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Chemical composition, antimicrobial and antioxidant activities of the essential
oil and the ethanol extract of Cleistocalyx operculatus (Roxb.) Merr and Perry buds
Nguyen Thi Dung a,b , Jung Min Kim c , Sun Chul Kang a, *
a Department of Biotechnology, Daegu University, Gyeongsan 712-714, Republic of Korea
b Department of Plant Biochemistry, Institute of Biotechnology, Vietnamese Academy of Science and Technology, Vietnam
c Department of Microbiology, School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea
article info
Article history:
Received 3 May 2008
Accepted 10 September 2008
Keywords:
Cleistocalyx operculatus
Essential oil
Antimicrobial activity
MRSA
VRE
Antioxidant assay
1. Introduction
abstract
The antimicrobial activity of plant oils and extracts has formed
the basis of many applications, including raw and processed potential
as natural agents for food preservation, pharmaceuticals, alternative
medicine and natural therapies (Cosentino et al., 1999;
Bakkali et al., 2008). In order to prolong the storage stability of
foods, synthetic antioxidants are mainly used in industrial processing.
But according to the toxicologists and nutritionists, the side effects
of some synthetic antioxidants used in food processing such
as butylated hydroxyltoluene (BHT) and butylated hydroxylanisole
(BHA) have been documented. For example, these substances can
show carcinogenic effects in living organisms (Botterweck et al.,
2000). For this reason, the search for antioxidants from natural
Abbreviations: ASA, L-ascorbic acid; ASE, L-ascorbic acid equivalents; BHA,
butylated hydroxyanisole; BHT, butylated hydroxyltoluene; DPPH, 1,1-diphenyl-2picrylhydrazyl;
DMSO, dimethylsulfoxide; FS, food spoilage; FB, food-borne; GC/MS,
Gas chromatography–mass spectrometry and mass spectrometer analysis; MIC,
minimum inhibitory concentration; MBC, minimum bactericidal concentration;
MARB, multiantibiotic-resistant bacteria; MRSA, methicillin-resistant Staphylococcus
aureus; VRE, vancomycin-resistant Enterococci; SP, skin pathogens; SEM,
scanning electron microscopic.
* Corresponding author. Tel.: +82 53 850 6553; fax: +82 53 850 6559.
E-mail address: sckang@daegu.ac.kr (S.C. Kang).
0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.09.013
Food and Chemical Toxicology 46 (2008) 3632–3639
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
In the present study, the essential oil isolated from the buds of Cleistocalyx operculatus by hydrodistillation
was analyzed by GC and GC/MS. A total of 55 compounds representing 93.71% of the oil were identified.
The oil significantly inhibited the growth of food spoilage (FS), food-borne (FB), skin pathogens
(SP), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and
multiantibiotic-resistant bacteria (MARB). The minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) of the oil against the tested microorganisms were found in the range
of 1–20 lL/mL. Whereas the ethanol extract exhibited potential antibacterial activity against the entire
tested Gram positive bacteria and one food spoilage Gram negative bacterium P. aeruginosa. The MIC
and MBC values of ethanol extract against the tested bacteria were found in the range of 0.25–32 mg/
mL. The scanning electron microscopic (SEM) studies demonstrated potential detrimental effect of the
essential oil on the morphology of MRSA-P249 and VRE-B2332 at the used MIC values, along with the
potential effect on cell viabilities of the tested bacteria. Moreover, the total antioxidant capacity and
the scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals of the essential oil and the ethanol
extract were also evaluated.
Ó 2008 Elsevier Ltd. All rights reserved.
source has received much attention and efforts have been into to
identifying compounds that can act as suitable antioxidants to replace
synthetic ones. In addition, these naturally occurring antioxidants
can be formulated as functional foods and can help to
prevent oxidative damage from occurring in the body (Dahanukar
et al., 2000).
Diets rich in selected natural antioxidants such as polyphenols,
flavonoids, vitamin C and vitamin E can reduce risk of incidence of
cardiovascular, other chronic diseases and certain types of cancer.
This has lead to the revival of interest in plant-based foods (Choi
et al., 2007; Majhenic et al., 2007; Mata et al., 2007). A large number
of plant species have already been tested for their potential
biological, therapeutic and pharmaceutical activities (Majhenic
et al., 2007; Mata et al., 2007; Alcaradz et al., 2000; Anthony
et al., 2002; Cosentino et al., 1999; Bakkali et al., 2008).
Plants contain a variety of substances called ‘‘phytochemicals”
that come from naturally occurring components present in plants.
The phytochemical preparations with dual functionalities in preventing
lipid oxidation and antimicrobial properties have tremendous
potential for extending the shelf life of food products.
