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Food and Chemical Toxicology 47 (2009) 18761883

Contents lists available at ScienceDirect

Food and Chemical Toxicology


journal homepage: www.elsevier.com/locate/foodchemtox

Chemical composition, antibacterial and antioxidant activities of leaf essential


oil and extracts of Metasequioa glyptostroboides Miki ex Hu
Vivek K. Bajpai a, Sharif M. Al-Reza a, Ung Kyu Choi b, Jong Hwi Lee c, Sun Chul Kang a,*
a

Department of Biotechnology, College of Engineering, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea
Pohang Center for Evaluation of Biomaterials, Pohang 790-834, Republic of Korea
c
Department of Chemical Engineering and Materials Science, Chung-Ang University, Seoul 156-756, Republic of Korea
b

a r t i c l e

i n f o

Article history:
Received 9 April 2009
Accepted 29 April 2009

Keywords:
Metasequioa glyptostroboides
Leaf essential oil
Food spoilage and food-borne pathogens
Antibacterial activity
Antioxidant activity
Phenolic content

a b s t r a c t
The aims of this study were to analyze the chemical composition of leaf essential oil of Metasequioa glyptostroboides Miki, and to test the efcacy of oil and extracts (hexane, chloroform, ethyl acetate and methanol) against food spoilage and food-borne pathogenic bacteria and their antioxidant activity. The GCMS
analysis revealed 49 compounds representing 94.62% of the total oil containing 2-butaneone (30.6%),
cyclopentane (15.1%), b-myrcene (13.29%), cyclobutane (7.67%), furan (3%), valeramide (2.81%), borneol
(1.2%), b-farnesene (1.67%), thymol (1.44%) and a-pinene (1.46%) as major components. The oil
(1000 lg/disc), and extracts (1500 lg/disc) exhibited promising antibacterial effect as a diameter of
zones of inhibition (1018 and 713 mm), respectively. MIC values of oil and the extracts were ranged
1252000 and 250 to <2000 lg/ml, respectively. Also the oil had strong antibacterial effect on the viable
counts. Scanning electron microscopic study demonstrated potential detrimental effect of the oil on the
morphology of S. aureus KCTC1916. The free radical scavenging activities of the oil and ethyl acetate
extract were found to be 11.32 and 19.12 lg/ml, respectively. Also the ethyl acetate extract revealed
the highest phenolic contents (85.17 mg/g of dry wt) as compared to the other extracts.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Global interest in bio-preservation of food systems has recently
been increased because of great economic costs of deterioration
and poisoning of food products by food pathogens and often
responsible for the loss of quality and safety. Also food-borne diseases pose a considerable threat to human health. Concern over
pathogenic and spoilage microorganisms in foods is increasing
due to the increase in outbreaks of food-borne disease. Currently,
there is a growing interest to use natural antibacterial compounds,
like extracts of herbs and spices for the preservation of foods. Plant
derived essential oils and extracts of various species have long
been used as natural agents for food preservation in food and beverages due to the presence of antimicrobial compounds (Nychas
et al., 2003). The United States and other industrialized countries
are experiencing an unprecedented increase in food-borne illness.
The Gram-positive bacterium Staphylococcus aureus is mainly
responsible for post-operative wounds infections, toxic shocks syndrome, endocarditis, osteomyelitis and food poisoning (Mylotte
et al., 1987). The Gram-negative bacterium Escherichia coli is present in human intestine and causes urinary tract infection, coleocystitis or septicemia (Singh et al., 2000).
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.04.043

With the increase of bacterial resistance to antibiotics, there is


considerable interest in investigating the antimicrobial effects of
essential oils and different extracts against a range of bacteria, to
develop other classes of safe and natural antimicrobials useful for
infection control or for the preservation of food (Bakri and Douglas,
2005). A range of synthetic antimicrobial agents have been used to
inhibit the growth of food spoilage and food-borne pathogens in
food systems, although concerns about the safety of these chemicals have increased consumer demand for naturally processed
food. Therefore, the identication and evaluation of natural products for the control of these pathogens, to assure consumers a safe,
wholesome, and nutritious food supply, can be considered as an
important international challenge.
Furthermore, there are ample evidences that reactive oxygen/
nitrogen species generated in the human body can cause oxidative
damages associated with many degenerative diseases such as atherosclerosis, coronary heart diseases, aging and cancer (Finkel and
Holbrook, 2000). It has been recognized that there is an inverse
association between consumption of some fruits and vegetables
and morbidity and mortality from degenerative diseases, which
could be partly attributed to their antioxidants (Gey, 1990; La
Vecchia et al., 2001). The health promoting effect of antioxidants
from plants is thought to arise from their potential effects on the
reactive oxygen/nitrogen species. In addition, antioxidants have
been widely used in food industry to prolong the shelf life.

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V.K. Bajpai et al. / Food and Chemical Toxicology 47 (2009) 18761883

