Abstract Thymus species essential oils are widely used in aromatherapy to treat several ailments. However, there is no report on their safety. In this study, we propose to investigate the acute toxicity of T. leptobotrys and T. broussonetii essential oils. These two species were selected on the basis of their frequency of medicinal use and commercial importance. Chemical analysis of these two species essential oil revealed that thymol, borneol, carvacrol and p-cymene were the main chemical constituents in T. broussonetii, whereas the essential oil of T. leptobotrys contains carvacrol (98 %) as the major component. In the acute toxicity assay, the animals showed no stereotypical symptoms associated with toxicity such as convulsion, ataxy, diarrhoea or increased diuresis. The calculated median lethal dose (LD 50 ) was estimated at 4.47 g/kg for T. broussonetii and 2.66 g/kg for T. leptobotrys

A Benharref

A Bensalah

A Chait

A Collado

A Zyad

H Ait Mouse

J Sanz

K Elhabazi

Mellal Maroc

R Aboufatima

CiteSeerX

Moroccan Journal of Biology 12-2012/N 8-9 
Acute toxicity of essential oils of two Moroccan endemic species: 
Thymus broussonetii and Thymus leptobotrys 
 
 
K. Elhabazi1, R. Aboufatima1, A. Bensalah1, A. Collado3, J. Sanz3, A. Zyad4, H. Ait 
Mouse4, A. Benharref 2, A. Chait1* 
 
1Laboratoire de Pharmacologie, Neurobiologie et Comportement. Equipe de Pharmacologie et 
Comportement, Faculté des Sciences Semlalia. Université Cadi Ayyad, Marrakech Maroc. 
2Laboratoire de Chimie des Substance Naturelles. Faculté des Sciences Semlalia, Université Cadi Ayyad, 
Marrakech Maroc. 
3Instituto de Ciencias Agrarias-CCMA, CSIC, c/Serrano 115-dpdo., 28006, Madrid, España. 
4Laboratoire de Génie Biologique, Faculté des Sciences et Techniques; Université Moulay Sliman, Béni 
Mellal Maroc. 
*Corresponding autor E-mail: chait@ucam.ac.ma 
 
 
Abstract 
Thymus species essential oils are widely used in aromatherapy to treat several ailments. 
However, there is no report on their safety. In this study, we propose to investigate the 
acute toxicity of T. leptobotrys and T. broussonetii essential oils. These two species 
were selected on the basis of their frequency of medicinal use and commercial 
importance. Chemical analysis of these two species essential oil revealed that thymol, 
borneol, carvacrol and p-cymene were the main chemical constituents in T. 
broussonetii, whereas the essential oil of T. leptobotrys contains carvacrol (98 %) as the 
major component. In the acute toxicity assay, the animals showed no stereotypical 
symptoms associated with toxicity such as convulsion, ataxy, diarrhoea or increased 
diuresis. The calculated median lethal dose (LD50) was estimated at 4.47 g/kg for T. 
broussonetii and 2.66 g/kg for T. leptobotrys. 
 
Key words: T. broussonetii, T. leptobotrys, Essential oil, Chemical analysis, Acute toxicity, LD50. 
 
 
Introduction 
Thymus species, the 
Mediterranean aromatic plants, are 
widely known and used in several 
countries as spice and food preservative 
as well as remedy for many health 
disorders. T. broussonetii and T. 
leptobotrys are two endemic species in 
Morocco. They are the most popular 
herbs used in folk medicine. 
Commonly, these plants were used as 
powder, decoction or infusion to treat 
digestive disorders, fever, cough, cold 
and dolorous processes (Bellakhdar, 
1997). 
Previous pharmacological studies 
have demonstrated analgesic (Elhabazi 
et al., 2006a), anti-inflammatory 
(Ismaili et al., 2002), anxiolytic and 
immunomodulators (Elhabazi et al., 
2006b) activities of the non volatiles 
extracts of T. broussonetii. While, it 
was shown that the essential oil of this 
species possesses antimicrobial activity 
due to the two phenolic compounds 
thymol and carvacrol (Lattaoui et al., 
1993). Whereas, there is no 
pharmacological and phytochemical 
investigations in the literature on T. 
leptobotrys extracts except our previous 
work demonstrating the analgesic effect 
of the non-volatiles extracts 
(unpublished data). 
K. Elhabazi et al. / Moroccan J. Biol. 8-9 (2012): 29-33 30
In this study, we have studied the 
acute toxicity and phytochemical profile 
of the essential oils extracted from two 
endemic Moroccan thyme species: T. 
leptobotrys and T. broussonetii. The 
essential oil of these two species are 
commercialised and used as medicine 
for several ailments and widely used in 
aromatherapy, but there is no 
investigation in the literature 
concerning their safety and acute 
toxicity. Such study is essential to 
prevent any overdose of this oil which 
may cause a serious health problem in 
humans. 
 
