Arch Pharm Res Vol 32, No 12, 1711-1719, 2009 DOI 10.1007/s12272-009-2208-8

Antibacterial Effects of Vulgarone B from Artemisia iwayomogi Alone and in Combination with Oxacillin Eun Young Chung1, Youn Hee Byun1, Eun Joo Shin2, Ha Sook Chung3, Yeon Hee Lee2, and Seungwon Shin1 1

College of Pharmacy, Duksung Women’s University, Seoul 132-714, Korea, 2College of Natural Sciences, Seoul Women’s University, Seoul 139-774, Korea, and 3College of Natural Sciences, Duksung Women’s University, Seoul 132-714, Korea (Received August 27, 2009/Revised October 13, 2009/Accepted October 13, 2009)

The antibacterial activities of vulgarone B, a component of Artemisia iwayomogi essential oil, were evaluated against some antibiotic-susceptible and -resistant human pathogens. Moreover, the effects of combining antibiotics, such as oxacillin, with vulgarone B were determined in this study. Significant inhibitory activities of Artemisia oils against antibiotic-susceptible and -resistant bacteria were confirmed by broth microdilution methods. The effects of vulgarone B on bacterial morphology and DNA were observed by scanning electron microscope and electrophoresis, respectively. In checkerboard microtiter tests, vulgarone B and A. iwayomogi oil combined with oxacillin resulted in synergism, or additive effects. Moreover, the safety of Artemisia oil and vulgarone B were confirmed in vivo. Both vulgarone B and the essential oil fraction of A. iwayomogi showed significant inhibitory activities against strains of antibioticsusceptible and -resistant bacteria. The oils showed synergism or additive effects when combined with oxacillin against two strains of Staphylococcus aureus. The antibiotic mechanism involved might be related to DNA cleavage. Thus, vulgarone B and the essential oil fraction of A. iwayomogi may be promising candidates for a safe, effective, natural agent active against antibiotic-resistant S. aureus, especially when combined with oxacillin. Key words: Artemisia iwayomogi, Combination effect, Essential oils, Oxacillin, Resistant bacteria, Vulgarone B

Selected by Editors INTRODUCTION The increase in antimicrobial resistance is an important public health issue worldwide. The development of resistance both in human and animal bacterial pathogens has been associated with the extensive therapeutic use of antimicrobials and with their administration as growth promoters in livestock feed. Staphylococcus aureus is the most common pathogen among the staphylococci. It is often present in food; because of this, it is among the leading causes of Correspondence to: Seungwon Shin, College of Pharmacy, Duksung Women’s University, Seoul 132-714, Korea Tel: 82-2-901-8384, Fax: 82-2-901-8386 E-mail: [email protected]

food-borne bacterial intoxications worldwide. The incidence of resistance in S. aureus to the antibiotics currently used therapeutically, which includes the antibiotics oxacillin and methicillin, has increased steadily over time (Acco et al., 2003; La Salandra et al., 2007; Forbes et al., 2008; Skinner et al., 2009). In many cases, the pathogen develops multi-drug resistance, which strictly limits the treatment options. Salmonella species comprise many of the common pathogens causing food-borne diseases. Similarly to S. aureus, there has been an increased emergence of antibiotic resistant Salmonella strains in recent times. This is thought to have largely resulted from the consumption of processed food and agricultural products that have been in contact with antibiotics. The emergence of antibiotic resistance in human pathogens has now become a global health problem (Varma et al., 2006; Hald et al., 2007; Andrysiak et al., 2008). In response to this situation, we propose the development of a new, safe agent from plant sources to

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act as an antibiotic substitute and minimize the present use of antibiotics. In this study, we evaluated the in vitro bacterial inhibitory activities of essential oils from Artemisia iwayomogi. A. iwayomogi is the representative Korean plant source for ‘Injin’ used for alleviation of various symptoms related to liver diseases in traditional therapy in Korea. The composition of this oil has been reported and was found to vary depending on the plant source (Miyazawa and Kameoka, 1977; Guo et al., 2004). Cha et al. (2005) reported the antibacterial activity of this oil against a range of bacteria, but S. aureus and Salmonella species were not included in their study. Although previous reports discuss the antibacterial activity and mode of action of essential oils (Sikkema et al., 1994; Ultee et al., 1999; Lambert et al., 2001), further in depth studies are needed. In this study, the antibacterial activity of the plant essential oil, vulgarone B, was characterized. The inhibitory activity of A. iwayomogi and its main components was evaluated against antibiotic-susceptible and -resistant strains of S. aureus, Salmonella serotype Enteritidis and Salmonella serotype Typhimurium. We also investigate the mechanism of activity by analyzing associated morphological changes, effects on viability, λ DNA and proteins in the bacterial cells were studied by treatment of the oil to S. aureus. Moreover, we determine the combined effect of the oils with oxacillin to cover the relatively mild activity compared to antibiotics, which has been commonly used for the treatment of Staphylococcal infections.

