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In vitro bioactivities of clove buds oil (Eugenia caryophyllata) and its effect on dermal fibroblast

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Abstract

The aim of this study was to investigate the antioxidant and antityrosinase activities of clove oil and its components, the correlation between their antioxidant and antityrosinase activity and to examine the effect of clove bud oil towards dermal fibroblasts. The results show that the major component presented in clove oil is eugenol (99.16%). Free radical scavenging capacity of clove oil using the DPPH method showed significant correlation with lipid peroxidation inhibition using TBARs method (r=0.804) but less correlation with tyrosinase inhibition (r=0.576). Linear regression analysis revealed that free radical scavenging capacity was more correlated to tyrosinase inhibition than lipid peroxidation inhibition. In addition, clove buds oil exhibited cytotoxicity at IC50 of 0.162 μl/ml and significantly increased collagen synthesis at as low as 0.0156 μl/ml. In conclusion, clove buds oil might possess whitening effect and anti-aging effect. However, due to its cytotoxicity, clove buds oil should be used in very low concentrations with care.
IN VITRO BIOACTIVITIES OF CLOVE BUDS OIL (Eugenia caryophyllata) AND ITS EFFECT ON
DERMAL FIBROBLAST
Research Article
WATCHAREE KHUNKITTI*1, PRAPHATSON VEERAPAN1 AND CHARIYA HAHNVAJANAWONG2
1Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002,Thailand, 2
Received: 3 Feb 2012, Revised and Accepted: 16 March 2012
Faculty of Medicine, Khon Kaen University, Khon
Kaen 40002,Thailand. Email: watkhu@kku.ac.th
ABSTRACT
The aim of this study was to investigate the antioxidant and antityrosinase activities of clove oil and its components, the correlation between their
antioxidant and antityrosinase activity and to examine the effect of clove bud oil towards dermal fibroblasts. The results show that the major
component presented in clove oil is eugenol (99.16%). Free radical scavenging capacity of clove oil using the DPPH method showed significant
correlation with lipid peroxidation inhibition using TBARs method (r=0.804) but less correlation with tyrosinase inhibition (r=0.576). Linear
regression analysis revealed that free radical scavenging capacity was more correlated to tyrosinase inhibition than lipid peroxidation inhibition. In
addition, clove buds oil exhibited cytotoxicity at IC50
Keywords: Clove oil, Antioxidant index, Tyrosinase inhibition, Collagen synthesis, Cytotoxicity
of 0.162 μl/ml and significantly increased collagen synthesis at as low as 0.0156 μl/ml. In
conclusion, clove buds oil might possess whitening effect and anti-aging effect. However, due to its cytotoxicity, clove buds oil should be used in very
low concentrations with care.
INTRODUCTION
Skin aging occurs with time and is caused by environmental factors.
In aged skin, slower turnover causes thinning of the epidermis that
gives aged skin a translucent appearance, resulting in dyschromic
skin changes. In the elderly dermis, the decrease of fibroblast cells
affects decrease of collagens and elastin fibers synthesis, resulting in
skin wrinkling and loss of elasticity1. In addition, photoaging skin
damage by reactive oxygen species (ROS) may reduce the strength
of skin cell walls, as well as degrading collagen and elastic fibers,
resulting in loss of skin humidity and elasticity leading to skin
wrinkling. Application of topical antioxidant cosmetic products may
prevent intrinsic and extrinsic skin aging1-2. However, sunlight can
induce skin tanning by ROS which may activate tyrosinase enzyme
to catalyze the hydroxylation of monophenols to o-diphenols and the
oxidation of o-diphenols to o-quinones. Then the o-quinones can
change L-tyrosine to L-DOPA and L-DOPA to L-dopaquinone,
resulting in melanin pigments3-4
Essential oils are commonly used in traditional medicine and widely
used in cosmetics. Many studies have demonstrated that essential
oils have anti-inflammatory activity, antibacterial activity and
antioxidant activity. Because inflammation is commonly related with
oxidative damage, substances which can inhibit inflammation may
also be antioxidants via free radical scavenging and lipid
peroxidation inhibition
. As a result, inhibition of tyrosinase
enzyme by antioxidants may reduce melanogenesis.
5-9. Clove (Eugenia caryophyllata) is the
aromatic dried flower buds of a tree in the family Myrtaceae. The
essential oil of clove is used as anti-mutagenic 10, anti-inflammatory
and antioxidant activities 11-13
MATERIAL AND METHODS
. The aims of this study were to
investigate in vitro bioactivities, the correlation of antioxidant
activity and tyrosinase inhibition of clove oil and its components as
well as the effect on dermal fibroblasts.
Materials
Clove oil was purchased from Thai China Flavours & Fragrances
Industry Co. (Thailand), butylated hydroxyl toluene (BHT) and
alpha-tocopherol acetate (vitamin E acetate), Eugenol and trans-
caryophyllene, were purchased from Sigma Chemicals (USA).
Thiobarbituric acid (TBA), Chloramine T reagent, Ehrlich’s reagent,
Trichloroacetic acid (TCA) Trans-caryophyllene, 2, 2-azole-(2-
aminopropane)-dihydrochloride (ABAP) were obtained from Sigma
Aldrich (Switzerland), calcium chloride (CaCl 2
Identification and quantification of clove oil components
), L-Tyrosine,
Mushroom tyrosinase enzyme, 2, 2-diphenyl-l-1-picrylhydrazil
(DPPH), tris-base buffer, sulforhodamine B (SRB) were purchased
from Fluka, Switzerland. All culture media and culture supplements
were purchase from Gibthai (Thailand).
