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

Abstract

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.

Full text

Molecules 2011, 16, 8273-8291; doi:10.3390/molecules16108273
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Helichrysum gymnocephalum Essential Oil: Chemical
Composition and Cytotoxic, Antimalarial and Antioxidant
Activities, Attribution of the Activity Origin by Correlations
Samia Afoulous 1, Hicham Ferhout 2, Emmanuel Guy Raoelison 3, Alexis Valentin 4,
Béatrice Moukarzel 4, François Couderc 1 and Jalloul Bouajila 1,*
1 Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique UMR CNRS 5623,
University of Toulouse, University of Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse,
France
2 Nat’Ex Biotech. Bat 7, 55 avenue Louis Breguet, 31400 Toulouse, France
3 Laboratoire de Phytochimie et Standardisation, IMRA, BP: 3833 Antananarivo 101, Madagascar
4 Faculté of Pharmacy, University of Toulouse, UMR 152 IRD-UPS Pharma-DEV, University of
Paul Sabatier Toulouse 3, 35 chemin des maraîchers, 31062 Toulouse Cedex 9, France
* Author to whom correspondence should be addressed; E-Mail: bouajila@cict.fr;
Tel.: +33-561558333; Fax: +33-561558155.
Received: 19 September 2011; in revised form: 23 September 2011 / Accepted: 23 September 2011 /
Published: 29 September 2011
Abstract: 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
OPEN ACCESS

Molecules 2011, 16 8274
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.
Keywords: Helichrysum gymnocephalum; GC-MS; antimalarial activity; cytotoxic activity;
antioxidant activity
1. Introduction
The flora of Madagascar is particularly rich, with a specific diversity and more importantly, it
contains endemic species. In Madagascar, more than 400 species described in other parts of the World,
especially in Southern Africa, Western Europe and Australia, and 115 species, all endemic of the large
Island, are currently known [1]. The gender Helichrysum (from the Greek "helios" sun and "chrysos"
gold) which belongs to Asteraceae family is one of the endemic species.
Helichrysum gymnocephalum (Immortal with naked head) is a shrub from 1 to 4 meters in height
whose ultimate branches are covered by a white and very fine tomentum. The leaves are lengthily
attenuated towards the base in short petioles, covered on the two faces by a tomentum and having
three quite visible principal veins in lower part. Flowering lasts from February till May, seeds are also
papillate [2].
Some medicinal properties of Helichrysum species have been reported, and various studies
have demonstrated the antimicrobial and antiseptic properties of many species from the genus
Helichrysum [3-5]. H. gymnocephalum has been traditionally used therapeutically as a tea or syrup
prepared from the leaves to treat gingivitis or buccal ulcers [6]. It is also used as ointment for
rheumatism or gout by mixing crushed leaves with bacon. The leaves and flowers have properties as
diuretics, stimulants and would relieve neuralgia [7]. Analgesic, aphrodisiac, antiseptic, antiscorbutic,
deodorant, tonic and anthelmintic applications have been reported for H. gymnocephalum [8,9].
H. gymnocephalum is also used in the manufacture of toothpaste for the local market. The leaves are
also used to embalm dead bodies.
The essential oil (EO), a complex mixture of compounds, is considered among the most important
antimicrobial agents [3] present in these plants, and may also have antioxidant and anti-inflammatory
activities. These last years, an overflow of information has appeared on the role of the oxidative stress
in the emergence of a certain number of diseases, such as cancers, cardiovascular diseases and
degenerative diseases related to ageing, in parallel the possible therapeutic role of antioxidants in these
diseases was emphasized [10].
More than a third of the World’s population (about two billion people) lives in malaria-endemic
areas. The majority of deaths caused by falciparum malaria occur in sub-Saharan Africa, primarily
among children younger than 5 years and pregnant women living in remote rural areas with limited
access to health services [11]. Nearly all malaria deaths and a large proportion of morbidity are caused
by Plasmodium falciparum. In the last decades, resistance to several antimalarial drugs became widely
disseminated, while the cost of effective treatments was prohibitive for the majority of the populations

