Cladanthus scariosus Essential Oil and Its Principal Constituents with Cytotoxic Effects on Human Tumor Cell Lines

Cladanthus is a small genus of the Asteraceae family comprising just five species that, apart from Cladanthus mixtus (L.) Chevall., has a large distribution in all the Mediterranean countries, mainly in the North Africa area. Several ethnopharmacological uses have been reported for species of this genus. Notably, Cladanthus scariosus (Ball) Oberpr. & Vogt is endemic to Morocco. Seeking to delve deeper into the phytochemistry and pharmacological aspects of this species, in this work, we investigated the essential oil (EO) obtained from the aerial parts of a locally sourced accession, hitherto unexplored, growing wild near Tizi n’Ticha, Morocco. The chemical composition of the EO, obtained by the hydrodistillation method, was evaluated by GC and GC-MS. The most abundant EO constituent was germacrene D (13.2%), the principal representative of the sesquiterpene hydrocarbons class (27.2%). However, the major class of constituents was monoterpene hydrocarbons (43.0%), with α-pinene (11.9%), sabinene (10.2%), p-cymene (8.5%), and α-phellandrene (5.2%) as the most abundant. The EO and its main constituents have been tested for their possible cytotoxic activity against three human tumor cell lines (MDA-MB 231, A375, and CaCo2) using the MTT assay, with corresponding IC50 values of 13.69, 13.21, and 22.71 µg/mL, respectively. Germacrene D and terpinen-4-ol were found to be the most active constituents with IC50 values between 3.21 and 9.53 µg/mL. The results demonstrate remarkable cytotoxic activity against the three human tumor cell lines studied, and in the future, further analyses could demonstrate the excellent potential of C. scariosus EO as an antitumor agent.

Actually, five species are accepted in the genus Cladanthus [3] and their distribution as well as their synonymous variants are reported in Table 1.Several ethnopharmacological uses have been reported for the species of this genus.C. eriolepis is endemic to Morocco and used by the local population against respiratory problems and as a poultice against bee stings [4,5].C. eriolepis is also used to heal various health conditions including gastrointestinal disorders, stomach ulcers, hypertension, and helminthiasis [6].C. arabicus, instead, is used in the treatment of diabetes in the High Atlas Central of Morocco [7] and of icterus [8].C. mixtus, known as Moroccan chamomile, is very abundant in the Gharb region [9][10][11], and the collected plants are used for the extraction of an essential oil used in perfumery and cosmetics, with Morocco being the only supplier of this product on the international market [9].Moroccan chamomile leaves and flowers have been largely used in traditional Moroccan medicine as an infusion to treat various ailments as an analgesic, antiallergic, anti-inflammatory, antispasmodic, carminative, digestive, febrifuge, fungicide, sudorific, vermifuge, and stimulant of leukocyte production [12][13][14].Several ethnomedicinal surveys report its peculiar uses in different parts of Morocco.In the region of Rabat, it is utilized to treat metabolic diseases [15], in Agadir for diabetes [16], in Sidi-Boughaba as a stomachic, anthelmintic, antidiabetic, and anxiolytic medicine and as a treatment for nervous breakdowns and hepatic and gastric insufficiencies [13], and in Fez for digestive and neurological troubles [17].
C. scariosus (synonyms: Chamaemelum scariosum (Ball) Benedi; Ormenis scariosa (Ball) Litard & Maire, Basionym = Santolina scariosa Ball) is endemic to Morocco.It is a perennial plant with a strong aromatic odor, ranging from 30 to 60 cm in height; the stems are erect and ended by flower heads with orange-yellow ligules (Figure 1).This species is quite common in open areas on sandstone and is found in the Moroccan High Atlas [18].Apart from C. flahaultii, which is totally devoid of any investigation, all the other four taxa have been studied for the composition of their essential oils (EOs) and for some biological activities as reported in Table 2.  C. scariosus, known in Morocco by several vernacular names ("Irezghi, Irezgui, Itzghi, Ifskin'uarras, Gartûfa"), is generally used to treat all disorders where spasms are important symptoms; it has tonic, stomachic, analgesic, and antispasmodic properties [18].Further-more, the tea of its leaves and inflorescences is used for gastrointestinal, gynecological, and pediatric troubles and for the treatment of diabetes [19,20].

