Anticancer Activity of Essential Oils and Other Extracts from Aromatic Plants Grown in Greece

Aromatic plants have a long and significant history in the traditional medicine of many countries. Nowadays, there is an increasing interest in investigating the biological properties of aromatic plant extracts mainly due to their diversity, high availability, and low toxicity. Greece is abundant in aromatic plants, which can be attributed to the country’s geographical position, the morphology of its landscape, and its numerous mountainous and insular areas. In the past 15 years, a number of aromatic plant extracts of Greek origin have been studied for their bioactivities, including their antiproliferative potential against different types of cancer. Although the pharmacological activities of specific species of Greek origin have been reviewed before, no gathered information on explicitly Greek species exist. In this review, we summarize existing data on the antiproliferative activity of extracts isolated from Greek aromatic plants and discuss their molecular mode(s) of action, where available, in order to identify promising extracts for future research and link chemical constituents responsible for their activity. We conclude that essentials oils are the most frequently studied plant extracts exhibiting high diversity in their composition and anticancer potential, but also other extracts appear to be worthy of further investigation for cancer chemoprevention.


Introduction
Aromatic plants take their name from the distinct aroma they produce, which is attributed to a mixture of volatile compounds extracted as essential oils. They constitute 10% of the plant kingdom (17,000 species) and are distributed all over the world, mostly in temperate and warm countries [1]. Greece is abundant in aromatic plants. Its geographical position, the morphology of its landscape, and the numerous mountainous and insular areas (109 out of 225 habitat types encountered in the European Union are found in Greece) [2] contribute to the country's high plant diversity and endemism (6600 species and subspecies) [3], explaining its long-documented ethnopharmacological history. Most of the plants collected for research purposes in the Greek area have been obtained from Central Greece and Crete and belong to the Lamiaceae family [4]. In recent years, increased interest has been focused on studying the biological properties of aromatic plant extracts mostly due to their high availability and few side-effects. Among their reported properties is their ability to affect proliferation of cancer cells and induce apoptosis, one of the most important features of cancer chemoprevention.
In Greece, cancer is the second leading cause of death after cardiovascular diseases. With a population of approximately 11,000,000, there was an expected number of 67,400 new cancer cases and 33,200 deaths in 2018 (~6% and 3% of the population, respectively) [5]. Although overall cancer rates have not increased since 2014 (~29,000 deaths), there has been an increase in the number Pistacia lentiscus var. Chia, commonly known as Chios mastic, is the plant mostly studied (nine studies) and is the only one with available and more detailed information on its molecular mode(s) of action on various cancer models. Pistacia lentiscus var. Chia is a variety endemic to Chios island, with high economic value as it is the only variety that produces resin when its bark is scored. When the essential oil of the resin was incubated with breast, colon, and ovarian adenocarcinoma cell lines (MCF-7, LoVo, 2008) for 3 h, followed by a 21 h recovery period, it demonstrated the weakest activity compared to other Pistacia species originating from other countries (EC 50 > 500 µg/mL) [13]. The essential oil has been also studied against human leukemic and melanoma cells and mouse lung carcinoma (K562, Lewis lung carcinoma cells, B16, respectively) and also in vivo in a lung cancer mouse model. These studies revealed that mastic oil reduces cell viability and tumor growth. In addition, it was shown that it induces apoptosis in cancer cells, reduces the expression of chemokines, limits their invasive and adhesive potential, and is an inhibitor of angiogenesis via the reduction of VEGF expression and neovascularisation [14][15][16][17]. When the oil was tested against colon carcinoma, it showed significant dose-and time-dependent cytotoxicity in vitro and tumor reducing capacity in vivo, which was higher than the activity of its major components, α-pinene and myrcene, tested either alone or in combination in the ratio found in the oil mixture. Furthermore, downregulation in the levels of the proliferation markers Ki-67 and survivin and attenuation of cancer cell migration was observed [18]. A hexane and an ethanol extract of mastic have been also studied for their anticancer potential in vitro against a human colon cancer cell line (HCT116) and in vivo in an experimental colon cancer mouse model. Both extracts caused dose-dependent inhibition of cell growth, while the hexane extract also caused tumor growth suppression. Furthermore, the extracts caused G1 cell cycle arrest, induction of apoptosis via the activation of caspases 3, 8 and 9, while it was found that the cell death caused by the ethanol extract is independent of p53, Bcl-2, and FADD [19][20][21].  Citrus medica (citron) is an evergreen shrub or small tree that produces citron, probably the only citrus fruit known in Europe in ancient times that has been extensively used for its medicinal properties as reviewed in detail in [41]. It was found that the oil of citron is cytotoxic against human cancer cell lines in vitro, especially against the colon carcinoma Caco2, while it affected a non-tumorigenic human cell line to a lesser extent. Its cytotoxicity was higher compared to its major component, limonene [24].
