*2.3. Evaluation of Cytotoxicity*

Both cell lines were exposed to increased concentrations of the extract ranging from 6.25–0.04 mg/mL. The extract exhibited its cytotoxic effect in a dose- and time-dependent manner. Significant differences were observed between the control group and the treated cells, quite at the same range of concentrations. For TE671 cells, the range of the concentrations that reduces cell growth and proliferation ranged from 6.25 mg/mL to 0.39 mg/mL. Note that at the concentration of 0.19 mg/mL, no significant differences were observed at 24 and 48 h of treatment, where proliferation seems to begin. On the contrary, this effect was not observed at 72 h (Figure 1A).

For the A172 cell line, this effect was evident at the concentrations from 6.25 mg/mL to 0.78 mg/mL while at the concentration of 0.39 mg/mL, no statistically significant differences were observed in comparison to the control group, while proliferation of cancer cells had begun.

Interestingly, regarding dose-dependent results and the concentration of 0.78 mg/mL, the effect of the extract was maximal at 72 h after treatment (Figure 1B).

In addition, common for both cell lines is the fact that, for TE671 cells and for the concentrations ranging from 6.25–0.39 mg/mL, the degree of the cytotoxic effect of the extract was the same. For A172 cells, the same was observed for the concentrations ranging from 6.25–1.56 mg/mL. Furthermore, when cells were treated with 0.78 mg/mL and at 24 and 48 h, although the extracts' cytotoxic activity was still evident, at the same time proliferation of cells had begun slightly. On the contrary, at 72 h of treatment, cancer-cells' viability had not considerably increased with respect to that of 24 and 48 h.

**Figure 1.** Dose-dependent and time-dependent effect of *R. officinalis* extract on TE671 (**A**) and A172 (**B**) cells. Data are presented as the mean ± standard error of the mean (SEM) (*n* = 8). The asterisk (\*) indicates significant differences between untreated and treated cells. The grey color corresponds to 24 h of treatment, the pink to 48 h and the light blue to 72 h.

IC50 value, thus, half of the maximal concentration of the tested extract required to inhibit growth and proliferation of cancer cells, was estimated. Dose–response curves regarding all the time points were constructed using a four-parameter logistic model. Normalized results are presented as log10 concentration in Figure 2A for the TE671 cancer cell line and in Figure 2B for the A172 cancer cell line. In the case of TE671 cells, the lowest IC50 value was estimated at 0.249 ± 1.09 mg/mL at 72 h after treatment with the extract. The IC50 values at 24 and 48 h were calculated at 0.287 ± 1.22 mg/mL and 0.274 ± 1.4 mg/mL, respectively. Regarding A172 cells, the lowest IC50 value was observed at 0.577 ± 0.98 mg/mL at 72 h after treatment. For the first 24 h, the IC50 value was calculated at 0.952 ± 1.11 mg/mL and after 48 h of treatment the corresponding value was found to be 0.871 ± 1.36 mg/mL. IC50 values decreased with increasing exposure time. The calculated values demonstrated that TE671 cells are more sensitive to the extract, since the IC50 value is lower than that of A172 cells. In addition, as is presented in Figure 2, the behavior of TE671 cells at all time points is almost the same, given the fact that IC50s do not differ considerably. On the contrary, for A172 cells, those values are rather close for the first 24 and 48 h; however, at 72 h, IC50 significantly decreases. This might be attributed to the population doubling time, which reached 80 h for TE671 cells and 40 h for A172 cells.

Microscopical investigation of TE671 is presented in Figure 3A–C. More precisely, in Figure 3A, cells are confluent since they have undergone any treatment, while at the concentration of 0.39 mg/mL cells are significantly reduced (Figure 3B). In Figure 3C, which corresponds to the concentration of 0.19 mg/mL, cells proliferation has begun. In the case of A172 cells, the same behavior was observed (Figure 4A–C). Figure 4A represents those cells that have received no treatment. At the concentration of 0.78 mg/mL, cells are less confluent (Figure 4B), while at the concentration of 0.39 mg/mL, proliferation of cells is evident (Figure 4C).

