Evaluation of Neuropharmacological Activities
of Methanolic and Aqueous Extracts of Citrus Reticulata (Rutaceae) Fruit Peels
Mohamed A Gbaj1, Inass A Sadawe1, Nisreen M Meiqal2, Salah M Bensaber2, Massaud Salem Maamar3, Anton Hermann4 and Abdul M Gbaj2*
1Department of Chemical Engineering, University of Tripoli, Libya
22Department of Medicinal Chemistry, University of Tripoli, Libya
3Zoology Department, Tripoli University, Libya
4Department of Biosciences, University of Salzburg, Austria
Submission: March 09, 2019; Published: May 02, 2019
*Corresponding author: Abdul M Gbaj, Associate Professor of Genetics and Biochemistry, Department of Medicinal Chemistry, Faculty of Pharmacy, University of Tripoli, Libya
How to cite this article: Md A Gbaj, Inass A S, Nisreen M M, Salah M B, Massaud S M et al. Evaluation of Neuropharmacological Activities of Methanolic
005 and Aqueous Extracts of Citrus Reticulata (Rutaceae) Fruit Peels. Glob J Pharmaceu Sci. 2019; 7(3): 555711. DOI: 10.19080/GJPPS.2019.06.555711.
Introduction:Herbal medicines are getting more interest because of their cost-effective, eco-friendly attributes and proper reprieve from disease condition. The present study was designed to evaluate the anxiolytic activity potentials in peels of Citrus reticulata (Family: Rutaceae) in Libya.
Methods:The peel of the fruits were separated and subjected to cold extraction using 99% Methanol and hot water extraction. The extracts obtained were screened for anxiolytic activity of extracts at 1.25 to 5mg/Kg body weight concentrations and were assessed by Elevated Plus Maze.
Results:The results revealed that, both extracts treated animals have shown significant anxiolytic activity. From the results it was evident that citrus peel extract of Citrus reticulata exhibited significant anxiolytic activity.
Conclusion:From the results it was evident that Citrus reticulata has prominent activity in terms of parameters assessed in a dose dependent manner.
Human brain is a speculate and is not explored entirely. It is a multifaceted gathering of interacting neurons and nuclei that control their own and each other’s activities in a dynamic manner, usually throughout chemical neurotransmission. Psychopharmacology is the scientific learning of the effect’s drugs encompass on mood, thinking, behaviour and sensation. It is different from neuropsychopharmacology, which studies drug-induced alterations in the functioning of cells in the nervous system [1,2]. Two‐thirds of the psychotic, depressed, or anxious patients react to the currently available treatments, but the enormity of improvement is not very significant.
The majority of the drugs for these conditions used these days have adverse side effects so the need for better-tolerated, newer and more efficacious treatments is enduring far above the ground [3,4]. The main use of sedative–hypnotic and anxiolytic drugs is to promote calmness (sedatives or anxiolytics) or to create sleep (hypnotics–sedative). Human are exposed to states of restlessness and emotional tension. Anxiety always accompanies many medical and surgical conditions, and it is frequently an indication of psychiatric illness. When the symptoms turn into insufferable or interfere with the treatment of the underlying disease, and if counseling is not enough, drug treatment can be used to help patients manage with their anxiety . There are many medicines are used to treat many psychotherapy and pharmacotherapy illness such as benzodiazepines, azapirones, and antidepressants and others .
Citrus reticulata (Rutaceae) is commonly known as narangi or santra (orange). It is a small spiny tree with thick top of slim branches, extensively grown in Egypt, Tunisia and Libya . Mandarin is a collection name for this class of orange with thin, loose peel. The name ‘tangerine might be applied as an interchange
name to the entire group, but in trade, it is usually limited to the
types with red-orange skin. The fruit has aphrodisiac, laxative,
tonic properties and astringent [8,9]. It is also used to alleviate
vomiting [10,11]. The fruit peel controls the skin moisture, rough
and softens hard skin and possess a cleaning effect on oily skin
. Chemical composition of the volatile oil of the fruit peels of
this species has been reported [13-17]. The effects of the volatile
oil of C. reticulata has been studied against Saccharomyces cerevisiae
, pathogenic fungi, Paenibacillus larvae, Schistosoma
mansoni, Aspergillus flavus, and other microorganisms [19-24].
