Or amounts) had been then separately extracted in 3 occasions the volume of 96 (v/v) methanol at area temperature (RT) with continuous shaking (740 0 T ingen; Edmun Buchler, Germany) at 100 r/min for 24 h. This approach was repeated till the color of extract wasPaula M. Kustiawan et al./Asian Pac J Trop Biomed 2014; four(7): 549-almost clear (maximum of 7 d) and the extracts were pooled.Table 1 Yields of the crude extracts of bee products.SourceCO2, seeding at 110 cells/25-cm flask in five mL CM, andrepassaging when at 70 -80 confluency. two.four. Cell countscontaining five (v/v) fetal calf serum) at 37 with five (v/v)5Initial IME yield(g)extractionaMethanolIME extractionb: The initial amount of sample extracted by methanol, and the yield of the obtained initial methanol extract (IME). b: The quantity of IME extracted with hexane/methanol and subsequent extraction with the methanol portion with ethyl acetate, using the yields obtained of your crude hexane extract (CHE), crude ethyl acetate extract (CEE) and crude methanol extract (CME).aT. incisa T. apicalis Propolis T. fuscibisca T. fuscobalteata T. incisa Bee T. apicalis pollen T. fuscibisca T. fuscobalteata T. incisa T. apicalis Honey T. fuscibisca T. fuscobalteata50 46 50 50 20 27 25 150 150 150 15045.eight (91.six) 39.two (85.two) 46.three (92.five) 43.7 (87.2) eight.1 (40.7) 7.3 (27.2) 8.two (33.0) 54.8 (36.five) 48.six (32.4) 58.two (38.eight) 59.2 (39.four) 7.6 (30.six)(g) ( )Initial CHE yield CEE yield5.0 2.0 10.0 1.0 3.7 2.7 28.0 28.0 28.0 28.0 three.5 five.(g)0.3 (16.0) 0.five (10.0) 0.1 (10.0) 0.1 (3.2) 0.2 (7.0) 0.1 (two.9) 0.three (1.1) 0.two (0.7) 0.3 (1.1) 0.three (1.1) 0.7 (6.9)0.2 (four.eight)(g) ( )two.five (50.eight) 1.1 (53.0) 3.6 (72.0) six.9 (68.five) 0.two (20.0) 0.2 (5.1) 0.two (7.8) eight.four (30.0) 3.3 (11.8) 3.three (11.8) three.9 (13.9) 0.1 (2.9)(g) ( )yield (g)1.9 (37.2) 0.6 (30.five) 0.9 (18.0) 1.eight (17.five) 0.2 (22.0) 2.three (62.four) 1.eight (65.two) 1.five (42.9) 2.9 (10.4) 3.7 (13.2) three.eight (13.6) 2.four (eight.6)( )CMEA dherent cells have been harvested by removing the CM , washing with 0.01 mol/L phosphate buffer saline (PBS (pH 2+ 2+ 7.four) with 0.01 EDTA but with out Ca and Mg ) then incubating with 1 – 1 . 5 m L of 0 . 05 ( w/v ) trypsin in the identical PBS at RT for 1-2 min. The trypsin remedy was then replaced with CM (1.5-2 mL) as well as the cells dissociated by gentle agitation, harvested and also the cell suspension further diluted as expected such that a ten aliquot may very well be counted employing a hematocytometer.2.five. Determination of your extract cytotoxicity by the surrogate MTT assayT he potential cytotoxicity ( anti-proliferative and/or lowered cell viability) of every crude extract was assayed using the surrogate cell viability 3-(four,5-dimethyl-thiazol2 -yl ) two , five -diphenyl-tetrazolium bromide ( MTT ) assay, as previously reported[16].1350518-27-2 structure In brief, cultured cells (5 000 cells) in 200 of CM have been transferred into each nicely of a flat 96 effectively plate then incubated at 37 in a humidified air atmosphere enriched with five (v/v) CO2 for 24 h as a way to let the cells attach for the bottom of every single effectively.Price of 4-Bromo-3-nitropyridine The cultured cells had been then treated with the test extract (triplicate wells per condition) at a final concentration of 1, 0.PMID:23664186 1, 0.01, 0.001 and 0 (solvent control) /mL by the addition of 2 of CM serial dilutions on the respective crude extract dissolved in dimethyl sulfoxide (stock concentration of 20 /mL). The cells were then cultured as above for another 48 h prior to the addition of 10 of a 5 mg/mL of MTT solution into each and every well. The incubation was continued for a further four h prior to the media was removed. A m.