Although it remains unclear which of the compounds of plants
are the active ones, essential oils and phenolics recently have received
increasing attention because of some interesting new findings
regarding their biological activities, and are widely distributed

in edible plants (Bakkali et al., 2008; Kamel et al., 2007; Archana
et al., 2005). Essential oils and extracts of plants are of growing
interest both in the industry and scientific research because of
their antioxidants, antibacterial, antifungal, antiviral and anti-parasitical
activities that make them useful as natural additives in
foods, cosmetic and pharmaceutical industries (Bakkali et al.,
2008; Kamel et al., 2007; Mabberley, 1997). The potential of the
essential oils for developing new drugs has largely been unexplored.
These constituents and their relative concentrations vary
from one plant oil to another, and essential oils from different
plants exhibit varied antimicrobial activity. In recent years due to
an upsurge in antibiotic-resistant infections, the search for new
prototype drugs to combat infections is an absolute necessity.
Use of essential oils and extracts may offer a great potential and
hope in this search.
Cleistocalyx operculatus (Roxb.) Merr & Perry, also known as
Eugenia operculata (Roxb.) Merr & Perry or Syzygium nervosum, is
a well-known perennial tree, widely distributed and propagated
in China, Vietnam and some other tropical countries. The leaves
and buds of C. operculatus have been used as an ingredient in various
beverages, common tea for gastrointestinal disorders and as
an antisepsis for dermatophytic disorders for many years (Loi,
1986). Previous attention to phytochemical led to the isolation of
oleanane type triterpene from its bark (Nomura et al., 1999), and
the presence of sterol, flavanone, chalcone, triterpene acid, b-sitosterol
and ursolic acid as the main constituents in the methanol extract
of the C. operculatus buds (Zhang et al., 1990; Ye et al., 2004,
2005a). GC and GC/MS analysis of C. operculatus leaves’ essential oil
has been also reported (Dung et al., 1994). Previous reports revealed
that the C. operculatus buds had various biological activities
in vitro and in vivo such as anticancer, antitumor, antihyperglycemic
and cardio tonic action (Loi, 1986; Ye et al., 2005a,b; Mai
et al., 2007; Anthony et al., 2002). The compositions of essential
oils and biological activities of other Cleistocalyx species such as
Eugenia caryophyllus, E. unifloral, E. malaccensis and E. jambolana
are well-known (Mabberley, 1997). However, to the best of our
knowledge, the chemical composition of essential oil of C. operculatus
buds has not been studied yet, as well as its antimicrobial and
antioxidant activities.
In the present investigation, we analyzed the chemical composition
of essential oil of C. operculatus buds, which was obtained
from hydrodistillation by GC–MS, and tested the efficacy of the
essential oil and ethanol extract against a diverse range of microorganisms
comprised of clinical isolates of food spoiling (FS), foodborne
pathogens (FB), skin pathogens (SP), methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci
(VRE) and multiantibiotic-resistant bacteria (MARB), along with
the effect of essential oil on the morphology and the cell viabilities
of tested microorganisms. We also evaluated the antioxidant potential,
which was assessed by the total antioxidant capacity and
the scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radicals of the essential oil and the ethanol extract.
2. Materials and methods
2.1. Plant materials and chemicals
The C. operculatus buds used in this experiment were purchased from a local
herb supplier in Hanoi, Vietnam and were identified by comparing its morphological
features with the specimen deposited at the Plant Laboratory, Institute of Biological
Ecology and Biological Resources, Vietnamese Academy of Science and
Technology, Hanoi, Vietnam.
n-Alkane standard solution C 8–C 20 (mixture no. 04070) was purchased from Fluka
Chemika (Buchs, Switzerland). Antimicrobial standards, L-ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and butylated hydroxyanisole (BHA) were
purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals and solvents
were of analytical grade.
N.T. Dung et al. / Food and Chemical Toxicology 46 (2008) 3632–3639 3633
2.2. Preparation of essential oil and ethanol extract
The air-dried C. operculatus buds were pulverized into powdered form (less than
150 lm form). The powder of sample (250 g) followed subjected to hydrodistillation
for 4 h using a Clevenger type apparatus to obtain the essential oil. The oil
was dried over anhydrous Na 2SO 4 and preserved in a sealed vial at 4 °C in the dark
until further analysis (yield 0.68%, w/w).
Further, the powder form of C. operculatus buds (50 g) was extracted with 70%
ethanol (200 ml 3 times) at room temperature. The ethanol extracts were combined
and evaporated by a vacuum rotary evaporator (EYELAN-1000, Japan) at
45 °C to the dried powdered form (yield 12.96%, w/w).
2.3. Essential oil analysis
2.3.1. Gas chromatography–mass spectrometry (GC/MS) and mass spectrometer
analysis
The GC/MS analysis of the essential oil was performed using a Shimadzu GC/MS
(GC-17A) equipped with a ZB-1 MS fused silica capillary column (30 m 0.25 mm
i.d., film thickness 0.25 lm). For GC/MS detection, an electron ionization system with
ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant
flow rate of 1 mL/min. Injector and MS transfer line temperature were set at
220 °C and 290 °C, respectively. The oven temperature was programmed from 50 °C
to 150 °Cat3°C /min, increase then held isothermal for 10 min and finally raised to
250 °Cat10°C /min. Diluted samples (1/100, v/v, in methanol) of 1.0 lL were injected
manually in the split less mode. The relative percentage amount of each component
was calculated by comparing its average peak area to the total areas.
