INVESTIGATION OF ANTI-INFLAMMATORY ACTIVITY OF BERGAMOT OIL

Citrus aurantium subsp. bergamia is a member of the Rutaceae. In Turkish folk medicine, Citrus species have several uses such as for their appetizing, diaphoretic, antiseptic, analgesic and anti-inflammatory effects (1). The Bergamot oil (BO) is a roughly pear-shaped citrus fruit. The fruit is produced in Italy, East Africa, Ivory Coast, Argentina, Brazil, Turkey and some other Mediterranean countries. The fruit is sour, and its aromatic peel is used to produce an essential oil that is used in Earl Grey tea, in perfumery, in candy-making (2). It is known empirically that inhalation of essential oils causes physiological and psychological changes in humans (3). Graham et al reported that aromatherapy during radiotherapy with bergamot oil and some other kinds of plant oil was not beneficial (4). The BO oil has been linked to several negative side-effects such as photosensitivity (5-7). The essential oil of BO is extracted from the peel of the fruit by a cold-pressing procedure or steam distillation. It consists of a volatile fraction, whose main components are, in approximate percentages, limonene (40%), linalool (8%), linalyl acetate INVESTIGATION OF ANTI-INFLAMMATORY ACTIVITY OF BERGAMOT OIL

In this study, essential oil extract of Bergamot oil was investigated for its antiinflammatory activity using carrageenaninduced rat paw oedema test (14).

Preparation of BO essential oil extract
Citrus aurantium L. var bergamia was collected in February, 2006 near Antalya (Turkey) and voucher specimens are kept in Yuzuncu Yil University, Faculty of Medicine (Specimen Nr: B-14).
The plant samples were kept at room temperature until they were processed.The fruits were peeled and the fresh pericarp of the fruit was sliced into small pieces and boiled in Clevenger apparatus (Ildam, TURKEY) to obtain essential oil of the Citrus aurantium var.bergamia (BO).BO was kept in test tubes and the yield was determined as 0.7%.

Drugs and chemicals
The following drugs were used; Lambda-Carrageenan and indomethacin (Sigma, Steinheim, Germany).Indomethacin was dissolved in ethyl alcohol (96 %).

Animals
Male and female Sprague-Dawley rats (180-220 g) were used in these experiments.The animals were housed in standard cages (48 cm x 35 cm x 22 cm) with food and water ad libitum, at room temperature (20 ± 2 0C) with artificial light from 7.00 am to 7.00 pm.The animals kept under controlled environment following the standard operating procedures of the animal house facility of the Faculty of Medicine, and provided with pelleted food Animal Feed Factory, Van-Turkey).The protocol for the study was approved of Yüzüncü Yıl University, Faculty of Medicine Animal Breeding and Research.Prior to administration of the drugs, the rats were fasted for 12 h with free access to tap water.

Carrageenan-induced rat paw oedema
Thirty-six rats of either sex were divided into six groups of six animals each.Group I, which served as control-I, received 0.2 mL isotonic saline solution (ISS), Group II, which served as control-II, received 0.2 mL ethanol.Group III received 3 mg/kg,indomethacin (14), group IV received 0.025 mL/kg BO,.Group V received 0.05 mL/kg BO.Group VI received 0.100 mL/kg BO.All agents were injected intraperitoneally.
The inhibitory activity of BO on carrageenan-induced rat-paw oedema was determined according to the method of Winter et al, with slight modification (15).The drugs were administered 1 h before the injection of 0.05 mL of carrageenan (1%) into the subcutaneous tissue of right hind paw.Since the hydration state of the animals can modify the intensity of swelling, the rats were fasted 12 h before the experiment with water ad libitum.The degree of oedema was measured 30 minutes before and 3 h after the injection of carrageenan.The difference between the volume of the paw before and after the injection of the oedema provoking agent indicated the severity of oedema.Volumes of right hind paw of the animals were measured with a plethysmometer (model 7140, Ugo Basile, Italy).
The percentage inhibition of the inflammatory reaction was determined for each animal by comparing with controls and calculated by the formula (16): x 100 where dt is the difference in paw volume in the drug-treated group and dc the difference in paw volume in the control group.

Statistical analysis
All data were represented as mean ± standard error of mean (SEM) or as percentages.The analysis of variance (ANOVA) was used for the statistical analysis of data.LSD test (Least significant difference test) was used for determining significance.Results with p<0.05 were considered as statistically significant.

RESULTS
Table 1 shows the results on antioedematous effect of intraperitoneally administered Bergamot oil on carrageenan paw oedema in rats.At doses of 0.025, 0.05 and 0.100 mL/kg BO caused a significant reduction in paw oedema (27.56%, 30.77% and 63.93%, respectively).In this model, indomethacin, a commonly used anti-inflammatory drug, produced a significant inhibition by 95.70%.As seen in Table 1, all doses of BO showed anti-inflamatory activity higher than that of the control groups, but it was not as strong as indomethacin.Anti-inflammatory activity of 0.100 mL/kg BO was stronger than those of 0.025 and 0.05 mL/kg BO.
Anti-inflammatory effect of Bergamot oil shown in this study confirms its use as an anti-inflammatory agent in traditional medicine.Median effective dose (ED50) value of BO was found to be 0.079 mL/kg.It was reported that main components of the volatile fraction of BO essential oil are limonene, linalool, linalyl acetate, bergapten, citropten, bergamottin, gamma-terpinen, alpha-pinene and beta-pinene (2,8).Antiinflammatory effect of BO essential oil could be due to one or several of these components.Linalool, linalyl acetate, limonene and alpha-pinene were shown to have strong antiinflammatory effects (11)(12)(13), which supports the results obtained in the current study.More studies are needed to show the mechanisms of the anti-inflammatory effects of these agents.Bergapten, citropten, bergamottin, gammaterpinen and beta-pinene, which are also found in BO essential oil, should be further studied for their anti-inflammatory effects.
The results of the present study showed that BO had anti-inflammatory effect and provided support to the traditional usage of BO in inflammation.