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Planta Medica
Journal of Medicinal Plant and Natural Product Research
www.thieme.de/fz/plantamedica l www.thieme-connect.com/ejournals
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Introduction
!
Malaria is unquestionably one of the most impor-
tant infectious diseases in the world along with
HIV/AIDS and tuberculosis. Roughly half of the
worldʼs human population lives in regions in
which malaria is endemic. There are 500 million
new cases and around 2 million deaths per year
[1] due to malaria. It is the main cause of econom-
ic loss, estimated at more than US$ 12 billion an-
nually [2] with a high morbidity in the endemic
tropical and subtropical areas worldwide. Ap-
proximately 99.8% of all malaria infections re-
corded in Brazil occur in the Amazon region [3].
The increase in Plasmodium falciparum strains
that are multiresistant to available antimalarial
drugs is of great concern and is a trend that re-
quires innovative strategies for controlling the
disease.
It is estimated that around 80 % of the tropical and
subtropical populations of the world depend on
herbal remedies for the treatment of disease [4].
Medicinal plants are a rich source for the discov-
ery of new drugs against malaria [5] and other in-
fectious diseases. This has already been proven by
the development of the antimalarials that are cur-
rently in use. Thus, the quinoline ring of quinine
(a natural product isolated from Cinchona spp. in
the 19th century) is the structural basis for many
synthetic antimalarials developed last century,
such as chloroquine, primaquine, and mefloquine.
The active principle of Artemisia annua, artemisi-
nin (a sequiterpene lactone) [6], and its semisyn-
thetic derivatives are effective drugs for treating
serious malaria caused by multidrug resistant P.
falciparum (resistant to chloroquine since 1980).
A very important class of antimalarial natural
products are lower molecular weight components
of essential oils (EOs), such as monoterpenes, ses-
Abstract
!
The prophylactic and therapeutic arsenal against
malaria is quite restricted and all the antimalar-
ials currently in use have limitations. Thus, there
is a need to investigate medicinal plants in the
search for phytochemicals which can be devel-
oped into drugs. In our investigation, essential oils
(EOs) were obtained from Vanillosmopsis arborea
(Gardner) Baker, Lippia sidoides Cham. and Croton
zehntneri Pax & K. Hoffm., aromatic plants abun-
dant in northeastern Brazil, which are found in
the caatinga region and are used in traditional
medicine. The chemical composition of these EOs
was characterized by GC‑MS, and monoterpenes
and sesquiterpenes were well represented. We
assessed the in vitro activity of these EOs and also
individual EO chemical components against the
human malaria parasite Plasmodium falciparum
(K1 strain) and the in vivo activity of EOs in mice
infected with Plasmodium berghei. The acute tox-
icity of these oils was assessed in healthy mice
and in vitro cytotoxicity was determined at differ-
ent concentrations against HeLa cells and mice
macrophages. The EO of V. arborea was partially
active only when using the subcutaneous route
(inhibited from 33 up to 47%). In relation to the
EOs, L. sidoides and C. zehntneri were active only
by the oral route (per gavage) and partially inhib-
ited the growth of P. berghei from 43 up to 55%
and showed good activity against P. falciparum in
vitro (IC50 = 7.00, 10.50, and 15.20 µg/mL, respec-
tively). Individual EO constituents α-bisabolol, es-
tragole, and thymol also exhibited good activity
against P. falciparum (IC50 = 5.00, 30.70, and
4.50 µg/mL, respectively). This is the first study
showing evidence for the antimalarial activity of
these species from northeastern Brazil and the
low toxicity of their EOs.
In Vitro and In Vivo Antimalarial Activity of Essential Oils
and Chemical Components from Three Medicinal Plants
Found in Northeastern Brazil
Authors Magaly L. Mota1, Lis Tavares Coelho Lobo 1, José Galberto M. da Costa2, Leandro S. Costa 3, Hugo A. O. Rocha 3,
Luiz F. Rocha e Silva4, 5, Adrian M. Pohlit5, Valter F. de Andrade Neto1
Affiliations The affiliations are listed at the end of the article
Key words
l
"Vanillosmopsis arborea
(Asteraceae)
l
"Lippia sidoides (Verbenaceae)
l
"Croton zehntneri
(Euphorbiaceae)
l
"estragole
l
"α‑bisabolol
l
"thymol
l
"antimalarial activity
received August 17, 2011
revised February 7, 2012
accepted February 10, 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1298333
Published online March 22,
2012
Planta Med © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Valter Ferreirade Andrade
Neto, Ph.D.
