Clove has
been used in ancient China as a spice and fragrance for more than 2,000 years. Medicinally, the well-known
traditional remedy of applying clove oil to treat a toothache was documented for the first
time in 1640 in ‘Practice of Physic’ in France. (1)

In Chinese traditional medicine clove oil has been
used as carminative, antispasmodic, antibacterial and antiparasitic agent. The buds
were used to treat dyspepsia, acute/chronic gastritis and diarrhea. (2). The name of the main constituent of clove oil, eugenol, is derived from
the species name Eugenia caryophyllata which contains a high level of eugenol
(45–90%) in addition to acetyleugenol, chavicol and humulenes (3). Eugenol was first
isolated in 1929 and commercial production commenced in the 1940s. Eugenol can
be produced synthetically, however, eugenol is predominantly prepared from
natural oil sources by fractional
distillation. (4). The major clove producers of the world are the
West Indies, Madagascar, Tanzania, India, Sri Lanka, Indonesia and Malaysia.

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PHYSICOCHEMICAL PROPERTIES OF EUGENOL

 

The
chemical structure of anethole is given in Fig. 2.

 

 

Fig. 2. Chemical structure
of eugenol

 

Eugenol
(C10H12O2), a phenylpropanoid, is an allyl chain-substituted guaiacol . Eugenol
is weakly
acidic, 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 stable
under ordinary conditions but is light sensitive.

 

Absorption, distribution and elimination

 

In humans
and rodents, orally administered eugenol and related allyihydroxyphenol derivatives
are rapidly absorbed from the gastrointestinal tract and undergo mainly phase-1
conjugation and subsequent excretion in the urine. To a lesser extent, eugenol
is metabolized to polar products, which are also conjugated and eliminated primarily
in the urine. Minute amounts (< 1%) of eugenol are excreted unchanged. The main urinary metabolites of eugenol are the glucuronic acid and sulfate conjugates of the phenolic hydroxyl group. Four healthy male and four female volunteers (weighing 52-86 kg) were given three gelatin capsules, each containing 50 mg of eugenol (total dose, 150 mg; 1.7-2.9 mg/kg bw) with a normal breakfast (tea and two biscuits). Urine was collected 3, 6, 12 and 24 h after administration, and venous 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 conjugated form. Within 3 h, 71 .3% (mean value for three volunteers) of the 150-mg dose was accounted for in the urine as conjugated eugenol or conjugated metabolites of eugenol. After 6 and 24 h, > 87% and 94%, respectively, of the dose had
been excreted in the urine (Fischer et al. , 1990).

Like
humans, rodents also rapidly absorbed, metabolized and excreted eugenol given
orally or by intraperitoneal injection. An unspecified number of female Wistar
rats were given 0.5, 5, 50 or 1000 mg/kg bw of 14C-ring-labelled
eugenol by stomach tube. More than 75% of the administered radiolabel was present
in pooled 72-h urine, while 10% was found in the faeces. The 24-h urine contained mainly glucuronic acid and sulfate
conjugates, the sulfate conjugates predominating at low doses and the
glucuronic acid conjugates at 1000 mg/kg bw (Sutton et al., 1985).

Excretion
of 50 mg/kg bw of 14C-eugenol was essentially complete within 24 h in four
female Wistar rats treated by intraperitoneal administration and in eight female
Fischer 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) far
exceeded that in the faeces (3.9 ± 1.6% and 7.4 ± 5.0%, respectively). The
pattern of absorption and excretion observed was similar to that in mice. Eight
mice 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).

Rapid
distribution to all organs, with tissue concentrations reaching 10-20 ng/mg of
tissue, was observed in male Wistar rats given a single dose of 450 mg/kg bw 14C-eugenol
by intraperitoneal injection. Higher levels of radioactivity were reached in
circulating erythrocytes than in sera, which showed a significant reduction in
radioactivity 4 h after dosing. Less than 1% of the total radioactivity administered
was eliminated as exhaled 14CO2 (Weinberg et al., 1972).

