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Antioxidant and protective effect of clove extracts and clove essential oil on hydrogen peroxide treated rats

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The antioxidant, liver-protective and kidney-protective effects of clove extract and clove essential oil were carefully investigated. Total phenolic compound and total flavonoids for water, ethanol and acetone extracts of clove were determined, reducing power assay was used to evaluate these extracts compared with clove essential oil. Acetone extract of clove showed the highest total phenolic, flavonids contents compared with water and ethanol extracts. Both clove essential oil and clove acetone extract appeared the best activities in reducing power assay. The liver-protective, kidney-protective effects and antioxidant activity potential of acetone extract of clove and clove essential oil was also evaluated in male Wistar rats against hydrogen peroxide- (H2O2). Rats dived into four groups, First group was kept as control and feed on Basel diet and water, Second group was treated by 0.5% in drinking water without any other treatment, third group was given acetone extract of clove at 500mg/kg body weight prior to H2O2 administration (0.5% in drinking water), while fourth group was given clove essential oil at 200mg/kg body weight prior to H2O2 administration (0.5% in drinking water). Rats treated with acetone extract of clove and clove essential oil remarkably prevented the elevation of plasma AST, ALT, while increased both plasma total protein and albumin compared with H2O2 treated rats. Also both third and fourth group showed significant decreased in kidney markers (urea and creatinine) compared with H2O2 trated group. Plasma antioxidant state (MDA content and catalase activity) was affected by H2O2 treatment in second group, and both third and fourth group improved this markers. This study suggests that acetone extract of clove and clove essential oil has a liver-protective and kidney-protective effects against H2O2induced oxidative stress and bad effects on both liver and kidney and possess in vitro antioxidant activities.
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International Journal of ChemTech Research
CODEN( USA): IJCRGG ISSN : 0974-4290
Vol.5, No.4, pp 1477-1485, April-June 2013
Antioxidant And Protective Effect Of Clove Extracts And Clove
Essential Oil On Hydrogen Peroxide Treated Rats
Medhat M., Abozid1* and S. M., EL-Sayed1
1Biochemistry Department, Faculty of Agriculture, Menofia University,
Shibin El-Kom, Egypt.
*Corres. author: medh_latef@yahoo.com,
Phone number: 01224208727
Abstract: The antioxidant, liver-protective and kidney-protective effects of clove extract and clove essential
oil were carefully investigated. Total phenolic compound and total flavonoids for water, ethanol and acetone
extracts of clove were determined, reducing power assay was used to evaluate these extracts compared with
clove essential oil. Acetone extract of clove showed the highest total phenolic, flavonids contents compared
with water and ethanol extracts. Both clove essential oil and clove acetone extract appeared the best activities in
reducing power assay. The liver-protective, kidney-protective effects and antioxidant activity potential of
acetone extract of clove and clove essential oil was also evaluated in male Wistar rats against hydrogen
peroxide- (H2O2). Rats dived into four groups, First group was kept as control and feed on Basel diet and water,
Second group was treated by 0.5% in drinking water without any other treatment, third group was given acetone
extract of clove at 500mg/kg body weight prior to H2O2 administration (0.5% in drinking water), while fourth
group was given clove essential oil at 200mg/kg body weight prior to H2O2 administration (0.5% in drinking
water). Rats treated with acetone extract of clove and clove essential oil remarkably prevented the elevation of
plasma AST, ALT, while increased both plasma total protein and albumin compared with H2O2 treated rats.
Also both third and fourth group showed significant decreased in kidney markers (urea and creatinine)
compared with H2O2 trated group. Plasma antioxidant state (MDA content and catalase activity) was affected
by H2O2 treatment in second group, and both third and fourth group improved this markers. This study suggests
that acetone extract of clove and clove essential oil has a liver-protective and kidney-protective effects against
H2O2induced oxidative stress and bad effects on both liver and kidney and possess in vitro antioxidant
activities.
Key words: Clove extract, clove essential oil, H2O2, antioxidant, liver, kidney.
Introduction And Experimental
In the past decades, oxidation mechanisms and free radical role in living systems have gained increased
attention (1). Oxygen uptake inherent to cell metabolism produces reactive oxygen species (ROS). The reaction
of this species with lipid molecules originates peroxyl radicals and their interaction with nucleic acids and
proteins conduces to certain alterations and, therefore, functional modifications (2). This lipid peroxidative
degradation of biomembranes is one of the principle causes of hepatotoxicity (3).
Exposure to H2O2 may cause elevation of superoxide anion and the dangerous hydroxyl (OH•) radical leading
to glomerular dysfunction and renal damage (4)
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1478
The harmful action of the free radicals can, however, is blocked by antioxidant substances, which scavenge the
free radicals and detoxify the organism (5). Antioxidants are compounds that can delay or inhibit the oxidation
of lipid or other molecules by inhibiting the initiation or propagation of oxidizing chain reactions (6)
Antioxidant compounds can scavenge free radicals and increase shelf life by retarding the process of lipid
peroxidation, which is one of the major reasons for deterioration of food and pharmaceutical products during
processing and storage (7). Antioxidants can protect the human body from free radicals and ROS effects.
At present, the most commonly used antioxidants are BHA, BHT, propyl gallate and tert butylhydroquinone.
Besides this BHA and BHT have been suspected of being responsible for liver damage and carcinogenesis (8).
Therefore, there is a growing interest on natural and safer antioxidants (9).
Consequently, the need to identify alternative natural and safe sources of food antioxidants arose and the search
for natural antioxidants, especially of plant origin, has notably increased in recent years (10, 11).
Clove water and ethanol extracts have powerful antioxidant activity against various antioxidant systems in vitro,
moreover, clove buds can be used as easily accessible source of natural antioxidants and as a possible food
supplement or in pharmaceutical applications (12) .