Laboratory of Malaria and
Toxoplasmosis Biology
Department of Microbiology
and Parasitology
Rio Grande do Norte Federal
University, Campus Universitário
Av. Senador Salgado Filho,
Lagoa Nova
CEP 69061-000 –Natal –RN
Brazil
Phone: + 55 84 32 15 34 37-226
Fax: +558432119210
aneto@cb.ufrn.br
Magaly LM et al. In Vitro and …Planta Med
Original Papers
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
b
quiterpenes [7], and phenylpropanoids [8]. Thus, a number of
highly active monoterpene components of EOs exhibit antiplas-
modial activity according to in vitro studies mainly in Plasmodi-
um falciparum [7]. Importantly, mechanistic studies have re-
vealed that low molecular weight monoterpenes, such as limo-
nene and linalool, and sesquiterpenes, such as farnesol and nero-
lidol, inhibit the biosynthesis of metabolites such as dolichols and
the terpenoid side chains of ubiquinones in the trophozoite and/
or schizont stages of Plasmodium falciparum [9]. Meanwhile, low
molecular weight volatile phenylpropanoids, such as isochavicol
and isochavicol propionate, have been found to exhibit antiplas-
modial activity [8]. Thus, medicinal plants whose utility and rel-
ative safety have been identified by ethnobotanical sources, and
which produce Eos, are of specific interest because of their po-
tential antiplasmodial and antimalarial activities.
Northeastern Brazil has a large medicinal plant biodiversity, a
rich traditional medicine heritage, and vast ethnobotanic knowl-
edge among its people. However, studies on the antimalarial ac-
tivity of the plants of this region are relatively unknown. The aim
of the present study was to investigate the antimalarial activity,
chemical composition, and toxicity of the EOs of three plant spe-
cies from the caatinga biome of Brazilʼs northeast for the first
time. These plants are used in folk medicine as infusions (stem
and leaves) and bark/leaf poultices, wherein their EOs are likely
important components in their medicinal effects. Thus, Vanillos-
mopsis arborea (Gardner) Baker (Asteraceae), Lippia sidoides
Cham. (Verbenaceae), and Croton zehntneri Pax & K. Hoffm. (Eu-
phorbiaceae) are commonly named “candeeiro”,“alecrim pimen-
to”,and“canelinha”, respectively, and are used by inhabitants of
the Araripe bioregion of Ceará State as anti-inflammatory, anti-
fungal, and antibacterial agents, and for the treatment of gastric
disorders. In addition, detailed analysis of these essential oils
showed monoterpenes and sesquiterpenes, which are chemical
classes with antimalarial activity well reported in the scientific
literature.
Materials and Methods
!
Collection of plant materials and extraction of EOs
Plant materials were collected (l
"Table 1) in the months of May,
July, and December of 2007. V. arborea and C. zehntneri were col-
lected, respectively, at Chapada do Araripe in the municipality of
Crato and at Morro do Chapéu in the municipality of Salitre,
Ceará State. L. sidoides was collected in the medicinal plant nurs-
ery of the Natural Product Research Laboratory –LPPN/Universi-
dade Regional do Cariri (URCA), Crato, Ceará State. A voucher
specimen of each species was classified and deposited at the Pris-
co Bezerra Herbarium (V. arborea) at the Universidade Federal do
Ceará and at the Dárdaro de Andrade Lima Herbarium (C. zehnt-
neri and L. sidoides) at URCA. The collected plant materials were
individually extracted using hydrodistillation for two hours in a
Clevenger apparatus to obtain the EO from each species. The EOs
were then dried with anhydrous Na2SO4and stored in the refrig-
erator until the analysis could be performed.
