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*Corresponding author e-mail: elsaad22@science.bsu.edu.eg & elsaad1@yahoo.com, (Abdelaziz SA Abuelsaad);
Received date 28 March 2024; revised date 09 May 2024; accepted date 15 May 2024
DOI: 10.21608/EJCHEM.2024.280108.9524
©2024 National Information and Documentation Center (NIDOC)
Egypt. J. Chem. Vol. 67, No. 8 pp. 665 - 687 (2024)
Extraction and characterization of essential oil of Lavandula dentata L.: Evaluation of its
cytotoxicity and anticonvulsant activities on an epileptic model
Aziza Antar1; Eman S. Abdel-Rehiem2, Areej A. Al-Khalaf3; Abdelaziz S. A. Abuelsaad4*; Gaber M.G.
Shehab5; Ayman M. Abdel-Aziz6 and Mohamed Abdel-Gabbar1
1Biochemistry Department, Faculty of Science, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt.
2Molecular Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef 62521,
Egypt.
3Plant Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428,
Riyadh 11671, Saudi Arabia
4Immunology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt.
5Department of Biochemistry, College of Medicine, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.
6Zoology Department, Faculty of Science, Fayoum University 63514, Fayoum, Egypt.
Abstract
The Labiatae (Lamiaceae, Mentaceae) plant family, includes many shrubs and herbs as well as trees and vines, is
economically significant. In the present study, aerial portions of Lavandula dentata L. (1753) were collected throughout the
vegetative phase and stored in the herbarium for identification, authentication, and description. The analysis was carried out
utilizing gas chromatography-mass spectrometry. The quantitative data were evaluated using peak area normalization without
correction factors. Moreover, in vitro, cytotoxicity assay was examined, and anticonvulsant activity was tested in vivo using
an epileptic rat model. The sulforhodamine B cell cytotoxicity assay was performed in vitro to assess cellular viability or
cytotoxicity produced by the ethanolic extract and oil of L. dentata against HepG2 and Vero cells. In addition, seizure
intensity scores and behavioral assessments were evaluaed and revealed that both essential oil and L. dentata extract reduced
seizures, decreased distance and latency time in open field experiment, increased both grooming, rearing, and frequency of
ambulation. Thus, the ethanolic extract or essential oil of L. dentata influences various behavioral domains and reduces
depression-like symptoms in rats by inhibiting convulsion and seizure activity.
Keywords: Lavandula dentata L – GC-MC – Cytotoxicity- Epilepsy- Seizure and Behavioral
1. Introduction
Natural products are common and gaining
popularity, and the use of plant extract, essential
oil(EO), and fixed oil in industrial applications is
increasing. Phytochemicals have been a crucial
source of drugs since ancient times. Phytochemicals
play a significant role in developing new synthetic
and manufactured drugs in various medical fields.
Currently, natural products are the main
components of approximately 50% of useful drugs.
When compared to conventional medicine, these
medications typically exhibit greater medicinal
value and lower adverse effects. [1]. Flavonoids
may have a moderating function in treating
neurodegenerative illnesses, which can affect
cellular oxidative processes in the central nervous
system [2]. Correspondingly, Lamiaceae is
considered an attractive plant family with a lot of
shrubs and herbs, and seldom trees. The species of
this family enjoy vital economic and horticulture
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A. Antar et.al.
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importance [3]. Globally, from two-hundred forty
to three hundred genera 7200–7500 species make
up the mint family[4]. Due to their fragrant
properties and ease of cultivation, several
Lamiaceae members are farmed in the desert of
North Africa and the cooling region of Asia and
Europe[5, 6]. Thousands of floral species in this
family are commonly used in landscapes and
butterfly gardens [5]. Essential oil (EO) is released
by the glandular hairs on the aerial vegetative and
reproductive organs in most family members [7].
Lavandula is a genus native to the Mediterranean
region and can treat digestive disorders and kidney
diseases [8]. Members of the genus produce large
amounts of phytochemicals [9]. Lavandula dentata
is one of five lavender species that grow naturally
in Saudi Arabia; the species is thought to represent
the origin of the genus. The species are distributed
in the mountains of Abaha, Asir, and Al-Taif
regions as herbaceous wild plants.
Certainly, some antiepileptic drugs have been
recognized by Shannon and Love [10] as
potentially causing cognitive impairment in healthy
individuals, besides sharing in the cognitive
abnormalities seen in patients with epilepsy
disorder. Therefore, the current investigation’s aim
is the extraction, isolation, and description of L.
dentata L., and the evaluation of its cytotoxicity
and anticonvulsant activity.
2. Materials and Methods:
2.1. Lavandula dentata L. Taif, Saudi
Arabia:
2.1.1. Collection of the plant:
The stem and leaves of Lavandula dentata L
were obtained during the vegetative stage from Taif
region, Saudi Arabia, in May 2018. A voucher
specimen was deposited in the herbarium, where it
was identified, authenticated, and described with
the aid of Agricultural Research Centre, Dokki,
Cairo, Egypt; as follows: The general climate of
Taif region, Saudi Arabia is Upland region, which
distinguished by rainy weather. It is characterized
by a cooling temperature in winter (10-12°C) and
worm weather in summer (21-28°C). Above sea
level at 1,879 m Taif is located on the Hejaz
Mountains slopes, that considered a vital portion of
the Sarawat Mountains[11].
2.1.2. Morphological Identification &
Classification:
The systematic position of Lavandula dentata L.
(1753) in A. Engler system [12], whereas, the latter
is a phylogenetic system of plant classification,
adopted in the Egyptian Flora books, and major
herbaria in Egypt [13-15].
2.2. Extraction and characterization of
Lavandula dentata L:
About 250 gm of the whole dried plant
material was coarsely milled and extracted with
70% ethanol extraction bath at room temperature
till exhaustion (3 times). The pooled ethanol extract
were evaporated under vacuum at 45 0C till
evaporation of the organic solvent, part of the
remaining aqueous part was frozen, and dried to
produce 47g of greenish residue, and will be used
as an ethanolic extract treatment group.
2.2.1. Essential oil extraction using GC/MS
Assay:
Another 1 Kg of dried plant parts were ground
into tiny bits and hydrodistilated for three hours
using a Clevenger-style equipment in order to
isolate the essential oil. In a nutshell, the plant was
submerged in water and brought to a boil before the
essential oil and water vapor evaporated and were
eventually collected in a condenser. After being
separated, the distillate was dried on anhydrous
sodium sulfate. The extracted oil was kept cold
until gas chromatography/mass spectrometry
(GC/MS) analysis. The yield of essential oil was
analysed by GC-MS analysis using Shimadzu
GCMS-QP2010 (Tokyo, Japan) at Ain Shams
University, Faculty of Pharmacy in Cairo, Egypt,
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and record mass spectra. Briefly, the temperature
was preserved at 45°C for 2 minutes in the starting
column, then the temperature was raised to three
hundred °C for an average of 5°C per minute and
preserved for 5 minutes. An injector temperature
was 250°C whereas the helium flow reached 1.41
ml/min. For registering all mass spectra some
conditions must be performed such as 70 ev voltage
of ionization, 200°C’, and current emission filament
with 60 mA and ion source. Then the split mode of
injections was carried out on a dilated sample (1%
v/v; split ratio: 1:15). For identifying the chemical
composition of the component we compared their
retention indices concerning alkanes (C8–C20) and
mass spectra with authentic standards from the
NIST/EPA/MSDC Mass Spectral Database. The
base of the quantitative data without the correction
of the unused factors was peak area normalization
[16-18].
2.2.2. Estimation of Total Phenolic and
Flavonoids
Regarding the gallic acid standards for total
phenolic compounds [19]. Briefly, gallic acid is a
solution of methanol (1 mg/ml) was prepared. After
that, we prepared 7 serial dile Cons (1000, 800,
600, 400, 200, 100 & 50 µg/ml). On the other hand,
rutin standards for total flavonoids are performed
[20]. We prepared the sample in 3mg/ml of
methanol. The seven typical samples and six
duplicates of a specimen were piped in the grid's
holes for the estimate of total phenolics and gallic
acid standards. The measurements were taken at
630 nm. Furthermore, six replicates of the seven
typical samples and one specimen were piped in the
grid’s holes for the estimate of total flavonoids and
rutin, and the measurements were made at 420 nm.