E. Y. Chung et al.

oven temperature was programmed as follows. Initial temperature was 50oC for 5 min, increased 2oC/min up to 180oC, sustained at 180oC for 5 min, and then increased 20oC/min up to 220oC

Isolation of vulgarone B Vulgarone B was isolated from the essential oil fraction (2 g) of A. iwayomogi by silicagel column chromatography with hexane-ethylacetate (5:95). Fraction 22-31 was subjected to rechromatography with hexanemethylene chloride (8:2-2:8 gradient) to give 0.28 g of 98.8% vulgarone B (a seaquiterpene ketone, MW 218). The chemical structure was elucidated on the basis of its UV, MS, 1H-NMR and 13C-NMR data. Vulgarone B Colorless oil, C15H22O; EI-MS m/z: 218 [M]+; UV (CH2Cl2) λmax nm: 250. 322; IR (KBr) νmax cm-1: 3038, 2867, 1676, 1374; 1H-NMR (300 MHz, CDCl3) δ : 5.75 (1H, br s, H-3), 2,78 (1H, d, J = 6.6, H-5), 2.56 (1H, d, J = 6.6, H-1), 2.04 (1H, s, H-7), 2.08 (3H, s, H-8), 2.021.43 (6H, m, H-10, H-11, H-12), 0.96 (6H, s, H-14, H15), 0.87 (3H, s, H-9); 13C-NMR (75 MHz, CDCl3) δ: 205.4 (C-4), 173.1 (C-2), 122.9 (C-3), 67.2 (C-5), 58.3 (C-7), 55.3 (C-6), 50.4 (C-1), 42.0 (C-12), 38.9 (C-10), 34.2 (C-13), 28.2 (C-15). 27.1 (C-14), 25.1 (C-9), 23.7 (C-8), 21.7 (C-11).

MATERIALS AND METHODS Preparation of samples tested for antibacterial activities Essential oils were obtained from flowers and leaves (1:1) of A. iwayomogi cultivated in Youngchun, Korea by steam distillation for five hours in a simultaneous steam distillation-extraction apparatus (Schultz et al., 1977). A voucher specimen was deposited at the herbarium of Duksung Women’s University (No. COMAC2). The composition of the oil fraction was analyzed using a previously described method (Shin and Kim, 2004). Analysis of the essential oil fraction by gaschromatography and mass spectrometry The essential oil fraction of A. iwayomogi were analyzed by the Hewlett-Packard 6890 GC and the Hewlett-Packard 5973 MSD apparatus (Agilent 5973 network mass selective detector, 280oC) with a fused silica capillary column (HP-5 MS, 30 m × 250 µm × 0.25 µm). The injector was adjusted to 260oC and the

Borneol (98%), camphor (96%0, ampicillin (N9890), erythromycin (A8586193), oxacillin (O1002; oxacillin sodium salt monohydrate), and penicillin G (13572), were purchased from Sigma Chemical Co. Kanamycin (sulfate salt) injection was produced by Donga Pharmaceutical Company.

Strains Clinically isolated S. aureus strains CCARM0027, CCARM3511 and CCARM3523, S. Enteritidis strains KCCM12201, CCARM8010 and CCARM8011 and S. Typhimurium strains KCCM11862, CCARM8007 and CCARM8009, were obtained from the Culture Collection of Antibiotic Resistant Microbes (CCARM) and the Korean Culture Center of Microorganisms (KCCM), and sub-cultured on Mueller Hinton II (BD, USA)

Vulgarone B Antibacterial Activity

agar plates with 5% sheep blood or trypticase soy agar plates.