Mass spectrometer: Shimadzu GC/MS (GC-17A) system recording at 70 eV; scan
time 1.5 s; mass range 40–300 amu. Software adopted to handle mass spectra and
chromatograms was a ChemStation.
2.3.2. Retention indices
Van den Dool and Kratz (1963) proposed a quasi-linear equation for temperature-programmed
retention indices as follows:
RIx ¼ 100n þ 100 ðtx tnÞ=ðtnþ1 tnÞ
where RIx is the temperature-programmed retention index of the interest, and tn, tn+1, tx are the retention times in minute of the two standard n-alkanes containing
n and n+1 carbons and the interest, respectively. This equation was used to calculate
retention indices in the present work, linear temperature-programmed GC operating
conditions (relative to C8–C20 on the ZB-1 column).
2.3.3. Identification of components
The components were identified by comparison of their mass spectra with those
in the Wiley and MassFinder Ver. 2.1 GC/MS library and those in the literature
(Adam, 2001), as well as by comparison of their retention indices with literature
data (Adam, 2001; Cosentino et al., 1999; Ramona et al., 2007).
2.4. Antibacterial activity
2.4.1. Microorganisms
Two food spoilage (Bacillus subtilis ATCC6633 and Pseudomonas aeruginosa
KCTC2004) and nine food-borne pathogens (Staphylococcus aureus ATCC6538, S.
aureus KCTC1916, Listeria monocytogenes ATCC19166, Enterobacter aerogenes
KCTC2190, Salmonella typhymurium KCTC, Salmonella enteritidis KCCM 12021, Escherichia
coli ATCC8739, E. coli O157:H7 (human) and E. coli O157:H7 ATCC43888)
were obtained from the Korea Food and Drug Administration. Besides that, four skin
infectious pathogens (S. aureus KCTC1621, Staphylococcus epidermidis KCTC1917,
E. coli KCTC1039 and a yeast Candida albicans KCTC7965) were obtained from the
Department of Microbiology, Daegu Oriental Medicine University. In addition, three
methicillin-resistant S. aureus (S. aureus P227, S. aureus P254 and S. aureus P249)
and three vancomycin-resistant Enterococci (Enterococcus faeccium A93, E. faeccium
B2332 and E. faeccium U914) were obtained from the Laboratory of Microbiology,
Medical School of Yonsei University. Also, 15 multiantibiotic-resistant bacteria
(MARB), used in this study, including 12 clinical isolates (Acinetobacter baumannii
05KA007, A. baumannii 05 KA31, E. coli 02K 276, E. coli 04K717, Enterobacter cloacae
04K453, E. cloacae 04K908, Klebsialla pneumoniae 05K279, K. pneumoniae 05K406, P.
aeruginosa 03K442, P. aeruginosa 03K 711, Serratia marcescens 03K188 and S. marcescens
03K201) and three reference strains (S. aureus ATCC25923, P. aeruginosa
ATCC27853 and E. coli ATCC25922) were obtained from Department of Microbiology,
Kyungpook National University, School of Medicine. The pattern profiles of
the antibiotic-resistant bacteria are described in Table 3. Culture of the food spoilage
(FS) and food-borne pathogens (FB) was maintained on Luria Broth (LB) agar
medium. The skin infectious pathogens (SP), methicillin-resistant S. aureus (MRSA),
vancomycin-resistant Enterococci (VRE) and multiantibiotic-resistant bacteria
(MARB) were maintained on Mueller Hinton Broth agar medium.
2.4.2. Determination of antimicrobial activity by the disc diffusion method
The agar diffusion method (Murray et al., 1995) was used for determining the
diameters of inhibition zones of essential oil and ethanol extract against the tested
bacteria. One mL of inoculums of each strain (10 6 –10 7 CFU/mL) was poured and

3634 N.T. Dung et al. / Food and Chemical Toxicology 46 (2008) 3632–3639
spread uniformly on Luria Broth or Mueller Hinton Broth agar medium. Whatman
No. 1 sterile filter paper discs (6 mm diameter) were impregnated with essential
oil or ethanol extract dissolved in 5% dimethylsulfoxide (DMSO, Carlo Erba, Italy).
Negative and positive controls (antibiotics) were prepared using the same solvent
employed to dissolve the samples (DMSO). The discs were applied on the agar surface,
and the plates were incubated at 37 °C for 24 h.