GC/MS analysis of the essential oil sample (1%v/v in
Dicholoromethane; DCM) was carried out on an Agilent
Technologies (China), Model CN 10402086, equipped with a column
DB-5ms (0.25mm x 30m x 0.25µm ID). The carrier gas used was
helium at a flow rate 1ml/min. The column temperature started with
70oC for 5 min, then programmed at 3oC /min to 120oC for 2 min and
at 5oC/min to 270oC. Sample (1µl) was injected neat with 1:100 split
ratios. Mass spectra were recorded in scan mode 35-550m/z with
scan rate 1388.2amu/s and the ion source temperature was 230oC.
The components were identified by their linear retention indices
and compared with their mass spectra with the NIST MS Search
Library and the reference compounds.
To determine the quantity of clove oil and reference compounds,
ethanolic solution of clove oil (0.2%v/v) or the synthetic reference
compounds (0.004-0.2 %v/v) in DCM were prepared and analyzed
using a gas chromatograph connected to a flame ionization detector
(FID) (GC 1850, Agilent). A HP-5 (30 m x 0.32 mm id. x 0.25 μm film
thickness) capillary column was used. The injector temperature was
250°C, The oven temperature was started at 100°C and held for 1
min. Temperature programming was increased from 100°C to 220°C
at 10°C/min and held for 1 min. The carrier gas was nitrogen at a
flow rate of 2 ml/min
Free radical-scavenging activity: 2,2-diphenyl-1-picrylhydrazyl
(DPPH) test
while the split ratio was 1:10. The effluent was
detected by FID at 280°C. A calibration curve was constructed by
plotting between concentrations of either reference compounds or
clove oil and their peak area.
The free radical-scavenging activity of essential oil was measured
according to the method modified from Lertsatitthanakorn et al.6
Antioxidant Index (%) = [1 – (A
.
Briefly, an equal volume (50µl) of a sample solutions which were the
ethanolic solution of clove oil (20 µl/ml), eugenol (20 µl/ml) and β-
caryophyllene (400µl/ml) was mixed with 0.156 mg/ml DPPH
ethanolic solution. 50µl of the solutions were two fold serially
diluted in absolute ethanol and mixed well. Then, a 50µl DPPH was
added to each well. The reaction mixture was mixed for 5min and
incubated in the dark for 25min, and then the OD measured at 490
nm on a microplate reader. α-tocopheryl acetate (vitamin E acetate)
and butylated hydroxyl toluene (BHT) were used as the positive
controls while ethanol alone served as a negative control.
samp- A blk)/ Acont ]*100
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 4, Suppl 3, 2012
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Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
557
Determination of antioxidant activity by inhibition of lipid
peroxidation (TBARs)
The thiobarbituric acid reactive species (TBARs) assay was used to
measure the potential antioxidant capacity of the compounds by
modifying the method from Ruberto and Baratta 14, using egg yolk
homogenates as lipid rich media. The stock solutions of tested
samples were prepared in absolute ethanol with the following
concentrations; 10 mg/ml of clove oil or eugenol, 100 mg/ml of β-
caryophyllene, 0.10 mg/ml of BHT and 100 mg/ml of vitamin E
acetate. Briefly, 50µl of 10% (w/v) egg yolk in 1.15% w/v KCl,
prepared immediately before use, was added into a 1.5-ml
Eppendrof. 10µl of the sample stock solution was added and 40µl of
distilled water was added to make a final volume of 100µl. 5 µl of
ABAP solution (0.07M) in water was added to induce lipid
peroxidation, and then the reaction was incubated at room
temperature for 30 min. Then, 150µl of 20% acetic acid (pH 3.5)
and 150 µl 0.8% (w/v) thiobarbituric acid (TBA) in 1.1% (w/v)
sodium lauryl sulphate (SLS) solution was added and the resulting
mixture was mixed well before heating at 95oC for 60 min. After
the mixture had cooled down, 500 µl of butan-1-ol was added and
centrifuged at 1200xg for 10min. After that, 200 µl of the organic
upper layer of each sample was removed and added into the 96-
well micro plate for measuring the absorbance at 550 nm. Vitamin
E acetate and BHT were used as the positive controls, absolute
ethanol was used as a negative control, and distilled water was
used as a blank.
Inhibition of lipid peroxidation (%) = [1 – (Asamp - A blk)/ Acont]*100
Determination of mushroom tyrosinase inhibition activity
The tyrosinase inhibition activity of clove oil was measured
according to the method modified from Marongiu et al.15. L-
tyrosine was used as the substrate in this experiment. Firstly, 30 µl
of clove oil and its component concentrations were dissolved in
10% v/v of DMSO. 30µl of the clove oil solutions were two fold
serially diluted in 0.1M phosphate buffer (pH 6.8). Then, 120µl of
the phosphate buffer was added to make the final volume 150µl
and it was mixed well. After that, 50µl of L-tyrosine solution
(0.30mg/ml of L-tyrosine in phosphate buffer pH 6.8) was added
to each well. Finally, 50µl of mushroom tyrosinase enzyme
solution (0.04mg/ml of mushroom tyrosinase enzyme in
phosphate buffer solution) were added. The resulting mixture was
then incubated at 30oC for 10 min and left to stand at room
temperature for 15min prior to measuring the absorbance at
490nm. Kojic acid solution was used as a positive control, and
phosphate buffer solution was as a negative control.
Inhibition of tyrosinase enzyme activity (%) = [1 – (Asamp- Ablk )/
Acont]*100
Where, Asamp , Ablk and Acont
Human skin fibroblast cell lines provided by Assistant Professor Dr.
Wilairat Leeanansaksiri, The School of Biology, Institute of Science,
Suranaree University of Technology, was used in this study. To
prepare the cell suspension, the dermal fibroblasts were cultured in
high glucose-DMEM supplemented with 10% fetal bovine serum,
1%w/v penicillin-streptomycin and maintained at 37
are the OD of the sample, blank and
negative control, respectively.