**Figure 2.** IC50 of *R. officinalis* extract on TE671 cells (**A**) and A172 cells at 24, 48 and 72 h (**B**). The lowest IC50 value for TE671 cell line was 0.249 ± 1.09 mg/mL, calculated at 72 h and 0.577 ± 0.98 mg/mL for A172 cell line, calculated at 72 h. Cancer -cell viability increases as concentration of the drug decreases.

**Figure 3.** Microscopic images of the TE671 rhabdomyosarcoma cells, grown for 72 h in DMEM with no other treatment (**A**), cells treated with 0.39 mg/mL of the extract (**B**) and cells treated with 0.19 mg/mL of the extract (**C**). Images were captured at ×200 magnification.

**Figure 4.** Microscopic images of the A172 glioblastoma cells, grown for 72 h in DMEM with no other treatment (**A**), cells treated with 0.78 mg/mL of the extract (**B**) and cells treated with 0.39 mg/mL of the extract (**C**). Images were captured at ×200 magnification.

#### **3. Discussion**

*R. officinalis* is a plant known for its potent antioxidant activity as it has been evaluated in many studies and with different antioxidant assays. Such activity is mainly attributed to the presence of rosmarinic acid, carnosic acid, carnosol and rosmanol [25,31,32]. It has been proposed that the catechol group of these compounds is responsible for their antioxidant activity [25].

*R. officinalis* belongs to the Lamiaceae family, a well-known family which includes a variety of plant species that contain a plethora of bioactive compounds [33]. *R. officinalis* leaves' extracts have been studied for their chemical composition and the presence of multiple compounds that belong to flavonoids (apigenin, genkwanin, scutellarein), phenolic diterpenes (carnosol, rosmanol, epirosmanol, carnosic acid), triterpenes (ursolic acid, betulinic acid) and caffeic acid esters (rosmarinic acid) has been reported [24,25].

Results reported in previous studies are in accordance with data presented here. In particular, in our study, chemical analysis of the hydroethanolic extract of *R. officinalis* showed the presence of rosmarinic acid, hydroxycinnamic acids, flavonoids and phenolic terpenes. According to the relative abundance as generated by the MassHunter software, carnosol was the compound presented in abundance followed by rosmarinic acid. Rosmanol, epirosmanol and rosmaridiphenol are metabolites derived from carnosic acid. Carnosol is an oxidized derivate of carnosic acid, produced via a non-enzymatic reaction [34]. References do report both the presence of carnosic acid and carnosol in *R.officinalis* plants [29,30,35]. Furthermore, many studies indicate that plants of the genus *Rosmarinus* grown in the Mediterranean, area are a very rich source of carnosic acid [25,36,37]. However, under extreme environmental conditions and in order for the plant to protect itself from various exogenous invasions, oxidative stress is unavoidable. That means that abiotic-induced stress was possibly the main reason for the oxidation of carnosic acid to carnosol, as well as for the presence of other oxidation metabolites, as previously reported [34,36].

Three are the most studied compounds isolated from *R. officinalis* extracts, carnosic acid, carnosol and rosmarinic acid. Carnosic acid is a compound commonly found in Lamiaceae species and was first isolated from *Salvia officinalis* [38]. Later, it was also found in abundance in rosemary which is yet considered as the richest source of all the Lamiaceae family plants. Chemically, carnosic acid is a phenolic diterpene and has been studied for its health-promoting properties, namely, antioxidant, antitumor, chemo-preventive, anti-inflammatory and hypoglycemic [39]. Carnosol belongs to phenolic diterpenes. It is a strong antioxidant, anticancer, chemo-preventive and anti-inflammatory agent [12,34,40]. The third well-studied compound of the plant is rosmarinic acid. Rosmarinic acid has been documented as a strong antioxidant and antimicrobial compound and it has also been tested against different cancer cell lines and against skin-irritating conditions such as atopic dermatitis [41–44]. Although the above three compounds are usually found in abundance in *R. officinalis* extracts, the therapeutic activity of the plant specifically regarding cancer, is not attributed only to these [45]. It has been observed that extracts from *R. officinalis* exert better antitumor activity with respect to its isolated compounds, precisely carnosol, ursolic and carnosic acid [46,47]. Interestingly, in the study of González-Vallinas et al., (2014) [47], a combination of carnosic acid and carnosol presented a better antiproliferative activity probably due to the synergistic effect of the two compounds. Given the cost advantages for a pharmaceutical company regarding the use of an extract rather than purified compounds and taking into account the above-mentioned findings, in this study a hydroethanolic extract derived from the leaves of the plant was used, to evaluate its cytotoxic activity against A172 and TE671 cancer cell lines. According to our knowledge, this is the first time that the cytotoxicity of *R. officinalis* has been evaluated against these two specific cell lines.