Clove oil is obtained by distillation of the flowers, stems and leaves of the clove tree (Eugenia aromatica or
Eugenia caryophyllata, Fam. Myrtaceae). Clove essential oils have been analyzed by GC-MS and 18
components found in essential oils. These components have been tested for antioxidant properties in an egg
yolk-based thiobarbituric acid reactive substance (TBARS) assay and also undiluted in a β-carotene agar
diffusion assay. The essential oils and the components tested in the TBARS assay have demonstrated some
degree of antioxidant activity (13).
Therefore, the present study was designed to explore the antioxidant effect of different clove extracts and the
protective effect of clove acetone extract and clove essential oil in biological experiment on plasma liver
functions, kidney functions and antioxidant status against H2O2 induced harmful effects.
Chemicals and procedures
1. Chemical reagents (Kits)
Kits for total protein, albumin, urea, creatinine and lipid peroxides (MDA) and ALT, AST, Catalase enzymes
activity were obtained from Diamond Company, Cairo, Egypt.
2- Clove extracts preparation
Clove (Eugenia caryophylata) was collected and dried. The dried plant materials were powdered using a
grinder. The extraction was done at room temperature. About 100 g of dried, ground plant materials were
soaked in each solvent ethanol, water, and acetone (1 L) for 5-7 days separately. The soaked material was stirred
every 18 h using a sterilized glass rod. The final extracts were passed through Whatman filter paper No.1. The
filtrates obtained were concentrated under vacuum on a rotary evaporator at 40oC and stored at 4oC for further
use.
3- Preparation of essential oil
Essential oil of clove was obtained by hydrodistillation method. The plant materials (about 100 g) were ground
into small pieces and were placed in a flask (2 L) together with double distilled water (1.5 L). The mixture was
boiled for 4 h. The extract was condensed in cooling vapour to collect the essential oil. The extracted oil was
dried over anhydrous sodium sulphate. All essential oils were kept at freezing temperature until used.
4- Determination of total phenolic compounds:
The amounts of phenolic compounds in different extracts of clove were determined with Folin-Ciocalteu reagent
using the method of (14). 2.5 ml of 10% Folin Ciocalteu reagent and 2 ml of Na2CO3 (2% w/v) was added to 0.5
ml of each sample of plant extract solution (1 mg/ml). The resulting mixture was incubated at 45°C with
shaking for 15 min. The absorbance of the samples was measured at 765 nm using UV/visible light. Results
were expressed as milligrams of Gallic acid dissolved in distilled water.
Medhat M. Abozid et al /Int.J.ChemTech Res.2013,5(4)
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5- Estimation of total flavonoids
Aluminum chloride colorimetric method was used for flavonoids determination according to (15). One millilitre
(1 ml) of sample (1 mg/ml) was mixed with 3 ml of methanol, 0.2 ml of 10% aluminum chloride, 0.2 ml of 1 M
potassium acetate and 5.6 ml of distilled water and remains at room temperature for 30 min. The absorbance of
the reaction mixture was measured at 420 nm with UV visible spectrophotometer. The content was determined
from extrapolation of calibration curve which was made by preparing quercetin solution in distilled water. The
concentration of flavonoids was expressed in terms of mg/ml.
6- GC/MS analysis of clove essential oil
The chromatographic procedure was carried out using a Finnegan Mat SSQ 7000-GC-MS with autosampler. A
methyl polysiloxane capillary column (DB-5, 50 m X 0.32 mm) was used. Helium was used as the carrier gas.
The oven temperature used was maintained at 50ºC for 8 min. The temperature was then gradually raised at a
rate of 3°C per min to 180°C per min and maintained at 180°C for 5 min. The temperature at the minjection port
was 250°C. Quantitative data were obtained from the electronic integration of the FID peak areas. The
components of the essential oils were identified by comparison of their mass spectra and retention indices with
those published in the literature (16) and presented in the MS computer library (WILEY275.L).
7- In vitro antioxidant activity by reducing power assay
The reducing power of different extracts was determined according to the method of Yen and Chen (17). 2.5 ml
of extract (25-800 μg/ml) in water were mixed with a phosphate buffer (2.5 ml, 0.2M, pH6.6) and potassium
ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50oC for 20 min. A portion (2.5 ml) of
trichloroacetic acid (10%) was added to the mixture to stop the reaction, which was then centrifuged at 3000
rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml,
0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated
increased reducing power. Vitamin C was used as a positive control.
8- invivo study for test the protective effect of best clove extract and essential oil against hydrogen
peroxide administration:
8.1. Animal experiments
Rats were obtained from Research Institute of Ophthalmology, Giza, Egypt. And the work was carried out at its
animal house. To study the protective effect of clove acetone extract and clove essential oil on hydrogen
peroxide oral administration in albino rats; twenty four male albino rats (weighing between 90 and 110 g) were
used for this investigation. The rats were fed ad libitum on a basal diet (BD) and water for 15 days as an
adaptation period. There were housed individually in stainless steel cages and divided into four groups of six.
All groups were fed the BD. Diet intake was monitored daily. The first group (C) was used as controls and
received tape water as drinking water. The other three groups; received tape water containing hydrogen peroxide
at a dose of (0.5%) in drinking water, daily for six weeks. The second group (H2O2 group) doesn’t have any
other treatment, while the third group (H2O2 + clove acetone extract group) was treated simultaneously by
stomach tube with clove acetone extract (500 mg/Kg body weight), while the last group (H2O2 + clove essintial
oil group) treated with cloove essintial oil (200 mg/Kg body weight). All rats fasted before blood sampling. The
blood samples were drawn from eye plexuses, after 6 weeks , the rats were anesthetized using diethyl ether. The
weight gain of the rats was recorded on a weekly basis.