Lippia citriodora (or Aloysia citriodora, Aloysia triphylla), commonly known as lemon verbena, belongs to the Verbenaceae family. It was cultivated in South and Central America and was brought to Europe in the 17th century [42]. Its leaves are mainly used for the preparation of infusions and decoctions, usually consumed for the relief of gastroinstestinal problems, but they have also been used as anti-spasmodic, diuretic, or sedatives [43]. There are published data on the antiproliferative activity of the essential oil from plants of different origin (Morocco and Colombia), but only one study on the bioactivities of the Greek oil exists. Lemon verbena oil demonstrated high antiproliferative activity against a panel of human cancer cell lines (A375 > Caco2 > HepG2 > MCF-7 > THP-1), which was lower than its major component, citral, possibly due to the lower DNA-damaging effect it was shown to possess [23].
Satureja parnassica spp. parnassica is an aromatic plant endemic to Central and Southern Greece. Although literature is sparse regarding its activity, existing studies reveal a potent antimicrobial and insecticidal profile of its essential oil [44,45]. Satureja thymbra (common names: Thyme-leaved savory, pink savory, thrubi) is an aromatic plant endemic to the Mediterranean region and is the most common specimen of the Satureja genus. It is regarded as oil-rich and its essential oil has been tested against several models and was shown to possess potent antioxidant, antifungal, antibacterial, antiviral, and insecticidal properties [44][45][46][47][48][49]. In a study published in 2016, the antiproliferative activity of the essential oils of S. thymbra and S. parnassica collected after their flowering season (August and September, respectively) and their major components were investigated [28]. It was shown that the oil of S. thymbra was more cytotoxic against MCF-7 cells, possibly due to its higher content in carvacrol, a phenol with significant bioactivities [50].
Origanum vulgare spp. hirtum (also known as Greek oregano) is considered the best for culinary purposes. It is the most common O. vulgare subspecies in the Greek area and has shown great variation in its chemical composition depending on the cultivation area [51]. Its essential oil was studied against two normal cell lines of monkey and rabbit origin (Vero, RSC, respectively) and two human cancer cell lines (Hep-2, HeLa) without exhibiting any cancer-specific cytototoxicity. In fact, the oil was found to be more cytotoxic against Vero cells compared to the other cell lines [29].
Another member of the Origanum genus, Origanum Onites L., is commercially known as Turkish oregano and can be mainly found in Turkey, but also in Greece and a small part of Sicily. Different extracts of the plant are used for the relief of common health problems and this has led to the investigation of their pharmacological properties, as well (reviewed in [52]). In a recent study, the essential oil of O. Onites isolated from plants collected in Northern Greece has shown dose-dependent antiproliferative activity in vitro, especially against the colon cancer cell line HT-29 (followed by A375, MCF-7 and HepG2 carcinomas). Treatment with the oil caused time-dependent cytotoxicity against HT-29 and CT26 cells (murine colon cancer cell line), attenuation of their migration, and apoptosis-like features, while oral administration for 13 days significantly inhibited the growth of CT26 tumors in mice [30].
Origanum dictamnus (dittany of Crete), is a perennial plant endemic to the island of Crete that grows at different altitudes. The essential oil of the plant demonstrated time-dependent cytotoxicity against the LoVo colon cancer cell line, while it reduced the viability of HepG2 cells [31,32]. A dichloromethane residue was almost two-fold more cytotoxic against the leukemic cells P-388 compared to the lung carcinoma NSCLC-N6; however, the ethanol extract appeared to be almost inactive against these cell lines. The difference in their activity could be attributed to the presence of ursolic acid in the dichloromethane residue, which was shown to be more cytotoxic than the residue against the cancer cell lines [33]. Finally, the herbal infusion of the aerial parts of the plant was the most cytotoxic against HT-29 and PC-3 cells compared to infusions from other Greek plants [34].
