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Study of Biological Activity

Cytotoxic activity

Cytotoxicity was assessed with the help of the survival test of brine shrimp larvae of Artemia salina (Leach). Experiments were carried out in 2-day-old larvae in cultivation conditions in vitro. The larvae were grown up by dipping of brine shrimp eggs Artemia salina (Leach) into the artificial seawater and by the 48 h incubation at a temperature of 37°C. A weighed portion of the test sample was dissolved in 2 ml of methanol, and then it was taken from that solution by 500 pi (3 parallels), 50 pi (3 parallels), 5 pi (3 parallels). After evaporation of methanol 5 ml of artificial seawater were added to each vial. Thus, if the initial sample weight was 2 mg, the final sample concentrations were 100, 10 and 1 pg/ml, respectively, of each concentration in 3 replications. Two-day-old brine shrimp larvae Artemia salina were put into each vial containing the sample using the Pasteur pipette. Thereafter, all the vials were left at room temperature exposed to light for 24 h. After 24 h survived and dead larvae were counted. Then, using the obtained data of the upper and lower toxic limits a half-toxic sample dose was calculated.

Antimicrobial Activity

The study of antimicrobial activity of the samples mentioned above was performed pertaining to Gram-positive bacteria strains Staphylococcus aureus, Bacillus subtilis, Gram-negative strains Escherichia coli and Candida albicans yeast fungus by the method of diffusion into the agar wells. Comparator agents were gentamicin for bacteria and nystatin for Candida albicans yeast fungus

Cultures were grown in a fluid medium with pH 7.3 ± 0.2 at a temperature of 30 to 35°C during 18-20 h. The cultures were diluted 1:1000 in a sterile 0.9% sodium chloride isotonic solution; then they were poured into cups with 1 ml of solution with relevant elective nutrient media clarify for the studied test-strains and inoculated according to the method of "continuous lawn”. After drying on the surface of the agar wells of 6.0 mm size were formed, which were filled with the solution of the test sample, gentamicin and nystatin. Ethanol in equivoluminar quantities was used as control. Thus, the sample was tested in quantity of 1 pg and the comparator agent in quantity of 1 mg. The inoculations were incubated at 37°C, the accounting of growing colonies was performed after 24 h

The antimicrobial activity of the samples was estimated by the diameter of test-strains growth inhibition zones (mm). The diameter of inhibition zones lower than 10 mm and the continuous increase in the Petri dish were estimated as the absence of the antibacterial activity, 10-15 mm—as a weak activity, 15-20 mm—as a moderate activity, over 20 mm—as an expressed one (REF). Each sample was tested in three parallel experiments. Statistical reporting was performed by methods of the parametric statistics with the calculation of arithmetic mean and standard error.

Analgesic Activity

The experimental objects were studied in the dose range from 25 to 100 mg/kg when administered intragastrically. The comparator agent diclofenac sodium was tested in the dose range from 25 to 100 mg/kg. The experimental objects and comparator agent were administered 30 min before administration of the 0.75% acetic acid solution.

Phagocytosis-Stimulating Activity

The comparator agent "Immunorm” (juice of Echinacea purpurea in alcohol, “Merkle,” Germany) which was tested at a dilution of 0.9% sodium chloride solution in the final concentration of 8%.

Blood sampling was taken from a healthy donor in the fasting state from the ulnar vein into heparinized test tubes. As an object of the phagocytosis the daily culture Staphylococcus aureus (strain 209) was used. At the microscopic examination (the magnification 15 * 90, oil immersion) the number of phagocytic cells (the phagocytic index, PI) out of 100 neutrophils (quantitative indicator) and the number of staphylococci, absorbed by one neutrophil—the phagocytic number (PN, qualitative indicator of the phagocytosis) were counted after 1 h of study.

75 mg of the dry sample with account of its solubility were diluted in 0.2 ml of 96% alcohol until fully dissolved and made up to 2 ml of 0.9% sodium chloride solution. The substances were tested at a concentration of 1 mg/ml in three parallel experiments. The smears of the blood incubated with the dilution medium (96% alcohol and saline 1:9) were served as controls.

Statistical results reporting was performed by methods of the non-parametric statistics with the calculation of the arithmetic mean (M) and its standard error (m).

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