Effect of clove oil and its components on dermal fibroblasts
Human skin fibroblast culture
oC in 5%
CO2/air until sub-confluences. The cell monolayer was trypsinised
with 0.25% trypsin/EDTA solution at 37 o
Cytotoxicity test using sulforhodamine B (SRB) colorimetric assay
C for 20 min and washed
with the culture media before use.
Cytotoxicity of clove oil and its major components were examined by
the SRB assay 16. 190µl the cell suspension was seeded at density of
104cells/well. After 24h of seeding cell suspension, 9 wells of cell
monolayer were fixed by 100µl 10% (w/v) trichloroacetic acid
(TCA) at 4oC for 1h after discarding the old medium, then clove oil
and eugenol concentrations were serially diluted from 10µl/ml to
0.15625µl/ml, and β-caryophyllene concentration were from 2µl/ml
to 0.03125µl/ml in absolute alcohol. 10µl of each concentration was
exposed to a 96-well plate containing confluent cell monolayers in
day1 (after 24 h of seeding cell suspension). 10µl ethanol and 10µl
media without serum were used as controls. After 72 h, the treated
cell monolayers were fixed by 100µl of 10% (w/v) TCA. After the 96-
well plates were dried, 100µl of 0.057% (w/v) SRB solution was
added to each well and the plates were shaken on a titer shaker for 5
min to dissolve the protein-bound dye and the absorbance measured
at 490nm using a microplate reader.
% cell killed = 100- (mean OD samp/ mean ODday0)*100
Quantification of collagen synthesis
A 10 µl of the stock sample solutions at various concentrations was
added into 190 µl of the cell suspension to make a final density of
104cells/well in a 48-well plate. The plate was kept at 37 oC in 5%
CO2/air for 3 days. Then, the cell layer was trypsinised at 37 oC for
20 min and collected as cell lysate solution. Collagen assay was
performed according to the study of Lin and Kuan 17 with some
modification. Briefly, coated 48-well plates were prepared
accordingly; 200 µl of 1%w/v sodium acetate in ethanolic solution
were added into each well and thoroughly coated the well before
air-dried. An equal amount of the cell lysate solution was mixed with
4N NaOH solution and hydrolyzed at 121 oC for 40 min. To
determination of collagen content, twenty microliters of the
hydrolysed solutions were mixed with 30 μl of 2 N NaOH solutions in
the coated 48-well plate. The mixture solution was then mixed with
450 μl of buffered chloromine T reagent for 25 min at room
temperature. Then, 500 μl of Ehrlich’s reagent was added and
incubated at 65 oC for 40 min. The incubated plates were gradually
cooled down at room temperature for 10 min then at 4 o
GC/MS chromatograms showed that the major component in clove
oil was eugenol (99.16%), followed by β-caryophyllene (0.30%) and
others (0.54%) (Table1). In this study, clove buds oil was practically
composed of a large amount of eugenol followed by a small amount
of β-caryophyllene. It was found that the quantity of eugenol was
greater than that found in the study of Dorman et al.
C for 15 min
and kept at room temperature for 20 min before measuring the
absorbance at 550 nm using microtiter plate reader (Biorad, USA). 4-
Hydroxyproline (4-hyp) solutions (0-100μg/ml) were prepared in the
same manner as test samples and used as a collagen standard curve.
The percentages of collagen synthesis on day 3 were calculated in
comparison with the collagen content of fibroblasts at day 0.
Statistical Analysis
All experiments were replicated three times and all measurements
were performed in triplicate. The differences of bioactivities among
clove oil, its major components and reference standards were
analyzed using one-way analysis of variance (ANOVA). Differences
were considered to be significant at <0.05. A linear regression
analysis was carried out to determine the correlation between
antioxidant index and tyrosinase inhibition.
RESULTS AND DISCUSSION
18
.
Table 1: The main components of clove oil
Chemical formula
Component
RI
Percent Area
Mode of Identification*
C10H12 O
eugenol
2
1350
99.16
a, b, c
C15H
β- caryophyllene
24
1416
0.30
a, b, c
*a = mass spectra; b = RI; c = authentic compounds
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
558
Table 2: Bioactivities of clove oil and its major components compared with reference standards
Substance
DPPH
TBARs
Tyrosinase Inhibition
Clove oil
134.2±4.9
942.0±49.6
9.6±1.5
a
Eugenol
145.2±44.3
382.0±35.4
8.2±4.2
a
β-caryophyllene
10449.0±1067.3
4917.8±60.8
a
17.0±0.2
b
BHT
a
84.4±25.6
63.1±15.6
ab
ND
ab
Vitamin E acetate
41332.4±7984.7
3651.5±395.4
a
ND
b
Kojic acid
ND
ND
0.007±0.001ab
a,b
ND: not determined
p< 0.05
The concentrations of samples resulting in a 50% inhibition of DPPH
free radical, TBARs and tyrosinase inhibition, IC50 , are shown in
Table 2. The results demonstrate that clove oil and eugenol possess
free-radical scavenging ability which is more pronounced than their
lipid peroxidation inhibition. Although the free-radical scavenging
activity of clove oil is not significantly different from eugenol, the
inhibition of lipid peroxidation of eugenol is more significantly
potent than clove oil. However, both clove oil and eugenol exhibit
less antioxidant activity than BHT. In addition, β-caryophyllene
exhibited weak antioxidant activity. In comparison with vitamin E
acetate, β-caryophyllene possessed more lipid peroxidation
inhibition than its free-radical scavenging activity. In the DPPH test,
free radical scavenging antioxidants act by donating hydrogen atoms
to DPPH radicals. The stable radicals obtained from antioxidants
then enable the stopping of the oxidation chain reaction. The
reaction mechanism between the antioxidant and DPPH radicals
depends on the structural conformation of the antioxidant5,19-20.