MIC (Minimum Inhibitory Concentration) test MIC values of the oils were determined using the broth microdilution method. A range of two-fold dilutions (32-0.5 mg/mL) of essential oils in medium containing 2% Tween-80 were prepared. The oil suspensions (100 µL) were added to 96-well plates. Antibiotics were similarly diluted in DMSO to generate a series of concentrations, ranging from 16 to 0.03 µg/mL per well. The turbidity of the bacterial suspensions was measured at 600 nm, and adjusted with medium to match the 0.5 McFarland standard (105-106 colony forming units/mL). Next, 100 µL of bacterial culture was inoculated into each well, and the plates were incubated at 36oC for 24 h. MIC values were determined in duplicate and re-examined where appropriate. Each organism was additionally cultured with a blank solution containing Tween-80 or DMSO at concentrations equivalent to those in test solutions to verify that these solutions did not affect growth.

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Scanning electron microscopy (SEM) Vulgarone B treated cells were prepared for SEM as follows. An overnight culture of S. aureus CCARM0027 grown on MH agar at 37oC, was dispersed in saline containing 0.05% Tween-80 to an optical density of three at A600. Vulgarone B at twice the MIC (1 mg/mL) was added, and this suspension was incubated at room temperature. After 8 h and 24 h, cells were centrifuged at 10,000 × g for 10 min. Cells were washed with 0.1 mol/L sodium cacodylate buffer (pH = 7.2), fixed in cacodylate buffer containing 1% glutaldehyde, freeze-dried and coated with gold. Each sample was observed by SEM (JSM 5410LV, JVULGARONE BL, Japan) at magnifications of 10,000X. The cell suspension in saline containing 0.05% Tween-80, without addition of vulgarone B was used as a negative control. Effect of vulgarone B on cell integrity of S. aureus CCARM0027 After treatment of S. aureus CCARM0027 with vulgarone B (1 mg/mL) for 8 h and 24 h prior to SEM, cells were collected by centrifugation at 10,000 × g for 10 min. Total DNA in the supernatant was analyzed on a 1% agarose gel and visualized by gel red (Biotium).

Checkerboard titer tests For checkerboard titer tests, 50 µL aliquots (64-1 mg/mL) of each dilution of A. iwayomogi essential oil fraction were added to the wells of 96-well plates in a vertical orientation, and 50 µL aliquots (64-1 µg/mL) of oxacillin were added in a horizontal orientation, so that the plate contained various concentration combinations of the two compounds. A 100 µL suspension of three S. aureus strains was added to each well, and cultured at 36oC for 24 h. Fractional inhibitory concentrations (FICs) were calculated as the MIC of the combination of A. iwayomogi oil fraction and oxacillin, divided by the MIC of the oil or oxacillin alone. The FIC index was obtained by adding both FICs. The FIC index was interpreted as representing a synergistic effect when it was >0.5, an additive or indifferent effect when it was >0.5 to 2.0 and an antagonistic effect when it was >2.0 (Davidson and Parish, 1989; Shin and Lim, 2004). A checkerboard experiment was also performed to test the effect of combining vulgarone B with oxacillin.

Effect of vulgarone B on viability of bacterial cells A cell viability assay was performed using the LIVE/ DEAD BacLight viability kit (L-13152, Molecular Probes) to verify cell viability after vulgarone B treatment (Berney et al., 2007). S. aureus CCARM0027 grown overnight in MH broth was dispersed in filter sterilized water to an optical density of 0.6 at A670 and mixed with vulgarone B to make a final concentration of 1 mg/mL. The mixture was incubated for 24 h at room temperature. The average percentage of viable and dead cells was calculated from the standard graph using various ratios of live:dead cells (0:100, 10:90, 50:50, 90:10 and 100:0). Cells were labeled using the LIVE/DEAD BacLight viability kit and fluorescence of live and dead cell densities was determined using a fluorescence microplate reader (FLx800, Bio-Tek instrument) (live cell, excitation 485 nm, emission 530 nm; dead cell, excitation 485 nm, emission 630 nm).

Inhibitory activity of vulgarone B on DNA One microgram of plasmid vector pBR322 (Takarabio) or λ DNA (Takarabio) was treated with 1 mg of vulgarone B at 37oC for 4 h. After DNA was electrophoresed on a 1% Tris-acetate/EDTA (TAE) agarose gel, DNA fragments were stained with ethidium bromide. The electrophoresis pattern of fragmented DNA was compared with that of untreated DNA.