2.4.3. Determinations of minimum inhibitory (MIC) and minimum bactericidal
concentration (MBC)
Micro-dilution susceptibility assay was performed using NCCLS method for
determination of the minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) (NCCLS, 1999). Bacteria or yeast were cultured overnight
at 37 °C. Dilutions were prepared in a 96 well microtitre plates to get final
concentration ranging from 0 to 50 lL/mL of the essential oil or 0–50 mg/mL of
the ethanol extract in the medium. Finally, 20 lL of inoculums (10 6 –10 7 CFU/
mL) were inoculated on to the microplates and the tests were performed in a volume
of 200 lL. Plates were incubated at 37 °C for 24 h. The lowest concentration
of tested samples, which did not show any visual growth after macroscopic evaluation,
was determined as MIC. Using the results of the MIC assay, the concentrations
showing complete absence of visual growth of bacteria were identified and
50 lL of each culture broth was transferred on to the agar plates and incubated for
the specified time and temperature as mentioned above. The complete absence of
growth on the agar surface in the lowest concentration of sample was defined as
the MBC.
2.4.4. Cell viability assay of MRSA and VRE
Each of the tubes containing bacterial suspension (approximately 10 6 CFU/mL)
of MRSA or VRE was inoculated with the minimum inhibitory concentration of C.
operculatus buds essential oil in 10 mL MHB, and kept at 37 °C. Samples for viable
cell counts were taken out at 0, 5, 10, 15, 20, 40 and 120 min time intervals. The viable
plate counts were monitored as followed: 0.1 mL sample of each treatment was
diluted and spread on the surface of MHB agar. The colonies were counted after 24 h
of incubation at 37 °C. The controls were inoculated without essential oil for each
bacterial strain with the same experimental condition as mentioned above.
2.4.5. Scanning electron microscopic (SEM) analysis
To determine the efficacy of essential oil and the morphological changes, SEM
studies were performed on MRSA–P249 and VRE–B2332 treated with MIC of
essential oil of C. operculatus buds. Controls were prepared without essential
oil. Further, to observe the morphological changes, the method of SEM was modified
from Kockro method (Kockro et al., 2000). The bacterial samples were
washed gently with 50 mM phosphate buffer solution (pH 7.2), fixed with
2.5 g/100 mL glutaraldehyde and 1 g/100 mL osmic acid solution. The specimen
was dehydrated using sequential exposure per ethanol concentrations ranging
from 30% to 100%. The ethanol was replaced by tertiary butyl alcohol. After dehydration,
the specimen was dried with CO2. Finally, the specimen was sputtercoated
with gold in an ion coater for 2 min, followed by microscopic examinations
(S-4300; Hitachi).
2.5. Antioxidant activities
2.5.1. Determination of total antioxidant capacity
The assay is based on the reduction of Mo (VI)–Mo (V) by the extract and subsequent
formation of green phosphate/Mo (V) complex at acidic pH (Archana et al.,
2005). The 0.1 mL extract was combined with 3 mL of reagent solution (0.6 M sulphuric
acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The
tubes were incubated at 95 °C for 90 min. This solution was allowed to cool at room
temperature and the absorbance of the solution was measured at 695 nm against a
blank. The antioxidant activity was expressed as the number of equivalents of
ascorbic acid.
2.5.2. Scavenging activity of DPPH radical
The antioxidant activities of the essential oil and ethanol extract were measured
on the basis of the scavenging activities of the stable 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical (Archana et al., 2005). Various concentrations of 100 lL of the
tested essential oil or extract were added to 2.9 mL of a 0.004% (w/v) ethanol solution
of DPPH. After 30 min of incubation period at room temperature, the absorbance
was measured against a blank at 517 nm. Inhibition of free radical DPPH in
percent (%) was calculated by:
% scavenging effect ¼½ðADPPH ASÞ=ADPPHŠ 100
where A DPPH is the absorbance of the control reaction (containing all reagents except
tested sample) and A S is the absorbance of the tested sample reaction. IC 50 values
(concentration of sample required to scavenge 50% of free radicals) were calculated
from the regression equation, prepared from the concentration of the essential oil or
the extract and the percentage inhibition of free radical formation/percentage inhibition
DPPH was assayed. Synthetic antioxidant reagents, butylated hydroxyanisole
(BHA) and L-ascorbic acid were used as positive controls.
3. Results and discussion
3.1. Chemical analysis of the essential oil
The hydrodistillation of dried C. operculatus buds gave yellowish
essential oil (yields 0.68%, w/w). The identified compounds, qualitative
and quantitative analytical results by GC and GC/MS are shown
in Table 1, according to their elution order on a ZB-1 capillary
column.