The volatile oil of C. reticulata also demonstrates the anticancer
activity [25-27]. The present paper describes the neuropharmacological
Activities of methanolic and aqueous extracts of Citrus
reticulata (Rutaceae) fruit peels of the essential oil of the fruit peel
of C. reticulata of Tripoli region.
The oranges were bought from a shop in Tripoli (February
2019). The Citrus reticulata was identified and authenticated by
a botanist. Orange rinds were peeled off carefully with the help of
a sharp razor blade. Each rind sample was cut into smaller pieces
and 30g mass of the sample was taken. The sample was initially
rinsed with distilled water. The fresh peels (30g) were added
to 30ml hot distilled water. In addition, another 30 g of the fresh
peels were macerated in cold 99% methanol for three hours. After
3 hours of maceration at room temperature (28°C), the mixture
was then was filtered under vacuum and the filtrate was stored at
4°C and used to treat animals as needed .
Swiss albino mice of either sex weighing about 18–28g
(2–2.6-month-old) used for experimental purpose. They were
housed in polypropylene cages in the air-conditioned room with
the temperature maintained at 25 ± 2°C, and 12h alternating light
and dark cycles. The mice were provided with a nutritionally adequate
diet and drinking water ad libitum throughout the study.
Approval by the Animal Ethics Committee for the experimental
Acute toxicity was generally carried out for the determination
of LD50 value in experimental animals. The aim of performing
acute toxicity study is for establishing therapeutic index of a
methanolic and aqueous extracts of Citrus reticulata and to ensure
safety in-vivo. Acute toxicity test was performed in mice. All animals
were fasted overnight before treatment and were given food
one hour after aqueous and methanolic extracts. General behavior
was also observed at 0.5, 1, 8, 12 and 24h after administration.
The number of animals that died after administration was traced
daily for 7 days [29,30].
Elevated plus-maze is simple apparatus to study neuroprotective
effects [31,32] and anxiolytic responses produced by the test
drugs. It is used to test almost all types of anxiolytic agents. Exposure
of animals to novel maze alley suggests an approach-avoidance
conflict which is stronger in open arm as compared to enclosed
arm. Rodents (rats and mice) have an aversion for high
and open space and prefer enclosed arm, consequently, spend a
greater amount of time in enclosed arm. When animals enter open
arm, they freeze, become immobile, defecate and show fear-like
The cortisol plasma level is increased, as a true reflection of
anxiety. Major advantages of this test procedure are:
a) It is less time consuming, simple and quick
b) no noxious stimuli (light or sound) or prior training is
c) it is conventional and consistent procedure for studying
anxiety response as well as anxiolytic action of drug [34,35]. Animals
were weighed, numbered, and divided into five groups, each
consisting 6 mice. One group was used as control (saline), second
for standard drug (diazepam) treatment; groups 3-6 for aqueous
Citrus reticulata extract treatment (1.25, 2.5, 3.75, 5.00mg/kg, intra-
peritoneally) and groups 7-10 for methanolic Citrus reticulata
extract treatment (1.25, 2.5, 3.75, 5.00mg/kg, intra-peritoneally).
Animals were placed individually in the center of the maze, head
facing toward open arm and stopwatch was started
The following parameters were noted for 5 min.
a) First favorite of mouse to open or closed arm.
b) Number of entries in open arm (an arm entry defined as
the entry of four paws into the arm).
c) Average time each animal spends in open arm (Average
time = total duration in the arm/number of entries) was estimated.
Saline and diazepam were injected to the control and standard
groups respectively. Citrus reticulata extracts were injected to the
test groups. After thirty minutes, animals were located individually
in the center of the maze. Lastly, a comparison of the preference
of the animals to open or enclosed arm, average time spent
in open arm and the number of entries in open arm in each group
were determined and recorded .
Data were expressed by mean ± standard error mean. For
comparison among the groups, we used analysis of variance with
multiple comparisons by post-hoc Dunnett t-test method. The statistical
significance of differences between the control and experimental
groups was assessed by Dunnett’s two-sided t-tests (posthoc
tests). Statistical analysis was done using Statistical Package
for the Social Sciences for windows (version 17.0, SPSS Inc., Chicago.,
USA). Statistical significance was considered P < 0.05 level.
