In vitro cytotoxic study for partially purified Lasparaginase from fresh leaves, unripe and ripe fruits of Withania somnifera plant

Abstract

This work aimed to study the cytotoxic effect of L-asparaginase isolated from local Withania somnifera plant on lymphocyte leukemia cells. To achieve this goal, L-asparaginase was purified from W. somnifera fruits by two purification steps, ion-exchange chromatography using DEAE-cellulose and gel filtration chromatography using Sephadex G-150, and the study utilized an in vitro evaluation for the cytotoxic effect of the partially purified L-asparaginase with concentrations ranging (12.5 to 100 μg/ml) in a two fold serial dilutions on some cell suspension culture including, acute lymphocyte leukemia and chronic lymphocyte leukemia culture at different concentrations (12.5 to 100 μg/ml) and different exposure time of treatment (24, 48 and 72 h). This two purification steps raised the specific activity from 1.73 U/mg in crude extract to 2.29 U/mg after ion-exchange and 10.5 U/mg after gel filtration; the purification fold was 1.32 after ion-exchange and 6.06 after gel filtration, the enzyme recovery was 56% after two purification steps and the results, pointed that acute lymphocyte leukemia culture showed highest sensitivity toward the cytotoxic effect (62.3±0.9%) of the partial purified L-asparaginase (100 μg/ml) than other culture after 48 h in a dose dependent manner, and highest cytotoxic inhibitory effect (73.2±1.6%) after 48 h of exposure on chronic lymphocyte leukemia culture, while healthy lymphocyte culture showed novel behavior. The lowest concentration of cell treatment gave the most significant (P< 0.01) inhibitory effect. The conclusion is that there is enough evidence to support the claim that Lasparaginase from W. somnifera may be considered chemotherapeutic agent against cancer, such as acute lymphoblastic leukemia and lymphosarcoma.

Keywords: Acute lymphocyte leukemia (ALL), chronic lymphocyte leukemia (CLL), L-asparaginase, cytotoxic assay.