The GC–MS analysis of the essential oil led to the identification of
55 different components, representing 93.71% of total oil constituents
(Table 1). The essential oil contains a complex mixture consisting
mainly oxygenated mono- (26.6%) and sesquiterpene (21.75%);
mono- (20.25%) and sesquiterpene hydrocarbons (14.9%). A portion
(6.29%) of total composition was not identified. The major monoterpenes
of the essential oil identified in the oil of C. operculatus buds
were c-terpinene (5.76%), cis-linalool oxide (5.21%), camphene
(4.12%), trans-carveol (3.93%), a-pinene (3.45%), b-pinene (3.07%),
terpinen-4-ol (2.58%) and myrcene (2.4%). The major sesquiterpenes
of the essential oil identified were globulol (5.61%), acorenol (5.12%),
b-himachalol (3.84%), cyclobazzanene (3.12%), 2,3-dehydro-1,4-cieol
(3.01%), trans-dihydrocarvone (2.58%), presilphiperfol-1-ene
(2.48%) and c-amorphene (2.12%).
In this study it was found that the percentage and compositions
of essential oil of C. operculatus buds were significantly different to
the essential oil obtained from C. operculatus leaves of Vietnamese
origin. Dung et al. (Dung et al., 1994) reported that in C. operculatus
leaves’ essential oil, major components found to be cis-b-ocimene
(32.1%), myrcene (24.6%), b-caryophyllene (14.5%) and trans-bocimene
(9.4%). Whereas, in C. operculatus buds’ essential oil we
were not found the presented of cis-b-ocimene, trans-b-ocimene
and b-caryophyllene. The chemical profiles of the essential oils
from leaves and buds differ not only in the number of molecules
but also in the stereochemical types of molecules extracted. These
could be attributed to the several factors such as climate, soil composition,
season, edaphic factors, plant organ, age, and vegetative
cycle stage (Angioni et al., 2006).
The compositions of solvent extract of C. operculatus buds have
already been reported with the presence of sterol, flavanone, chalcone,
triterpene acid, b-sitosterol and ursolic acid, as the major
constituents of its own (Zhang et al., 1990; Ye et al., 2004, 2005a).
3.2. Antimicrobial activity
The in vitro antimicrobial potential of C. operculatus buds’ essential
oil and ethanol extract against the employed microorganisms
was quantitatively assessed by the presence/absence of inhibition
zones, MIC and MBC values (Table 2). As can be seen from Table
2, essential oil showed moderate in vitro antimicrobial activity
against the all the tested microorganisms including Gram positive,
Gram negative and Candida albicans with diameter zones of inhibition
8–16 mm, along with MIC and MBC values ranging from 1 to
20 lL/mL. Whereas, the ethanol extract were found inhibited antimicrobial
activity against the entire tested Gram positive bacteria,
and only one of the Gram negative bacterium namely P. aeruginosa
KCTC 2004. The diameter of inhibition zones found in the range of
8 to 22 mm, along with MIC and MBC values ranging from 0.25 to
32 mg/mL.
In the comparison of microbial sensitivity to both essential oil
and ethanol extract, FS and FB seem to be more sensitive than human
infectious pathogens such as SP, MRSA, VRE and MARB. The VRE–A93
and VRE–U914 strains were the most resistant bacteria to ethanol
extract (MIC and MBC values were found to be 32 mg/mL).
In fact, essential oils of many Cleistocalyx species were known to
exhibit antimicrobial activity against several microorganisms
(Kamel et al., 2007; Alessandra et al., 2005; Djoukeng et al.,

Table 1
Volatile components (%) of the essential oil from Cleistocalys operculatus buds
Compound Retention
index
Molecular
formula
Area Identification
Monoterpene hydrocarbon 20.25
a-Pinene 0936 C10H16 3.45 MS, RI
Camphene 0950 C10H16 4.12 MS, RI
b-Pinene 0978 C10H16 3.07 MS, RI
Myrcene
a-Terpinene
0987
1013
C10H16 C10H16 2.40
1.19
MS, RI
MS, RI
Limonene 1025 C10H16 0.26 MS, RI
c-Terpinene 1051 C10H16 5.76 MS, RI
Oxygenated monoterpines 26.46
cis-Linalool oxide
a-Pinene Expoxide(isomer 1)