Chemical composition of EOs using GC‑MS
Chemical composition analyses on the EOs obtained in the
above procedure were performed using a GC‑MS system (Shi-
madzu CG-17A gas chromatograph/MS-QP5050A spectrometer):
DB-5HT capillary column (30 m × 0.251 mm); car rier gas: heli-
um = 1.0 mL/min; column pressure = 72.3 kPa; linear velocity =
37.2 cm/sec; total flow = 85 mL/min; carrier f low = 85 mL/min;
injector temperature = 280 °C; detector temperature = 280 °C;
column temperature = 60 (2 min) –180 °C (1 min) at 4 °C/min, then
180–260 °C at 10 °C/min (10 min), operating under ionization en-
ergy of 70 eV. Standard hydrocarbon reference samples were in-
jected and Kovats indices were corrected by a linear equation.
Component identification was based on spectral fragmentation,
using a computer library (Wiley 229), on retention indices and
comparison with literature data [12].
Chromatographic separation of the EO of V. arborea
and isolation of α-bisabolol
Column chromatography (CC) was performed using a glass col-
umn (10 × 5 cm) having a 0.3 mm glass thickness. The column
was packed with 20.0 g of silica gel 60 (Vetec Química Fina Ltda).
The EO of V. arborea (2.0 g) was loaded onto the column head and
the column was eluted with petroleum ether, pure dichlorometh-
ane, and chloroform, or in binary combinations of these solvents.
All the solvents used were purchased from Labsynth Prod. Lab.
Ltda. Fractions measuring 5.0 mL were collected in test tubes.
Monitoring by thin-layer chromatography (TLC) was used to
compare and combine the fractions obtained. Combined frac-
tions 48–80 yielded 0.78 g of a colorless liquid, which was shown
to be pure by TLC.
Monoterpene and phenylpropanoid substances
As described above, pure α-bisabolol was isolated from the EO of
V. arborea. Samples of pure thymol and estragole were kindly do-
nated by Kaapi. The purity of these samples based on GC‑MS
analysis was > 99.9 %.
Animals and ethics committee approval
Adult Swiss albino mice were used for the acute toxicity and anti-
malarial assays. The animals were housed in standard cages and
received water and food ad libitum. The use of the animals was
approved by the Ethics Committee of the Universidade Federal
do Rio Grande do Norte (CEUA 043/2010).
In vivo acute toxicity assay
The acute toxicity of the EOs was determined in healthy mice us-
ing a modified version of the procedure described by Lorke [11].
Briefly, this involved gavage administration of different doses of
the EO. This route was chosen because it is the same administra-
tion route used in antimalarial tests. Groups of three mice (fe-
Table 1 Plant species from the Araripe bioregion used to assess antimalarial activity.
Species (family) Voucher specimen number Part of the plant (g) Essential oil extraction (hydrodistillation)
Amount obtained (mL) Output (%)
Vanillosmopsis arborea (Asteraceae) 43291 shredded stem (1700) 5.00 0.29
Lippia sidoides (Verbanaceae) 464 dry leaves (750) 3.70 0.49
Croton zehntneri (Euphorbiaceae) 1619 fresh leaves (480) 4.20 0.87
Magaly LM et al. In Vitro and …Planta Med
Original Papers
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
b
males) weighing 20± 2g were given oral doses of 0.15, 0.31, 0.6,
0.12, 0.25, 0.5, and 10 g/kg. The EOs were diluted in a 2% Tween-
20 solution in distilled water, and administered in a single 200µL
dose. The negative control group received 2% Tween-20 in dis-
tilled water. The mortality, weight, and overall aspects of the ani-
mals were monitored for eight consecutive days. Mortality was
expressed in accumulated percentage per group and median le-
thal doses (LD50) were established [12]. The surviving animals
were reutilized in the toxicity assay and received 200 µL oral
doses of EO suspension per animal for four consecutive days.
The mortality rate and overall aspects of these animals were
monitored daily for twenty consecutive days.