In our study, we observed that treatment with *R. officinalis* manifested a threshold-like mechanism, as it appeared that up to certain concentrations the extract manifested similar toxicity as the control sample and, on the other hand, after a certain "step" (0.39 mg/mL) the extract becomes effective. This phenomenon was not only dose-dependent but also time-dependent, as it manifested the same behavior at 24 h, 48 h and 72 h. This type of action is reported for the first time. Studies concerning the effects of *R.officinalis* on prostate cancer cells [48,49], melanoma [50] and in hematopoietic, epithelial, and mesenchymal tumor cell types [51] manifested a gradual dose-dependent type of action. Thus, the most interesting conclusion from these observations is that *R. officinalis* acts on tumor cell survival differently, depending on the cell type. All studies agree that the extract is effective against tumor cells, yet the fact that it acts in a cell-dependent manner urges towards a more in-depth investigation into its mechanics.

*R. officinalis* anticancer effects against glioblastoma cell lines have been previously described. U87MG has been used and it was shown that an aqueous extract of the plant (1/75 *v*/*v* dilution) managed to inhibit cancer cell proliferation by 42%. On the contrary, rosemary extract boosted the viability of mouse embryonic fibroblasts cells (MEF) by 9.5%. Authors compared the efficacy of the extract with that of etoposide, a highly toxic agent that causes myelosuppression. Etoposide reduced cell viability to a higher degree with respect to *R. officinalis*. However, authors also showed that co-treatment with the extract and etoposide does not influence the chemotherapeutic agent toxicity but increases cells rate inhibition. Nevertheless, rosemary extract does not seem to inhibit growth in MEF cells to the same degree as etoposide [52].

Carnosol was examined for its potent cytotoxic activity on the U87MG glioblastoma cell line. Using a range of concentrations between 100 nM–60 μM, carnosol not only significantly inhibited in a dose-dependent manner cancer cell viability at 48 and 72 h of treatment, but also its anti-proliferative effect continued even after washing the substance. Furthermore, the compound did not promote the metastasis of cancer cells. The same effect was also observed when cells were treated with a mixture of carnosol and temozolomide, an alkylating agent, used to treat brain tumors. In addition to this, carnosol potentiated the cytotoxic effect of temozolomide. Of note, also, is that carnosol did not affect the proliferation of healthy cells. In addition, since U87MG cell lines express the p53 gene, the possible involvement of carnosol in the p53-activation pathway was investigated. A re-activation of p53 and the concomitant activation of BAX protein and deactivation of Bcl-2 were observed [53]. The results of our study are in accordance with the abovementioned studies. The extract of *R. officinalis* inhibited the growth and proliferation of A172 glioblastoma cells. In contrast to the study of Giacomelli et al. (2016) [53], in our study, the extract exhibited its antiproliferative effect after 24 h of treatment and the peak

of its effect was observed at 72 h. Nonetheless, its cytotoxic potency did not outweigh that of carnosol expressed as IC50 values.

Chemotherapeutic treatment of rhabdomyosarcoma includes the use of agents such as doxorubicin, vinblastine, and etoposide. Combination of these drugs with rosemary extracts allowed to diminish the concentration of the chemical agent, thus reducing its toxic effects [45,54]. Regarding the cytotoxic activity of *R. officinalis* on rhabdomyosarcoma cell lines, there is lack of literature data. According to our results, the extract used is capable of inhibiting cancer cell proliferation by exerting its best activity at 72 h after treatment.

In general, many studies report the use of secondary metabolites against cancer [55–60].

