Solution of each concentration (500��L) was transferred into clea

Solution of each concentration (500��L) was transferred into clean 24-well plates via a pipette, and aerated seawater having 10�C20 nauplii (500��L) was added. A check count was performed and the number alive noted after thing 24h. The mortality end point of the bioassay was determined as the absence of controlled forward motion during 30 seconds of observation. The controls used were seawater and berberine hydrochloride (LC50 = 26��g/mL). Lethality percentage was determined and LC50 calculated based on probit analysis with 95% of confidence interval [11].2.5. Cell Cultures and Cytotoxicity AssayFour cancerous cell lines HT29/219 (human, colon, epithelial-like, carcinoma), Caco2 (human, colon, adenocarcinoma), NIH-3T3 (Swiss NIH mouse, embryo fibroblast), and T47D (human, breast, ductal carcinoma) were purchased from the Pasteur Institute, Tehran, Iran.

The cells were maintained in RPMI 1640, supplemented with 10% fetal bovine serum, 0.28 units/mL insulin, 100��g/mL streptomycin, 100 units/mL penicillin, and 0.3mg/mL glutamine. The cells were grown at 37��C in a humidified atmosphere of 5% CO2, in air.The cytotoxicity of the compounds isolated from S. spicigera was assayed using the MTT cytotoxicity assay. The cells (3 �� 10 4) were plated in 500��L of medium/well in 48-well plates (NUNC Cell Culture Flasks, Denmark). After an overnight incubation at 37��C, in 5% CO2, and a humidified atmosphere, the samples were added to the cells to a final concentration of 500��g/mL. ��Methotrexate�� (positive control) and pure compounds were examined at concentrations ranging from 5, 10, 20, 40, 80, and 100��g/mL.

The plates were incubated at 37��C, in 5% CO2, humidified atmosphere, for 48 hours. After 48 hours, 50��L of 5mg/mL MTT (dissolved in PBS) was added per well. After three hours of incubation, the MTT solution was removed and the cells were washed with 100��L of PBS, twice. One hundred and fifty microlitres of DMSO was added per well, to solubilize the formazan crystals. The optical densities of the wells were then measured at 570nm (690nm reference wavelength). By referring to the control (medium with DMSO), the cell survival was assessed [20].3. ResultsDried aerial parts of S. spicigera, collected during full flowering stage, were successively extracted with ethyl acetate and methanol. These extracts were concentrated and examined with brine shrimp lethality assay (BSA).

Both extracts showed cytotoxic activity against Artemia salina larvae, so were used for further isolation on silica gel and sephadex column chromatography to obtain compounds 1�C9 (Figure 1). Isolated compounds, 1�C7, were identified as thymoquinone (1), thymol (2), Entinostat carvacrol (3), ��-sitosterol (4), ursolic acid (5), oleanolic acid (6), and nubigenol (7) by comparison of their spectral data with those reported in literature [11, 21�C23].

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