In vitro cytotoxicity assays
HeLa cells (human cervical carcinoma) and mice macrophages
were maintained in continuous culture in DMEM (Dulbeccoʼs
modified Eagle medium) supplemented with 10 % bovine fetal se-
rum (BFS) in a low oxygen atmosphere (5% CO2,2%O
2,andN
2
balance) at 37 °C. The cytotoxicity of the plant EOs was deter-
mined using the methylthiazoletetrazolium colorimetric assay
(MTT) [13]. For the assays, the cells were trypsinized, washed,
suspended in DMEM, and distributed into 72 wells per plate
(5 × 103cells per well) then incubated for 18 h at 37 °C. The EO
from the stems of V. arborea, the leaves of L. sidoides.andthe
leaves of C. zehntneri were separately diluted in DMEM and
tested in duplicate at the following concentrations: 10, 50, 75,
125, 250, and 500 µg/mL. The positive control group used DMEM
with 10% BFS and a negative control group used DMEM without
BFS. After 24 and 48 h of incubation at 37 °C, 100 µL of MTT (1 mg/
mL DMEM) was added to each well. After 4 h of incubation at
37 °C, the supernatant was removed and 100 µL of isopropyl alco-
hol was added to 0.04 M hydrochloric acid in each well. The ab-
sorbance of each well was obtained from a spectrophotometric
reading at 562 nm. The minimum lethal doses that inhibited
50% of cell growth were obtained from the drug concentration-
response curves. Results are expressed in mean ± standard devia-
tion.
Parasite culture and in vitro antimalarial tests
The parasite used for the in vitro tests was the chloroquine, pyri-
methamine, and cycloguanil-resistant Plasmodium falciparum K1
strain which was acquired from MR4 (Malaria Research and Ref-
erence Reagent Resource Center). Parasites were maintained in
continuous culture in human erythrocytes (blood group A+, us-
ing RPMI 1640 medium supplemented with 10 % human serum).
The antiparasitic effects of the EOs were measured by the percent
inhibition of parasite growth in relation to the negative control
(parasites cultivated in drug-free medium). Briefly, the samples
tested were diluted in RPMI 1640 culture medium (with 0.02%
Tween-20). These stock solutions were further diluted in com-
plete medium (RPMI 1640 plus 10% human serum) to give each
of the test concentrations used (seven dilutions from 100 to
0.13 µg/mL). The cultures used in the tests exhibited trophozoites
in sorbitol-synchronized blood [14] at 1 to 1.5% parasitemia and
2.5% hematocrit, and were incubated with extracts, fractions, or
isolated compounds for a total of 48h at 37 °C. A positive control
containing the reference antimalarial drug chloroquine diphos-
phate, 97% (Sigma-Aldrich), and a negative control with medium
and the Tween-20 solution were used in each experiment. The
50% inhibitory concentrations (IC50) as compared to the drug-
free control responses were estimated by linear interpolation us-
ing Microcal Origin®software. Each experiment was performed
in triplicate and each experiment was repeated three times.
Blood smears were prepared from each test well and read in a
double-blind manner.
In vivo antimalarial assays
The traditional suppression test [15] with modifications [16] was
used. Br iefly, adult Swiss albino mice weighing 20 ± 2 g were in-
fected intraperitoneally with infected blood containing 1 × 105
Plasmodium berghei NK65. The mice were randomly allocated to
groups of three to five animals per cage. The EOs were tested in
different experiments using the gavage technique and, when
necessary, by subcutaneous administration. For oral treatment,
the EOs of the stem of V. arborea, the leaves of L. sidoides,and
the leaves of C. zehntneri were diluted in a Tween-20 solution
with distilled water (final concentration of 2% Tween-20), and
200 µL of the concentration of test solution was administrated
orally to each animal in doses of 100, 500, and 1000 mg/kg/day.
Chloroquine diphosphate, 97% (Sigma-Aldrich), was dissolved in
distilled water and was administered orally as an antimalarial
control reference at a dose of 10 mg/kg/day. A negative control
group consisted of a 2% Tween-20 solution in distilled water that
was orally administered to each animal. On the 5th and 7th day
after parasite inoculation, blood smears from all the mice were
prepared, fixed with methanol, stained with Giemsa, and exam-
ined microscopically (1000 × magnification).
