Chemical Constituents of Clove (Syzygium aromaticum, Fam.

Myrtaceae) and their Antioxidant Activity

Mahmoud I. Nassar1*, Ahmed H. Gaara1, Ahmed H. El-Ghorab2, Abdel-Razik H. Farrag3, Hui Shen4,
Enamul Huq4 and Tom J. Mabry4
(Received: April 2007; Accepted October 2007)

Abstract

Sixteen volatile compounds were identified from the n-hexane extract of the buds of
Syzygium aromaticum by using gas chromatography-mass spectroscopy (GC-MS).
The major components were eugenol (71.56 %) and eugenol acetate (8.99 %). The
dichloromethane extract of the buds yielded limonin and ferulic aldehyde, along with
eugenol. The flavonoids tamarixetin 3-O-b-D-glucopyranoside, ombuin 3-O-b-D-glu-
copyranoside and quercetin were isolated from the ethanol extract; identifications
of all these compounds were established by chemical and spectroscopic methods
including 1D and 2D NMR. This is the first report of limonin, ferulic aldehyde and
these flavonoids from this plant. All extracts and the isolated flavonoids showed
strong antioxidant activity against 1, 2-diphenyl picrylhydrazyl (DPPH). Among the
tested extracts, the ethanol extract of the clove buds showed remarkable scaven-
ging activity, as compared with synthetic antioxidants such as butylated hydroxyl
toluene (BHT). The ethanol extract of clove showed remarkable hepatoprotective
activity against paracetamol-induced liver injury in female rats.

Keywords: Syzygium aromaticum, Myrtaceae, volatile compounds, limonin, ferulic

aldehyde, flavonoids, antioxidant activity, hepatoprotective activity.

INTRODUCTION 2002). In addition to fruits and vegetables

Chemical constituents with antioxidant ac-
tivity found in high concentrations in plants
(Velioglu et al, 1998) determine their con-
siderable role in the prevention of various
degenerative diseases (Challa et al, 1997;
Diplock et al, 1998; Willett and Willett,
that are recommended at present as opti-
mal sources of such components, the sup-
plementation of human diet with spices or
herbs, containing especially high amounts
of compounds capable of deactivating free
radicals (Madsen and Bertelsen, 1995). The
benefits resulting from the use of natural

1
Natural Compounds Chemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt.
2
Flavors and Aromatic Department, National Research Centre,12311 Dokki,Cairo, Egypt.
3
Pathology Department, National Research Centre,12311 Dokki, Cairo, Egypt.
4
Molecular Cell and Developmental Biology, University of Texas at Austin, Austin 78712, USA. *Correspond-
ing author: Tel: 202-22337651, Fax: 202-33370931, Email: mnassar_eg@yahoo.com

