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FOOD MICROBIOLOGY
Food Microbiology 22 (2005) 139144

www.elsevier.nl/locate/jnlabr/yfmic

Short Communication

Volatile constituents from the leaves of Polygonum cuspidatum S. et Z. and their anti-bacterial activities
Yong-Suk Kima, Cheol-Seung Hwangb, Dong-Hwa Shinb,*
b a Research Center for Industrial Development of BioFood Materials, Chonbuk National University, Republic of Korea Faculty of Biotechnology (Food Science & Technology Major), Chonbuk National University, Dukjin-Dong, Jeonju, Chonbuk 561-756, Republic of Korea

Received 27 August 2003; accepted 26 January 2004

Abstract Volatile substances extracted from leaves of Polygonum cuspidatum S. et Z. by simultaneous steam distillation and solvent extraction (SDE) were investigated in terms of their inhibitory activities against six foodborne micro-organisms using Bioscreen C. The SDE extracts from P. cuspidatum S. et Z. obtained after 1.5 or 2.0 h at pH 4.5 exhibited strong growth inhibition upon the six micro-organisms tested; their volatile contents were 5.74 and 8.89 ml/100 g, respectively. Anti-bacterial activities against the bacterial strains examined increased upon reducing SDE pH from 6.5 to 3.5 and by increasing the extraction time from 0.5 to 2.0 h. The major volatile components of the SDE extracts obtained after 1.5 h at pH 4.5 were 2-hexenal (73.36%), 3-hexen-1-ol (6.97%), n-hexanal (2.81%), 1-penten-3-ol (2.55%), 2-penten-1-ol (2.21%), and ethyl vinyl ketone (1.13%) by Gas chromatography. The addition of 10% (v/v) of the SDE extracts to broth completely inhibited the growth of Bacillus cereus and of Vibrio parahaemolyticus for 72 h. r 2004 Elsevier Ltd. All rights reserved.
Keywords: Polygonum cuspidatum S. et Z.; Anti-bacterial; Volatile constituents; Simultaneous steam distillation and solvent extraction; Foodborne micro-organisms

1. Introduction Preservatives are designed to prevent food decay and the growth of foodborne pathogens, and to increase the shelf-lives of foods. Currently, many food additives, i.e., benzoic acid and sorbic acid are commercially produced for the food industry (Cherry, 1999). Although these synthetic preservatives are effective, they can be detrimental to human health (Cho et al., 1995), and consequently an increasing number of consumers required food products, which are preservative free or contain only trace amounts. Volatile plant extracts commonly used for antibacterial tests are usually produced by simultaneous steam distillation and solvent extraction (SDE). Moreover, it has been found that the essential oils of Picea
*Corresponding author. Tel.: +82-63-270-2570; fax: +82-63-2702572. E-mail addresses: dearkim@prumail.co.kr (Y.-S. Kim), violettuna@simmani.com (C.-S. Hwang), dhshin@moak.chonbuk.ac.kr (D.-H. Shin). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.01.016

excelsa (Canillac and Mourey, 2001), oregano (Skandamis et al., 2002), and mustard seeds (Kim et al., 2001) have anti-bacterial activities upon putrefactive and pathogenic bacteria. The bactericidal effect of allyl isothiocyanate on Listeria monocytogenes has also been investigated (Ahn et al., 2001). Polygonum cuspidatum S. et Z. belongs to the Polygonaceae perennial herb group found in Korea, Japan, Taiwan and China. In its dried form, it is commonly known as knotweed root (Park and Lee, 2000). The roots of the P. cuspidatum are used as a laxative, a diuretic, and for the treatment of suppurative dermatitis in the oriental medicine (Chi et al., 1982). In addition, the powder of the dried roots has been used in Asia to treat atherosclerosis (Kimura et al., 1985), and other medical ailments, such as coughs, asthma, hypertension, and cancer (Su et al., 1995). And, the cytotoxicity of P. cuspidatum have been tested on HL-60 cells (Yeh et al., 1988). The active compounds of P. cuspidatum have been identied as 2-methoxy-6-acetyl-7-methyljuglone (Kimura et al., 1983), emodin (Jayasuriya et al., 1992),