For subcutaneous treatment, the EO from the stem of V. arborea
was suspended in saline phosphate buffer (SPB) solution, and
200 µL was administered to each animal in doses of 25, 50, 100,
and 500 mg/kg/day. Chloroquine diphosphate was dissolved in
SPB at a dose of 10 mg/kg/body weight, and administered subcu-
taneously. The negative control group consisted of the subcuta-
neous administration of 200 µL of SPB to each animal. Blood
smears were obtained from all the mice on the 5th and 7th day
after parasite inoculation as shown above. The parasitemia of all
the experiments was determined by counting the number of par-
asitized erythrocytes out of 1000–3000 in random fields of the
microscope. Parasite growth inhibition in the drug-treated
groups was defined as the parasitemia in the non-treated control
group minus the parasitemia in the treated control group, divid-
ed by the parasitemia in the non-treated control group, ex-
pressed in percentages. The EOs were considered partially active
if parasitemia decreased by 30% or more [16]. Mortality was
monitored in all the groups for four weeks after inoculation.
Statistical analyses
ANOVA and the Tukey test were used for parasitemia analysis in
the antimalarial assays, and the Studentʼs t-test was used in the
analysis of the reduction in parasitemias between the groups,
and cytotoxicity in the assays with HeLa cells and mice macro-
phages. The statistics were significant when p ≤0.05.
Results
!
This is the first report on the antimalarial properties of EOs from
V. arborea, L. sidoides,andC. zehntneri. In this study, chemical
composition, acute toxicity, cytotoxicity, and the antimalarial ac-
tivity of the EOs of these plant species were investigated.
Results of chemical composition analysis of the EOs are summar-
ized in l
"Table 2. The EOs contained a variety of monoterpenes
and sesquiterpenes. The hexane-chloroform fractions were ob-
tained from successive column chromatographic analyses. The
Magaly LM et al. In Vitro and …Planta Med
Original Papers
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
b
main component of the EO from the stem of V. arborea was the
sesquiterpene α-bisabolol. Using GC‑MS analysis, it was estab-
lished that this component was present in ca. 80.4 % of the fraction
[10]. Analysis of the EO obtained from the leaves of L. sidoides re-
vealed a chemical composition consisting of monoterpenoids and
sesquiterpenoids, and thymol as the main component (84.9%).
Analysis of the EO from the leaves of C. zehntneri revealed the
presence of monoterpenoids and sesquiterpenoids, and estragole
as the main component (76.80%).
The EOs of all three medicinal plants exhibited significant inhibi-
tion of the human malaria parasite in vitro as well as individual
major chemical components which are found in these EOs (l
"Ta-
ble 3). The antimalarial activity of EOs and individual compo-
nents against the P. falciparum K1 strain were similar based on
IC50 values, which were 7.00 to 15.20µg/mL for EOs and 4.50 to
30.70 µg/mL for isolated compounds. The sensitivit y of the P. fal-
ciparum strain toward the EOs tested was similar and reproduci-
ble in assays in duplicate on separate occasions. The in vitro cyto-
toxicity assay results for EOs and estragole, thymol, and α-bisabo-
lol are shown in l
"Table 3.Thein vitro cytotoxicity of the EOs
from these plants to the HeLa cell line and mice macrophage
was evaluated in three different experiments. The minimum le-
thal dose that inhibits 50% of cell growth for the EOs exhibited
values ranging from 340–500 µg/mL.
In vivo antimalarial assay results for the EO from the stem of
V. arborea and from the leaves of L. sidoides and C. zehntneri using
Plasmodium berghei-infected mice are summarized in l
"Tables 4
and 5. In all the experiments conducted, the control drug chloro-
quine diphosphate, 97%, assayed in parallel, reduced parasitemia
by 98 to 100%, and no mortality was observed in the groups that
received chloroquine diphosphate after 30 days of observation.
When the EO from the stem of V. arborea was administered orally
at doses of 500 and 1000 mg/kg, it did not significantly reduce
parasitemia compared to the untreated control. When adminis-
tered subcutaneously for eight days, the EO from the stem of
V. arborea at a dose of 100 mg/kg reduced parasitemia by 47 and
42%, on the 5th and 7th day after infection, respectively. At a dose
of 500 mg/kg, the reduction was higher on the 7th day. The mean
survival of the mice was 30 and 29 days at doses of 100 and
500 mg/kg, respectively, and 26 days for the untreated control.
