## 3 Results and Discussion
### Evaluation of Cytotoxicity
In addition, crude extracts of various plants have been tested as monotherapy in both in vitro and in vivo experiments, and their cytotoxic activity has been proved [38; 39]. These extracts contain a vast number of active compounds endowed with strong antioxidant activity. Nevertheless, there is still a controversial debate to clarify the actual role of antioxidants in human cancer progression. Some studies support the idea that antioxidants desensitize cancer cells against chemotherapy. On the other hand, many studies acknowledge the beneficial, protective role of these compounds against cancer and also relate them to a better survival rate [40]. _S. aspera_ cytotoxic activity on glioblastoma has not been previously reported. Thus, we tried to explain the cytotoxic effect of the extract based on the compounds presented in abundance, namely rutin and luteolin. Rutin, a glycoside of the flavonoid quercetin, is of great pharmacological importance as apart its antioxidant activity it possesses among others also anticancer activity [37; 41; 42; 43]. For example, rutin, isolated from _Dimorphandra mollis_ seeds, was studied for its cytotoxic activity against GL-15 glioblastoma cell line. Rutin when tested at the range of concentrations between 50 and 100 uM caused a dose-dependent inhibition of growth and proliferation of the treated cells, while the maximum inhibitory effect was observed at 72 h. Apoptosis and cell cycle arrest at the G2/M phase was proposed by the authors as the mechanism of action by which rutin exerts its cytotoxic activity [44]. Similarly, [37], examined the effect of rutin alone or in combination with temozolomide (TMZ) on U87-MG, DS4-MG, and U251-MG glioblastoma cells (concentration range 25-400 uM). Rutin reduced the survival of cancer cells. When cells were treated with a combination of rutin and TMZ, rutin at a low dose managed to increase the cytotoxic activity of TMZ. Apart from the in vitro experiments, the same combination of drugs reduced the volume of the tumor in xenograft mice. The possible suggested mechanism of action of rutin was that the flavonoid inhibited autophagy and c-Jun N-terminal kinase (JNK) activity. Moreover, the cytotoxicity of rutin was also examined on CHME cells. The maximum concentration of the flavonoid used was 20 uM and its cytotoxic activity was exerted via upregulation of the p53 gene [43]. On the other hand, luteolin, a flavone, also plays an important role in cancer progression since it influences several signaling pathways. Types of glioblastoma cell lines including LN229, U251, U251 MG and U87 MG, have been examined for their sensitivity against treatment with luteolin. A dose and time-dependent effect of cell viability were observed after treatment with luteolin at the concentration of 0-80 uM of the flavonoid, via activation of apoptosis, autophagy, death receptor (FADD) expression, stimulation of MAPK signaling pathway [45] and interference with cell cycle and EGFR protein [46]. A similar effect of luteolin was also observed for A172 and U373 MG cancer cell lines, in which 200 uM of the flavonoid were needed to decrease cell viability for the first 24 h which was further decreased at 50 uM for the next 48 and 72 h of treatment [47]. In this study, the extract of _S. aspera_ compounds that do not belong to the flavonoid family was also identified. Taking into account that the concentration of both rutin and luteolin in the extract used to estimate cytotoxicity of cancer cells were calculated at uM levels, which means much lower than those used in the literature, we deduce that a synergistic effect of all the compounds that the extract contains is responsible for its cytotoxic activity. Hence, we mention here that apart from flavonoids also, steroidal saponins have been evaluated for their cytotoxic activity [48]. For example, the cytotoxic effect of dioscin on C6 rat glioma cells was investigated and it was found that dioscin enhances the cytotoxicity of treated cells in a dose and time-dependent manner.