However, there is widespread agreement that some synthetic antioxidants such as butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) need to be replaced with natural antioxidants due to
their potential health risks and toxicity (Safer and Al-Nughamish,
1999). Therefore, it is very important to nd out new sources of
safe and inexpensive antioxidants of natural origin.
In addition to this, the presence of phenolic compounds (phenolic acids, polyphenols and avonoids) in plants, herbs and spices, is
gaining increasing attention because of their various functions,
such as antioxidant activity and avouring properties (Sacchetti
et al., 2005). Consumption of food containing natural aromatic
plant extracts is expected to prevent the risk of many free radical-mediated diseases (Young and Woodside, 2001).
Metasequoia glyptostroboides Miki ex Hu is a deciduous conifer
of the redwood family of Cupressaceae. It is only living species in
the genus Metasequoia propagated and distributed in many
parts of Eastern Asia and North America as well as in Europe.
However, there is no report available in the literature on the
analysis of the leaf essential oil of M. glyptostroboides, and antibacterial and antioxidant properties. Hence, efforts have been
made, to investigate the role of leaf essential oil and various leaf
extracts of M. glyptostroboides as natural antibacterial and antioxidant potential.
Previously, we reported the chemical composition and antimicrobial properties of the essential oil and various organic extracts
derived from the oral cone of M. glyptostroboides (Bajpai et al.,
2007a,b). In the present investigation, we examined the chemical
compositions of the leaf essential oil of M. glyptostroboides, and
tested the efcacy of leaf essential oil and various leaf extracts
against a diverse range of food spoilage and food-borne pathogens
comprising Gram-positive and Gram-negative bacteria as well as
their antioxidant activity.
2. Materials and methods

1877

erichia coli ATCC8739, Escherichia coli O157:H7 ATCC43888, Escherichia coli O157
(Human), Salmonella enteritidis KCTC2021, Staphylococcus aureus ATCC6538 and
Staphylococcus aureus KCTC1916, were obtained from the Korea Food and Drug
Administration (KFDA), Daegu, Republic of Korea. Cultures of each bacterial strain
were maintained on LuriaBertani (LB) agar medium at 4 C.

2.6. Gas chromatographymass spectrometry (GCMS) analysis


The GCMS analysis of the essential oil was performed using a Shimadzu GC
MS (GC-17A, Kyoto, Japan) equipped with a ZB-1 MS fused silica capillary column
(30 m  0.25 mm i.d., lm thickness 0.25 lm). For GCMS detection, an electron
ionization system with ionization energy of 70 eV was used. Helium gas was used
as the carrier gas at a constant ow 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 C at 3 C/min, then held isothermal for
10 min and nally raised to 250 C at 10 C/min. Diluted samples (1/100, v/v, in
methanol) of 1.0 ll were injected manually in the splitless mode. The relative percentage of the oil constituents was expressed as percentage by peak area normalization. The identity of the components of the essential oil was assigned by
comparison of their retention indices (RI), relative to a series n-alkane indices
on the ZB-1 capillary column and GCMS spectra from the Wiley 6.0 MS data
and literature data and whenever possible, by co-injection with authentic compounds (Adams, 2001). The relative amounts (RA) of individual components of
the essential oil were expressed as percentages of the peak area relative to the total peak area.

2.7. Assay for antibacterial potential


Standard agar diffusion method was used for antibacterial assay (Murray et al.,
1995). Petri plates were prepared by pouring 20 ml of LB medium and allowed to
solidify. Plates were dried and 1 ml of standardized inoculum containing
1067 CFU/ml of bacterial suspension was poured and uniformly spread, and the
inoculum was allowed to dry for 5 min. A Whatman No. 1 sterile lter paper disc
(6 mm diameter) was impregnated with 1000 lg/disc of leaf essential oil, and
1500 lg/disc of leaf extracts of hexane, chloroform, ethyl acetate and methanol.
Negative controls were prepared using the same solvent employed to dissolve the
samples. Standard reference antibiotics, streptomycin and tetracycline (10 lg/disc)
were used as positive controls for the tested bacteria. The plates were incubated at
37 C for 24 h. Antibacterial activity was evaluated by measuring the diameter of
the zones of inhibition against the tested bacteria. Each assay in this experiment
was replicated three times.

2.1. Chemicals and reagents


Gallic acid, L-ascorbic acid (AA), FolinCiocalteaus phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and butylated hydroxyanisole (BHA) were obtained
from SigmaAldrich (St. Louis, MO). All other chemicals and solvents were of highest commercial grade.
2.2. Plant materials
The leaves of M. glyptostroboides were collected from the Pohang City, Republic
of Korea, in NovemberDecember 2008 and initially identied by the morphological features and the data base present in the library at the Department of Biotechnology. A voucher specimen number has been deposited in the Herbarium of
College of Engineering, Department of Biotechnology, Daegu University, Republic
of Korea.
2.3. Isolation of the leaf essential oil
The air-dried leaves of M. glyptostroboides (200 g) were subjected to hydrodistillation for 3 h, using a modied Clevenger-type apparatus. The oil was dried over
anhydrous Na2SO4 and preserved in a sealed vial at 4 C prior to further analysis
with a yield of 0.26% (w/w).
2.4. Preparation of leaf extracts
The air-dried leaves of M. glyptostroboides were pulverized into powdered form.
The dried powder (50 g) was extracted with 70% methanol (MeOH), hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) separately at room temperature and the solvents from the combined extracts were evaporated by vacuum rotary evaporator
(EYELA N1000, Japan). The extraction process yielded in hexane (3.8 g), chloroform
(4.5 g), ethyl acetate (6.3 g) and methanol (6.1 g) extracts.
2.5. Microorganisms (food spoilage and food-borne pathogens)
The bacterial pathogens including food spoilage bacteria, such as Bacillus subtilis
ATCC6633 and Pseudomonas aeruginosa KCTC2004, and food-borne pathogens,
namely Enterobacter aerogenes KCTC2190, Salmonella typhimurium KCTC2515, Esch-

