1. Introduction

Citrus is a genus of fruit-trees of the family Rutaceae. It is Asiatic origin and are cultivated throughout the warmer parts of the world (5). The trees are typically aromatic because of the presence of glands in the leaves, flowers and fruits which produce essential oils. These oils are extracted for use in perfumes, to flavour drinks and used in confectionary. These oils are also used in the production of organic chemicals (6). It also utilised for culinary, ceremonial and medication purposes amongsts the Malays and also been claimed to be a snake deterant and possess aesthetic properties as hair shampoo amongst Malay and Malenasians.This study is focused to the 3 members of the Citrus genus:Citrus hystrix (DC), Citrus microcarpa (Bunge) and Citrus aurantifolia (Swingle) (also namedC. acida, Roxb.) (5).

1.1 Citrus hystrix

Citrus hystrix, locally known as ‘limau purut’, is a small tree, with a pear-shaped fruit, the skin intensely green, or ultimately upon ripening yellowish and wrinkled. Before the fruit is ripe the juice is gummy, but with ripeness becomes thin and watery, though never abundant (5). It has a characteristic sweet lemony smell whereas the flowers are small, white in colour and sweet smelling. The leaves, about 7.5 to 10 cm long, shiny and looks oily. The essential oil from the leaves is a light yellowish green liquid.

1.2 Citrus microcarpa

Normally C. microcarpa is consume as a refreshing fruit drink or they are commonly pickled with salt and can be preserved with sugar or salt as dried fruits. This is a common plant grown in the home, either as a potted plant or planted in the garden. It grows to about 3 to 4 m tall. Immature fruits are green but turn yellow or orangy when ripe. The fruits are very small compared to other Citrus. It is roundish (about 2.5 to 3.7 cm across) with a smooth, shiny, thin skin. They are very juicy but sour (7). C. microcarpa has a peculiar musky fragrance which gives it its name.

1.3 Citrus aurantifolia

It is normally consume as a fruit juice and used for cooking and garnishing. Leaves are traditionally used for poulticing, both against evil spirits and for skin complaints. It grows to about 5 m tall. In the early years, it is a thorny shrub. Flowers are about 1 inch across with 4 waxy petals (8). The fruits are roundish, about 2.5 to 5.0 cm in diameter, green when immature but turning yellow at maturity and very juicy but sour. The skin is shiny and smooth, about 0.3 to 1.2 mm thick (7). Citrus aurantifolia is really common and many varieties are available and can be found throughout the country.

2. Methods and Materials

2.1 Essential Oil

Essential oil of C. hystrix, C. microcarpa and C. aurantifolia were distilled from the leaves by vapour distillation method where distillation from 3 kg of leaves produces 0.4 to 0.7 ml of essential oil.

2.2 Animals

Male Swiss albino mice weighing between 22 to 26 g were used. Animals were bred at the USM animal house and keep in controlled conditions (light or dark cycle and temparature at 22°C) with food and water ad libitum.

2.3 Method

Mice were divided into 4 groups (6 mice per group). One group act as the control and the others were treated with Citrus spp essential oils. For the treated group, 0.1 ml essential oils given over 30 minutes in standardised animal container by vapourising the oil inside it. The container is a perspex container 22 cm x 12 cm x 15 cm fabricated at our laboratory. All groups undergo the Forced Swimming Test (antidepression) and Elevated Plus-maze test (anxiety). For the control group, each mouse is given 0.1 ml distilled water by the same method.

2.4 The Tests

2.4.1 The Force Swimming Test

This test is popularly used in screening anti-depressive effect. It is an animal depression model developed by Porsolts et al (9), validated by Nomura et al (10) and Sunal et al (11), adepted by Abdul Razak et al (12). The Force Swimming Test is readily accepted as the standard test with a degree of sensitivity and reability for the screening of anxiolytic and anxiogenic subtances. The apparatus is a cylindrical chamber made of glass with 25.3 cm diameter and 35.5 cm height. The height of water in chamber is 23.7 cm and it is at room temperature. The test is single blinded and conducted within a period of 15 minutes. Recordings and/or observation start on placement of the mouse into the chamber.

The parameters as follows:

1. Number of rounds of swimming within a 3 minutes duration (SR).

2. Time in immobility (total time in state immobility at 3rd, 4th, 5th and 6th minute (TI).

3. Time of latency ( time to reach complete immobility within 15 minutes (TL).

Summary of parametric indication:

1. SR if increases, it shows antidepressive effect.

2. TI if decreases, it shows antidepressive effect.

3. TL if decreases, it shows antidepressive effect.

2.4.2 The Elevated Plus-maze Test

This test is used in the screening of anxiolytic effects of drugs. It is an animal anxiety model developed by Montgomery (13), validated by Pellow et al (14), Lister (15) and Cruz et al (16). This test is a standard test with an acceptable degree of sensivity and reability for the screening of anxiolytic and anxiolgenic substances. Some modification has been made in the mode of recording the parameters and in the apparatus by adopting recommendations of Lister (15) and Cruz et al (16) by Abdul Razak et al (12). The apparatus is made of black perspex with two sets of arms, the open and the enclosed arm opposing each other. The test were conducted within 5 minutes and recording and/or observation start upon placement of the mouse at the centre of the plus-maze facing forward of the enclosed arm.

