**1. Introduction**

*Rosmarinus officinalis* L. (Lamiaceae), commonly known as rosemary, is a muchbranched, evergreen small shrub, usually 50–100 cm tall. It is native in the Mediterranean region and widely cultivated elsewhere for its essential oil, as well as ornamental purposes. Most Greek populations are probably naturalized and originated from cultivated plants, but at least some populations are considered native in the country [1].

Rosemary is considered a typical spice of the Mediterranean diet and it has been characterized as a functional ingredient [2,3]. Traditionally, rosemary leaves have been used against muscle, joint and rheumatism pain [4], as a stimulant and diaphoretic and for its flatulence-relieving properties [5,6]. Headaches, epilepsy, dysmennorhea, inflammation and spasmolytic conditions were also treated with rosemary [7,8]. Nowadays rosemary is among the most studied medicinal plants and its essential oil and extracts' therapeutic activity has been evaluated against various diseases [9,10]. In particular, *R. officinalis* extracts

**Citation:** Kakouri, E.; Nikola, O.; Kanakis, C.; Hatziagapiou, K.; Lambrou, G.I.; Trigas, P.; Kanaka-Gantenbein, C.; Tarantilis, P.A. Cytotoxic Effect of *Rosmarinus officinalis* Extract on Glioblastoma and Rhabdomyosarcoma Cell Lines. *Molecules* **2022**, *27*, 6348. https:// doi.org/10.3390/molecules27196348

Academic Editor: Nour Eddine Es-Safi

Received: 30 August 2022 Accepted: 21 September 2022 Published: 26 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have been studied for their antioxidant, anticancer, anti-inflammatory, and antimicrobial activity. Ameliorating the status of metabolic and central nervous system (CNS) disorders has also been evaluated [11–14].

Glioblastoma is an aggressive, malignant cancer of the CNS that originates from the glial cells, characterized by poor survival rate. One reason may be an intrinsic or acquired resistance to radiation and chemotherapy, as many brain tumors could intrinsically manifest a multidrug resistance (MDR) phenotype, thus resulting in relapses or disease progression [15,16]. Rhabdomyosarcoma forms at the soft tissues and more frequently affects the skeletal muscle tissue. It is generally considered a disease of childhood, as most cases are observed between the ages of 0–18 years old. Localized disease is associated with a good prognosis and an overall 5-year survival rate of over 80% with combined surgery, radiation therapy, and chemotherapy. However, in metastatic disease, prognosis is poor with a 5-year event-free survival rate of less than 30% [17,18]. It is the third most common extracranial tumor of the pediatric population, accounting for 4.5% of all cases of childhood cancers.

Both are considered rare types of cancers. Rare-type cancers comprise 22% of the reported cases of cancer [19]. Generally, among the difficulties that accompany a rare disease is the reluctance of pharmaceutical industries to invest time and, of course, a considerable amount of money for the development of a specific therapeutic treatment, since it will be addressed only to a small population. Therefore, one of the ongoing challenges is the continuous gaining of experimental data that will significantly contribute and facilitate the design of specific pharmaceutical formulations. However, independent of the cost required for the research of new pharmacologically active compounds, it should be taken under consideration that many cancer cells are resistant to current therapy due to mutations. Although current therapeutic approaches aim to alleviate symptoms, increase life expectancy and maintain the progression of the disease in remission, they are not few the cases of synthetic formulations leading to severe side effects that are not associated to the disease itself. Consequently, because of limitations regarding the many side effects that impair quality of daily life, cancer drug resistance, rapid increase in the percentage of cancer mortality and numerous new cases diagnosed, scientists are driven towards the development of new therapeutic agents, with fewer or no side effects, to be used as monotherapy or together with current available treatment. To this end, natural products and, in particular, those found in abundance in nature or are easy to cultivate, consist a new area of research, since most of the times the cost of the raw material is affordable and the side effects are usually minimized [20,21].

Given the acquired knowledge from Traditional medicine and the continuous interest in *R. officinalis* as a potential therapeutic agent, the present study aims to evaluate the cytotoxic effect of a hydroethanolic extract of *R. officinalis* against A172 glioblastoma and TE671 rhabdomyosarcoma cell lines, since its effect against these two cancer types has not been previously reported. The biologic activity of a plant is attributed to its chemical profile. However, the chemical profile is strongly dependent on many parameters [22,23]. Therefore, although the chemical characterization of *R. officinalis* extracts has already been given in previous studies [24–26], here, we present again the chemistry of the extract used, alongside its total phenolic content and its antioxidant activity.

#### **2. Results**

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

The extract contains a considerable number of phenolic compounds that corresponds to 35.65 ± 0.03 GAE/g. The extract also exhibited a notable antioxidant activity. The IC50 value calculated for the DPPH assay was 12.8 ± 2.7 μg/mL while for the ABTS assay the IC50 value was estimated at 6.98 ± 1.9 μg/mL.

#### *2.2. Identification of Secondary Metabolites by LC/Q-TOF/HRMS Analysis*

Although the chemistry of *Rosmarinus officinalis* is known, we report again its chemical profile, since not only does it depends upon the area, season, and extraction method but it also is essential for explaining its biological activity on A172 and TE671 cell lines.

Characterization of the compounds presented in *R. officinalis* extract was performed with the LC/Q-TOF/HRMS analysis. Most of the compounds identified were flavonoids and phenolic terpenes. Data obtained from the ESI (+) and the ESI (−) ionization mode are summarized in Table 1. Information regarding the generated ms/ms fragmentation process is given as Supplementary Materials. Identification of the compounds detected was based on data obtained from the MassHunter Workstation Software and literature data [26–30].

**Table 1.** Tentatively identified compounds of *Rosmarinus officinalis* leaves at the positive and negative ionization mode.



n.d: not detected.
