Plant material
B. cordata leaves were collected in the forested areas of Pedregal de San Angel, near the campus of the Universidad Nacional Autónoma de México (UNAM). The campus is located on the south side of Mexico City. The collection occurred in March 2010 and was authenticated by the corresponding author. A voucher specimen (No. 42663) was deposited at the Izta Herbarium of the Universidad Nacional Autónoma de México. The plant material was air dried indoors at room temperature before extraction.
BCME preparation
Leaves of B. cordata (253 g) were dried, ground and extracted with hexane and methanol in succession. The methanolic portion was evaporated under reduced pressure at 55°C to obtain a syrup. The resulting dry residue (29.55 g) was stored at 4°C and is hereafter referred to as Buddleja cordata methanolic extract (BCME).
UV absorption analysis of BCME
The UV absorption spectrum of BCME was obtained using a UV/VIS spectrophotometer (Perkin-Elmer, Lambda 2S UV/VIS). BCME and the commercial organic filter octyl methoxycinnamate (Parsol, ISP VAN DIK) were dissolved in ethanol at a concentration of 60 μg/mL and 10 μg/mL respectively. The samples were placed in a standard quartz cuvette with a 1 cm path length and then quickly scanned with UV light in the range of 200–400 nm.
HPLC analysis of BCME
The HPLC system consisted of a Hewlett Packard Series 1100 HPLC instrument with a UV detector set at 320 nm. The column was obtained from Supelco Technologies C18 (150 mm × 4.6 mm, 5 μm). The eluent was a mixture of 4% tetrahydrofuran in acetonitrile and water (35:65, v/v) and contained 0.04% phosphoric acid. The flow rate was 1 mL/min, the column temperature and pressure were 23°C and 149 bar, respectively, and the injection volume was 20 μL. Verbascoside, linarin and syringin served as standards.
Determination of the total polyphenol content of BCME
The total polyphenol content of the methanolic extract was determined using Folin Ciocalteu’s phenol reagent according to the method described previously [21]. The total polyphenols were expressed as mg of gallic acid equivalents per gram of extract.
In vitro antioxidant assays
Scavenging of DPPH free radicals was measured using a modification of Lohézic-Le Dévéhat method [22] in which BCME (5–300 μg/mL), gallic acid (0.5-10 μg/mL) and DPPH (250 mM) were dissolved in methanol. At least six different dilutions of extract were tested and allowed to incubate for 20 min in the dark before absorbance was measured at 517 nm in an ELISA lector spectrophotometer (Thermo Scientific, USA). The experiment was conducted in triplicate. The scavenging activity of each concentration was calculated as a percentage of reduction in DPPH concentration as follows:
Where Abs DPPH is the absorbance of the DPPH in methanol, and Abs Sample represents the absorbance of each sample in the presence of each concentration of BCME and gallic acid. Antioxidant activity was expressed as an IC50 value (inhibitory concentration in μg/mL of sample or positive control necessary to reduce the absorbance of DPPH by 50% compared to the negative control). The positive control was gallic acid. A lower IC50 value represents a higher antioxidant activity.
Scavenging of superoxide radical (O2
•-) was measured [22] using 96-well microplates and a non-enzymatic technique. The reaction mixture in the sample wells consisted of NADH (78 μM), nitro-blue tetrazolium (NBT) (50 μM), phenazine methosulfate (PMS) (10 μM) and BCME (500, 250, 125, 62.5, 31.25 μg/mL). The reagents were dissolved in 16 mM tris-hydrochloride buffer (pH = 8), except for BCME, which was dissolved in DMSO. After 5 min of incubation at room temperature, the spectrophotometric measurement (Thermo Scientific. USA) was performed at 560 nm against a blank lacking PMS and sample. Gallic acid was used as a positive control. The percentage inhibition at a steady state was used to calculate the IC50 values for each dilution. This measurement determined the amount of antioxidant required (measured as the concentration of the stock solution added to the reaction mixture) to scavenge 50% of O2
•-, with the lowest values representing the best efficiency for scavenging O2
•-. All tests were performed in triplicate, and the results were averaged.
Hydroxyl radical scavenging was measured according to the Halliwell method [23]. Hydroxyl radical (•OH) was generated by a Fenton system (ascorbic acid/FeCl3EDTA/H2O2). Deoxyribose (DR) is degraded to malonaldehyde when exposed to hydroxyl radicals, which generates a pink compound in the presence of thiobarbituric acid at low pH under heat. The reaction mixtures contained the following reagents at the indicated final concentrations (in a final volume of 1 mL): potassium phosphate buffer, pH 7.4 (10 mM), DR (2.8 mM), H2O2 (1.42 mM), BCME at different concentrations, FeCl3–EDTA (20 and 100 μM) and gallic acid was used as a positive control. The iron salt was premixed with the chelator and dissolved in water before addition to the reaction mixture. All other components were dissolved in potassium phosphate buffer, pH 7.4 (10 mM). After incubation at 37°C for 1 h, 1 mL of 2.8% (w/v) trichloroacetic acid and 1 mL of 1% (w/v) TBA were added, and the mixture was heated in a water bath at 100°C for 15 min. The absorbance of the resulting solution was measured at 532 nm.
Experimental animals and protocol
The SKH-1 hairless mouse strain, a widely used model for human photocarcinogenesis, was used in our study. Female SKH-1 mice at 5–6 weeks of age (weighing 26 ± 5 g) were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a climate-controlled environment with a 12 h light/dark cycle. Five mice were housed per cage and acclimatised for two weeks before starting the experiment. Throughout the experimental period, mice had free access to food and water that were provided through the food chamber on top of the cages. The Bioethical Committee/FES Iztacala, UNAM approved all animal protocols.
