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NPC

Natural Product Communications

Anti-oxidant, Anti-inflammatory and Anti-proliferative Activities of

Moroccan Commercial Essential Oils

2014
Vol. 9
No. 4
587 - 594

Smail Aazzaa,b, Badiaa Lyoussib, Cristina Megasc, Isabel Corts-Giraldoc, Javier Vioquec,
A. Cristina Figueiredod and Maria G. Miguele*
a

Universidade do Algarve, Faculdade de Cincias e Tecnologia, Departamento de Qumica e Farmcia,

Campus de Gambelas 8005-139 Faro, Portugal
b
Laboratory of Physiology, Pharmacology and Environmental Health, Faculty of Sciences Dhar El Mehraz,
BP 1796 Atlas, University Sidi Mohamed Ben Abdallah, Fez 30 000, Morocco
c
Instituto de la Grasa (C.S.I.C.), Avda. Padre Garca Tejero 4, 41012-Sevilla, Spain
d
Universidade de Lisboa, Faculdade de Cincias de Lisboa, Departamento de Biologia Vegetal,
Instituto de Biotecnologia e Bioengenharia, Centro de Biotecnologia Vegetal, C2, Piso 1, Campo Grande,
1749-016 Lisboa, Portugal
e
Universidade do Algarve, Faculdade de Cincias e Tecnologia, Departamento de Qumica e Farmcia,
Instituto de Biotecnologia e Bioengenharia, Centro Biotecnologia Vegetal, Campus de Gambelas 8005-139 Faro,
Portugal
mgmiguel@ualg.pt
Received: December 12th, 2013; Accepted: January 5th, 2014

Essential oils (EO) possess antimicrobial, anti-inflammatory, insect repellent, anti-cancer, and antioxidant properties, among others. In the present work, the
antioxidant, anti-inflammatory and anti-proliferative activities of Moroccan commercial EOs (Citrus aurantium, C. limon, Cupressus sempervirens, Eucalyptus
globulus, Foeniculum vulgare and Thymus vulgaris) were evaluated and compared with their main constituents. T. vulgaris EO showed the best free radicals
scavenging capacity. This EO was also the most effective against lipid peroxidation along with C. limon and F. vulgare EOs. C. sempervirens EO was the most
effective in scavenging NO free radicals, whereas C. limon EO showed the best chelating power. Not all of the major compounds of the EO were responsible
for the whole activity of the EOs. T. vulgaris EO showed the best anti-proliferative activity against THP-1 cells in contrast to that of F. vulgare. The
antioxidant and anti-inflammatory activities of the EOs were plant species dependent and not always attributable to the EOs main components. Nevertheless,
the EOs anti-proliferative activities were more related to their main components, as with T. vulgaris, C. limon, E. globulus and C. sempervirens.
Keywords: Chemical composition, Biological properties, Essential oils, MTT.

For food and beverage consumption, the essential oil products used
are those on the Generally Recognised as Safe (GRAS) list
approved by the Food and Drug Administration (FDA). For medical
purposes essential oils need to fulfil national and international
Pharmacopoeia recommendations. The maximum quantities and
uses of some essential oils, as well as their single components, are
regulated by the International Fragrance Association (IFRA), the
Bundesinstitut fr Risikobewertung (BfR), the Research Institute
for Fragrance Materials (RIFM) and the Scientific Committee
Consumers Safety (SCCS). Physical standards of essential oils are
also specified by the Association Franaise de Normalisation
(AFNOR), as well as the International Organization for
Standardization (ISO). The need for this control and standardization
lies in the fact that the chemical composition varies depending on
plant health, growth stage, edaphic and climate factors, harvesting
time, part of plant used, and agronomic conditions, among other
factors [1,2].
The use of essential oils in the pharmaceutical, agricultural and
nutritional fields are due to their antimicrobial, antiviral,
nematicidal, antifungal, insecticidal, antioxidant and antiinflammatory activities [3].
In the present work, six traded essential oils belonging to the first
{Citrus aurantium L., C. limon (L.) Burman f. and Eucalyptus
globulus Labil.) and second (Thymus vulgaris L.) groups of global

production, along with Cupressus sempervirens L., and Foeniculum

vulgare Mill. were chemically analyzed. Also, antioxidant and antiproliferative activities of these essential oils were investigated.
Essential oils are composed of many compounds and their
biological activities can be attributed to the major components
and/or to minor ones. Hence, in the present work, the biological
activities of the essential oils were compared with those of their
main components in order to ascertain if the activities of these
compounds reflect the biological activities of the respective
essential oils.
Essential oils composition: The detailed composition of the
essential oil (EO) components of Citrus aurantium (leaves), C.
limon (peel), Cupressus sempervirens, Eucalyptus globulus,
Foeniculum vulgare and Thymus vulgaris are listed in Table 1, in
order of their elution from a DB-1 column. In a previous work, only
the main components (>10%) were reported [4].
The volatile profile of some of the studied species was in general in
accordance with previous studies, namely those of C. aurantium [5],
C. sempervirens [6,7] and F. vulgare [8]. On the contrary, other
essential oils showed some differences from those previously
reported, such as those of C. limon [9], although some authors [10]
also reported high percentages of limonene in some lemon taxa
essential oils (81% to 96%); E. globulus [11] and T. vulgaris [12].

588 Natural Product Communications Vol. 9 (4) 2014

The EO yields can vary considerably, as well as the exact

composition of any EO will be variable depending, among other
factors, on the particular plant part material used in the isolation
procedure, on the cultivation conditions, cultivar, harvesting time
and distillation [2,13]. This chemical variability may contribute to
the existence of EO chemotypes, which may partly explain the
contrasting properties of the essential oils isolated from the same
species.
Antioxidant activity: The antioxidant capacity of the studied
commercial EOs was determined by evaluating their ability to
scavenge free radicals (ABTS, hydroxyl, peroxyl and NO), lipid
peroxidation (liposomes) and chelating ability (Table 2). T. vulgaris
EO showed the best capacity for scavenging ABTS, hydroxyl and
peroxyl radicals. Nonetheless, it was not as effective for either
scavenging nitric oxide radicals or for chelating iron metal (Table
2). C. sempervirens EO was the most effective in scavenging NO
free radicals, and C. limon EO showed the most effective chelating
power. The antioxidant properties of T. vulgaris essential oils have
been reported in previous studies using either the same methods or
different ones from those used in the present work [4,14,15].

