Azadirachta indica A. Juss (neem) phenolic extract inhibits human B-lymphoblastoid cells growth via cell cycle arrest, apoptosis induction, and DNA damage

Abstract

Aim: As far as is known, the pharmaceutical effects of neem on human B-lymphoblastoid (TK6) cells have not been studied until now. Hence, the present study aimed to obtain neem phenolic extracts for inhibits the proliferation of TK6 cells and explore some possible underlying mechanisms involved in these effects. Methods: Hexane extract (HE) was obtained in the first step. After that, the residual hexane was removed from the neem. The dried neem sample was used in a new extraction for obtaining the ethyl acetate extract (EAE). Total phenolic compounds (TPC) and total flavonoid contents (TFC) were determined by spectrophotometric methods. Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate the cytotoxicity in TK6 cells. The stop at G0/G1 cell cycle and inducing apoptosis in the TK6 cells were analyzed by flow cytometry. For deoxyribonucleic acid (DNA) damage evaluation, the alkaline comet test was used. Results: The higher TFC (65.50 mg/g of extract ± 1.17 mg/g of extract) and TPC (52.08 mg of extract ± 0.88 mg of extract) were obtained in EAE compared to HE that was obtained TFC of 14.61 mg/g of extract ± 0.60 mg/g of extract and TPC of 3.20 mg/g of extract ± 1.20 mg/g of extract. EAE was more significantly cytotoxic to TK6 cells than HE. The apoptosis induction was higher after exposure to 15.0 µg/mL of EAE (11.29%) in comparison to 15.0 µg/mL of HE (2.52%). The G0/G1 phase increased from 72% negative control (NC) to 83% after treatment with neem extracts (15 µg/mL). Neem extracts were also able to cause DNA strand breaks in TK6 cells. Conclusions: The extraction residue from neem leaf after hexane extraction is a source important of cytotoxic and genotoxic molecules against TK6 cells, the results also can suggest that the toxic effects in TK6 cells can be provided most likely due to the presence of high content of TPC from neem extracts.

Keywords

• medicinal plant
• bioactive extracts
• sequential pressurized fluids
• antiproliferative effect
• human b-lymphoblastoid cells