Molecules 2011, 16 8275
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.
2. Results and discussion
2.1. 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.
Twenty three components have been identified in H. gymnocephalum essential oil by GC-MS
(Figure 1 and Table 1). Sesquiterpene hydrocarbons and oxygenated monoterpenes were the major
groups of compounds (32.4% and 54.0%, respectively), while oxygenated sesquiterpenes were not
present. The major monoterpenes found were 1,8-cineole (47.4%), p-cymene (4.3%), (E)-β-ocimene
(2.4%), 2,3-dihydro-1,8-cineole (2.1%) and α-terpinolene (1,3%). Among the sesquiterpene hydrocarbons
bicyclosesquiphellandrene (5.6%), γ-curcumene (5.6%), α-amorphene (5.1%) and bicyclogermacrene
(5%) were present in appreciable quantities. On the other hand, this essential oil contained a phenolic
compound (2,3-di-tert-butylphenol) at 0.5%.
The chemical composition of H. gymnocephalum essential oil has been reported elsewhere.
Mollenbeck et al. [26] have shown that 1,8-cineole (66.7%) was the major compound of the essential
oil, a level which was higher than in our work (47.7%). Also, in our work, the major compounds were
β-caryophyllene (3.3%), γ-terpinene (3.1%), β-pinene (2.7%), α-pinene (2.5%) and ρ-cymene (2.3%).

Molecules 2011, 16 8276
Figure 1. Chromatograms GC-MS of H. gymnocephalum essential oil (1: α-thujene;
2: sabinene; 3: β-pinene; 4: 2,3-dihydro-1,8-cineole; 5: α-terpinene; 6: p-cymene;
7: limonene; 8: 1,8-cineole; 9: (E)-β-ocimene; 10: α-terpinolene; 11: α-phellandrene;
12: terpinen-4-ol; 13: α-terpineol; 14: α-copaene; 15: aromadendrene; 16: bicyclo-
sesquiphellandrene; 17: γ-curcumene; 18: β-selinene; 19: bicyclogermacrene;
20: α-amorphene; 21: 2,3-di-tert-butylphenol; 22: calamenene; 23: δ-cadinene).
Table 1. Chemical composition of H. gymnocephalum essential oil.
N RI Compounds (%)
1 928 α-Thujene 1.0
2 967 Sabinene 0.3
3 971 β-Pinene 1.1
4 984 2,3-Dihydro-1,8-cineole 2.1
5 1010 α-Terpinene 1.3
6 1018 p-Cymene 4.3
7 1022 Limonene 0.5
8 1026 1,8-Cineole 47.4
9 1052 (E)-β-Ocimene 2.4
10 1084 α-Terpinolene 1.3
11 1164 α-Phellandrene 0.2
12 1175 Terpinen-4-ol 2.7
13 1187 α-Terpineol 1.8
14 1373 α-Copaene 0.4
15 1438 Aromadendrene 2.0
1 2
3
4 5
6
7
8
9 10 11
1213 14
15-21
22
23

Molecules 2011, 16 8277
Table 1. Cont.
N RI Compounds (%)
16 1470 Bicyclosesquiphellandrene 5.6
17 1473 γ-Curcumene 5.6
18 1485 β-Selinene 3.3
19 1494 Bicyclogermacrene 5.0
20 1497 α-Amorphene 5.1
21 1502 2,3-di-tert-butylphenol 0.5
22 1512 Calamenene 1.8
23 1521 δ-Cadinene 3.6
Identified components 99.3
Monoterpene hydrocarbons 8.1
Monoterpenes oxygenated 54.0
Sesquiterpenes hydrocarbons 32.4
Others 4.8
Cavalli et al. [27] have shown that the chemical composition of the essential oil of
H. gymnocephalum was dominated by 1,8-cineole (59.7%), p-cymene (6.3%), α-pinene (4.9%),
β-pinene (3.3%) and terpinen-4-ol (2.6%). These published compositions of H. gymnocephalum
essential oil indicated the presence of oxygenated sesquiterpenes, which were not present in our work.
On the other hand, we found more α-thujene (1%) and δ-cadinene (3.6%) compared to Cavalli et al. [27]
0.5% and 0.5%, respectively. For terpinen-4-ol, we obtained 2.6% which was similar to the result of
Cavalli et al. [27] (2.7%) but higher than the result of Mollenbeck et al. [26] (1.3%). An intermediate
concentration (4.3%) of p-cymene was obtained compared to Cavalli et al. [27] and Mollenbeck et al. [26],
with 6.3% and 2.3%, respectively. On the other hand, β-pinene (1.1%) was obtained in the lowest
concentration compared to these two studies of Cavalli et al. [27] and Mollenbeck et al. [26] (3% and
2.7%, respectively).
We identified some new compounds which were not reported in these two previous studies. The
total yield of these new compounds varied between 2.1 and 5.6% (Figure 2). The presence of
2,3-dihydro-1,8-cineole (2.1%), (E)-β-ocimene (2.4%), β-selinene (3.3%), bicyclogermacrene (5.0%),
α-amorphene (5.1%), bicyclosesquiphellandrene (5.6%) and γ-curcumene (5.6%) was evidenced.
Figure 2. Structures of new abundant compounds identified in H. gymnocephalum
essential oil compared to other studies of same essential oil.
O
2,3-dihydro-1,8-cineole
(E)-β-ocimene