Chemical Composition of C. scariosus EO
Hydrodistilled C. scariosus EO, obtained from fully flowering aerial parts of the plant, had a dark blue color.Overall, forty-seven different compounds were identified and listed in Table 3.
The EO was found to be very rich in monoterpene hydrocarbons (43.0%) with αpinene (11.9%), sabinene (10.2%), p-cymene (8.5%), and α-phellandrene (5.2%) as the most abundant compounds.However, the major constituent was germacrene D (13.2%), the principal constituent of sesquiterpene hydrocarbons (27.2%), whereas terpinen-4-ol (8.8%) was the principal component among the oxygenated monoterpenes (11.7%).Chamazulene, responsible for the blue color of the EO, was present at 4.0%.Fortunately, the response factor obtained from the appropriate calibration lines confirmed the percentage of α-pinene, p-cymene, germacrene D, and terpinene-4-ol compounds from the integrals of the areas of the GC-TOF/MS chromatogram (Table 2).The data obtained in this work basically agreed with the compositions of the EOs from other accessions of C. scariosus previously published [18,48,49], although some differences should be noted.As can be seen from Table 2, the major compounds identified in C. scariosus, such as α-pinene, sabinene, chamazulene, and germacrene D (Table 3), are present in all the other accessions collected in different places in the Moroccan territory.They could be considered exactly as markers of the species in question.

Effects of C. scariosus EO on Tumor Cell Viability
In the literature, no data are available about the cytotoxic activity of C. scariosus EO.For the first time, we here report the effect of this EO against a panel of human cancer cells and a non-tumor cell line.Three human cancer cell lines, a human breast adenocarcinoma (MDA-MB 231), a human malignant melanoma (A375), and a human colon adenocarcinoma (CaCo2), and one human endothelial cell line (EA.hy926) were treated with different concentrations of EO and its main compounds such as germacrene D, α-pinene, terpinen-4-ol, and p-cymene for 72 h.An MTT assay was used to evaluate the cytotoxic activity of EO, and the results obtained are shown in Table 4 and Figure 2. C. scarious EO showed antiproliferative activity against all three cell lines with IC 50 values of 13.69, 13.21, and 22.71 µg/mL for MDA-MB 231, A375, and CaCo2, respectively.Based on the criteria of the American National Cancer Institute for considering a crude extract promising for further purification, the IC 50 value being lower than 30 µg/mL [50] suggests that the EO shows excellent activity against tumor cells.Such a cytotoxic activity of EO could also be attributed to its main components.Indeed, in our experiments, germacrene D and terpinen-4-ol were the most active on all three cell lines, with IC 50 values ranging from 3.21 to 5.23 µg/mL for terpinen-4-ol and from 4.41 to 8.57 µg/mL for germacrene D on MDA-MB 231 and CaCo2, respectively.
Noteworthy, the cytotoxic activity of terpinen-4-ol is comparable with that of cisplatin, showing IC 50 values of 2.54 and 3.07 µg/mL on MDA-MB 231 and CaCo2 cell lines, respectively.Germacrene D showed IC 50 values slightly higher than IC 50 values for terpen-4-ol, and α-pinene showed remarkable activity on MDA-MD 231 (IC 50 value, 12.40 µg/mL; 91 µM) and on A375 (IC 50 value 17.04, 125 µM).p-Cymene exerted lower cytotoxic activity.The data are in good agreement with the reported antiproliferative activity of terpinen-4-ol against the human acute promyelotic leukemia cell line HL60 [51], human non-small-cell lung cancer cell line HNCLC [52], and human leukemic MOLT-4 cell line [53], where terpinen-4-ol induced apoptotic and macrophagic cell death.Terpinen-4-ol was active on melanoma, as reported in the literature [54]; it potently induces cell cycle arrest, apoptosis, and necrotic cell death [55].Germacrene D was reported to be active on human breast adenocarcinoma (MDA-MB 231 and MCF-7), human ductal carcinoma (Hs578T), and human hepatocellular carcinoma (HepG2) [56].α-Pinene has also been reported to exhibit apoptotic and antimetastatic activity on melanoma cells [57].In the literature, sabinene also exerts cytotoxic activity against liver (HepG2), colon (HCT116), and breast (MCF7) cancer cell lines [58].From the data reported above on the antiproliferative activities of the main compounds of the EO, it could be hypothesized that the EO activity may involve the activation of apoptotic processes, a study project that could be explored further later.