 
Materials and methods 
 
Animals 
Swiss mice (25-35) g were used in 
these experiments. Animals were 
housed in standard cages with food and 
water ad libitum on 12-h light/12-h dark 
cycle. The air temperature was 
maintained at 22±2 ◦C.  
 
Plant material and preparation of 
essential oil 
The plants were collected in 
March 2003, in two regions in Morocco 
as followed: T. broussonetii: Essaouira, 
T. leptobotrys: Tiznite. Taxonomic 
identification of the plants was 
confirmed by Pr. Ouyahya Aicha from 
the Scientific Institute (Rabat). The 
plants specimens with their localities 
and other required fields records were 
recorded at the herbarium of the 
Scientific Institute, Mohamed V 
University, Rabat, Morocco. The 
essential oil was extracted by 
hydrodistillation using a Clevenger 
apparatus. 
 
GC-MS analysis 
The constituents of the essential 
oils were identified by gas 
chromatography/mass spectrometry 
(GC-MS) using an Agilent 6890 with 
5973A detector. We used a 
methylpolisiloxane capillary column 
(25 m x 0.2 mm x 0.33 um), injecting 1 
µl of a 1:100 oil solution in the split 
mode. The temperature program used 
was 70 to 250ºC, at 5º/ min.  
 
Toxicity assay 
Mice were randomly assigned to 
several groups with 10 animals in each 
group. The essential oil of each species 
was administered orally to the animals 
in increasing dosages 1, 2, 3, 4 and 5 
g/kg body weight in a single dose. The 
control group was given an equal 
volume of water. The animals were 
observed for 7 days; the number of dead 
mice in each group was recorded daily 
to calculate the effective lethal dose 
(LD50). The mice were also observed 
for other signs commonly associated 
with toxicity such as behavioural 
changes (excitation, twitches), motor 
coordination, piloerection, convulsion 
and diarrhoea. An evolution of body 
weight was also determined for groups 
receiving the dose 1 g/kg. 
 
Data analysis 
Data were analysed using SPSS 
program. The percentage of mortalities 
was converted to probits. The probit 
analysis was used to determine LD50 
and LD90 (with their confidence limits) 
using the Leitchfield and Wilcoxon 
method (Leitchfield and Wilcoxon, 
1949). 
 
 
Results 
 
Essential oils constituents 
Table 1 presents the qualitative 
and quantitative results obtained from 
the GC-MS analysis of volatiles in T. 
broussonetii and T. leptobotrys. 
Relative concentrations were calculated 
directly from the total ion current trace, 
K. Elhabazi et al. / Moroccan J. Biol. 8-9 (2012): 29-33 31
and expressed as % of total volatile 
content. 13 compounds which represent 
a total of 88.4 % were identified in T. 
broussonetii, thymol (36.7 %) and 
borneol (21.9 %) being the two major 
constituents. Other important 
components are carvacrol (9.0 %) and 
p-cymene (7.56 %). In T. leptobotrys 
essential oil, carvacrol is the most 
abundant constituent and represents a 
96.8 %. This oil also contains p-cymene 
(2.6 %) and α pinene (0.6 %). Other 
components listed in Table 1 such as 
thymol and borneol are present in low 
concentrations. 
 
Table 1. Constituents of T. broussonetii and T. 
leptobotrys essential oil with their relative 
proportion in the pure oil 
 
Constituents % of total volatile content 
 T. broussonetii 
T. 
leptobotrys 
Aromadendrene 
Borneol 
Camphene 
Carvacrol 
Caryophylene 
p-Cymene 
Spathulenol 
Thymol methylether 
Limonene 
Myrcene 
α-Pinene 
ß-Pinene 
Thymol 
α-Terpinene 
δ-Terpinene 
α-Terpineol 
1.2 
21.9 
2.8 
9.0 
- 
7.6 
1.9 
- 
0.7 
1.2 
- 
0.7 
36.7 
0.7 
2.8 
1.2 
- 
traces 
- 
96.8 
traces 
2.6 
- 
traces 
- 
- 
0.6 
- 
traces 
- 
traces 
traces 
 
Acute toxicity of T. broussonetii 
At 1 g/kg, the treated mice were 
immobilised approximately for 1 hour. 
After that, they find their normal 
activity. For the dose 2 mg/kg, there are 
cases of death whose the number 
depends on the time. Mostly, the death 
occurs at 2 h after treatment. Whereas, 
there is also cases of death occurring at 
2 days after treatment. For the lethal 
doses, the animals remained 
immobilised until death. Nevertheless, 
the animals didn’t show any additional 
clinical symptoms of toxicity such as 
convulsion, diarrhoea or motor 
incoordination. On the other hand, the 
survivors did not feed and therefore lost 
body weight (Table 2).  After three or 
four days, the survivors recuperated the 
movement and recovered their normal 
state. The LD50 and LD90 were 
estimated at 4.47 (3.6-6.72) g/kg and 
7.31 (5.64-13.54) g/kg. 
 