In the same study, Rosmarinus officinalis (rosemary), Thymus vulgaris (thyme), Origanum majorana (marjoram), Salvia officinalis (sage), and Hypericum perforatum (St John's Wort) were also studied. The infusions of marjoram and St John's wort were the least cytotoxic against HT-29 cells, while PC-3 were the least sensitive against thyme. St John's Wort in HT29 and thyme in PC3 cells caused reduction of the levels of the p65 NF-kB subunit and all infusions decreased the levels of the pro-inflammatory interleukin 8, in an order depending on the cell line. The authors could not make a correlation between the antiproliferative profile of the extracts and their composition.
A similar study was conducted a year before, in 2013, where the antiproliferative activity of herbal infusions from leaves and flowers of the plants Origanum vulgare, Origanum microphyllum, Satureja thymbra (pink savory), Sideritis syriaca (mountain tea), Mentha pulegium (pennyroyal), and Matricaria chamomilla (chamomile) was tested [39]. The authors showed that pink savory was the most cytotoxic against HT-29 and PC-3 cells, while chamomile infusion and chamomile together with mountain tea were the least effective against HT-29 and PC-3 cells, respectively. However, the infusion of chamomile was the only one that caused statistically significant reduction of the p65 NF-kB subunit in HT-29 cells. In addition, all infusions were able to decrease the levels of interleukin-8 in the following order: HT-29: mountain tea > pennyroyal > chamomile > Origanum microphyllum > pink savory> Origanum vulgare, PC-3: Origanum microphyllum > pennyroyal > pink savory > Origanum vulgare > chamomile > mountain tea.
St. John's Wort is mainly known for its use against depression and anxiety; however, there is emerging evidence for its potential against cancer, too [53]. The aqueous and methanolic solution (AS and MS) as well as the methanolic and petroleum ether extracts (ME and PEE) of St John's Wort from Epirus were tested against the urinary bladder carcinoma T24 and the rat urinary bladder carcinoma NBT-II. The MS was more potent than the AS against T24 cells and the same applied for the PEE compared to the ME. The PEE was also more cytotoxic than the ME against the NBT-II cell line, which is attributed to yet unidentifiable components of the herb. PEE was also shown to induce apoptosis in both cell lines using the TUNEL assay [36]. A methanolic extract of the plant together with methanolic extracts from four other species of the genus Hypericum (H. empetrifolium, perfoliatum, rumeliacum, triquetrifolium) showed diverse antioxidant activity and cytotoxicity against Caco2 and HepG2 cells, with the extracts of H. empetrifolium Wild from three different locations being the most cytotoxic; however, the concentrations used were very high [37]. Methanolic extracts from aerial parts of different St John's wort's chemotypes from different locations (cultivated and wild) were tested against Caco2 cells [35]. The highest activity was presented by one of the cultivated chemotypes, followed by one of the wild populations, while the other extracts did not affect cell viability at all. They also included a commercial sample in their analysis, but it was not of Greek origin. The authors found positive correlation between the antiproliferative activity and the content in rutin and hyperforin, two main components of Hypericum species. Differences in their content were attributed to different environmental conditions at the growing sites of the plants.
The genus Teucrium can be found worldwide but mostly in the Mediterranean region. Menichini and her colleagues in 2009 studied the antiproliferative potential of four Teucrium species from Greece [38]. More specifically, the essential oil from the aerial parts of T. brevifolium, T. flavum, T. montbretii ssp. Heliotropiifolium, and T. polium ssp. capitatum were isolated and studied against lung carcinoma COR-L23, colorectal adenocarcinoma Caco2, and amelanotic melanoma C32. They observed that T. brevifolium oil was the most potent against COR-L23 cells, while Teucrium polium ssp. capitatum was the most potent against Caco2 and C32 cells. The authors attributed the diverse activity to the different classes of compounds present in the oils (different classes of sesquiterpenes). All oils also exhibited anti-inflammatory activity, being able to inhibit LPS-induced NO production, with T. brevifolium possessing the highest capacity.
