eng Oriental Scientific Publishing Company Biomedical and Pharmacology Journal 0974-6242 2025-12-30 18 4 2873 2879 10.13005/bpj/3301 68599 article Asha Khanna 1 Ayushi Verma 3 Daya Shankar Gautam 4 Pradeep Singour 5 Ameesha Nigam 6 AVIKA Biological research Foundation Lab, Jabalpur, India Department of Zoology and Biotechnology, Govt. Science College, Jabalpur, India Department of Biological Sciences, Rani Durgawati Vishwavidyalaya, Jabalpur, India Department of Zoology, St. Aloysius’ College (Autonomous), Jabalpur, India Department of Biotechnology, St. Aloysius’ College (Autonomous), Jabalpur, India Department of Biological Sciences and Engineering (BSBE), IIT Kanpur, India Methotrexate (MTX) is frequently used in the treatment of leukemia and several other types of cancer. Its effects are antiproliferative on malignant cells. Withania sominifera (Withania leaf extract: WE) is widely used for its antibacterial, anti-inflammatory & anticancer effects in traditional medicine systems. In this investigation the effects of MTX alone & MTX + WE on the proliferation of normal human peripheral blood mononuclear cells have been studied. The antiproliferative effects were studied by MTT Assay and DNA breakage studies due to MTX were done by florescent Comet Assay. MTX was used in doses of 20µl per well. The concentrations of MTX used in this study were 75, 125 & 250 µg/ml. In some reaction wells 20 µl of WE were also added. The results were recorded after incubation for 4 & 18 hrs. A dose dependent decrease in viability % was found when the cells were treated with MTX alone both at 4 & 18 hrs of incubation but a greater fall in viability at every stage was noted after addition of WE. At 4hrs of incubation the drop-in viability was maximum at 250 µg/ml. The average viability% at this concentration with only MTX was 82.62±4.15 (significant p˂ 0.05). This dropped to 58.13± 4.45 when WE was also added. At 18hrs the average viability was 66.98± 12.35 with only MTX (250 µg/ml), but when WE was also added the viability dropped to 40.96±6.94.  The study shows that the antiproliferative effect of MTX was enhanced by Withania sominifera leaf extract. https://biomedpharmajournal.org/vol18no4/in-vitro-evaluation-of-methotrexate-cytotoxicity-and-antiproliferative-effects-of-withania-somnifera-on-human-peripheral-blood-mononuclear-cells-using-mtt-and-comet-assay/ Antiproliferative effect Comet assay Methotrexate MTT assay Withania somnifera