Aazza et al.

capacity of limonene-rich essential oils detected in the present work

was also reported by Gursoy et al. [19], this activity being dosedependent.
The capacity for either scavenging ABTS radicals or preventing
lipid peroxidation by C. aurantium EO cannot be attributed to
linalool and linalyl acetate, the dominant monoterpenes in this EO,
since the activities were much lower than that of C. aurantium EO.
The weak ability of linalool to prevent oxidation either by inhibiting
lipid peroxidation or scavenging free radicals was also previously
reported [16,20]. Hence other EO components may be responsible
for the activity of C. aurantium EO and/or their synergism.
All EOs showed metal chelating activity and the capacity for
scavenging peroxyl radicals, although linalool and linalyl acetate
had higher activities, suggesting that these monoterpenes and/or
other components present in the EO showed an antagonistic effect,
lowering the antioxidant capacity of the Citrus EO. Linalool, one of
the main components of Hedychium coronarium and Diplazium
squamigerum EOs exhibited moderate ferrous chelating activity
[21], as well as a capacity for scavenging peroxyl radicals [22].

C. limon, along with F. vulgare and T. vulgaris EOs showed the

highest prevention of liposome peroxidation, in contrast to that of C
aurantium in which the IC50 was not possible to determine and E.
globulus EO (Table 2). This EO also had a weak capability for
scavenging free peroxyl radicals.

Only E. globulus EO was able to scavenge ABTS free radicals

(Table 2). 1,8-Cineole had very little ability to scavenge these free
radicals. Hence, the activity may be the result of several
components of the EO and not only to one of the major compounds.

C. sempervirens EO was the most effective for scavenging NO

radicals and, along with F. vulgare EO, was a good chelator of
metal ions (Table 2).

Limonene had the best capacity for scavenging peroxyl radicals in

contrast to that of 1,8-cineole (Table 2). The combination of 1,8cineole and p-cymene or other combinations with minor
components seemed to determine the activity of E. globulus EO.

As mentioned, EOs are complex mixtures of lipophilic and volatile

compounds at different concentrations. The activity can be
attributed to major EO compounds, but also minor compounds may
reveal antioxidant activity. Moreover, the association between
major and minor compounds may result in an antagonistic result. In
the present work, the antioxidant activities of entire EOs were
compared with those of their main components to determine the
potential correlation between the EOs and their major constituents.

1,8-Cineole was also the worst in terms of prevention of lipid

peroxidation, in contrast to limonene (Table 2). E. globulus EO was
better as an antioxidant than 1,8-cineole, but the worst when
compared to the remaining samples (Table 2). This compound also
had very little ability for preventing lipid peroxidation when egg
yolk was used as the lipid model in the thiobarbituric acid reactive
substances assay [16].

The activity of T. vulgaris EO, which was one of the best

antioxidants in almost all the assays used, seems to be
predominantly due to thymol and carvacrol. Borneol and p-cymene
had only very weak activity (Table 2), which is in accordance with
previous studies [16].
The capacity of T. vulgaris EO for scavenging hydroxyl radicals
was the best, whereas in the remaining assays thymol was generally
the best antioxidant (Table 2). The antioxidant activities of thymol
and carvacrol have already been demonstrated by Puertas-Meja et
al. [17], both alone and in combination. The authors concluded that
these phenols combined in a 1:1 ratio had a synergistic effect.
The antioxidant activity C. limon EO may be attributed to limonene,
independently of the method used for assay. Limonene was
practically the sole terpene in this EO (Table 1). The low capacity
of limonene for scavenging ABTS free radicals (<50%) was also
reported by Roberto et al. [18] when using DPPH as free radicals.
Limonene, as well as C. limon EO, had the capacity for preventing
lipid peroxidation of liposomes. Ruberto and Baratta [16] found a
low capacity for preventing lipid peroxidation of limonene through
the modified thiobarbituric acid reactive species and the rate of
conjugated diene formation from linoleic acid. The chelating

Neither 1,8-cineole nor p-cymene showed NO radical scavenging

activity. Only E. globulus EO and limonene showed this property,
although the EO activity was poorer than that of limonene. Such
results indicate that an antagonistic effect may have occurred that is
responsible for the lower activity of the eucalyptus EO (Table 2).
In contrast to the results reported in the other assays, 1,8-cineole
was the best for scavenging hydroxyl radicals. The EO presented an
intermediate value between 1,8-cineole and limonene. 1,8-Cineoles
low IC50 was quite different from that reported by Singh et al. [23].
According to them, E. tereticornis EO was better for scavenging
hydroxyl radicals than the pure constituents, such as 1,8-cineole.
Comparing the antioxidant and chelating activities of C.
sempervirens EO with its main components (Table 2), -pinene was
the least active in almost all the assays, with the exception of lipid
peroxidation prevention. In most cases, it was even impossible to
calculate the IC50 for -pinene due to its very low antioxidant
activity (TEAC, hydroxyl and nitric oxide scavenger and chelator).
-Pinene was reported as having DPPH free radical scavenging
ability, although lower than that of 1,8-cineole, myrcene and
thymol, but better than rosemary EO, in which that monoterpene
prevailed [24].

Biological properties of essential oils

Natural Product Communications Vol. 9 (4) 2014 589

Table 1: Percentage compositions of the commercial essential oils isolated by hydrodistillation.

