Signature: Med Sci Monit, 2003; 9(9): BR331-335 WWW.MEDSCI MONIT.

COM
PMID: 12960922 Basic Research BR

Received: 2003.07.16
Accepted: 2003.07.30 Antioxidant, antidiabetic, antihyperlipidemic,
Published: 2003.09.08
reproduction stimulatory properties and safety
of essential oil of Satureja Khuzestanica in rat in vivo:
a toxicopharmacological study
Authors Contribution: Mohammad Abdollahi acdefg, Alinazar Salehnia ag, Seyed Hamid Reza Mortazavi b,
A Study Design
B Data Collection
Mohsen Ebrahimi b, Ahmad Shafiee b, Fatemeh Fouladian b,
C Statistical Analysis Ketayoun Keshavarz b, Saheleh Sorouri b, Reza Khorasani b, Alireza Kazemi b
D Data Interpretation
E Manuscript Preparation Department of Toxicology & Pharmacology, Pharmaceutical Sciences Research Center and Faculty of Pharmacy,
F Literature Search Tehran University of Medical Sciences, Tehran, Iran
G Funds Collection
Source of support: This study was supported by the Khorraman Herbal Company under the supervision of the
Tehran University of Medical Sciences Research Council.

Summary
Background: Satureja Khuzestanica is an endemic plant of Iran that is widely distributed in the southern part
of the country. It is famous for its medical uses as an analgesic and antiseptic in folk medicine.
The present study was designed to explore the toxicological and pharmacological effects of
essential oil of Satureja Khuzestanica (SKEO) in vivo.
Material/Methods: The intraperitoneal LD50 of SKEO was determined. Teratogenicity was determined by admin-
istration of SKEO at doses of 500, 1000 and 1500 ppm to pregnant rats during days 0 to 15 of
gestation. FRAP and TBARS assays were used to determine total antioxidant power and lipid
peroxidation respectively. Diabetes and hyperlipidemia were induced by administration of
streptozocin and lipid regimen in rats. SKEO (1000 ppm) was administered in drinking water
for 10 days.
Results: SKEO is not lethal up to a dose of 2 gkg1 in rats. In the teratogenicity test, dams of the treated
group were active and did not show any signs of toxicity. A significant increase in the number of
implantation and live fetuses were observed with SKEO (500 and 1000 ppm) in comparison to
the control group. SKEO treatment decreased the normal blood lipid peroxidation level and
increased total antioxidant power. Significant decreases in fasting blood glucose and triglyceride
levels were observed with SKEO in diabetic and antihyperlipidemic rats respectively.
Conclusions: This preliminary study indicates the safety and interesting stimulatory effect of SKEO on
reproduction. The antioxidant properties of SKEO may explain its antidiabetic and trigly-
ceride-lowering effects.

key words: Satureja Khuzestanica essential oil lipid peroxidation antioxidant power diabetes hyper-
lipidemia reproduction teratogenicity

Full-text PDF: http://www.MedSciMonit.com/pub/vol_9/no_9/3991.pdf

Word count: 1745
Tables: 2
Figures:
References: 32
Authors address: Prof. Mohammad Abdollahi, Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran University
of Medical Sciences, Tehran 14155-6451, Iran, email: mohammad.abdollahi@utoronto.ca