a-Pinene Expoxide(isomer 2)
1072
1096
1116
C10H16O2 C10H16O C10H16O 5.21
1.14
1.04
MS, RI
MS, RI
MS, RI
cis-Verbenol 1132 C10H16O 1.85 MS, RI
Isomenthome 1146 C10H16O 0.87 MS, RI
Terpinen-4-ol 1164 C10H16O 2.58 MS, RI
trans-Dihydrocarvone 1177 C10H16O 1.58 MS, RI
trans-Carveol 1200 C10H16O 3.93 MS, RI
Nerol 1210 C10H16O 1.24 MS, RI
2,3-Dehydro-1,4-cineol 1219 C10H16O 3.01 MS, RI
Lavandulyl acetate 1275 C12H20O2 0.65 MS, RI
cis-Carvyl acetate 1345 C12H20O2 0.68 MS, RI
Geranyl acetate 1362 C12H20O2 0.76 MS, RI
Methyl jasmonate 1611 C12H20O3 1.92 MS, RI
Sesqutterpene hydrocarbons 14.59
Presilphiperfol-l-ene
a-Copanene
1325
1379
C15H24 C15H24 2.48
0.70
MS, RI
MS, RI
Sesquithujene 1399 C15H24 0.44 MS, RI
b-Cedrene 1424 C15H24 0.17 MS, RI
6-Epi-b-cubene 1449 C15H24 1.17 MS, RI
Aromadendr-9-ene 1463 C15H24 1.25 MS, RI
c-Muurolene 1474 C15H24 1.65 MS, RI
c-Amorphene 1492 C15H24 2.12 MS, RI
Cyclobazzanene
a-Cadinene
1514
1534
C15H24 C15H24 3.12
1.47
MS, RI
MS, RI
Oxygenated desqutterpene 21.75
1.8-Oxidocadin-4-ene 1551 C15H24O 1.84
(+)-Marsupellol 1564 C15H24O 1.47 MS, RI
Globulol 1589 C15H26O 5.61 MS, RI
b-Himachalol 1638 C15H26O 3.84 MS, RI
Acorenol
6a-Hydxoxygermacra-1(10).4-
Diene
1667
1687
C15H26O C15H26O 5.12
1.07
MS, RI
MS, RI
trans-Nuciferol
a-Santalol acetate
a-Vetivone
1715
1756
1821
C10H22O C10H26O C10H24O2 0.69
0.78
0.42
MS, RI
MS, RI
MS, RI
trans-Epoxypseudoisoeugenyl-
2-methylbutyrate
1871 C10H26O 0.31 MS, RI
Ethyl hexadecanoate 1990 C10H26O4 0.11 MS, RI
Falcarinol 2028 C10H26O2 0.49 MS, RI
Diterpene 1.43
Axinissene 1860 C10H32 0.23 MS, RI
Rimuene 1907 C10H32 0.78 MS, RI
Cembrene 1938 C10H32 0.04 MS, RI
16-Kaurene 2056 C10H32 0.27 MS, RI
Abieta-7,13-diene 2084 C10H32 0.11 MS, RI
Other 9.23
1-Hexanol 0837 C10H14O 2.59 MS, RI
2-Heptanene 0871 C10H14O 2.03 MS, RI
5,7,-Dimethylocta-1,6-diene 0911 C10H18 1.78 MS, RI
3-Methylbutyl isobutyrate 8 0994 C10H18O2 0.93 MS, RI
Lavender lactone 1006 C10H18O2 0.31 MS, RI
Nanonic acid 1263 C10H18O 0.60 MS, RI
Benzyl benzoate 1730 C10H12O 0.99 MS, RI
Total identified 93.71 MS, RI
2005). The antibacterial activity of the oil of C. operculatus buds
could, in part, be associated with major constituents such as c-terpinene,
cis-linalool oxide, camphene, trans-carveol, a-pinene,
b-pinene, terpinen-4-ol and myrcene, globulol, 2,3-dehydro-1,4cieol,
trans-dihydrocarvone, presilphiperfol-1-ene, and c-amorph-
N.T. Dung et al. / Food and Chemical Toxicology 46 (2008) 3632–3639 3635
ene. These components have been reported to display antibacterial
effects (Alessandra et al., 2005; Djoukeng et al., 2005). Terpenes
were active against bacteria (Djoukeng et al., 2005). As described
previously by other authors, that essential oils containing terpenoids
are more active against Gram positive bacteria than against
Gram negative ones (Cosentino et al., 1999). In addition, the components
in lower amount may also contribute to antibacterial
activity of the oil, involving probably some type of synergism with
other active compounds.
The NCCLS method (NCCLS, 2000) was using to determine the
antibiotic-resistance and sensitivity pattern profile of the antibiotic-resistant
bacteria. The susceptibility of these bacteria to multi-antibiotics
has shown in Table 3. This susceptibility was
independent from microorganisms examined to essential oil and
ethanol extract of C. operculatus buds.
According to the recent studies, community-acquired or outpatient
MRSA and VRE infections are increasing in both children and
adults. They are responsible for worldwide outbreaks of nosocomial
infections (Suller and Russell, 1999). Further, the effect on
the cell viabilities of MRSA and VRE demonstrated that exposure
of essential oil at MBC had a potential antibacterial effect on the
viabilities of MRSA and VRE strains. The exposure time of essential
oil for complete inhibition of cell viability of MRSA and VRE were
found to be as 10–40 and 10–20 min, respectively (Fig. 1a and b).
