Natural products are used for the treatment of various diseases since the beginning of human history. It has been estimated that approximately 80–85 % of the world population rely on traditional medicines for their primarily health care and it is known that a major part of these therapies involves the use of plant extracts or their active components [
17,
33,
34]. Most of the chemotherapy medicines to treat the cancer diseases are the molecules identified and isolated from plant materials or their synthetic analogs [
35,
36]. Though many researches have been explored for the advancement to protect cancer diseases, still there is a need to develop new drugs to improve the efficacy. The major disadvantages of available synthetic drugs are their association with the side effects [
37]. Natural therapy, such as use of the plant products have proved beneficial against the cancer diseases [
38,
39]. Thus, there is a constant demand to develop effective new anticancer drugs at affordable cost [
36,
40]. Over the last 30 years, natural products have received increasing attention for their potential as new cancer preventive agents [
41,
42]. Bearing these facts in mind, we aimed to examine the anticancer potential of
V. encelioides against three human cancer cell lines, i.e. lung cancer (A-549), breast cancer (MCF-7), and liver cancer (HepG2). Further, oxidative stress (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), cell cycle arrest, and DNA damage were also assessed against liver cancer (HepG2) cells. The results of MTT and NRU assay showed that the
V. encelioides extract exhibited a concentration-dependent cytotoxic response. The MTT and NRU assays are commonly used endpoints for cytotoxicity assessments, since they evaluate different aspects of cellular functions and, therefore, can be useful to examine the potential cytotoxic effects of plant extracts. Estimation of cytotoxicity is generally based on uptake or exclusion of dye and are an indicator of the integrity of the plasma membrane or some intracellular organelles. However, the MTT assay indicates the mitochondrial function based on the enzymatic reduction of a tetrazolium salt by the mitochondrial dehydrogenase of viable cells [
43]. NRU is a measure of lysosomal integrity since it reflects the capacity of viable cells to incorporate vital dye into these organelles [
44].
The decrease in the cell viability found was specifically towards MCF-7 and HepG2 cells, whereas no effect was observed on A-549 cells. Further, the cytotoxic response was more in HepG2 cells as compared to MCF-7 cells. The differential cytotoxic response of the
V. encelioides might be due to the specificity of plant towards different cancer cells, as has been reported previously [
45,
46]. Our results are well in ordinance with the other findings where these kinds of effects are due to the presence of active components [
47]. Our present study demonstrated that
V. encelioides extract showed promising anti-cancer activity. However, activity was less in MCF-7 cells as compared to HepG2 cells. Thus, HepG2 cells were selected as a model to further investigate the underlying mechanism (s) responsible, for this cytotoxic response. An observable concentration dependent statistically significant increase in lipid peroxidation occurred. An increase of 17, 39, and 49 % was observed at 250, 500, and 1000 μg/ml of
V. encelioides extract, respectively. Oxidative stress is involved in normal cellular processes of cell signaling [
48]. Many studies have shown that exposure to natural products promotes cellular oxidative stress which includes lipid peroxidation and depletion in glutathione levels [
49,
50]. Data from this study also showed that
V. encelioides induced oxidative stress in HepG2 cells in a concentration-dependent manner. An increase in the level of lipid peroxidation and a decrease in the antioxidant enzyme GSH were observed. These findings suggest that oxidative stress may be the primary mechanism of the cell death in HepG2 cells when exposed to
V. encelioides extract. Previous reports, suggesting the role of oxidative stress in the cell death induced by plant extracts [
51,
52] firmly support our results. One of the most common cytotoxic effects in cancer cells is due to the induction of reactive oxygen species (ROS) generation [
53]. We found that HepG2 cells exposed to
V. encelioides extract significantly increased intracellular ROS generation in a concentration-dependent manner. Our results are also in agreement with previous findings, where researchers showed ROS induction due to the treatment of plant extracts [
54,
55]. We found an induction in the MMP level in HepG2 cells treated with
V. encelioides extract for 24 h. Reports suggest that high levels of ROS can lead to cellular damage by resulting in mitochondrial damage, which can then induce cell death [
32]. Induction in MMP level, based on cationic fluorescent probe Rh123 indicate the role of oxidative stress and ROS generation in cell death of HepG2 cells, due to the generation of free radicals during the mitochondrial respiration. The results from this study confirmed that HepG2 cells treated with
V. encelioides extract for 24 h significantly activate the G2/M cell cycle checkpoint as observed by flow-cytometry. Reports reveal that p53 protein is regarded as the guardian of the cell genome and is able to activate cell cycle checkpoints, DNA repair and apoptosis to maintain stability of genome [
56]. Flow cytometry analysis of
V. encelioides extract treated HepG2 cells suggests the activation of DNA repair process as observed by cell cycle arrest in G2/M phase at 250, 500, and 1000 μg/ml concentration of
V. encelioides. It is also known that the DNA repair mechanisms in the cells are highly conserved, thus the extensive DNA damage may lead to cell-cycle arrest and cell death as observed in this investigation. We have highlighted the DNA damaging potential of
V. encelioides extract in HepG2 cells by comet assay. The results of Table
1 revealed that the
V. encelioides extract induced a concentration-dependent significant DNA damage as observed by the induction in the fold change of olive tail moment (OTM), tail length, and tail DNA intensity. This
V. encelioides extract induced DNA damage can be explained on the basis of the experimental evidence of genotoxic potential in HepG2 cells. The DNA damage may either lead to apoptotic cell death or disrupt the factionalizing of cells [
57‐
59]. The DNA damage induced in HepG2 cells indicates the presence of bioactive components in
V. encelioides extract that are capable of oxidative DNA damage in cancer cells through their pro-oxidant mechanism.