Molecules 2011, 16 8278
Figure 2. Cont.
β-selinene
bicyclogermacrene
α-amorphene
bicyclosesquiphellandrene
γ-curcumene
As shown in Table 1, the differences between our work and previously published results concerned
extraction yield, the number of identified compounds and their respective contents. In our
investigation, H. gymnocephalum plants were harvested in July 2008, and we treated only leaves for
the essential oil extraction. Indeed, H. gymnocephalum was harvested in March 1997 by the team of
Mollenbeck et al. [26] and between November–December 1994 by Cavalli et al. [27].
2.2. 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 (IC50 value > 1,000 mg/L) and ABTS+
(IC50 = 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 (IC50 > 100 mg/L) [28].
Table 2. Anticancer, antimalarial and antioxidant activities (IC50 (mg/L)) of
H. gymnocephalum essential oil.
Sample Anticancer
activity
Antiplasmodial
activity
Antioxidant activity
(DPPH assay)
Antioxidant activity
(ABTS assay)
Essential oil 16 ± 2 25 ± 1 >10000 1487.67±47.70
Control 0.218
a ± 0.04 0.10
b
± 0.09 3.75 c ± 0.01 1.84 c ± 0.03
a Doxorubicin; b Chloroquine; c Ascorbic acid.

Molecules 2011, 16 8279
2.3. 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 (IC50 = 16 ± 2 mg/L) was reported for the first time.
Data in Table 3 shows the anti-cancer activity against MCF-7 cells (IC50 mg/L) of several essential
oils reported in the literature and their main components. Essential oils were extracted from
H. gymnocephalum, Schefflera heptaphylla [30], Laurus nobilis, Origanum syriacum, Origanum vulgare,
Salvia triloba [31], Heteropyxis dehniae [32], Schinus molle, Schinus terebenthifolius [33], Salvia
officinalis [34], Melaleuca alternifolia [35], Citrus limon, Citrus medica, Citrus sinensis [36] and
Talauma gloriensis [37].
Based on the bibliographical review, we can consider that the IC50 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).
2.4. 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
(IC50 values) was 25 ± 1 mg/L. Since the value of IC50 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 IC50 compared to that of chloroquine (IC50 = 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 [IC50 (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 IC50 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).

