Professional Documents
Culture Documents
Department of Biotechnology
Daegu University
Kyoungsan, Kyoungbook 712-714, Korea
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ABSTRACT
The antibacterial potential of essential oil and methanolic extracts of
Erigeron ramosus (Walt.) B.S.P. was evaluated. Thirty-one components representing 95.3% of the total oil were identified, of which b-caryophyllene
(24.0%), a-humulene (14.5%), 1,8-cineole (9.0%), eugenol (7.2%), globulol
(7.1%), caryophyllene oxide (5.2%), d-cadinene (5.0%), a-copaene (4.9%)
and widdrol (2.0%) were the major components. The antibacterial activity of
essential oil and methanolic extracts of E. ramosus was determined in vitro
using the agar diffusion method and minimum inhibitory concentration determination test against 14 (seven gram-positive and seven gram-negative) foodborne bacteria. The essential oil (5 mL/mL, corresponding to 1,000 ppm/disc),
methanol extract and its different organic subfractions (7.5 mL/mL, corresponding to 1500 ppm/disc) of E. ramosus displayed a great potential of
antibacterial activity against all gram-positive bacteria: Staphylococcus
aureus (ATCC 6538 and KCTC 1916), Listeria monocytogenes (ATCC 19116,
ATCC 19118, ATCC 19166 and ATCC 15313) and Bacillus subtilis ATCC
6633 and four gram-negative bacteria: Pseudomonas aeruginosa KCTC 2004,
Enterobacter aerogenes KCTC 2190 and Escherichia coli (0157:H7 ATCC
43888 and ATCC 8739). The zones of inhibition of different concentrations of
essential oil and methanolic extracts against the tested bacteria were found in
the range of 10.1~22.3 mm, and MIC values were recorded between 62.5 and
500 mg/mL.
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PRACTICAL APPLICATIONS
The use of essential oil and organic extracts of Erigeron ramosus (Walt.)
B.S.P. as antibacterial agents will be suitable for applications on the food
industry as natural preservatives or flavoring to control foodborne pathogens.
They can be used as growth inhibitors of Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes and
Pseudomonas aeruginosa, some important foodborne pathogens and spoiling
bacteria. The main reason for their suitability is their natural origin, which
consumers find comforting and which is beneficial 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 beneficial characteristics could increase food
safety and shelf life.
INTRODUCTION
Illness caused by the consumption of contaminated foods has a wide
economic and public health impact worldwide (Mead et al. 1999). Many
pathogenic microorganisms such as Listeria monocytogenes, Staphylococcus
aureus, Bacillus subtilis, Escherichia coli, Salmonella sp. and Pseudomonas
aeruginosa have been reported as the causal agents of foodborne diseases
(McCabe-Sellers and Samuel 2004). A variety of different chemical and synthetic compounds have been used as antimicrobial agents to inhibit bacteria in
foods. Due to the identified and potential toxicity of chemical food preservatives, there have been increased demands for food preservatives from natural
sources. The demands for more natural antimicrobials have driven food
scientists to investigate the effectiveness of inhibitory compounds, such as
essential oils (Nguefack et al. 2004), and extracts from plants (Nasar-Abbas
and Halkman 2004; Shin et al. 2004). Essential oils are a complex mixture of
compounds, mainly monoterpenes, sesquiterpenes and their corresponding
oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols
and oxides) from plants, which are widely known for their scents and flavors.
Plant-derived essential oils have been long used as flavoring agents or preservatives in foods, beverages and confectionary products, and also have a broad
spectrum of in vitro antimicrobial activities (Conner 1993). In general, plantderived essential oils are considered as nonphytotoxic compounds and potentially effective against microorganisms (Pandey et al. 1982). Thus, essential
oils and plant extracts are promising natural antimicrobial agents with potential applications in food industries for controlling of foodborne pathogens and
spoiling bacteria.
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with 80% methanol (200 mL 3) at room temperature, and the solvents from
the combined extracts were evaporated by a vacuum rotary evaporator (EYELA
N-1000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The methanol extract (5.7 g)
was suspended in water and extracted successively with hexane, chloroform and
ethyl acetate to give hexane (1.9 g), chloroform (1.2 g) and ethyl acetate (0.8 g),
and residual methanol fractions (0.7 g), respectively. Solvents (analytical
grade) for extraction were obtained from commercial sources.
