1 Antiproliferative activity of neem leaf extracts 2 obtained by a sequential pressurized liquid extraction 3

Azadirachta indica A. Juss (neem) extracts have been used in pharmaceutical applications as 17 antitumor agents, due to their terpenes and phenolic compounds. To obtain extracts from neem 18 leaves with potential antiproliferative effect, a sequential process of pressurized liquid extraction was 19 carried out in a fixed bed extractor at 25 oC and 100 bar, using hexane (SH), ethyl acetate (SEA) and 20 then ethanol (SE) as solvents. An extraction using only ethanol (EE) was also conducted to compare 21 the characteristics of the fractionated extracts. The results obtained by liquid chromatography22 electrospray ionization mass spectrometry suggested the highest concentration of terpenes for SEA 23 extract in comparison to SH, SE and EE extracts. Therefore, antiproliferative activity showed SEA 24 extracts were the most efficient inhibitors to human tumor cells MCF-7, NCI-H460, HeLa, and HepG2 25 between all other extracts studied. However, hepatocellular normal cells were more resistant to SH, 26 SEA, SE, and EE compared to malignant cells of breast, lung, hepatocellular, and cervical. Neem 27 fractioned extracts obtained in the present study seem to be more selective for malignant cells 28 compared to the normal cells. 29

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