Effects of C. scariosus EO on Tumor Cell Viability
In the literature, no data are available about the cytotoxic activity of C. scariosus EO.For the first time, we here report the effect of this EO against a panel of human cancer cells and a non-tumor cell line.Three human cancer cell lines, a human breast adenocarcinoma (MDA-MB 231), a human malignant melanoma (A375), and a human colon adenocarcinoma (CaCo2), and one human endothelial cell line (EA.hy926) were treated with different concentrations of EO and its main compounds such as germacrene D, αpinene, terpinen-4-ol, and p-cymene for 72 h.An MTT assay was used to evaluate the cytotoxic activity of EO, and the results obtained are shown in Table 4 and Figure 2.Although the presence of germacrene D (13.2%), α-pinene (11.9%), sabinene (10.2%), terpinen-4-ol (8.8%), and p-cymene (8.5%) may explain the cytotoxic activity of C. scariosus EO, it cannot be excluded that even the presence of lower-concentration components might contribute to the final cytotoxic activity observed.
Although the cytotoxic activity of the essential oil is significantly relevant to tumor cell lines, its specificity is not very high.In fact, its cytotoxic activity on the normal vascular endothelial line EA.hy926 is only slightly lower than that of MDA-MB 231 and A375.Perhaps a better comparison can be made with a normal cell line of ectodermal derivation given that the three tumor lines tested are all of epithelial origin.

Plant Material
Aerial parts from several fresh individuals of C. scariosus, at the full flowering stage, were collected near Tizi n'Ticha, Morocco, at about 2170 m a.s.l., 31

Isolation of EO
Fresh aerial parts (660 g) were subjected to hydrodistillation for 3 h, according to the standard procedure described in European Pharmacopoeia [59].The EO was dried over anhydrous Na 2 SO 4 and preserved at 4 • C prior to further analysis (up to one month).Samples yielded 0.15% of EO.Hydrodistilled C. scariosus EO, obtained from fully flowering aerial parts of the plant, had a dark blue color (Figure 3).Although the cytotoxic activity of the essential oil is significantly relevant to tumor cell lines, its specificity is not very high.In fact, its cytotoxic activity on the normal vascular endothelial line EA.hy926 is only slightly lower than that of MDA-MB 231 and A375.Perhaps a better comparison can be made with a normal cell line of ectodermal derivation given that the three tumor lines tested are all of epithelial origin.

Plant Material
Aerial parts from several fresh individuals of C. scariosus, at the full flowering stage, were collected near Tizi n'Ticha, Morocco, at about 2170 m a.s.l., 31°17′18″ longitude N and 7°22′55″ latitude W, in May 2023.One of the samples, identified by Prof. Vincenzo Ilardi, has been stored in the University of Palermo Herbarium (No. PAL 109762).

Isolation of EO
Fresh aerial parts (660 g) were subjected to hydrodistillation for 3 h, according to the standard procedure described in European Pharmacopoeia [59].The EO was dried over anhydrous Na2SO4 and preserved at 4 °C prior to further analysis (up to one month).Samples yielded 0.15% of EO.Hydrodistilled C. scariosus EO, obtained from fully flowering aerial parts of the plant, had a dark blue color (Figure 3).

GC-MS Analysis and Quantitative Determination
Analysis of EOs was carried out according to the procedure reported by Lauricella et al. [60].The composition of the EO was determined by GC-MS analyses.They were achieved on an Agilent Technologies 7890 GC equipped with FID and mass spectrometer detectors using a DB-5MS (5% phenylmethylpolysiloxane) capillary column (30.00 m × 0.25 mm, 0.25 µm film thicknesses; J & W Scientific, Folsom, CA, USA).The carrier gas was helium at a flow rate of 0.8 mL/min.The initial column temperature was 60 °C and programmed to increase up to 280 °C at a rate of 4 °C/min.The split ratio was 40:1.The injector temperature was set at 300 °C.The acquisition range was 50-550 m/z in electronimpact (EI) mode using an ionization voltage of 70 eV.The EO was diluted 1:100 in nhexane, and then 0.1 µL was injected into the GC system.
Six calibration solutions containing α-pinene, terpinen-4-ol, germacrene D, and pcymene were prepared in hexane at a concentration (w/w %) of 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0%.These calibration solutions contained the compounds, except for the unavailable sabinene constituent, present in greater abundance in the EO sample.Linear retention indices (LRIs) were calculated using a mixture of pure n-alkanes (C8-C40), and all the peaks' compounds were identified by comparison with MS and by comparison of their relative retention indices with WILEY275, NIST 17, ADAMS, and FFNSC2 libraries.