Tests for cytotoxicity and cultivating cells (in
vitro):
Cell line for carcinoma of the liver (HepG2)
was provided by Nawah Scientific Inc. in
Mokatam, Cairo, Egypt. 10% inactivated fetal
bovine serum as well as 100 mg/mL streptomycin
was added by Dulbecco's Modified Eagle's Medium
(DMEM) and also supplemented 100 units/mL
penicillin for maintaining the cellular structure at
37 °C in a humidified 5% (v/v) CO2 surroundings.
Considerably, the viability of cells and toxicity of
cells caused by drugs can be investigated by the
Sulforhodamine B (SRB) cell cytotoxicity test. This
technique makes use of the bright-pink
aminoxanthene dye SRB, that responsible for
protein binding stoichiometrically in a slightly
acidic atmosphere and can be taken out in basic
surroundings. Therefore, the quantity of bound dye
can be utilized as a measurement for cell mass as a
standard and its value could be calculated at
wavelength 565 nm for measurement proliferation
of cells [21, 22]. Shortly, the cellular viability could
be determined by the SRB technique: 96-grid’s hole
was treated in complete habitat for all day with
aliquots of hundred microlitre cell suspension of
green monkey kidney (Vero, 5x103 cells) or
hepatocellular carcinoma (HepG2; 5x103 cells) cell
lines. Another addition of standard equal one-
hundred microlitre with serial dilutions of
Lavanduladentata L. ethanolic extract or its
essential oil (0.01, 0.1, 1.0, 10 &100 ug/ml) was
added to the cells for treatment. After the cells were
exposed to the extract for seventy-two hours, the
fixation occurred by the addition of one-hundred-
fifty microlitre of ten percent of the solution of
TCA to the cultural medium for 60 minutes at a
temperature of four °C. The next step is washing
with distilled water 5 times to remove the excess of
the TCA. By adding seventy microlitres of SRB
(0.4% w/v) the next step was incubation for 10
minutes at normal temperature. After three 1%
CH3COOH washes, the plates were dried for 12
hours. When we added one-handred-fifty microlitre
of TRIS (10mM), the SRB protein-bound dissolved
and also the registration of wavelength was at
540nm a BMG LABTECH®-FLUO star Omega
microplate reader (Ortenberg, Germany).
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2.3. Seizure and Behavioral assays (in Vivo):
2.3.1. Experimental animals:
Animals were purchased from National
Research Centre, Eldoki, Giza, Egypt a total of 40
mature male Dawley rats, weighing between 150 -
180 g, were acquired. They were kept in separate
metabolic cages with a 12-hour light/dark cycle in a
regulated environment (23± 1°C; humidity, 55±
5%). Dietary requirements were freely available.
The Beni-Suef University animal research ethics
committee authorized all methods, which were
carried out following the rules for the care and use
of laboratory animals. The committee's permission
number was BSU/FS/2021/021/138.
2.3.2. Chemical component :
The supplier of pilocarpine hydrochloride
(PILO) (99%) was Acros Organics in New Jersey,
USA. In addition, Nanjing Chemical Reagent Co.,
Ltd. (Nanjing, China) was the supplier of diazepam,
chloral hydrate, and methylscopolamine. Sanofi
Aventis CO., Depakine Chrono® 500mg.
2.3.3. Induction of Epilepsy (in vivo):
The method of Turskiet al. [23]& Abdel-
Reheimet al.[24]was used to experimentally induce
epilepsy. Before receiving an intraperitoneal dose
of pilocarpine hydrochloride (300mg/ Kg.B.wt), the
rats under investigation take a 30-minute of 1
mg/kg methylscopolamine to infused
intraperitoneally. The behavior of the animal was
then examined for signs of seizure activity using a
set of standards. These requirements for modeling
success include the following: indications of an
epileptic seizure (sluggishness, salivation, tremors,
convulsions, etc.). After receiving pilocarpine
hydrochloride, the experimental rats exhibited these
behaviors for 120 minutes, but rats in the control
group behaved as usual. The rats were diagnosed
with seizure episodes when they consistently
displayed seizure activity in a general way without
exhibiting the activity of control rats during each
phase. The rats exhibited seizures for a duration of
one hour, with attacks happening every two to five
minutes. Diazepam (4 mg/kg, i.p.) was injected
every 20 minutes to stop the activity of seizures at
the time of need. The identical approach was used
for the normal rats, with the exception that
pilocarpine was replaced with an injection of
phosphate-buffered saline (PBS, pH 7.4; 0.2
ml/rat), and diazepam was given one hour later.
2.3.4. Animal grouping:
In general, the current experiment included 40
mature male Dawley rats, weighing between 150 -
180 g. They were split up into the following five
groups (8 rats/group):
(1) Control group (C): received routine food,
unrestricted access to clean water, and oral
ingasterically intubed at periods equivalent to the
other groups (PBSS, pH 7.4; 0.2 ml/rat).
(2) The epileptic control group (EP): received an
i.p. injection of pilocarpine hydrochloride (300
mg/kg bw), then fed orally via gastric intubation
with PBSS (pH 7.4; 0.2 ml/rat) by intra-
abdominal injection at durations that were
consistent with those of the other groups.
(3) The Depakine®-treated epileptic group:
received an i.p. injection of pilocarpine
hydrochloride (300 mg/kg bw) as had been
mentioned. After that, rats were given 500 mg/kg
bw. of Depakine® dissolved in PBS (pH 7.4; 0.2
ml/rat) then fed orally via gastric intubation for
equal periods at the same time as other groups.
Depakine treatment was carried out two times a
week for successive four weeks.
(4) Pure essential oil of L. dentata-treated
epileptic group: received an i.p. injection of
pilocarpine hydrochloride (300 mg/kg bw), and it
was subsequently fed orally with 100 μg of L.
dentata oil per kg of body weight of rats, suspended
in PBSS (pH 7.4; 0.2 ml/rat). By oral ingasterically
intubation method for equal periods at the same
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time as other groups, oil administration was carried
out every day for successive four weeks.
(5) L. dentata ethanolic extract-treated epileptic
group: received an i.p. injection of pilocarpine
hydrochloride with a dose of 300 mg/kg, then fed
an oral gastric intubation dose containing 300 mg/
kg. bw. of dissolved L. dentata in PBSS (pH 7.4;
0.2 ml/rat). By oral ingasterically intubation method
for equal periods at the same time as other groups,
feeding was carried out every day for
successive four weeks.
2.3.5. Assessment of Seizure and Behavior
Alterations:
The scale modification of values registered for
the various stages [25] was used to evaluate the
seizure intensity value in the following phases:
phase(0):no response; phase(I): (twitching of the
eye, ear, and face); phase(II): axial convulsive
waves through the body); phase(III): myoclonic
and axial convulsive waves through the body;
phase(IV): generalized colic convulsions that turn
over into the side position); phase(V): generalized
convulsions with tonic extension episode and status
epileptic) and phase VI: mortality.
Every behavioral examination, such as the
forced swimming (FS), hot plate (HP), and open-
field test (OFT), was conducted at two different
times six hours after the lights were turned on or
off, or at 3:00 p.m. and 03:00 a.m., successively.
The animals were placed into the isolated chamber
at least thirty minutes prior to every examination to
conduct the behavioral experiments there. Rats that
displayed unexpected recurrent seizures (SRSs) one
hour prior to the commencement of the
examinations were not allowed to continue with the
experiment. Rats with disorders of OF, FS, and HP
were observed also their behavior was noted.
2.3.5.1. Open-field test (OFT):
The Open Face Test (OFT) is a behavioral
assessment that has been confirmed. It is an
effective way to evaluate exploration and motor
skills as well as anxiety levels in stressful or
frightening settings, but it is not a model of anxiety
disorders [26]. The gray polystyrene Plexiglas box
was the open-field apparatus. It was separated into
two zones: the inner square (center) and the outside
square (periphery), which each rat would discover
to be equal squares. For the rat, the central area was
unpleasant. An entry into the corresponding zone
was determined by the rat placing all four paws
there. The following were the standard measures
that were computed: The first four factors are
latency time, followed by grooming count, rearing
count, and ambulation frequency. After being put in
the middle of the box, each rat was given five
minutes to investigate. To remove unfavourable
smell we washed with 0.1% acetic acid solution
after each test.