Eugenol is a natural phenolic compound which more easily donates
hydrogen than β-caryophyllene which is a sesquiterpene
hydrocarbon, and vitamin E acetate, which is an ester compound,
requires esterase enzyme to convert into vitamin E which is an
active antioxidant. As a result, eugenol possesses more potent free
radical scavenging capacity than β-caryophyllene and vitamin E
acetate. However, antiradical action of eugenol was less effective
than that of BHT. The results can be explained as proposed by
Bondet et al. 19
In the TBARs assay, antioxidants react with peroxyl radicals to
inhibit the propagation cycle of lipid peroxidation. The ability of
antioxidants on inhibition of lipid peroxidation reaction in lipid-rich
substrate was examined by measuring the formation of
malonaldehyde which is the secondary oxidative product of the lipid
substrate
that eugenol reacts with DPPH radicals in a
dimerization mechanism with a stoichiometry of 1.9 whereas BHT
reacts with DPPH radicals in three different pathways including
complexation, hydrogen atom delocalization and dimerization with a
stoichiometry of 2.8.
5,18. In this study, inhibition of lipid peroxidation of
eugenol was much greater than that of clove oil. Although clove oil is
composed of eugenol as a major component, the lipid peroxidation
inhibition of the component was concentration dependent. Besides,
clove oil also contains a small amount of β-caryophyllene which
possesses lesser inhibition capacity. Moreover, the correlation
between DPPH and lipid peroxidation of eugenol was 0.639 while
that of clove and β-caryophyllene was 0.804 and 0.922, respectively
(Table 3). As a result, antioxidant activity of eugenol and clove oil in
lipid-rich substrate was significantly different. Furthermore, free
radical scavenging capacity of eugenol and clove oil was stronger
than inhibition of lipid peroxidation activity suggesting that eugenol
might be able to donate hydrogen atoms in an aqueous environment
more effectively than in lipid. On the contrary, β-caryophyllene
reacts with peroxyl radicals better than donated hydrogen atoms.
This might be due to its oil soluble property. Furthermore, the
tyrosinase inhibition of clove oil and eugenol appeared to have the
same extent whereas that of β-caryophyllene was approximately
twice less than that of clove oil and eugenol. All tested oils were
significantly less effective than kojic acid. The correlation among
free radical scavenging activity, lipid peroxidation inhibition and
tyrosinase inhibition of clove oil is illustrated in Figure 1 and the
correlation coefficient between percent antioxidant index and
percent inhibition of mushroom tyrosinase enzyme is shown in
Table 3. At the same concentrations, inhibition of tyrosinase enzyme
is more pronounced than antioxidant activity. When the correlations
between percent tyrosinase inhibition and antioxidant index using
DPPH test and TBARs of the main components were examined, it
was found that tyrosinase inhibition of clove oil was less correlated
with antioxidant indices. These findings are supported by the study
of Arung et al. 21 that eugenol and clove buds oil can inhibit melanin
formation in B16 melanoma cells. In addition, the results also
suggest that antityrosinase of clove oil and its components may
involve not only antioxidative action but also other mechanism of
actions22-23. However, antioxidative activity of clove buds oil may be
used in a skin-lightening agent based on the hypothesis that the skin
pigmentation occurs by photo-oxidation of pre-existing melanin, the
oxidative effect of reactive oxygen species in the skin which is
induced by UV radiation and the oxidative polymerization of
melanin intermediates by tyrosinase which is a copper-containing
enzyme 3
.
Table 3: Correlation between antioxidant activity and tyrosinase inhibition
Substance
DPPH
TBAR
Clove oil
DPPH
1
.804**
TYROSINASE
.576*
.421*
Eugenol
DPPH
1
.639*
TYROSINASE
.386*
.757**
Trans-caryophyllene
DPPH
1
.922**
TYROSINASE
.923**
.960**
**Correlation is significant at the 0.01 level;
* Correlation is significant at the 0.05 level
Table 4: Cytotoxicity of clove oil and its major components against skin human fibroblasts
Substance
IC50 of SRB cytotoxicity test (µl/ml)
Clove oil
0.162
±
0.005
Eugenol
0.167
±
0.003
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
559
β-caryophyllene
> 2.0
0
10
20
30
40
50
60
70
80
90
100
0.03125 0.0625 0.25 0.5
% Antioxidant index
DPPH TBARs %Tyrosinase inhibition
Concentration (μg/ml)
Fig. 1: Correlation between antioxidant index and tyrosinase inhibition of clove buds oil.
Table 4 demonstrates that β-caryophyllene was well tolerate at
very high concentration without any toxic effect to dermal
fibroblasts while clove oil and eugenol exhibited the cytotoxicity
to fibroblasts at IC50 of 0.162 µl/ml and 0.167 µl/ml, respectively.
However, The IC50 of clove oil and eugenol in this finding were less
cytotoxicity than the study of Prashar et al.24, whereas β-
caryophyllene did not exhibit cytotoxic activity. This might be due
to the method of testing as well as the different source of tested
oils. In addition, according to the study of Arung et al.21, clove oil
and eugenol at a concentration of 100µg/ml exhibited melanin
inhibition with less cytotoxicity towards B16 melanoma cells
while β-caryophyllene did not inhibit melanogenesis and was toxic
to the cells. As shown in Figure 2, at all tested concentrations, the
oil treated cells significantly stimulated collagen synthesis greater
than the untreated cells. Clove oil, eugenol and β-caryophyllene
possessed a maximum collagen synthesis at the concentrations as
low as 0.0156, 0.0625, and 0.0031μl/ml, respectively. As the oil
concentrations increased greater than those, the amount of
collagen gradually decreased. Although the fibroblasts were
treated with the oils at the concentrations higher than their IC50
values of cytotoxicity test, the collagen synthesis in the treated
groups were significantly greater than in control group. As
reported by Khorshid et al.25
, the similar findings were also
observed in essential oil of Plectranths tenuiflorus and thymol. In
addition, the wound healing effect of these ethanolic solutions in
rat wound model was also demonstrated.