When administered subcutaneously for four days, the EOs from
L. sidoides and C. zehntneri were inactive; they became active on-
ly when administered orally. The EO from the leaves of L. sidoides
inhibited parasitemia by 40 to 55%; the mean antimalarial activ-
ity is shown in l
"Table 5. At doses of 100 and 500 mg/kg, parasite
growth was inhibited by 55 and 47%, respectively, on the 5th day,
and by 45 and 40%, respectively, on the 7th day after infection. In
additional experiments, the 100 mg/kg dose showed similar ac-
tivity to that of the 500 mg/kg dose (no signif icant statistical dif-
ference). The 1000 mg/kg dose exhibited activity on the 5th day
(48%) and 7th day (49%). The mean survival of the mice was 25,
39, and 23 days for 100, 500, and 1000mg/kg, respectively, and
24 days for the untreated control.
Table 3 In vitro biological activity of the essential oils from the stem of V. arborea, and from the leaves of L. sidoides and C. zehntneri.
EOs/compounds Biological assay
The minimum lethal dose 50 % (µg/mL) Inhibition of P. falciparum
HeLa Macrophages IC50 (µg/mL)
24 h 48 h 24 h 48 h
V. arborea EO ≥500 ≥500 ≥500 ≥500 7.00 ± 3.50
L. sidoides EO 480 ± 23 340 ± 34 455 ± 28 415 ± 30 10.50 ± 2.80
C. zehntneri EO ≥500 ≥500 ≥500 ≥500 15.20 ± 3.30
Estragole ≥500 –≥500 –30.70 ± 3.10
Thymol ≥500 –≥500 –4.50 ± 1.70
α-Bisabolol ≥500 –≥500 –5.00 ± 2.30
Values are expressed as mean ± standard error of the mean (SEM). Chloroquine diphosphate, 97 %, was tested in parallel (control) and showed an IC50 = 0.30 ± 0.04 µg/mL
Table 2 Essential oil compounds,
determined by chromatographic
analyses, from the stem of V. ar-
borea, and from the leaves of L. si-
doides and C. zehntneri.
Compounds** Retention time (min) Relative peak areas (%)
V. arborea*Lippia sidoides C. zehntneri
α-Bisabolol 34.04 80.43 ––
Estragole 29.1 ––76.80
1.8-Cineol 22 ––7.0
Eugenol 35 ––5.3
Bicyclogermacrene 38 ––1.7
β-Ocimene 22.2 ––1.6
Sabinene 19.2 ––0.61
Myrcene 19.7 ––4.4
Thymol 11.779 –84.87 –
p-Cymene 4.288 –5.33 –
Carvacrol 11.918 –0.41 –
Limonene 4.416 –1.68 –
α-Terpinene 5.024 –1.32 –
Thymyl-methyl-ether 9.781 –3.01 –
Total 80.43 96.62 97.41
* Proportion present in the hexane-chloroform fraction; ** chromatographic analyses conducted by GC‑MS
Magaly LM et al. In Vitro and …Planta Med
Original Papers
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
b
In the experiments conducted with the EO from the leaves of C.
zehntneri, only the dose of 500 mg/kg/day was effective and sig-
nificant in all the biological assays. This dose reduced parasitemia
by 53.5% on the 5th day after infection. On the 7th day, inhibition
was 43%. The other doses used were inactive (≤30 %). The mean
survival of the mice was 23 days, quite similar to that of the un-
treated control.
Discussion
!
Terpene compounds are responsible for the antimalarial activity
of many plants [17,18]. Lopes et al. [19] isolated the sesquiter-
pene nerolidol and demonstrated that it exhibited antimalarial
activity. In the Katzin group at the University of São Paulo, it has
been shown that the terpenoid compounds farnesol, nerolidol,
limonene, and linalool inhibited dolichol biosynthesis in the
trophozoite and schizont stages of P. falciparum in vitro, while
farnesol, nerolidol, and linalool exhibited stronger inhibitory ac-
tivity on the biosynthesis of the isoprenic side chain of the benzo -
quinone ring of ubiquinones in the schizont stage.
The main component, α-bisabolol, is an unsaturated hydroxy ses-
quiterpene that is present at high levels in the EO from the stem
of V. arborea [20].
The monoterpene thymol is present in the essential oil from the
leaves of Lippia sidoides [21]. In this work, all EOs and individual
components were considered to be active according to the crite-
ria established by Andrade-Neto et al. (IC50 < 50 µg/mL) [22].
