Eugenol: Antipyretic activity in rabbits

Neur~pha?mac~log~ Vol. 26, No. Printed in Great Britain

~28-3~8/87 $3.00+ 0.00

12, pp. 1?7>1778, I987

EUGENOL:

Pergamon

ANTIPYRETIC

ACTIVITY

Journals

Ltd

IN RABBITS

JIAIX FENG* and J. M. LIPTON Physiology and Anesthesiology Departments, University of Texas Health Science Center at Dallas, Dallas, Texas 75235, U.S.A.

Summary-The analgesic agent eugenol reduced fever when given intravenously in small doses to rabbits made febrile by interleukin I. To test this observation further, the drug was given intragastrically and its antipyretic effect was compared with that of a molar equivalent dose of a~taminophen. Eugenol was more effective in reducing fever than acetaminophen. Tests with intracerebrovent~cular injections indicated that eugenol could act centrally to reduce fever in doses much smaller than those which were effective peripherally. Other tests with the presumed fever mediators sodium archidonate and prostaglandin E, (PGE,), suggest that the central action of eugenol is similar to that of acetaminophen. Eugenol, a derivative of clove oil, has marked antipyretic activity when given intravenously, intragastrically and centrally and may reduce fever primarily through a centraI action that is similar to that of common antipyretic drugs such as a~taminophen. Key words: eugenol, antipyretic drugs, fever, interleukin-1, CNS, antipyretic action.

Eugenol, used as an analgesic agent in dentistry, is reported to have antiseptic and anodyne activities (Dobbs, 196 1; Kay, 1972) as well as myorelaxant and anticonvulsant actions (Dallmeier and Carlini, 1981; Engelbrecht, Long, Nichols and Barkfnecht, 1972). It is also one of the main ~mponents of Bupi~ur~m (Hubei Provincial Institute of Pharmaceutical Industry, 1982), a medicinal herb used in China to treat febrile diseases. There is strong evidence that eugenol inhibits the synthesis of prostaglandins (Dewhirst and Goodson, 1974) and that it reduces vasocons~ctor responses to norepinephrine, histamine and stimulation of peri-arterial sympathetic nerves, responses that are associated with pain (Hume, 1983). The effects of intravenous and intracerebroventricular injections of eugenol were examined in rabbits made febrile by interleukin 1, (IL-l) and compared the antipyretic effects of intragastric administration of single equimolar doses of eugenol and acetaminophen. The results indicate that eugenol has significant antipyretic activity and they suggest a possible mechanism of action.

Surgery The animals were pretreated with 40mg/kg ketamine hydrochloride (Vetalar, ParkeDavis) and 4 mg/kg promazine (Acepromazine, Ayerst) and anesthesia was induced and maintained by inhalation of methoxyflurane (Metofane, P&man-Moore, Inc.) and a N&I-Oz mixture. Rabbits were placed in a modified Kopf rabbit stereotaxic instrument (Crawford, Kennedy and Lipton, 1977; Lipton and Romans, 1976), equipped for gaseous anesthesia and a stainless-steel injection cannula (David Kopf Instruments, no. 201) was implanted into a lateral cerebral ventricle (co-ordinates: 0 mm anterior to bregma, 4 mm lateral to the midline, 6 mm below the dura). Stainless-steel screws and dental acrylic were used to anchor the cannula to the calvarium. A postoperative period of at least 7 days elapsed prior to the first experiment. During this period penicillin G (150,000 U, i.m., Crysticillin, Squibb Laboratories) was given to each rabbit daily for 6 days.

SOLUTIONS AND INJECTIONS

METHODS

Animals Twenty-four adult male New Zealand white rabbits, weighing 34 kg, were used. Each animal was individually caged in a 22 + 1°C environment with food and water available ad libitum except during the period of experimentation. Lights were on 12 hr per day and all experiments were run during the light phase. *Present address: University of Illinois, Champaign, Illinois, U.S.A.

Eugenol (2-methoxy-4-(2-propenyl) phenol); C,,,H,,O,; mol. wt 164.2; Sigma Chemical Company) was suspended in non-pyrogenic, isotonic saline (Abbott Laboratories). The dose differed with the aims of the experiment: 7.6 x 10e3 mmol for intracerebroventricular injections; 1.3 mmol or 0.33 mmol pr kg for intragastric administration; three different doses (1.5 x lo-*, 3 x lo-‘, 6 x lo-* mmol/kg) for the intravenous tests. Interleukin 1, prepared from leucocytes from the rabbit, incubated with Salmoneiia typhosa endotoxin (Difco, no. 0901), using a method reported previously

1775

JIADI FENG and J. M. LIPTON

1776

(Glyn-Ballinger, Bernardini and Lipton, 1983), was injected intravenously in a dose selected to cause a 0.8-1.2”C rise in temperature. A given rabbit always received the same volume from the same batch of pyrogen. Acetaminophen (Eastman Organic Chemicals) and sodium arachidonate (Sigma Chemical Co.) were also dissolved in saline. The former was given by gavage as stated above; the latter was injected intracerebroventricularly. Prostaglandin E, (Sigma Chemical Co.) was initially dissolved in ethanol and stored at 4°C. The ethanol was evaporated with a stream of nitrogen gas and the prostaglandin was redissolved in saline immediately prior to injection. The volume for this and other intracerebroventricular injections was 100 ~1. The doses of prostaglandin E, (1 pg) and arachidonate (1OOpg) were chosen so that the resulting hyperthermia would be comparable, in maximum increase and duration, to the effect of interleukin 1.

prostaglandin or arachidonate (Clark and Cumby, 1975), and this index was used in statistical tests of differences (ANOVA, t-test with Bonferroni correction for individual comparisons).

