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Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) and their antioxidant activity


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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.
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Mahmoud I. Nassar1*, Ahmed H. Gaara1, Ahmed H. El-Ghorab2, Abdel-Razik H. Farrag3, Hui Shen4,
Enamul Huq4 and Tom J. Mabry4
1Natural Compounds Chemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt.
2 Flavors and Aromatic Department, National Research Centre,12311 Dokki,Cairo, Egypt.
3Pathology Department, National Research Centre,12311 Dokki, Cairo, Egypt.
4Molecular Cell and Developmental Biology, University of Texas at Austin, Austin 78712, USA. *Correspond-
ing author: Tel: 202-22337651, Fax: 202-33370931, Email:
(Received: April 2007; Accepted October 2007)
Sixteen volatile compounds were identied 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 avonoids tamarixetin 3-O-b-D-glucopyranoside, ombuin 3-O-b-D-glu-
copyranoside and quercetin were isolated from the ethanol extract; identications
of all these compounds were established by chemical and spectroscopic methods
including 1D and 2D NMR. This is the rst report of limonin, ferulic aldehyde and
these avonoids from this plant. All extracts and the isolated avonoids 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, avonoids, antioxidant activity, hepatoprotective activity.
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,
2002). In addition to fruits and vegetables
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
benets resulting from the use of natural
48 MahMoud I. Nassar, et al.
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
(Sha et al, 2002) and anti-inammatory
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
ofcinalis 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-β-
D-glucopyranoside, biorin, 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, avonoide 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
benecial 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 avonoids) via solvent extraction,
from S. aromaticum as well as determine
the antioxidant activity of the extracts and
isolated avonoids by using 2, 2-diphenyl-
2-picrylhydrazyl (DPPH) and the hepatopro-
tective activity.
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 puried on Sepha-
dex LH-20 columns to give the avonoids
tamarixetin 3-O-β-D-glucopyranoside (3),
ombutin 3-O-β-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 δ
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. Quím. 35/3 (2007) 49
Fig. 1. Chemical structures of isolated compounds.
Table 1: The volatile compounds identied in the n-hexane extract of Syzygium aromaticum buds by using
No. Compound Conc. (%) Type Identication 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
(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, conrmed by comparison with mass spectrum; KI, conrmed by comparison with Kovat’s index on a
DB5 column (Adams 1995); AU: authentic compound; Conc. (%) based on peak area integration
50 MahMoud I. Nassar, et al.
ve methylenes and nine methine groups.
Limonin is reported here from S. aromati-
cum for the rst 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 δ 193.5 and was attri-
buted to an aldehydic group and another one
appeared at δ 56.01 assigned to a methoxyl
group. The spectrum also showed in the
downeld region, six peaks of a trisubsti-
tuted benzene ring together with two reso-
nances for two olenic 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 avonol glycoside. Acid hydrolysis
of 3 afforded glucose and tamarixetin. The
1H-NMR spectrum of 3 showed signals
pattern of a quercetin moiety in the down
eld region, a methoxyl singlet appeared
at δ 3.83 and a doublet at δ 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 δ 177.5 and another was for a methoxyl
at δ 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 δ 133.0, whereas the methoxyl group
showed correlation with C-4’ at δ 146.9.
These data conrmed that compound 3
was tamarixetin 3-O-b-D-glucopyranoside
(Harborne, 1999), which was also isolated
for the rst 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 avonol glycoside. Acid hydrolysis
of 4 afforded glucose and ombutin. The 1H-
NMR spectrum of 4 showed a signal pattern
very similar to that of 3 with an extra me-
thoxyl singlet at δ 3.85. Also, the 13C-NMR
of 4 was similar to that of 3 with an extra
methoxyl carbon resonance at δ 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 conrmed that compound 4 is
ombutin 3-O-b-D-glucopyranoside, also
isolated for the rst 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. Quím. 35/3 (2007) 51
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 avonoids
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 avo-
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
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 avonoids 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 signicant increase (P < 0.05) of ALT
(41.1 ± 0.8 u/L), AST (44 ± 2.7 u/L), and
Fig. 2: Free radical scavenging (%) of different extracts and isolated avonoids of Syzygium aromaticum buds.
All the values of DPPH inhibition % aree means ± standard deviation (n=3).
52 MahMoud I. Nassar, et al.
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 ndings were conrmed
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 inltration
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-
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 ndings 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 ndings, 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. Quím. 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 inltration 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, et al.
