Collection and proper identification of plant Ixora nigricans R. Br. was collected from Chittagong Hill near Mimi super market area, Chittagong. The sample was identified by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong. (12th October, 2013).
Chemicals and drugs The chemicals used were bovine serum albumin (BSA), diclofenac sodium, absolute methanol (99.50%) and vincristine sulfate (VS) were purchased from Sigma-Aldrich, Munich, Germany. All chemicals in this investigation were of analytical reagent grade.
Preparation of extract Plant materials were dried and ground (Moulinex Blender AK-241, Moulinex, France) into powder (40 to 80 mesh, 500 g) and soaked for 7 days with 2 to 3 days interval in 2.0 L of methanol at room temperature (23 ± 0.5°C). Filtrate obtained through cheese cloth and Whatman filter paper No. 1 was concentrated under reduced pressure at a temperature below 50°C using a rotary evaporator (RE 200, Sterling, UK). The extracts (yield 4.4 to 5.6% W/W) were all placed in glass petri dishes (90 × 15 mm, Pyrex, Germany). A 100 mg each of the extracts was suspended in 10 ml distilled water and the suspension was shaken vigorously on a vortex mixer. In this way, the concentration (10 mg/ml) of extracts was prepared for screening the cytotoxic properties.
Inhibition of protein denaturation in vitro For the inhibition of protein denaturation, in vitro diclofenac sodium was used as standard. The test solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of methanol extract of Ixora nigricans. The control solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of distilled water. Product control (0.5 ml) consists of 0.45 ml of distilled water and 0.05 ml of methanolic extract of Ixora nigricans. Standard solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of diclofenac sodium. Various concentrations (31.25, 62.5, 125, 250, 500, and 1000 µg/ml) of methanol extract of Ixora nigricans (IN) and diclofenac sodium (standard) were taken, respectively. All the solutions were adjusted to pH 6.3 using 1 N HCL. Samples were incubated at 37°C for 20 min and the temperature was increased to keep the samples at 57°C for 3 min. After cooling, 2.5 ml of phosphate buffer was added to the previous solutions. The absorbance was measured using UV-Visible spectrophotometer at 416 nm. The control represents 100% protein denaturation. The results were compared with diclofenac sodium. The percentage inhibition of protein denaturation of different concentrations is tabulated. The percentage inhibition of protein denaturation can be calculated as:
% inhibition = [100 - (OD of test solution - OD of product control)] ×100
Where OD = optical density.
The control represents 100% protein denaturation. The results were compared with diclofenac sodium.
Cytotoxicity screening Cytotoxicity of the methanol extracts of Ixora nigricans was evaluated by the brine shrimp lethality bioassay, which is widely used for screening bioactive compounds (Meyer et al., 1982; Zhao et al., 1992). In this study, a simple zoological organism (Artemiasalina) was used as a convenient monitor for the experiment. The eggs of the brine shrimp were collected from an aquarium shop (Dhaka, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) for 48 h to develop into larval shrimp called nauplii. The cytotoxicity assay was performed on the brine shrimp nauplii using the Meyer method. The test samples (extract) were prepared by dissolving them in DMSO (not more than 50 μl in 5 ml solution) plus seawater (3.8% NaCl in water) to attain concentrations of 10, 50, 100, 150, 200 and 300 µg/ml. A vial containing 50 µl DMSO diluted to 5 ml was used as a control. Standard vincristine sulfate was used as a positive control. Mature shrimps were placed into each of the experimental vials. After 24 h, the vials were inspected using a magnifying glass, and the number of surviving nauplii in each vial was counted. From these data, the percentage lethality of the brine shrimp nauplii was calculated for each concentration using the following formula:
% Mortality = N1 / N0
Where N1 = Number of dead nauplii after a 24 h incubation; No = Number of total nauplii transferred (10).