To visualize the effects of the essential oil on the morphology of
MRSA–P249 and VRE–B2332, SEM analyses were performed and
demonstrated to the altered cell morphology as compared to control
group (Fig. 2). Control cells in the absence of the essential oil
showed a regular, smooth surface (Fig. 2a and c). In contrast, cells
inoculated with the essential oil at MIC value (8 lL/mL) revealed
severe detrimental effect on the morphology of cell membrane,
showing disruption and lysis of the membrane integrity (Fig. 2b
and d). In fact, initial exposure of essential oil to MRSA–P249 revealed
large surface collapse on the morphology of the cells, and
wrinkled abnormalities on the cells of VRE–B2332 with numerous
small clefts were observed (Fig. 2b and d). Such morphological features
in bacterial cells might be due to the lysis of outer membrane
and the transformation by weak peptidoglycan followed by the
loss of cellular electron dense material on surface of the treated
cells, resulting in the release of inner cell materials (Fig. 2b and
d). Such morphological abnormalities mainly occurred due to the
disruption of membrane structure as evident by the previous findings
(Koyama et al., 1997; Shin et al., 2007).
3.3. Determination of total antioxidant capacity and scavenging
activity of DPPH radical
Total antioxidant activity of the C. operculatus buds was expressed
as the number of equivalents of ascorbic acid (ASE). The
antioxidant capacity was estimated from the regression equation
prepared from concentration versus optical density of sample
and ascorbic acid. The extraction by ethanol showed equivalents
62.40 ± 3.16 mg ASE/g dried C. operculatus buds (data not shown).
The DPPH free radical is a stable free radical, which has been
widely used as tool to estimate free radical-scavenging activity of
antioxidants. Antioxidants, on interaction with DPPH, either transfer
electrons or hydrogen atoms to DPPH, thus neutralizing the free
radical character (Archana et al., 2005). The color of the reaction
mixture changes from purple to yellow, and its absorbance at
wavelength 517 nm decreases. Table 4 shows the DPPH radicalscavenging
activities of essential oil and ethanol extract of C. operculatus
buds. The IC50 values were compared with the IC 50 values of
butylated hydroxyanisole (BHA) and ascorbic acid. A lower IC50
value indicates greater antioxidant activity. The IC 50 values of
essential oil and ethanol extract were found to be 807.00 ± 74.25
and 38.58 ± 2.08, respectively. The scavenging effects of BHA and

3636 N.T. Dung et al. / Food and Chemical Toxicology 46 (2008) 3632–3639
Table 2
Antimicrobial activity of the essential oil and ethanol extract from Cleistocalys operculatus buds
Bacterial strains Gram Essential oil Ethanol extract
ascorbic acid were found to be 2.11 and 6.06 times greater than
ethanol extract of C. operculatus buds, respectively.
The ursolic acid, a main constituent present in the ethanol extract
of C. operculatus buds (Ye et al., 2004), is well-known for its
hepatoprotective, anti-inflammatory, antiallergic, antiviral and
cytotoxic activities (Alcaradz et al., 2000; Flávia et al., 2006; Cocchietto
et al., 2002). Flavanone and chalcone, presented in C. operculatus
buds, could also be responsible for antibacterial and
antioxidant activity with some synergism of minor or major components
present (Alcaradz et al., 2000). The key role of phenolic
compounds as antimicrobial and scavengers of free radicals is
emphasized in several reports (Rauha et al., 2000; Archana et al.,
2005). Here we propose, for the first time, the use of essential oil
and ethanol extract of C. operculatus buds as the potential antimicrobial
and antioxidant sources. According to Borchers, Keen and
Inhibition zone (mm) MIC (lL/mL) MBC (lL/mL) Inhibition zone (mm) MIC (mg/mL) MBC (mg/mL)
Food spoilage(FS)
Bacillus subtilis ATCC 6633 + 12 ± 0.0 a
2 4 8.0 ± 0.8 d
0.5 1.0
Pseudomonas arginosa KCTC 2004 15 ± 0.5 2 2 18.0 ± 0.0 0.5 1.0
Food borne pathogens (FB)
Listeria monocytogenes ATCC 19166 + 10 ± 0.8 a
4 4 13.0 ± 0.8 d
0.25 1.0
Staphylococcus aureus KCTC 1916 + 16 ± 0.5 1 2 12.0 ± 0.0 0.5 1.0
Staphylococcus aureus ATCC 6538 + 10 ± 0.8 4 4 9.0 ± 0.0 1.0 2.0
Escherichia coli 0157:H7 (Human) 13 ± 0.0 4 8 – – –
Escherichia coli 0157:H7 ATCC 43888 10 ± 0.0 4 4 – – –
