Antianxiety and Antidepressive Effects of Essential Oils of Citrus Spp in Mice


1. Introduction				2.6 Ethical committee

Citrus is a genus of fruit-trees of the family				This study was approved by the USM Health
Rutaceae. It is Asiatic origin and are cultivated				Campus Animal Ethics Committee.
throughout the warmer parts of the world (5).
The trees are typically aromatic because of the				3. Results
presence of glands in the leaves, flowers and
fruits which produce essential oils. These oils are				3.1 The Force Swimming Test
extracted for use in perfumes, to flavour drinks
and used in confectionary. These oils are also				The results of parameters obtained are shown in
used in the production of organic chemicals (6). It				Figure 1 to Figure 3. Figure 1 represents group of
also utilised for culinary, ceremonial and medication				mice versus SR. Kruskal-Wallis test showed no
purposes amongsts the Malays and also been				significant differences between control group and
claimed to be a snake deterant and possess				treated groups of C. hystrix, C. microcarpa and C.
aesthetic properties as hair shampoo amongst				aurantifolia (p > 0.05). In Figure 2 (group of
Malay and Malenasians.This study is focused to the				mice versus TI), P value for Mann-Whitney U
3 members of the Citrus genus:Citrus hystrix				statistic test showed significant differences
(DC), Citrus microcarpa (Bunge) and Citrus				between treated group of C. aurantifolia
aurantifolia (Swingle) (also namedC. acida, Roxb.)				compared to the control group (p = 0.05). Thus,
(5).				C. aurantifolia shows antidepressive properties (p
				= 0.05). In Figure 3 (group of mice versus TL),
1.1 Citrus hystrix				Mann-Whitney U statistic test showed that C.
				aurantifolia has significant differences compared to
Citrus hystrix, locally known as ‘limau				the control group (* p = 0.05). C. aurantifolia
purut’, is a small tree, with a pear-shaped				shows antidepressive properties.
fruit, the skin intensely green, or ultimately upon
ripening yellowish and wrinkled. Before the fruit is				3.2 The Elevated Plus-maze Test
ripe the juice is gummy, but with ripeness
becomes thin and watery, though never abundant				The results of parameters obtained are shown in
(5). It has a characteristic sweet lemony smell				Figure 4 to Figure 9. Figure 4 represents group of
whereas the flowers are small, white in colour and				mice versus EOA. There were significant
sweet smelling. The leaves, about 7.5 to 10 cm				differences between the control group and
long, shiny and looks oily. The essential oil from				treated group of C. hystrix, C. microcarpa and C.
the leaves is a light yellowish green liquid.				aurantifolia (p < 0.05, Kruskal-Wallis test).
				Mann-Whitney U statistic test showed significant
1.2 Citrus microcarpa				differences when C. microcarpa was compared to
				the control group (* p < 0.05). Therefore C.
Normally C. microcarpa is consume as a refreshing				microcarpa shows antianxiety properties. Figure 5
fruit drink or they are commonly pickled with salt				represents group of mice versus TOA. There
and can be preserved with sugar or salt as dried				were significant differences between the control
fruits. This is a common plant grown in the home,				group and treated group of C. hystrix, C.
either as a potted plant or planted in the garden.				microcarpa and C. aurantifolia (p < 0.05,
It grows to about 3 to 4 m tall. Immature fruits				Kruskal-Wallis statistic test). P value for
are green but turn yellow or orangy when ripe.				Mann-Whitney U statistic test showed significant
The fruits are very small compared to other				differences between treated group of C. hystrix
Citrus. It is roundish (about 2.5 to 3.7 cm across)				compared to the control group (* p = 0.05). C.
with a smooth, shiny, thin skin. They are very				hystrix shows antianxiety properties. In Figure 6
juicy but sour (7). C. microcarpa has a peculiar				(group of mice versus EEA), P value for
musky fragrance which gives it its name.				Kruskal-Wallis statistic test showed no significant
				differences between the control group and
1.3 Citrus aurantifolia				treated group of C. hystrix, C. microcarpa and C.
				aurantifolia (p > 0.05). In Figure 7 (group of
It is normally consume as a fruit juice and used				mice versus TEA), P value for Mann-Whitney U
for cooking and garnishing. Leaves are traditionally				statistic test showed significant differences of C.
used for poulticing, both against evil spirits and for				hystrix compared to the control group (* p =
skin complaints. It grows to about 5 m tall. In the				0.05). C. hystrix shows antianxiety properties.
early years, it is a thorny shrub. Flowers are				Figure 8 represents group of mice versus TRA. P
about 1 inch across with 4 waxy petals (8). The				value for Kruskal-Wallis statistic test showed no
fruits are roundish, about 2.5 to 5.0 cm in				significant differences between treated group and
diameter, green when immature but turning				the control group (p > 0.05). Figure 9
yellow at maturity and very juicy but sour. The				represents group of mice versus TS. P value for
skin is shiny and smooth, about 0.3 to 1.2 mm				Kruskal-Wallis statistic test showed no significant
thick (7). Citrus aurantifolia is really common and				differences between all groups (p > 0.05).
many varieties are available and can be found
throughout the country.				4. Discussion