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 purication 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 puried 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).
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-β-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
Ombuin 3-O-β-D-glucopyranoside (4):
ESI/MS m/z (rel. int.), 493 (45)
13C-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. Quím. 35/3 (2007) 55
ing to Miliauskas et al, 2004, as described
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:
AB absorption of blank sample (t=0 min);
A absorption of tested extract solution
(t=30 min).
An HP model 6890 GC interfaced to an HP
5791A mass selective detector (GC/MS)
was used for mass spectral identication
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 Scientic) was used
for GC. The linear velocity of the helium
carrier gas was 30 cm/s. The injector and
the detector temperatures were 250 ºC. The
oven temperature was programmed from
35 to 220 ºC at 3 ºC /min and held for 40
min. Kovat’s 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.
The separated components were identi-
ed by matching with NIST mass spectral
library data, and by comparison of Kovat’s
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
Animals were then sacriced and dis-
sected. Their livers were taken out, washed
with water, dried gently with lter paper
and preserved in 10% formalin. Sections
(4–5 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).
% Inhibition = [(AB-AA)/AB]X 100,
56 MahMoud I. Nassar, et al.
Student’s t-test was used to calculate sta-
tistical signicance by SPSS software. P <
0.05 and <0.01 were considered statisti-
cally signicant.
T. J. M. acknowledges support from the
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... Similarly, several studies demonstrated that trans-cinnamaldehyde constitutes the major compound in cinnamon bark EOs, ranging from 31 to 44.2% (Möllenbeck et al. 1997;Jantan et al. 2008;Ribeiro et al. 2020). Likewise, eugenol is widely mentioned in literature as clove EOs main constituent (percentage > 70%) (Nassar et al. 2007;Xu et al. 2016;Maimulyanti et al. 2019). On the other hand, many investigations showed different percentages of these compounds in respective EOs (LALAMI Abdelhakim EL OUALI et al. 2013;Purwanti et al. 2018;Alizadeh Behbahani et al. 2020). ...
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In the food industry, the development of microbial biofilms is a serious problem that leads to the contamination and deterioration of food products. To overcome that, our aim consists of searching for natural antimicrobial and non-toxic compounds (essential oils EOs), which might be used alone or adsorbed on natural biopolymer films (chitosan). In this work, the antioxidant activity of eight EOs was evaluated by DPPH radical-scavenging method while their antibacterial activity was determined by diffusion on agar and microdilution methods. Among all tested EOs, Eugenia caryophyllus, Cinnamomum zeylanicum Blume and Thymus satureioides Cosson showed high antioxidant activities at the concentration of 25.6 mg/mL, with respective values of (86.26%, 81.75%, and 76%), and strong antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus hirae, with (MIC) values ≤ 4 µL/mL. At the concentration of 1 µL/mL, these EOs tested alone, showed values of antibiofilm-forming activity ranging from 79.43 to 99.33% and from 44.18 to 94.17%, when they are adsorbed onto chitosan film. These promising results confirm that these three EOs have a good potential for an eventual application in the food industry, as antimicrobial and antioxidant agents, or as active biodegradable food packaging, if combined with chitosan.
... Six sesquiterpenes, α-cubebene (1.3%), α-copaene (0.4%), β-humulene (9.1%), β-caryophyllene (64.5%), γ-cadinene (2.6%) and δ-cadinene (2.6%) have been characterized from the hydrocarbon fraction of the freshly distilled Indian clove bud oil (Gopalakrishnan et al., 1984). Nassar et al. (2007) evaluated the chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) using GC-MS, chemical and spectroscopic methods including 1D and 2D NMR for identifications of the compounds. ...
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Spices-dried aromatic parts of plants (leaves, seeds, bark, roots, rhizomes, buds, etc) used to enhance flavour, taste and colour (sensory quality) of foods, are increasingly finding other useful roles in healthcare beyond their primary use as culinary organoleptic enhancers. Several spices are currently being investigated for their potential health benefits, because of the failing efficacy, toxicity and high cost associated with conventional drugs. One such spice: Syzygium aromaticum (L.) Merr. and L.M.Perry [Myrtaceae] (Clove), has a multi-dimensional role in diet, medicine, functional foods and nutraceuticals, agriculture, among other industries. Peer-reviewed articles, mostly from PubMed and Google Scholar, were consulted for the purpose of this review. The nutritional and phytochemical contents, selected biological activities as well as some functional foods and beverages of clove and their uses for human health are presented. Although these observations are largely empirical, the efficacious attributes have led to their pharmacological applications in the indigenous system of medicine all over the world and bridge between food, diet and medicine. Considering the GRAS status of clove, more studies on bioavailability, accumulation, toxicity, dosage and efficacy of clove as a spice drug or functional foods in biological systems especially in humans are required. Meanwhile, clove and its products can be used as co-adjuvants in the prevention, treatment and management of chronic diseases. Further, many applications of clove in food, health, cosmetics, pharmaceutics, nanoparticles and agricultural industries are still open for investigations.