Single dose oral toxicity study This study was carried out to evaluate the singledose oral toxicity of the volatile oil fraction of A. iwayomogi in male and female specific pathogen free (SPF; Koatec Co. Ltd) ICR mice. The test substance was administered to male and female mice at doses of 500, 1,000 and 2,000 mg/kg. A group of mice were treated with each dose. Each group consisted of five

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mice of each gender, representing 10 animals per group. Mortalities, clinical signs and body weight changes were monitored for 14 d compared to the vehicle control group. At the end of the 14 d observation period, all animals were sacrificed according to the animal experiment guidelines of the Korean Food and Drug Administration, and necropsy findings were noted.

RESULTS To develop a new strategy for dealing with the everworsening situation regarding the increase of antibiotic-resistant pathogens based on Korean plant resources, we examined the activities of the essential oil from A. iwayomogi and its main components, in particular vulgarone B. Vulgarone B is the major, active compound in this plant essential oil. We analyzed the activity of vulgarone B against antibiotic-susceptible and -resistant strains of four species of pathogenic bacteria.

Analysis of composition of the essential oils from A. iwayomogi As listed in Table I, by GC and GC-MS analysis in combination with a Wiley 275 Library Search, 99 compounds were detected in the essential oil fraction of A. iwayomogi. Vulgarone B was the main component and represented 19.07% of this oil, followed by camphor (10.22%) and borneol (6.67%). MIC tests The results of the MIC tests are listed in Table II. The oil fraction and its main components, including vulgarone B, displayed distinct patterns of activity Table I. Constituents of essential oils from A. iwayomogi identified by GC-MS Rt (min)

Area (%)

7.43 8.58 9.16 9.38 9.88 10.02 10.22 10.44 10.63 10.76 11.05 11.26 11.89

0.01 0.29 0.11 0.01 0.04 0.01 0.08 0.03 0.08 0.13 0.01 0.19 0.09

Compound α-pinene camphene benzaldehyde ethylbutyl acetylene β-pinene artemiseole 1-octen-3-ol hexanoic acid 2-dehydro-1,8-cineole β-myrcene vinylcyclohexane sabinene α-terpinene

RI 902 932 947 953 966 969 975 980 985 989 996 1001 1014

Table I. Constituents of essential oils from A. iwayomogi identified by GC-MS (Continued) Rt (min)

Area (%)

12.30 12.59 13.03 13.27 13.63 13.77 14.06 14.30 14.47 15.22 16.39 16.84 18.32 19.05 19.25 19.47 19.84 20.05 20.77 20.95 21.35 21.60 23.11 23.34 23.81 23.97 26.17 26.38 26.62 26.90

0.49 5.65 0.40 0.23 0.03 0.02 0.23 0.19 0.08 0.04 1.87 0.03 10.22 0.41 0.38 6.67 0.42 2.01 0.57 1.04 1.97 0.76 0.63 0.35 0.44 0.72 0.58 0.14 0.05 0.22

27.93 28.36 28.76 28.96 29.06 29.30 29.50 29.96 30.60 30.74 31.01 31.43 31.86 32.05 32.90 33.61 33.97

0.14 0.61 0.27 0.04 0.09 0.03 0.78 0.09 0.02 0.15 0.07 3.78 0.13 0.53 0.75 0.18 0.26

Compound cymol p-cineole 3-methyl-2,4-hexadiene benzene acetaldehyde santolina triene n-dodecanal γ-terpinene artemisia ketone sylvestrene myrcenyl acetate α-thujone trans-limonene oxide camphor sabina ketone pinocarvone borneol isocamphopinone 4-terpineol α-terpinolene benihinal isoborneol 1-verbenone cumic aldehyde 4-methyl-1,3-heptadiene piperitone carone santene carvacrol piperitenone 2-ethyl-5,5-dimethyl-1,3cyclopentadiene eremophilene α-cubebene eugenol aromadendrene α-bisabolene γ-himachalene α-copaene β-damascenone cis-jasmone tetradecane seychellene β-caryophyllene germacrene D coumarin α-humulene junipene α-amorphene

RI 1022 1027 1036 1041 1048 1051 1056 1061 1064 1079 1102 1111 1140 1155 1158 1163 1170 1174 1188 1192 1200 1205 1237 1242 1252 1256 1302 1307 1312 1318 1340 1349 1357 1362 1364 1369 1373 1383 1397 1400 1406 1416 1426 1431 1451 1468 1476

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Table I. Constituents of essential oils from A. iwayomogi identified by GC-MS (Continued) Rt (min)

Area (%)

34.12 34.98 35.38 35.54 35.96 36.30 36.51 36.67 37.55 37.80 38.34 38.70 39.01 39.74

