Clove hasbeen used in ancient China as a spice and fragrance for more than 2,000 years. Medicinally, the well-knowntraditional remedy of applying clove oil to treat a toothache was documented for the firsttime in 1640 in ‘Practice of Physic’ in France. (1) In Chinese traditional medicine clove oil has beenused as carminative, antispasmodic, antibacterial and antiparasitic agent.
The budswere used to treat dyspepsia, acute/chronic gastritis and diarrhea. (2). The name of the main constituent of clove oil, eugenol, is derived fromthe species name Eugenia caryophyllata which contains a high level of eugenol(45–90%) in addition to acetyleugenol, chavicol and humulenes (3).
Eugenol was firstisolated in 1929 and commercial production commenced in the 1940s. Eugenol canbe produced synthetically, however, eugenol is predominantly prepared fromnatural oil sources by fractionaldistillation. (4). The major clove producers of the world are theWest Indies, Madagascar, Tanzania, India, Sri Lanka, Indonesia and Malaysia.
PHYSICOCHEMICAL PROPERTIES OF EUGENOL Thechemical structure of anethole is given in Fig. 2. Fig. 2. Chemical structureof eugenol Eugenol(C10H12O2), a phenylpropanoid, is an allyl chain-substituted guaiacol . Eugenolis weaklyacidic, slightly soluble in water and very soluble in organic solvents.
It is a colorless to pale yellow oily liquid with a characteristic and pleasant odour of cloves and a spicy pungent taste. Melting point is -9 and boiling point is 254 C.Specific gravity is 1.06.
Solubility in water is less than 1mg/ml. Eugenol is stableunder ordinary conditions but is light sensitive. Absorption, distribution and elimination In humansand rodents, orally administered eugenol and related allyihydroxyphenol derivativesare rapidly absorbed from the gastrointestinal tract and undergo mainly phase-1conjugation and subsequent excretion in the urine.
To a lesser extent, eugenolis metabolized to polar products, which are also conjugated and eliminated primarilyin the urine. Minute amounts (< 1%) of eugenol are excreted unchanged. Themain urinary metabolites of eugenol are the glucuronic acid and sulfate conjugatesof the phenolic hydroxyl group. Four healthy male and four female volunteers(weighing 52-86 kg) were given three gelatin capsules, each containing 50 mg ofeugenol (total dose, 150 mg; 1.
7-2.9 mg/kg bw) with a normal breakfast (tea andtwo biscuits). Urine was collected 3, 6, 12 and 24 h after administration, andvenous blood was sampled at O, 15, 20, 25, 30, 40, 50, 60, 80, 100 and 120 min.In all body fluids analyzed, eugenol was found predominantly in the conjugatedform. Within 3 h, 71 .3% (mean value for three volunteers) of the 150-mg dosewas accounted for in the urine as conjugated eugenol or conjugated metabolitesof eugenol.
After 6 and 24 h, > 87% and 94%, respectively, of the dose hadbeen excreted in the urine (Fischer et al. , 1990).Likehumans, rodents also rapidly absorbed, metabolized and excreted eugenol givenorally or by intraperitoneal injection. An unspecified number of female Wistarrats were given 0.5, 5, 50 or 1000 mg/kg bw of 14C-ring-labelledeugenol by stomach tube.
More than 75% of the administered radiolabel was presentin pooled 72-h urine, while 10% was found in the faeces. The 24-h urine contained mainly glucuronic acid and sulfateconjugates, the sulfate conjugates predominating at low doses and theglucuronic acid conjugates at 1000 mg/kg bw (Sutton et al., 1985). Excretionof 50 mg/kg bw of 14C-eugenol was essentially complete within 24 h in fourfemale Wistar rats treated by intraperitoneal administration and in eight femaleFischer rats given the compound by gavage. Excretion in the urine (91.
2 ± 4.3%and 75.1 ± 9.4% for the intraperitoneal and oral routes, respectively) farexceeded that in the faeces (3.9 ± 1.6% and 7.4 ± 5.