47
48 Mahmoud I. Nassar,
products rich in bioactive substances has
promoted the growing interest of phar-
maceutical, food and cosmetic industries.
Syzygium species (Fam. Myrtaceae) have
been reported to possess antibacterial
(Shafi et al, 2002) and anti-inflammatory
activity (Muruganadan et al, 2001). It was
reported that the buds of Syzygium aromati-
cum (L.) Merr. & Perry (clove) were used in
folk medicine as diuretic, odontalgic, sto-
machic, tonicardiac, aromatic condiment
properties and condiment with carmina-
tive and stimulant activity (Boulos, 1983).
The antimicrobial activity of the essential
oils from clove and rosemary (Rosmarinus
officinalis L.) has been tested alone and in
combination (Fu et al, 2007). In addition,
the antimicrobial activity of clove essentials
oil have been studied against a large num-
ber of multi-resistant Staphylococcus epi-
dermidis as well as the composition of the
oil by GC/MS analysis (Chaieb et al, 2007).
The antioxidant activity of commercial
clove leaf essential oil (Eugenia carophyl-
lus) and the main constituent eugenol was
tested (Jirovetz et al, 2007). Cytotoxicity
of clove oil and its major components has
been investigated (Prashar et al, 2006). Se-
veral compounds from S. aromaticum have
been found to possess growth inhibitory
activity against oral pathogens, namely
5, 7-dihydroxy-2-methylchromone-8-C--
D-glucopyranoside, biflorin, kaempferol,
rhamnocitrin, myricetin, gallic acid, ellagic
acid and oleanolic acid (Cai and Wu, 1996).
Also, an orsellinic acid glucoside has been
isolated from S. aromaticum (Charles et al,
1998). Recently, flavonoide triglycosides
have been isolated (Nassar, 2006).
The evaluation of antioxidant properties
of the raw material allows the determina-
tion of its suitability as high quality food
beneficial for human health and therefore
is of considerable importance. The aim of
this study was to isolate and identify the
volatile components and nonvolatile com-
pounds (limonin and ferulic aldehyde and
several flavonoids) via solvent extraction,
et al.
from S. aromaticum as well as determine
the antioxidant activity of the extracts and
isolated flavonoids by using 2, 2-diphenyl-
2-picrylhydrazyl (DPPH) and the hepatopro-
tective activity.
RESULTS AND DISCUSSION
The n-hexane extract of the buds of S. aro-
maticum gave an orange oil with a charac-
teristic clove odor. The volatile compounds
of the hexane extract were determined by
GC/MS analyses (Table 1). Because the
authentic compounds of the most of these
components were available, the quantitative
calculations were based upon the relative
areas of the corresponding GC signals. GC/
MS analyses also established the percent-
age composition of the 16 volatiles detected
in the n-hexane extract of the buds (Table
1); eugenol (71.56%) and eugenol acetate
(8.99%) were the major components. Frac-
tionation of the methylene chloride extract
of the buds on silica gel and Sephadex LH-
20 columns afforded a tetrahentriterpene,
limonin (1) and an aromatic aldehyde, fe-
rulic aldehyde (2), along with eugenol. The
ethanol extract was subjected to polyamide
column chromatography eluted with wa-
ter/methanol step gradient. The obtained
fractions were further purified on Sepha-
dex LH-20 columns to give the flavonoids
tamarixetin 3-O--D-glucopyranoside (3),
ombutin 3-O--D-glucopyranoside (4) and
quercetin (5) (Fig. 1).
The CI/MS spectrum of 1 showed a
molecular ion peak [M+1]+ at m/z 471,
C26H30O8. HRCI showed an ion peak at
m/z 471.20113 (calc. 471.20189). 1H-
NMR and 13C-NMR spectral data of 1 were
identical to that previously reported for
limonin (Patra et al, 1988). The 13C-NMR
spectrum showed 26 nonequivalent carbon
resonances, three of which appeared at
206, 169 and 166.6 ppm and represented
three carbonyl groups. A DEPT experiment
showed the presence of four methyl groups,
Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) Rev. Latinoamer. Qum. 35/3 (2007) 49

Table 1: The volatile compounds identified in the n-hexane extract of Syzygium aromaticum buds by using
GC-MS

No. Compound Conc. (%) Type Identification method

1 p-Cymene 0.9 M MS & KI
2 5-Hexene-2-one 0.67 LOC MS & KI
3 Thymol 0.87 LOC MS & KI
4 Eugenol 71.56 LOC MS & KI & AU
5 Eugenyl acetate 8.99 LOC MS & KI & AU
6 Caryophyllene oxide 1.67 LOC MS & KI
7 Guaiol 0.90 HOC MS & KI
8 Benzene-1-butylheptyl 0.55 LOC MS
9 Nootkatin 1.05 S MS & KI
10 Isolongifolanone (trans) 0.86 S MS & KI
11 Hexadecanoic acid 0.50 LOC MS
12 9,17-Octadeca-dienal 0.24 HOC MS
13 Octadecanoic acid butyl ester 0.33 HOC MS
14 Phenol-4-(2,3-dihydro-7 0.98 HOC MS
-methoxy-3-methyl-5-
(1-propenyl)-2 -benzofurane
15 Dodecatrienoic acid-3,7, 0.38 HOC MS
11- trimethylethyl ester
16 Vitamin E acetate 0.43 HOC MS
M, monoterpene; S, sesquiterpene; LOC, lightly oxygenated compound; HOC, Heavily oxygentaed compound;
MS, confirmed by comparison with mass spectrum; KI, confirmed by comparison with Kovats index on a
DB5 column (Adams 1995); AU: authentic compound; Conc. (%) based on peak area integration