Although the compounds tested are less active than chloroquine
diphosphate in vitro, the data reported here provide some ratio-
nal evidence to support studies for their improvement. This fact
should be assessed in terms of the balance between the ineffec-
tiveness of antimalarial drugs available against P. falciparum-re-
sistant strains and prototypes of new drugs, as well as the risks
of drug toxicity and the benefits of the pharmacological action.
The EO from the stem of V. arborea showed effective antimalarial
activity only when administered subcutaneously, which may
limit its usefulness as an antimalarial substance. This may be the
result of slow uptake or rapid elimination of the active metabo-
lites due to intracellular compartmentalization, or deactivation
of the compound in vivo as has been shown for other molecules
[23]. Factors such as absorption indices and bioavailability are
likely acting to make the subcutaneous route the most effective
route of administration of the EO of V. arborea, where the speed
of disintegration and dissolution of the drug occurred more rap-
idly [24–26]. This outcome means that the animal organism ab-
sorbed a larger amount of the drug, while plasma levels remained
stable, thus generating the expected effect. However, by the oral
route, there were likely losses in bioavailability, consequently in-
terfering with distribution [27]. It was observed that, in almost
all the doses of EOs tested, there is little evidence of a dose-re-
sponse relationship, a fact often observed when using crude ex-
tracts/fractions and outbred mice. These factors are likely related
to the time elapsed for the drug to reach its action site, a time pe-
riod of around five to seven days. In our study, the sample popu-
lation consisted of genetically variable animals, which may have
been a factor in the activity of the drug. This occurs in the human
population, where the phenomena of latency and duration of
drug activity are altered by individual variations [27].
The EO from the leaves of L. sidoides showed effective antimalarial
activity in all the experiments, but with variations in parasitemia
reduction among the days analyzed. This fact, which was not sta-
tistically significant, is likely related to the individual variation of
animals as a response to the treatment performed, as well as to
oral administration, which may be associated with the absorp-
tion and metabolization of active components. In just one experi-
ment (data not shown), only the higher doses showed borderline
Table 4 Antimalarial activity of the essential oil from the stem of Vanillosmopsis arborea administered orally (per gavage) and subcutaneously.
Administration route Dose (mg/kg/day) % Parasitemia on day 5 or 7 ± SEM* (% inhibition of parasite growth)**
5th 7th
Oral 1000 1.83 ± 0.27 (20) 4.96 ± 1.02 (0)
500 3.48 ± 1.56 (16) 6.80 ± 1.60 (0)
Subcutaneous 500 2.17 ± 1.16 (40) 6.20 ± 3.04 (47)
100 2.93 ± 1.60 (47) 6.80 ± 3.50 (42)
50 3.50 ± 1.30 (33) 6.70 ± 2.35 (28)
25 4.20 ± 1.50 (29) 6.27 ± 2.80 (20)
* Mean ± standard error of the mean (four independent experiments). Parasitemia reduction compared to untreated control mice.** EO dose that reduced parasitemia by ≥30 % is
considered active. Chloroquine diphosphate, 97% (CQ), administered in parallel (10 mg/kg/day) reduced parasitemia by 98–100%
Table 5 Antimalarial activity of the essential oils from the leaves of Lippia sidoides and Croton zehntneri by the oral route (per gavage).
EOs Dose (mg/kg/day) % Parasitemia on day 5 or 7 ± SEM* (% inhibition of parasite growth)**
5th 7th
L. sidoides 1000 0.81 ± 0.23 (48) 2.41 ± 0.06 (49)
500 0.60 ± 0.14 (47) 3.80 ± 0.55 (40)
100 0.41 ± 0.10 (55) 2.55 ± 0.40 (45)
C. zehntneri 1000 0.68 ± 0.09 (25) 4.03 ± 0.53 (20)
500 0.12 ± 0.08 (53.50) 0.77 ± 0.23 (43)
100 0.56 ± 0.25 (28) 2.90 ± 0.17 (22)
* Mean ± standard error of the mean (four independent experiments). Parasitemia reduction compared to untreated group control. ** EO dose that reduced parasitemia by ≥30%
is considered active. Chloroquine phosphate, 97% (CQ), administered in parallel (10 mg/kg/day) reduced parasitemia by 98–100%
Magaly LM et al. In Vitro and …Planta Med
Original Papers
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b
antimalarial activity on the 5th day after infection, and on the 7th
day its activity increased significantly, which would suggest a
slower pharmacological effect. The toxicity was considered low
for V. arborea and C. zehntneri, and moderate for L. sidoides,at
the highest concentrations tested (up to 500 µg/mL) since it was
nearly 20–30 times higher than the dose inhibiting 50 % of P. fal-
ciparum growth (16.4 µg/mL). The isolated compounds showed
cytotoxicity similar to the EOs.