2.8. Determination of minimum inhibitory concentrations (MICs)


Minimum inhibitory concentrations (MICs) of essential oil and the extracts
were tested by standard NCCL method (NCCLS, 2000). Active cultures for MIC
determination were prepared by transforming a loopful of cells from stock cultures to asks and inoculated in LB medium and incubated at 37 C for 24 h.
The cultures were diluted with fresh LB broth to achieve optical density of
107 CFU/ml for the test organisms at 600 nm by UV/Vis Spectrophotometer Optizen 2120UV and Optizen III [10]. Dilutions, to get the nal concentration ranging
from 04000 lg/ml of oil and the extracts of hexane, chloroform, ethyl acetate
and methanol in LuriaBertani (LB) broth medium were prepared in a 96 well
microplates. Finally 20 ll inoculum of each bacterial strain (107 CFU/ml) was
inoculated onto the microplates and the tests were performed in a volume of
200 ll. The plates were incubated at 37 C for 24 h. The lowest concentrations
of the test samples, which did not show any visual growth of test organisms
after macroscopic evaluation, were determined as MICs, which were expressed
in lg/ml.
2.9. Effect of the leaf essential oil on viable count of the tested bacteria
Active cultures for viable count assay were prepared in LB broth medium (Shin
et al., 2007). For each strain, 1 ml of active stock solution was transferred to 4 ml of
LB broth. All treated cultures were kept at 37 C for 2 h. The cultures were then centrifuged at 10,000 rpm for 10 min. The pellets were retained and resuspended with
1 ml of phosphate-buffered saline. For viable counts, each of the tubes containing
resuspended bacterial suspension (approximately 107 CFU/ml) of B. subtilis
ATCC6633, S. aureus KCTC1916, S. aureus ATCC3538 and P. aeruginosa KCTC2004
was inoculated with the minimum inhibitory concentration of the essential oil in
10 ml LB broth, and kept at 37 C. Samples for viable cell counts were taken out
at 0, 20, 40, 60, 80, 100 and 140 min time intervals. The viable plate counts were
monitored as followed: After incubation, 1 ml of the resuspended culture was diluted into 9 ml buffer peptone water, there by diluting it 10-fold. About 0.1 ml sample of each treatment was diluted and spread on the surface of LB 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.

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2.10. Scanning electron microscopic (SEM) analysis


To determine the efcacy of the leaf essential oil on the morphology of S. aureus
KCTC1916, SEM study was performed using minimum inhibitory concentration of
the oil. Controls were prepared without essential oil. Further, to observe the morphological changes, the method of SEM was modied from Kockro method (Kockro
et al., 2000). The bacterial sample was washed gently with 50 mM/l phosphate buffer solution (pH 7.2), xed 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 30100%. The ethanol was replaced by tertiary butyl
alcohol. After dehydration, the specimen was dried with CO2. Finally, the specimen
was sputter-coated with gold in an ion coater for 2 min, followed by microscopic
examinations (S-4300; Hitachi).
2.11. Antioxidant activity assay
The antioxidant activity of the leaf essential oil and the leaf extracts (n-hexane,
chloroform, ethyl acetate and methanol) of M. glyptostroboides was measured on the
basis of the scavenging activities of the stable 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical (Archana et al., 2005). IC50 values (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation, prepared from the concentration of the oil and the extracts, and percentage
inhibition of free radical formation/percentage inhibition DPPH was assayed. Synthetic antioxidant reagents, butylatedhydroxyanisole (BHA) and L-ascorbic acid
were used as positive controls and all tests were carried out in triplicate.
2.12. Determination of total phenolic contents
The amount of total phenolics was determined with the FolinCiocalteu reagent
(Lister and Wilson, 2001). First, a standard curve was plotted using gallic acid as a
standard. Different concentrations of samples were prepared in 80% of methanol.
100 ll of sample was dissolved in 500 ll (1/10 dilution) of the FolinCiocalteu reagent and 1000 ll of distilled water. The solutions were mixed and incubated at
room temperature for 1 min. After 1 min, 1500 ll of 20% sodium carbonate solution
was added. The nal mixture was shaken and then incubated for 2 h in the dark at
room temperature. The absorbance of samples was measured at 760 nm and the results were expressed in mg of gallic acid/g (GAE) of dry weight of samples.
2.13. Statistical analysis
The data were statistically analyzed by using Students t-test and analysis of
variance for individual parameters was performed by Duncans test on the basis
of mean values to nd out the signicance at p < 0.05. Correlation between antioxidant activity and total phenolic content was carried out using the correlation and
regression in the EXEL program.

3. Results
3.1. Chemical composition of the leaf essential oil
The hydrodistillation of the leaves of M. glyptostroboides gave
brownish oil. GCMS analyses of the oil led to the identication
of forty nine different compounds, representing 94.62% of the total
oil. The identied compounds are listed in Table 1 according to
their elution order on a ZB-1 capillary column. The oil contained
a complex mixture mainly of monoterpene hydrocarbons and oxygen containing mono- and sesquiterpenes along with some other
essential phytochemicals. As shown in Table 1, the major compounds detected in the oil were 2-butaneone (30.6%), cyclopentane
(15.1%), b-myrcene (13.29%), cyclobutane (7.67%), furan (3%), valeramide (2.81%), borneol (1.2%), b-farnesene (1.67%), thymol
(1.44%), a-pinene (1,46%), 2-hydroxypropanoic acid (1%) and 3cyclohexen-1-ol (1%). Limonene (0.22%), pyrolidine (0.4%), linalool
oxide (0.96%), verbenol (0.43%), a-terpineol (0.17%), myrtenol
(0.18%), farnesol (0.91%), eugenol (0.3%), caryphyllene oxide
(0.17%) and veridiorol (0.53%) were also found to be the minor
components of M. glyptostroboides leaf oil (Table 1).
3.2. Antibacterial activity
The in vitro antibacterial activities of leaf essential oil and the
leaf extracts of M. glyptostroboides, against the employed bacteria,
were qualitatively and quantitatively assessed by the presence or