2.3.5.2. Hot Plate (HP):
The hot plate test is an accepted model utilized
to assess a compound's antinociceptive (acute pain
model) performance concerning short thermally
sensory perception [27] .In a nutshell, the apparatus
was made up of an electrically warmed platform
and an exposed Plexiglas tube that served as the
animals' confinement. A temperature range of 56.0
± 0.1 °C was chosen. A stopwatch was used to time
how long it took for the rats to hop off, shake their
hind paws, or lick their front or rear paws after they
were placed on the warming surface. To ascertain
whether the medication had a therapeutic impact,
the time it took to respond to sucking was accepted
as a pain response time.
2.3.5.3. Forced Despair Swim (FDS) Test:
A standard forced despair swimming (FDS) test
was used to assess despair-like behavior [28, 29].
This test is useful for assessing depression-like
performance as well as for antidepressant drug
screening. Almost all antidepressants that are
currently on the market can be acutely administered
to reverse the increase in immobility time required
for this test. The test was conducted in an obvious,
transparent tube. It was filled with 24 °C tap water
until it was 30 cm from the bottom. Rat water was
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Egypt. J. Chem. 67, No. 8 (2024)
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substituted for rat water in the device. On the first
day, there were two swimming experiments, lasting
15 minutes each and 5 minutes on the second. The
rat was dried and heated for ten minutes using an
electric heater following each test. Two
experienced experimenters who were not informed
of the treatment conditions observed behavior on
the second day (test). Excessive immobility, in
which the rat only moves to maintain his snout
above water to avoid drowning, is a sign of a
depressive mood in animals. The rat was immobile
for a set amount of time when it either stayed still
or moved only as much as was required to maintain
its head above water, and was measured in terms of
seconds.
2.4. Statistical Analysis:
The method of Tukey-Kramer was utilized for
post-Hoc analysis of the data for comparison of
different sections with one another. There were
findings in the form of mean ± SD. The statistical
significance interval is defined for all the data in the
form of P < 0.05. We use (the SPSS) version 20
program to analyze all of the data (IBM Corp.,
2011).
3. Results and Discussion:
The plant material was collected, and its
systematic position as L. dentata L. was identified
using A. Engler system [12], for phylogenetic plant
classification. This system is adopted in Egyptian
flora books and major herbaria in Egypt [13-15]
and is as follows: Division: Angiospermae; Class:
Dictoyldoneae; Subclass: Sypetalae; Order:
Tubiflorae (Solanales); Suborder: Boraginineae;
Family: Labiatae (Lamiaceae, Mentaceae);
Subfamily: Lavanduloideae; Genus: Lavandula
and Species: Lavandula dentata L.
Gas chromatography–mass spectrometry data
from total EO components identified approximately
95.73% of the constituents (Table 1). The oil
contained various monoterpenes, including
oxygenated monoterpenes (81.94%), monoterpene
ketones (58.29%), monoterpene aldehydes (49.5%),
monoterpene esters (31.93%), monoterpene ethers
(7.85%), and monoterpene hydrocarbons (4.59%).
Other constituents included phenols (52.1%) and
diterpenes (2.9%). Aromatic hydrocarbons were
poorly represented (1.2%). Moreover, the unknown
content of L. dentata extract was 0.752 ± 0.049 at
an absorbance of 630 nm (Table 2). Substitution in
the linear regression equation suggested a phenolic
content of 261.04 ± 16.03 µg/ml. Therefore, the
total phenol (gallic acid equivalents) was 87.01±
5.3 mg/g extract (Table 4).
Estimation of total flavonoids (Table 4) showed
unknown content in L. dentata extract of 0.263 ±
0.019 at an absorbance of 420 nm. Total flavonoid
content by linear regression was 74.77 ± 6.35
µg/ml; therefore, average total flavonoid content
(rutin equivalents) was 24.92 ± 2.1 g. Per 100 g of
extract, the total polyphenolic content was
proportional to gram weight equivalents of gallic
acid (%). Constituent of phenol accounted for a
relative fifteen percent of L. dentata extract [8].
They accounted for the characteristics of the
biology of the extract of hydroalcoholic in
inflammation of the experimental rat [30].
Disparities could be caused by qualitative and
quantitative differences in polyphenol content.
EO of L. dentata contained many groups of
chemicals (Tables 1–2). Forty-eight components
were registered, which represented 96.15% of the
sample. Correspondingly, the profile of the floral
family, oxygenated monoterpenes (81.94%) were
the most prevalent, then came hydrocarbons
monoterpene (4.59%), sesquiterpenes (3.62%),
aromatic hydrocarbons (1.20%), monoterpene
ethers (1.05%), monoterpene esters (1.02%),
monoterpene ketones (0.88%), phenols (0.72%),
monoterpene aldehydes (0.71%), and diterpenes
(0.42%). The main compound was (1S-(4beta,
1Alpha & 2alpha)) 2-cyclohexane diol(46.09%), -1-
isopropenyl-4-methyl-1, followed by menthone
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Egypt. J. Chem. 67, No. 8 (2024)
671
(18.69%), fenchol (9.42%), borneol (3.35%), renal
(1.72%), cis-dihydro-occidental (1.49%), linalool
(1.4%), p-Cymene (1.04%), and trans-linalool
oxide (1.03%). Six compounds were identified each
accounting for 3.65%. The 33 remaining volatile
organic compounds accounted for 8.27% of the oil.
Previous reports stated the construction of the
Essential oil (EO) based on multiple causes like
geographical location, genetic variability, biotic and
abiotic stresses, plant age, phonological stage and
chemotype, time of harvest [31], and methods of
drying and extracting oil [32]; hence, there are
differences in constituent composition. Thus, the
constituent contents observed in the present study
differ from those described in some previous
reports mainly in constituents such as monoterpene
hydrocarbon content (4.59%), which was greater in
other studies: 17.89% in L. multifida from Tunisia
[33], 17.60% in aerial parts of L. dentata[34],
15.64% in L. dentata from Mexico [35], and
14.80% in L. dentata inflorescence [34].
In contrast, higher levels of monoterpene
hydrocarbons have been reported in other studies:
0.23% in L. dentata from Palestine[36], 0.25% in L.
dentata [33], and2.44% in L. stoechas[33].
Aromatic hydrocarbons in the present L. dentata oil
accounted for 1.20% (Taif, SA) of the total content,
which was less than 1.77% in L. multifida from
Tunisia [33] and more than 0.04% in L. dentata
[33], 0.06% of L. stoechas[33], and0.5% in the
aerial parts and inflorescence of L. dentata [34].
Table (1): Comparison between chemical compositions of the essential oil (%) of Lavandula spss.
Chemical
Compound/
Family
L. dentate
(Taif, SA)
Present
L.dentata(T
unisia) [33]
L.stoechas
(Tunisia)
[33]
L.multifida
(Tunisia)
[33]
L. dentata
(Palestine)
[36]
L. dentata
(Mexico)
[35]
L.dentata(in
florescence)
[34]
L. dentata
(aerial part)
[34]
Monterpene
Hydrocarbons
4.59
0.25
2.44
17.89
0.23
15.64
14.80
17.60
Aromatic
Hydrocarbons
1.20
0.04
0.06
1.77
0.00
0.00
0.50
0.50
Oxygenated
Monoterpene
81.94
81.94
81.94
81.94
81.94
81.94
81.94
81.94
Phenols
52.10
52.10
52.10
52.10
52.10
52.10
52.10
52.10
Monoterpene
Esters
31.93
31.93
31.93
31.93
31.93
31.93
31.93
31.93
Monoterpene
ketones
58.29
58.29
58.29
58.29
58.29
58.29
58.29
58.29
Monoterpene
Aldehydes
49.50
49.50
49.50
49.50
49.50
49.50
49.50
49.50
Monoterpene
ethers
7.85
7.85
7.85
7.85
7.85
7.85
7.85
7.85
Diterpenes
2.90
2.90
2.90
2.90
2.90
2.90
2.90
2.90
Total Identified
95.73
89.66
100.00
99.98
99.19
93.96
98.90
99.20
L. dentata in the present study showed the
highest content of oxygenated monoterpene
(81.94%), which was more than 58.29% and
52.10% in L.multifida and L. dentata from Tunisia,
respectively [33],49.50% in L. dentata [36],
31.93% in L. stoechas[33], 7.85% in L. dentata
from Mexico [35], and 3.40% and 2.90% in L.
dentata aerial parts and inflorescence, respectively
[34]. The present phenolic compounds accounted
for 0.72% of the total content, which was less than
1.33% and 0.82% for L. multifida and L. dentata
from Tunisia, respectively [33] and higher than
0.01% in L. dentata[36] and 0.03% in L.
stoechas[33]. L. dentata from Mexico [35] and L.
dentata inflorescence and aerial parts [34]
exhibited no phenolic compounds.
