Antinociceptive Effects of the Essential Oil of Croton Zehntneri in Mice

Croton zehntneri is an aromatic plant native to Northeastern Brazil, where it is often used in folk medicine. In the present study the antinociceptive effects of the essential oil of Croton zehntneri (EOCz) were evaluated in mice. EOCz administered orally at doses of 100 and 300 mg/kg reduced paw licking time in the second phase of the formalin test from the control value of 41.61 ± 8.62 to 12.01 ± 7.97 and 6.57 ± 3.42 s, respectively. During the first phase of the formalin test only 300 mg/kg induced a significant alteration (from 58.2 ± 7.02, control, to 28.7 ± 4.73 s). The number of contortions in response to intraperitoneal injections of acetic acid did not differ significantly between controls (80.6 ± 9.01) and experimental (300 mg/kg body weight) animals (89.1 ± 9.53% of the control numbers; P³0.05, Student t-test). In the hot-plate test, EOCz at doses ³ 100 mg/kg significantly increased the latency time with respect to controls (11.2 ± 0.80). At 100 and 300 mg/kg this increase persisted for 180 and 240 min, respectively. The data show that EOCz is effective as an antino-ciceptive agent.

• Croton zehntneri Croton zehntneri is an aromatic plant native to Northeastern Brazil, where it is popularly called "Canela de Cunhã" and used in folk medicine principally as a sedative, as an appetite-stimulating antianorexigen, and for the relief of gastrointestinal disturbances (1).Despite this, little pharmacological investigation has been carried out on the effects of C. zehntneri.
Published research on plant extracts has focused primarily on the central nervous system (2,3) and on muscle (4)(5)(6)(7).We have previously shown that the essential oil of C. zehntneri (EOCz) exerts antispasmodic effects on guinea pig-isolated ileum (4,6), pos-sibly explaining the use of C. zehntneri in the treatment of gastrointestinal problems.However, it is not currently known whether such effects of EOCz originate exclusively from antispasmodic activity or also due to the contribution of other effects.
Because many essential oils possess potent analgesic activity, in the present study we investigated whether EOCz possesses antinociceptive activity.The data suggest that EOCz also has an analgesic action.
C. zehntneri leaves were collected in September, 1998, near the city of Viçosa, MG, Brazil.The botanical identity of the plant was confirmed by Dr. Francisco J.Abreu Matos (Natural Products Laboratory, Universidade Federal de Ceará, UFC) and Dr. Raymond Harley (Royal Botanic Garden, Kew, UK).A voucher specimen (#27477) is deposited in the Prisco Bezerra herbarium (UFC).
EOCz was extracted from freshly chopped plant leaves by steam distillation and analyzed at the Natural Products Laboratory of UFC.Freshly chopped leaves were placed in a glass flask connected at one end to a glass vessel with water and at the other end to a water-cooled condenser.When the water was boiled, steam percolated through the leaves and was collected in the condenser.After condensation, the essential oil separated from the aqueous phase with its solutes.The composition (w/w) of EOCz, determined by gas chromatography and mass spectrometry, was 85.7% anethole, 4.8% estragole, 2.95% 1,8cineole, 2.2% ß-myrcene, 1.22% anisaldehyde, 0.9% trans-caryophyllene, and 2.23% unidentified.
EOCz solutions were prepared each day by vigorous manual shaking (3-5 min) or by vortexing EOCz in vehicle (1% Tween 80 in distilled water).EOCz was administered orally in a volume of 0.1 ml solution/10 g.
All experiments were conducted on male Swiss mice (25-30 g) deprived of food, but with free access to drinking water during the 12 h prior to experiments.EOCz solubilized in vehicle (1% Tween 80, aqueous solution) or vehicle only (control) was administered orally with an orogastric cannula, 60 min prior to nociceptive testing.Animals were kindly provided by the vivarium of UFC.The number of animals in each control and experimental group varied from 6 to 12.
The formalin test was done as described by Hunskaar et al. (8).Briefly, 20 µl of a 0.1% (v/v) aqueous formalin solution was injected into the plantar region of the right hind paw.The time that the animal spent licking the paw during the first 5 min (early phase) and from 20 to 25 min (late phase) post-injection served as a measure of sensitivity.The test was done at an ambient temperature of 22-26 o C and care was taken to exclude environmental disturbances that might interfere with animal response (9).