The parameters measured as follows:

1. Number of entries into the open arm (EOA) and enclosed arm (EEA).

2. Time spent in open arm (TOA). Mouse is considered in open arm when all four limbs of the mouse are in the open arm.

3. Time spent in the enclosed arm (TEA). Mouse is considered in enclosed arm when all of four limbs of the mouse are in the enclosed arm.

4. Time of risk assessment (TRA). Recording starts when the mouse in the enclosed arm starts to protrude its face at the entrance until all four limbs are in the open or retreat back to the enclosed arm.

5. Time scanning (TS). Time when the mouse is in the open arm while scanning the edge of open arm.

Summary of parametric indication:

1. EOA if increases, it shows antianxiety effect.

2. TOA if increases, it shows antianxiety effect.

3. EEA if decreases, it shows antianxiety effect.

4. TEA if decreases, it did not show antianxiety effect.

5. TS if increases, it shows antianxiety effect.

6. TRA if increases, it shows antianxiety effect.

2.5 Statistical analysis

Statistical Package for Social Sciences (SPSS) Statistical Sofeware (version 11.0, SPSS) was used for the analysis of data in this study. The normality of each variable was tested by normality test and the homogeneity by homogeneity varian test (Levene’s test). All data were analyzed by using non-parametric statistic programme because most of the data were not normally distribution and were not homogenous. Kruskal-Wallis stastic test were used to compare median (IR) of data for more than two groups and if significant, it is followed by Mann-Whitney U test in order to determined the differences between groups. Level of significance was set at 0.05 and p value < 0.05 was accepted as significant.

2.6 Ethical committee

This study was approved by the USM Health Campus Animal Ethics Committee.

3. Results

3.1 The Force Swimming Test

The results of parameters obtained are shown in Figure 1 to Figure 3. Figure 1 represents group of mice versus SR. Kruskal-Wallis test showed no significant differences between control group and treated groups of C. hystrix, C. microcarpa and C. aurantifolia (p > 0.05). In Figure 2 (group of mice versus TI), P value for Mann-Whitney U statistic test showed significant differences between treated group of C. aurantifolia compared to the control group (p = 0.05). Thus, C. aurantifolia shows antidepressive properties (p = 0.05). In Figure 3 (group of mice versus TL), Mann-Whitney U statistic test showed that C. aurantifolia has significant differences compared to the control group (* p = 0.05). C. aurantifolia shows antidepressive properties.

3.2 The Elevated Plus-maze Test

The results of parameters obtained are shown in Figure 4 to Figure 9. Figure 4 represents group of mice versus EOA. There were significant differences between the control group and treated group of C. hystrix, C. microcarpa and C. aurantifolia (p < 0.05, Kruskal-Wallis test). Mann-Whitney U statistic test showed significant differences when C. microcarpa was compared to the control group (* p < 0.05). Therefore C. microcarpa shows antianxiety properties. Figure 5 represents group of mice versus TOA. There were significant differences between the control group and treated group of C. hystrix, C. microcarpa and C. aurantifolia (p < 0.05, Kruskal-Wallis statistic test). P value for Mann-Whitney U statistic test showed significant differences between treated group of C. hystrix compared to the control group (* p = 0.05). C. hystrix shows antianxiety properties. In Figure 6 (group of mice versus EEA), P value for Kruskal-Wallis statistic test showed no significant differences between the control group and treated group of C. hystrix, C. microcarpa and C. aurantifolia (p > 0.05). In Figure 7 (group of mice versus TEA), P value for Mann-Whitney U statistic test showed significant differences of C. hystrix compared to the control group (* p = 0.05). C. hystrix shows antianxiety properties. Figure 8 represents group of mice versus TRA. P value for Kruskal-Wallis statistic test showed no significant differences between treated group and the control group (p > 0.05). Figure 9 represents group of mice versus TS. P value for Kruskal-Wallis statistic test showed no significant differences between all groups (p > 0.05).

4. Discussion

Porsolt et al (9) observed that rats or mice in the Behavioral Despair test or the Forced Swimming test, when forced to swim in a restricted space from which they cannot escape, cease to struggle and quickly maintain a characteristic immobile posture. They correlated this despair behavior, expressed as immobility to clinical state of mental depression (11). When antidepressants were given, its reduces the immobility of the rats or mice and this suggested that the test is usefulness in the screening for anti-depressive substance (10). In our study, for time in immobility (SR), P value fot statistic test Mann-Whitney U showed significant differences between treated group of C. aurantifolia (p = 0.05) and for time in latency (TL), Mann-Whitney U statistic test showed that C. aurantifolia has significant differences compared to the control (* p = 0.05). These results taken together indicate that C. aurantifolia appears to show an anti-depressive effect. The decrease in time of immobility is the positive results in screening anti-depressive using the Behavioral Despair test. The results using BDT shows C. aurantifolia has decreased for time in immobility and time of latency.