Components
Tricyclene
-Thujene
-Pinene
Camphene
Sabinene
1-Octen-3-ol
-Pinene
-Myrcene
-Phellandrene
-3-Carene
-Terpinene
p-Cymene
1,8-Cineole
-Phellandrene
Limonene
Z--Ocimene
E--Ocimene
-Terpinene
n-Octanol
Z-Linalool oxide
Fenchone
2,5-Dimethyl styrene
E-Linalool oxide
Terpinolene
Linalool
Isopentyl isovalerate
Z-Limonene oxide
Camphor
E-Pinocarveol
E-Limonene oxide
Borneol
Terpinen-4-ol
Z-Dihydrocarvone
-Terpineol
Estragole (= Methyl chavicol)
Verbenone
n-Decanal
E-Carveol
Bornyl formate
Anisaldehyde
Z-Carveol
Nerol
Pulegone
Carvone
Methyl carvacrol oxide
Geraniol
Linalyl acetate
E-Anethole
Bornyl acetate
Thymol
Carvacrol
Limonene-1,2-diol
-Terpenyl acetate
Neryl acetate
Geranyl acetate
-Copaene
-Caryophyllene
-Copaene
-Humulene
-Muurolene
Germacrene-D
Valencene
Bicyclogermacrene
-Cadinene
Calamenene
-Cadinene
-Caryophyllene oxide
Cedrol
T-Cadinol (= epi--Cadinol)
% of Identification
Grouped components
Monoterpene hydrocarbons
Oxygen-containing monoterpenes
Sesquiterpene hydrocarbons
Oxygen-containing sesquiterpenes
Phenylpropanoids
Others

RI
921
924
930
938
958
961
963
975
995
1000
1002
1003
1005
1005
1009
1017
1027
1035
1045
1045
1050
1059
1059
1064
1074
1084
1095
1102
1106
1112
1134
1148
1159
1159
1163
1164
1180
1189
1199
1200
1202
1206
1210
1210
1224
1236
1245
1254
1265
1275
1286
1314
1334
1353
1370
1375
1414
1426
1447
1469
1474
1484
1487
1500
1505
1505
1561
1574
1616

Apiaceae

Cupressaceae

Lamiaceae

Myrtaceae

Foeniculum
vulgare

Cupressus
sempervirens
t
t
47.3
0.7
0.4

Thymus
vulgaris
0.2
0.1
3.4
4.7
t
t
0.6
0.2
t

Eucalyptus
globulus

t
9.0
t
0.7
0.7
1.7
t

0.8
0.2

t
3.5
t
0.1
0.1
0.4
1.8

Rutaceae
Citrus
aurantium

Citrus
limon

0.1

0.3

0.2

2.5
1.3

0.1
t

17.6
0.3
0.3
0.3
8.3
t
t
0.1

t
t
31.4
t
t
t

0.3
24.6
0.5
0.5
1.5

37.8
29.3
t
26.1

1.2

t
t
0.3
0.3
1.9
t

96.7

t
9.1
t
0.4
t

t
3.4

t
t

t
0.3
54.0

0.3
0.5

0.7
0.4
0.3
15.5
t
t

0.9
3.5

0.1

8.5

0.1

t
t
0.1
0.3

t
0.1
t
t

t
0.6
5.4

1.4
3.1
21.1
69.4
0.7
11.8
15.4
0.2
0.7

t
1.4
3.3
0.1
2.9

0.2

0.8

0.1

0.4

0.1
0.1
t
t
0.1
t
t
0.1

0.1
t
t

0.1
t
99.9

99.9

99.7

99.8

99.9

99.3

21.1
9.4

98.8
0.7
0.3
0.1

37.3
59.3
3.1
t

69.8
29.7
0.3

6.7
92.0
1.2

97.3
1.8
0.1

69.4

RI, in lab calculated retention index relative to C9C17 nalkanes on a DB1 column; t, trace (<0.05%)

0.1

590 Natural Product Communications Vol. 9 (4) 2014

Aazza et al.

Table 2: Antioxidant and anti-inflammatory activities of essential oils and their main constituents.
Plant / Standard
Thymus vulgaris
Carvacrol
Thymol
Borneol
p-Cymene

TEAC
IC50 (mg mL-1)
0.0100.000aF
0.0030.000b
0.0020.000cc
-

Hydroxyl
IC50 (g mL-1)
0.400.00cF
1.100.00a
0.900.00b
-

NO
IC50 (mg mL-1)
28.70.4aC
11.20.4b
9.90.4b
-

Citrus limon
Limonene

28.000.53A
-

4.000.00aC
4.200.00a

19.61.0aD
18.71.0a

Citrus aurantium
Linalool
Linalyl acetate

22.540.23B
-

1.600.00bE
4.100.00b
237.400.00a

ORAC
TE* (mol mg-1)
1788.0244.5cA
24891.4244.5b
27156.5244.5a
7.2244.5d
3.7244.5d

Chelating
IC50 (mg mL-1)
-

Liposomes
IC50 (mg mL-1)
0.460.01bC
0.040.01c
0.040.01c
0.990.01a

Lipoxygenase
IC50 (mg mL-1)
0.190.00aC
0.110.00b
0.200.00a
-

57.13.9aC
51.13.9a

0.030.00aD
0.030.00a

0.540.02aC
0.510.02a

0.300.01aB
0.290.01a

78.014.4aA
51.914.4b
-

161.92.1aB
31.52.1b
23.02.1c

0.060.00aA
0.030.00c
0.040.00b

0.940.25bA
1.370.25a

Eucalyptus globulus
1,8-Cineole
Limonene
p-Cymene

1.430.05E
-

2.900.00bD
0.060.00c
4.200.0001a
-

60.422.9aB
18.722.9b
-

5.90.5bD
1.80.5d
51.10.5a
3.70.5c

0.050.00bB
0.100.00a
0.030.00c
-

3.660.00bA
4.200.00a
0.510.00d
1.000.00c

0.160.07bC
0.290.07a
-

Cupressus sempervirens
-Pinene
Limonene

3.620.12D
-

0.010.00aA
0.0040.00b

6.56.7bE
18.76.7a

26.00.6bCD
1.20.6c
51.10.6a

0.040.01aC
0.030.01b

2.170.03aB
1.020.03b
0.510.03c

0.170.07bC
0.290.07a

Foeniculum vulgare
E-Anethole

9.280.08C
-

0.010.007bB
0.030.007a

16.81.7aD
14.31.7b

58.930.4bC
114.330.4a

0.040.02aC
0.010.02b

0.470.01aC
0.460.01a

0.040.01aD
0.020.01b

-: without activity or very poor activity. TE*: Trolox Equivalent.