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Basic Research
BACKGROUND
Satureja Khuzestanica is an endemic plant of Iran that is
widely distributed in the southern part of the country.
It is famous for its therapeutic value as an analgesic and
antiseptic in folk medicine [1]. The genus Satureja
belongs to the family Lamiaceae, subfamily Nepetoideae
and the tribe Mentheae. One of the specific identifying
characteristics of the subfamily Nepetoideae is that its rep-
resentatives contain more than 0.5% essential oil [2]. It
has been reported that there are marked differences in
the composition of this essential oil between and within
the subspecies of Satureja [3].
In recent years, antiviral [4], antinociceptive and antiin-
flammatory [5], antibacterial and antifungal [68], anti-
spasmodic and antidiarrhetic [9], and vasodilatory [10]
effects have been reported for different species of
Satureja growing in different parts of the world, but no
study has yet been undertaken on Satureja Khuzestanica.
In studies of the constituents of different species of
Satureja growing in various parts of the Middle East,
carvacrol and flavonoids have been reported as the
main components [6,9,11,12]. Both carvacrol and
flavonoids have been found to have antioxidant proper-
ties [1316]. Lipid peroxidation and consequent oxida-
tive stress are increased in diabetes and hyperlipidemia,
and have been proposed to play roles in the pathogene-
sis of these diseases [1720]. Therefore, we were inter-
ested in investigating the antidiabetic, antihyperlipi-
demic and anti-oxidative stress effects of the essential oil
of Satureja Khuzestanica (SKEO) on rat blood in vivo.
Primary acute toxicity and teratogenicity tests were also
performed to confirm its safety.
MATERIAL AND METHODS
Hydrogen peroxide, triton X-100, 2,4,6-tripyridyl-s-tri-
azine (TPTZ), 2-thiobarbituric acid (TBA), 1,1,3,3-
tetraethoxypropan, trichloroacetic acid (TCA), potassi-
um monohydrogen phosphate, potassium dihydrogen
phosphate, ethylenediaminetetra acetic acid as disodium
salt (Na2EDTA), streptozotocin from the Merck
Chemical Company (Germany) were used in this study.
Diagnostic kits for the measurement of blood glucose,
cholesterol, and triglycerides were purchased from
Zistshimi Diagnostic Kits Production (Tehran). Animals
were provided from Institute de Pasteur of Tehran.
The aerial parts of the Satureja Khuzestanica plant were
collected during the flowering stage in June 2000 from
Khoramabad in the Lorestan province of Iran. The
plant was identified by the Department of Botany of the
Research Institute of Forests and Rangelands (TARI) in
Tehran. A voucher specimen (No. 58416) has been
deposited at the TARI Herbarium. The plant was culti-
vated in Khoramabad and the aerial parts of the plant
were collected during the flowering stage.
The aerial parts were air dried at ambient temperature in
the shade and hydrodistilled using a Clevenger type appa-
ratus for 5 h, giving yellow oil in a 0.9% yield. The oils we-
re dried over anhydrous sodium sulfate and stored at 4C.
BR332
Med Sci Monit, 2003; 9(9): BR331-335
In order to determine the acute toxicity (LD50) of
SKEO, doses of 10, 100, 1000 and 2000 mgkg1 were
injected intraperitoneally (ip) into rats. The animals
were observed for 48 h, and any mortality was recorded
at the end of this period [21].
Healthy three-month-old female white Sprague-Dawley
rats weighing 180200 g were mated with healthy
mature males. Day zero of pregnancy was confirmed by
the presence of sperm in their vaginas the next morn-
ing. Pregnant females were randomly divided into four
groups of 6 each. They were provided with feed and
water ad libitum. Females in groups 13 received drink-
ing water containing 100, 500, and 1000 ppm of SKEO
as low, medium and high doses respectively from the
0 to 15th day of gestation. Females in group 4 were sup-
plemented with tap water only. All pregnant animals
were assessed daily for any sign of toxicity, while their
body weights were recorded every other day.
One day before delivery date, the dams were anes-
thetized and sacrificed. The ovaries from each rat were
examined and the number of corporea lutea was record-
ed. The total number of uterine implantations, pre- and
post-implantation losses, resorbed, dead, and live fetuses
per litter were recorded. Dead and live fetuses were
weighed and examined for external gross abnormalities.
One third of both dead and live fetuses were fixed in
Bouins solution for visceral examination. The others
were fixed in ethanol, eviscerated and stained by alizarin
red stain for skeletal examination [21].
In the in vivo study protocol, Male Sprague-Dawley rats
weighing 180200 g were randomly distributed into
control and treated groups with 6 animals in each
group. The animals in the treated group received
SKEO (1000 ppm) dissolved in drinking water. The
control animals received only tap water. All treatments
were continued for 10 days, after which blood samples
were collected following an overnight fast.
Plasma lipid peroxidation was determined by measure-
ment of malonedialdehyde (MDA) with the thiobarbituric
acid (TBA) test. The absorption of the TBA-MDA com-
plex was determined spectrophotometrically at 532 nm.
The calibration curve was provided by using 1,1,3,3-
tetraethoxypropan as standard [22]. The antioxidant
capacity of plasma was determined by measuring its abili-
ty to reduce Fe3+ to Fe2+. The complex between Fe2+ and
TPTZ gives a blue color with absorbency at 593 nm [23].
In order to measure the antihyperlipidemic effect, male
Sprague-Dawley rats weighing 180200 g were random-
ly distributed into control and treated groups with 6
animals in each group. The treated animals received
SKEO (1000 ppm) dissolved in drinking water, while
the control animals received only tap water. For induc-
tion of hyperlipidemia, both treated and control animals
were placed on a special high fat diet until the end of
the experiment [24]. All treatments were continued for
10 days, after which blood samples were collected fol-
lowing an overnight fast. Serum samples were analyzed
for cholesterol and triglyceride using commercial diag-
Med Sci Monit, 2003; 9(9): BR331-335 Abdollahi M et al Toxicopharmacological effects of Satureja Khuzestanica