0%, respectively). Thepattern of absorption and excretion observed was similar to that in mice. Eightmice given 50 mg/kg bw of eugenol by intraperitoneal injection excreted 76.3 ±4.1% and 4.9 ± 2.7% in 24-h urine and faeces, respectively (Sutton, 1986). Rapiddistribution to all organs, with tissue concentrations reaching 10-20 ng/mg oftissue, was observed in male Wistar rats given a single dose of 450 mg/kg bw 14C-eugenolby intraperitoneal injection.
Higher levels of radioactivity were reached incirculating erythrocytes than in sera, which showed a significant reduction inradioactivity 4 h after dosing. Less than 1% of the total radioactivity administeredwas eliminated as exhaled 14CO2 (Weinberg et al., 1972). When 500mg of eugenol in sesame oil were administered to rats by gavage (about 1250mg/kg bw), the compound was detected in the stomach, intestines and faeces,with lesser amounts in the liver and kidneys. Similar results were obtained after1500, 2500 or 5000 mg of eugenol in sesame oil (about 750, 1250 and 2500 mg/kgbw, respectively) were administered to rabbits by gavage. Eugenol was detectedmainly in the stomach, intestines and urine of rabbits at all dose, and in the lungs,liver, kidneys, muscle and blood of animals at 2500 mg/kg bw (Schröder & Vollmer,1932). Groups ofeight male rats were given a single oral dose of O or 200 mg eugenol (about 500mg/kg bw) in olive oil, and urine was collected at 12-h intervals.
The 0-12-hand 12-24-h urine samples contained more glucuronides (45.4 ± 13.2 and 42.
9 ± 9.1mg of total glucuronic acid/12 h per rat) than the O-12-h urine sample from controlrats (10.9 ± 4.9 mg/12 h per rat). The excess amount of glucuronides wasconsidered to be due to excretion of eugenol glucuronides. Therefore, orally administeredeugenol undergoes rapid glucuronic acid conjugation and excretion in rats(Yuasa, 1974).
Absorption, distribution and elimination Eugenoland other hydroxyallylbenzene derivatives have several metabolic options fordetoxication. The results of studies in humans indicate that most eugenol israpidly conjugated with glucuronic acid or sulfate (Sutton, 1986; Fischer etal., 1990). To a much lesser extent, eugenol undergoes (1) isomerization toyield isoeugenol, which can then undergo allylic oxidation and reduction of thedouble bond; (2) epoxidation of the allyl double bond to yield an epoxide,which is hydrolysed to the corresponding diol and, subsequently, can beoxidized to the corresponding lactic acid derivative; (3) conjugation ofglutathione (GSH) with a quinone-methidetype intermediate and (4) hydroxylationat the allyl position to yield 1 ‘-hydroxyeugenol. As all these metaboliteshave a free phenolic OH group or other polar oxygenated functional groups, theyreadily conjugate with glucuronic acid or sulfate and are excreted in urine.
Inhumans, 95% of ingested eugenol is excreted in conjugated form in the urinewithin 24 h (Fischer et al., 1990). Two malevolunteers (weighing 93 and 95 kg) received 0.6 mg of 14C-eugenol (about6.4 µg/kg bw) in the form of agelatin capsule, which was taken orally with water.
Within 24 h, 94-103% of theradioactivity was accounted for in the urine; none was found in faeces. Over85% of the radioactivity in 24-h urine was accounted for by glucuronic acid andsulfate conjugates of eugenol, the glucuronic acid conjugates predominating.Minor amounts (2% each) of the corresponding diol 3- (4-hydroxy-3-methoxyphenyl)propane-1 ,2-diol and alcohol 3-(4-hydroxy-3-methoxyphenyl) propane-2-ol were alsodetected. Unlike other hydroxyallylbenzene derivatives, which generally undergooxidative metabolism at the allyl moiety, eugenol undergoes reductivemetabolism and its conjugates are rapidly eliminated, which could explain itslack of toxicity(Sutton, 1986). Inrodents, the metabolic fate of eugenol appears to be similar to that in humans.The 24-h urine of eight female Wistar rats given 0.