Crocus sativus L. is an aromatic flowering plant, which produces saffron spice, the most expensive spice in the world, known as "red gold" [54]. The herb has been widely used by ancient Indian, Arabic, and Chinese civilizations as medicine, but is also studied nowadays for its therapeutic potential against several pathological conditions including depression, diabetes, and cancer [55]. In a recent study, Giakoumettis and his colleagues investigated the antiproliferative effect of an ethanolic saffron extract against rat glioma cells C6 alone or in combination with the chemotherapeutic drug temozolomide (TMZ). They showed that the saffron extract is cytotoxic against the cell line after 48 h of treatment and in some combinations, it enhances the cytotoxicity of the drug, too. In addition, they demonstrated that the extract does not induce apoptosis, but calpain-dependent death, possibly through the activation of autophagy and also impairs the colony formation potential of the cells [22].
The genus Marrubium consists of approximately 50 species native to temperate regions of the world. The methanolic extract of the species Marrubium thessalum Boiss. & Heldr from Olympus Mountain was tested against four commonly diagnosed cancer types: Breast adenocarcinoma MCF-7, cervix adenocarcinoma HeLa, colon carcinoma HCT116, and melanoma FM3. It was found to be more cytotoxic against HeLa cells, while exhibiting similar but lower cytotoxic potency in the other cell lines. In addition, the methanolic extract was also tested against human peripheral blood mononuclear cells (PBMCs) where it was found to be the most cytotoxic compared to some of its phenolic compounds (acteoside, leucosceptoside A, forsythoside B, alyssonoside, isoacteoside decaffeoyl-acteoside) indicating that it does not selectively kill cancer cells, in contrast to its components, which are promising agents for further studies [27].
Another member of the Lamiaceae family, the genus Ocimum consists of 30 species that are cultivated all around the world. Ocimum basilicum, also known as sweet basil, is one of the most popular food spices in the world and has been studied for its antimicrobial and pharmaceutical properties [56]. In 2014, the essential oil and ethanolic extract of sweet basil from Greece were studied for their action against the cervix adenocarcinoma HeLa, melanoma FemX, chronic myelogenous leukemia K562, and ovary adenocarcinoma SKOV3 cells lines [26]. The researchers observed that both extracts showed similar activity against SKOV3 and K562 cells, while the oil was two-fold more cytotoxic against HeLa and FemX cells. The oil exhibited the highest and similar activity against HeLa and FemX cells, followed by K562 and SKOV3. The ethanolic extract, on the other hand, was more cytotoxic against K562 cells, followed by HeLa, FemX, and SKOV3. Their activities were considered "mild but notable" compared to their main components. In general, with the exception of few cases, the components exhibited more significant activity and were considered to be responsible for the activity of the extracts. Those components were caffeic and rosmarinic acids for the ethanolic extract and eugenol and isoeugenol for sweet basil oil. Furthermore, the chemical composition of basil oil together with the essential oils isolated from the plants Fortunella margarita (kumquat), Mentha spicata (spearmint), and Pimpinella anisum (anise) was analyzed and their biological properties (antimicrobial, antioxidant, antiproliferative) were determined [25]. All of these plants are widely used for culinary purposes in Greece and as flavorings in beverages, gums, ice creams, etc. None of the essential oils exhibited significant antioxidant activity in vitro, whereas their cytotoxicity depended on the cell line used (HepG2, MCF-7, Caco2, THP-1). In general, sweet basil oil was the most cytotoxic against most of the cancer preclinical models used in the study, while kumquat and anise oils were the most efficient against THP-1 cells. Spearmint oil demonstrated an intermediate effect and it was the least cytotoxic against THP-1 cells.

Conclusions
Cancer incidence and mortality are constantly increasing, and novel strategies are required for its prevention and treatment. Products of natural origin have been used for their medicinal properties since ancient times and in the last years interest in these properties has increased. Currently, numerous studies exist investigating their pharmaceutical potential, including their chemopreventive and chemotherapeutic effect. Although the number of aromatic plant extracts of Greek origin that have been studied for their antiproliferative activity is limited, existing data against several cancer types are promising and encourage further exploitation of the country's unique flora diversity. Undoubtedly, further exploration of the molecular mechanisms of action of the extracts and their components is required not only in vitro but in vivo as well, to verify and establish their activity for their future use in the battle against cancer.