Table 1. Embryofetal abnormalities in pregnant rats receiving essential oil of Satureja Khuzestanica (SKEO). BR
SKEO SKEO SKEO
Control 100 ppm 500 ppm 1000 ppm
Gestation weight gain/mother (g) 241.6 24.871.82 32.122.4 33.963.54
No. of corpora lutea/litter 4 4 7* 7*
No. of pre-implantation loss/litter 0 0 0 0
No. of post-implantation loss/litter 0 0 0 0
No. of resorbed fetus/litter 0 0 0 0
No. of dead fetus/litter 0 0 0 0
No. of live fetus/litter 4 4 7* 7*
Fetal weight (g) 4.120.21 3.980.12 4.260.36 3.960.2
No. of visceral anomalies 0 0 0 0
No. of skeletal anomalies 0 0 0 0
Pregnant females in groups 13 received drinking water contained 100, 500 and 1000 ppm respectively of SKEO from the 0 to the 15th day of gestation. Females in the
control group (4) were supplemented with tap water only. One day before delivery date, the dams were anesthetized and sacrificed. The ovaries from each rat were exam-
ined for teratogenicity parameters. One third of both dead and live fetuses were fixed in Bouins solution for visceral examination. The others were fixed in ethanol, eviscer-
ated and stained by alizarin red stain for skeletal examination

Table 2. Effects of essential oil of Satureja Khuzestanica on plasma cant. Fischers exact test was used to analyze non-para-
lipid peroxidation, antioxidant power, glucose, cholesterol metric teratogenicity data.
and triglyceride levels.
RESULTS
Treated Control
The acute toxicity test (LD50) demonstrated that SKEO
Lipid peroxidation (nmol/ml) 1.660.25* 2.10.33 is not lethal up to a dose of 2 gkg1 after ip injection
Antioxidant power (mol/ml) 98.254.81* 322.86 into rats, and is nontoxic.
Glucose (mg/dl) 25818.1* 32521.5
Total cholesterol (mg/dl) 1469.6 15614.1 The teratogenicity test results indicated that the dams in
Triglyceride (mg/dl) 16614.9* 20117.6 the treated groups were active and did not show any
Diabetes was induced in both groups of rats by a single ip injection of 75 mg/kg signs of toxicity or any notable changes in their behav-
of streptozotocin. Hyperlipidemia was induced in both groups of rats using a ior. No significant differences in food intake and water
special high fat diet. Lipid peroxidation and antioxidant power were measured
consumption were recorded among the treated and
in non-diabetic normolipidemic rats. Data are meanSE of 6 animals in each
control groups during gestation. As shown in Table 1,
group;
the groups treated with medium and high doses of
* means that the difference between treated and control values is significant at
SKEO (500 and 1000 ppm) showed a significant
P<0.01
increase in the number of corpora lutea and live fetuses
per litter in comparison to the control group.