8.2. Blood sampling and analysis
Blood samples were collected after six weeks in tubes contain heparin as an anticoagulant from the eye plexuses
under diethyl ether anesthesia and then centrifuged at 3000 rpm for 20 min. to obtain plasma, which was kept
frozen until analysis. The both of alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST)
activities were measured according to the method described by (18). Total protein was determined according to
(19). And albumin was determined according to (20). The content of malondialdehyde (MDA) was determined
spectrophotometrically at wave length 532 nm according to the method of Draper and Hadley (21). Catalase
(CAT) activity was determined at wave length 510 nm according to the method described by Beers and Sizer
(22). Urea was determined according to (23) and creatinine was determined according to (24).
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1480
9- Statistical analysis
The results of the animal experiments were expressed as the Mean ± SD and they were analyzed statistically
using the one-way analysis of variance ANOV A followed by compare means with Duncan’s multiple range
test. In all cases p≤0.01 was used as the criterion of statistical significance.
Results And Discussion
1- Total phenolic compounds and total flavonids inclove extracts:
Data in Table (1) showed that total phenolic content of acetone, ethanol and water clove extracts were (31.88,
10.06, 28.7 mg gallic acid equivalent/100g respectively). The total flavonoid of acetone, ethanol and water clove
extracts were (10, 3.44, 8.3 mg gallic acid equivalent/100g respectively). Phenols and polyphenolic compounds,
such as flavonoids, are widely found in food products derived from plant sources and they have been shown to
possess significant antioxidant activities (25). Studies have shown that increasing levels of flavonoids in the diet
could decrease the occurrence of certain human diseases (26).
Acetone extract showed high total phenol and flavonoid contents. This result is consistent with the findings of
(27) who studied the total phenolic content and antioxidant activity of three lentil seed extracts acetone,
methanol, and hexane, and found the highest extraction rate of phenolic compounds for lentil seeds was
obtained by acetone.
Table (1): total phenolic compounds and total flavonids in different ginger extracts.
Phenolic content (mg/100g )
Flavonids content(mg/100g)
Acetone extract
31.88
10
Ethanol extract
10.06
3.44
Water extract
28.7
8.3
2- Essential oil chemical composition:
Table (1) showed the identified compounds in clove essential oil, Eugenol and eugenol acetate were the main
components in clove essential oil, our data are agree with (28, 29).
Table (2): chemical composition of clove essential oil
Component
%
%
Eugenol
83.661
0.014
Eugenol acetate
12.118
0.01
Caryophyllene oxide
1.572
0.013
2,3,4 trimethoxy acetophenone
1.12
0.035
Benzyl benzoate
0.185
0.012
Ledol
0.496
0.017
Eucalyptol
0.068
0.029
Linalool
0.078
0.052
Camphor
0.044
0.022
2-Nonanone
0.028
0.008
α- Pinene
0.036
0.026
Total identified
99.644
22
2- In vitro evolution of antioxidant activity of different clove extracts and clove essential oil:
Fe (III) reduction is often used as an indicator of electron- donating activity, which is an important mechanism
in phenolic antioxidant action (30). In this assay, the presence of reductants (antioxidants) in the samples would
result in the reduction of Fe+3 to Fe+2 by donating an electron. The amount of Fe+2 complex can be then be
monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Increasing absorbance at 700 nm
indicates an increase in reductive ability. Fig. 1 shows the dose response curves for the reducing powers of the
Medhat M. Abozid et al /Int.J.ChemTech Res.2013,5(4)
1481
clove extracts and clove essential oil. It was found that the reducing powers of extracts also increased with an
increase in their concentrations. At the highest concentration (200 ug/ml) of all tested materials clove essential
oil showed highest activity (0.504) followed by acetone extract (0.411) , then water extract (0.401) and finally
ethanol extract (0.344).
However, the inhibitory action of herb extracts could be enhanced by more recovery of phenolic compounds
using suitable solvents because the connection of phenolics complex is not the same for all types of solvents
used (31). It can be concluded that the clove essential oil were considerably more effective as antioxidant in
reducing power assay followed by acetone extracts.
Clove essential oil showed the highest antioxidant activity compared with 16 essential oils tested by ferric
reducing power assay (32)
Clove essential oil has been reported in previous studies as one of the strongest antioxidants, even higher than
some synthetic antioxidants like BHT or butylated hydroxyanisole (33 35). The strong activity of clove
essential oil can be due to the presence of eugenol, the main constituent of this essential oil, which is known to
have antioxidant activity (35, 36).
Reducing power assay for clove essential oil and
extracts
0
0.1
0.2
0.3
0.4
0.5
0.6
25 50 100 200
Conc. ug/ml
Absorbance at 700 nm
Essential oil
Acetone
water
Ethanol
Fig. 1: Reducing power of clove extracts and clove essential oil.
8- In vivo study for test the protective effect of best clove extract and essential oil against hydrogen
peroxide administration:
8.1. Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on liver functions:
Table (1) illustrates the effect of H2O2 and/or supplemented clove extract and clove essential oil on plasma
liver functions parameters. In comparison with control group in group treated with 0.5% H2O2 revealed
significantly increased AST and ALT activities, and decreased total protein and albumin. In rats subjected to
H2O2 and supplemented with clove extract and clove essential oil, the enzyme liver marker indicate a decrease
of AST (53.24±2.99 and 44.39±1.33) ALT(50.41±1.23 and 41.54±1.89) and increases the level of total protein
(3.72±0.04 and 3.83±0.057) albumin (2.073±0.519 and 2.22±0.0935) as compared with group treated with
H2O2 only.