5, 5, 50 or 1000 mg/kg bw of14C-ring-labelled eugenol in trioctanoin by stomach tube containedglucuronic acid and sulfate conjugates of eugenol, the O-demethylationmetabolite, 3,4- dihydroxypropylbenzene and the reduced metabolite,3-methoxy-4-hydroxypropylbenzene. At the three lower doses, sulfate conjugateswere the main metabolites, while at the highest dose glucuronic acid conjugatespredominated. No reduction or demethylation metabolites (i e.3,4-dihydroxypropylbenzene and 3-methoxy-4- hydroxypropylbenzene) were detectedat the highest dose (Sutton et al. , 1 985; Sutton, 1986). Toinvestigate the origin of the reduction and O-demethylation metabolites, 10 mgof 14C-eugenol were incubated with rat caecal contents underanaerobic conditions. Formation of both reduction and O-demethylationmetabolites suggested that the gut microflora are involved.
Furthermore, thefact that no O-demethylation metabolites were found when eugenol wasadministered to germ-free Fischer 344 or Wistar rats pre-treated withantibiotics supports the conclusion that reduction and O-demethylation aremediated by gut microflora in rats (Sutton, 1986). In a studyto determine species-specific metabolism, 50 mg/kg bw of 14C eugenol were administered bygavage to female Wistar rats or injected intraperitoneally to CD-i mice.Analysis of the urine showed that mice and rats excreted> 80% as glucuronicacid and sulfate conjugates.
Mice excreted 27 ± 2.7% as sulfate conjugates and53 ± 3.5% as glucuronic acid conjugates, while rats excreted 55 ± 3.3% assulfate conjugates and 25 ± 3.8% as glucuronic acid conjugates (Sutton, 1986). Studieshave been undertaken to evaluate the involvement of cytochrome P450 (CYP45O)oxidation and subsequent GSH conjugation in the metabolism of eugenol. Most ofthese experiments were intended to evaluate the mechanism of action andpotential toxicity of eugenol. When male ddY mice were treated with 400 or 600mg/kg bw of eugenol in olive oil by gavage, there was no evidence ofhepatotoxicity, as indicated by the absence of changes in relative liverweight, liver blood volume and serum alanine aminotransferase (ALT) activity.
Mice receiving 4 mmol/kg bw of the GSH inhibitor buthionine sulfoximine (BSO)by intraperitoneal injection i h before administration of 400 or 600 mg/kg bwof eugenol, however, showed significant increases in relative liver weights,serum ALT activity and volume of blood in the liver (indicative of hepatic congestion)3 h after administration of 600 mg/kg bw in comparison with a control groupreceiving saline or olive oil. Additionally, the mortality of rats treated withBSO and 600 mg/kg bw eugenol was increased, although not statisticallysignificantly. Gross examination showed marked enlargement and uniform orspotted dark-reddish coloration of the livers of mice receiving BSO and 600mg/kg bw eugenol. Histologically, the centrilobular sinusoidal spaces werecongested and vacuolation was observed near Glisson capsule. The livers of micethat survived 24 h after treatment showed marked necrosis in the centrilobularregion. The livers of mice receiving eugenol at 600 mg/kg bw alone or BSO alone showed no liver pathological changes (Mizutani et al.,1991).