nostic kits (Zistshimi Kit Production Co. Tehran) by
spectrophotometer.
For purposes of assessing the antidiabetic effect, male
Sprague-Dawley rats weighing 180200 g were random-
ly distributed into control and treated groups, with 6
animals in each group. Diabetes was induced in both
groups by a single ip injection of 75 mg/kg of streptozo-
tocin [25] freshly dissolved in saline. The treated ani-
mals received SKEO (1000 ppm) dissolved in drinking
water for ten days, starting the day after streptozotocin
injection. The control animals received only tap water.
Blood sugar was measured in blood samples obtained
from the tail of the rats using a diagnostic kit. The rats
average blood glucose was 7080 mg/dl prior to the
experiment and increased to 300350 mg/dl. Water
intake and urination were also drastically increased.
Statistical analysis
Values are reported as meanSE. Statistical significance
between groups was computed using the Student t-test.
P values greater than 0.05 were considered insignifi-
Blood lipid peroxidation and antioxidant power were
also affected by SKEO treatment. SKEO treatment
(1000 ppm) decreased the normal lipid peroxidation
level and increased antioxidant capacity significantly
(1.660.25 vs 2.10.33 and 98.25 vs 322.86 respec-
tively). Significant decreases in blood glucose (25818.1
vs 32521.5) and triglycerides (16614.9 vs 20117.6)
levels were also observed. Blood total cholesterol levels
were not affected by SKEO treatment (1000 ppm).
DISCUSSION
Taken together, the results presented here point to the
antioxidant, antidiabetic and triglyceride-lowering
effects of SKEO and the safety of its use during gesta-
tion in rats. Reactive radicals such as superoxide anions,
hydroxyl and hydroperoxyl can extract atoms of hydro-
gen from the polyunsaturated fatty acid side-chain in
biological membranes and produce lipid peroxidative
damage. Mammalian cells possess nonenzymatic and
enzymatic antioxidant defenses to cope with deleterious
free radicals. The nonenzymatic defenses include vita-
min E, beta-carotene and vitamin C, while the enzymes

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Basic Research
involved include superoxide dismutase, catalase, and
glutathione peroxidase. Increased free radical forma-
tion can cause oxidative stress and resulting cell damage
if antioxidant defenses are overwhelmed [26]. Oxidative
stress plays a role in the pathogenesis and complications
of many chronic diseases, including diabetes and hyper-
lipidemia [1720]. The data in Table 2 show that SKEO
increased the total antioxidant power of blood and
decreased the production of thiobarbituric reactive sub-
stances (TBARS). Supporting this property of SKEO,
there is evidence that Satureja hortensis from Greece con-
tains phenolic antioxidant compounds with significant
antioxidant capacities [12]. Previous studies on other
species of Satureja have indicated carvacrol and flavo-
noids as the main components of Satureja essential oil
[6,9,11,12]. Flavonoids are a class of secondary plant
phenolics with significant antioxidant and chelating
properties. Their cardioprotective effects stem from the
ability to inhibit lipid peroxidation, chelate redox-active
metals, and attenuate other processes involving reactive
oxygen species. The propensity of a flavonoid to inhibit
free-radical-mediated events is governed by its chemical
structure [16]. In addition, carvacrol has been found to
have significant antioxidant properties [1315]. Thus
the effects of SKEO in lowering normal lipid peroxida-
tion and increasing the total antioxidant power of blood
(Table 2) suggest the presence of these antioxidants in
SKEO. This ability of SKEO also explains its actions in
the reduction of blood glucose and TG levels. A number
of studies have suggested that enhanced oxidation is the
underlying abnormality responsible for some of the
complications of diabetes, a chronic disease associated
with serious complications.
The results of the present study also indicate that SKEO
can safely be used during gestation in rats. One of the
interesting findings in this study was the stimulation of
reproduction, as indicated by the increased number of
live fetuses per litter in dams treated with SKEO (Table
1). This effect of SKEO may also be traced back to its
antioxidant properties. Oxidative stress has been found
to decrease normal reproduction in both males [27] and
females [28,29], which is counteracted by the adminis-
tration of antioxidants.
CONCLUSIONS
Our recent studies have indicated that most of the
herbal products that have been used in Iranian folk
medicine exhibit significant pharmacological properties,
thus the importance of this kind of study cannot be
ignored [3032]. This preliminary study demonstrates
beneficial properties of SKEO that could be of value in
the treatment of many diseases in which oxidative stress
plays a role in pathogenesis, including diabetes and
hyperlipidemia. However, further studies are needed to
determine the possible mechanisms of action, establish
complete safety profiles, and evaluate the potential
value of SKEO for the management of diabetes and
hyperlipidemia in the clinic. The stimulatory effect of
SKEO on female rats reproduction seems the most
interesting result of this study, and its mechanism
remains to be elucidated by further studies.
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Med Sci Monit, 2003; 9(9): BR331-335
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