A. Antar et.al.
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Egypt. J. Chem. 67, No. 8 (2024)
672
Monoterpene esters in the present study of L.
dentata (Taif, SA) were poorly represented at
1.02%, which was less than 64.45% in L.
stoechas[33], 44.20% in L. dentata [36], and
28.83% and 10.56% in L. dentata and L. multifida
from Tunisia, respectively [33]. In contrast, L.
dentata from Mexico [35] and inflorescence and
aerial parts of L. dentata[34] contained no
monoterpene esters. Monoterpene ketones were
also poorly represented in L. dentata (Taif, SA) at
0.88%, which was more than L. stoechas (0.14%)
and L. multifida (0.60%) from Tunisia [33] but less
than 1.88% in L. dentata [35], 1.90% in L.
dentata[36], 2.34% in L. dentata [33], and 30.40%
and 30.8% in L. dentata aerial parts and
inflorescence, respectively [34].
Monoterpene aldehydes made up 0.71% of L.
dentata (Taif, SA) oil, a value greater than L.
stoechas and L. dentata from Tunisia (0.06% and
0.11%, respectively) [33]. Further, this value was
less than that in L. multifida (1.77%; [33]. L.
dentata from Palestine [36], L. dentata from
Mexico [35], and L. dentata inflorescence and
aerial parts [34] produced no monoterpene
aldehydes. Monoterpene ethers (1.05%) of L.
dentata (Taif, SA) exceeded the content in L.
stoechas (0.10%) and L. dentata (0.54%)from
Tunisia [33] but was less than 1.40% in L.
dentata[36], 1.46% in L. multifida[33], and 40.40%
and 46.30% in L. dentata aerial parts and
inflorescence, respectively [34]. The highest level
of esters was 68.59% for L. dentata from
Mexico[35].
Table (2): Comparison between individual chemical compositions of the essential oil (%) of Lavandula spss.
Compound
L. dentate
(Taif, SA)
Present
L.dentata(T
unisia) [33]
L. stoechas
(Tunisia)
[33]
L.multifida
(Tunisia)
[33]
L. dentata
(Palestine)
[36]
L. dentata
(Mexico)
[35]
L.dentata(i
nflorescenc
e) [34]
L. dentata
(aerial
part) [34]
Monterpene Hydrocarbons
Tricyclene
0.04
0.02
0.67
0.11
N.D.
N.D.
N.D.
N.D.
α-Thujene
N.D.
0.02
0.08
3.83
N.D.
N.D.
N.D.
N.D.
α-Pinene
0.50
0.06
0.96
0.45
0.10
2.87
3.70
3.70
Camphene
0.92
0.01
0.33
10.06
N.D.
N.D.
1.30
1.20
Sabinene
N.D.
0.01
0.02
0.95
0.10
N.D.
0.70
0.80
Santolina
triene
N.D.
N.D.
N.D.
N.D.
0.02
N.D.
N.D.
N.D.
Beta-
Phellandrene
0.11
N.D.
N.D.
N.D.
0.01
N.D.
N.D.
N.D.
β-Pinene
0.73
0.01
0.32
0.34
N.D.
11.53
5.30
6.10
Limonene
0.04
0.08
0.03
0.10
N.D.
N.D.
3.20
5.20
D-Limonene
2.25
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Citronelol
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.30
0.30
Terpinolene
N.D.
0.04
0.03
2.05
1.24
N.D.
N.D.
Total
4.59
0.25
2.44
17.89
0.23
15.64
14.50
17.30
Aromatic Hydrocarbons
p-Cymene
1.04
0.04
0.06
1.77
N.D.
N.D.
0.50
0.50
o-cymene
0.16
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Total
1.20
0.04
0.06
1.77
0.00
0.00
0.50
0.50
Oxygenated Monoterpene
1-Octen-3-ol
0.02
0.13
0.02
0.25
N.D.
N.D.
N.D.
N.D.
Myrtenal
1.72
0.48
0.46
0.80
0.40
0.55
0.60
1.10
Linalool
1.40
47.30
20.25
50.05
40.80
1.63
0.30
0.30
β-Thujone
0.21
0.02
8.97
0.14
N.D.
N.D.
N.D.
N.D.
(1S-
(1Alpha,2alph
a,4beta))-1-
isopropenyl-4-
methyl-1,2-
cyclohexanedi
46.09
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Extraction and characterization of essential oil of Lavandula dentata L.: Evaluation of its..
________________________________________________________________________________________________________________
Egypt. J. Chem. 67, No. 8 (2024)
673
Compound
L. dentate
(Taif, SA)
Present
L.dentata(T
unisia) [33]
L. stoechas
(Tunisia)
[33]
L.multifida
(Tunisia)
[33]
L. dentata
(Palestine)
[36]
L. dentata
(Mexico)
[35]
L.dentata(i
nflorescenc
e) [34]
L. dentata
(aerial
part) [34]
ol
Borneol
3.35
0.07
0.07
0.21
0.50
N.D.
N.D.
N.D.
Plinol C
N.D.
N.D.
N.D.
N.D.
2.60
N.D.
N.D.
N.D.
Lavandulol
N.D.
0.02
0.23
1.29
N.D.
N.D.
N.D.
N.D.
3-Octanol
N.D.
N.D.
N.D.
N.D.
0.10
N.D.
N.D.
N.D.
Menthone
18.69
0.04
0.02
0.37
N.D.
N.D.
N.D.
N.D.
Terpinene-4-
ol
0.04
0.82
0.09
0.24
0.80
0.57
N.D.
N.D.
α-Terpineol
0.55
0.67
0.97
0.26
4.30
1.11
0.40
0.50
Myrtenol
0.03
0.12
0.02
0.27
N.D.
1.01
0.90
0.80
δ-Terpineol
N.D.
1.47
0.03
1.64
N.D.
1.83
N.D.
N.D.
trans-
Pinocarveol
0.09
N.D.
N.D.
N.D.
N.D.
1.15
0.70
0.70
Nerol
N.D.
0.04
0.53
2.01
N.D.
N.D.
N.D.
N.D.
Geraniol
N.D.
0.02
0.25
0.25
N.D.
N.D.
N.D.
N.D.
Fenchol
9.42
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Dehydrolinalo
ol
0.33
tr
tr
N.D.
N.D.
N.D.
N.D.
N.D.
(E)-Isoeugenol
N.D.
tr
tr
0.27
N.D.
N.D.
N.D.
N.D.
Total
81.94
52.10
31.93
58.29
49.50
7.85
2.90
3.40
Phenols
Thymol
0.10
0.79
0.01
0.20
N.D.
N.D.
N.D.
N.D.
Carvacrol
0.62
0.03
0.02
1.13
0.01
N.D.
N.D.
N.D.
Total
0.72
0.82
0.03
1.33
0.01
0.00
0.00
0.00
Monterpene Esters
bornyl
formate
0.15
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Fenchyl
acetate
0.78
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Linalyl
acetate
N.D.
28.65
64.30
7.30
42.10
N.D.
N.D.
N.D.
Hexyl
ethanoate
N.D.
lptol
N.D.
N.D.
0.40
N.D.
N.D.
N.D.
Lavandulyl
acetate
N.D.
0.14
0.02
0.12
0.40
N.D.
N.D.
N.D.
P-Menth-8-
en-1-olacetate
N.D.
