## 4 Discussion
vulgar_ extract to induce DNA damage in cancer cells and alter the expression of checkpoints, stress-response and repair genes or proteomics analysis to detect differentially expressed proteins would be necessary to identify the exact pathways and mechanisms involved. Examining the chemical composition of the extracts of the in vitro cultivated and wild plants shows that the total content of both flavonoids and polyphenols, as well as the antioxidant activity, are higher for the wild plants compared to the in vitro cultivated plants, which cannot explain the higher cytotoxic effect of the in vitro plant extract on the HeLa cell line at the 24 th h and the consistently higher cytotoxic effect of the same extract on all cancer cell lines at the 48 th h. However, our previous research [26] has shown that it is only the in vitro cultivated plants (leaves and flowers) and not the wild-growing plants that contain catechin. Furthermore, neochlorogenic acid (flowers), quercetin (stems) and apigenin (stems) are significantly higher in the in vitro cultivated plants than in the wild plants [26]. Many studies have shown the protective and anticarcinogenic effects of catechins against a number of cancers, amongst which are gynecological cancers such as ovarian and endometrial cancer, breast cancer, oropharyngeal and laryngeal cancer, digestive tract cancers such as stomach and colorectal cancer, incident glioma, liver and gallbladder cancers, lung cancer and others [40]. For example, epigallocatechin gallate (EGCG), the most abundant catechin in fresh tea leaves [41], has been reported to inhibit tumor invasion and angiogenesis essential for tumor growth and metastasis [42]. Similarly, neochlorogenic acid or extracts rich in it have been reported to possess strong anticancer activity against several types of human cancers, like gastric cancer and human glioma, with some of the underlying mechanisms being the loss of mitochondrial membrane potential, reactive oxygen species generation and the induction of apoptosis partly through DNA damage and the regulation of epigenetic factors in human cancer cells [43; 44]. Regarding apigenin and quercetin, their anticancer potential has been confirmed through their ability to modulate various cell signaling pathways related to cancer, including tumor suppressor genes and the PI3K/AKT, NF-\(\kappa\)B, MAPK/ERK and the STAT3 pathways [45; 46]. Thus, for example, quercetin has been demonstrated to exert an anti-tumor immune effect in breast cancer by regulating the IL-6/JAK2/STAT3 signaling pathway to deplete regulatory (Treg) cells, which in the tumor microenvironment are reprogrammed to acquire higher immunosuppressive capacity, and a large number of infiltrated Treg cells in the tumor microenvironment is often related to poor prognosis [46]. It could be that one of the above biologically active substances, or rather their combination, is responsible for the better cytotoxic efficiency of the extract of the in vitro cultivated plants compared to the wild-growing plants. The tendency toward the use of total plant extracts for anticancer activity screening [14; 47; 48] is justified and useful as the pharmacological (protective) effect of the extract is very often the result of the complex, multi-targeted action of the different active substances and, frequently, of the synergistic relations between them. The specific mechanisms of action of the active substances are variable and dependent on their structure and concentration in the extract as well as on the different physicochemical environmental conditions. Thus, although in a previous study [26] we examined the antioxidant and antitumor activity of different parts (stems, leaves and flowers) of in vitro cultivated and wild _C. vulgare_ plants, in the present research, we turned our attention to evaluating the cytotoxicity and anticancer potential of total extracts, comprising the entire aerial part, of wild and in vitro cultivated _C. vulgare_ plants.