0
200
400
600
800
1000
1200
0
0.0031
0.0063
0.0125
0.0156
0.025
0.0313
0.05
0.0625
0.1
0.125
0.25
0.5
Concentrations (µl/ml)
% Collagen synthesis
Clove oil
Eugenol
Trans caryophyllene
Concentration (μl/ml)
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
560
Fig. 2: Effect of clove oil and eugenol at various concentrations on collagen synthesis
CONCLUSION
Clove oil and eugenol might be a potential anti-aging substance by
prevent aging of skin via oxidative processes and inducing collagen
synthesis. Moreover, they also possess antityrosinase activity,
suggesting a skin whitening effect. Besides other findings on their
antimicrobial effects particularly antibacterial, antifungal and anti-
acne, other activities on inhibition of 5-Lipoxygenase as well as
formation of LTC4 in human PMNL suggesting anti-inflammatory
action have also been reported26-27
1. McCullough JL, Kelly KM. Prevention and Treatment of Skin
Aging. Ann N Y Acad Sci 2006;1067:323-31.
. Therefore, these properties of
clove oil might have good implications for skin care applications.
However, due to its cytotoxicity, clove buds oil should be used in
very low concentrations with care and clinical trial should be further
investigated.
ACKNOWLEDGEMENT
This study was financial supported by the Graduate School, Khon
Kaen University, Thailand.
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... 15 Clove oil also possesses anticarcinogenic and antiallergic activities. 16 The major constituent of clove oil is eugenol (78−99%), but it also contains a variety of other active constituents such as β-caryophyllene (0.3−13%), eugenyl acetate (6%), benzyl alcohol (34%), humulene (0.19%), humulenol (0.27%), calcorene (0.11%), etc. 14,16,17 Clove bud oil as well as eugenol was found to exhibit free radical scavenging activity, and lipid peroxidation inhibition activity and induce collagen synthesis by dermal fibroblasts. 17 They can also inhibit reactive oxygen species and nitric oxide production and myeloperoxidase activity in human neutrophils. ...
... 16 The major constituent of clove oil is eugenol (78−99%), but it also contains a variety of other active constituents such as β-caryophyllene (0.3−13%), eugenyl acetate (6%), benzyl alcohol (34%), humulene (0.19%), humulenol (0.27%), calcorene (0.11%), etc. 14,16,17 Clove bud oil as well as eugenol was found to exhibit free radical scavenging activity, and lipid peroxidation inhibition activity and induce collagen synthesis by dermal fibroblasts. 17 They can also inhibit reactive oxygen species and nitric oxide production and myeloperoxidase activity in human neutrophils. 18 Eugenol has also been found to possess anti-inflammatory, antiviral, antifungal, insecticidal, antitumor, and anesthetic activities. ...
Article
Infections are a leading cause of mortality and amputations among patients with burns and chronic wounds, respectively. Moreover, the extensive use of antibiotics has led to the rapid spreading of drug resistance among microorganisms. Alternatively, plant-derived natural products, which have been used as traditional therapies for several centuries, are recently gaining popularity as they are relatively affordable and easily available in many developing countries where modern medications are expensive or unavailable. In this study, clove essential oil is used for its antimicrobial property and is further incorporated into cryogels to increase its bioavailability and prolong its bioactivity. The oil-incorporated cryogels are macroporous, biodegradable, possess mechanical properties similar to commercial skin substitutes, are cytocompatible, antibacterial, and allow long-term sustained release of oil for up to at least 14 days. Additionally, clove oil aids the faster closure of in vitro scratch wounds by improving the migration of fibroblasts. This work presents a novel, bioactive scaffold that has the potential to be used as a dermal substitute and serves as an alternative to commercial skin substitutes.
... Clove essential oil fabricated as NFs with various polymers show strong antibacterial properties, especially against Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens, and Bacillus subtilis [8][9][10]. The wound-healing effect of clove oil in nanoemulsion form was studied, and it was confirmed that the oil in nanoemulsion has a marked wound-healing capacity and reduced the incidence of inflammatory cells at the site of a wound when compared to pure clove oil and enhanced cell viability [11,12]. Poly(ε-Caprolactone)/Gelatin NFs loaded with clove oil showed good antibacterial as well as marked healing properties [10]. ...
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Wound-healing is complicated process that is affected by many factors, especially bacterial infiltration at the site and not only the need for the regeneration of damaged tissues but also the requirement for antibacterial, anti-inflammatory, and analgesic activity at the injured site. The objective of the present study was to develop and evaluate the natural essential oil-containing nano-fiber (NF) mat with enhanced antibacterial activity, regenerative, non-cytotoxic, and wound-healing potential. Clove essential oil (CEO) encapsulated in chitosan and poly-ethylene oxide (PEO) polymers to form NFs and their morphology was analyzed using scanning electron microscopy (SEM) that confirmed the finest NFs prepared with a diameter of 154 ± 35 nm. The successful incorporation of CEO was characterized by Fourier transform infra-red spectroscopy (FTIR) and X-ray diffractom-etry (XRD). The 87.6 ± 13.1% encapsulation efficiency and 8.9 ± 0.98% loading of CEO was observed. A total of 79% release of CEO was observed in acidic pH 5.5 with 117% high degree of swelling. The prepared NF mat showed good antibacterial activity against Staphylococcus aureus and Escherichia coli and non-cytotoxic behavior against human fibroblast cell lines and showed good wound-healing potential.