Escherichia coli ATCC 8739 10 ± 0.5 4 8 – – –
Enterobacter aerogenes KCTC 2190 13 ± 0.8 4 8 – –
Salmonella typhimurium KCTC 2515 14 ± 0.8 4 4 – – –
Salmonella enteritidis KCTC 12021 13 ± 0.0 4 4 – – –
Skin pathogens
Staphylococcus aureus KCTC 1621 + 13.0 ± 0.0 a
10 10 15.0 ± 0.0 e
2.5 2.5
Staphylococcus epidermidis KCTC 1917 + 16.0 ± 0.0 10 10 15.0 ± 0.0 2.5 2.5
Echerichia coli KCTC 1039 11.0 ± 0.5 10 10 – – –
Candida albicans KCTC 7965 Yeast 10.8 ± .00 5 5 – – –
Methyicillin resistant Staphylococcus aureus (MRSA)
Staphylococcus aureus P 227 + 12 ± 0.0 b
8 16 12.5 ± 0.0 f
4 4
Staphylococcus aureus P 254 + 11 ± 0.0 8 16 10.0 ± 0.0 16 16
Staphylococcus aureus P249 + 16 ± 0.0 8 8 11.5 ± 0.0 4 4
Vancomycin resistant Enterococci
Enterococcus faeccium A 93 + 9 ± 0.0 b
8 16 9 ± 0.0 f
32 32
Enterococcus faeccium B 2332 + 9 ± 0.0 8 16 9 ± 0.0 16 16
Enterococcus faeccium U 914 + 10 ± 0.0 8 16 10 ± 0.0 32 32
Multiantibiotic–resistant bacteria (MARB)
Acinetobacter baumannii 05 KA 007 + 14.0 ± 0.0 c
10 20 12.0 ± 0.0 f
5 10
Acinetobacter baumannii 05 KA 31 + 10.5 ± 0.5 10 10 8.0 ± 0.0 10 20
Staphylococcus aereus ATCC 25923 + 12.0 ± 0.0 5 10 8.0 ± 0.0 10 20
Escherichia coli 02 K 276 9.0 ± 0.0 20 20 – – –
Escherichia coli 04 K 717 8.0 ± 0.0 10 10 – – –
Enterobacter cloacae 04 K 453 10.0 ± 0.0 10 10 – – –
Enterobacter cloacae 04 K 908 9.0 ± 0.0 10 20 – – –
Klebsiella pneumoniae 05 K 279 9.0 ± 0.0 10 10 – –
Klebsiella pneumoniae 05 K 406 10.5 ± 0.5 10 20 – – –
Pseudomonas aeruginosa 03 K 442 8.0 ± 0.0 20 20 – –
Pseudomonas aeruginosa 03 K 711 8.0 ± 0.0 20 20 – – –
Serratia marcescens 03 K 188 9.0 ± 0.0 5 5 – – –
Serratia marcescens 03 K 201 9.0 ± 0.0 20 20 – – –
Pseudomonas aeruginosa ATCC 27853 8.0 ± 0.0 10 10 – – –
Echerichia coli ATCC 25922 9.0 ± 0.0 10 10 – – –
MIC (minimum inhibition concentration) and MBC (minimum bactericidal concentration) as lL/mL or mg/mL of essential oil or ethanol extract respectively; (–) no
antimicrobial activity; (±) standard deviation.
Values are average of triplicate.
a
As 1 lL/disc.
b
As 2 lL/disc.
c
As 5 lL/disc.
d
As 0.5 mg/disc.
e
As 2 mg/disc.
f
As 5 mg/disc.
Geratiwin (Borchers et al., 2004), food extracts may be more beneficial
than isolated constituents, because other compounds present
in the extracts can be change the properties of bioactive
individual component.
In this research, we found that essential oil and ethanol extract
of C. operculatus buds severely inhibited the growth of food spoilage,
food-borne pathogens, skin pathogens, methicillin-resistant
S. aureus, vancomycin-resistant Enterococci and multiantibioticresistant
bacteria. Beside that, the essential oil and the extract also
exhibited strong scavenging effect on DPPH free radicals. Therefore,
from the above results, it can be concluded that the essential
oil and ethanol extract derived from C. operculatus buds could be
considered as potential alternatives for synthetic bactericides and
natural antioxidants for using in the food industry along with their
possible applications in pharmaceutical industry for the prevention

Table 3
Antibiotic resistance and sensitivity pattern of tested bacteria and the criterion of sensitivity using disc and dilution method a
Initial Antibiotics Antibiotics applied (lg/disc) Inhibition zone (mm) MIC (lg/mL)
Resistant Intermediate Sensitive Resistant Sensitive
Ak Amikacin 30 614 15–16 P17 P64 616
Ap Ampicillin 10 613 14–16 P17 P32 68
Az Azteronam 30 627 – P27 P2

3638 N.T. Dung et al. / Food and Chemical Toxicology 46 (2008) 3632–3639
Fig. 2. Scanning electron micrographs of Staphylococcus aureus P249 (a and b) and Enterococcus faeccium B2332 (c and d) after 10 min incubation at 37 °C in the absence (a
and c) and present (b and d) of essential oil from Cleistocalys operculatus buds as treated at MIC values of each strains (8 mg/mL for S. aureus P249 and 16 ll/mL for E. faeccium
B2332).
Table 4
Scavenging activity of DPPH radical of the essential oil and ethanol extract from
Cleistocalys operculatus buds
Samples Concentration
(lg/mL)
or treatment of severe skin diseases caused by emerging antibioticresistant
microorganisms and free radicals.
Conflict of interest statement
The authors declared there are no conflicts of interest.
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