Molecules 2011, 16 8280
Table 3. Anticancer activity and chemical composition of essential oils (I: Helichrysum gymnocephalum; II: Schefflera heptaphylla [30];
III: Laurus nobilis; IV: Origanum syriacum; V: Origanum vulgare; VI: Salvia triloba [31]; VII: Heteropyxis dehniae [32]; VIII: Schinus
molle; IX: Schinus terebenthifolius [33]; X: Salvia officinalis [34]; XI: Melaleuca alternifolia [35]; XII: Citrus limon; XIII: Citrus medica;
XIV: Citrus sinensis [36]; XV: Talauma gloriensis [37]).
Essential oil I II III IV V VI VII VIII IX
Anticancer activity (IC50 mg/L) 16 ± 2 7.3 101.7 ± 7.9 130 ± 52.2 30.1 ± 1.14 174.3 ± 73.04 150 * 54 ± 10 47 ± 9
Component
α-Thujene 1.0 0.62 0.47 0.54 0.38
Sabinene 0.3 6.92 0.35 5.56 0.24 0.02 0.02
β-Pinene 1.0 22.24 4.55 0.6 1.3 8.89 4.96 3.09
2,3-Dihydro-1,8-cineole 2.0
α-Terpinene 1.2 0.7 0.37 4.46 0.44
p-Cymene 4.2 0.74 30.22 3.83 0.34 1.5 2.49 7.34
Limonene 0.5 3.61 2.1 1.68 0.9
1,8-Cineole 47.4 40.91 0.27 45.16 0.4
(E)-β-Ocimene 2.4
α-Terpinolene 1.3 0.35 0.43
α-Phellandrene 0.2 46.52 34.38
Terpinen-4-ol 2.7 1.55 1.09 0.07 0.03
α-Terpineol 1.8 0.62 3.6 8.38 5.6
α-Copaene 0.4 0.6 0.11 0.19
Aromadendrene 2.0 0.49 0.19
Bicyclosesquiphellandrene 5.6
γ-Curcumene 5.6
β-Selinene 3.3 0.3 1.1
Bicyclogermacrene 5.0 1.05
α-Amorphene 5.1
2,3-Di-tert-butylphenol 0.5
Calamenene 1.8
δ-Cadinene 3.6 2.8 0.27 0.69

Molecules 2011, 16
8281
Table 3. Cont.
Essential oil X XI XII XIII XIV XV
[37]
Anticancer activity (IC50 mg/L) 554.4 ± 1.5 310 * 10 1 0.5 14.1
Component
α-Thujene 0.31
Sabinene 0.41 0.1
β-Pinene 2.57 0.91 16.3 3.7
2,3-Dihydro-1,8-cineole
α-Terpinene 0.2 5.76 0.1
p-Cymene
Limonene 1.7 98.4 56.6 98.4 0.8
1,8-Cineole 17.52 19.29
(E)-β-Ocimene 0.1
α-Terpinolene
α-Phellandrene
Terpinen-4-ol 1.01 42.62
α-Terpineol 0.27 11.3
α-Copaene 0.1
Aromadendrene
Bicyclosesquiphellandrene
γ-Curcumene
β-Selinene
Bicyclogermacrene 2.1
α-Amorphene
2,3-Di-tert-butylphenol
Calamenene
δ-Cadinene 3.3

Molecules 2011, 16 8282
Table 4. Antipaludic activity and chemical composition of essential oils (I: Helichrysum gymnocephalum; II: Xylopia phloiodora;
III: Pachypodanthium confine; IV: Antidesma laciniatum; V: Xylopia aethiopica; VI: Hexalobus crispiflorus [40]; VII: Salvia stenophylla;
VIII: Salvia runcinata; IX: Salvia repens [41]; X: Salvia albicaulis; XI: Salvia dolomitica[42]; XII: Lippia multiflora [43]; XIII: Helichrysum
cymosum [44]; XIV: Artemisia gorgonum Webb [45]; XV: Arnica longifolia; XVI: Aster hesperius; XVII: Chrysothamnus nauseosus [46];
XVIII: Essential oil [39]). * IC50 was calculated from percentage; na: no activity.
Essential oil I II III IV V VI VII VIII IX X XI
Antipalaudic activity (IC50 mg/L) 25 ± 1 17.9 16.6 29.4 17.8 2.0 4.38 ± 1.07 1.23 ± 0.31 1.68 ± 0.26 6.4 ± 2.0 4.8 ± 0.7
Component
α-Thujene 1.0 0.59 0.61
Sabinene 0.3 0.59 0.46 0.1 0.2 0.1
β-Pinene 1.0 0.68 10.07 0.12 0.7 0.8 3.0
2,3-Dihydro-1,8-cineole 2.0
α-Terpinene 1.2 0.48 0.43 0.3 0.2
p-Cymene 4.2 0.35 0.32 1.72 0.2 0.7 2.5 0.2
Limonene 0.5 5.3 0.6 9.8 9.4 0.6
1,8-Cineole 47.4 2.0 9.4
(E)-β-Ocimene 2.4 1.93 1.13 0.8 1.5
α-Terpinolene 1.3
α-Phellandrene 0.2
Terpinen-4-ol 2.7 0.16 0.49 0.16 0.8
α-Terpineol 1.8 4.99 2.7 6.2
α-Copaene 0.4 0.53 7.06 2.2 4.07 13.27 0.1
Aromadendrene 2.0 1.08 2
Bicyclosesquiphellandrene 5.6
γ-Curcumene 5.6
β-Selinene 3.3 0.28 2.2
Bicyclogermacrene 5.0
α-Amorphene 5.1
2,3-Di-tert-butylphenol 0.5
Calamenene 1.8 2.32 0.93 1.09
δ-Cadinene 3.6 15.11 8.06 1.3 4.3 10.07 0.5 0.3