The effectof microsomal P450-dependent monooxygenase inhibitors on the hepatotoxicity ofa high dose of eugenol (600 mg/kg bw) was evaluated in mice treated with theCYP45O inhibitors carbon disulfide methoxsalen or piperonyl butoxide togetherafter administration of BSO. Treatment with carbon disulfide (50 mg/kg bw) resultedin complete protection against the hepatotoxicity seen in mice pre-treated withBSO and then given eugenol. Methoxsalen (50 mg/kg bw) partially prevented theincrease in serum ALT activity reported after combined BSO and eugenol treatment,and it completely protected against increases in relative liver weight and hepaticcongestion. Piperonyl butoxide (400 mg/kg bw) also suppressed BSOeugenol- inducedhepatotoxicity, although not as effectively as the other two inhibitors (Mizutaniet al., 1991).Pre-treatmentof mice with phenobarbital, an inducer of CYP45O enzymes, enhanced the toxicityof eugenol administered in combination with BSO, increasing the relative liverweights and causing hepatic congestion and increased serum ALT activity. In theabsence of BSO, phenobarbital-pre-treated mice showed no significant increasesin any of the indicators of hepatotoxicity after dosing with 400 mg/kg bw ofeugenol.
Pre-treatment of mice with the CYP45O inhibitor b-naphthoflavone preventedan increase in serum ALT activity in mice treated with BSO and subsequentlywith 400 mg/kg bw eugenol; however, none of the indicators of hepatotoxicitywas suppressed at 600 mg/kg bw of eugenol. The authors suggested thatb-naphthoflavone acts by stimulating detoxicating pathways of eugenol metabolismat lower doses of this compound. These results suggest that a metabolite ofeugenol formed by a CYP45Q-mediated reaction conjugates with GSH (Mizutani etal., 1991). In otherstudies of the CYP45O oxidation-GSH conjugation pathway (see Figure 3),concentration- and time-dependent indicators of cytotoxicity were reported whenfreshly isolated rat hepatocytes were incubated with 0, 0.5, 1 or 1.
5 mmol/l ofeugenol. At each concentration tested, onset of cell death was observed after 2h, preceded by blebbing of cellular membranes. Celldeath was inhibited when 1 mmol/l of eugenol was incubated with rat hepatocytesin the presence of i mmol/l of Nacetylcysteine. Cellular GSH was depleted byeugenol to less than 30% of control values by 2 h, while control cells showedsignificant depletion of GSH only after 4 h; until that time, GSH levels weremaintained at 90%. Addition of i mmol/l of Nacetylcysteine prevented theeugenol-induced depletion of OSH. In hepatocytes depleted of GSH by theaddition of diethylmaleate, cytotoxicity was observed 2 h before the onset ofcytotoxicity ¡n control cells exposed to eugenol only.
Covalent binding ofradiolabelled eugenol to cellular protein occurred at a linear rate up to 3 h afterincubation; however, addition of N-acetylcysteine inhibited covalent bindingfor up to 3 h. Thereafter, N-acetylcysteine was depleted, allowing covalentbinding to occur. The three metabolites isolated after 5 h of incubation of immol/l of eugenol with rat hepatocytes were identified as the glucuronic acid,GSH and sulfate conjugates, the glucuronic acid conjugate predominating (i.
e.200 nmol of eugenol glucuronide formed and 25 nmol of each of the other twoconjugates) (Thompson et al., 1991). To study theeffect of eugenol on drug-metabolizing enzymes such as CYP45O and UDPGT, maleWistar rats were given 250, 500 or 1000 mg/kg bw per day of eugenol ¡n corn oilby gavage for 10 days. The animals were necropsied 24 h after the last dose,their livers were excised, blood samples were collected and liver microsomesand cytosolic fractions were isolated. No statistically significant changes inbody weight, relative liver weight, haematological indices, plasma ALT oraspartate aminotransferase activities or total liver CYP45O content were seenin comparison with controls. The authorsconcluded that, while eugenol does not effectively induce CYP45O enzymeactivity, it can induce phase-Il biotransformation enzymes (Rompelberg et al.,1996a).
Thepotential of eugenol to be oxidized to a reactive intermediate was investigatedin a series of studies in vitro. In one experiment, 500 µmol/l of eugenol wereincubated with hydrogen peroxide and myeloperoxidase isolated from human polymorphonuclearleukocytes. Spectral evidence indicated the formation of a quinone methidemetabolite in an enzyme-dependent manner. Formation of this metabolite wascompletely inhibited when reduced GSH (10-50 µmol/l) was present in thereaction mixture at the beginning of the reaction; however, when GSH (50 µmol/l)was added to the reaction mixture after formation of the metabolite had begun,the metabolite reacted directly with the GSH. The authors proposed that eugenol forms a phenoxy radical under these conditions,which is reduced back to eugenol through the formation of oxidized GSH (i.e.