For each plant, values in the same column followed by the same upper letter case are not significant by the Tukeys multiple range test (p<0.05). Data are the mean of at least three
replicates.
For each plant/standard, values in the same column followed by the same lower letter case are not significant by the Tukeys multiple range test (p<0.05) or by the Paired Student t
test (no differences at 5% significance). Data are the mean of at least three replicates.

The capacity of -pinene for preventing lipid peroxidation was

worse than that of limonene, as already reported [16], although
using a different lipid substrate. In the present work, the lower
capacity for inhibiting lipid oxidation of C. sempervirens EO
suggests the influence of either other minor components or the
involvement of limonene + -pinene in the EO in the antagonism
processes, which were not evaluated. The iron chelating inability of
-pinene agrees with data from Boulanouar et al. [25] that showed a
lack of chelating activity in the -pinene-rich Juniperus phoenicea
EO. The chelating ability of C. sempervirens EO may be attributed
to other minor components.
The weak capacity for scavenging free radicals of E-anethole and Eanethole-rich fennel EO has already been reported [8,17]. The
capacity for inhibiting peroxidation of lipids, measured through the
TBRAS method, was reported by Miguel et al. [8]. However, the
authors found that high concentrations of E-anethole-rich fennel EO
had a pro-oxidant activity. In the present study, the activity was
measured using liposomes as the lipid substrate and differences
between the EO and the phenylpropanoid were not observed. In
contrast, E-anethole was significantly better than the respective EO
for scavenging peroxyl radicals and nitric oxide, as well as metal
chelating (Table 2). The capacity for iron binding of the EO from
some cultivars of fennel, particularly those richest in E-anethole,
was previously reported [26]. In the present study, E-anethole was a
significantly better chelating agent than fennel EO. The iron binding
property can be attributed to this compound, although other
components of fennel EO may antagonize its action. The same may
explain the lowest capacity of the essential oil for scavenging
peroxyl (Table 2).
Anti-inflammatory activity: The lipoxygenase assay was used as
an indication of the anti-inflammatory and antioxidant activities of
the EOs. Lipoxygenase catalyses the addition of molecular oxygen
to fatty acids containing a Z,Z-1,4-pentadiene system originating
from unsaturated fatty acid hydroperoxides. Compounds which are
able to inhibit this enzyme, which is responsible for the production
of these peroxides, can be considered as antioxidants. At the same
time, those products are converted into others that play a key role in

inflammatory processes. F. vulgare EO had the highest activity in

contrast to the lowest observed with C. aurantium EO (Table 2).
Several works have shown that EOs may possess anti-inflammatory
activity. This has been attributed to limonene, 1,8-cineole,
-terpinene, and -pinene, among other components [27,28]. In the
present work, 1,8-cineole, -pinene, p-cymene, linalool and borneol
either did not show anti-inflammatory activity or it was very low,
which did not allow IC50 determination (Tables 2 and 3). F. vulgare
EO activity may be attributed to its major compound, E-anethole.
Nevertheless, other EO components may partially antagonize this
phenylpropanoid activity, since the EO showed less activity than Eanethole. -Pinene, limonene, and fenchone at >8% may have
contributed to the lower activity of fennel EO when compared with
that of E-anethole (Table 2). It is noteworthy that, among the pure
compounds tested, this phenylpropanoid had the best antiinflammatory activity.
The EO of C. limon and limonene standard, which dominates the
EO, showed similar activities, which allowed us to conclude that the
activity of the whole EO may be attributed to this monoterpene. On
the other hand, limonene was also a dominant component of E.
globulus and C. sempervirens EOs, but never reaching percentages
as elevated as those observed in C. limon EO. In both cases, the
activities of the EOs were always higher than that of limonene,
which may mean that other EO components are also responsible for
the activity, acting by synergism with limonene.
In C. aurantium EO, linalyl acetate, along with other components,
contributed, by synergism, to the activity of the EO since the
activity of linalyl acetate was lower than that of the respective EO
(Table 2).
Anti-proliferative activity: The cytotoxic activities of C.
aurantium, C. limon, C. sempervirens, E. globulus, F. vulgare and
T. vulgaris EOs and their main constituents were studied on the
THP-1 leukemia cell line by treating these cells with increasing
doses of either the EOs or their main components for 24 or 96 h.

Natural Product Communications Vol. 9 (4) 2014 591

120

120

100

100

80

80
% Control

% Control

Biological properties of essential oils

60
40

60
40
20

20

0
0

100
Foeniculum vulgare
Eucalyptus globulus
Citrus aurantium

200
300
Concentration (g mL)

400

500

Citrus limon
Cupressus sempervirens
Thymus vulgaris

100
Foeniculum vulgare
Eucalyptus globulus
Citrus aurantium

200
300
Concentration (g mL)

400

500

Citrus limon
Cupressus sempervirens
Thymus vulgaris

Figure 1: Antiproliferative activity of the essential oils on THP-1 cell line with (A) 24 h exposure and (B) 96 h exposure. The mean absorbance values for the negative control
(DMSO treated cells) was standardized as 100% absorbance (i.e. no growth inhibition) and results were displayed as absorbance (% of control) vs. essential oil concentration.