Table 1
Chemical composition of leaf essential oil of Metasequioa glyptostroboides Miki ex Hu.
No

Compound

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
21
21
22
23
24
25
26
27
23
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49

2-Butanone
2-Hydroxypropanoic acid
Cyclopentane
Cyclobutane
Butylcarbinol
l-Dodecyn-4-ol
Valeramide
a-Pinene
Benzenol
Amyl vinyl carbinol
Geranyl bromide
b-Myrcene
1,6,10-Dodecatriene
Benzyl alcohol
Furan
Limonene
Pyrrolidine
Imidazole
Guaiacol
Linalool oxide
Fenchol
9,12-Tetradecadien-1-ol
Verbenol
2-pinen-4-ol
Octanoic acid
7-octen-2-ol
Borneol
Thymol
3-Cyclohexen-l-ol
3-Caren-4-ol
a-Terpineol
Myrtenol
2,3-Dimethy 1-3-isopropylpyrazine
Chavicol
Famesol
a-Bergamotene
2,3-benzopyrrole
Pyrindine
Ocimene
Isobomyl acetate
Acetophenone
Eugenol
b-Famesene
Caryphyllene oxide
a-Bisabolol
Veridiorol
Aromadendrene oxide
a-Bisabolene epoxide
6,9,12,15-Docosatetraenoic acid
Total

717
838
783
810
697
1374
918
948
901
969
1282
958
1440
1036
831
1018
1175
894
1090
1164
1138
1672
1136
1136
1173
1064
1088
1262
1137
1131
1143
1191
1141
1203
1710
1430
1174
1023
1164
1277
1218
1392
1440
1507
1625
1530
1462
1531
2507
94.62%

a
b

RI

RA (%)

30.6
1.00
15.10
7.67
0.15
0.55
2.81
1.46
0.70
0.25
0.61
13.29
0.31
0.16
3.00
0.22
0.40
0.23
0.23
0.96
0.34
0.74
0.43
0.26
0.89
0.18
1.20
1.44
1.00
0.57
0.17
0.18
0.27
0.15
0.91
0.19
0.34
0.27
0.39
0.18
0.50
0.30
1.67
0.17
0.42
0.53
0.56
0.52
0.24

Retention index relative to n-alkane on ZB-1 capillary column.


Relative area (peak area relative to the total peak area).

absence of inhibition zones. As shown in Table 2, the essential oil


at 1000 lg/disc exhibited potent inhibitory effect against the
tested bacterial pathogens. S. aureus KCTC1916, B. subtilis
ATCC6633, S. aureus ATCC6538, P. aeruginosa KCTC2004 and
E. coli O157:H7 ATCC43888 were found to be the most inhibited
bacterial pathogens by the oil, with their respective diameter zones
of inhibition of 18, 16, 17, 14 and 12 mm. Rest of the bacterial
strains (E. coli ATCC8739, E. coli O157 (Human), E. aerogenes
KCTC2190, S. typhimurium KCTC2515 and S. enteritidis KCTC2021)
were inhibited moderately, with diameter of zones of inhibition
ranging from 10 to 11 mm (Table 2).
Also the methanol and ethyl acetate extracts exerted potential
effect of antibacterial activity against the tested bacteria. The
diameters of zones of inhibition of methanol and ethyl acetate extracts against the tested bacteria were found in the range of 711
and 713 mm, respectively (Table 2). However, chloroform extract
exhibited moderate effect of antibacterial activity against some of

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Table 2
Antibacterial activity of essential oil and organic extracts derived from the leaves of Metasequoia glyptostroboides against food spoilage and food-borne pathogens.
Microorganism

Diameter of zones of inhibition


A

Essential oil

S. aureus (KCTC1916)
B. subtilis (ATCC6633)
S. aureus (ATCC6538)
P. aeruginosa (KCTC2004)
E. coli 0157:H7 (ATCC43888)
E. coli (ATCC8739)
E. coli O/51 (Human)
E. aerogenes (KCTC2190)
S. typhimurium (KCTC2515)
S. enteritidis (KCTC2021)

18.0 1.2ab
16.0 1.7ac
17.0 1.9ac
14.0 1.1c
12.0 0.7c
11.0 1.3c
10.0 1.2c
11.0 1.7c
11.0 1.3c
11.0 1.7c

Leaf extracts

Standard

MeOH

Hexane

CHCl3

EtOAc

SM

TC

10.0 1.1ab
11.0 1.9a
11.0 1.7a
10.0 1.2ab
7.0 0.9c
7.0 0.3c
8.0 0.7c
7.0 1.1c
7.0 1.9bc
7.0 1.9bc

7.0 1.3ab
7.0 0.7bc
7.0 0.7bc
D
nd
nd
nd
nd
nd
nd
nd

9.0 0.9ab
8.0 1.5bc
9.0 1.7ac
9.0 0.5b
7.0 0.7c
8.0 0.7bc
9.0 0.7a
nd
nd
nd

13.0 0.7ab
11.0 1.2c
11.0 1.6bc
11.0 1.3bc
9.0 1.7c
9.0 0.7c
9.0 0.7c
7.0 1.2c
8.0 1.9c
8.0 1.9c

14 0.9b
14 0.2b
14 0.6b
19 0.4A
15 0.9b
24 0.7c
15 0.7b
13 0.2b
13 0.2b
14 0.6b

18 0.5bc
18 0.5bc
19 0.6b
20 1.0ab
20 0.5ab
17 1.1bc
19 1.2b,c
20 0.6ab
21 0.6E
21 1.0ab

