analysis and expressed as the mean and its 95% confidence interval.