Clove or Syzygium aromaticum (L.) Merr. & L.M.Perry is a valuable spice that has been used for centuries. The flower buds are commercially produced mainly from Indonesia, Madagascar, Tanzania, Sri Lanka, and Comoros. Besides comprising fiber, vitamins, and minerals, the buds possess various biologically active secondary metabolites such as phenylpropanoids, terpenoids, chromone glycosides, tannins, and phenyl glycosides. This chapter reviews the botanical aspect of clove buds and their compositions. Additionally, since the clove buds have been used directly for many purposes, such as ingredients in food and traditional medicine recipes and kretek cigarettes, clove buds’ applications are also scoped in these fields.
In this chapter, the composition and biofunctional characteristics of clove (Syzygium aromaticum) extracts were reviewed. Extraction is a key process for gaining valuable ingredients from plants and plant-derived products. There are several extraction techniques, including conventional and novel methods in the literature. Solvent and Soxhlet extraction, maceration, hydro- or steam-distillation, squeezing, and cold processing are the most known and used techniques. In contrast, microwave, pulsed electric field, ultrasound-assisted extraction, pressurized liquid extraction, and supercritical fluid extraction methods are novel techniques that have been intensively used in the plant extraction process. Syzygium aromaticum (syn. Eugenia caryophyllata Thunb., Eugenia aromaticum), commonly known as clove, is a tree from the Myrtaceae family and the second most important spice in the world trade. The plant has been used as medicine traditionally and flavoring agent in different parts of the world. The leaves, buds, and stems of the cloves can be used to prepare extracts and are generally used as whole plants. The clove extracts comprise the major compounds such as eugenol, eugenyl acetate, and caryophyllene. Among these, eugenol is the active ingredient of clove and is a phenolic and aromatic compound. The antibacterial, antifungal, antioxidant, anticancer, anti-inflammatory, antidiabetic, and antistress potentials of eugenol were reported in several studies. As well as eugenol, clove extracts have also been studied for their health-promoting effects. Clove extracts could also be utilized in the food industry as a natural antioxidant for better oxidative stability of food substances and as an anticarcinogenic agent in dentistry. In this chapter, extraction techniques used for the clove, the compositional and functional properties of clove extracts, and also using the potential for different industries are discussed.
The clove (Syzygium aromaticum) is an aromatic evergreen plant that belongs to the family of Myrtaceae. Different parts of this plant have been used in the traditional system of medicine for the treatment of various kinds of ailments. Clove is a conventional spice that has been used for food preservation and exhibited a wide range of pharmacological activities, including antibacterial, antioxidant, antifungal, anti-inflammatory, anticancer, nematicidal, anesthetic, herbicidal, and insecticidal activities. The phytochemical analysis of the plant has reported the presence of various classes of phytoconstituents, including triterpenes, flavonoids, chromones, tannins, and steroids. The gas chromatography coupled with mass spectrometry (GC-MS) analysis of n-hexane extract of buds of the plant has reported the presence of 16 volatile compounds with eugenol (71.5%) and eugenol acetate (8.99%) as the major constituents. The terpenoids, limonin, and ferulic aldehyde, along with eugenol, are also reported from the dichloromethane extract of buds of S. aromaticum. The ethanol extract of the buds of S. aromaticum reported the presence of some bioactive flavonoids such as tamarixetin 3-O-β-d-glucopyranoside, ombuin 3-O-β-d-glucopyranoside, and quercetin. Some other important bioactive phytoconstituents in clove included oleanolic acid lactone, β-sitosterol, nigricin, flavaellagic acid, 2α-hydroxyolanolic acid, 3β-hydroxy-11-oxo-olean-12-en-28-oic acid, and β-sitosterol 3-O-β-d-glucopyranoside. A ferulic acid derivative, sabrinic acid, benzophenone derivative, 4,4′-diacetoxybenzophenone, and two existing compounds, kaempferol-3,5-dimethyl ether and 4-methyl benzoic acid, have also been isolated from S. aromaticum. Some of these isolated compounds have shown promising biological activities.