Values are given as mean SD of triplicate experiment and considered to be signicantly different at p < 0.05.
A
Diameter of inhibition zones of essential oil (tested volume 1000 lg/disc).
B
Diameter of inhibition zones of leaf extracts of MeOH (methanol), Hexane, CHC13 (chloroform), EtOAc (ethyl acetate) (tested volume 1500 lg/disc).
C
Standard antibiotics: SM, streptomycin; TC, tetracycline (tested volume 10 lg/disc).
D
nd, antibacterial activity not detected.

the bacterial pathogens tested with diameters of zones of inhibition ranging from 7 to 9 mm. Ethyl acetate extract exhibited potent
inhibitory effect of antibacterial activity against the tested bacteria
as compared to chloroform and methanol extracts. Hexane extract
did not reveal signicant effect of antibacterial activity against the
tested bacteria except S. aureus KCTC1916, B. subtilis ATCC6633 and
S. aureus ATCC6538 with diameter of zones of inhibition of 7 mm
for each bacterial strain.
In this study, in some cases, the essential oil exhibited higher
antibacterial activity than that of standard streptomycin in regard
to Gram-positive bacteria, while, tetracycline showed higher antibacterial effect, in some other cases, than those of leaf essential oil
and leaf extracts (Table 2). Hexane extract did not show the significant results against the tested bacterial strains as compared to the
standard antibiotics.
3.3. Minimum inhibitory concentrations (MICs)
As shown in Table 3, the minimum inhibitory concentration values of the leaf essential oil against the employed bacterial strains
were found lower as compared to the leaf extracts. The oil displayed remarkable antibacterial effect as minimum inhibitory concentration against all three Gram-positive bacteria such as S.
aureus KCTC1916, B. subtilis ATCC6633 and S. aureus ATCC6538,
and two Gram-negative bacteria namely P. aeruginosa KCTC2004

and E. coli O157:H7 ATCC43888, with MIC values of 250, 125,


250, 250 and 500 lg/ml, respectively. The oil also exhibited moderate antibacterial effect against E. aerogenes KCTC2190, S.
typhimurium KCTC2515, E. coli ATCC8739, E. coli O157 (Human),
S. Enteritidis KCTC2021 with MIC values ranging from 1000 to
2000 lg/ml. On the other hand, methanol, chloroform and ethyl
acetate extracts exhibited strong antibacterial effect as compared
to hexane extract. However, methanol and ethyl acetate extracts
exhibited better results of antibacterial effect against some of the
bacterial strains (S. aureus KCTC1916, B. subtilis ATCC6633, S. aureus ATCC6538 and P. aeruginosa KCTC2004) as minimum inhibitory
concentrations compared to chloroform extract, which were ranged from 500 to 1000 and 250 to 1000 lg/ml, respectively. Chloroform extract exhibited mild to moderate antibacterial effect
against all three Gram-positive and one Gram-negative bacterium
P. aeruginosa KCTC2004 with MIC values ranging from 500 to
2000 lg/ml. No antibacterial effect of the hexane extract was observed as a minimum inhibitory concentration (<2000 lg/ml)
against any of the bacterial strains tested (Table 3).
3.4. Effect of the leaf essential oil on viable count
Based on the susceptibility, further study was carried out to
evaluate the effect of leaf essential oil on the viable count of B. subtilis ATCC6633, S. aureus KCTC1916, S. aureus ATCC3538 and P.

Table 3
Minimum inhibitory concentrations of essential oil and organic extracts derived from the leaves of Metasequoia glyptostroboides against the growth of food spoilage and foodborne pathogens.
Microorganism

MICs

MeOH

Hexane

CHCI3

EtOAc

250
125
250
250
500
1000
2000
2000
1000
1000

1000
500
500
1000
<2000
<2000
<2000
<2000
<2000
<2000

<2000
<2000
<2000
<2000
<2000
<2000
<2000
<2000
<2000
<2000

500
500
2000
2000
<2000
<2000
<2000
<2000
<2000
<2000

500
250
500
1000
2000
2000
<2000
<2000
<2000
<2000

Essential oil

S. aureus (KCTC1916)
B. subtilis (ATCC6633)
S. aureus (ATCC6538)
P. aeruginosa (KCTC2004)
E. coli 0157:H7 (ATCC43888)
E. coli (ATCC8739)
E. coli O/51 (Human)
E. aerogenes (KCTC2190)
S. typhimurium (KCTC2515)
S. enteritidis (KCTC2021)
a
b
c

Leaf extracts

Minimum inhibitory concentration (MIC).


MIC of essential oil (values in lg/ml).
MIC of various leaf extracts of MeOH (methanol), Hexane, CHC13 (chloroform), EtOAc (ethyl acetate) (values in lg/ml).

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Fig. 1. Effect of M. glyptostroboides leaf essential oil (MIC concentration) on viability of the tested bacterial pathogens CT. Control without treatment.

aeruginosa KCTC2004. The effects of essential oil on the growth of


tested bacteria demonstrated the reduced viability at MIC concentration. At 40 min exposure, nearly 90% inhibition of all the tested
strains was observed. Also the steep decline in CFU numbers was
observed at 20 min exposure against the tested bacteria. Exposure
of 60 min of the leaf essential oil revealed complete inhibition of
CFU numbers against all the tested bacterial pathogens (Fig. 1).
3.5. Scanning electron microscopy (SEM)
Elaborative study of SEM was carried out to visualize the effects
of the leaf essential oil of M. glyptostroboides on the morphology of
S. aureus KCTC1916 and demonstrated altered cell morphology as
compared to control group (Fig. 2). Control cells in the absence of
the oil showed a regular, smooth surface (Fig. 2a). In contrast, cells
inoculated with the oil at MIC concentration (250 lg/ml) revealed
severe detrimental effect on the morphology of cell membrane,
showing disruption and lysis of the membrane integrity (Fig. 2b).
Initial exposure of the oil to S. aureus KCTC1916 revealed large surface collapse and wrinkled abnormalities on the morphology of the
cells along with some small clefts (Fig. 2c and d).