N.D.
N.D.
N.D.
1.30
N.D.
N.D.
N.D.
Methyl
citronellate
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.30
0.30
Bornyl acetate
0.08
0.02
0.08
3.03
N.D.
N.D.
N.D.
N.D.
α-Terpenyl
acetate
0.01
0.02
0.05
0.11
N.D.
N.D.
N.D.
N.D.
Total
1.02
28.83
64.45
10.56
44.20
0.00
0.30
0.30
Monoterpene ketones
Fenchone
N.D.
tr
0.03
0.20
0.10
N.D.
15.80
13.40
Verbenone
0.41
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Camphor
0.15
2.32
0.04
0.30
1.80
1.03
15.00
17.00
Carvone
0.32
0.02
0.07
0.10
N.D.
0.85
N.D.
N.D.
3-octanone
N.D.
0.90
0.02
0.24
N.D.
N.D.
N.D.
N.D.
Total
0.88
3.24
0.16
0.60
1.90
1.88
30.80
30.40
Monoterpene Aldehydes
Neral
N.D.
0.03
0.03
1.61
N.D.
N.D.
N.D.
N.D.
cumenal
0.13
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
a-
Campholenal
0.58
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Geranial
N.D.
0.08
0.03
0.16
N.D.
N.D.
N.D.
N.D.
Total
0.71
0.11
0.06
1.77
0.00
0.00
0.00
0.00
Monoterpene ethers
1,8-Cineole
N.D.
0.44
0.04
0.19
1.40
68.59
46.30
40.40
cis-Linalool
oxide
0.02
0.02
0.05
1.23
N.D.
N.D.
N.D.
N.D.
A. Antar et.al.
_____________________________________________________________________________________________________________
________________________________________________
Egypt. J. Chem. 67, No. 8 (2024)
674
Compound
L. dentate
(Taif, SA)
Present
L.dentata(T
unisia) [33]
L. stoechas
(Tunisia)
[33]
L.multifida
(Tunisia)
[33]
L. dentata
(Palestine)
[36]
L. dentata
(Mexico)
[35]
L.dentata(i
nflorescenc
e) [34]
L. dentata
(aerial
part) [34]
trans-Linalool
oxide
1.03
0.08
0.01
0.04
N.D.
N.D.
N.D.
N.D.
Total
1.05
0.54
0.10
1.46
1.40
68.59
46.30
40.40
Diterpenes
Andrographol
ide
0.42
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Sesquiterpene
s
β-
Caryophellene
N.D.
0.14
0.09
2.13
1.90
N.D.
0.30
0.70
α-bisabolene
0.03
N.D.
N.D.
N.D.
N.D.
N.D.
0.30
0.70
β-selinene
0.59
N.D.
N.D.
N.D.
N.D.
N.D.
0.90
1.00
α-selinene
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.60
1.20
β-Farnesene
N.D.
N.D.
nr
0.13
N.D.
N.D.
N.D.
N.D.
γ-Gurjunene
0.01
0.04
0.18
0.32
N.D.
N.D.
N.D.
N.D.
Germacrene-
D
N.D.
0.11
0.02
0.84
N.D.
N.D.
0.90
2.40
2-Carene
N.D.
N.D.
N.D.
N.D.
0.02
N.D.
N.D.
N.D.
1,3,8-p-
Menthatriene
N.D.
N.D.
N.D.
N.D.
0.03
N.D.
N.D.
N.D.
Bicyclogerma
crene
N.D.
3.40
0.02
0.14
N.D.
N.D.
N.D.
N.D.
Cuparene
0.02
tr
0.02
0.54
N.D.
N.D.
N.D.
N.D.
γ-Cadinene
N.D.
0.13
0.03
0.20
N.D.
N.D.
N.D.
N.D.
δ-Cadinene
N.D.
0.02
0.02
0.43
N.D.
N.D.
N.D.
N.D.
Nootkatene
0.53
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
cis-dihydro-
Occidentalol
1.49
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Curcumene
0.03
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
8-
Isopropenyl-
1,3,3,7tetrame
thyl-
bicyclo[5.1.0]o
ct-5-en-2-one
0.31
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Cadalene
0.24
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Sesquisabinen
e hydrate
0.14
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
alpha-
alaskene
0.21
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Elemol
N.D.
0.04
0.02
0.14
N.D.
N.D.
N.D.
N.D.
Caryophyllen
e oxide
0.02
0.26
N.D.
0.10
N.D.
N.D.
0.60
0.90
T-Cadinol
N.D.
0.04
0.08
0.41
N.D.
N.D.
N.D.
N.D.
α-Eudesmol
N.D.
0.34
0.26
0.35
N.D.
N.D.
N.D.
N.D.
Cadinol
N.D.
0.08
0.03
0.43
N.D.
N.D.
N.D.
N.D.
epi-α-Cadinol
N.D.
0.03
0.02
0.15
N.D.
N.D.
N.D.
N.D.
Total
3.62
4.63
0.79
6.31
1.95
0.00
3.60
6.90
Total
Identified
95.73
89.66
100.00
99.98
99.19
93.96
98.90
99.20
The current L. dentata (Taif, SA) oil was the
only extract that showed diterpene content (0.42%)
(Table 3). Moreover, the sesquiterpene content of
the present L. dentata (Taif, SA) oil (3.62%)
exceeded the 0.79% content in L. stoechas[33],
1.95% in L. dentata[36], and 3.60% in L. dentata
inflorescence[34]. However, the content was
greater in L. dentata at 4.63% [33], L. multifida at
6.31% [33], and aerial parts of L. dentata at 6.90%
[34]. L. dentata from Mexico [35] had no
Extraction and characterization of essential oil of Lavandula dentata L.: Evaluation of its..
________________________________________________________________________________________________________________
Egypt. J. Chem. 67, No. 8 (2024)
675
sesquiterpene content. Among the more important
and effective compounds (Table 3),linalool
(1.42%) is a monoterpene in the EO of coriander.
The N-methyl-D-aspartate (NMDA) receptor is
considered a vital competitive antagonist that
displays antitumor and anticardiotoxic activity.
PPARα ligand is the compound that is responsible
for the reduction of plasma triglyceride rate and
modulates the hepatic determination of the genes
working on or off and the quantification of the
entire metabolites in the plasma [37, 38]. Further,
linalool may act as a nonsteroidal anti-
inflammatory agent (Chu and Kemper 2005) and
display neuroprotective properties associated with
the inhibition of NMDA glutamate receptors[36].
Moreover, borneol, which accounted for 3.35% of
the present oil content, at this time is a naturally
occurring bicyclic monoterpene that has analgesic
and anesthetic properties in conventional Chinese
medication. Borneol has an EC50 of 248 μM and
increases GABA receptor activation. Borneol
makes direct stimulation to GABAA receptors with
high doses (>1.5 mM), resulting in maximal
response of GABA (84%). This feature revealed a
weakening action of the agonist. At α1β2γ2L
GABAA receptors, borneol generates a positive
modulation of Cl− conductance dose that is
stimulated by low-dose GABA [39] (Table 3).
Moreover, thymol (a monoterpene phenol) L.
dentata (0.1%) is primarily found in EO-
represented plant isolation related to a family of
Lamiaceae. Thymol has antioxidant, anti-
inflammatory, antibacterial, and antifungal
properties [40]. Despite an IC50 of 1.32 mM, β-
pinene, a significant turpentine component found in
L. dentata (0.7%), inhibits the spread of the viral
bronchitis virus [41, 42]. The activities of the
antimicrobial and bactericidal are possessed by β-
Pinene [43].Andrographolide represents about
0.42% of the EO.Andrographolide is a bicyclic
diterpenoid lactone produced by
Andrographispaniculate. This small antagonist for
NF-κB activation covalently modifies cysteine 62
of p50. Andrographolide can reduce the stimulation
of NF-κBg during the activation of endothelial
cells. This role inhibits leukocyte adherence
mediated by the cell adhesion molecule E-selectin
by reducing its expression. The molecule does not
affect IκBα degradation or p50 and p65 nuclear
translocation [45]. Despite the NFκB pathway
being regulated negatively in the stimulation of
RAW 264.7 cells in LPS, the Alpha-cyperone
(0.03% in the present study) shows anti-
inflammatory activity by reduction of IL-6 and
COX-2 [46]. The deficiency of Rac1, COX,
Cdc42-2, IL-6, & Nck-2 significantly impacts
Alpha-cyproterone and, thereby reduces
inflammation. This property could be extremely
useful in treating cellular inflammation that occurs
in the disease of Alzheimer's. In vitro
Sulforhodamine B (SRB) cell cytotoxicity assay of
both ethanolic extract and the essential oil of
Lavandula dentata (Taif, Saudi Arabia) against the
hepatocellular carcinoma (HepG2) cell line (Fig.