Liver plays a major role in transforming and clearing the chemicals form the body, but same time it is susceptible to the toxicity of these chemicals. Liver diseases are now become major public health problem worldwide, beside viral infection, autoimmune diseases, infectious agents, toxic chemicals and many drugs at higher doses can cause hepatic injury. The drugs used for the treatment of hepatic disorders have many side effects since all drugs detoxified in the liver with the help of cytochrome p-450 system. Several plants are being used for treatment of liver diseases among people in different areas of the world since ancient time. In the current review we have summarized the information regarding the causes of liver diseases and plants used for the treatment of liver diseases.
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Researchers are increasingly looking to plants as sources of novel ingredients active against vector-borne diseases. Medicinal plant extracts and their metabolites are an attractive source for such products. This study investigated the insecticidal activity of five essential oils extracted from the most common medicinal herbs in Jazan province, Kingdom of Saudi Arabia. Extracted oils and nanoemulsions synthesized from those oils were characterized before application at different concentrations to laboratory-reared fourth-stage larvae of Culex pipens. Basil ( Ocimum bascilicum ) and cumin ( Cuminum cyminum ) essential oils showed moderate larvicidal effect with LC50 81.07 ug/mL and 96.29 ug/mL, respectively. That activity was improved in their nanoemulsion forms, as evidenced by a reduction in the LC50 to 65.19 ug/mL for basil and 64.50 ug/mL for cumin. Clove ( Syzygium aromaticum ), henna ( Lawsonia inermis ) and ginger ( Zingiber officinalis ) oils showed weaker insecticidal activity, with LC50 values of 394 ug/mL, 306 ug/mL, and 494 ug/mL, respectively. Moreover, the nanoemulsion forms of those essential oils did not show any improvement in their insecticidal activity. In conclusion, of the studied plants, the nanoemulsions of basil and cumin showed significant larvicidal activity.
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The green synthesis of silver nanoparticles (AgNPs) and their applications have attracted many researchers as the AgNPs are used effectively in targeting specific tissues and pathogenic microorganisms. The purpose of this study is to synthesize and characterize silver nanoparticles from fully expanded leaves of Eugenia roxburghii DC., as well as to test their effectiveness in inhibiting biofilm production. In this study, at 0.1 mM concentration of silver nitrate (AgNO3), stable AgNPs were synthesized and authenticated by monitoring the color change of the solution from yellow to brown, which was confirmed with spectrophotometric detection of optical density. The crystalline nature of these AgNPs was detected through an X-Ray Diffraction (XRD) pattern. AgNPs were characterized through a high-resolution transmission electron microscope (HR-TEM) to study the morphology and size of the nanoparticles (NPs). A new biological approach was undertaken through the Congo Red Agar (CRA) plate assay by using the synthesized AgNPs against biofilm production. The AgNPs effectively inhibit biofilm formation and the biofilm-producing bacterial colonies. This could be a significant achievement in contending with many dynamic pathogens.
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Increase in food-borne outbreaks has become public health concern worldwide. Exploitation of the antimicrobial properties of dietary spices has become important pharmaceutical tool for controlling food-borne pathogens. This study aimed at evaluating the antibacterial potentials of Ghanaian spices against Listeria monocytogenes and other prevalent food-borne pathogens. In preliminary studies, Listeria was isolated from some food samples. The overall prevalence of Listeria spp. was 23% (13/56). Of the 56 samples examined, 7% showed pathogenic potential for L. monocytogenes. Different solvent extracts of thirteen spices namely Calabash nutmeg, West African black pepper, Aidan, Grains of paradise, Negro pepper, Aniseed, African locust bean, Cinnamon, Black pepper, Clove, Cayenne, Basil, and Rauvolfia were tested for their potential to inhibit clinical and isolated strains of L. monocytogenes using qualitative and quantitative antimicrobial assay methods. Only clove and negro pepper among the thirteen different solvent extracts showed bacteriostatic and bactericidal activity against L. monocytogenes indicated by minimum inhibitory concentrations ranging from 0.05% to 0.4% and minimum bactericidal concentrations ranging from 0.1 to >0.4% under experimental conditions. Time-kill study demonstrated listericidal activity of ethanolic clove and negro pepper extracts indicated by absolute mortality of more than 3 log units at 2x MIC and 4x MIC. GC-MS analysis revealed three and eight major chemical components present in clove and negro pepper respectively. Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus cereus, and B. subtilis also showed satisfactory susceptibilities to ethanolic extracts with MIC ranging from 0.025% to >0.8%. In general, negro pepper showed broad activity eliciting inhibitory effects against all the tested pathogens. The findings suggest that clove and negro pepper may be promising antibacterial candidates for the decontamination and control bacterial pathogens in food and food supply chain.