The Elevated Plus-maze test was developed from work of Montgomery (13). Pellow et al and co-workers (14) performed an extensive series of studies validating the procedure as an animal model of anxiety in rats and Lister et al (15) had validated the test in mice. Preferences to stay the open arm indicate the anxiolytic effect and test substances influency a longer stay in the open arm suggest anxiolytic properties. The results using the Elevated Plus-maze test shows C. hystrix has preferences to spent time in open arm and did not prefer to spent time in the enclosed arm. C. microcarpa treated mice have preferences to entry into the open arm. Essential oils of C. hystrix and C. microcarpa have antianxiety where C. hystrix has more effect compared to C.microcarpa. C. hystrix and C. microcarpa show anxiolytic properties based on the results obtained.

Conclusion

The present study provide some evidence to indicate that the smelling of essential oils (0.1 ml) of C. hystrix and C. microcarpa confer anxiolytic effect, while C. hystrix shows stronger anxiolytic effect, and C. aurantifolia showed antidepressive effect. It can be concluded that essential oils of the Citrus family may affect behavior. However, the mechanism of actions of these essential oils are not known. Further studies will needed to explore in the possible mechanism of action. These will require neurological test and related test to ascertain the effects observed in this study.

Acknowledgement

I would like to thank Mr. Abdul Razak Ahmad, Mr. Norman Mukiar, Mr. Rosliza Haron, Mr. Mohd. Aminorddin Darus Mohd. Noor, Mrs. Halijah Miran, Mrs. Norzihana Ramli for their technical assistance and to all friends at Laboratory of the Department of Pharmacology, USM Medical School.

References

1. Azhar Md. Zain dan Syed Mohsin Syed Sahil Jamalullail (2003). Effect of Taking Chicken Essence on Stress and Cognition of Human Volunteers. Malaysian Journal Nutrition. 9(1): 19.

2. Umezu T.(2000). Behavioral Effects of Plant-Derived Essential Oils in the Geller Type Conflict Test in Mice. Japan Journal Pharmacology. 83(2): 150.

3. Ilmberger J., Heuberger E., Mahrhofer C., Dessovic H., Kowarik D., Buchbauer G. (2001). The influence of Essential Oils on Human Attention. I: Alertness. Chemistry Senses. 26(3): 239.

4. Che Rugayah Che Awang dan Syed Mohsin Syed Sahil Jamalullail (2001). Effects of Essential Oil of Citrus hystric on the Blood Pressure and Heart Rate of Anaesthetised Rats. Asia Pacific Journal of Pharmacology. Volume 15(2): S50.

5. Burkill R.H. and Watson J.G. (1966). A Dictionary of the Economic Products of the Malay Peninsula. Jilid 1 (A – H). Ministry of Agriculture and Co-operatives, Kuala Lumpur.568, 575.

6. Jones D.T. (1984). Citrus In Malaysia. Nature Malaysiana. Tropical Press Sdn. Bhd., Kuala Lumpur, Malaysia. 4(9). 8,10.

7. Hoong Fong C. dan Hoi-Sen Y. (1982). Malaysian Fruit in Colour. Tropikal Press Sdn. Bhd., Kuala Lumpur, Malaysia. 46-47.

8. Allen B.M. (1975). Common Malaysian Fruits. Longman Malaysia Sdn. Bhd., Kuala Lumpur. 36.

9. Porsolt R.D., Bertin A., Jalfre (1977). Behavioral despair in mice: A primary screening test for antidepressants. Arch. Int. Pharmacodyn. 229: 327-336.

10. Nomura S., Shimizu J., Kinjo M., Kametani H., Nakazawa T. (1982). A new behavioral test for antidepressant drugs. European Journal of Pharmacology. 83: 171-175.

11. Sunal R., Gumusel B., Kayaalp S.O. (1994). Effect of changes in swimming area on results of ‘Behavioral Despair Test’. Pharmacology Biochemistry and Behavior. 49(4): 891-896.

12. Abdul Razak Kasmuri, Md. Lukmi Ismail, Siti Amrah Sulaiman, Syed Mohsin Sahil Jamalullail (1998). Psychopharmacological aspect of post partum jamu. Program and abstract of the 14th scientific meeting of Malaysian Natural Products Society.

13. Montgomery K.C. (1955). The relation between fear induced by novel stimulation and exploratory behavior. J. Compr. Physiol. Psychol. 48: 254-260.

14. Pellow S., Chopin P., File S., Briley M. (1985). Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Meth. 14: 149-167.

15. Lister R.G. (1987). The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology. 92: 180-185.

16. Cruz A.P.M., Frei F., Graeff F.G. (1994). Ethopharmacological analysis of rat behavior on the Elevated Plus-maze. Pharmacology Biochemistry and Behavior. 49 (1): 171-176.

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CHE RUGAYAH BT. CHE AWANG
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