Fig. 1. Chemical structures of isolated compounds.

50 Mahmoud I. Nassar,
five methylenes and nine methine groups.
Limonin is reported here from S. aromati-
cum for the first time.
The CI/MS of 2 showed a molecular ion
peak [M+1]+ at m/z 179, C10H10O3. 1H-NMR
spectral data of 2 were identical to that previ-
ously reported for ferulic aldehyde (Grande
et al, 1985). 13C-NMR of compound 2 showed
10 nonequivalent carbon resonances, one
of which appeared at 193.5 and was attri-
buted to an aldehydic group and another one
appeared at 56.01 assigned to a methoxyl
group. The spectrum also showed in the
downfield region, six peaks of a trisubsti-
tuted benzene ring together with two reso-
nances for two olefinic carbons.
The ESI/MS of 3 showed a molecular
ion peak [M+1]+ at m/z 479, C22H22O12. It
showed UV spectral data with diagnostic
reagent identical to those of 4- substi-
tuted flavonol glycoside. Acid hydrolysis
of 3 afforded glucose and tamarixetin. The
1
H-NMR spectrum of 3 showed signals
pattern of a quercetin moiety in the down
field region, a methoxyl singlet appeared
at 3.83 and a doublet at 5.55 (J = 7.6
Hz) was assigned to an anomeric proton
of glucopyranosyl moiety. The 13C-NMR of
3 showed 22 nonequivalent carbon reso-
nances, one of which was for a carbonyl
at 177.5 and another was for a methoxyl
at 55.75, in addition to six peaks were
attributed to the glucose moiety (Agrawal
and Banzal, 1989). The protonated carbons
were assigned using HMQC. In HMBC, the
anomeric proton showed cross peak with
C-3 at 133.0, whereas the methoxyl group
showed correlation with C-4 at 146.9.
These data confirmed that compound 3
was tamarixetin 3-O-b-D-glucopyranoside
(Harborne, 1999), which was also isolated
for the first time from S. aromaticum.
The ESI/MS of 4 showed a molecular
ion peak [M+1]+ at m/z 493, C23H25O12. It
showed UV spectral data with diagnostic
reagent identical to those of 7, 4- disub-
stituted flavonol glycoside. Acid hydrolysis
of 4 afforded glucose and ombutin. The 1H-
et al.
NMR spectrum of 4 showed a signal pattern
very similar to that of 3 with an extra me-
thoxyl singlet at 3.85. Also, the 13C-NMR
of 4 was similar to that of 3 with an extra
methoxyl carbon resonance at 56.1. The
protonated carbons were assigned using
HMQC. Similarly, the locations of the two
methoxyl groups at C-7 and at C-4 as well
as the location of glucose moiety at C-3
were all established by HMBC correlations.
These data confirmed that compound 4 is
ombutin 3-O-b-D-glucopyranoside, also
isolated for the first time from S. aromati-
cum, previously reported only from Gyn-
ostemma yixingense (Fam. Cucurbitaceae)
(Si et al., 1994).
Antioxidant activity
Many aromatic plants and spices especially
clove buds and their essential oils have
been known to support various biological
activities such as antimicrobial and antioxi-
dant properties (Fu et al, 2007). The radical
scavenging effects (percentage of quenched
radicals) were determined for clove buds
extracts and their constituents. The clove
buds extracts or their constituents when
mixed with DPPH decolorized it due to
hydrogen donating ability. All the tested
samples (n-hexane, methylene chloride
and ethanol extracts as well as quercetin,
compound 3 and 4) revealed scavenging
effects on DPPH (10 to 93 %) as shown in
Fig. (2).
Antioxidants are believed to neutralize
the free radicals in lipid chains by con-
tributing a hydrogen atom usually from
a phenolic hydroxyl group, which in turn
converts phenolic groups into stable free
radicals that do not intiate or propagate
further oxidation of lipids.
It was observed that the scavenging
activity of volatile extract of clove buds at
all concentrations from 50 to 400 mg/ml
is rather strong (42 -83 %). The remar-
kable antioxidant activity of hexane extract
Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) Rev. Latinoamer. Qum. 35/3 (2007) 51