Table (3): Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on liver functions
Total protein (g/dl)
Albumin (g/dl)
AST (U/L)
ALT (U/L)
Control
3.94 ± 0.16 a
2.225 ± 0.179 a
30.96 ± 1.92 d
21.28 ± 1.43 d
H2O2 group
3.25 ± 0.17 b
1.902 ± 0.0879 b
92.32 ± 4.3 a
73.4 ± 3.05 a
H2O2 + acetone clove extract
3.72 ± 0.04 a
2.073 ± 0.519 ab
53.24 ± 2.99 b
50.41 ± 1.23 b
H2O2 + clove essential oil
3.83 ± 0.057 a
2.22 ± 0.0935 a
44.39 ± 1.33 c
41.54 ± 1.89 c
LSD 0.01
0.227
0.209
5.296
3.747
Each value is the mean ± SD. Means have different superscript letters indicate significant variation at (P 0.01),
while the same letters indicate non significant variation.
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1482
H2O2 can be a potential source of damage to cells if it is decomposed through reduction to the highly reactive
hydroxyl radical. As shown in the well-characterized Fenton reaction (Equation 1), this reduction requires the
presence of unchelated ferrous iron:
Fe+2 + H+ + H2O2---------------- Fe+3 + .OH + H2O
Lipid peroxidation is an auto-catalytic, free-radical mediated, destructive process, whereby polyunsaturated fatty
acids in cell membranes undergo degradation to form lipid hydroperoxides (37, 38).
This lipid peroxidative degradation of biomembranes is one of the principle causes of hepatotoxicity (3). This is
evidenced by an elevation in the serum marker enzymes namely AST, ALT, ALP, total bilirubin, GGTP and
decrease in albumin and total protein.
Free radical mediated process has been implicated in pathogenesis of most of the diseases. The protective
effects of clove extract and clove essential oil on H2O2 induced hepatotoxicity in rats appears to be related to
inhibition of lipid peroxidation in addition to free radicals scavenging action. Preliminary phytochemical studies
reveal the presence of Polyphenolic compound and flavonids present in clove extract (12) and clove essential oil
(13). Polyphenolic compounds and flavanoids are hepatoprotectives (39). The observed antioxidant and
hepatoprotective activity of clove may be due to the presence of polyphenolic compounds and flavanoids.
8.2. Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on kidney
functions:
Data in Table (4) appeared a significant elevation (P<0.01) in serum urea and creatinine concentration in H2O2
treated group comparing to control group with mean value of (41.506 ± 1.92, 1.001 ± 0.042), (21.48 ± 0.7,
0.536 ± 0.046) respectively. However, acetone extract of clove and clove essential oil groups caused significant
decrease (P<0.01) in mean value of previous parameters compared to H2O2 group.
Table (4): Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on kidney
functions
Urea (mg/dl)
Creatinine (mg/dl)
Control
21.48 ± 0.7 d
0.536 ± 0.046 d
H2O2 group
41.506 ± 1.92 a
1.001 ± 0.042 a
H2O2 + acetone clove extract
33.097 ± 1.13 b
0.854 ± 0.4 b
H2O2 + clove essential oil
28.27 ± 1.04 c
0.756 ± 0.0365 c
LSD 0.01
2.366
0.0768
Each value is the mean ± SD. Means have different superscript letters indicate significant variation
at (P 0.01), while the same letters indicate non significant variation.
The role of oxidative stress as important contributing cofactors to cellular dysfunction including kidney, has
substantially increased over the last years (40, 41). We can hypothesized that exposure to H2O2 may cause
elevation of superoxide anion and the dangerous hydroxyl (OH•) radical leading to glomerular dysfunction (4)
with subsequent elevation in serum creatinine, blood urea, and serum uric acid concentrations (42). H2O2
exposure may lead to activation of a wide variety of inflammatory response like cytokines (43), thus diverse
deleterious renal damage may occur with subsequent decrease in glomerular function which may result in
elevation of kidney biomarkers.
Polyphenolic compound present in clove extract (12) and clove essential oil(13) may be responsible for the
antioxidant capability of the plant and has protective effect against oxidative damage induced by H2O2.
8.3. Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on plasma
antioxidants:
Hydrogen peroxide (H2O2) has been shown to induce oxidative stress in both human and animal models,
leading to the generation of potent reactive oxygen species (ROS), such as hydroxyl radical (OH_). Oxidative
stress results when generation of reactive oxygen and/or nitrogen species and activity of the antioxidant
defenses are unbalanced.
Medhat M. Abozid et al /Int.J.ChemTech Res.2013,5(4)
1483
Lipid peroxidation is an auto-catalytic, free-radical mediated, destructive process, whereby polyunsaturated fatty
acids in cell membranes undergo degradation to form lipid hydroperoxides (38,39). These latter compounds then
decompose to form a wide variety of products, including low molecular mass hydrocarbons, hydroxy aldehydes,
fatty acids, ketones, alkenals and alkanals, in particular malonaldehyde (MDA) (44), Thus, reduction of MDA
production would indicate inhibition of lipid peroxidation.
Plasma MDA were significantly increased in rats treated with H2O2 as compares with control group (Table 5).
Clove extract and clove essential oil resulted significant reduction of lipid peroxidation product induced by
H2O2. Clove essential oil supplementation kept value of MDA in plasma with in the normal limit.
Table (5): Effect of acetone extract of clove and clove essential oil against hydrogen peroxide on plasma
antioxidants
MDA (nmol/dl)
Catalase (IU/ml)
Control
16.66 ± 1.097 c
34.76 ± 0.289 c
H2O2 group
36.56 ± 2.17 a
51.36 ± 1.97 a
H2O2 + acetone clove extract
24.016 ± 2.76 b
42.62 ± 2.267 b
H2O2 + clove essential oil
19.72 ± 1.48 c
35.32 ± 3.56 c
LSD 0.01
3.663
4.311
Each value is the mean ± SD. Means have different superscript letters indicate significant variation at (P 0.01),
while the same letters indicate non significant variation.
