Helichrysum gymnocephalum Essential Oil: Chemical Composition and Cytotoxic, Antimalarial and Antioxidant Activities, Attribution of the Activity Origin by Correlations

Helichrysum gymnocephalum essential oil (EO) was prepared by hydrodistillation of its leaves and characterized by GC-MS and quantified by GC-FID. Twenty three compounds were identified. 1,8-Cineole (47.4%), bicyclosesquiphellandrene (5.6%), γ-curcumene (5.6%), α-amorphene (5.1%) and bicyclogermacrene (5%) were the main components. Our results confirmed the important chemical variability of H. gymnocephalum. The essential oil was tested in vitro for cytotoxic (on human breast cancer cells MCF-7), antimalarial (Plasmodium falciparum: FcB1-Columbia strain, chloroquine-resistant) and antioxidant (ABTS and DPPH assays) activities. H. gymnocephalum EO was found to be active against MCF-7 cells, with an IC50 of 16 ± 2 mg/L. The essential oil was active against P. falciparum (IC50 = 25 ± 1 mg/L). However, the essential oil exhibited a poor antioxidant activity in the DPPH (IC50 value > 1,000 mg/L) and ABTS (IC50 value = 1,487.67 ± 47.70 mg/L) assays. We have reviewed the existing results on the anticancer activity of essential oils on MCF-7 cell line and on their antiplasmodial activity against the P. falciparum. The aim was to establish correlations between the identified compounds and their biological activities (antiplasmodial and anticancer). β-Selinene (R² = 0.76), α-terpinolene (R² = 0.88) and aromadendrene (R² = 0.90) presented a higher relationship with the anti-cancer activity. However, only calamenene (R² = 0.70) showed a significant correlation for the antiplasmodial activity.

in the endemic regions. Thus, rapid development of resistance by P. falciparum to the conventional drugs necessitates search for new antimalarial drugs [11].
Breast cancer is second only to lung cancer as the most common cancer in women. Roughly 180,000 women are diagnosed with this disease each year, of which 44,000 or almost 20% will die [12]. With increased awareness and increased use of routine mammograms, more women are diagnosed in the earlier stages of this disease, at which time a cure may be possible. The disease is more common in women after the age of 40. It is also more frequent in women of a higher social-economic class [12]. Cancer diseases are characterized by an uncontrolled proliferation of cells. They constitute the second cause of mortality behind cardiovascular diseases in developed countries and the third after infectious and cardiovascular diseases in developing countries [13]. The use of plant extracts and derived products in the treatment of cancer is of exceptional value in the control of malignancies, due to the fact that most of the anticancer drugs severely affect the normal cells [13,14].
The aim of the present study was the evaluation of H. gymnocephalum leaves essential oil for its possible antioxidant and biological activities. In this study, we: (i) examined the chemical components of the essential oil by GC-MS and GC-FID; (ii) evaluated their cytotoxic, antimalarial and antioxidant activities and (iii) reviewed researches for essential oils having an activity against P. falciparum and/or on MCF-7 cell line in order to identify, by correlation, the main active compounds.

Chemical Composition
The essential oil yield of H. Gymnocephalum obtained from hydrodistillation of leaves was 0.40%. Two previous works have quantified the yield of this essential oil. They studied the shoot part of the plant mainly bark and leaves; extraction yields for essential oil were 0.5% and 0.41% according to Mollenbeck et al. [26] and Cavalli et al. [27], respectively. In the present work, only the leaves were used and a similar essential oil yield was obtained.

Antioxidant Activity
Data presented here is the first bibliographical report on the antioxidant activity of H. gymnocephalum. Furthermore, our results (Table 2) demonstrated that the essential oil of H. gymnocephalum has poor antioxidant activity against DPPH (IC 50 value > 1,000 mg/L) and ABTS + (IC 50 = 1,487.67 ± 47.70 mg/L). These results may be attributed to a low antioxidant activity (in these two tests) of the compounds identified in the essential oil of H. gymnocephalum. Four other Helichrysum species (H. dasyanthum, H. excisum, H. felinum and H. petiolare) were also reported to exhibit also a low antioxidant activity against test DPPH (IC 50 > 100 mg/L) [28].