T. vulgaris EO showed the best anti-proliferative activity (Figure

1A) after 24 h of cell treatment, in contrast to F. vulgare EO. After
96 h of cell exposure, the trend was similar (Figure 1B). All samples
inhibited cell proliferation in a concentration-dependent manner. T.
vulgaris EO at >200 g mL-1 practically prevented the growth of
THP-1 cells after 96 h of exposure, and at 100 g mL-1 only T.
vulgaris EO had significantly higher anti-proliferative capacity. At
this concentration <40 % of the cells survived. T. vulgaris EOs
main components are p-cymene, borneol, carvacrol and thymol
(Table 1). Comparing the anti-proliferative activities of these
monoterpenes with that of the EO showed that the activity of the oil
may be attributed to carvacrol and thymol (Figure 2A). p-Cymene
was the least effective since more than 80% cell survival was
observed at > 400 g mL-1 (Figure 2A). p-Cymene and borneol
might be responsible for the lower activity of the EO compared with
carvacrol and thymol (Figure 2A).
Limonene also showed relatively high anti-proliferative activity
(Figures 2B and 2C), being better for preventing THP-1 cell growth
than C. limon and E. globulus EOs. C. limon EO is almost entirely
dominated by limonene. Maybe for this reason, its cytotoxic activity
(Figure 2B) was higher than that of E. globulus EO (Figure 2C),
that also possesses p-cymene and 1,8-cineole in relatively high
amounts (Table 1). p-Cymene and 1,8-cineole were less active than
limonene and are probably responsible for the weaker activity of E.
globulus EO. The anti-proliferative activities of both EOs, as well as
those of the standards, were dose-dependent. Limonene alone or as
an EO component has been reported to inhibit colon cancer
(SW480) cell proliferation [29], MCF-7 breast tumor cells [30], and
a lymphoma cell line [18], as well as acting against carcinogeninduced mammary tumors in rats [31]. Nevertheless, -pinene, terpineol, -terpinene and trans--bergamotene, along with
limonene in some Citrus fruit peel EOs, were critical to obtain a
better anti-proliferative activity on MCF-7 and HeLa cell lines [32].
The present results demonstrated that p-cymene and 1,8-cineole
may also interfere with the activity of limonene on the growth of
THP-1 cells.
C. sempervirens EO also had anti-proliferative activity on THP-1
cells (Figure 2D), similar to that of limonene, one of the major
constituents of the EO, in contrast to that of -pinene, which only
possessed weak activity. The cytotoxic activity of -3-carene, also
in relative high percentage in the EO, on THP-1 cells was not
evaluated. However, the similar anti-proliferative activity of the EO
and limonene may reveal a weak cytotoxic activity of -3-carene
and -pinene on THP-1 cells, or at least there was not an
antagonistic effect between these two monoterpenes and limonene.

C. aurantium EO had a higher capacity to reduce THP-1 cell growth

than the main oil components, linalool and linalyl acetate (Figure
2E). Linalyl acetate was more effective than linalool, at
concentrations >400 g mL-1, although lower than the EO. O.
vulgare, a linalool and linalyl acetate-rich EO, was reported as
possessing considerable cytotoxicity against breast cancer MCF-7
and androgen-sensitive human prostate adenocarcinoma cell lines
[33]. Linalool, a minor component of Platycladus orientalis,
Prangos asperula and Cupressus sempervirens ssp. pyramidalis
EOs was cytotoxic to amelanotic melanoma C32 and renal cell
adenocarcinoma cells, in contrast to -pinene, the major
component, which was inactive on tumor cell population growth
and proliferation [34]. However, Tundis et al. [35] found that
linalool was inactive against LNCaP and MCF-7 cell lines, despite
its cytotoxic efficacy against tamelanotic melanoma C32 and renal
cell adenocarcinoma cells. These findings demonstrate that one
compounds cytotoxic activity largely depends on the evaluated cell
type. According to Prashar et al. [36], linalyl acetate present in
lavender EO was more cytotoxic to human skin cells in vitro
(endothelial cells and fibroblasts) than linalool, another lavender EO
component. Using non-cancer cells, Prashar et al. [36] showed that
the acetate group (linalyl acetate) would be responsible for higher
cytotoxicity than the respective alcohol, linalool. The results
obtained by Prashar et al. [36] and those obtained in the present
study support the view that linalyl acetate may show antiproliferative activity against normal cells and THP-1 leukemia cells.
E-Anethole, fennel EOs main component, showed low antiproliferative activity, even at high concentrations (Figure 2F). This
may explain the weak action of fennel EO on THP-1 cell growth.
These results contrast with those of al-Harbi [37], in which Eanethole was active against Erlich tumor (EAT)-cells in Swiss
albino mice paw. However, the present results agree with those
reported by Firuzi et al. [38] since the E-anethole-rich Heracleum
persicum EO was inactive against human cervical adenocarcinoma
(HeLa), human colon adenocarcinoma (LS180) and human B
lymphoma (Raji) cell lines. Therefore, the cytotoxicity of EOs
and/or their components depend on the type of cancer cell lines.
Conclusion: The chemical composition of the commercial EOs did
not fit within the typical composition recognized by Food and
Health Institutions, which reinforces the need to control the EO
trade. T. vulgaris EO was the best for scavenging ABTS, hydroxyl
and peroxyl radicals, whereas C. sempervirens EO was the best for
scavenging NO radicals. The best chelating activity was found in C.
limon EO. T. vulgaris, F. vulgare and C. limon EOs had similar
capacities for preventing lipid peroxidation. Fennel EO had the best

592 Natural Product Communications Vol. 9 (4) 2014

Aazza et al.

120

100

100

% Control

140

120

% Control

140

80
60

40

20

20
0
0

100

Thymus vulgaris

200
300
Concentration (g mL)
p-Cymene

Borneol

400

500

Thymol

Carvacrol

200

Citrus limon

140

140

120

120

100

100

80

80

60
40
20

300

400

500

Limonene

60
40
20

0
0

100

Concentration (g mL)

% Control

% Control

60

40

100

Eucalyptus globulus

200
300
Concentration (g mL)
1,8-Cineole

400

Limonene

500

p-Cymene

140

140

120

120

100

100

80

80

60
40

100

200
300
Concentration (g mL)

Cupressus sempervirens

% Control

% Control

80

Limonene

400

500

-Pinene

60
40
20

20

0
0

100

Citrus aurantium

200
300
Concentration (g mL)
Linalool

400

500

Linalyl acetate

100

200
300
Concentration (g mL)
Foeniculum vulgare

400

500

E-Anethole

Figure 2. Anti-proliferative activity of (A) T. vulgaris, (B) C. limon, (C) E. globulus, (D) C. sempervirens, (E) C. aurantium and (F) F. vulgare EOs and their main components on
THP-1 cell line (96 h exposure). The mean absorbance values for the negative control (DMSO treated cells) was standardized as 100% absorbance (i.e. no growth inhibition) and
results are displayed as absorbance (% of control) vs EOs concentration.