Fig. 2: Free radical scavenging (%) of different extracts and isolated flavonoids of Syzygium aromaticum buds.
All the values of DPPH inhibition % aree means standard deviation (n=3).

might be due to the higher concentration
of phenolic compounds such as euge-
nol (71.56%), eugenol acetate (8.99 %)
and thymol (0.87 %). These results are
in accord with previous literature (Lee
and Shibamoto, 2001; El-Ghorab and El-
Massry, 2003).
The dichloromethane and ethanol ex-
tracts as well as of the isolated flavonoids
of S. aromaticum buds were found to act as
strong free radical scavengers in compari-
son with commercial antioxidants BHT as
indicated by DPPH assays (Fig. 2).
All the extracts and the isolated flavo-
noids exhibited potential antioxidant activity
against DPPH radicals at different concen-
trations (50 to 400 mg/mL). All extracts of
clove buds at higher concentration (400
mg/ml) have remarkable inhibition of DPPH
radical scavenging activity (45 to 93 %) in
comparison with 400 mg/ml BHT (95 %) (Fig.
2). It is well known that free radicals play
an important role in autoxidation of unsatu-
rated lipids in food stuffs (Kaur et al, 1991).
Quercetin has a moderate antioxidant ac-
tivity (46 %) at 400 mg/mL in comparison
with BHT (70 %) at 50 mg/mL. Our results
are consistence with Miller (1996), who
found that quercetin scavenges oxygen-free
radicals, and inhibits the enzyme xanthine
oxidase.
Among the tested extracts, the ethanol
extract of the clove buds showed remar-
kable scavenging activity (93%), as compared
with synthetic antioxidants such as BHT
(95%). These results demonstrated that
the extracts of S. aromaticum buds and the
isolated flavonoids have effective activity as
hydrogen donors and as primary antioxi-
dants by reacting with lipid radicals.
Hepatoprotective study:
As the ethanol extract of clove showed the
high antioxidant activity, this study evalu-
ates the hepatoprotective activity of it on
the paracetamol- induced liver injury.
The serum biochemical analysis indi-
cated that paracetamol treatment resulted
in a significant increase (P < 0.05) of ALT
(41.1 0.8 u/L), AST (44 2.7 u/L), and
52 Mahmoud I. Nassar,
alkaline phosphatase levels (199.8 21 u/
L) as compared with those of control (29.5
2.3, 36 1.6 and 130 8 u/L respectively).
On the other hand, pretreatment the etha-
nol extract of clove succeeded in restoring
all the biochemical parameters towards the
normal values of the controls (ALT (26.1
1.3 u/L), AST (33 3.1 u/L), and alkaline
phosphatase levels (164.6 4.6 u/L). The
activities of enzymes AST, ALT and ALP
in serum are used routinely to assess the
functional status of the liver in both clinical
and experimental settings. They are used
as serum markers of hepatic damage. Ele-
vated levels of these enzymes in serum in
paracetamol-treated group point to liver
dysfunction. These findings were confirmed
by histological observations of liver. Liver
sections from control rats showed normal
lobular architecture and normal hepatic
cells with a well-preserved cytoplasm and