GENERAL PROCEDURES

RESULTS

The animals were divided into 4 groups of 6 rabbits, 1 group for each test: a test of the effects of the intravenous administration of eugenol on fever caused by interleukin 1; a test of the effects of intragastric administration of eugenol and acetaminophen on fever; a test of the influence of eugenol, given intravenously, on fever induced by the intracerebroventricular administration of prostaglandin E, or sodium arachidonate; a test of the influence of the central injection of eugenol on fever induced by interleukin- 1. The animals were restrained in conventional holders and placed in a temperature-controlled chamber maintained at 22 f 1°C. A thermistor probe (Yellow Springs International, No. 701) was inserted approximately 10 cm into the rectum and taped to the tail. Measurements of temperature were made automatically at 10 min intervals through a digital temperature recorder (Datalogger, United Systems, Inc.) connected to a MINC 11 on-line computer (Digital Electronics Corporation). Baseline measurements of temperature were recorded for at least 1 hr prior to the injection of interleukin 1, prostaglandin E, or sodium arachidonate. A marginal ear vein was used for the intravenous injection of interleukin 1 or eugenol. A feeding tube (Bardic Feeding Tube, C. R. Bard, Inc.) was used for the intragastric administration of eugenol or acetaminophen, given 30min after interleukin 1, during the chill phase of the febrile response. All injections of pyrogen were separated by at least 72 hr. In each series of experiments a randomized crossover design was used so that each animal served as its own control. The thermal response index, the area under the fever curve, expressed as “C x hr, relative to the baseline temperature, was calculated for the 3-hr period following the administration of pyrogen,

To test for the antipyretic activity of eugenol, saline or one of three doses of eugenol was injected intravenously into 6 rabbits 30min after the intravenous injection of interleukin 1. All three doses reduced the febrile response to interleukin 1 (Fig. 1): 1.5 x 10e2 mmol/kg eugenol caused a 25% reduction in the thermal response index (P < 0.05); 3.0 x 10m2mmol/kg caused a 43% reduction (P < 0.05) and the largest dose (6.0 x 10m2mmol/kg) reduced fever by 54% (P < 0.01). Increased respiratory rate and vasodilation in the ears were apparent in all animals about 5-12min after the injection of eugenol and lasted 7&150min. Eugenol and acetaminophen both reduced fever when given intragastrically in a dose of 1.3 mmol/kg. Eugenol not only abolished interleukin l-induced fever but caused slight hypothermia, whereas the same dose of acetaminophen reduced fever by only 68% (Fig. 2). In subsequent tests, a smaller dose of

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Fig. 1. Dose-related decreases in IL-l-induced fever after the intravenous injection of eugenol. Scores in this and following figures are means (f SEM). An injection of eugenol or saline shown at arrow.

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Fig. 2. Eugenol(0.33 mM/kg), given intragastrically, caused antipyresis similar to that after a large dose of acetaminophen (1.3 mM/kg). A dose of eugenol equivalent to the dose of acetaminophen not only reduced fever but also caused hypothermia.

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eugenol (0.33 mmol/kg) reduced fever by approximately the same amount as 1.3 mmol acetaminophen. To find whether eugenol had a central antipyretic action, either eugenol (7.6 x 10M3mmol) or saline was given intracerebroventricularly to febrile rabbits. Fever was reduced 54% by eugenol, (P < 0.01; Fig. 3). This antipyretic effect was comparable to that of the largest intravenous dose of eugenol (Fig. 2). No unusual behavior or signs of discomfort were seen after intracerebroventricular injection of the drug. To determine whether the mechanism of antipyretic action of eugenol might be similar to that of certain antipyretic drugs, rabbits were given intracerebroventricular injections of prostaglandin E,, sodium arachidonate or saline (100 ~1) and 30 min later eugenol was injected intravenously. In these tests a large dose (6.0 x lO~*mmol) did not influence the hyperthermia caused by prostaglandin E, (Fig. 4, P < 0.2), whereas a smaller dose (3 x 10m2mmol/kg) of eugenol did reduce the hyperthermia caused by arachidonic acid (Fig. 5, P < 0.05). DISCUSSION

The four experiments on eugenol yielded new evidence on the antipyretic activity of this drug. The aim in the first experiment was to learn whether eugenol could reduce fever after intravenous administration in febrile rabbits. Dose-related reductions in fever were noted and, together with the short latency to onset (5-12min) and the duration of action (7&150min), indicated that eugenol was a potent

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Fig. 4. Lack of effect of eugenol given intravenously PGE,-induced hyperthermia.