Gas chromatography-mass spectrometry (GC-MS)/MS analysis
The GC-MS analysis of the essential oil was performed using a Shimadzu
GC-MS (GC-17A, Shimadzu, Kyoto, Japan), equipped with a ZB-1 MS fused
silica capillary column (30 m 0.25 mm i.d., film thickness 0.25 mm). For
GC-MS detection, an electron ionization system with an 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 and
290C, respectively. The oven temperature was programmed from 50 to 150C
at 3C/min, then held isothermal for 10 min and finally raised to 250C at
10C/min. Diluted samples (1/100, v/v, in methanol) of 1.0 mL was injected
manually in the splitless mode. The relative percentage of the oil constituents
was expressed as percentages by peak area normalization.
Identification of compounds of the essential oil was based on GC retention time on a ZB-1 capillary column, computer matching of mass spectra with
those of standards (Wiley 6.0 data of GC-MS system), and, whenever possible,
by co-injection with authentic compounds (Adam 2001).
Microorganisms
The following food-spoiling and foodborne bacterial strains were used in
the antimicrobial tests: S. aureus ATCC 6538, S. aureus KCTC 1916, L.
monocytogenes (ATCC 19116, ATCC 19118, ATCC 19166 and ATCC 15313),
B. subtilis ATCC 6633, P. aeruginosa KCTC 2004, Enterobacter aerogenes
KCTC 2190, E. coli ATCC 8739, E. coli 0157:H7 ATCC 43888, E. coli 0157
(human), Salmonella enteritidis KCTC 12021 and S. typhimurium KCTC
2515. The strains were obtained from the Korea Food and Drug Administration (KFDA), Daegu, South Korea. Listeria monocytogenes strains were maintained on BHI agar (brain heart infusion, Becton Dickinson, Sigma, St. Louis,
MO) medium at 4C. The other strains were maintained on Luria Broth (LB)
agar medium (Acumedia Manufacturers, Inc., Lansing, MI) at 4C.
Antibacterial activity assay
The agar diffusion method (Murray et al. 1995) was used for antibacterial
assay. Petri plates were prepared by pouring 20 mL of BHI agar and LB agar
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(1.0% trypton, 0.5% yeast extract, 1.0% NaCl, 1.5% agar) medium and allowed
to solidify. Plates were dried, and 1 mL of standardized inoculum suspension
was poured and uniformly spread. The excess inoculum was drained out and
the inoculum was allowed to dry for 5 min. A Whatman No. 1 sterile filter
paper disc (6 mm diameter) was impregnated with 5 mL/mL of essential oil
(1,000 ppm/disc) and 7.5 mL/mL of MeOH extract and its derived subfractions
(1500 ppm/disc). Negative controls were prepared using the same solvent
employed to dissolve the samples. Standard reference antibiotics, tetracycline
and streptomycin (10 mg/disc, each from Sigma-Aldrich Co., St. Louis, MO),
were used as positive controls for the tested bacteria. 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.
Minimum inhibitory concentration (MIC)
MIC of essential oil, methanol, and methanol-derived subfractions of
hexane, chloroform and ethyl acetate, was tested by the twofold serial dilution
method (Chandrasekaran and Venkatesalu 2004). The test samples of oil,
methanol extract and its derived subfractions were incorporated into 1 mL BHI
broth and LB medium to get a concentration of 1,000 mg/mL, and serially
diluted to achieve 500, 250 125, 62.5 and 31.25 mg/mL, respectively. A 10-mL
standardized suspension of each tested organism (108 cfu/mL) was transferred
to each tube. The control tubes contained only bacterial suspension and were
incubated at 37C for 24 h. The lowest concentration of the test samples, which
did not show any growth of tested organism after macroscopic evaluation, was
determined as MIC.
RESULTS
Chemical composition of the essential oil
The hydrodistillation of the air-dried flower parts of E. ramosus (Walt.)
B.S.P. gave the dark yellowish oil with a yield of 0.4% (w/w). GC-MS analyses
of the oil led to the identification of 31 different components, representing
95.3% of the total oil. The identified compounds are listed in Table 1 according
to their elution order on a ZB-1 capillary column. The oil contains a complex
mixture consisting of mainly oxygenated mono- and sesquiterpene hydrocarbons. The major compounds detected were b-caryophyllene (24.0%),
a-humulene (14.5%), 1,8-cineole (9.0%), eugenol (7.2%), globulol (7.1%),
caryophyllene oxide (5.2%), d-cadinene (5.0%), a-copaene (4.9%) and
widdrol (2.0%). Geraniol (1.9%), spathulenol (1.4%), viridiflorol (1.3%),
nerolidol (0.8%), trans-b-farnesene (0.6%), ledol (0.6%), b-selinene (0.5%)
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TABLE 1.