1). and against Green monkey kidney (Vero) cell
line (Fig. 2) showed that The standard curve for the
sulforhodamineB cytotoxicity assay was plotted
(Fig.2) and supported an IC50 of L. dentata L.
extract, or its oil, as >100 µg with R% (NA). L.
dentata is widely used for itsanticonvulsant and
antidepressant [47], sedative [48], and antioxidant
properties [49]. A positive impact on teaching and
memory appeared by the plant [50]. When taken
orally, EO formulations have calming and
anxiolytic effects that happen more quickly than
with first-choice anxiety medications like
benzodiazepines and serotonin reuptake
inhibitors[51]. Oral preparations of the EO show
anxiolytic and calming effects with a faster onset
than first-choice anxiety treatments, such as
serotonin reuptake inhibitors and benzodiazepines
[52].
A. Antar et.al.
_____________________________________________________________________________________________________________
________________________________________________
Egypt. J. Chem. 67, No. 8 (2024)
676
Table (3): Review for different biological functions of the most common active constituents of Lavandula dentata
(Taif, Saudi Arabia)
Compound
%
Biological Functions
References
α-Pinene
0.5
α-Pinene is a monoterpene enhances sleeping, binds directly to GABAA-
benzodiazepine (BZD) receptors, and acts as a partial modulator at the
BZD binding site
[44]
β-Pinene
0.73
β-Pinene consider as vital component of turpentine, which has
antimicrobial and antiviral activities, whereas it can inhibit infectious
bronchitis virus (IBV) with an IC50 of 1.32 mM.
[41, 42]
D-Limonene
2.27
A monoterpene found in many pine-needle oils and in turpentine. (-)-
Limonene can induce a mild bronchoconstrictive effect
[57]
linalool
1.42
Linalool is a natural monoterpene component in essential olis of coriander.
Linalool acts as a competitive antagonist of Nmethyl d-aspartate (NMDA)
receptor, with anti-tumor, and anti-cardiotoxicity activity. Linalool is a
PPARα ligand reducing plasma TG levels and rewires the hepatic
transcriptome and plasma metabolome.
[37, 38]
borneol
3.35
Borneol represents a natural bicyclic monoterpene used for analgesia and
anesthesia in traditional Chinese medicine. Also, it enhances GABA
receptor activity with an EC50 of 248 μM. At high concentrations (>1.5
mM). It activates GABAA receptors producing 84% of the maximal
GABA response indicative of a weak partial agonist action. Borneol
produces dose-dependent positive modulation of the Cl- conductance
generated by extremely low dose GABA at α1β2γ2L GABAA receptors.
[39]
Caryophyllene
oxide
0.02
Caryophyllene oxide, isolated from Annona squamosa L. bark. It poses
analgesic and anti-inflammatory activity.
[58]
α-Terpinene[
0.16
α-Terpinene (Terpilene) is natural monoterpene of the essential oils in
various foods and aromatic plants such as Mentha piperita. α-Terpinene
acts against Trypanosoma evansi . It also a potential treatment for
trypanosomosis. In addition, it poses antioxidant and antifungal activities.
[59-62]
Andrographolide
0.42
The plant andrographis (Andrographis paniculate) is a key producer of
Andrographolide.
Andrographolide (Andro) suppresses NF-κB activation by modifying
reduced cysteine 62 of p50, the expression of cell adhesion molecule E-
selectin, and E-selectin-mediated leukocyte adhesion.
[45]
Cedrol
0.05
Cedrol represents a bioactive sesquiterpene, a potent competitive inhibitor
of cytochrome P-450 (CYP) enzymes. Cedrol is found in cedar essential
oil and possess anti-septic, anti-inflammatory, anti-spasmodic, tonic,
astringent, diuretic, sedative, insecticidal, and anti-fungal properties.
[63, 64]
Isolongifolene,
4,5,9,10-
dehydro-
0.04
A tricyclic sesquiterpene isolated from Murrayakoenigii. Isolongifolene
reduces induced oxidative stress, mitochondrial dysfunction, and apoptosis
through the regulation of P13K/AKT/GSK-3β signaling pathways.
Isolongifolene has antioxidant, anti-inflammatory, anticancer and
neuroprotective activities.[65]
[65]
alpha-Cyperone
0.03
alpha-Cyperone associates with the downregulation of COX-2, IL-6, Nck-
2, Cdc42 and Rac1, which reduce inflammation., It may be a potential
treatment of inflammatory diseases such as AD.
[46]
alpha-Cyperone has anti-inflammatory activity. It is correlated with the
down-regulation of COX-2 and IL-6 by down regulation of the NFκB
pathway in LPS-stimulated RAW 264.7 cells.
β-Bisabolene
0.03
β-Bisabolene is a sesquiterpene isolated from opoponax
(Commiphoraguidotti). β-Bisabolene, has anti-cancer activities, can be
used for the study of breast cancer.
[66]
β-Elemene
0.01
β-Elemene is isolated from natural plant Curcuma wenyujin with an
antitumor activity. β-Elemene may induce cell apoptosis.
[67]
Thymol
0.1
Thymol represents the major monoterpene phenol of essential oils isolated
from plants belonging to the Lamiaceae family, and other plants such as
those belonging to the Verbenaceae, Scrophulariaceae, Ranunculaceae and
Apiaceae families. Thymol poses antioxidant, anti-inflammatory,
antibacterial and antifungal activities.
[40]
Carvacrol
0.12
Carvacrol represents a monoterpenoid phenol isolated from Lamiaceae
family plants, with antioxidant, anti-inflammatory and anticancer
properties. Carvacrol arrests cell cycle in G0/G1, downregulates Notch-1,
and Jagged-1, and induces apoptosis.
[55, 68]
Bornyl acetate
0.08
Bornyl acetate is a major odorants of fresh ginger juice, as it exhibits one
of the highest FD factors. Bornyl acetate has a pivotal sensory roles in the
[69]
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Egypt. J. Chem. 67, No. 8 (2024)
677
Table (3): Review for different biological functions of the most common active constituents of Lavandula dentata
(Taif, Saudi Arabia)
Compound
%
Biological Functions
References
aroma of fresh Japanese ginger.
Carvone
0.32
Carvone (D Carvone) is a natural compound found in various foods and
can be used in flavoring foods.
[70]
Verbenone
0.41
Verbenone is a natural terpene in tree
leaves, Suregadazanzibariensis Verdc.. Verbenone has anti-aggregation
property and interrupts the attraction of bark beetles to their aggregation
pheromones.
[71, 72]
fenchylacetate
0.78
Fenchyl alcohol is a monoterpene in the essential oils isolated from
Douglas fir needles, acts as a fragrance. Fenchyl alcohol inhibits the rumen
microbial activity of both sheep and deer.
[73]
Isoborneol
0.04
Isoborneol is a monoterpene in the essential oils of various medicinal
plants posing antioxidant and antiviral properties. Isoborneol is a potential
inhibitor of herpes simplex virus type 1.
[74, 75]
Valencene
0.05
Valencene is a sesquiterpene isolated from Cyperus rotundus, posing
antiallergic, antitumor, anti-inflammatory, and antioxidant properties.
Valencene suppress the exaggerated expression of Th2 chemokines and
proinflammatory chemokines through blocking of the NF-κB pathway.
