2.1. Preparation of crude extract The roots of Hemidesmus indicus and the stem barks of Polyalthia longifolia and Aphanamixis polystachya were extracted with 80% ethanol:water in cold extraction process. The first plant yielded 8.9% extract, the second, 6.87%, and the third, which resulted in two extracts, an oily phase and a solid mass, yielded 2.54 and 8.91%, respectively. The stem bark of Oroxylum indicum the, fruits of Tribulus terrestris and the seeds of Nigella sativa were extracted in a soxhlet apparatus using absolute ethanol as solvent. The first two species yielded 15.24 and 17.94% extracts, respectively. The extract of Nigella sativa was further partitioned in diethylether and yielded 7.65%. The other plants, Cuscuta reflexa (whole plant), Paederia foetida (whole plant), Emblica officinalis (fruits), Moringa oleifera (roots), and Aegle marmelos (stem bark), were extracted with absolute ethanol in a cold extraction process. Cuscuta reflexa, Paederia foetida and Emblica officinalis yielded 10.32, 11.92 and 15.0% extracts, respectively. The crude extract of Moringa oleifera, which yielded 14.54%, was further partitioned in n-hexane (7.39% yield), chloroform (5.94%), methanol (9.46%) and water (9.48%), and the extract derived from Aegle marmelos was partitioned in diethylether (9.87% yield) (Khan et al., 2002). The extracts were dried in a rotary vacuum evaporator and the residue reconstituted in DMSO before testing. The vehicle was used as negative control. All of these plants were collected from various localities, including Chittagong, Chittagong Hill Tract, Tangail and Dhaka, of Bangladesh, in different months of 1996.
2.2. Brine shrimp assay Brine shrimp (Artemia salina Leach) eggs were hatched in a becker filled with seawater under constant aeration. After 48 h the nauplii were collected by pipette and were counted macroscopically in the stem of the pipette against a lighted background. Ten nauplii were transferred to each well of 24-multiwell plates containing the samples. The extract concentration ranged from 10 to 1000 µg/ml. The plates were maintained under illumination. Survivors were counted after 24 h of incubation and the percentage of deaths at each dose and control (seawater plus vehicle) were determined.
2.3. Assay on sea urchins The test was performed in 24-well plates following the method described by Costa-Lotufo et al. (2002). Adult sea urchins (Lytechinus variegatus) were collected at Pecem´ beach, on the northeastern coast of Brazil. The gamete elimination was induced by injecting 3.0 ml of 0.5 M KCl into the urchins coelomic cavity via the peristomial membrane. The eggs were washed twice using filtered seawater to remove the jelly coat surrounding the cells. Concentrated sperm was collected with a Pasteur pipette and maintained under low temperatures until the moment of fertilization. For fertilization, 1 ml of a sperm suspension (0.05 ml of concentrated sperm in 2.45 ml of filtered seawater) was added to every 50 ml of egg solution. Each well received 1 ml of fertilized egg suspension. The extracts were added immediately after fertilization (within 2 min) to get concentrations of 10, 30, 100, 300 and 1000µg/ml in a final volume of 2 ml. Doxorubicin (0.058–58.0µg/ml) was used as the positive control. The plates were then shaken on a constant temperature water bath at 26 ± 2 0C. At appropriate intervals, aliquots of 200µl were fixed in the same volume of 10% formaldehyde to obtain first and third cleavages and blastulae. One hundred eggs or embryos were counted for each concentration of test substance to obtain the percentage of normal cells.
2.4. MTT assay The cytotoxicity of the extracts was tested against B16 (murine melanoma), HCT-8 (human colon carcinoma), CEM and HL-60 (leukemia) tumor cell lines (Children’s Mercy Hospital, Kansas City, MO, USA). Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 100µg/ml streptomycin and 100 U/ml penicillin at 37 0C with 5% CO2. For experiments, cells were plated in 96-well plates (105 cells/well for adherent cells or 0.3 × 106 cells/well for suspended cells in 100µl of medium). After 24 h, the extracts (2–125g/ml) dissolved in DMSO (1%) was added to each well and incubated for 3 days (72 h). Control groups received the same amount of DMSO. Doxorubicin (0.01–0.58g/ml) was used as positive control. Growth of tumoral cells was quantitated by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product (Mosmann, 1983). At the end of 72 h incubation, the medium in each well was replaced by fresh medium (200µl) containing 0.5 mg/ml of MTT. Three hours later, the formazan product of MTT reduction was dissolved in DMSO, and absorbance was measured using a multi-plate reader (Spectra Count, Packard, Ont., Canada). Drug effect was quantified as the percentage of control absorbance of reduced dye at 550 nm.
2.5. Hemolytic assay The test was performed in 96-well plates following the method described by Costa-Lotufo et al. (2002). Each well received 100l of 0.85% NaCl solution containing 10 mM CaCl2. The first well was the negative control that contained only the vehicle (distilled water or DMSO 10%), and, in the second well, 100µl of test substance that was diluted in half was added. The extracts were tested at concentrations ranging from 10 to 2500g/ml. The serial dilution continued until the 11th well. The last well-received 20µl of 0.1% Triton X100 (in 0.85% saline) to obtain 100% hemolysis (positive control). Then, each well received 100µl of a 2% suspension of mouse erythrocytes in 0.85% saline containing 10 mM CaCl2. After incubation at room temperature for 30 min and centrifugation, the supernatant was removed and the liberated hemoglobin was measured spectroscopically as absorbance at 540 nM.
2.6. Statistical analysis Data are presented as mean ± S.E.M. The IC50 or EC50 values and their 95% confidence intervals (CI 95%) were obtained by nonlinear regression using the GRAPHPAD program (Intuitive Software for Science, San Diego, CA). LC50 on brine shrimp was obtained from the 24 h counts using the probit analysis method described by Finney (1971).