Clove oil reduced tissue oxidative stress, shown by the significantly (p < 0.05) reduced MDA levels in the
hearts of diabetic rats and the kidneys of normal rats (45).
Catalase, an enzyme predominantly located in peroxisomes and\ enriched in hepatocytes and erythrocytes,
catalyzes the dismutation of hydrogen peroxide, forming O2 and H2O. (46)
CAT activity changes in the blood plasma implied that H2O2 treatment increased CAT levels, and that clove
extract and clove essential oil tended to decrease CAT levels. These results could be deduced from the
assumption that anti-oxidative clove extract and clove essential oil contributed to removing H2O2, the CAT
substrate, as by ascorbic acid. Nevertheless, this reversion of the CAT level increase by the clove extract and
clove essential oil suggests that the anti-oxidative of clove should protect the rats from cellular damage caused
by ROS, similar to ascorbic acid.
Our findings are similar with (47) who suggested that the plant extracts decreased CAT levels in rats through the
same mechanism as that of the antioxidant ascorbic acid and that they have potential as antioxidants.
Conclusion
Our present study indicates that clove essential oil and clove extracts is devoid of genotoxicity and pro-oxidant
property. The enhanced liver functions, kidney functions, and antioxidant status observed in clove treated rats
and its protective role against H2O2 induced cell damages might be due to the effect of active compounds which
found in essential oil and plant extract.
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... The oxidative stress causes many diseases, especially liver diseases 6 . Hydrogen peroxide (H 2 O 2 ) is one of the most commonly used hepatotoxins in the experimental study of liver diseases; it has been proved that it induces oxidative stress in experimental animals 7 . ...
... In the present work the activities of liver enzymes (ALT, AST, ALP and GGT) in H 2 O 2 treated group increased significantly, relative to the normal control group (Table 3). These results are in line with Abozid and El-Sayed, (2013); Kaplowitz et al., (1986) and Sevanian, (1985) 7,32,33 ; who confirmed the bad influence of H 2 O 2 on liver function especially of their remarkable role in raising the liver enzymes activities in the blood. However, after the intervention of green tea extracts, the (AST, ALT, ALP and GGT) activities in all green tea treated groups reduced significantly and these results showed green tea extracts have hepatoprotective effect in the rats treated with H 2 O 2 because adequate amounts of antioxidants help protect the liver against free radical damage (Table 3) ...
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The study aims to identify green tea flavonoids and evaluate the protective effect of green tea water extracts against liver changes induced by H2O2 in male albino rats. Both two extracts showed a good amount of phenols and flavonoids; HPLC analysis of the different green tea flavonoids showed that epicatechin gallate, gallocatechin, caffeine, epicatechin and catechin are the main components in all green tea extracts. Rats were treated with H 2 O 2 (0.5% in drinking water) and water extracts of green tea by stomach tube (80 mg/kg b.wt) for 30 days. Elevated levels of plasma total bilirubin, MDA and AST, ALT, ALP, GGT, SOD, CAT enzymes activity with decrease in plasma total protein and albumin levels in H 2 O 2 treated rats compared with normal control group, all these parameters were significantly decreased by using different green tea extracts except total protein and albumin levels were significantly increased. Histopathological examination revealed degeneration of hepatocytes of rat liver treated with H 2 O 2. Green tea extracts supplement improve the harmful effects of H 2 O 2 in rats liver. Our results suggests that treatment with green tea extracts containing large amounts of phenolic compounds acting as natural antioxidants to reduce the harmful effect of treatment with H 2 O 2 .
... The active compounds constituting the CEO, particularly eugenol, can donate hydrogen atoms or electrons to stabilize the phenoxyl radicals and thereby hinder the propagation of oxidation reactions [5]. Such antioxidant capability justifies the role of CEO in enhancing liver and kidney functions in H 2 O 2 [40] and methotrexate [41] treated rats, its ability to restore memory and learning deficiencies in scopolamine treated mice [42], and the analgesic effect of the oil and eugenol against spasm, toothache, and joint pain by activating chloride and calcium channels in ganglion cells and as capsaicin agonist [5]. ...
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Aromatic plants embrace volatile compounds with efficiency in treating different diseases. In Jordan, Syzygium aromaticum flower buds (clove) are extensively used as folk medicine without awareness of its bio-safe dosage. Herein, clove buds were hydrodistilled using the Clevenger apparatus, and the resulting essential oil (CEO) was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The antibacterial activity was evaluated against tested bacterial strains by agar diffusion test and micro-broth dilution assay. The antioxidant capacity was assessed using DPPH radical scavenging assay, while the cytotoxic potency was unraveled by determination of its anti-proliferative activity against MDA-MB-231 breast adenocarcinoma and normal Vero cell lines. CEO yield was 5.7 ± 1.3% (w/w); encompassed 24 volatile ingredients with eugenol as the principal compound (73.41%). The CEO inhibited the growth of both Gram-positive and Gram-negative bacterial test strains, causing the formation of 13.7 ± 1.5–17.3 ± 0.6 mm and 11.7 ± 1.5–20.7 ± 1.2 mm inhibition zones, respectively with MIC 1.25–5 μL/mL. Moreover, it showed antioxidant activity with IC 50 0.0016 ± 0.0001 μL/mL (1.6 ± 0.1 μg/mL, 2.98 ± 0.4 µg Trolox ® /µg CEO). Intriguingly, the CEO was cytotoxic against both cancerous and noncancerous cell lines at IC 50 of 0.25 ± 0.02 μL/mL and 0.18 ± 0.01 μL/mL, respectively. Herein results unveil the potential application of CEO as a pharmaceutical remedy with considering its bio-safe dosage.