GC-MS Analysis and Quantitative Determination
Analysis of EOs was carried out according to the procedure reported by Lauricella et al. [60].The composition of the EO was determined by GC-MS analyses.They were achieved on an Agilent Technologies 7890 GC equipped with FID and mass spectrometer detectors using a DB-5MS (5% phenylmethylpolysiloxane) capillary column (30.00 m × 0.25 mm, 0.25 µm film thicknesses; J & W Scientific, Folsom, CA, USA).The carrier gas was helium at a flow rate of 0.8 mL/min.The initial column temperature was 60 • C and programmed to increase up to 280 • C at a rate of 4 • C/min.The split ratio was 40:1.The injector temperature was set at 300 • C. The acquisition range was 50-550 m/z in electron-impact (EI) mode using an ionization voltage of 70 eV.The EO was diluted 1:100 in n-hexane, and then 0.1 µL was injected into the GC system.
Six calibration solutions containing α-pinene, terpinen-4-ol, germacrene D, and pcymene were prepared in hexane at a concentration (w/w %) of 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0%.These calibration solutions contained the compounds, except for the unavailable sabinene constituent, present in greater abundance in the EO sample.Linear retention indices (LRIs) were calculated using a mixture of pure n-alkanes (C 8 -C 40 ), and all the peaks' compounds were identified by comparison with MS and by comparison of their relative retention indices with WILEY275, NIST 17, ADAMS, and FFNSC2 libraries.

MTT Assay
Cell viability was examined by the ability of the cells to cleave the tetrazolium salt MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide] using the mitochondrial enzyme succinate dehydrogenase following the procedure described earlier [62].Briefly, cells were seeded at a density of 2 × 10 4 cells/mL.After 24 h, samples were exposed to different concentrations of essential oil and standards (0.78-200 µg/mL) and incubated for 72 h in a humidified atmosphere of 5% CO 2 at 37 • C. The anticancer drug cisplatin (0.01-50 µg/mL) was used as the positive control.At the end of incubation, each well received 10 µL of MTT (5 mg/mL in phosphate-buffered saline, PBS) and the plates were incubated for 4 h at 37 • C.Then, the supernatant was removed, and DMSO (dimethyl sulfoxide) was added to dissolve the formazan crystals.The plates were placed on a shaker for 15 min and the optical density was determined at 540 nm using a microplate spectrophotometer FLUOstar Omega (BMG Labtech, Milan, Italy).Experiments were conducted in triplicate.The cell survival curves were calculated after comparing with the vehicle (Et-OH).The 50% cytotoxic concentration (IC 50 ) was defined as the compound concentration required to reduce the cell viability by half.The IC 50 values were determined with the GraphPad Prism 5 computer program (GraphPad Software, San Diego, CA, USA).

Conclusions
In this study, the chemical profile of C. scariosus EO's aerial parts was examined.The most abundant class was monoterpene hydrocarbons (43.0%), in which the main compounds were α-pinene (11.9%), sabinene (10.2%), p-cymene (8.5%), and α-phellandrene (5.2%).This composition was found to be very similar to the other five Moroccan C. scariosus accessions, not only in terms of the main components but also the different sub-divisions of metabolite classes.To explore the anticancer potential of the EO and its main components, their effects on several cancer cell lines were tested.The remarkable cytotoxic activity is promising for use in cancer prevention and treatment or in combination with conventional chemotherapy drugs to reduce the toxicity of the latter.Further in vitro and in vivo studies on the anticancer mechanisms exerted by EO and the components are required to validate these preliminary data.In addition, studies are necessary to demonstrate the non-toxicity of EO on non-tumor cells as well as the elucidation of the molecular mechanisms governing the anticancer properties of this EO and their major constituents.

Figure 3 .
Figure 3. Close-up photo of C. scariosus EO isolated by the Clevenger apparatus.

Figure 3 .
Figure 3. Close-up photo of C. scariosus EO isolated by the Clevenger apparatus.

Table 1 .
Distribution and synonymous of all the taxa of genus Cladanthus.

Table 2 .
Main constituents (>3%) and several biological properties of the EOs of all the taxa of Cladanthus studied so far.

Table 2 .
Main constituents (>3%) and several biological properties of the EOs of all the taxa of Cladanthus studied so far.

Table 4 .
In vitro cytotoxic activity of C. scariosus EO and its main constituents.
a IC 50 = The concentration of compound that affords a 50% reduction in cell growth (after 72 h of incubation).b Human breast adenocarcinoma cell line.c Human malignant melanoma cell line.d Human colon adenocarcinoma cell line.e Human vascular endothelial cells.f Confidence interval.ND: not determined.