Molecules 2011, 16
8283
Table 4. Cont.
Essential oil XII XIII XIV XV XVI XVII XVIII
Antipalaudic activity (IC50 mg/L) 2–4 1.25 ± 0.77 5.2 ± 0.77 na na na 40.15 * 205.19 * 10.82 * 72.68 *
Component
α-Thujene 0.8 0.8
Sabinene 0.3 0.2
β-Pinene 3.7 96.32
2,3-Dihydro-1,8-cineole
α-Terpinene 1.2 1.0
p-Cymene 0.2 2.4 0.1 0.1 0.6 93.19
Limonene 0.2 7.2 4.7 92.98
1,8-Cineole 3.1 20.4 0.1 0.1 93.13
(E)-β-Ocimene 3.6 0.3
α-Terpinolene
α-Phellandrene 1.8
Terpinen-4-ol 0.2
α-Terpineol 0.3 2.6 0.2 0.2 0.1 0.3
α-Copaene 0.3 1.2 0.2
Aromadendrene 1.5
Bicyclosesquiphellandrene
γ-Curcumene 0.5
β-Selinene 0.3
Bicyclogermacrene
α-Amorphene
2,3-Di-tert-butylphenol
Calamenene 0.1 0.2
δ-Cadinene 0.2 0.4 0.8

Molecules 2011, 16 8284
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 IC50 on the tested biological target (MCF-7 cell line or
P. falciparum).
2.5. 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).
Figure 3. Structures of compounds which have good correlation for anticancer (a–c) and
antimalarial (d) activities.
α-terpinolene (a) aromadendrene (b)
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.
Based on this correlation study, we noticed that six components were present only in our essential
oil, namely bicyclosesquiphellandrene (5.6%), α-amorphene (5.1%), 2,3-dihydro-1,8-cineole (2%),
limonene (0.5%), and 2,3-di-tert-butylphenol (0.5%). In addition, for bicyclogermacrene (5.0%) and
(E)-β-ocimene (2.4%), the correlation has not been established because we did not have enough data
(only two essential oils).
A deep study of the anticancer activity of aromadendrene, α-terpinolene, β-selinene, bicyclo-
sesquiphellandrene, α-amorphene, 2,3-dihydro-1,8-cineole, calamenene, 2,3-di-tert-butylphenol,
bicyclogermacrene and (E)-β-ocimene will have to be carried out to determine the origin of the
anticancer activity of H. gymnocephalum. On the same target, we should evaluate the synergy between
these compounds and their own contribution to the final anticancer activity.