When 500
mg of eugenol in sesame oil were administered to rats by gavage (about 1250
mg/kg bw), the compound was detected in the stomach, intestines and faeces,
with lesser amounts in the liver and kidneys. Similar results were obtained after
1500, 2500 or 5000 mg of eugenol in sesame oil (about 750, 1250 and 2500 mg/kg
bw, respectively) were administered to rabbits by gavage. Eugenol was detected
mainly 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 of
eight male rats were given a single oral dose of O or 200 mg eugenol (about 500
mg/kg bw) in olive oil, and urine was collected at 12-h intervals. The 0-12-h
and 12-24-h urine samples contained more glucuronides (45.4 ± 13.2 and 42.9 ± 9.1
mg of total glucuronic acid/12 h per rat) than the O-12-h urine sample from control
rats (10.9 ± 4.9 mg/12 h per rat). The excess amount of glucuronides was
considered to be due to excretion of eugenol glucuronides. Therefore, orally administered
eugenol undergoes rapid glucuronic acid conjugation and excretion in rats
(Yuasa, 1974).

 

Absorption, distribution and elimination

 

Eugenol
and other hydroxyallylbenzene derivatives have several metabolic options for
detoxication. The results of studies in humans indicate that most eugenol is
rapidly conjugated with glucuronic acid or sulfate (Sutton, 1986; Fischer et
al., 1990). To a much lesser extent, eugenol undergoes (1) isomerization to
yield isoeugenol, which can then undergo allylic oxidation and reduction of the
double bond; (2) epoxidation of the allyl double bond to yield an epoxide,
which is hydrolysed to the corresponding diol and, subsequently, can be
oxidized to the corresponding lactic acid derivative; (3) conjugation of
glutathione (GSH) with a quinone-methidetype intermediate and (4) hydroxylation
at the allyl position to yield 1 ‘-hydroxyeugenol. As all these metabolites
have a free phenolic OH group or other polar oxygenated functional groups, they
readily conjugate with glucuronic acid or sulfate and are excreted in urine. In
humans, 95% of ingested eugenol is excreted in conjugated form in the urine
within 24 h (Fischer et al., 1990).

Two male
volunteers (weighing 93 and 95 kg) received 0.6 mg of 14C-eugenol (about
6.4 µg/kg bw) in the form of a
gelatin capsule, which was taken orally with water. Within 24 h, 94-103% of the
radioactivity was accounted for in the urine; none was found in faeces. Over
85% of the radioactivity in 24-h urine was accounted for by glucuronic acid and
sulfate 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 also
detected. Unlike other hydroxyallylbenzene derivatives, which generally undergo
oxidative metabolism at the allyl moiety, eugenol undergoes reductive
metabolism and its conjugates are rapidly eliminated, which could explain its
lack of toxicity
(Sutton, 1986).

In
rodents, 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 of
14C-ring-labelled eugenol in trioctanoin by stomach tube contained
glucuronic acid and sulfate conjugates of eugenol, the O-demethylation
metabolite, 3,4- dihydroxypropylbenzene and the reduced metabolite,
3-methoxy-4-hydroxypropylbenzene. At the three lower doses, sulfate conjugates
were the main metabolites, while at the highest dose glucuronic acid conjugates
predominated. No reduction or demethylation metabolites (i e.
3,4-dihydroxypropylbenzene and 3-methoxy-4- hydroxypropylbenzene) were detected
at the highest dose (Sutton et al. , 1 985; Sutton, 1986).

To
investigate the origin of the reduction and O-demethylation metabolites, 10 mg
of 14C-eugenol were incubated with rat caecal contents under
anaerobic conditions. Formation of both reduction and O-demethylation
metabolites suggested that the gut microflora are involved. Furthermore, the
fact that no O-demethylation metabolites were found when eugenol was
administered to germ-free Fischer 344 or Wistar rats pre-treated with
antibiotics supports the conclusion that reduction and O-demethylation are
mediated by gut microflora in rats (Sutton, 1986).

In a study
to determine species-specific metabolism, 50 mg/kg bw of 14C eugenol were administered by
gavage to female Wistar rats or injected intraperitoneally to CD-i mice.
Analysis of the urine showed that mice and rats excreted> 80% as glucuronic
acid and sulfate conjugates. Mice excreted 27 ± 2.7% as sulfate conjugates and
53 ± 3.5% as glucuronic acid conjugates, while rats excreted 55 ± 3.3% as
sulfate conjugates and 25 ± 3.8% as glucuronic acid conjugates (Sutton, 1986).