Cytotoxic Activity
In recent years, considerable attention has been focused to identifying naturally occurring substances able to inhibit, delay or reverse the process of multistage carcinogenesis. Plant essential oils are believed to reduce the risk of cancer when used in prevention [29].
In this work, the cytotoxic activity of H. gymnocephalum leaf essential oil against human breast cancer cells MCF-7 (IC 50 = 16 ± 2 mg/L) was reported for the first time.
Based on the bibliographical review, we can consider that the IC 50 of our essential oil (16 mg/L ± 2) is very well positioned among these studied oils in the Table 3. Among the studied essential oils only five showed higher anti-cancer activities than H. gymnocephalum's essential oil. Indeed, nine essential oils had lower activities for the same test (Table 3).

Antimalarial Activity
Many studies on the antiplasmodial activity of crude essential oils have been reported [38,39]. The in vitro antiplasmodial activity of H. gymnocephalum essential oil was determined against the FcB1 chloroquine-resistant strain of P. falciparum ( Table 2). The antimalarial activity of the essential oil (IC 50 values) was 25 ± 1 mg/L. Since the value of IC 50 was found between 5 and 50 mg/L, it can be considered that H. gymnocephalum essential oil has a good activity against P. falciparum [24]. This rather high value of IC 50 compared to that of chloroquine (IC 50 = 0.1 ± 0.09 mg/L) can be explained by the low concentration of the active compound(s) since the essential oil is a multi-components mixture.
Data in Table 4 summarize the antimalarial activity [IC 50 (mg/L)] of all essential oils cited in the literature and their components. These EO have been obtained from H. gymnocephalum, Xylopia phloiodora, Pachypodanthium confine, Antidesma laciniatum, Xylopia aethiopica, Hexalobus crispiflorus [40], Salvia stenophylla, Salvia runcinata, Salvia repens [41], Salvia albicaulis, Salvia dolomitica [42], Lippia multiflora [43], Helichrysum cymosum [44], Artemisia gorgonum Webb [45], Arnica longifolia, Aster hesperius and Chrysothamnus nauseosus [46]. The results based on this bibliographical review (Table 4) showed that the IC 50 of our essential oil (25 mg/L ± 1) was at an interesting level, quite well positioned among these studied oils. We found seven essential oils which have higher antimalarial activity than that of H. gymnocephalum essential oil. In addition, 11 essential oils had lower activities in similar tests (Table 4).    To study the role of the various components of an essential oil in the biological activities obtained, we performed a complete survey of the activities and the percentage of compounds present in order to assign the origin of the activity. The aim of the study was to find correlations between each component present in our literature search of all essential oils of various plants tested for biological activities (anticancer and antimalarial). Correlations between our essential oil, other oils and their components are listed in Tables 3 and 4, with the IC 50 on the tested biological target (MCF-7 cell line or P. falciparum).

Cytotoxic Activity Correlations
We established correlations between compound contents and anti-cancer activity against the MCF-7 cells. Aromadendrene, α-terpinolene and β-selinene showed good correlations, respectively R² = 0.90, 0.88 and 0.76 (chemical structures are presented in Figure 3). β-selinene (c) calamenene (d) To our knowledge, no literature has cited any anticancer activity of these three compounds (β-selinene, aromadendrene and α-terpinolene). We will carry out a deeper study to focus on their specific biological activity against MCF-7 cell line.

Extraction of the Essential Oil
The leaves of H. gymnocephalum were collected in Antananarivo, Madagascar (July 2008). Steamdistillation was used to extract the essential oil according to the European Pharmacopoeia protocol [15]. The essential oil was dried with anhydrous sodium sulphate, filtered and stored in sealed vials at 4 °C prior to analyses.

Chemicals
All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich-Fluka (Saint-Quentin, France).