COMPONENTS OF ESSENTIAL OIL OF ERIGERON
RAMOSUS (WALT.) B.S.P. IDENTIFIED BY GC-MS
Peak no.
Components
Percentage
in total oil
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
2-phenethyl alcohol
Eugenol
b-chamigrene
a-humulene
Nerolidol
g-cadinene
a-copaene
Aromadendrene
b-caryophyllene
d-cadinene
Trans-b-farnesene
Aromadendrene epoxide
Caryophyllene oxide
3-p-menthen-9-ol
(Z)-5-pentadecen-7-yne
b-selinene
Spathulenol
(Z)-6-hexadecen-4-yne
Widdrol
Globulol
Viridiflorol
Ledol
n-nonanal
Ledane
Geraniol
Myristic acid
Patchulane
1,8-cineole (eucalyptol)
Dibutyl phthalate
Stearic acid
Palmitic acid
Total
0.6
7.2
0.6
14.5
0.8
0.6
4.9
1.3
24.0
5.0
0.6
0.5
5.2
0.4
0.5
0.5
1.4
0.4
2.0
7.1
1.3
0.6
0.5
0.5
1.9
1.0
0.2
9.0
0.3
0.7
1.2
95.3
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employed bacteria was qualitatively and quantitatively assessed by the presence or absence of inhibition zones. According to the results given in Table 2,
a total of 14 food spoilage and foodborne bacterial strains, including seven
gram-positive and seven gram-negative bacteria, were tested. The oil exhibited antibacterial activity against all seven gram-positive and four gramnegative bacteria at the concentration of 5 mL/mL (1,000 ppm/disc). The oil
exhibited a potent inhibitory effect against S. aureus ATCC 6538, S. aureus
KCTC 1916, L. monocytogenes (ATCC 19116, ATCC 19118, ATCC 19166
and ATCC 15313), B. subtilis ATCC 6633, P. aeruginosa KCTC 2004, E.
aerogenes KCTC 2190, E. coli ATCC 8739 and E. coli 0157:H7 ATCC 43888,
with diameter zones of inhibition of 22.3~12.2 mm, as shown in Table 2.
Methanol extract of E. ramosus (Walt.) B.S.P. and its derived subfractions also
revealed a great potential of antibacterial activity against all seven grampositive and four gram-negative bacteria, at the concentration of 7.5 mL/mL,
corresponding to 1500 ppm/disc (Table 2). Methanol extract showed the
strongest antibacterial effect against S. aureus (ATCC 6538 and KCTC 1916),
L. monocytogenes (ATCC 19116, ATCC 19118 and ATCC 19166), B. subtilis
ATCC 6633, P. aeruginosa KCTC 2004 and E. coli ATCC 8739, with the
diameter of inhibition zones ranging from 16.1~21.4 mm, as compared with
standard drug streptomycin. On the other hand, hexane, chloroform and ethyl
acetate subfractions showed interesting antibacterial effect, with inhibition
zones in the range of 10.1~20.2 mm. In this study, in some cases, the oil,
methanol extract and organic subfractions (chloroform and ethyl acetate)
exhibited higher antibacterial activity compared with streptomycin, while
tetracycline showed higher activity in some other cases than the essential oil
and solvent fractions. Hexane fraction displayed a moderate inhibitory effect.
However, the residual methanol subfraction did not show any activity against
all the bacterial strains tested (data not shown). The blind control did not
inhibit the growth of the bacteria tested. No inhibitory effect was observed
against E. coli 0157 (human), S. enteritidis KCTC 12021 and S. typhimurium
KCTC 2515, in all cases.