Acute toxicity of T. leptobotrys 
At 800 mg/kg, death cases were 
recorded. Just after treatment, the mice 
were immobilised and some signs of 
toxicity occurred such as acceleration of 
respiratory changes and piloirection. 
For all cases, the mice were 
immobilised until death. Additionally, 
the survivors also lost body weight 
(Table 2). The normal activity of the 
survivors was recuperated after four 
days. The LD50 and LD90 calculated 
were estimated at 2.66 (1.98-3.77) g/kg 
and 5.32 (4.09-8.66) g/kg. 
 
 
Discussion 
Examination of the two essential 
oils analysis make possible to highlight 
wide significant differences in the 
chemical composition between the two 
species. Carvacrol varies from 96.76 % 
in T. leptobotrys to 8.96 % in T. 
broussonetii. The two major 
components presents in T. broussonetii 
(thymol: 36.72 % and borneol: 21.9 %) 
are at very low concentration in T. 
leptobotrys. These results are in 
accordance with previous work which 
has found an extraordinary diversity in 
the composition of essential oil of 
Moroccan thyme species (Richard et al., 
1985). This variability could be 
explained by the fact that essential oil 
composition depends not only on the 
genetic factors but also on the biotopes 
K. Elhabazi et al. / Moroccan J. Biol. 8-9 (2012): 29-33 32
Table 2. Body weight evolution. 
 
 Body weight (g) 
j0 j1 j2 j3 j4 j5 j6
Lot 1 
(control) 26±0.6 26.07±0.6 25.8±0.3 26.9±0.1 27.12±0.5 28.2±0.4 28.8±0.2 
Lot 2 
(T. broussonetii) 24.8±0.4 24.92±0.7 23.61±0.5* 24.38±0.5 24.89±0.6 25.15±0.3 25.82±0.7 
Lot 3 
(T. leptobotrys) 26.02±0.3 26.85±0.5 24.64±0.1* 25.16±0.4 26.86±0.9 27.08±0.9 27.8±1.4 
 
(climatic conditions and soil 
composition). Besides, Thymus sp. are 
known to present chemical varieties 
(chemotypes) which differ markedly in 
their chemical composition (Thompson 
et al., 1998; Ložienė et al., 2002). On 
the other hand, our results showed that 
T. leptobotrys is more toxic than T. 
broussonetii. Knowing that T. 
leptobotrys essential oil contains 
predominantly carvacrol (96.8 %), we 
can suppose that this component is 
mainly responsible for this toxic effect. 
Whereas, T. broussonetii essential oil 
contains predominantly thymol (36.7 
%) and borneol (21.9 %). It’s known 
that carvacrol is more toxic which 
explains the high toxicity of T. 
leptobotrys essential oil. Additionally, 
our results showed that after treatment 
with the essential oil of both species, 
the mice remain immobilised during 
some time. This finding indicates that at 
high doses, the essential oil of the two 
thyme species exerted in animals a 
sedative effect. In a similar study, 
Ismaili et al., (2002) have already 
showed that the administration of T. 
broussonetii total extract exerted a 
sedative action. Furthermore, it was 
shown that thymol modulates in a 
positive way the gabaergic receptors 
(Garcia et al., 2006; Priestly et al., 
2003). So, we can suppose that thymol 
could be responsible for the sedative 
effect observed after administration of 
thyme extracts at high doses. 
In conclusion, this study showed 
that T. broussonetii essential oil 
contains 22 compounds. Monoterpenes 
are the most common. The main 
constituents are thymol,  borneol, 
carvacrol and p-cymene. In T. 
leptobotrys essential oil, carvacrol is the 
most abundant compound. The acute 
toxicity assay revealed that T. 
leptobotrys is the most toxic possessing 
the low median lethal dose. 
Additionally, our finding showed that 
high doses of the two species essential 
oil produced a sedative effect and a loss 
in average body weight. 
 
Acknowledgements 
We gratefully acknowledge Pr. 
Ouyahya Aicha for the botanical 
identification of the two thyme species. 
 
 
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Acute toxicity of essential oils of two Moroccan endemic species: Thymus broussonetii and Thymus leptobotrys

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Acute toxicity of essential oils of two Moroccan endemic species: Thymus broussonetii and Thymus leptobotrys

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