well-defined nucleus and nucleoli (Fig.
3a). Histopathological examination of the
livers of animals given only the ethanol
extract of clove showed no significant
morphological changes, as compared to
animals in the control group (Fig. 3b). Liver
sections from animals administered with
paracetamol showed marked hepatocytes
necrosis especially in the centrolobular,
sinusoidal congestion, broad infiltration
of the lymphocytes loss of cell boundaries
and hepatic architecture, and ballooning
degeneration. Some cells showed loss of
nucleus and nucleoli. Also, areas of edema
were found (Fig. 3c-e). Pretreatment with
the ethanol extract of clove showed normal
lobular structure with hardly ascertainable
regenerative activity in paracetamol-treated
animals (Fig. 3-f).
The rise in serum levels of ALP, AST and
ALT has been attributed to the damaged
structural integrity of the liver (Chenoweth
and Hake, 1962); because these are cyto-
plasmic in location and are released into
circulation after cellular damage (Sallie et
al., 1991). The extent of hepatic damage is
assessed by the level of increased cytoplas-
et al.
mic enzymes (AST and ALT) in circulation
(Sallie et al., 1991). Zimmerman and Seeff,
(1970) reported that due to liver injury,
the transport function of the hepatocytes
gets disturbed, resulting in the leakage of
plasma membrane, thereby causing an
increased enzyme level in the serum.
The reversal of increased serum en-
zymes in paracetamol-induced liver dama-
ge by the ethanol extract of clove may be
due to the prevention of leakage of the
intracellular enzymes by its membrane
stabilizing activity, which in agreement with
the commonly accepted view that serum
levels of transaminases return to normal
with healing of hepatic parenchyma and
the regeneration of hepatocytes (Thabrew
et al, 1987).
From the foregoing findings it can be
speculated that the observed increasing
effect of ALT, AST and ALP levels in serum
in rats treated with paracetamol alone
were due to hepatocellular damage and the
ethanol extract of clove afforded protection
from such paracetamol-induced liver da-
mage. A possible mechanism for protection
by clove against paracetamol-induced liver
damage could involve clove components
acting as free radical scavengers intercep-
ting those radicals involved in paracetamol
metabolism by microsomal enzymes. Thus,
by trapping oxygen related free radicals
clove extract could hinder their interaction
with polyunsaturated fatty acids and would
abolish the enhancement of lipid peroxida-
tive processes leading to MDA formation.
Therefore, clove extract may be a useful
agent for the normalization of paracetamol
induced impaired membrane function.
Thus, from the foregoing findings, it was
observed that the ethanol extract of clove
is a promising hepatoprotective agent and
this hepatoprotective activity may be due
to its antioxidant and normalization of im-
paired membrane function activity.
Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) Rev. Latinoamer. Qum. 35/3 (2007) 53