... However, at fixed and high amounts of the clove extract, by rising amount of zinc salt, the antioxidant activity of the resulted ZnO NPs significantly (p < 0.05) increased. Obtained results can be designated by the point that, at high values of the clove extract, the concentration of the main bioactive compounds such as Caryone and Eugenol, which played a key role in zinc ions' reduction and converting them into NPs, is high [7,26]. Therefore, higher amounts of the clove extract could result in ZnO NPs with a higher concentration, as compared to those fabricated using lower amounts of the prepared extract. ...
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Green approach in fabrication of photocatalytic, antimicrobial, and antioxidant zinc oxide nanoparticles-hydrothermal synthesis using clove hydroalcoholic extract and optimization of the process Abstract: Zinc oxide nanoparticles (ZnO NPs) were hydrothermally fabricated, using hydroalcoholic clove extract. GC-MS analysis demonstrated that Eugenol is the main bioactive compound of the prepared extract. Experiments were designed, based on the central composite design. The effects of different amounts of zinc nitrate (2-6 g) and clove extract (10-30 mL) were evaluated for antioxidant and bactericidal properties of the formed ZnO NPs using the response surface methodology. The attained results demonstrated that more desirable NPs with maximum antioxidant activity (85.23%) and bactericidal effect, against Escherichia coli and Staphylococcus aureus, as manifested in the diameter of formed clear zones of 11.12 and 12.11 mm, respectively, were resulted using 3.98 g of the zinc salt and 20.30 mL of the clove extract. Furthermore, XRD and SEM analysis results revealed that the fabricated ZnO NPs had a hexagonal shape with a particle size of 50 nm and could degrade 70% of methylene blue during UV radiation.
... Plasma antioxidant state (malondialdehyde content and catalase activity), also showed the same ameliorative property against hydrogen peroxide toxicity. Abozid et al suggested that extract of clove or clove essential oil has hepatoprotective and renoprotective effects against hydrogen peroxide-induced oxidative stress on both liver and kidney (13). Similarly to examine the potency of Clove on malondialdehyde and superoxide dismutase in the liver of Rabbits under hypercholesterolemia situation, Mu'nisa et al found that clove leaf extract could overwhelm superoxide dismutase and malondialdehyde elevations in rabbits' liver tissue under situation of hypercholesterolemia. Accordingly, Clove kept superoxide dismutase activity (14). ...
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Introduction: The administration of iodinated contrast medium is extensive in various imaging techniques including coronary angiography. Objectives: The present investigation was designed to examine the ameliorative effect of clove buds (Syzygium aromaticum) on contrast-induced acute kidney injury (CI-AKI) in rats. Materials and Methods: Forty male Wistar rats with a mean body weight of 200-250 g were studied. Rats randomly assigned into four groups, 10 rats for each; Group 1; normal rats (control group; sham group); they did not receive any drugs. Group 2; rats were received 10 mL/kg as a single dose of iodixanol (contrast media) by intravenous (IV) injection. Group 3; rats received 10 mg/kg clove by intraperitoneal (IP) injection for three days, while in day forth, they received a single dose of iodixanol (10 mL/kg). Group 4; rats of this group first received a single dose of iodixanol (10 mL/kg). Then rats treated by clove (10 mg/kg) by intraperitoneal (IP) injection for three days (days 2, 3 and 4th). Kidney sections were examined for degeneration, flattening, and necrosis of renal tubular cells and also dilatation of tubular lumen. Results: We found a significant difference between groups regarding the sum of injury (degeneration, flattening, necrosis and dilatation). A significant difference in types of injury (degeneration, flattening, necrosis and dilatation) among the groups (P=0.001) was seen too. We detected a significant difference between groups II (contrast media) and III (rats pretreated by clove; P<0.001). Accordingly, we detected a significant difference between groups II (contrast media) and IV (rats post-pone treated by Clove buds; P<0.001) too. There was not a significant difference between groups of III and IV (P> 0.05). Conclusion: In this animal study, we found that post-pone treatment of clove was as effective as pretreatment to mitigate the injury induced by contrast media.
... That ciprofloxacin leads mild damage in hepatocyte, our finding were similar to those obtained by Rang and Dale (1993), and Shefaa (2009). Also it showed significant decrease in total protein, albumin and globulin at 1 st and 2 nd week Fig. (7) Rang and Dale (1993), and shefaa (2009) showed non -significant changes at the 3 rd week, non-significant increase in creatinine and urea at the end of experiment present in Fig. (8). 4 th group which treated with clove extract only showed significant increase in ALT & AST in 1 st and 2 nd week but in 3 rd weeks showed non-significant increase as in Fig. (6), It also showed significant decrease in total protein, albumin and globulin in 1 st and 2 nd weeks, but in 3 rd week was showed highly significant decrease in total protein, albumin and globulin Fig. (7), this due to partial hydrolysis of protein, Velisek et al., (2005) suggest that it was a significant increase (p < 0.01) in blood plasma immediately after 10 min exposer of carp to clove oil, this increasing returned to normal after 24 hours Fig. (8) it showed non-significant increase in creatinine in 1 st week, significant increase in 2 nd and 3 rd weeks, it also showed significant decrease in urea in 1 st week and highly significant decrease in 2 nd and 3 rd weeks.This revealed due to focal areas of coagulative necrosis in the hemopsietic element, the glomeruli showed slightly shrunken glomerular this result similar to these reported by Medhat and El-sayed (2013). 5 th group which treated with clove extract & ciprofloxacin showed significant increase In 1 st week and non -significant in 2 nd and 3 rd week also showed significant decrease in total protein, albumin and globulin during experimental period ,it due to that liver showed intact parenchyma with slight congestion and few interstitial and portal lymphocytes infiltrations, it also showed highly significant decrease in urea in 1 st & 2 nd weeks, but non -significant result in 3 rd week, and In creatinine showed significant increase at period of experiment due to focal vaculation in tubular epithelium and glomeruli appeared by percellular and really congested. ...