2. Methods and Materials				Porsolt et al (9) observed that rats or mice in the
				Behavioral Despair test or the Forced Swimming
2.1 Essential Oil				test, when forced to swim in a restricted space
				from which they cannot escape, cease to
Essential oil of C. hystrix, C. microcarpa and C.				struggle and quickly maintain a characteristic
aurantifolia were distilled from the leaves by				immobile posture. They correlated this despair
vapour distillation method where distillation from 3				behavior, expressed as immobility to clinical state
kg of leaves produces 0.4 to 0.7 ml of essential				of mental depression (11). When antidepressants
oil.				were given, its reduces the immobility of the rats
				or mice and this suggested that the test is
2.2 Animals				usefulness in the screening for anti-depressive
				substance (10). In our study, for time in immobility
Male Swiss albino mice weighing between 22 to 26				(SR), P value fot statistic test Mann-Whitney U
g were used. Animals were bred at the USM				showed significant differences between treated
animal house and keep in controlled conditions				group of C. aurantifolia (p = 0.05) and for time in
(light or dark cycle and temparature at 22°C)				latency (TL), Mann-Whitney U statistic test
with food and water ad libitum.				showed that C. aurantifolia has significant
				differences compared to the control (* p = 0.05).
2.3 Method				These results taken together indicate that C.
				aurantifolia appears to show an anti-depressive
Mice were divided into 4 groups (6 mice per				effect. The decrease in time of immobility is the
group). One group act as the control and the				positive results in screening anti-depressive using
others were treated with Citrus spp essential oils.				the Behavioral Despair test. The results using BDT
For the treated group, 0.1 ml essential oils given				shows C. aurantifolia has decreased for time in
over 30 minutes in standardised animal container				immobility and time of latency.
by vapourising the oil inside it. The container is a
perspex container 22 cm x 12 cm x 15 cm				The Elevated Plus-maze test was developed from
fabricated at our laboratory. All groups undergo				work of Montgomery (13). Pellow et al and
the Forced Swimming Test (antidepression) and				co-workers (14) performed an extensive series
Elevated Plus-maze test (anxiety). For the control				of studies validating the procedure as an animal
group, each mouse is given 0.1 ml distilled water				model of anxiety in rats and Lister et al (15) had
by the same method.				validated the test in mice. Preferences to stay
				the open arm indicate the anxiolytic effect and
2.4 The Tests				test substances influency a longer stay in the
				open arm suggest anxiolytic properties. The
2.4.1 The Force Swimming Test				results using the Elevated Plus-maze test shows
				C. hystrix has preferences to spent time in open
This test is popularly used in screening				arm and did not prefer to spent time in the
anti-depressive effect. It is an animal depression				enclosed arm. C. microcarpa treated mice have
model developed by Porsolts et al (9), validated				preferences to entry into the open arm. Essential
by Nomura et al (10) and Sunal et al (11), adepted				oils of C. hystrix and C. microcarpa have
by Abdul Razak et al (12). The Force Swimming				antianxiety where C. hystrix has more effect
Test is readily accepted as the standard test with				compared to C.microcarpa. C. hystrix and C.
a degree of sensitivity and reability for the				microcarpa show anxiolytic properties based on
screening of anxiolytic and anxiogenic subtances.				the results obtained.
The apparatus is a cylindrical chamber made of
glass with 25.3 cm diameter and 35.5 cm height.				Conclusion
The height of water in chamber is 23.7 cm and it
is at room temperature. The test is single blinded				The present study provide some evidence to
and conducted within a period of 15 minutes.				indicate that the smelling of essential oils (0.1 ml)
Recordings and/or observation start on placement				of C. hystrix and C. microcarpa confer anxiolytic
of the mouse into the chamber.				effect, while C. hystrix shows stronger anxiolytic
				effect, and C. aurantifolia showed antidepressive
The parameters as follows:				effect. It can be concluded that essential oils of
				the Citrus family may affect behavior. However,
1. Number of rounds of swimming within a 3				the mechanism of actions of these essential oils
minutes duration (SR).				are not known. Further studies will needed to
				explore in the possible mechanism of action.
2. Time in immobility (total time in state immobility				These will require neurological test and related
at 3rd, 4th, 5th and 6th minute (TI).				test to ascertain the effects observed in this
				study.
3. Time of latency ( time to reach complete
immobility within 15 minutes (TL).				Acknowledgement