Fig. 3: Photomicrographs of liver of rats show A) control with normal structure, B) rats given the extract of
clove (500 mg/kg b. wt.) with normal structure, C, D, E) rats treated with paracetamol (650 mg/kg b.wt)
showing different lesions, loss of cell boundaries and hepatic architecture and marked hepatocytes necro-
sis, ballooning degeneration and loss of nucleus and nucleoli (c) broad infiltration of the lymphocytes (D)
sinusoidal congestion and area of edema (E) and F) rats given the extract and paracetamol with normal
structure (H & E X 300).
54 Mahmoud I. Nassar,
EXPERIMENTAL
Plant material
The buds of S. aromaticum were obtained
from a local market (Harraz Company for
Medicinal Plants, Cairo, Egypt). A voucher
specimen has been deposited in the National
Research Centre Herbarium, Cairo, Egypt.
Extraction and isolation
The dry powdered buds of S. aromaticum
(500 g) were subjected to successive extrac-
tions, using n-hexane, dichloromethane
and ethanol to give 14, 27 and 30 mg of
extracts, respectively. The n-hexane extract
was analyzed for its volatile components
using GC/MS. The dichloromethane ex-
tract was subjected to Si-gel CC eluted by
CH2Cl2/ethyl acetate step gradient. The
eluted fractions were subjected to repetitive
separation and purification on Sephadex
LH-20 columns eluting with CH2Cl2-MeOH
(1: 1) to give 12 mg of limonin, 8 mg of
ferulic aldehyde and 10 mg eugenol. The
ethanol extract was subjected to polyamide
column chromatography eluted with wa-
ter/methanol step gradient. The obtained
fractions were further purified on Sephadex
LH-20 columns to give 22 mg of tamarix-
etin 3-O-b-D-glucopyranoside (3),36 mg of
ombutin 3-O-b-D-glucopyranoside (4) and
14 mg of quercetin (4).
Limonin (1):
CIMS m/z (rel. int.), 471 (100), 13C-NMR
(CDCl3, 125 MHz): 79.2 (C-1), 35.6 (C-2),
169.0 (C-3), 80.3 (C-4), 60.6 (C-5), 36.4 (C-
6), 206.0 (C-7), 51.3 (C-8), 48.1 (C-9), 45.9
(C-10), 18.9 (C-11), 30.8 (C-12), 37.9 (C-13),
65.7 (C-14), 53.9 (C-15), 166.6 (C-16), 77.8
(C-17), 65.3 (C-18), 119.9 (C-19), 141.1 (C-
20), 109.7 (C-21), 143.2 (C-22), 20.7 (C-23),
17.6 (C-24), 21.4 (C-25), 30.2 (C-26).
et al.
Ferulic aldehyde (2):
CI/MS m/z (rel. int.), 179(100) [M+1]+, 13C-
NMR (CDCl3, 125 MHz): 146.9 (C-1), 148.9
(C-2), 109.4 (C-3), 126.7 (C-4), 124.0 (C-5),
114.9 (C-6), 152.8 (C-7), 126.5 (C-8), 193.5
(C-9), 56.0 (OMe).
Tamarixetin 3-O--D-glucopyranoside (3):
ESI/MS m/z (rel. int.), 479 (60), 13C-NMR
(DMSO-d6, 125 MHz): 156.4 (C-2), 133.0
(C-3), 177.5 (C-4), 161.3 (C-5), 98.8 (C-6),
164.2 (C-7), 93.8 (C-8), 156.5 (C-9), 104.1
(C-10), 121.1 (C-1`), 113.5 (C-2`), 149.5 (C-
3`), 146.9 (C-4`), 115.2 (C-5`), 122.1 (C-6`),
55.75 (3`-OMe),100.8 (C-1``), 74.4 (C-2``),
76.5 (C-3``), 70.0 (C-4``), 77.5 (C-5``), 60.7
(C-6``).
Ombuin 3-O--D-glucopyranoside (4):
ESI/MS m/z (rel. int.), 493 (45)
13
C-NMR (DMSO-d6, 125 MHz): 156.3 (C-
2), 133.2 (C-3), 177.5 (C-4), 160.9 (C-5),
97.9 (C-6), 165.1 (C-7), 92.3 (C-8), 156.6
(C-9), 105.0 (C-10), 120.9 (C-1`),113.5 (C-
2`), 149.5 (C-3`), 146.9 (C-4`), 115.2 (C-5`),
122.2 (C-6`), 56.1 (7-OMe), 55.7 (4`-OMe),
100.7 (C-1``), 74.3 (C-2``), 76.4 (C-3``), 69.8
(C-4``), 77.5 (C-5``), 60.6 (C-6``).
DPPH radical scavenging assay:
Radical scavenging activity of clove buds
extracts and their constituents against
stable DPPH (2, 2-diphenyl-2-picrylhydra-
zyl hydrate was determined spectropho-
tometrically. When DPPH reacts with an
antioxidant compound, which can donate
hydrogen, it is reduced. The changes in
color (from deep violet to light yellow) were
measured at 517 nm on a UV/visible light
spectrophotometer. Radical scavenging
activity of extracts was measured accord-
Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) Rev. Latinoamer. Qum. 35/3 (2007)