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2.6. Correlations for Antimalarial Activity
We established correlations between both major and minor components and IC50 (antimalarial
activity). Calamenene showed the highest correlation R2 = 0.7 (Figure 3). Our results are in accordance
with those of Van Zyl et al. [39]; the former showed that p-cymene, limonene and β-pinene had low
antiplasmodial activity (IC50 = 205.20, 72 and 40.16 mg/L, respectively). Indeed, they also showed that
there are significant synergies (the sum of the fractional inhibitory concentrations (∑FIC) < 0.5)
between p-cymene and other compounds such as E- and Z-(±)-nerolidol, carvacrol and γ-terpinene
(0.09, 0.02 and 0.37, respectively). We noticed that bicyclosesquiphellandrene (5.6%), α-amorphene
(5.1%), bicyclogermacrene (5.0%), 2,3-dihydro-1,8-cineole (2.0%), and 2,3-di-tert-butyl-phenol
(0.5%) were present only in our essential oil. It is possible that these compounds may be involved in
the activity of our essential oil.
3. Experimental
3.1. Extraction of the Essential Oil
The leaves of H. gymnocephalum were collected in Antananarivo, Madagascar (July 2008). Steam-
distillation 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.
3.2. Chemicals
All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-
Aldrich-Fluka (Saint-Quentin, France).
3.3. Gas Chromatography and Gas Chromatography-Mass Spectrometry
Quantitative and qualitative analysis of the essential oil was carried out by gas chromatography-
flame 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 C5-C24 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)

Molecules 2011, 16
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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.
3.4. 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:
% inhibition = 100 (A(blank) − A(sample)) / A(blank)
Antioxidant activities of test compounds or the essential oil were expressed as IC50 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.
3.5. 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 NaH2PO4,
5 mmol/L Na2HPO4 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 IC50 (mg/L). IC50 values were calculated as the concentration required for
scavenging 50% of ABTS radicals. The capacity of free radical scavenging (IC50) 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.

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3.6. Antiplasmodial Activity
The chloroquine-resistant FcB1-Columbia strain of Plasmodium falciparum (IC50 for chloroquine:
186 nM) was cultured continuously according to Trager and Jensen [18], with modifications [19]. The
IC50 values for chloroquine were checked every 2 months, and we observed no significant variations.
The parasites were maintained in vitro in human red blood cells (O±; EFS; Toulouse, France), diluted
to 4% hematocrit in RPMI 1640 medium (Lonza, Emerainville, France) supplemented with 25 mM
Hepes and 30 M NaHCO3 and complemented with 7% human AB+ serum (EFS).
Parasite cultures were synchronized by combination of magnetic enrichment [20] followed by
D-sorbitol lysis (5% of D-sorbitol in sterile water) as described by Lambros and Vanderberg [21]. The
antimalarial activity of essential oil was evaluated by a radioactive micromethod described elsewhere [22].
Tests were performed in triplicate in 96-well culture plates (TPP) with cultures mostly at ring stages
(synchronisation interval, 16 h) at 0.5–1% parasitemia (hematocrit, 1.5%). Parasite culture was
incubated with each sample for 48 h. Parasite growth was estimated by [3H]-hypoxanthine (Perkin-
Elmer, Courtaboeuf, France) incorporation, which was added to the plates 24 h before freezing. After
48 h incubation, plates were frozen-defrosted and each well was harvested on a glass fiber filter.
Incorporated (3H)-hypoxanthine was then determined with a β-counter (1450-Microbeta Trilux,
Wallac-Perkin Elmer). The control parasite cultures, free from any sample, was referred to 100% growth.
IC50 were determined graphically in concentration versus percent inhibition curves. Chloroquine
diphosphate was used as positive control. The antimalarial activity of sample was expressed by IC50,
representing the concentration of drug that induced a 50% parasitaemia decrease compared to the
positive control culture referred to as 100% parasitaemia [23]. According to the literature concerning
plant antiplasmodial activities a sample is very active if IC50 < 5 mg/L, active if IC50 between 5 and
50 mg/L, weakly active if IC50 between 50 and 100 mg/L and inactive if IC50 > 100 mg/L [24].
3.7. 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 × 104 cells/well in 100 µL, and then 100 µL of
culture medium containing sample at various concentrations were added. Cell growth was estimated by
(3H)-hypoxanthine incorporation after 48h incubation exactly as for the P. falciparum assay. The
(3H)-hypoxanthine incorporation in the presence of sample was compared with that of control cultures
without sample (positive control being doxorubicin) [25].
3.8. 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.

Molecules 2011, 16
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4. 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.
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Sample Availability: Samples of the compounds are available from the authors.
© 2011 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 license
(http://creativecommons.org/licenses/by/3.0/).