3.6. Antioxidant activity


The DPPH free radical scavenging activity of leaf essential oil
and the leaf extracts (hexane, chloroform, ethyl acetate and methanol) of M. glyptostroboides has been shown in Fig. 3. The IC50 values of essential oil and the extracts were compared with the
standards ascorbic acid (AA = 6.55 lg/ml) and butylated hydroxyanisole (BHA = 18.27 lg/ml). A lower IC50 value indicates a greater
antioxidant activity. The IC50 values of essential oil and the extracts
were recorded in the range of 11.32192.32 lg/ml. The free radical
scavenging activities of essential oil and ethyl acetate extract
(IC50 = 11.32 and 19.12 lg/ml) were found superior to all other extracts. However, low polar extracts exhibited low DPPH scavenging
activity.
3.7. Total phenolic contents
The amount of total phenolic contents of the leaf extracts (nhexane, chloroform, ethyl acetate and methanol) of M. glyptostroboides was tested, and occurred in the range of 11.2385.17 mg
GAE/g of dry sample (Fig. 4). The total phenolic contents of the leaf

Fig. 2. Scanning electron micrographs of Staphylococcus aureus KCTC1916 treated with leaf essential oil MIC concentration of M. glyptostroboides Miki ex Hu. (a) Control (b)
disruption and lysis of membrane integrity (c) wrinkled abnormalities and cleft formation (d) abnormal breaking of cell.

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Fig. 3. DPPH radial scavenging activity of leaf essential oil and the leaf extracts of M. glyptostroboides Miki ek Hu. EO, essential oil; HXE, hexane extract; CHE, chloroform
extract; ETE, ethyl acetate extract; MNE, methanol extract; AA, ascorbic acid (control); BHA, butylated hydroxyanisole (control). Data are represented as the mean SD of
triplicate experiments.

Fig. 4. The amount of total phenolic content (mg/g dry weight) in leaf organic extract of M. glyptostroboides Miki ek Hu.

extracts of methanol, ethyl acetate, chloroform and hexane were


noted to be 61.26, 85.17, 43.12 and 11.23 mg GAE/g of dry sample,
respectively. The results showed that the total phenolic contents in
ethyl acetate extract (85.17 mg GAE/g of dry sample) were the
highest as compared to the other extracts.
4. Discussion
Plant based secondary metabolites such as essential oil and extracts are widely used in the food industry and considered GRAS
(Generally Recognized as Safe). Various publications have documented the antimicrobial activity of the essential oils and plant extracts (Morris et al., 1979). Also, several researchers reported
mono- and sesquiterpenoids as the major components of essential
oils, which are phenolic in nature. It seems reasonable to assume
that their antimicrobial mode of action might be related to the
phenolic compounds present (Cakir et al., 2004). Most of the studies on the mechanism of phenolic compounds have focused on
their effects on cellular membranes. Phenolic compounds not only
attack cell walls and cell membranes, thereby affecting their permeability and release of intracellular constituents (e.g. ribose, Na
glutamate) but they also interfere with membrane functions (electron transport, nutrient uptake, protein, nucleic acid synthesis and
enzyme activity). Thus, active phenolic compounds might have
several invasive targets which could lead to the inhibition of bacterial pathogens.
Also the results of the antibacterial screening showed that the
leaf essential oil and the leaf extracts of methanol, ethyl acetate
and chloroform have potential antibacterial effect against S. aureus
KCTC1916, S. aureus ATCC6538, B. subtilis ATCC6633, P. aeruginosa
KCTC2004 and E. coli O157:H7 ATCC42888. This might be the result
of the major component such as b-myrcene (13.29%), cyclobutane
(7.67%), furan (3.00%), valeramide (2.81%), borneol (1.20%), b-farnesene (1.67%), thymol (1.44%) and a-pinene (1,46%) present in

the leaf essential oil of M. glyptostroboides and these ndings are


in agreement with previous reports (Mevy et al., 2007; Maria-Rose
et al., 2004). Essential oils, which are odorous and volatile products
of plant secondary metabolism, have wide applications in the food
avouring and preservation industries (Smith-Palmer et al., 2001).
In addition, it is also possible that the minor components such as
limonene, linalool oxide, verbenol, a-terpineol, myrtenol, farnesol,
eugenol, caryphyllene oxide and veridiorol might be involved in
some type of antibacterial synergism with other active components of essential oil, as evident by the work of Marino et al.
(2001). The results from viable count assay revealed that exposure
of the MIC concentration of the leaf essential oil had a severe effect
on the cell viability of the tested bacteria. All the strains of B. subtilis ATCC6633, S. aureus KCTC1916, S. aureus ATCC3538 and P.
aeruginosa KCTC2004 were found sensitive to the leaf essential
oil. The oil exerted its maximum bactericidal activity as evident
by the signicant reduction in microbial counts at 20 min exposure
and complete inhibition of cell viable counts at 60 min exposure.
Similar to our ndings, one of n-6 essential oils also exerted an
inhibitory effect against some food spoilage and food-borne pathogenic bacteria (Giamarellos-Bourboulis et al., 1998). Further, the
scanning electron microscopic study showed potential detrimental
effect of the oil on the morphology of S. aureus KCTC1916. These
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 as
evident by our previous ndings and the literature reported by
others (Shin et al., 2007; Koyama et al., 1997).
Deans and Ritchie (1987) observed that the susceptibility of
Gram-positive and Gram-negative bacteria to plant volatile oils
had little inuence on growth inhibition. However, some oils appeared more specic, exerting a greater inhibitory activity against
Gram-positive bacteria. It is often reported that Gram-negative