Regarding the family of phenolic compounds, those belong to the most studied biomolecules. The anticancer activity of phenolic compounds has been demonstrated in a variety of malignant cell lines such as HT-1080 fibrosarcoma cell line, HT-44 melanoma cells, HT-20, HT-29 and DLD-1 colon cancer cells, MCF-7, MDA-MB 468 and 231, T47D breast cancer cells, PC-3 and LNC prostate cancer cells, HS-22 lung cancer cells, SGC-7901 gastic cancer cells, cervical cancer cells (HeLa), human leukemia (HL-60) and NB-4 promyelocytic leukemia cells, adenocarcinomic human alveolar basal epithelial cells (A549) and OAW adenocarcinoma cancer cells [58,61].

In our study, visual observation of microscopic images of the cells demonstrated that the extract exerts its cytotoxic activity by reduction in cell population. In addition, reduction in cell size was observable, as well as a nuclei fragmentation, which confirmed the observed cytotoxicity through the photometric method. This observation gave us a hint for the type of cell death caused by the extract, yet with more investigations in need to confirm.

A lot of mechanisms have been proposed to explain the cytotoxic effect of phenolic compounds. For example, phenolics chemoprotective/ anticancer activity is mainly due to their antioxidant and anti-inflammatory properties and many studies relate a phenolic rich diet with minor incidence of cancer development [62–65]. Many researchers have pointed out the potential of these molecules to interfere with crucial signaling pathways of the proliferation, migration, differentiation, apoptosis and angiogenesis of cancer cells [61,66–68]. For example, cinnamic and benzoic acid induce their antiproliferative effect on melanoma and breast cancer cells by interrupting the S and G2/M phase, respectively. Furthermore, caffeic acid, 5-caffeoylquinic acid, di-caffeoylquinic acid, ferulin and p-coumaric acid exert a potent antiproliferative effect against various cancer cell types [61]. In addition, cell death is another point that has been evaluated using phenolic compounds. Arrest of the cell cycle at Go/G1 phage, morphological changes in cancer cells; activation of apoptosis regulators such as caspaces and Bax protein and p53 and p21 genes; downregulation of transcription factors, such as transcription factor kappa B (NF-kB) and Bcl-2 (B-cell lymphoma 2) gene; and inhibition of enzymes vital for DNA transcription are some examples that confirm the potential of phenolic compounds to accelerate cancer cell death [63,69].

#### **4. Materials and Methods**

#### *4.1. Plant Material*

Plant material of *R. officinalis* was collected from the Botanical Garden of Philodassiki Enossi Athinon, at the foothills of Mt Hymettus (Attica, Greece). The living collection established in the Botanical Garden originated from a native population located in Ritsona area (eastern Sterea Ellas, Greece). Voucher specimen was deposited at the Herbarium of the Agricultural University of Athens (ACA), with the following label: Greece, Sterea Ellas, prefecture of Attiki, Botanical Garden of Philodassiki Enossi Athinon, alt. 360 m, 37◦57 N, 23◦47 E, 20.09.2016, Trigas 6327, ACA.

#### *4.2. Sampling Extraction*

Four grams of dried *R. officinalis* leaves were extracted as previously described by Kakouri et al., (2019) [70], in an ultrasonic water bath using a hydroethanolic solution (70% *v*/*v*). Extraction took place in triplicate.

#### *4.3. Total Phenolic Content and Antioxidant Activity*

Total phenolic content was performed using Folin–Ciocalteu reagent (0.2N) and gallic acid to construct the calibration curve. The experiment took place as previously described by Kakouri et al., (2019) [70]. Results were expressed as mg of gallic acid equivalents (GAE) per gram of dry material, derived from threefold measurements and according to the following equation:

$$\mathbf{y} = 0.0012\mathbf{x} + 0.012 \text{ (r = 0.998)}\tag{1}$$

The antioxidant activity was estimated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) and the 2,2 -azinobis [3-ethylbenzthiazoline-6-acid] (ABTS•+) radical scavenging assays. The experimental procedure followed that of Kakouri et al., 2019 [70]. For both the assays trolox was used as standard antioxidant. Results were expressed as IC50 values and according to the following equation:

$$\% \text{ Inhibition} = \text{(Acontroll} - \text{Asample)} / \text{Acontroll} \times 100 \tag{2}$$