... Moreover, its antibacterial activity has been demonstrated to be particularly effective against bacterial strains such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas fluorescens [13]. On the other hand, the effective dose of CLV has enhanced cell viability [14,15]. According to our knowledge, a few studies related to EOs' influence on in vitro cell migration have been carried out. ...
Article
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The objective of this study was to produce antibacterial poly(ε-caprolactone) (PCL)-gelatin (GEL) electrospun nanofiber mats containing clove essential oil (CLV) using glacial acetic acid (GAA) as a “benign” (non-toxic) solvent. The addition of CLV increased the fiber diameter from 241 ± 96 to 305 ± 82 nm. Aside from this, the wettability of PCL-GEL nanofiber mats was increased by the addition of CLV. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of CLV, and the actual content of CLV was determined by gas chromatography–mass spectrometry (GC-MS). Our investigations showed that CLV-loaded PCL-GEL nanofiber mats did not have cytotoxic effects on normal human dermal fibroblast (NHDF) cells. On the other hand, the fibers exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. Consequently, PCL-GEL/CLV nanofiber mats are potential candidates for antibiotic-free wound healing applications.
... Eugenol possesses a potent antioxidant activity as documented by several reports 1,6,7,14,16 . In consistence with these reports, our data demonstrated that administration of eugenol at two doses (10 and 100 mg/kg) increased the activity of serum SOD and CAT. ...
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Eugenol, a component of essential oils of medicinal and food plants, has a hypolipidemic effect in experimental animals although its mechanism of action is still unclear. This study aims to explore the mechanism of the hypolipidemic effect of eugenol in rats fed a high cholesterol and fat diet (HCFD). Eugenol significantly reduced total cholesterol (TC), low-density lipoproteins (LDL), atherogenic index (AI) but not high-density lipoproteins (HDL) or triglycerides (TG). Eugenol also decreased steatosis and hepatic inflammation in liver sections, decreased hepatomegaly, and the hepatic marker enzymes alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activity and increased the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) activity in hypercholesterolemic rats. Eugenol did not inhibit hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase but caused down-regulation of transient receptor potential vanilloid (TRPV1) channels in the liver. Docking simulation using fast, rigid exhaustive docking (FRED) software indicated a tail-up/head-down interaction of eugenol with TRPV1 channel. Data indicate that eugenol does not inhibit HMG-CoA reductase but rather induces its action by interaction with TRPV1 channels.
... Pure eugenol had an IC 50 value of 129.69 μg mL -1 . Mahapatra and Roy [84] found an IC 50 value of 242.47 μg mL -1 for eugenol and Khunkitti et al. [85] and Politeo et al. [86] reported IC 50 values 145.2 μg mL -1 and IC 50 96.0 μg mL -1 , respectively, for this phenylpropene. ...
Article
In this study, the effect of operational conditions on the extraction of clove leaves using CO2 on eugenol yield and content was investigated. The conditions used in the supercritical extraction with CO2 included temperatures of 40, 50, and 60 °C and pressures of 150, 185 and 220 bar. The highest yield (1.08 wt%) and highest eugenol content were obtained at 40 °C and 220 bar. The main components identified in the clove extract were eugenol (29.73%), chavicol (13.31%), n-pentacosane (11.66%), hexacosanal (11.37%), and vitamin E (9.30%). The extracts were evaluated for their antioxidant capacities by the DPPH and FRAP methods, and quantification of the phenolic compounds was done by the Folin-Ciocalteu method. The best results among the conditions were obtained at the lowest pressure and temperature, 150 bar and 40 °C. The Sovová model satisfactorily described the extraction kinetics in all the studied conditions.
... caryophyllene (0.3-36.94%) and αhumulene (1.9-4.41%) [74,[129][130][131][132][133][134] Low-molecular-weight phenolic compounds are among the most typical components of essential oils with antimicrobial activity [135]. They could have an activating or inhibiting effect on microbial growth according to their structure and concentration [136][137]. ...
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Aspergillus ostianus Wehmer was a new record to the Mycological Laboratories in Egypt, isolated form cabbage seeds and tested for aflatoxins and ochratoxin A production. Aflatoxins B 1 and G 1 and ochratoxin A were demonstrated in the crude extracts of Aspergillus ostianus cultivated on different media for mycotoxins production. Both of essential oil and ethanol extract of clove were tested in order to control Aspergillus ostianus. The minimum inhibitory concentration (MIC) was 46.77 and 500 mg/ml for essential oil and ethanol extract, respectively. Also, qualitative phytochemical screening of the extracts and their chemical components using GC/MS analysis were determined.
... Essential oil of clove (clove oil) is one such example which has exhibited biological activity on a wide range of organisms ranging from microorganisms to humans (Kumar et al., 2011). 60% -90% of clove oil is constituted by eugenol which is regarded to be the source of its antifungal, anaesthetic and antiseptic properties (Rani et al., 2012) and also its cytotoxicity (Watcharee et al., 2012) . Clove oil is exempted from pesticide registration requirements and pesticide residue tolerance requirements (Hollingsworth et al., 2012 ). ...
