Cytotoxic Activity of Essential Oils of Aerial Parts and Ripe Fruits of Echinophora spinosa (Apiaceae) Daniele Fraternale, Donata Ricci, Cinzia Calcabrini, Michele Guescini, Chiara Martinelli and Piero Sestili Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy piero.sestili@uniurb.it Received: June 24 th , 2013; Accepted: September 3 rd , 2013 The cytotoxic effects of the essential oils obtained from the flowering aerial parts (APO) and ripe fruits (RFO) of Echinophora spinosa L. (Apiaceae) from central Italy toward human U937 promonocytoid cells were studied; the contribution of each of the major constituents to the whole cytotoxic activity of either APO or RFO was also characterized. The major components of APO were β-phellandrene (34.7%), myristicin (16.5%), p-cymene (16.3%), δ 3 -carene (12.6%), α-pinene (6.7%) and α-phellandrene (6.2%); those of RFO p-cymene (50.2%), myristicin (15.3%), α-pinene (15.1%) and α-phellandrene (8.1%). Both oils tested were toxic to U937 cells, but RFO was much more cytotoxic: indeed, the IC50 values calculated from the linear regression curves of RFO and APO were 14.5 ± 0.85 and 43.4 ± 2.81 μg/mL, respectively. α-Pinene and α-phellandrene were identified as the most toxically relevant constituents: however, they did not completely account for the toxic effects of genuine APO and RFO. Interestingly, we found that p-cymene, although per se devoid of toxicity within the tested range of concentrations, was capable of significantly sensitizing U937 cells to the cytotoxic activity of α-pinene and α-phellandrene, and that specific mixtures of these three terpenes were as toxic as genuine APO and RFO. Keywords: Apoptosis, Cytotoxicity, Echinophora spinosa, Essential oil, p-Cymene, U937 cells. Echinophora spinosa L. (Apiaceae) is an herbaceous perennial plant from the Mediterranean region, growing mainly on maritime sands. The plant is edible with a pleasant taste: thornless young and tender leaves are used for salads and the roots as carrots [1]. The literature shows very few studies of this species; one concerned the alleged content of phytosterols in the roots [2], and much more recently Kubeczka et al. [3] characterized, for the first time, the chemical composition of the essential oils of E. spinosa of unspecified origin. They reported that the main constituents of root oil were terpinolene (77.2%), myristicin (9.1%), limonene (4.3%) and falcarinol (2.9%), a widespread acetylenic compound in Apiaceae, while those of the aerial parts were α-phellandrene (36.8%), p-cymene (27.3%), α- pinene (15.0%), β-phellandrene (6.9%), limonene (2.6%) and cosmene (5.0%). Subsequently, only two works - namely those of Glamoclija et al. [4] and Fraternale et al. [5] - dealt with the biologically relevant effects of the essential oils of this plant, reporting their chemical composition and antimicrobial activity against some human and animal pathogens (bacteria and fungi). Indeed, several plants of the genus Echinophora are used in folk medicine to heal wounds and to treat gastric ulcers [6]. Interestingly, over the last decade, some essential oils from plants belonging to the Apiaceae have been tested for their cytotoxicity against tumor cell lines [7-10]. Ridolfia segetum and Oenanthe crocata oils were cytotoxic toward K562 cells [7]; the oil of Bupleurum marginatum, a herb indigenous to southern and southwestern China, exhibited a strong cytotoxicity to different cancer cell lines (CCRF, CEM, HePG2) [8]; Prangos asperula oil was active against human renal adenocarcinoma [9]; the essential oils from the fruits of four different chemotypes of Angelica archangelica growing in Iceland were shown to exert cytotoxic activity toward PANC-1 human pancreas cells [10]. Interestingly, with A. archangelica, this last study reported that the oils from two very similar chemotypes showed a markedly different cytotoxic activity [10], a finding suggesting that the cytotoxic capacity was independent of the specific amount of their main constituents, which were very similar in these two oils. In an attempt to interpret these data, the authors hypothesized that more components act together, synergistically or cumulatively. Such a hypothesis implies that, with regard to the cytotoxic activity, complex interactions may occur between the constituents of a given essential oil. Since the essential oils from some Apiaceae plants seem to have some potential in anticancer therapy, we investigated the cytotoxic activity of the hydrodistilled APO and RFO of Echinophora spinosa collected in central Italy on the beach (Fano-Marche) of the Adriatic sea, as well as that of their major components. The composition of these essential oils has been reported in a recent work [5] and is shown in Table 1. Twenty-three and twenty-six compounds, representing 98.7% of the total in both cases, were identified in APO and RFO oils, respectively. The major compound in APO is β-phellandrene (34.7%), followed by myristicin (16.5%), p-cymene (16.3%), δ 3 -carene (12.6%), α-pinene (6.7%) and α-phellandrene (6.2%), while the major compound in RFO is p-cymene (50.2%), followed by myristicin (15.3%), α-pinene (15.1%) and α-phellandrene (8.1%); Both oils were toxic to human promonocytoid U937 cells (Figure1A-1B): 48 h exposure to increasing concentrations of APO caused a marked decrease of U937 cell survival and, under the same conditions, RFO was much more cytotoxic. Indeed, the IC50 values calculated from the linear regression curves of RFO and APO cytotoxicity were 14.5 ± 0.85 and 43.4 ± 2.81 μg/mL, respectively. It is worth noting that the numbers of viable cells in samples treated for 48 h with the highest doses of RFO (9.2 ± 1.11 /10 -4 ) and APO (1.42 ± 0.13 /10 -5 ) were lower as compared to those at the beginning of the treatment stage (4.0/10 -5 ): such an extensive cell demise suggests that high doses of both oils are capable of causing cell death in treated cultures, rather than a simple growth arrest. The increased release of LDH, which depends on cell membrane disruption and might be suggestive of cell necrosis, in cultures exposed to the highest concentration (25 μg/mL) of RFO for 48 h (165.6% ± 7.2 as compared with control cells) strengthens this hypothesis. Using the Fast Halo Assay (FHA) [11] we next tested NPC Natural Product Communications 2013 Vol. 8 No. 11 1645 - 1649