Summary of parametric indication:				I would like to thank Mr. Abdul Razak Ahmad, Mr.
				Norman Mukiar, Mr. Rosliza Haron, Mr. Mohd.
1. SR if increases, it shows antidepressive effect.				Aminorddin Darus Mohd. Noor, Mrs. Halijah Miran,
				Mrs. Norzihana Ramli for their technical assistance
2. TI if decreases, it shows antidepressive effect.				and to all friends at Laboratory of the
				Department of Pharmacology, USM Medical School.
3. TL if decreases, it shows antidepressive effect.				References

2.4.2 The Elevated Plus-maze Test				1. Azhar Md. Zain dan Syed Mohsin Syed Sahil
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This test is a standard test with an acceptable				Plant-Derived Essential Oils in the Geller Type
degree of sensivity and reability for the screening				Conflict Test in Mice. Japan Journal Pharmacology.
of anxiolytic and anxiolgenic substances. Some				83(2): 150.
modification has been made in the mode of
recording the parameters and in the apparatus by				3. Ilmberger J., Heuberger E., Mahrhofer C.,
adopting recommendations of Lister (15) and Cruz				Dessovic H., Kowarik D., Buchbauer G. (2001). The
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sets of arms, the open and the enclosed arm
opposing each other. The test were conducted				4. Che Rugayah Che Awang dan Syed Mohsin
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start upon placement of the mouse at the centre				of Citrus hystric on the Blood Pressure and Heart
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The parameters measured as follows:				5. Burkill R.H. and Watson J.G. (1966). A Dictionary
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1. Number of entries into the open arm (EOA) and				Jilid 1 (A - H). Ministry of Agriculture and
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mouse are in the open arm.				Malaysia. 4(9). 8,10.

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enclosed arm.				despair in mice: A primary screening test for
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5. Time scanning (TS). Time when the mouse is in				327-336.
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1. EOA if increases, it shows antianxiety effect.
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4. TEA if decreases, it did not show antianxiety				12. Abdul Razak Kasmuri, Md. Lukmi Ismail, Siti
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2.5 Statistical analysis				13. Montgomery K.C. (1955). The relation between
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Statistical Package for Social Sciences (SPSS)				behavior. J. Compr. Physiol. Psychol. 48: 254-260.
Statistical Sofeware (version 11.0, SPSS) was
used for the analysis of data in this study. The				14. Pellow S., Chopin P., File S., Briley M. (1985).
normality of each variable was tested by				Validation of open: closed arm entries in an
normality test and the homogeneity by				elevated plus-maze as a measure of anxiety in
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data were analyzed by using non-parametric
statistic programme because most of the data				15. Lister R.G. (1987). The use of a plus-maze to
were not normally distribution and were not				measure anxiety in the mouse.
homogenous. Kruskal-Wallis stastic test were used				Psychopharmacology. 92: 180-185.
to compare median (IR) of data for more than
two groups and if significant, it is followed by				16. Cruz A.P.M., Frei F., Graeff F.G. (1994).
Mann-Whitney U test in order to determined the				Ethopharmacological analysis of rat behavior on
differences between groups. Level of significance				the Elevated Plus-maze. Pharmacology
was set at 0.05 and p value < 0.05 was				Biochemistry and Behavior. 49 (1): 171-176.
accepted as significant.

Rubicon Med

Antianxiety and Antidepressive Effects of Essential Oils of Citrus Spp in Mice