Valencene is used in flavoring foods and drinks
[76-78]
Table (4): Estimation of Total Phenolic and Flavonoids of Lavandula dentata (Taif, Saudi Arabia):
TotalPhenolic
TotalFlavonoids
Gallic acid
(µg/ml)
Absorbance
/630 nm
Rutin
(µg/ml)
Absorbance
/ 420 nm
1000
2.9552
1000
3.253
800
2.5155
600
1.943
600
1.739
400
1.368
400
1.109
200
0.7758
200
0.5218
100
0.372
100
0.2324
50
0.1537
50
0.1686
10
0.0248
Seizure Intensity Scale:
Seizure intensity measurements (Table 5)
indicated that some natural products increase the
latency between the uncontrolled seizures that recur
and lessen the frequency of those seizures while
receiving treatment. The behavior observed during
pilocarpine-induced status Epileptic (SE) in rats
composed of initial shaking of wet-dog, head
bowing, and facial and ear shaking. These
behaviors progressed to Phase II with axial
rhythmic waves that were constant throughout the
body. Phases III represented by involuntary
twitching of the muscle (myoclonic) and IV
constituted a generalized repetitive jerking
movement of arms and legs (clonic convulsions)
followed by the assumption of aside status,
rhythmic stretch segments, and widespread
convulsions together with SE (phase V).
Total distance traveled and vertical activity in
control rats showed diurnal fluctuations in
locomotor activity (grooming, rearings counting,
frequency of standing on hind limbs, and
ambulation frequency). In open-field examinations,
the hyperactivity of epileptic rats was reduced to
control levels through E-DK® and ethanolic extract
and Essential oil (EO) of L. dentata (p< 0.01). The
frequency of seizures was considerably decreased
and the intervals of the latency period was
prolonged in all epileptic stages, particularly phase
V, which included widespread seizures and rigid
extension episodes, by both ethanolic extract and
oil (17.353 ± 0.167 and 16.837 ± 0.505 sec,
respectively).
All treatments attenuated pilocarpine-induced
anxiety mostly by reducing the spending time and
distance in the central portion. Control rats
exhibited less immobility time and lack of
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variations during the diurnal time by applying
melatonin treatment. Besides, the rat treatment with
various degenerative models [53-55]. Moreover,
Alvi et al. [55] indicated that either ethanolic
extract or essential oil (EO) of L. dentatadisplayed
depressing conduct during mandatory swimming
exams (FSTs) [56].
Antiepileptogenic treatment with either Depakine®
or L. dentata oil or ethanolic extract was assessed
via evaluation of latency between involuntary
jerking movement of muscle, interval between
seizures that are tonic-clonic in general, numbers of
these seizures, and severity of seizures after each
subconvulsant PTZ injection during kindling [79,
80]. The present data are consistent with studies by
Singh et al. [80], Löscher [81], who confirmed that
AEDs alleviated exacerbation of seizure severity
during kindling. Therefore, a possible role of crude
ethanolic extract or essential oil (EO) is the
modulation of changes in neurochemistry, which
might be responsible for depression in epilepsy
[80]. Many reports indicate a significant role of
monoterpenes as agents had protective, antioxidant,
and anti-inflammatory roles incarveol, a
monoterpene, can decrease seizure intensity and
frequency and delay seizure onset.
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Egypt. J. Chem. 67, No. 8 (2024)
679
Table (5): Changes in the seizure intensity was evaluated using the following modified scale of different
groups: epileptic (EP), Depakine®-treated (EP_DK®), L. dentata oil–treated (EP_LO) and L. dentata
extract–treated (EP_LX). Values were represented as Mean ± SD & n = 8 animals. Means within the same
parameter and not sharing a common superscript symbol(s), are differ significantly at P < 0.05. This scale is
applied for the occurrence of seizure-related behavioral changes and its severity. Each number represents
latency and intervals of the seizure severity.
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680
Healthy control Rats' overall distance traveled and
vertical activity indicated typical fluctuations in
their movement activity (rearing) (p < 0.05) in
some behavioral assessments, e.g., open field tests
(Table 6). The rats with epileptic disorders were
less troubled than control animals, as demonstrated
by a longer delay period spent in the negative
center zone of the field and by decreased
locomotion of frequency. This was minimized by
injection of oil or L. dentata extract, enhancing
grooming, parenting, and frequency of moving
around while lowering the distance and latency’s
intervals in the central region. Depakine® lowers
the duration of grooming even when locomotion,
rearing, and excretion are not modified. The
anxiety-reducing properties of valproate may be the
source of this activity[82].This study evaluated
behavioral effects caused by ethanolic extract and
essential oil (EO) of L. dentata were performed to
explore their role with antiepileptic drugs in vivo..
In earlier models of epilepsy, changed behavioral
reactions were thought to represent different
feelings outputs. The present model used sub
convulsive pilocarpine doses to induce epileptic
seizures. In earlier models of epilepsy, changed
behavioral reactions were thought to represent
different feelings outputs [55, 81, 83].
Generally, the open field test (Table 6) is an
acceptable model to measure anxiety status in
stressful or threatening situations. The epileptic
(EP) group showed a long latency time to react
with the open field (154.00± 13.53 sec) when
compared to controls (12.00± 1.00 sec).
Conversely, epilepsy-DK® exhibited a
significantly (p< 0.05) shorter latency time
(41.667± 2.887 sec) than the Epileptic group. Both
essential oil (EP_LO) and extract of L. dentata
(EP_LX) exhibited a highly significant decrease in
entering the central part of the apparatus (P<0.01)
(11.15± 0.787 and 12.77± 2.36 sec) in contrast to
epilepsy group. Increased time spent in the central
zone or decreased latency for entering this zone are
indications of anxiolysis [84, 85]. The frequency
of entries, time spent in latency, and the central
square are all indicators of the behavior of
exploration and fear [84]. The grooming tasks in
the open area exam, involve quickly wiping the
front legs toward the body or face. Epileptic
animals showed a significantly decreased activity
(5.667± 1.528 /3 min, P<0.05) relative to controls
(13.00± 2.00 /3 min). Conversely, the epilepsy_DK
section, which exhibited hyperactivity significantly,
recording (15.33± 4.16 with P<0.05) was regarding
to epilepsy group. Similarly, a corresponding
elevation (P<0.05)in the movement of the forelegs
towards the face and the body was recorded in
EP_LO and EP_LX (17.333± 2.082 and 13.000±
1.000 /3 min, respectively) comparable to EP
(5.667± 1.528 / 3 min) Table (6). Any new setting
is likely to elicit grooming, which is a migration
behavior. Anxiety can be reduced in difficult times
by grooming oneself. Compared to grooming
replies, the importance of grooming as a behavioral
sign of depression has received substantially less
research[86]. There were no differences seen in the
research rats' latency of getting into the central
zone, falling in locomotion action, or rearing. Rats
that were administered extract or Essential oil (EO)
of L. dentata displayed significantly increased
grooming activity compared with Depakine®
treatment (p< 0.01). Depakine® (valproic acid)
displays anxiolytic properties [87]. Therefore, the
rats with higher anxiety are distinguished by
locomotion and grooming.
Concerning there was frequency of standing
on hind limbs (rearing) of the open field test, Table
(6) illustrated a significant hypoactivity in the
epilepsy group (EP)(P<0.05) (5.00± 2.00 /3 min)
comparable to the control group (11.00± 2.65 /3
min). On the other hand, rearing of rats in EP_DK,
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Egypt. J. Chem. 67, No. 8 (2024)
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EP_LO, and EP_LX sections revealed higher
activity (P<0.05) also registered 9.00± 1.00, 11.00±
1.00 and 13.00± 1.00, respectively, as compared
with EP group. Moreover, a significant decrease in
the ambulation frequency (i.e. total number of
entered squares/3min)(P<0.05) in the epilepsy
group (EP, 19.00± 1.00) is comparable to normal
rats (33.33± 5.93). Moreover, however, the EP_DK
group showed a no significant increase
(25.33±3.51). While EP_LO and EP_LX animals
showed significant hyperactivity and entered more
squares (P<0.05), as recorded at 37.33± 4.29, and
43.33± 3.51, respectively, in contrast to the EP
group.
Some antiepileptic drugs (AEDs) such as
Depakine® benefit some patients with epilepsy and
cause a variety of adverse consequences, such as
irritation, anger, and violence. Moreover,
Depakine® strongly inhibits seizure-induced
neurogenesis [88].