ing to Miliauskas et al, 2004, as described
below.
Extract solutions (volatile oils, dichlo-
romethane and ethanol extracts) were pre-
pared by dissolving 0.1 g of dry extract in
10 ml of methanol. The solution of DPPH
in methanol (6x10-5 M) was prepared daily,
before UV measurements. Various concen-
trations of each extract (50, 100, 200 and
400 mg/mL) were added to solutions (1 ml)
of DPPH in methanol. The mixtures were
shaken vigorously and left to stand at room
temperature for 30 min; the absorbances
of the resulting solutions were measured
spectrophotometrically at 517 nm. In this
assay, the percentage of DPPH reduction
by different extracts of clove was compared
to that of BHT.
The experiment was carried out in
triplicate. Radical scavenging activity was
calculated by the following formula:
% Inhibition = [(AB-AA)/AB]X 100,
AB absorption of blank sample (t=0 min);
A absorption of tested extract solution
(t=30 min).
Instrumentation:
An HP model 6890 GC interfaced to an HP
5791A mass selective detector (GC/MS)
was used for mass spectral identification
of the GC components at MS ionization
voltage of 70 eV. A 30 m x 0.25 mm i.d. (df
= 0.25 lm) DB-5 bonded-phase fused-silica
capillary column (J&W Scientific) was used
for GC. The linear velocity of the helium
carrier gas was 30 cm/s. The injector and
the detector temperatures were 250 C. The
oven temperature was programmed from
35 to 220 C at 3 C /min and held for 40
min. Kovats indices were determined by
co-injection of the sample with a solution
containing homologous series of n-hydro-
carbons (C8-C26) under the same condi-
tions as described above.
55
The separated components were identi-
fied by matching with NIST mass spectral
library data, and by comparison of Kovats
indices with those of authentic components
and with published data (Adams, 1995).
The quantitative determination was carried
out based on peak area integration.
Hepatoprotective assay
Paracetamol hepatotoxicity was induced
in female albino rats of Sprage Dewally
strain weighing between 170 and 200 g.
Animals bred and maintained in the Lab
Animal House, National Research Centre,
Cairo. Four groups of animals (6 rats each)
were used in this study. Group 1, control,
group 2 treated with the ethanol extract
of clove (500 mg/kg b.wt.) for seven days,
group 3, treated with paracetamol (i.p.
at a dose of 650 mg/kg b.wt according to
Parmar et al, (1995) and group 4, treated
with the extract for seven days and at the
eighth day injected with paracetamol as
in group 3. After 24 h of the last treat-
ment blood samples were withdrawn
from ratino bulber venous plexus with
under light anaesthesia and were kept at
room temperature to coagulate. The blood
samples were centrifuged and the sepa-
rated serum was used for the estimation
of AST, ALT, and ALP. The activity of AST
and ALT were measured according to the
method described by Reitman and Frankel
(1957). The estimation of ALP was carried
out by the methods of King and Armstrong
(1980).
Animals were then sacrificed and dis-
sected. Their livers were taken out, washed
with water, dried gently with filter paper
and preserved in 10% formalin. Sections
(45 mm thick) were prepared and then
stained with hematoxylin and eosin dye for
microscopic examination.
All data were expressed as means
standard errors, and analyzed with
one-way analysis of variance (ANOVA).
56 Mahmoud I. Nassar, et al.

Students t-test was used to calculate sta- Acknowledgments

tistical signicance by SPSS software. P <
0.05 and <0.01 were considered statisti- T. J. M. acknowledges support from the
cally signicant. Robert A. Welch Foundation (Grant F-
130).

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