Extract of Syzygium aromaticum (Clove) is a lipophilic substance with low solubility in aqueous solution. As this may compromise its handling, bioavailability, and bio-absorption, oil-in-water (O/W) emulsion is formulated. Briefly, dried flower buds of cloves were grounded into a fine dry powder and macerated in absolute ethanol solvent for 48 h duration with intermittent shaking at the room temperature. The extracts of the cloves were filtered before being evaporated by a rotary evaporation method (200-75 Mbar; 60°C, 90 rpm) in order to isolate the solvent from the extracts. An O/W emulsion system was prepared by mixing various surfactants (Tween 80, Tween 20, and Span 20) to emulsify the clove oil in the aqueous phase. Initial screening was done on the prepared formulations with centrifugation (3000 rpm, 3 min) to detect for any phase separation in these formulations. The most stable formulation of O/W emulsion of the cloves (no phase separation) which consisted of 34.48% distilled water (aqueous phase), 34.48% clove oil (oil phase), 27.59% Tween 20 (surfactant), and 3.45% Tween 80 (surfactant) were tested for its stability at different storage conditions (8°C [in refrigerator], 25°C [in temperature controlled room], and 40°C [in oven]) at different time points (24 h, 48 h and 72 h). Stability evaluation consists of organoleptic characteristics, centrifugation tests, particle size measurements, microscopic observation, pH determination, and freeze thaw cycle tests. Results showed that the O/W emulsions samples kept at 8°C displayed good stability over 72 h of observation period and could be used for tests in which stability, ease of handling, and improved pharmaceutical characteristics are of importance.
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Flavonoids occur in most plant species, and account for a significant percentage of the chemical constituents of some; e.g. dried green tea leaves contain approximately 30% flavonoids by weight. Flavonoids have been shown to have antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory activity. The potent antioxidant activity of flavonoids-their ability to scavenge hydroxyl radicals, Superoxide anions, and lipid peroxy radicals-may be the most important function of flavonoids, and underlies many of the above actions in the body. Qxidative damage is implicated in most disease processes, and epidemiological, clinical, and laboratory research on flavonoids and other antioxidants suggest their ; use in the prevention and treatment of a number of these. Catechin and its derivatives, oligomeric proanthocyanidins, quercetin and quercetin chalcone, Ginkgo flavone glycosldes, silymarin, and others can be utilized in preventative and treatment protocols for cardiovascular disease, cancer, inflammatory conditions, asthma, periodontal disI ease, liver disease, cataracts and macular degeneration.
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Adams, R. P. 2007. Identification of essential oil components by gas chromatography/ mass spectrometry, 4th Edition. Allured Publ., Carol Stream, IL Is out of print, but you can obtain a free pdf of it at
We examined the effect of paracetamol treatment (650 mg/kg) on the function of the ATPase from rat hepatic mitochondria. The drug treatment caused an overall 35% decrease in ATPase activity, with a complete loss of the high affinity component as determined by substrate kinetic studies. The Km for the intermediate and low affinity components decreased by about 30% without change in Vmax, which may represent a compensatory mechanism. The drug treatment also resulted in a dramatic decrease in the phase transition temperature by about 19°C without affecting the energies of activation of the enzyme. Mitochondrial total phospholipid content increased significantly with a reciprocal decrease in the cholesterol content. The total phospholipid/cholesterol molar ratio increased by 50% after paracetamol treatment. However, phospholipid composition (as % of total) of the mitochondria was unaltered.
The buds of Syzygium aromatica yielded two triterpenoid acids, oleanolic acid and crategolic acid, and a new phenolic glucoside, orsellinic-2-O-β-d-glucopyranoside, from its methanolic extract. The structure was elucidated by spectroscopic methods and acid hydrolysis.