1.71 0.37 0.29 0.26 1.39 0.19 0.21 0.67 0.24 0.66 5.99 0.71 3.86 0.51

39.90 40.19 40.73 41.05 41.32 41.87 42.10 42.45 43.41 43.94 44.14 44.79 44.17 45.34 46.20 46.42 46.57 47.91 48.31 48.72 49.80 50.41 50.84 51.13 51.44

0.22 1.08 0.99 19.07 3.35 0.64 0.45 1.52 0.48 0.39 0.48 0.33 0.25 0.56 0.77 0.29 0.65 0.41 0.59 0.17 0.03 0.14 0.09 0.03 0.18 96.060

Compound β-cubebene α-muurolene β-bisabolene γ-cadinene δ-cadinene cadina-1,4-diene aromadendrene phlorol α-calacorene E-farnesol caryophyllene oxide vulgarol A vulgarone A 1-methylcyclohexene-4carboxaldehyde eremophilene α-cedrene acorenone B vulgarone B β-selinene valerenol α-guaiene α-ylangene N-(carboxymethyl)valine isoaromadendrene epoxide tremetone β-costal dehydroaromadendrene methylpentylacetylene valerenic acid α-copaene-8-ol β-oplopenone isobarbatene ketone 3-cyclohepten-1-one neopentylidene cyclohexane β-ocimene-X δ-nerolidol farnesyl acetone methyl caprinate α-farnesene

RI 1480 1500 1510 1514 1524 1532 1537 1541 1561 1567 1580 1589 1596 1615 1619 1627 1641 1650 1657 1671 1678 1687 1712 1726 1731 1749 1762 1763 1786 1792 1796 1834 1846 1858 1889 1907 1919 1928 1937

In total

GC retention indices (RI) calculated against C9 to C24 nalkanes on a HP-5MS column (0.25 mm × 30 m × 0.25 um).

against the species tested, as exemplified by the differential MIC values. The MICs of five antibiotics against the test strains were estimated to compare their resistance patterns.

The essential oil fractions of A. iwayomogi and its main components exhibited the highest inhibitory activities against the antibiotic-susceptible and -resistant strains of S. aureus, with MIC values ranging from 0.5 mg/mL to 4 mg/mL. Among the tested oils, vulgarone B showed the highest inhibitory activity against all three of the tested S. aureus strains with MICs of 0.5 mg/mL to 1 mg/mL. No remarkable differences were evident between the antibiotic-susceptible and -resistant strains. Against both of the Salmonella species tested, the A. iwayomogi oil fraction and vulgarone B showed relatively weak activity compared with S. aureus strains, resulting in MICs between 1.00 and >16.00 mg/mL. Vulgarone B exhibited the highest activity among the oil components. As in experiments with S. aureus, no remarkable differences were evident between the antibiotic-susceptible and -resistant strains in these experiments.

Checkerboard titer tests As illustrated in Table III, the MIC values of oxacillin against S. aureus CCARM0027 (an oxacillinsusceptible strain) decreased from 0.25 µg/mL to 0.06 µg/mL in combination with A. iwayomogi oil, resulting in a FIC of 0.25. The MICs of the A. iwayomogi oil fraction decreased from 2 mg/mL to 0.5 mg/mL (FIC = 0.25). Thus, the combination of oxacillin and the A. iwayomogi oil fraction produced fractional inhibitory concentration indices (FICI) of 0.50 indicating the presence of additive effects. Similarly, in a parallel experiment with vulgarone B, an additive effect was seen when combined with oxacillin, with a FICI of 0.75. In a similar experiment with S. aureus CCARM3511 (an oxacillin resistant strain) (MIC = 4) the combinatorial treatment of oxacillin with the A. iwayomogi oil fraction induced significant synergism with an FICI of 0.18. In this experiment the MIC of oxacillin was lowered from 4 µg/mL to 0.25 µg/mL. The additive effect was additionally observed with a FICI of 0.50 in a similar experiment with vulgarone B and oxacillin. Here, the MIC of oxacillin combined with vulgarone B was 1 µg/mL. Effect of vulgarone B on cell morphology and bactericidal activity SEM revealed that vulgarone B induced significant changes in cell morphology. Cells of S. aureus were bloated after 8 h of treatment (Fig. 1C and D), while they were crushed and aggregated after 24 h of treatment (Fig. 1E and F) with vulgarone B. The untreated control cells (Fig. 1A and B) did not have any de-