Gas Chromatography and Gas Chromatography-Mass Spectrometry
Quantitative and qualitative analysis of the essential oil was carried out by gas chromatographyflame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Gas chromatography analyses were carried out on a Varian Star 3400 Cx chromatograph (Les Ulis, France) fitted with a fused silica capillary DB-5MS column (5% phenylmethylpolysyloxane, 30 m × 0.25 mm, film thickness 0.25 µm). Chromatographic conditions were 60 °C to 260 °C temperature rise with a gradient of 5 °C/min and 15 min isotherm at 260 °C. A second gradient was applied to 340 °C at 40 °C/min. Total analysis time was 57 min. For analysis purposes, the essential oil was dissolved in petroleum ether. One microliter of sample was injected in the split mode ratio of 1:10. Helium (purity 99.999%) was used as carrier gas at 1 mL/min. The injector was operated at 200 °C. The mass spectrometer (Varian Saturn GC/MS/MS 4D) was adjusted for an emission current of 10 µA and electron multiplier voltage between 1,400 and 1,500 V. Trap temperature was 150 °C and that of the transfer line was 170 °C. Mass scanning was from 40 to 650 amu.
Compounds were identified by comparison of their Kovats indices (KI) obtained on a nonpolar DB-5MS column relative to C 5 -C 24 n-alkanes, with those provided in the literature, by comparison of their mass spectra with those recorded in NIST 08 (National Institute of Standards and Technology) and reported in published articles and by co-injection of available reference compounds. The samples were analyzed in duplicate. The percentage composition of the essential oil was computed by the normalization method from the GC peak areas, assuming identical mass response factors for all compounds. Results were calculated as mean values of two injections from essential oil, without using correction factors. All determinations were performed in triplicate and averaged.

Antioxidant Activity
Free Radical Scavenging Activity: DPPH Test Antioxidant scavenging activity was studied using the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) assay as described by Blois [16] with some modifications. Various dilutions of EO (1.5 mL) were mixed with a 0.2 mmol/L methanolic DPPH solution (1.5 mL). After an incubation period of 30 min at 25 °C, the absorbance at 520 nm (the wavelength of maximum absorbance of DPPH) were recorded as A (sample) , using a Helios spectrophotometer (Unicam, Cambridge, UK). A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A (blank) . The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation: Antioxidant activities of test compounds or the essential oil were expressed as IC 50 values, defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

ABTS Radical-Scavenging Assay
The radical scavenging capacity of the samples for the ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined as described by Re et al. [17] with some modifications. ABTS was generated by mixing a 7 mmol/L solution of ABTS at pH 7.4 (5 mmol/L NaH 2 PO 4 , 5 mmol/L Na 2 HPO 4 and 154 mmol/L NaCl) with 2.5 mmol/L potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using a spectrophotometer (Helios). For samples, solutions of the essential oil in methanol (100 μL) were allowed to react with fresh ABTS solution (900 μL), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed by IC 50 (mg/L). IC 50 values were calculated as the concentration required for scavenging 50% of ABTS radicals. The capacity of free radical scavenging (IC 50 ) was determined using the same previously used equation for the DPPH method. All measurements were performed in triplicate. All data of antioxidant activity were expressed as means ± standard deviations (SD) of the triplicate measurements. The confidence limits were set at P < 0.05. SD did not exceed 5% for the majority of the values obtained.

Cytotoxicity Evaluation
Cytotoxicity of sample was estimated on human breast cancer cells (MCF-7). The cells were cultured in the same conditions as those used for P. falciparum, except for the 10% human serum, which was replaced by 10% foetal calf serum (Lonza). For the determination of pure compound activity, cells were distributed in 96-well plates at 3 × 10 4 cells/well in 100 µL, and then 100 µL of culture medium containing sample at various concentrations were added. Cell growth was estimated by ( 3 H)-hypoxanthine incorporation after 48h incubation exactly as for the P. falciparum assay. The ( 3 H)-hypoxanthine incorporation in the presence of sample was compared with that of control cultures without sample (positive control being doxorubicin) [25].

Statistical Analysis
All data were expressed as mean ± standard deviation of triplicate measurements. The confidence limits were set at P < 0.05. Standard deviations (SD) did not exceed 5% for the majority of the values obtained.

Conclusions
In conclusion, we have identified all volatile constituents of H. gymnocephalum leaf essential oil and evaluated its anticancer, antimalarial, and antioxidant activities. Our results clearly showed that this essential oil was active against the tumor cell lines MCF-7 and the FcB1 strain of P. falciparum. Based on established correlations, compounds such as α-terpinolene, aromadendrene and β-selinene against MCF-7, could be the best candidates for further analysis. Purification of these compounds is under development to test them separately. In addition, an in depth study could determine the mechanisms by which these compounds exert their biological activities. The results presented here can be considered as the first information on the anticancer and antimalarial properties of H. gymnocephalum. This may also contribute to our knowledge of the genus Helichrysum. These in vitro results provide some scientific validation for the widespread use of plants from this genus in traditional medicine.