In vivo acute toxicity assays on the EOs under study showed low
or moderate toxicity [28]. The EO from the stem of V. arborea,
when administered only once at different doses (0.15–10 g/kg),
produced mainly ruffled fur in the mice, resulting in the death
of some of the animals. The lethal dose for 50% of the animals
(LD50) was 7 g/kg (no acute toxicity). When the EO was adminis-
tered for four consecutive days, the signs of toxicity observed
were: weight loss at the start of the experiment, a loss that was
recovered after dose suspension, and ruffled fur in all the ani-
mals. However, no deaths occurred in any of the groups. It was
not possible to assess the toxicity of the doses higher than
0.12 g/kg (highest dose tested in this experiment) owing to the
small amount of material available. The EO from the leaves of
L. sidoides, when administered in a single dose (0.6–10 g/kg), pro-
duced toxic effects such as tachycardia, weight loss, and ruffled
fur at all the doses tested, and death at doses between 0.25 and
10 g/kg. The LD50 determined was 7 g/kg (no acute toxicity). When
administered for four consecutive days, the following toxic as-
pects were observed: weight loss, ruffled fur, and death. The
LD50 was 1.8 g/kg (low acute toxicity).
Mendonça et al. [29], in preclinical acute toxicological assays,
demonstrated the low toxicity of the hydrosoluble components
carried by water vapor during the extraction of the EO from the
leaves of L. sidoides. The contact reaction test, performed with the
external application of 1% EO in animals, showed no hypersensi-
tive reactions. In acute toxicity assays with mice, it was demon-
strated that the EOs of L. sidoides and C. zehntneri showed no
toxic effects up to 3 g/kg [30].
This study shows, for the first time, partial antimalarial activity in
P. berghei-infected mice for the EOs of V. arborea, L. sidoides,and
C. zehntneri, as well as important activity against P. falciparum in
vitro for its active components. In the present study, preliminary
in vivo acute toxicity and in vitro cytotoxicity assays on the EOs
enabled a more thorough analysis of the toxic effects, suggesting
no or low acute toxicity.
Acknowledgements
!
We thank Dr. L.H. Carvalho from René Rachou/FIOCRUZ Research
Center for the donation of Plasmodium berghei NK 65 strains. We
are deeply indebted to Eduardo Mattoso of Kaapi (Campinas, São
Paulo State, Brazil) for the generous donation of pure compounds
used in this study. This study was part of a Masterʼs thesis for M. L.
Mota (a student of the Post-graduate Program Biology Science –
PPgCB) in the Federal University of Rio Grande do Norte, Brazil.
This study was supported by a grant (561559/2008–2) from the
CNPq. The authors also thank CAPES for fellowships for M. L. Mota.
Conflict of Interest
!
The authors have no conflicts of interest concerning the work re-
ported in this paper.
Affiliations
1Laboratório de Biologia da Malária e Toxoplasmose, Departamento de
Microbiologia e Parasitologia, Universidade Federal do Rio Grande do Norte,
Natal, Brazil
2Laboratório de Pesquisa de Produtos Naturais, Universidade Regional do
Cariri, Crato, Brazil
3Laboratório de Biopolímeros, Universidade Federal do Rio Grande do Norte,
Natal, Brazil
4Pós-graduação em Biotecnologia, Universidade Federal do Amazonas,
Manaus, Brazil
5Laboratório de Princípios Ativos da Amazônia, Instituto Nacional de Pesquisas
da Amazônia, Manaus, Brazil
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Magaly LM et al. In Vitro and …Planta Med
Original Papers
This is a copy of the authorʼs personal reprint
This is a copy of the authorʼs personal reprint