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Table II. Minimal inhibitory concentrations (MICs) of the essential oils of A. iwayomogi against antibiotic-resistant and -susceptible bacterial strains estimated by the broth dilution method Samples A. iwayomogi Borneol Camphor Vulgarone B Amphicillin Erythromycin Kanamycin Oxacillin Penicillin G

Strains Sa27

Sa11

>02 >04 >02 >00.5 >00.12(s) >00.25(s) >16(r) >00.25(s) >00.12(s)

>04 >04 >04>0 >01 >01(r†) >16(r) >02(s) >04(r) >00.5(r)

Sa23 >04 >02 >02 >01 >02(r) >16(r) >16(r) >16(r) >02(r)

Se21

Se10

>16 >16 >16 0>2 >00.5(s‡) >02 >02(s) >16 -*

>16 >16 >16 0>2 >16(r) >16 >04(s) >16 -

Se11 >08 >16 >16 0>2 >00.12(s) >16 >01(s) >00.5 -

St62

St07

St09

>02 >02 >02 >01 >16(r) >16 >16(r) >08

>08 >04 >04 0>2 >16(r) >16 >08(s) >08 -

>08 >08 >04 >02 >16(r) >16 >16(r) >16 -

*Not tested. † Resistant and ‡ susceptible to the corresponding antibiotics discriminated by the criteria of the Clinical and Laboratory Standards Institute, USA (2007). The values are the means from triplicate experiments. The values for A. iwayomogi, borneol, camphor and vulgarone B are shown in mg/mL, the values for amphicillin, erythromycin, kanamycin, oxacillin and penicillin G are shown in µg/mL. Sa27: S. aureus CCARM0027, Sa11: S. aureus CCARM3511, Sa23: S. aureus CCARM3523, Se21: S. enteritidis KCCM12201, Se10: S. enteritidis CCARM8010, Se11: S. enteritidis CCARM8011, St62: S. typhimurium KCCM11862, St07: S. typhimurium CCARM 8007, St09: S. Typhimurium CCARM 8009. Table III. Fractional inhibitory concentrations (FICs) and fractional inhibitory concentration indices (FICIs) of oxacillin combined with the A.iwayomogi oil fraction or vulgarone B against two strains of S. aureus Strain Combination

Sa27

Sa11 †

MIC

FIC

Oxacillin

MICo MICoca

0.25 0.06

- A. iwayomogi oil

MICao MICaco

2 0.5

Oxacillin

MICo MICocv

0.25 0.125

- Vulgarone B

MICvo MICvco

0.5 0.125

‡

FICI

MIC

FIC

MICo MICoca

4 0.25

0.06

0.25

MICao MICaco

4 0.5

0.12

0.50

MICoo MICocv

4 1

0.25

MICvo MICvco

1 0.25

0.25

0.25 0.50

0.75 0.25

FICI

0.18

0.50

†

Fractional inhibitory concentration (MIC of the combined samples/MIC of the sample alone), ‡Fractional inhibiting concentrations indices (sum of the FICs of combined samples), MICo: MIC of oxacillin alone, MICoca: MIC of oxacillin (µg/mL) combined with A. iwayomogi essential oil fraction, MICao: MIC of A. iwayomogi essential oil fraction (mg/mL) alone, MICaco: MIC of A. iwayomogi essential oil fraction combined with oxacillin, MICocv: MIC of oxacillin combined with vulgarone B, MICv: MIC of vulgarone B (mg/mL) alone, MICvco: MIC of vulgarone B combined with oxacillin.

tectable morphological changes. Treatment with vulgarone B for 24 h caused 99.62% death of S. aureus CCARM0027. This result indicated that vulgarone B has strong bactericidal effects on S. aureus.

Effect of vulgarone B on cell membrane integrity and DNA At a vulgarone B concentration of 1 mg/mL, DNA was leaked from S. aureus cells and decomposed depending on the treatment time (Fig. 2), as detected on agar gel plate. As shown in Fig. 3, vulgarone B treatment of plasmid vector pBR322 or λ DNA produced one linear DNA fragment larger than the original circular

DNA, suggesting that vulgarone B treatment resulted in a single nick in the DNA. The DNA mobility shift observed after addition of vulgarone B indicated that vulgarone B is able to interact with or damage bacterial DNA.