The SKH-1 mice were randomly divided into five groups of five mice each: untreated (U), negative control (C-), and positive control with UV (C+UV), BCME and BCME irradiated with UV (BMC+UV).
The mice in the negative and positive control groups were treated topically on the dorsal skin with 200 μL of ethanol (exposed area: 6 cm2). BCME was dissolved in ethanol at a concentration of 2 mg/mL [20]. The BCME groups were treated topically on the dorsal skin with 200 μL of the respective test solutions.
Mice that were untreated and pre-treated with ethanol (without UVB irradiation) were used as the normal and negative controls for analysis of normal skin, respectively. A treatment with ethanol and UVB irradiation served as the UVB-induced skin damage positive control. Treatments with BCME served as the experimental groups (without UVB and with UVB irradiation). Fifteen minutes after application of the substances, the C+UV and BCME+UV groups were irradiated with UV-B lamps (302 nm, UVP. UVM-26, 6 W) positioned 15 cm above their backs. Irradiation at this distance produced a dose of 6 mJ/cm2, which was measured with a Spectroline model DM-300HA research radiometer. The irradiation-exposure time in acute experiments was ten minutes. After 24 h of irradiation, erythema was measured. Animals were subsequently sacrificed by carbon dioxide asphyxiation, and the dorsal skin was dissected.
Erythema measurements
The erythema in mice was calculated by measuring the skin redness of the dorsal area of mice using a colour analyser (Lutron, Mod. RGB-1002). Among the various methods, colourimetry has the advantage of being the simplest and most reproducible method. Redness values were measured 24 hours after irradiation. The results are expressed as redness values for each treatment [24].
Histological observation of the skin
Each dorsal skin sample was fixed with 2% paraformaldehyde in phosphate buffer solution (pH 7.2) for 24 h in the tissue embedding cassette, dehydrated with a sequence of ethanol solutions (70%, 80%, 95% and 100%, v/v) and embedded in paraffin. All serial sections were cut to a thickness of 5 μm, de-paraffinised, and stained with haematoxylin-eosin (HE). The histopathological changes of each section were observed using multiple microscopic fields and photographed with a photomicroscope (Nikon). Measurements included the epidermal thicknesses and the number of sunburned cells (400X). The histological diagnosis was performed comparing the untreated and C- mouse skin samples with UVB irradiated groups in accordance with the number of sunburns reported [25].
In vivo BCME penetration study: tape stripping
Female SKH-1 mice at 5–6 weeks of age (n=5, weigh on 26 ± 5 g) were placed in a laminar flux chamber at 21°C and 62% relative humidity for 30 min. Application zones measuring 2 × 2 cm2 was marked for each mouse in dorsal area. The BCME was applied to the surface of the skin at 2 mg/cm2. 15 min after an application, the excessive substance on the applied area was wiped off with a cotton swab, followed by washing with ethanol 96% and drying. Stratum corneum of the treated areas was removed by four successive tape strip-pings using Scotch tape strips (19 mm × 32.9 m, 3 M, MN, USA). Each strip was taken in a controlled way, i.e. a 10 g rubber weight was rolled over it 10 times. The first strip was discarded and the three following strips were collected and deposited in a 20 mL beaker. 5 mL of ethanol were added at each sample and then stirred with a magnetic bar for 30 min. The absorbance was measured at 320 nm using a double-beam spectrophotometer (Perkin-Elmer, Lambda 2S UV/VIS) because the BCME main components absorb that wavelength. BCME penetration was obtained in accordance with concentration measurements calculated by regression analysis [26, 27].
Genotoxicity assay: micronucleus test
Genotoxicity was determinate according to the Ma method [28]. Seeds of Vicia faba var. minor that had been previously stored at 4°C were used for this study. Dry seeds of Vicia faba were soaked for 24 h in distilled water, the seed coats were removed, and the seedlings were allowed to germinate between two layers of moist cotton. After seven days, the primary roots (approximately 1–2 cm in length) were selected randomly, and 4 seeds were used per treatment. Growing roots were treated for 2, 4, or 8 h with BCME at various concentrations (0.1, 0.2 and 0.4% w/v), followed by a 44 h recovery period. Tap water and 1% glycerine were used as negative controls. The exposure time was 48 h for the negative controls. Each batch was incubated at 22°C. At the termination of the exposure time for each treatment, the roots were cut to approximately 0.5 cm and placed into a Farmer solution (1:3 acetic acid and absolute methanol) at 4°C for 24 hours. The roots were subsequently hydrolysed in 1 N HCl at 55°C to break the bonds of the plant cell wall. The samples were rinsed three times with distilled water to remove the HCl and placed in 70% alcohol at 4°C. The roots were macerated in 45% acetic acid and were then stained for 5 min with 1% orcein acetate for observation via an optical microscope (Nikon). Three thousand cells per experimental group were scored to determine the mean frequency of micronuclei and the mitotic index. Each experiment was repeated three times. To calculate genotoxicity, the following equations were used: Mitotic Index (MI) TOTAL = number of cells from each phase × 100/1000 and Micronuclei (MCN) = Number of interphase cells with MCN × 100/1000.
Statistical analysis
A statistical analysis was performed on the collected data. The mean values of the negative and positive controls and the BCME groups were obtained from descriptive analyses, and a One-Way ANOVA test was conducted to obtain F values and MS errors. Dunnett’s test was used to determine the level of significance in comparison to the negative and positive control values in each experimental series.