capacity for inhibiting lipoxygenase activity, thus showing the best

anti-inflammatory activity, although greatly test dependent. The
EOs antioxidant activity was also greatly dependent on several
compounds and not only on their major components. T. vulgaris EO
had the best anti-proliferative activity on THP-1 cells in contrast to
F. vulgare EO. T. vulgaris EOs activity may be attributed to
thymol and carvacrol, whereas its weak anti-proliferative activity
may be attributed to E-anethole. Limonene, present in relative high
amounts in Citrus limon, E. globulus and Cupressus sempervirens
EO also showed good anti-proliferative activity on THP-1 cells.
Experimental
Essential oils provenance and analysis: Citrus aurantium (leaves),
Citrus limon (peel), Cupressus sempervirens, Eucalyptus globulus,
Foeniculum vulgare and Thymus vulgaris essential oils were

provided by the Zaraphyt Company from Rabat, Morocco. The EOs

were analyzed by gas chromatography, and gas chromatography
coupled to mass spectrometry, as previously detailed [39].
Antioxidant activity
ABTS radical cation scavenging capacity: The ABTS radical
cation decolorization assay was carried out using the method
described in [25]. The capability to scavenge the ABTS + was
calculated using the formula: ABTS.+ scavenging activity (%) =
[(A0-A1)/ A0] 100 (%), where A0 is the absorbance of the control
(without sample) and A1 is the absorbance in the presence of the
sample. The sample concentration providing 50% inhibition (IC50)
was obtained by plotting the inhibition percentage against EOs
concentrations.

Biological properties of essential oils

Natural Product Communications Vol. 9 (4) 2014 593

Hydroxyl radical scavenging activity: OH-scavenging activity (%)

was assessed as in Boulanouar et al. [25], and calculated using the
equation: Inhibition = [(A0-A1)/ A0] 100 (%) where A0 is the
absorbance of the control (without sample) and A1 is the absorbance
in the presence of the sample. Tests were carried out in triplicate.
The sample concentration providing 50% inhibition (IC50) was
obtained by plotting the inhibition percentage against EOs
concentrations.

Anti-inflammatory activity
5-Lipoxygenase inhibitory activity: Lipoxygenase is known to
catalyze the oxidation of unsaturated fatty acids containing 1-4
diene structures. The conversion of linoleic acid to 13-hydroperoxy
linoleic acid was followed spectrophotometrically by the
appearance of a conjugate diene at 234 nm on a UV/visible
spectrophotometer, as in [42]. The concentration giving 50%
inhibition (IC50) was calculated as above.

Nitric oxide scavenging capacity: The nitric oxide (NO) scavenging

activity was measured as in [40a]. Fifty L of a serially diluted
sample of each EO and its major components were added to 50 L
of 10 mM sodium nitroprusside in phosphate buffer saline (PBS) in
a 96-well plate, which was incubated at room temperature for
90 min. Finally, an equal volume of Griess reagent was added to
each well and the absorbance was read at 546 nm. Several
concentrations of samples were made and the percentage inhibition
calculated from the formula: [1 - (Asample - Asample blank)/( Acontrol Acontrol blank)]x100, where (Asample - Asample blank) is the difference in
the absorbance of a sample, with or without 10 mM sodium
nitroprusside, and (Acontrol - Acontrol blank) is the difference in the
absorbance of the PBS control, with or without 10 mM sodium
nitroprusside. The sample concentration providing 50% inhibition
(IC50) was obtained by plotting the inhibition percentage against
EOs concentrations.

Anti-proliferative activity
Cell culture: THP-1 cells were cultured in Dulbeccos Modified
Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum, 1% (v/v) non-essential amino acids, 100 U mL-1 penicillin,
and 100 g mL-1 streptomycin. Cells were incubated at 37C in a
humidified 5% CO2 atmosphere.

Oxygen radical absorbance capacity (ORAC): The procedure was

as reported by [25] for EOs. The results were expressed as
micromoles Trolox equivalent (TE).
Chelating metal ions: EOs and their major components ferrous ion
chelating degree was evaluated as in [40b]. The chelating ability
percentage was determined according to: [(A0 A1)/A0 x 100], in
which A0 is the absorbance of the control and A1 the absorbance of
sample. The values of IC50 were determined as above.
Inhibition of lipid peroxidation of lecithin liposomes: Lipid
peroxidation (LP) was measured according to [41]. Liposomes were
obtained from 0.4 g lecithin in 80 mL chloroform. This solution was
dried under vacuum in a rotary evaporator (<50C) to yield a thin,
homogenous film, which was submitted to a nitrogen flux for 30 s.
Liposomes were then submitted to vacuum for at least 2 h until
complete dryness. The film was then dispersed in 80 mL of
phosphate saline buffer 0.01 M, pH 7.0. The mixture was sonicated
to obtain a homogeneous suspension of liposomes and kept at 4C
until the assay. The percentage of LP inhibition was calculated by: I
(%) = (A0-A1)/A0 x 100, where A0 was the absorbance of the control
reaction (full reaction, without the test compound) and A1 was the
absorbance in the presence of the inhibitor.