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Nowadays consumers are increasingly demanding food with fewer synthetic preservatives, which makes antimicrobial essential oils (EOs) from plants promising alternatives. In this work, surfactant-free emulsions were successfully fabricated from Cinnamon cassia oil (C. cassia oil) with partially deacetylated chitin nanofiber (ChNF) adopted as a Pickering stabilizer. The storage stability and microstructures of the emulsions with different concentrations of ChNF were studied in detail. As ChNF concentration increased, the emulsion droplet size decreased while the emulsion stability increased with stable periods as long as 90 days. This could be attributed to the Pickering stabilization realized by irreversible adsorption of the ChNF at the oil−water interface (revealed by fluorescent microscopy) and subsequent formation of an interdroplet ChNF network in the continuous phase, which was further strengthened in the presence of the aldehyde moiety in the C. cassia oil (verified by FTIR spectra). The rheological data and SEM images provided further evidence for network formation in the emulsions with increased ChNF concentration. Furthermore, the antimicrobial activity of the emulsion against Escherichia coli and the release patterns of EOs from emulsions were also investigated. The emulsions showed prolonged antibacterial activities but enhanced diffusion efficiency with the introduction of ChNF, which turned out to be a good encapsulation system for the controlled release of EOs. This work evidences the promising advantages of ChNF-stabilized Pickering emulsions as a facile EOs delivery system for application in food preservation and related fields.
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The antioxidant effects of individual natural compounds have been extensively investigated. However, studies on their interactions are still lacking. This study aimed to observe the interaction effect of ginger extract (GE) combined with ascorbic acid on antioxidant activity by applying response surface methodology. The results from the quadratic model showed that individually tested DPPH free radical scavenging capacities of GE and ascorbic acid manifested positive effects on the antioxidant index (AI%). However, interaction of their combination demonstrated an antagonistic effect that negatively influenced the AI%. An optimization process revealed that the highest AI% could be achieved by applying maximum effective concentrations of GE (0.49 mg/mL) and ascorbic acid (0.82 mg/mL). Although, the mechanisms behind these interactions need to be further explored, the results of this study suggested that antagonistic interaction between the combination of ginger or other herbal extracts with ascorbic acid should be taken into consideration to prevent any potential health complications.
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Bovine mastitis is an inflammation of the mammary gland due to bacterial infection. Contaminations of milk with chemical residues from disinfectant solutions during the milking process have been of great concern. The aims of this study were to investigate the antioxidant activity and antibacterial action of eight essential oils, namely citronella grass, lemongrass, kaffir lime, holy basil, sweet basil, plai, turmeric and betel vine against clinical isolate pathogens commonly found in dairy cows. None of the selected essential oils were effective against Pseudomonas aeruginosa. Among the tested essential oils, lemongrass possessed the strongest antibacterial against clinical isolate mastitis pathogens.The MIC/MBC values were in a range of 0.13 to 0.54 ul/ml. Free radical scavenging activity of lemongrass oil at 10 μl/ml was 46.0% and lipid peroxidation inhibition at I μl/ml was 32.1%, while those of holy basil oil were 89.9 and 67.5 %, respectively, which indicated the strongest antioxidant activity. In conclusion, lemongrass oil and holy basil oil might be promising alternative agents to chemical disinfectants for preventing mastitis causing by bacterial infection and reducing the oxidative stress due to tissue damage of the mammary gland. Further studies on formulation development are in progress.
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Nitrogen containing heterocyclic compounds such as oxindoles especially isatins (β‐lactams) and their derivatives have excellent biological properties such as anti‐inflammatory, antimicrobial, anticancer activity. In the present study the antioxidant activity of the said class of compounds was investigated by DPPH method with respect to ascorbic acid. To determine the antioxidant activity, a number of methyl/chlorinated isatins (2af), their Schiff‐bases (2a', 2d', 2e' and 2f'), spiro‐thiadiazolines (1gi), and optically active phenolics (1af) of different isatins were synthesized by both microwave and conventional heating methods. The present findings revealed that some of the synthesized compounds (1b, 1c, 1e, 1f, 2ab, 2d, 2a' and 2b') exhibited a marked scavenging effect on DPPH radical.
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The aim of this study was to investigate the effect of citronella oil (Java type) (Cymbopogon winterianus Jowitt) on Propionibacterium acnes DMST 14916. Citronella oil compositions were determined by gas chromatography mass spectrometry (GC/MS). Anti-P. acnes activity of citronella oil and its major components were also investigated. After exposure with P. acnes at various concentrations of citronella oil (0–100 μL/mL), time-kill profiles, pentose leakage and electron microscopic characteristics were determined. The GC/MS results revealed that the major constituents of the oil were citronellal, geraniol and citronellol. At all concentrations above Minimum Bactericidal Concentrations (MBCs; 0.625μL/mL), this essential oil killed P. acnes in a dose-dependent manner. The intracellular materials leakage and electron microscopic characteristics evidenced that the major target sites of antibacterial activity appeared to be cell wall, cytoplasmic membrane and intracellular materials. Citronella oil exerted satisfactory bacteriostatic and bactericidal actions against P. acnes.
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Nineteen compounds have been identified and quantified from the bark oil of Gliricidia sepium by GC-MS analysis. The major components are methyl-3(E)-pentenyl ether (11.55%), 3-methyl-2-butanol (10.65%), 3-methoxy hexane (10.14%), 1-(1-ethoxyethoxy)-2-hexene (9.72%), 2- decanol (8.97%), coumarin (8.07%) and hexadecanoic acid (5.16%). The antibacterial activity of the essential oil was checked against various pathogenic bacteria such as Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Streptococcus faecalis, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens by 'agar well diffusion' method. The bark oil exhibited pronounced activity against all tested microorganisms and its activity is quite comparable with the standard antibiotics screened under similar conditions. The remarkable antibacterial activity exhibited by the bark oil can be attributed to the long chain fatty acids and alcohols present in it. This study shows that the Gliricidia sepium bark essential oil can be used as a potential external antiseptic and can be incorporated into the drug formulations.