The writhing test was performed according to Koster et al. (10).Briefly, 0.1 ml/10 g body weight of an aqueous acetic acid solution (0.6%, v/v) was administered by intraperitoneal injection and the number of abdominal contortions was counted from 10 to 30 min post-injection.
The hot-plate test was done according to Carlini (11).Briefly, a mouse was placed on a plate maintained at 50.0 ± 0.5 o C and the latency of the reaction to this nociceptive stimulus (number of seconds before it started licking the hind paw or jumping) was quantified.Only mice that had responded in a pretest with a reaction time £20 s were employed in these experiments.Mice were tested every 30 min for up to 3 h starting 1 h after EOCz treatment.
Results are reported as mean ± SEM, with N indicating the number of animals.Values were analyzed using the Student ttest, ANOVA, or a nonparametric test as appropriate, and were considered to differ significantly at P£0.05.
In the formalin test, the number of seconds that control mice spent licking their paws in the first and second phases of the response to formalin injection was 58.2 ± 7.02 (N = 6) and 41.6 ± 8.62 (N = 6), respectively.EOCz significantly reduced this time (P£0.05,ANOVA, Dunn's test) in the second phase, at doses of 100 and 300 mg/kg to 12.01 ± 7.97 (N = 6) and 6.57 ± 3.42 (N = 10), respectively.During the first phase of the formalin test only 300 mg/kg induced a significant alteration (from 58.2 ± 7.02, control, to 28.7 ± 4.73 s, N = 10) (Figure 1).
In the acetic acid-induced writhing test, the number of contortions occurring in control mice was 80.6 ± 7.01 (N = 6).In animals that received 300 mg/kg EOCz the number of contortions (89.1 ± 9.53% of control, N = 6) was not significantly altered relative to controls (P³0.05,Student t-test).
EOCz showed an antinociceptive effect in the hot-plate test and in both phases of the formalin test, although its effect during the first phase of the formalin test was significant only with 300 mg/kg.EOCz also showed no effect in the acetic acid-induced writhing test.The hot-plate test results suggest that this analgesic agent acts primarily at the spinal cord and/or higher central nervous system levels, or by an indirect mechanism (12).The two phases of response to the formalin test have been attributed to different mechanisms (9,13,14).The first phase of the formalin test, not pronouncedly altered by EOCz, is attributed to peripheral mechanisms.The second phase, which is conspicuously affected by EOCz, is considered to be due to alteration of central processing.This agrees with the hypothesis of a central mechanism as the major causative factor for EOCz induction of antinociceptive effects.On the other hand, contortions induced by intraperitoneal injections of acetic acid are said to originate from the pain of inflammation mediated by prostaglandins (15)(16)(17).Experiments not included in the results of this study showed that 300 mg/kg EOCz did not alter mouse motor performance in the rotorod test.This effect and the lack of EOCz-induced effect in the writhing test suggest that doses lower than 300 mg/kg EOCz have no general depressant effect.
In the present study we did not attempt to elucidate the contribution of the known con-  In conclusion, EOCz is likely to be acting through a central nervous system mechanism or via an indirect, perhaps anti-inflammatory effect and the elucidation of its mechanism of action deserves further investigation.Unpublished experiments performed in this laboratory indicate that EOCz has an oral LD 50 >2.5 g/kg.Since EOCz displays an antinociceptive effect at doses well below the LD 50 , this effect is potentially useful in therapeutics, and this essential oil deserves further pharmacological investigation.

Figure 1 .
Figure 1.Effects of the essential oil of Croton zehntneri (EOCz) on nociceptive responses to intraplantar injection of formalin (1%).Ordinate values refer to the time (in s) mice spent licking the paw.Data are reported as means ± SEM (N = 6).*P<0.05 compared to control (ANOVA, Dunn's test).
it should be noted that at 300 mg/kg EOCz was effective during both phases of the formalin test, as expected for an agent acting on opioid receptors.It has also been demonstrated (19) that ß-myrcene-induced antinociception is partially blocked by naloxone.Further investigation is necessary to elucidate this effect.