Studies
have been undertaken to evaluate the involvement of cytochrome P450 (CYP45O)
oxidation and subsequent GSH conjugation in the metabolism of eugenol. Most of
these experiments were intended to evaluate the mechanism of action and
potential toxicity of eugenol. When male ddY mice were treated with 400 or 600
mg/kg bw of eugenol in olive oil by gavage, there was no evidence of
hepatotoxicity, as indicated by the absence of changes in relative liver
weight, 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 bw
of 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 group
receiving saline or olive oil. Additionally, the mortality of rats treated with
BSO and 600 mg/kg bw eugenol was increased, although not statistically
significantly. Gross examination showed marked enlargement and uniform or
spotted dark-reddish coloration of the livers of mice receiving BSO and 600
mg/kg bw eugenol. Histologically, the centrilobular sinusoidal spaces were
congested and vacuolation was observed near Glisson capsule. The livers of mice
that survived 24 h after treatment showed marked necrosis in the centrilobular
region. 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 effect
of microsomal P450-dependent monooxygenase inhibitors on the hepatotoxicity of
a high dose of eugenol (600 mg/kg bw) was evaluated in mice treated with the
CYP45O inhibitors carbon disulfide methoxsalen or piperonyl butoxide together
after administration of BSO. Treatment with carbon disulfide (50 mg/kg bw) resulted
in complete protection against the hepatotoxicity seen in mice pre-treated with
BSO and then given eugenol. Methoxsalen (50 mg/kg bw) partially prevented the
increase in serum ALT activity reported after combined BSO and eugenol treatment,
and it completely protected against increases in relative liver weight and hepatic
congestion. Piperonyl butoxide (400 mg/kg bw) also suppressed BSOeugenol- induced
hepatotoxicity, although not as effectively as the other two inhibitors (Mizutani
et al., 1991).

Pre-treatment
of mice with phenobarbital, an inducer of CYP45O enzymes, enhanced the toxicity
of eugenol administered in combination with BSO, increasing the relative liver
weights and causing hepatic congestion and increased serum ALT activity. In the
absence of BSO, phenobarbital-pre-treated mice showed no significant increases
in any of the indicators of hepatotoxicity after dosing with 400 mg/kg bw of
eugenol. Pre-treatment of mice with the CYP45O inhibitor b-naphthoflavone prevented
an increase in serum ALT activity in mice treated with BSO and subsequently
with 400 mg/kg bw eugenol; however, none of the indicators of hepatotoxicity
was suppressed at 600 mg/kg bw of eugenol. The authors suggested that
b-naphthoflavone acts by stimulating detoxicating pathways of eugenol metabolism
at lower doses of this compound. These results suggest that a metabolite of
eugenol formed by a CYP45Q-mediated reaction conjugates with GSH (Mizutani et
al., 1991).

 

In other
studies of the CYP45O oxidation-GSH conjugation pathway (see Figure 3),
concentration- and time-dependent indicators of cytotoxicity were reported when
freshly isolated rat hepatocytes were incubated with 0, 0.5, 1 or 1.5 mmol/l of
eugenol. At each concentration tested, onset of cell death was observed after 2
h, preceded by blebbing of cellular membranes. Cell
death was inhibited when 1 mmol/l of eugenol was incubated with rat hepatocytes
in the presence of i mmol/l of Nacetylcysteine. Cellular GSH was depleted by
eugenol to less than 30% of control values by 2 h, while control cells showed
significant depletion of GSH only after 4 h; until that time, GSH levels were
maintained at 90%. Addition of i mmol/l of Nacetylcysteine prevented the
eugenol-induced depletion of OSH. In hepatocytes depleted of GSH by the
addition of diethylmaleate, cytotoxicity was observed 2 h before the onset of
cytotoxicity ¡n control cells exposed to eugenol only. Covalent binding of
radiolabelled eugenol to cellular protein occurred at a linear rate up to 3 h after
incubation; however, addition of N-acetylcysteine inhibited covalent binding
for up to 3 h. Thereafter, N-acetylcysteine was depleted, allowing covalent
binding to occur. The three metabolites isolated after 5 h of incubation of i
mmol/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 two
conjugates) (Thompson et al., 1991).

To study the
effect of eugenol on drug-metabolizing enzymes such as CYP45O and UDPGT, male
Wistar rats were given 250, 500 or 1000 mg/kg bw per day of eugenol ¡n corn oil
by gavage for 10 days. The animals were necropsied 24 h after the last dose,
their livers were excised, blood samples were collected and liver microsomes
and cytosolic fractions were isolated. No statistically significant changes in
body weight, relative liver weight, haematological indices, plasma ALT or
aspartate aminotransferase activities or total liver CYP45O content were seen
in comparison with controls. The authors
concluded that, while eugenol does not effectively induce CYP45O enzyme
activity, it can induce phase-Il biotransformation enzymes (Rompelberg et al.,
1996a).