With the growing amount of research about naringin as a
component of the orange and its potential utilize within the pharmacological
and food industries, illuminating its toxicological
outline becomes increasingly significant. In the present study, the
Citrus reticulata extracts were found to be safe up to 200mg/kg
orally. This present study is compared with other previous studies
in which an oral single dose of 16g/kg of naringin did not produce
acute oral toxicity in rats . (Tables 1 & 2) summarized
the number of entries (open and total) of mice in elevated plus
maze and the time spent by mice on elevated plus maze in open
and closed arms, respectively.
Recently, interests in powerful pharmacological properties
and clinical applications of natural products for replacing synthetic
drugs are rising. Despite of chief scientific and technological
development in combinatorial chemistry, drugs obtained
from natural products still create a huge contribution to drug
discovery today. The citrus plant belongs to the family Rutaceae,
comprising of about seventeen species found all over the tropical,
subtropical and temperate regions [38,39]. Among the species,
Citrus indica, Citrus ichangenesis Citrus latipes, Citrus megaloxycarpa,
Citrus macroptera, Citrus jambhiri, Citrus aurantium, and
Citrus reticulate are the most common. The genus citrus includes
special essential fruits such as orange, mandarins, limes, lemons,
and citrons grapefruits . Even though, there are many groups
of plants that are crucial in phytochemistry, citrus plantation has
been assumed to be a valuable target for commercial agricultural
and industrial practices in the world . About 37 major components
were identified from Citrus reticulata [41,42], and the major
components were geranial (19.0%), geranyl acetate (3.9%), limonene
(46.7%), neral (14.5%), nerol (2.3), β-caryophyllene (2.6),citronellal (1.3%), geraniol (3.5%), and neryl acetate (1.1%). Citrus
oil also composed of aldehydes and esters being the lowest
percentage components and about 97% monoterpenes with alcohols,
with values ranging between 1.8 to 2.2%. It has been reported
that the identify of about 16-27 chemical constituents in the
peel essential oil of C. reticulate was allocated including limonin
and naringin and others [43-46].
Naringin is found in many plants. It is a flavonoid and was believed
to relieve anxiety at the dose level below 3mg/kg when given
intra-peritoneally. At high doses it has been reported to cause
sedation but no muscle relaxant property . The anxiolytic effect
of naringin has been studies using mice 6-8 weeks old and
30g to 35g weight and the anxiolytic effect was confirmed using
elevated plus maze and locomotors activity and these results are
in consistency with the results obtained in (Table 1 & 2)  It has
been reported that the diazepam 2 and 10mg/kg dose were used
which increased the number of open arm entries and percent of
time spent in open arm as compared to saline control  and
these results also are in consistency with the results obtained in
(Tables 1 & 2).
The results obtained in (Tables 1 & 2) are also in agreements
with studies done by Marder et al. [48,49], which have shown that
naringin flavones (which is the main component of Citrus reticulata)
at the level of dose 3 to 10mg/kg to have excellent anxiolytic
potential with no myorelaxation, sedation, or significant
reduction in locomotor activity. It is reported that at high doses
of narigine shows increase open arm exploration and decrease locomotor
activity as shown from reduction in close arm eateries.
Naringin and has anxiolytic and sedative potential at high doses.
Naringin at dose 30mg/kg show a slight myorelaxant effect in the
horizontal wire test . It has been reported that naringin and
its derivatives are mediators of GABA receptors and are supportive
in relieving anxiety. This flavonoid also has some other targets
such as Human Ether-a-go-go-Related Gene (hERG) voltage-dependent
potassium channels and Inwardly Rectifying Potassium
Channels (GIRK). This flavonoid has positive modulating effect on
GIRK channels .
The present study demonstrated that the aqueous extract of
peeled Citrus reticulata possess dose-dependent anxiolytic activity.
Further, there is need to isolate, characterize, and screen the active
principles that are responsible for its anxiolytic activity. Furthermore,
there is need to find out the exact mechanism by which
the Citrus reticulata extract exerts above effects. Further studies
are needed to separate and confirm the active components and its
effect on anxiety.