Table (6): Assessment of behavioral tests (open field, hot plat and despair forced swim tests) between different
experimental group: Control; epileptic (EP), Depakine®-treated (EP_DK®), L. dentata oil–treated (EP_LO) and
L. dentata extract–treated (EP_LX). Values were represented as Mean ± SD & n = 8 animals. Means within the
same parameter and not sharing a common superscript symbol(s), are differ significantly at P < 0.05.
Behavioral
Test
Open Field Test
Hot
Plate
Despair Swim
Latency time
(Sec.)
Grooming
/3 min.
Rearing
/3 min.
Ambulation
Frequency
/3 min.
Latency
time (Sec.)
Latency
time
(Sec.)
Control
12.00±
1.00a
13.00±
2.00b
11.00±
2.65bc
33.33±
5.93bc
13.00±
1.00a
33.33±
6.69a
EP
154.00±
13.53c
5.67±
1.528a
5.00±
2.00a
19.00±
1.00a
70.00±
5.00c
111.67±
10.41d
Ep_DK®
41.67±
2.88b
15.33±
4.163b
9.00±
1.00b
25.33±
3.51ab
33.333±
2.89b
68.33±
4.163c
EP_LO
11.15±
0.78a
17.33±
2.082b
11.00±
1.00bc
37.33±
4.29c
16.67±
4.76a
35.67±
3.512ab
EP_LX
12.77±
2.36a
13.00±
1.00b
13.00±
2.00c
43.33±
3.51c
12.33±
2.08a
46.00±
2.65b
F value
286.326
10.075
8.118
5.478
146.658
83.842
P<
0.000
0.002
0.003
0.013
0.000
0.000
The present data demonstrate that grooming
behavior is a response to any unfamiliar
environment and helps calm anxiety caused by
stress. It is a better marker for anxiety than
depression because grooming represents a state of
pleasure or comfort. Therefore, in conflict
situations, grooming activity or, more commonly,
discrete grooming fragments are observed[89].In
Table (6), the test of the hot plate is a behavioral
design of nociception and a mirror for analgesic
impact. The present data revealed a higher
obviously increased latency time to paw
withdrawal latency time in reaction to thermal
noxious stimulation as heat pain in epilepsy group
EP (70.00± 5.00sec, P<0.001) in contrast to
controls (13.00± 1.00sec).Conversely, a shorter
paw withdrawal latency time was registered by the
group administered with Depakine® (EP_DK) and
also had a notably more sensitive response (33.33±
2.89sec, P<0.05), in comparison with the epilepsy
section. There was a significantly greater feeling
and a shorter paw withdrawal delay time to respond
in both the EP_LO and EP_LX groups (16.67±
4.73 and 12.333± 2.082 sec, respectively, P<0.001)
in contrast to epileptic animals. The pain action can
be measured by the delay period of licking [90] and
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Egypt. J. Chem. 67, No. 8 (2024)
682
for evaluation of analgesic effects. The HPT
measures a complex response to a non
inflammatory acute nociceptive input and is
normally used for assessing central nociception
[91, 92]. With the treatment of extraction or EO of
L. dentata, the pain was reduced and passivity in
HPTs was higher significantly (p< 0.001) which
suggests analgesic potential via prostaglandin
pathways. Multiple noxious-evoked patterns were
described by Espejo and Mir [93] and responses of
exploration and self-care in rats during HPTs.
Finally, rats reacted to heat stimuli and attempted
escape according to sequential behavior structures
based on the first occurrence. Various behaviors
were used to quantify nociceptive thresholds, e.g.,
hindpaw-licking and stamping latency. Such
patterns are initial reactions to heat as a noxious
stimulus. Moreover, valproic acid decreases the
phases of inflammation and acute pain. The drug
shortened the duration of licking during the stage of
inflammation [94, 95].
Forcing swim (FST) test was performed on the
experimental rats with epilepsy disorder (Table 6)
showed distinct oscillations together with
prolonged immobility (111.67± 10.41sec) (P<0.05)
when corresponding with the behavior of rats in the
control group (33.33± 6.67sec). These diurnal
alterations were improved by the injection of oil
and extract of L. dentata and significantly
decreased with the Depakine®-treated group
(EP_DK®), L. dentata oil–injected section
(EP_LO), and L. dentata extract–treated group
(EP_LX) (68.33± 4.16, 35.67± 3.512 and 46.00±
2.65 sec respectively) as compared to the EP group.
When enclosed in an impenetrable container in
FSTs, treated rats had an immovable expression.
This mobile action was not registered in most
pilocarpine-treated rats. Instead, these animals
showed less time despairing as they continued to
swim, trying to escape from the cylinder. The
present results are consistent with those of Ilbay et
al. [96], who reported that caffeine-treated animals
explored significant immobilization delay and
prolonged continuous swimming in FSTs compared
with untreated controls.
Generally, the "behavioral despair" test, is
known as FST, and used in clinical forms of mental
disorder to assess behavior resembling depression.
[97, 98]. FST causes alterations in neurochemical
and endocrine release [99, 100]. Moreover,
neurochemical changes coincided with endocrine
and immune alterations associated with rats in
FSTs. Furthermore, a stress reaction to
antidepressant medication may account for the
present higher latency period for animals receiving
treatment in FSTs as contrasted with second-group
epileptic animals. On the other hand, during forced
swimming, post-pilocarpine SE mice remained
motionless for a prolonged duration, suggesting a
condition akin to despair (Mazarati et al.[101].
Similar researchers proposed that serotonergic
circuit changes are not the exclusive cause of
depression in SE. The pathophysiological
relationship between depression and seizures is due
to impairments in serotonergic conduction[102]. In
contrast, Gröticke et al. [103] explained enhanced
performance under different adverse environments
of FST in SE in mice compared with controls.
Epileptic animals showed significantly increased
anxiety-related behavior. Moreover, motor activity
was increased in FST in SE animals.
SE rats' swimming trials may be indicative of
cognitive decline in a learning test that depends on
the hippocampal region. Rather than hippocampal
damage, this disorder could be linked to aberrant
regeneration in the dentata gyrus in SE mice.
Whereas the animals receiving treatment did not
actively search for the concealed platform, the SE
rats just swam within a comparatively small area of
the maze or in rings around its perimeter[88].
Components of L. dentata that contain
flavonoids or saponins have anticonvulsant effects
Extraction and characterization of essential oil of Lavandula dentata L.: Evaluation of its..
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Egypt. J. Chem. 67, No. 8 (2024)
683
[104]. Additionally, monoterpenes and flavonoids
have been shown to protect versus seizures caused
by picrotoxin, PTZ, and NMDA [105]. Numerous
therapeutic effects on the CNS, such as
anticonvulsant and anxiolytic effects, have been
linked to flavonoids, sterols, and terpenoids
[106].Sterols and flavonoids have neuromodulatory
and central inhibitory effects [107]. Ethanolic
extract and Essential oil (EO) of L. dentata
increased seizure thresholds and inhibited
pilocarpine-induced convulsions. Extract and
Essential oil (EO) of L. dentata show antiepileptic
properties, likely due to some phytoconstituents
that facilitate GABA transmission. Extract and
Essential oil (EO) of L. dentata also show some
motor impairment and decreased spontaneous
locomotor activity at anticonvulsant doses. The
impairment in muscle movement may be caused by
different phytoconstituents, such as phenols,
saponins, terpenoids, and flavonoids [108].
Analgesic medication concepts working through
prostaglandin routes may be the source of a
substantial decrease in pain following treatment
[92].
In conclusion, the present study provides
evidence that ethanolic extract and Essential oil
(EO) of L. dentata modulate several behavioral
domains by controlling seizures in conjunction with
antiseizure medications and via shorter latency time
and greater ambulation frequency. Therefore,
reduced depression-like symptoms in the EP_LO
and EP_LX animals might be a consequence of
reduced convulsion and seizure activity.
Conflict of Interest & Disclosure Statement:
The authors have no potential conflict of
interest with any groups.
Acknowledgement
The authors thank Princess Nourahbint
Abdulrahman University Researchers supporting
Project Unit.
Funding Support:
The present work was supported by Princess
Nourah Bint Abdulrahman University Researchers
supporting Project [grant number
PNURSP2024R37].
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