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5. Eugenol given intravenously (IV) reduced fever caused by the central injection of arachidonic acid (AA).

Fig.

Fig. 3. Eugenol(7.6 x lo-’ mmol) reduced fever when given intracerebroventricularly (ICV).

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antipyretic compound with predictable effects. After intravenous injection the only apparent changes in thermoeffector activity were increased respiration and vasodilation in the ears. The increased respiration was presumed to reflect an action upon the CNS since peripherally eugenol can directly inhibit activity in the phrenic nerve (Fordin and Roed, 1984), an effect that would tend to depress respiration. Peripheral administration of eugenol may also directly reduce heat-production capacity since it has been found to depress cell respiration in concentrations of 0.1 mmol and more (Hume, 1984). However, the primary site of action appears to be the CNS as indicated by the results of other experiments (cited below). Once it was established that eugenol could reduce fever when given peripherally, it became important to learn whether the agent was effective when it was given intragastrically and to learn how its activity compared with that of a well-known antipyretic such as acetaminophen. A standard fever was reduced 68% when acetaminophen (1.3 mM/kg) was given by gavage, whereas an equivalent amount of eugenol totally reversed the febrile response; a much smaller dose of eugenol reduced the fever by about as much as acetaminophen. Although these results with limited doses do not allow firm conclusions, it may be that eugenol is generally more potent than acetaminophen in reducing fever. The third experiment, concerned with a possible site of action of eugenol in the CNS, showed that the drug reduced fever when given intracerebroventricularly in doses considerably smaller than those required for antipyresis after peripheral administration. Thus, eugenol has a characteristic in common with antipyretic drugs that are known to reduce fever through an action within the CNS. These results suggest that the effects on thermoeffectors observed after intravenous administration, as noted above, could be due primarily to a central effect of eugenol. The central mechanism of action of common antipyretic drugs is not clear and the results with eugenol do not indicate a specific mechanism of action. However, the lack of effect of eugenol on hyperthermia induced by PGE, and the marked reduction of arachidonate-induced hyperthermia indicate that eugenol has an action similar to that of ace-

JIADI FENG and J. M. LIPTON

1778 taminophen.

If prostaglandins are involved in mediation of fever in the CNS, a role that has not received unqualified acceptance, eugenol and acetaminophen may inhibit fever by inhibition of the synthesis of prostaglandin E through the arachidonic acid cascade. Whether this is the mechanism of action or not, it is clear that the two antipyretic agents, eugenol and acetaminophen, have a similar effect on hypothermia induced by sodium arachidonate. To summarize, eugenol, a Bupleurum derivative, long recognized for its analgesic effect, also has antipyretic properties. It has marked effects on fever when given peripherally and centrally and it appears to reduce fever through an action within the CNS that is not different from that of common antipyretic drugs. Acknowledgement-This study was supported by Grant NS10046 from the National Institute of Neurological Communications Disorders and Stroke.

No. and

REFERENCES

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mic, myorelaxant and anticonvulsant effects of synthetic eugenol derivatives and natural analogues. Pharmacology 22: 113-127. Dewhirst F. E. and Goodson J. M. (1974) Prostaglandin synthetase inhibition by eugenol, guaicol and other dental medicaments. J. Dem. Res. Suppl. 53: 104. Dobbs E. C. (1961) Pharmacology and Oral Therapeutics. 12th edn. n. 448. The C. V. Mosbv Co.. St. Louis. Engelbrecht J. A., Long J. P., Nicholsb. E.‘and Barkfnecht C. F. (1972) Pharmacologic evaluation of 3,4-dimethoxyphenylpropenes and 3,4-dimethoxyphenylpropanediols. Archs int. Pharmacodyn. Thbr. 199: 226-244. Fordin P. and Roed A. (1984) Effects of eugenol on rat phrenic nerve and nerve-diaphragm preparation. Archs oral Biol. 29: 611615. Glyn-Ballinger J. R., Bemardini G. L. and Lipton J. M. (1983) a-MSH injected in the septal region reduces fever in rabbits. Peptides 4: 199-203. Hume W. R. (1983) Effect of eugenol on constrictor resuonses in blood vessels of rabbit ear. J. Dent. Res. 62: 1‘013-1015. Hume W. R. (1984) Effect of eugenol on respiration and division in human pulp, mouse fibroblasts, and liver cells in vitro. J. Dent. Res. 63: 1262-1265. Hubei Provincial Institute of Pharmaceutical Industry. Analysis of the antipyretic and antiseptic compounds in Chinese DC. Chinese Pharm. BUN. 17: Bupleurum 202-205 (1982). Kay L. W. (1972) Drugs in dentistry. Dental Practitioner’s Handbook Series (Derrick A., Ed.), pp. 156202. Wright, Bristol. Lipton J. M. and Romans W. E. (1976) Modification of rabbit head holder to increase speed and accuracy of stereotaxic surgery. Brain Res. Bull. 1: 159-160.