IN VITRO BIOACTIVITIES OF CLOVE BUDS OIL (Eugenia caryophyllata) AND ITS EFFECT ON
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: email@example.com
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.
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
. 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.
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
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
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
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
Determination of antioxidant activity by inhibition of lipid
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 )/
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.
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
Table 1: The main components of clove oil
Mode of Identification*
a, b, c
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
Table 2: Bioactivities of clove oil and its major components compared with reference standards
Vitamin E acetate
ND: not determined
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
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
Table 3: Correlation between antioxidant activity and tyrosinase inhibition
**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
IC50 of SRB cytotoxicity test (µl/ml)
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
0.03125 0.0625 0.25 0.5
% Antioxidant index
DPPH TBARs %Tyrosinase inhibition
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.
% Collagen synthesis
Khunkitti et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 556-560
Fig. 2: Effect of clove oil and eugenol at various concentrations on collagen synthesis
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
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However, due to its cytotoxicity, clove buds oil should be used in
very low concentrations with care and clinical trial should be further
This study was financial supported by the Graduate School, Khon
Kaen University, Thailand.
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