Anti-proliferative activity: The growth-inhibitory effect of EOs was

measured using the standard 3-(4,5-dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (MTT) assay adapted from Mosmann
[43]. THP-1 cells were seeded in a 96 well plate at 5.103 cells well-1
and exposed to different EO concentrations (10-500 g mL-1) for 1
and 4 days at 37C in 5% CO2/95% air in complete medium
containing serum. All test substances were dissolved in dimethylsulfoxide (DMSO). The solvent concentration in the incubation
medium never exceeded 0.5%. Control cultures received the
equivalent concentration of DMSO. After treatment, cells were
incubated for 1 h in the usual culture conditions after addition of the
same volume of medium containing MTT (2 mg mL-1). After this
incubation, 150 L HCl (0.1 M) in isopropanol was added to
dissolve the blue formazan crystals formed by reduction of MTT.
Absorbance at 570 nm using a background reference wavelength of
630 nm was measured using a dual-wavelength plate reader. The
mean absorbance values for the negative control (DMSO treated
cells) were standardized as 100% absorbance (i.e. no growth
inhibition) and results were displayed as absorbance (% of control)
vs essential oil concentration.
Statistical analysis: Data were analyzed by one-way analysis of
variance (ANOVA) using IBM SPSS Statistics version 20. Tukey
test was used to determine the difference at the 5% significance
level. Paired Student t test was used in some tests to determine
differences at 5% significance.
Acknowledgements - Partially funded by Fundao para a Cincia
e a Tecnologia (FCT) under Pest-OE/EQB/LA0023/2011. Cristina
Megias is recipient of a JAE-Doc (C.S.I.C.) contract from the "Junta
para la Ampliacin de Estudios" program (cofinanced by the
European Social Fund). Isabel Corts-Giraldo is recipient of a JAEPre (C.S.I.C.) fellowship from the "Junta para la Ampliacin de
Estudios" program (cofinanced by the European Social Fund)

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Natural Product Communications Vol. 9 (4) 2014

Published online (www.naturalproduct.us)

Quantification of -Aminobutyric Acid in Sri Lankan Tea by Means of Ultra Performance Tandem Mass Spectrometry
Elisabete Carvalho, P.A. Nimal Punyasiri, H.P.P Sudarshana Somasiri, I. Sarath B. Abeysinghe and Stefan Martens
5-(Hydroxymethyl)-2-furaldehyde Inhibits Adipogenic and Enhances Osteogenic Differentiation of Rat Bone
Mesenchymal Stem Cells
Xiang-ling Tan, Yan-Hong Zhang, Jian-Ping Cai, Li-Hua Zhu, Wen-Jie Ge and Xian Zhang
Variation of Glucosinolate Accumulation and Gene Expression of Transcription Factors at Different Stages of Chinese
Cabbage Seedlings under Light and Dark Conditions
Yeon Bok Kim, Jin-Hyuk Chun, Hye Ran Kim, Sun-Ju Kim, Yong Pyo Lim and Sang Un Park
Identification of the Hydroxamate Siderophore Ferricrocin in Cladosporium cladosporioides
Nina Pourhassan, Ren Gagnon, Thomas Wichard and Jean-Philippe Bellenger
Structure Characterization and Adhesive Ability of a Polysaccharide from Tendrils of Parthenocissus heterophylla
Li Zhang and Wenli Deng
Two Peptides, Cycloaspeptide A and Nazumamide A from a Sponge Associated Marine Actinobacterium Salinispora sp.
Utpal Bose, Mark P. Hodson, P. Nicholas Shaw, John A. Fuerst and Amitha K. Hewavitharana
Full Assignments of the 1H, 13C and 15N Magnetic Resonance Spectra of Two Porphyrin Compounds
Qi-Feng Chen, Yao-Nan Wang, Ling Wang, Xi-Xian Jian, Dong-Lin Chen, Ming Zhao and Feng-Peng Wang
The Effects of Salacia reticulata on Anti-Cellular Oxidants and Melanogenesis Inhibition in -MSH-stimulated and
UV Irradiated B16 Melanoma Cells
Prasit Suwannalert, Ryusho Kariya, Ikuko Suzu and Seiji Okada
Korean Propolis Suppresses Angiogenesis through Inhibition of Tube Formation and Endothelial Cell Proliferation
Seon-Il Park, Toshiro Ohta, Shigenori Kumazawa, Mira Jun and Mok-Ryeon Ahn
ETAS, an Enzyme-treated Asparagus Extract, Attenuates Amyloid -Induced Cellular Disorder in PC12 Cells
Junetsu Ogasawara, Tomohiro Ito, Koji Wakame, Kentaro Kitadate, Takuya Sakurai, Shogo Sato, Yoshinaga Ishibashi,
Tetsuya Izawa, Kazuto Takahashi, Hitoshi Ishida, Ichiro Takabatake, Takako Kizaki and Hideki Ohno
An Integrated Approach to the Evaluation of a Metabolomic Fingerprint for a Phytocomplex. Focus on Artichoke
[Cynara cardunculus subsp. scolymus] Leaf
Giada Fodaroni, Michela Burico, Anna Gaetano, Anna Maidecchi, Rita Pagiotti, Luisa Mattoli, Pietro Traldi and Eugenio Ragazzi
Cytotoxicity and Antimicrobial Activity of the Essential Oil from Satureja montana subsp. pisidica (Lamiceae)
Tatjana Kundakovi, Tatjana Stanojkovi, Branka Kolundija, Stevan Markovi, Branka ukilovi, Marina Milenkovi and
Branislava Lakui
Chemical Composition of Essential Oils of Grindelia squarrosa and G. hirsutula
Katalin Veres, Orsolya Roza, Eszter Laczk-Zld and Judit Hohmann
Seasonal Influence on the Essential Oil of Eucalyptus microcorys
Flvia N. M. Oliveira, Gilmara A. C. Fortes, Jos R. Paula, Pedro H. Ferri and Suzana C. Santos
Composition of the Essential Oil of Wild Grown Caraway in Meadows of the Vienna Region (Austria)
Remigius Chizzola
Volatile Compounds from Roots, Stems and Leaves of Angelica acutiloba growing in Taiwan
Hsin-Chun Chen, Yi-Jr Tsai, Li-Yun Lin, Chin-Sheng Wu, Shan-Pao Tai, Yu-Chang Chen and Hsiu-Mei Chiang
Anti-oxidant, Anti-inflammatory and Anti-proliferative Activities of Moroccan Commercial Essential Oils
Smail Aazza, Badiaa Lyoussi, Cristina Megas, Isabel Corts-Giraldo, Javier Vioque, A. Cristina Figueiredo and Maria G. Miguel

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Natural Product Communications