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Clove and nutmeg essential oils were analysed by GC and GC-MS. These oils, together with 16-18 components found to be present, were tested for antioxidant properties at final concentrations of 0.05-2.5 x 104 ppm in an egg yolk-based thiobarbituric acid reactive substances (TBARS) assay and also undiluted in a β-carotene agar diffusion assay. Both the essential oils and the components tested in the TBARS assay demonstrated some degree of antioxidant activity. Only the clove oil, the nutmeg oil, eugenol and terpinolene demonstrated any ability to inhibit the oxidative bleaching of the β-carotene agar. The ability of the oil components to inhibit malondialdehyde formation, and therefore lipid peroxidation, in the TBARS assay, yet apparently to possess no activity in the β-carotene agar diffusion assay, demonstrates the importance in the screening of plant material for bioactivity of using a bank of assays in vitro before assigning bioactivities. By using a number of assays, not only should the number of false positives and negatives be greatly reduced, but evidence pertaining to the mechanism of action may be obtained.
Article
Clove and nutmeg essential oils were analysed by GC and GC–MS. These oils, together with 16–18 components found to be present, were tested for antioxidant properties at final concentrations of 0.05–2.5×104 ppm in an egg yolk-based thiobarbituric acid reactive substances (TBARS) assay and also undiluted in a β-carotene agar diffusion assay. Both the essential oils and the components tested in the TBARS assay demonstrated some degree of antioxidant activity. Only the clove oil, the nutmeg oil, eugenol and terpinolene demonstrated any ability to inhibit the oxidative bleaching of the β-carotene agar. The ability of the oil components to inhibit malondialdehyde formation, and therefore lipid peroxidation, in the TBARS assay, yet apparently to possess no activity in the β-carotene agar diffusion assay, demonstrates the importance in the screening of plant material for bioactivity of using a bank of assays in vitro before assigning bioactivities. By using a number of assays, not only should the number of false positives and negatives be greatly reduced, but evidence pertaining to the mechanism of action may be obtained. Copyright © 2000 John Wiley & Sons, Ltd.
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Essential oils are used in skincare products for perfuming and aromatherapy purposes. In this study, the bioactivities of seven essential oils commonly used and claimed for skincare namely citronella grass (Cymbopogon nardus L.), lemongrass (Cymbopogon citratus DC), Kaffir lime (Citrus hystrix DC), holy basil (Ocimum sanctum L.), sweet basil (Ocimum basilicum L.), plai (Zingiber cassumunar Roxb) and ginger (Zingiber officinale Roscoe) were investigated. Investigation of the in vitro susceptibility of the oils against Propionibacterium acnes (P. acnes) using the broth microdilution technique revealed that citronella grass oil exhibited the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at 0.005–0.3 and 0.6–1.2 μl/ml, respectively. The MIC and MBC values of lemongrass oil were 0.6 μl/ml and those of kaffir lime oil and holy basil oil were 5 μl/ml. Antioxidant activity using the DPPH free radical scavenging assay showed that the IC50 values of holy basil oil (0.03 μl/ml), plai oil (6.9 μl/ml) and citronella grass oil (2 μl/ml) were lower than that of ascorbic acid (7.9 μl/ml). Anti-inflammatory activity of the oils determined using the 5-lipoxygenase inhibition assay found that IC50 values of holy basil oil (0.04 μl/ml), kaffir lime oil (0.05 μl/ml) and citronella grass oil (0.15 μl/ml) were less than that of nordihydroquaretic acid (1.7 μg/ml). Since P. acnes has a role in the inflammation of acne leading to scar formation, citronella grass oil may help to relieve acne blemishes. However, further investigation in the form of clinical studies would be necessary.
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4-Hydroxyproline (4-Hyp) is a specific amino acid of collagen and widely used as a factor to estimate the collagen content in biological specimens. The authors have developed an analysis kit with the ability to detect collagen on microwells. The method includes chromophore formation without solvent transfers, that allows the analysis of multiple specimens with excellent sensitivity, high specificity at low cost with shorter analysis time. The calibration curve of 4-HYP kit exhibiting a high positive relationship (R2 = 0.999) while showing a very low detection limit of (1.0 μg/ml). Specificity of 4-HYP kit was decreased with increasing hydrolysed non-collagenous biomolecules (HNCB), however this was negligible since only a few collagen specimens have a high amount of HNCB. The 4-HYP kit was successfully applied to commercial collagen quantification, measuring the collagen content of connective tissue and collagen synthesis of fibroblast with high satisfactory results; therefore, this is a more suitable alternative to previous analysis methods.
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The reaction mechanisms of three antioxidants are proposed in order to explain experimental results obtained from a kinetic study using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH.) method, previously adapted in our laboratory. In its radical form, DPPH.shows an absorbance maximum at 515 nm which disappears upon reduction by an antiradical compound. BHT, a synthetic antioxidant, slowly reacts with DPPH.reaching a steady state within 5 h. This 2.8-stoichiometric complete reaction follows a 1.5-order with respect to DPPH.and 0.5 to BHT. The kinetic rate constant, k, is estimated to be 5.0 L/(mol·s) at 20 °C and the energy of activation,Ea, is equal to 35 kJ/mol in methanol. Eugenol reacts with DPPH.reaching a steady state within 2 h. This 1.9-stoichiometric reaction follows a 2-order with respect to both DPPH.and eugenol, k andEaare estimated to be 5.4 × 1010L3/(mol3·s) at 20 °C and 30 kJ/mol, respectively. The eugenol mechanism may involve a dimerization between two phenoxyl radicals. The reaction with isoeugenol is rapid and reversible, with a stoichiometry of 1.1. It is first order with respect to isoeugenol with k (direct reaction) equal to 8.9 × 10−2s−1at 10 °C. This reaction is consistent with a pseudo-monomolecular mechanism.