MIC
As shown in Table 3, the MIC values for the oil were found more
susceptible to S. aureus ATCC 6538, L. monocytogenes ATCC 19116 and
B. subtilis ATCC 6633 (62.5 mg/mL for each) than those of S. aureus KCTC
1916, L. monocytogenes ATCC 19118, P. aeruginosa KCTC 2004 and E. coli
ATCC 8739 (125 mg/mL for each). On the other hand, MIC values of the
methanol extract and its derived subfractions of hexane, chloroform and ethyl
acetate against the tested bacteria were found in the range of 62.5~500 mg/mL
(Table 3). Methanol extract and its chloroform fraction showed higher
MeOH extract
20.2 1.1
18.2 1.5
16.1 1.2
18.3 1.6
16.2 1.1
15.2 1.1
21.4 0.7
17.1 1.4
15.4 0.7
16.4 0.7
12.2 1.7
nd
nd
nd
Essential oil*
22.3 1.6
20.0 1.4
18.4 1.2
17.3 0.6
15.0 1.5
16.3 1.2
20.3 0.7
16.3 1.2
14.3 1.2
16.3 1.2
12.2 1.2
nd
nd
nd
14.3 0.7
13.2 0.5
14.1 1.1
13.5 1.0
12.3 1.4
10.2 1.2
14.2 0.8
10.1 0.6
11.3 0.7
nd
nd
nd
nd
nd
Hexane
20.2 1.2
18.1 1.2
16.1 0.5
15.1 1.2
14.0 1.2
15.3 0.5
17.3 1.2
15.0 1.1
14.2 0.9
13.5 0.7
12.2 1.5
nd
nd
nd
CHCl3
16.2 1.5
17.3 1.2
15.2 1.0
15.1 1.2
14.3 1.6
12.0 1.1
15.2 0.6
14.2 1.1
12.2 0.6
13.2 0.6
nd
nd
nd
nd
EtOAc
Diameter of inhibition zones of essential oil including diameter of disc 6 mm (tested at a volume of 1,000 ppm/disc).
MeOH extract (1,500 ppm/disc).
Subfractions of MeOH extract (1,500 ppm/disc).
Standard antibiotics: TC, tetracycline and SM, streptomycin (10 mg/disc).
nd, not detected. Values are given as mean SD (n = 3).
Microorganism
19.3 0.7
18.1 0.6
19.5 1.1
18.4 0.5
17.3 1.6
20.5 1.3
18.3 0.5
20.3 1.2
20.1 1.0
20.3 0.5
17.3 1.2
18.1 0.5
22.1 1.2
21.3 0.6
TC
Antibiotics
14.3 0.7
14.1 0.5
14.4 0.9
16.2 1.2
15.3 0.6
14.3 1.1
14.3 0.6
19.0 0.5
14.0 0.5
15.3 1.1
24.0 0.7
15.4 1.3
15.2 0.8
13.3 0.6
SM
TABLE 2.
ANTIBACTERIAL ACTIVITY OF ESSENTIAL OIL, MeOH EXTRACT AND SUBFRACTIONS OF MeOH EXTRACT OF ERIGERON RAMOSUS
(WALT.) B.S.P. AGAINST FOODBORNE PATHOGENS AND SPOILING BACTERIA
ANTIBACTERIAL ACTIVITY OF ERIGERON RAMOSUS
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TABLE 3.
MINIMUM INHIBITORY CONCENTRATION OF ESSENTIAL OIL, MeOH EXTRACT AND
SUBFRACTIONS OF MeOH EXTRACT OF ERIGERON RAMOSUS (WALT.) B.S.P. AGAINST
FOODBORNE PATHOGENS AND SPOILING BACTERIA
Microorganism
MIC*
Essential
oil
62.5
125
62.5
125
250
250
62.5
125
250
125
500
nd
nd
nd
MeOH
extract
62.5
62.5
125
125
250
250
62.5
125
250
250
500
nd
nd
nd
CHCl3
EtOAc
250
250
250
500
500
500
125
500
500
nd
nd
nd
nd
nd
62.5
125
125
125
250
250
62.5
125
250
250
500
nd
nd
nd
125
125
125
250
250
500
125
125
500
500
nd
nd
nd
nd
DISCUSSION
Since ancient times, the aromatic plant extracts have been used for many
purposes, such as food, drugs and perfumery. Historically, many plant oils and
extracts have been reported to have antimicrobial properties (Hoffman 1987).
Also, the renewal of interest in food industry and increasing consumer
demands for effective, safe and natural products means that quantitative data
on plant oils and extracts are required. A number of publications documented
the antimicrobial activity of essential oil constituents and plant extracts
(Morris et al. 1979; Nasar-Abbas and Halkman 2004). In recent years, several
researchers have also reported that mono- and sesquiterpene hydrocarbons and
their oxygenated derivatives are the major components of essential oils from
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