The
potential of eugenol to be oxidized to a reactive intermediate was investigated
in a series of studies in vitro. In one experiment, 500 µmol/l of eugenol were
incubated with hydrogen peroxide and myeloperoxidase isolated from human polymorphonuclear
leukocytes. Spectral evidence indicated the formation of a quinone methide
metabolite in an enzyme-dependent manner. Formation of this metabolite was
completely inhibited when reduced GSH (10-50 µmol/l) was present in the
reaction 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 minor
extent, in an oxidation pathway leading to a quinone methide intermediate;
however, at the concentrations of the quinone methide intermediate present in
liver, effective detoxication by GSH conjugation is expected. In addition, the
extensive conjugation of eugenol greatly limits formation of this quinone
methide intermediate.

Eugenol is partly (10%) metabolized to an epoxide,
which undergoes hydrolysis catalysed by epoxide hydrolase. Hydrolysis yields a
diol that can be either conjugated and excreted or oxidized to a lactic acid
derivative and then excreted. When eugenol was incubated with rat epithelial
cells or rat liver microsomes, 2,3’- eugenol epoxide was formed (Delaforge et
al., 1978). After treatment of adult male Wistar rats with a single
intraperitoneal dose of 200 mg/kg bw of eugenol in corn oil, eugenol epoxide,
the corresponding diol (dihydrodihydroxy eugenol), allylcatechol epoxide and
dihydrodihydroxy allylcatechol were identified after 24 h in urine collected every
2 h (Delaforge et al., 1980). The 24-h liver homogenates obtained from the same
rats also contained eugenol epoxide and the corresponding diol. When liver microsomes
obtained from rats pre-treated with phenobarbital (80 mg/kg bw; intraperitoneally)
for 3 days were incubated with 1 µmol (164 µg) of eugenol, the resulting
metabolites were identified as eugenol epoxide, the corresponding diol, allylcatechol
epoxide3 and dihydrodihydroxy allylcatechol. In contrast, cultured adult rat
liver cells incubated with 1 mg of eugenol formed only eugenol epoxide and dihydrodihydroxy
eugenol and not allylcatechol derivatives. Eugenol epoxide is presumed to be
rapidly detoxicated by the formation of eugenol-2,3-diol by epoxide hydrolase,
as the epoxide was not detected at any appreciable concentration in vivo (Luo
et aI., 1992; Guenthner & Luo, 2001).

 

Spasmolytic activity

 

In a study to investigate the effect of eugenol on
smooth muscle activity in the intestines, groups of 8-12 male Wistar-Nossan
rats were given 0, 25, 50, 100 or 200 mg/kg bw of eugenol 1 h before
administration of 1 ml of an aqueous suspension of 10% charcoal and 5% acacia
gum. The small intestine was removed 20 min later, and the distance that the
charcoal had travelled from the pylorus was measured. A significant
dose-dependent decrease in the total length of the small intestine travelled by
the charcoal suspension was observed, indicating a decrease in smooth muscle
action. When 100 mg/kg bw of eugenol were administered 30, 60, 120, 180 or 240
min before administration of the charcoal suspension, maximum transit distance
was observed after 60 min. The authors suggested that eugenol at the doses used
in this study might inhibit the spontaneous activity of the longitudinal gut
muscle, possibly by inhibiting prostaglandin synthesis (Bennett et al., i 988).

 

Antiinflammatory  activity

 

 

To investigate the potential of eugenol to inhibit
prostaglandin synthesis, homogenized human colon mucosa was incubated with 0,
1, 10 or 100 µg/ml of eugenol. Concentration-dependent inhibition of prostanoid
formation was found, with statistically significant inhibition at
concentrations of i 0 and 100 µg/ml and as low as 1 µg/mI for inhibition of
thromboxane B2. Human polymorphonuclear leukocytes incubated with 14C-arachidonic
acid in the presence of 0, 1, 10 or 100 µg/ml of eugenol showed marked
inhibition (approximately 85%) in the formation of 5-hydroxyeicosatetraenoic acid
at the highest concentration tested (100 ig/ml) (Bennett et al., 1988).

 

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