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Extract of six plant species that are available from local market in sharkia governorate, Cinnamon (Cinnamomum verum), Clove (Syzygium aromaticum), Thymus (Thymus vulgrais), Nutmeg (Myristica fragrans), Rosemary (Rosmarinus officinalis) and Hibiscus (Hibiscus sabdariffa), were screened for antibacterial activity against ten isolates of pathogenic Clarias gariepinus which bacteria including Aeromonas spp, Pseudomonas spp, Vibrio spp, staphylococcus spp,. Clove extract showed strong inhibitory effect against all bacterial isolates, also Ciprofloxacin showed highly inhibition zone for all bacterial isolates. One hundred and fifty Nile catfish Clarias gariepinus were divided into five equal groups each of 30 fishs, 1 st group was neither challenged nor treated as control group, but 2 nd ,3 rd ,4 th and 5 th groups were challenged with Aeromonas sobria which predominant isolates, the 2 nd group was challenged only while 3 rd , 4 th and 5 th groups were treated with Ciprofloxacin, clove extract and Ciprofloxacin & clove extract combination, respectively. The antibacterial activity of antibiotic and extract in their therapeutic doses decrease the mortality rate as (20, 16 & 13%) respectively compared with non-medicated 2 nd group which recorded 83%. Results confirmed significant (p≤ 0.05) improvement of productive performance parameter, increase in levels of liver function as well as significant (p≤ 0.05) while decrease in total protein, albumin, globulin and kidney function test in fish challenged with Aeromonas sobria and treated with Ciprofloxacin , clove extract compared with challenged non treated fish, more ever the best significant (p≤ 0.05) were detected in 5 th group treated with combination in comparison with those treated with single treated. It could be concluded that, mixture of clove extract with Ciprofloxacin was effective for treatment of Aeromonas sobria infection of Clarias gariepinus.
... International Journal of Organic Chemistry use its essential oils for medical purpose, since it active against oral associated bacteria and fungi [2]. Previous studies have reported the antifungal [3] [4] [5], antioxidant [6] [7] [8], antibacterial [9] [10] [11] and anti-inflammatory [12] [ 13] properties of clove oil. The main component of clove oil, eugenol, has been used as an anti-cancer [14] [15] and as a starting material for synthesis analog L-α-metil DOPA [16]. ...
... The hepatotoxicity resulting from TCA treatment can be explained by the production of ROS, which have been observed to increase in the liver tissues of mice treated with TCA [8,33,34]. Dietary intake of different plant extracts (which contain polyphenolics and flavonoids) grants repressing effects versus oxidative damage in animals [35] and humans [36]. In this context, the current study affirms that AER modulates significantly the liver functions against the harmful effects of TCA. ...
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Objective: The current study was designed to estimate the potential protective role of the aqueous extract of rosemary (AER) (Rosmarinus officinalis) against trichloroacetic acid (TCA)-created hepatotoxicity in male albino rats.Methods: Forty male albino rats were separated into four groups of ten: Group I served as control; Group II was given AER (200 mg/kg/day) by gavage; Group III received TCA at the dose 50 mg/kg/day, and Group V was treated with AER (200 mg/kg/day) and received TCA (50 mg/kg/day). The experiment was carried out for 2 months.Results: The toxicity of TCA for rats was revealed by an elevation in liver marker enzymes activities (gamma-glutamyl transferase [GGT], alkaline phosphatase [ALP], aspartate transaminase [AST], alanine aminotransferase [ALT]) and conjugated bilirubin (CB) level, and a decrease in albumin and total protein (TP) levels. The TCA administration also caused a significant increase in the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), and also malondialdehyde (MDA) level in liver tissues. These biochemical effects were accompanied by histological indicators of liver damage. Treatment with ARE recovered the liver damage instigated by TCA, as showed by perfection of liver enzyme markers (GGT, ALT, AST, ALP), CB, TP and albumin; as well as antioxidant parameters (CAT, SOD, GPx) and lipid peroxidation (MDA) and amelioration of histopathology changes in the liver tissues.Conclusion: It could be concluded that AER supplementation for 2 months in TCA-induced toxicity in rats benefited hepatic antioxidant status and improved liver injury and damage in male albino rats exposed to TCA.
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This study was designed to evaluate the modulating effect dietary clove essential oil (CL) has on the antioxidant and immunological status of Nile tilapia following Streptococcus iniae (Si) infection. Fish were placed on either control or (1.5 and 3%) CL-supplemented diets for 4 weeks. After sampling, the remaining fish in the control group were divided into 2 groups: an unchallenged (negative control) and an Si-challenged positive control. On the other hand, the remaining fish in CL-supplemented groups were challenged with Si, and mortality was checked for two weeks before the final sampling. Serum immunological parameters, tissue antioxidants, and oxidative stress markers were determined. Moreover, hepatic hepcidin expression was also measured in different groups. The obtained results showed improvements in blood phagocytic, bactericidal, lysozyme, and respiratory burst activities in CL-supplemented fish before and after the Si challenge. Si-challenge caused a remarkable increase in tissue malondialdehyde (MDA) levels that was inhibited by CL supplementation. The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in tissues were significantly elevated in a dose-dependent manner in CL-supplemented groups in both pre- and post-challenge experiments; renal SOD did not show any differences. Hepatic nitric oxide (NO) level was significantly decreased in CL-supplemented fish in a dose-dependent manner. In the post-challenge experiment, nitrosative stress was apparent in the liver and kidney; however, CL supplementation was sufficient to reverse it. Interestingly, a remarkable induction of the hepatic hepcidin expression was observed in all CL-supplemented groups in the pre-challenge experiment and Si-challenged fish, underscoring the role of CL as an antibacterial through inducing hepatic hepcidin expression to combat S. iniae infection. CL-supplementation was associated with lower mortality rates after Si-challenge, which was more pronounced in CL-3% supplemented fish. In conclusion, our results demonstrate that CL has a potent antioxidant role via increasing antioxidant enzymes’ activities and antagonizing lipid peroxidation. Moreover, CL has an immune-stimulant effect by inducing the hepatic hepcidin expression and immunological markers in response to S. iniae infection.