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bacteria are more resistant to the plant-based essential oils (Reynolds, 1996). The hydrophilic cell wall structure of Gram-negative
bacteria is constituted essentially of a lipo-polysaccharide (LPS)
that blocks the penetration of hydrophobic oil and avoids the accumulation of essential oils in target cell membrane (Bezic et al.,
2003). For the above reason, the Gram-positive bacteria were
found to be more sensitive to the essential oil and the extracts derived from the leaves of M. glyptostroboides than were Gram-negative bacteria.
Furthermore, the DPPH radical is a free radical, which has been
widely used as a 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). In the present study, in
some cases, essential oil and ethyl acetate extract showed higher
or similar antioxidant activities as compared to the standard BHA
and ascorbic acid. This is due to most bioactive compounds such
as polyphenols including tannins, avonoid existed in higher polar
extracts (Karmanoli, 2002). Polyphenols are one of the major plant
compounds with antioxidant activity. The antioxidant activity of
phenolic compounds is reported to be mainly due to their redox
properties (Galato et al., 2001), which can play an important role
in adsorbing and neutralizing free radicals, quenching singlet and
triplet oxygen, or decomposing peroxides. Organic extracts may
be more benecial than isolated constituents, because other compounds present in the extracts can change the chemical or biological properties of bioactive individual component (Borchers et al.,
2004).
Besides, phenolics were also found to be one of the most plentiful classes of constituents in ethyl acetate extract of M. glyptostroboides leaves. This is due to the presence of high bioactive
compounds in ethyl acetate extract as compared to other organic
extracts. The key role of phenolic compounds to scavenge free radicals has been emphasized in several reports (Rauha et al., 2000;
Archana et al., 2005).

5. Conclusion
The results obtained in this study support the possible use of M.
glyptostroboides mediated leaf essential oil and extracts in the food
industry, where bacterial pathogens cause severe destruction by
hampering the quality of food and consumer demand as well as
for using as a source of natural antioxidants. We hope that natural
compounds such as essential oil and extracts might be suitable for
using as preservatives to control food-borne pathogens and as natural antioxidants to reduce oxidative stress in human beings. The
main reason for their suitability is their natural origin, which consumers nd comforting and which is benecial for the environment and the very low risk that pathogens will develop
resistance to the mixture of components that make up the oil
and extracts with their apparent diversity of antibacterial mechanisms. These benecial characteristics could increase food safety
and shelf life. On the other hand, the use of plant essential oils in
consumer goods is expected to increase in the future due to the
risk of green consumerism, which stimulates the use and development of products derived from plants, as both consumers and
regulatory agencies are more comfortable with the use of natural
antimicrobials (Tuley de Silva, 1996). This not only applies to the
food and cosmetic sectors but also to medicinal products. Thus,
from the above results it can be concluded that the use of natural
antibacterial and antioxidant agents derived from the leaves of M.
glyptostroboides will be suitable for applications in the food industry because plant derived natural antimicrobials have been consumed by mankind for centuries with lesser or no signs of any
toxicity.