Single dose oral toxicity study In the single dose oral toxicity study with male and female SPF ICR mice, there was no death of any animal relating to the toxicity of Artemisia oil during the experimental period. No clinical signs or body weight changes were found in relation to the tested samples.

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Fig. 3. Effects of vulgarone B on pBR322 plasmid DNA and λDNA. One µg of pBR322 or λ DNA was treated with 1 mg vulgarone B at 37oC for 4 h. Then DNA was electrophoresed on a 1% agarose gel and DNA was stained with ethidium bromide (Lane 1, 1 kb DNA marker; 2, pBR322 1 µg; 3, pBR322 1 µg and vulgarone B 1 mg; 4, λDNA 1 µg; 5, λDNA 1 µg and vulgarone B 1 mg; 6, λDNA 1 µg and norfloxacin (0.1 mg); 7, 1 kb DNA marker.

Fig. 1. Electron micrographs of S. aureus CCARM0027 treated with vulgarone B taken at a magnification of 10,000X (A and B, S. aureus treated for 8 h and 24 h in saline; C and D, scanning electron micrographs of S. aureus treated for 8 h with vulgarone B; E and F, S. aureus treated for 24 h with vulgarone B).

Fig. 2. Effect of vulgarone B on cell integrity. Staphylococcus aureus CCARM0027 was treated with vulgarone B as described in Materials and Methods. Supernatants were electrophoresed on a 1% agarose gel (M, 1 Kb DNA marker; 1, S. aureus alone, 0 h; 2, S. aureus alone, 8 h; 3, S. aureus alone, 24 h; 4, S. aureus treated with 1 mg of vulgarone B, 8 h; 5, S. aureus treated with 1 mg of vulgarone B, 24 h).

DISCUSSION In MIC tests, vulgarone B, the main component of A. iwayomogi essential oil, showed higher activity against all of the bacterial strains tested than the

other main components of the oil fraction. It therefore appears that vulgarone B is predominantly responsible for the antimicrobial activity of A. iwayomogi essential oil. Results showed that vulgarone B had higher inhibitory activity on S. aureus (Gram positive) than on both of the Salmonella species tested (Gram negative). Under these experimental conditions, the MICs of the oil were ca. 1,000-10,000 fold higher than those of antibiotics, as shown in Table I. The antibacterial activity of most plant essential oils has been shown to be low compared to that of antibiotics; however, they still have promising applications as novel, safe alternatives, especially in foods (Burt, 2004). Vulgarone B could potentially be used as an anti S. aureus agent in food. As there are several potential targets in the cell, the antibacterial activity of essential oils cannot attribute to any one specific mechanism. Indeed the permeability of cell membranes is dependent on the hydrophobicity of the solutes that have to cross the membrane and the membrane composition (Sikkema et al., 1994). It is widely believed that essential oils interact with and disrupt the cytoplasmic membrane, leading to leakage of essential molecules and resulting in cell death (Lambert et al., 2001). A large amount of cellular component leakage was not observed with vulgarone B treatment; however, changes in cellular morphology were evident and DNA was cleaved (Kuhn et al., 2002). In recent years, combining plant essential oils with antibiotics has emerged as a new strategy for combating microbial growth because it produces an additive effect or synergy (Shin and Kim, 2004; Shibata et al., 2009). In checkerboard titer tests, to evaluate the

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combination effects of mixing the oil with oxacillin against S. aureus strains CCARM0027 and CCARM3511, FICIs were 0.18 and 0.57, indicating an additive or synergistic effect. In the experiment with S. aureus CCARM3511, an oxacillin resistant strain, Artemisia oil fraction or vulgarone B in combination with oxacillin lowered the MIC of oxacillin eight or four times, which is below the susceptibility criterion of oxacillin (≥2 µg/mL) as specified by the Clinical and Laboratory Standards Institute, USA (2007). Thus, we confirmed that the antibiotic-susceptible and -resistant strains of the important human pathogens S. aureus, S. enteritidis and S. typhimurium are inhibited by essential oils of A. iwayomogi, especially vulgarone B. Furthermore, the resistance of S. aureus oxacillin-resistant strains to oxacillin could be modulated by adding vulgarone B or Artemisia oils. The mechanism of vulgarone B activity might be related to its ability to cleave DNA and potentially cause cell leakage. Further investigation of other possible bactericidal mechanisms is underway.

ACKNOWLEDGEMENTS This study was supported by a grant (KOSEF; R012006-000-10732-0) from the Korea Science and Engineering Foundation.

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