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Feeding Effects of Hydroalcoholic Extract of Clove Flower and Priformospora Indica Fungi in chick broiler challenge by dexamethasone on blood hematocrit and hemoglobin concentration Abolfazl Farmani Kesajini 1 Mahdi Hedayati * 2 Mojtaba Yari 3 Mehdi ghaboli 4 1 Master of Science (MSc), Department of Animal Sciences, Malayer University 2 Assistant Professor, Department of Animal Sciences, Malayer University (responsible author: (hedayati@malayeru.ac.ir) 3 Assistant Professor, Department of Animal Sciences, Malayer University 4 Assistant Professor, Department of Biotechnology, Malayer University Abstract The aim of this experiment was to investigate the comparative effects of hydroalcoholic extract of clove flower with Priformospora Indica, plant-specific fungus, separately and co-administered on blood hematocrit and hemoglobin concentration, in broiler chickens challenge by dexamethasone. Chicks (n=192; Ross 308) were randomly assigned to 8 experimental groups which each group had 3 pen as replicate and in each replicate, 8 chicks were fed for 6 weeks with diet based on corn and soybean meal. Experimental groups were 1, control which fed with basal diet; 2, feeding basal diet along with 0.2 mg/kg injection of dexamethasone; 3, feeding basal diet along with 300 mg/kg hydroalcoholic extract of clove flower solved in drinking water; 4, feeding basal diet along with 10 cc P .Indica culture solved in drinking water; group 5; feeding basal diet along with 300 mg/kg hydroalcoholic extract of clove flower solved in drinking water along with injection of 0.2 mg/kg dexamethasone ; 6; basal diet along with 10 cc P.Indica culture in drinking water and 0.2 mg/kg injection of dexamethasone; 7; feeding basal diet ration along with solved 300 hydroalcoholic extract of Clove and 10 cc P.Indica in drinking water and 8, feeding basal diet with solved 300 mg/kg hydroalcoholic extract of clove flower along with 10 cc P.Indica in drinking water and injection of 0.2 mg/kg dexamethasone). Feeding hydroalcoholic extract of clove and P.Indica as a prebiotic increased blood hematocrit and hemoglobin significantly concentration (P <0.05). Dexamethasone injection at days of 35 and 42 of study influnced blood hemoglobin (P <0.05). There was no significant difference in blood hemoglobin and hematocrit concentration in chicks fed with prebiotic and injection of dexamethasone at 35 and 42 days of age. Interaction effects of clove extract and dexamethasone at 42 day of age on blood hemoglobin was not significant. At 42 days of age, dexamethasone, Prebiotic and clove extract had no significant effect on hemoglobin. In conclusion, feeding solved hydroalcoholic extract of Clove and Priformospora Indica culture, plant-specific fungus, in drinking water of broiler chicks can change blood factors such as red blood cells, hematocrit, and hemoglobin concentration. Keywords: Broiler chick, Clove, Hemoglobin, Hematocrit, Priformospora Indica.
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In this study, the changes in phenolic compounds and antioxidant activity of lentil seeds before and after germination were investigated. Lentil seeds were germinated in dark chambers maintained near 100% relative humidity at 20°C. Three different solvents were employed to extract the phenolic compounds present in lentil seeds and sprouts. Total content of phenolic compounds were measured by Folin-Ciocalteau method and antioxidant activity determined by two different methods: assay of hydroxyl radical scavenging activity method and oven test method. For the later method, two different concentrations of extracts (0.02 and 0.1% w/w) were added to sheep tallow and the stabilities of the treatments were determined. Peroxide value and induction period measurements were used to evaluate the in vivo antioxidant activities. The results indicated that germination modifies the quantity and quality of phenolic composition of lentil, and the solvent used for extraction influence the antioxidant activity. The evaluation of in vivo antioxidant activity of the extracts measured by delay in tallow oxidation indicated that the activity was dependent on the phenolics concentration of extract which increased when higher concentrations of the extracts were applied. These data suggested that lentil sprout flour or extract can be used as a source of natural antioxidants in functional foods.
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There has been an increasing interest during the last ten years or so in the contributions of free radical reactions to the overall metabolic perturbations that result in tissue damage and disease. For a historical overview of free radical-mediated disturbances see Slater (1971); more recent references and comments can be obtained from Slater (1984).
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Traditionally corn silk (CS) has been used as diuretic, antilithiasic, uricosuric, and antiseptic. It is used for the treatment of edema as well as for cystitis, gout, kidney stones, nephritis, and prostatitis. In the present study, the antioxidant properties of ethanol-water extract from CS were estimated by different methods. Also phenol and flavonoid content of the extract were measured by Folin Ciocalteu and AlCl3 assays. CS extract contained a significant amount of phenol and flavonoids. The percentage of DPPH radical scavenged by CS extract was 92.6 at a concentration of 1.6 mg ml -1. IC50 of the extract and the standard compounds butylated hydroxytoluene (BHA) and quercetin was 0.59, 0.053, and 0.025 mg ml-1, respectively. Iron chelating activity of the extract was less than the standard compounds. CS extract showed nitric oxide-scavenging effect less than the reference agent (quercetin). The extract showed a high reducing ability. According to ferric thiocyanate (FTC) method, the extract showed more than 88% inhibition of linoleic acid peroxidation. It might be concluded that some of the properties of CS in traditional medicine is due to its antioxidant ability.