Conict of interest statement


None declared.
References
Adams, R.P., 2001. Identication of Essential Oil Components by Gas
Chromatography/Quadrupole
Mass
Spectroscopy.
Allured
Publishing
Corporation, Carol Stream, Illinois.
Archana, B., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of
Syzygium cumini fruit. Food Chem. 90, 727733.
Bajpai, V.K., Rahman, A., Choi, U.K., Youn, S.J., Kang, S.C., 2007a. Inhibitory
parameters of the essential oil and various extracts of Metasequoia
glyptostroboides Miki ex Hu to reduce food spoilage and food-borne
pathogens. Food Chem. 105 (3), 10611066.
Bajpai, V.K., Rahman, A., Kang, S.C., 2007b. Chemical composition and anti-fungal
properties of the essential oil and crude extracts of Metasequoia glyptostroboides
Miki ex Hu Ind. Crops Prod. 26, 2835.
Bakri, I.M., Douglas, C.W.I., 2005. Inhibitory effect of garlic extract on oral bacteria.
Arch. Oral Biol. 50, 645651.
Bezic, N., Skocibusic, M., Dinkic, V., Radonic, A., 2003. Composition and
antimicrobial activity of Achillea clavennae L. essential oil. Phytother. Res. 17,
10371040.
Borchers, A.T., Keen, C.L., Gerstiwin, M.E., 2004. Mushrooms, tumors, and immunity:
an update. Exp. Biol. Med. 229, 393406.
Cakir, A., Kordali, S., Zengin, H., Izumi, S., Hirata, T., 2004. Composition and
antifungal activity of essential oils isolated from Hypericum hyssopifolium and
Hypericum heterophyllum. Flavour Frag J. 19 (1), 6268.
Deans, S.G., Ritchie, G., 1987. Antibacterial properties of plant essential oils. Int. J.
Food Microbiol. 5, 165180.
Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing.
Nature 408, 239247.
Galato, D., Ckless, K., Susin, M.F., Giacomelli, C., Ribeiro Do Vallle, R.M., Spinelli, A.,
2001. Antioxidant capacity of phenolic and related compounds: correlation
among electrochemical, visible spectroscopy methods and structureantioxidant activity. Redox. Rep. 6, 243250.
Gey, K.F., 1990. The antioxidant hypothesis of cardiovascular disease: Epidemiology
and mechanisms. Biochem. Soc. Trans. 18, 10411045.
Giamarellos-Bourboulis, E.J., Grecka, P., Dionyssiou-Asteriou, A., Giamarellou, H.,
1998. In vitro activity of polyunsaturated fatty acids on Pseudomonas
aeruginosa: Relationship to lipid peroxidation. Prostag. Leukotr. Ess. Fatty
Acids 58, 283287.
Karmanoli, K., 2002. Secondary metabolites as allelochemicals in plant defence
against microorganisms of the phyllosphere. In: Reigosa, M., Pedrol, N. (Eds.),
Allelopathy from Molecules to Ecosystems. Science Publishers Inc., NH, USA, pp.
277288.
Kockro, R.A., Hampl, J.A., Jansen, B., Peters, G., Scheihing, M., Giacomelli, R., 2000.
Use of scanning electron microscopy to investigate the prophylactic efcacy of
rifampin-impregnated CSF shunt catheters. J. Med. Microbiol. 49, 441
450.
Koyama, S., Yamaguchi, Y., Tanaka, S., Motoyashiya, J., 1997. A new substance
(yoshixol) with an interesting antibiotic mechanism from wood oil of Japanese
traditional tree (kiso-hinoki), Chamaecyparis obtuse. Gen. Pharmacol. 28, 797
804.
La Vecchia, C., Altieri, A., Tavani, A., 2001. Vegetables, fruits, antioxidants and
cancer: a review of Italian studies. Eur. J. Nutr. 40, 261267.
Lister, E., Wilson, P., 2001. Measurement of Total Phenolics and ABTS Assay for
Antioxidant Activity. Crop Research Institute, Lincoln, New Zealand (personal
communication).
Maria-Rose, J.R.A., Elnatan, B.S., Maria-Usileide, D.S.L., Nadja, A.P.N., Telma-Leda,
G.L., Edilberto, R.S., 2004. Composition and antimicrobial activity of the
essential oil from aerial parts of Baccharis trinervis Lam., Pers. ARKIVOC 6, 59
65.
Marino, M., Bersani, C., Comi, G., 2001. Impedance measurements to study the
antimicrobial activity of essential oils from Lamiaceace and Compositae. Int. J.
Food Microbiol. 67, 187195.
Mevy, J.P., Bessiere, J.M., Dherbomez, M., Millogo, J., Viano, J., 2007. Chemical
composition and some biological activities of the volatile oils of a chemotype of
Lippia chevalieri Moldenke. Food Chem. 101, 682685.
Morris, J.A., Khettry, A., Seitz, E.W., 1979. Antimicrobial activity of aroma chemicals
and essential oils. J. Am. Oil Chem. Soc. 56, 595603.
Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H., 1995. ASM: Manual
of Clinical Microbiology, sixth ed. Washington.
Mylotte, J.M., McDermott, C., Spooner, J.A., 1987. Prospective study of 114
consecutive episodes of Staphylococcus aureus bacteria. Rev. Infect. Dis. 9,
891907.
NCCLS, (National Committee for Clinical Laboratory Standards), 2000. Methods for
dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.
Approved Standard, M7-A5.
Nychas, G.J.E., Tassou, C.C., Skandamis, P., 2003. Antimicrobials from herbs and
spices. In: Roller, S.M. (Ed.), Natural Antimicrobials for the Minimal Processing
of Foods. CRC Press, Woodhead Publishers, New York, pp. 176200.
Rauha, J.P., Remes, S., Heinonen, M., Hopia, A., Kahkonen, M., Kujala, T., Pihlaja, K.,
Vuorela, H., Vuorela, P., 2000. Antimicrobial effects of Finnish plant extracts

Author's personal copy

V.K. Bajpai et al. / Food and Chemical Toxicology 47 (2009) 18761883


containing avonoids and other phenolic compounds. Int. J. Food Microbiol. 56,
312.
Reynolds, J.E.F., 1996. Martindale the Extra Harmacopoeia, 31st ed. Royal
Pharmaceutical Society of Great Britain, London.
Safer, A.M., Al-Nughamish, A.J., 1999. Hepatotoxicity induced by the anti-oxidant
food additive butylated hydroxytoluene (BTH) in rats: an electron microscopical
study. Histol. Histopathol. 14, 391406.
Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., 2005.
Comparative evaluation of 11 essential oils of different origin as functional
antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621632.
Shin, S.Y., Bajpai, V.K., Kim, H.R., Kang, S.C., 2007a. Antibacterial activity of
bioconverted eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against
foodborne pathogenic bacteria. Int. J. Food Microbiol. 113, 233236.

1883

Shin, S.Y., Bajpai, V.K., Kim, H.R., Kang, S.C., 2007b. Antibacterial activity of
eicosapentaenoic acid (EPA) against foodborne and food spoilage
microorganisms. LWT 40, 15151519.
Singh, R., Chandra, R., Bose, M., Luthra, P.M., 2000. Antibacterial activity of Curcuma
longa rhizome extract on pathogenic bacteria. Curr. Sci. 83, 737740.
Smith-Palmer, A., Stewart, J., Fyfe, L., 2001. The potential application of plant
essential oils as natural food preservatives in soft cheese. Food Microbiol. 18,
463470.
Tuley de Silva, K., 1996. A Manual on the Essential Oil Industry. United Nations
Industrial Development Organization, Vienna.
Young, I.S., Woodside, J.V., 2001. Antioxidants in health and disease. J. Clin. Pathol.
54 (3), 176186.