Collection and identification of plant material
The aerial parts of the plant Cestrum diurnum L. (Family: Solanaceae) was collected from Dumuria area of Khulna district, Bangladesh (Coordinate 22.8083° N, 89.4250° E). The species was identified by the experts at Bangladesh National Herbarium (voucher specimen no. DACB 38792).
Young Swiss albino mice of either sex, 3-4 weeks old, weighing 20-25 g were purchased from the Animal Resources Branch of the International Centre for Diarrhoeal Diseases and Research, Bangladesh (ICDDR,B) and used for in vivo anti-inflammatory activity screening. The animals were kept in the animal house of Manarat International University under standard laboratory conditions (relative humidity 55-65%, room temperature 25.0 ± 2.0 °C and 12 h light-dark cycle) for adaptation. They were fed with ICDDR,B formulated pelleted standard diet and water ad libitum.
The collected aerial part of the plant was separated from undesirable materials and shade-dried. The dried plant materials were ground into a coarse powder with the help of a grinder. The powdered sample was stored in an airtight container and kept in a cool, dark and dry place until extraction commenced. Powered plant material (135 g) was taken in a clean, dry, flat-bottomed glass container and soaked with 400 ml of methanol (Merck Germany). The container was sealed and kept for 3 days with occasional shaking or stirring. It was filtered through a cotton plug and evaporated to dryness using a rotary vacuum evaporator to get the crude extract (yield 7.4% of the dry powder).
Carrageenan-induced paw oedema test
Anti-inflammatory activity was tested by carrageenan induced paw oedema test in mice [15, 16]. In brief, 0.1 ml of 1% w/v carrageenan suspended in 1% CMC was injected into the sub-plantar tissue of the left hind paw of each mouse. Mice were divided into four groups containing six animals in each. Test groups received plant extract at the doses of 150 and 300 mg/kg body weight, while control and positive control group received vehicle and diclofenac sodium (10 mg/kg body weight), respectively. The paw thickness was measured using a calliper at 0, 60, 120 and 180 min after the carrageenan injection. The anti-inflammatory activity was calculated as percentage inhibition of oedema in the animals treated with extract/standard as compared to the control. Percentage (%) inhibition of oedema was calculated using the following formula where Tt and T0 are the thickness of paw oedema in test/positive control and control group, respectively.
[Please see the supplementary files section to view the equation.]
Formalin induced paw licking test
Mice were grouped in control, test and positive control receiving vehicle, extract (150 and 300 mg/kg) and diclofenac sodium (10 mg/kg), respectively. Formalin solution (0.2 ml of freshly prepared 5% v/v formalin in distilled water) was injected into the dorsal surface of the right hind paw 30 min after the treatments. The mice were observed for the time spent licking the injected hind paw during the early phase (0-5 min) and late phase (15-30 min) of post formalin injection with the help of a stop watch and recorded [17, 18].
TNF-α induced NF-κB activation assay
RAW 264.7 cells (Sigma-Aldrich) were cultured in RPMI supplemented with 10% foetal bovine serum, penicillin G (100 µg/ml) and streptomycin (100 µg/ml). Cells were maintained at 37°C with 5% CO2 in a humidified incubator. NF-κB activation kit (Thermo Scientific) was used as previously described . RAW 264.7 cells were seeded in 96-well plate overnight and the cells were stimulated with tumour necrosis factor α (TNF-α) for 1 h. The extract was added into the medium and further incubated for 4 h. Cells were fixed, permeabilised and incubated with NF-κB p65 antibody for 1 h. Staining solution (containing DyLight 488 Goat Anti-Rabbit and Hoechst dye) was then added and further incubated for 1 h. The plate with stained cells was evaluated using a Cellomics ArrayScan HCS Reader (Cellomics, PA, USA). Data were captured, extracted and analysed with ArrayScan II Data Acquisition and Data Viewer version 3.0 (Cellomics).
The HR-ESIMS spectra were recorded on 6530 Accurate-Mass Q‑TOF LC/MS spectrometer (Agilent Technologies) equipped with ZORBAX Eclipse XDB-C18 Rapid Resolution column (HT 4.6 mm i.d. × 50 mm × 1.8 µm). The extract was injected at a volume of 10 μl (1 mg/ml) with a flow rate of 1 ml/min. A gradient elution with solvent A (1% trifluroacetic acid in water) and B (MeOH) starting at 95%A5%B to reach 100%B in 40 min was adopted. Other parameters include capillary voltage of 3500 V, nebuliser pressure 35 psi, drying gas flow 8 L/min, drying gas temperature 200ºC. The mass recorded in the range of m/z 100-1000 and expressed as total ion chromatogram (TIC).
Values are expressed as mean ± SEM. Results were analysed using one-way ANOVA followed by Dunnet’s test. Differences were considered as statistically significant at p<0.05.