GSH disulfide) (Thompson et al., 1989). In conclusion, eugenol may participate, to a minorextent, in an oxidation pathway leading to a quinone methide intermediate;however, at the concentrations of the quinone methide intermediate present inliver, effective detoxication by GSH conjugation is expected. In addition, theextensive conjugation of eugenol greatly limits formation of this quinonemethide intermediate.Eugenol is partly (10%) metabolized to an epoxide,which undergoes hydrolysis catalysed by epoxide hydrolase. Hydrolysis yields adiol that can be either conjugated and excreted or oxidized to a lactic acidderivative and then excreted.
When eugenol was incubated with rat epithelialcells or rat liver microsomes, 2,3′- eugenol epoxide was formed (Delaforge etal., 1978). After treatment of adult male Wistar rats with a singleintraperitoneal dose of 200 mg/kg bw of eugenol in corn oil, eugenol epoxide,the corresponding diol (dihydrodihydroxy eugenol), allylcatechol epoxide anddihydrodihydroxy allylcatechol were identified after 24 h in urine collected every2 h (Delaforge et al., 1980). The 24-h liver homogenates obtained from the samerats also contained eugenol epoxide and the corresponding diol. When liver microsomesobtained from rats pre-treated with phenobarbital (80 mg/kg bw; intraperitoneally)for 3 days were incubated with 1 µmol (164 µg) of eugenol, the resultingmetabolites were identified as eugenol epoxide, the corresponding diol, allylcatecholepoxide3 and dihydrodihydroxy allylcatechol.
In contrast, cultured adult ratliver cells incubated with 1 mg of eugenol formed only eugenol epoxide and dihydrodihydroxyeugenol and not allylcatechol derivatives. Eugenol epoxide is presumed to berapidly detoxicated by the formation of eugenol-2,3-diol by epoxide hydrolase,as the epoxide was not detected at any appreciable concentration in vivo (Luoet aI., 1992; Guenthner & Luo, 2001). Spasmolytic activity In a study to investigate the effect of eugenol onsmooth muscle activity in the intestines, groups of 8-12 male Wistar-Nossanrats were given 0, 25, 50, 100 or 200 mg/kg bw of eugenol 1 h beforeadministration of 1 ml of an aqueous suspension of 10% charcoal and 5% acaciagum.
The small intestine was removed 20 min later, and the distance that thecharcoal had travelled from the pylorus was measured. A significantdose-dependent decrease in the total length of the small intestine travelled bythe charcoal suspension was observed, indicating a decrease in smooth muscleaction. When 100 mg/kg bw of eugenol were administered 30, 60, 120, 180 or 240min before administration of the charcoal suspension, maximum transit distancewas observed after 60 min. The authors suggested that eugenol at the doses usedin this study might inhibit the spontaneous activity of the longitudinal gutmuscle, possibly by inhibiting prostaglandin synthesis (Bennett et al.
, i 988). Antiinflammatory activity To investigate the potential of eugenol to inhibitprostaglandin synthesis, homogenized human colon mucosa was incubated with 0,1, 10 or 100 µg/ml of eugenol. Concentration-dependent inhibition of prostanoidformation was found, with statistically significant inhibition atconcentrations of i 0 and 100 µg/ml and as low as 1 µg/mI for inhibition ofthromboxane B2. Human polymorphonuclear leukocytes incubated with 14C-arachidonicacid in the presence of 0, 1, 10 or 100 µg/ml of eugenol showed markedinhibition (approximately 85%) in the formation of 5-hydroxyeicosatetraenoic acidat the highest concentration tested (100 ig/ml) (Bennett et al., 1988).