2014
Volume 9, Number 4
Contents
Original Paper

Page

Activation of Cell-mediated Immunity by Morinda citrifolia Fruit Extract and Its Constituents
Kazuya Murata, Yumi Abe, Megumi Futamura-Masuda, Akemi Uwaya, Fumiyuki Isami, and Hideaki Matsuda
A New Pyrrolosesquiterpene from the Terrestrial Streptomyces sp. Hd7-21
Dong-Ze Liu and Bo-Wen Liang
Structure-Activity Relationships of Tanshinones in Activating Nrf2. A DFT Study and Implications for Multifunctional
Antioxidant Discovery
You-Min Sun, Zheng-Tao Xiao and Hong-Yu Zhang
Chemical Modifications of Cinchona Alkaloids Lead to Enhanced Inhibition of Human Butyrylcholinesterase
Daniela Karlsson, Adyary Fallarero, Pravin Shinde, Anju CP, Igor Busygin, Reko Leino, C. Gopi Mohan and Pia Vuorela
Alkaloids from Marine Sponges as Stimulators of Initial Stages of Development of Agricultural Plants
Mikhail M. Anisimov, Elena L. Chaikina and Natalia K. Utkina
Crinane Alkaloids of the Amaryllidaceae with Cytotoxic Effects in Human Cervical Adenocarcinoma (HeLa) Cells
Jerald J. Nair, Lucie Rrov, Miroslav Strnad, Jaume Bastida, Lee Cheesman and Johannes van Staden
Alkaloids from Xylariaceae sp., a Marine-derived Fungus
Xu-Hua Nong, Xiao-Yong Zhang, Xin-Ya Xu, Yun-Lin Sun and Shu-Hua Qi
Occurrence of a Taurine Derivative in an Antarctic Glass Sponge
Marianna Carbone, Laura Nez-Pons, M. Letizia Ciavatta, Francesco Castelluccio, Conxita Avila and Margherita Gavagnin
Two New Thyminenol Derivatives from the Marine Sponge Haliclona sp.
Bin Wang, Yaocai Lin, Yinning Chen and Riming Huang
Decorosides A and B, Cytotoxic Flavonoid Glycosides from the Leaves of Rhododendron decorum
Mostafa E. Rateb, Hossam M. Hassan, El-Shaimaa A. Arafa, Marcel Jaspars and Rainer Ebel
In vitro Cultures of Bituminaria bituminosa: Pterocarpan, Furanocoumarin and Isoflavone Production and Cytotoxic
Activity Evaluation
Francesca DAngiolillo, Laura Pistelli, Cecilia Noccioli, Barbara Ruffoni, Simona Piaggi, Roberto Scarpato and Luisa Pistelli
In Vitro Antioxidant Activity and Phenolic Content of Cedrus brevifolia Bark
Elena Cretu, Juha-Pekka Salminen, Maarit Karonen, Anca Miron, Christiana Charalambous, Andreas I. Constantinou and
Ana Clara Aprotosoaie
Evaluation of Bioactive Components and Antioxidant and Anticancer Properties of Citrus Wastes Generated During
Bioethanol Production
Soon Jae Im, Jae-Hoon Kim and Min Young Kim
A New Coumarin and Cytotoxic Activities of Constituents from Cinnamomum cassia
Tran Minh Ngoc, Nguyen Xuan Nhiem, Nguyen Minh Khoi, Doan Cao Son, Tran Viet Hung and Phan Van Kiem
Coumarin Compounds in Coronilla scorpioides Callus Cultures
Anna Piovan, Raffaella Filippini and Gabbriella Innocenti
A New Isocoumarin from Cajanus cajan (Fabaceae)
Virginia F. Rodrigues, Rodrigo R. Oliveira and Maria Raquel G. Vega
Styryllactones and Acetogenins from the Fruits of Goniothalamus macrocalyx
Quy Hung Trieu, Huong Doan Thi Mai, Van CuongPham, Marc Litaudon, Franoise Gueritte, Pascal Retailleau,
Isabelle Schmitz-Afonso, Olinda Gimello, Van Hung Nguyen and Van Minh Chau
Potent Acetylcholinesterase Inhibitory Compounds from Myristica fragrans
To Dao Cuong, Tran Manh Hung, Hyoung Yun Han, Hang Sik Roh, Ji-Hyeon Seok, Jong Kwon Lee, Ja Young Jeong,
Jae Sue Choi, Jeong Ah Kim and Byung Sun Min
Clastogenic Effect of Atranorin, Evernic acid, and Usnic Acid on Human Lymphocytes
Gordana S. Stojanovi, Miroslava Stankovi, Igor . Stojanovi, Ivan Pali, Vesna Milovanovi and Sofija Rani
MAO-A Inhibition Profiles of Some Benzophenone Glucosides from Gentiana verna subsp. pontica
Duygu Kaya, Anna K. Jger, Funda N. Yaln and Tayfun Ersz
Phytochemical Investigations of Lonchocarpus Bark Extracts from Monteverde, Costa Rica
Caitlin E. Deskins, Bernhard Vogler, Noura S. Dosoky, Bhuwan K. Chhetri, William A. Haber and William N. Setzer
Immune Enhancing Effects of Echinacea purpurea Root Extract by Reducing Regulatory T Cell Number and Function
Hyung-Ran Kim, Sei-Kwan Oh, Woosung Lim, Hyeon Kook Lee, Byung-In Moon and Ju Young Seoh
Isocorilagin, a Cholinesterase Inhibitor from Phyllanthus niruri
Yee-Hui Koay, Alireza Basiri, Vikneswaran Murugaiyah and Kit-Lam Chan
Antioxidant Activity and Phenolic Content of Bergenia crassifolia, B. x ornata and B. ciliata
Helena Hendrychov, Anna Vildov, Nina Koevar-Glava, Lenka Tmov, Elnura Abdykerimova Kanybekovna and Ji Tma
Antioxidant Activity and Total Phenolic Contents of Three Bupleurum Taxa
Hyeusoo Kim, Sea Hyun Kim and Kyeong Won Yun
Continued inside backcover

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