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polygonin, emodinmono ethylether, and chrysophano (Park and Lee, 2000). Glycosides, reynoutrin, avicularin and hyperin were found in its the leaves (Huang, 1993), and mainly stilbenes (Vastano et al., 2000) in its roots. The hot-water extract of P. cuspidatum rhizome is known to inhibit the growth of Staphylococcus aureus, a, and b-Streptococcus, Escherichia coli and Pseudomonas aeruginosa. In addition, the taste of P. cuspidatum has been reported to be bitter and its toxicity to be low (Park and Lee, 2000). The roots of the P. cuspidatum have been used as an agent for medicinal purposes for centuries, but studies on the anti-bacterial effects of the essential oil from its leaves are scarce. As part of an ongoing investigation into the potential medical uses of traditional remedies, we undertook this study to extract the essential oil of P. cuspidatum leaves under different conditions, to identify the volatile present, and to conduct tests upon their antibacterial activities, with a view towards increasing the shelf-life of foods without articial preservatives.

round-ask. The mixture was heated at 100 C under different conditions, as follows. The extraction condition used for P. cuspidatum tree leaves were varied by using SDE times from 0.5, 1.0, 1.5, to 2.0 h at pH 4.5. The pH was then adjusted in all cases to 3.5, 4.5 (control), 5.5 and 6.5 and extracted for 1.5 h. Anhydrous sodium sulfate (about 5 g) was added, and incubated for 12 h at 20 C. The mix was then concentrated to 1 ml under a nitrogen ow. The concentrates obtained were used to identify the volatile components and to test for anti-bacterial activity. As internal standard, 10 ml of 1-pentanol (n-amyl alcohol) was added to the extracts as an internal gas chromatography (GC) standard.

2.3. Analysis and identication of the volatile constituents GC (GC-17A V3) and GC-MS (QP5050, Shimadzu Co., Kyoto, Japan); both using a Supelcowax 10 fused silica capillary column (60 m 0.25 mm; 0.25 mm lm thickness); were used for this purpose. Helium was used as a carrier gas at a ow rate of 1 ml/min. The oven temperature was maintained at 50 C for 5 min and then increased to 230 C at rate of 2 C/min, and then held for 10 min. The temperature of the injector was 250 C and of the ame ionization detector, 260 C. The split ratio used was 1:60 and a volume of 0.5 ml of extracts was injected for each run. The mass spectra ranged from 28 to 400 m=e; and the ionizing voltage was 70 eV. Detected components were identied by comparing the spectra obtained with a mass spectrum library (Wiley NBS 139), and by comparing GC retention indices versus those of using known standards.

2. Materials and methods 2.1. Micro-organisms and cultures Six different types of foodborne micro-organisms were used in this research. Bacillus cereus (ATCC 11778) and Salmonella Typhimurium (ATCC 14028) strains were grown at 30 C in Nutrient Broth or Nutrient Agar (Oxoid Ltd., Basingstoke, Hampshire, England). E. coli O157:H7 (ATCC 43894) and Staphylococcus aureus (ATCC 25923) were was grown at 37 C, and Listeria monocytogenes (ATCC 19111) at 30 C in Tryptic Soy Broth or Tryptic Soy Agar (Difco, Detroit, MI, USA). Vibrio parahaemolyticus (ATCC 33844) strain was grown at 37 C in Tryptic Soy Broth or Tryptic Soy Agar supplemented with 3% (w/v) NaCl. The bacteria were grown for 24 h in sterilized broth medium. A portion of each culture (0.1 ml) was transferred into new broth medium 9.9 ml and grown for 18 h for the antibacterial experiments. 2.2. Extraction of volatile components The leaves of P. cuspidatum S. et Z. were collected from Jeonju Arboretum (Jeonju, Korea) in September, 2001, and washed and stored at 20 C. Extracts of the volatile compounds in the leaves were obtained by simultaneous steam distillation and solvent extraction (SDE) method using the improved LikensNickerson unit (Parliment, 1997). After circulating 50 ml of the extracting solvent (redistilled diethyl ether) through the apparatus at 36 C, 100 g of the leaves ground using a Waring blender (Waring, New Hartford, Connecticut, USA) and added to 1000 ml of distilled water in a

2.4. Anti-bacterial activities of SDE extracts P. cuspidatum leaf extract (5.74 ml from 100 g of leaves) of was obtained after completely removing the diethyl ether portion under a nitrogen atmosphere, and 10% (v/v) Tween 80 (Showa Chemical Co. Ltd., Tokyo, Japan) in water was added up to a volume of 0.5 ml. The extract was then sterilized by passing it through a membrane lter (0.2 mm) (Kim et al., 1995; Naigre et al., 1996). To determine its anti-bacterial activity, 5.82 ml of media was supplemented with 0.12 ml of the sterilized leaf extract or with 10% Tween 80 as a control, and then inoculated with 0.06 ml (105106 cfu/ml) of each bacterial strain. The concentration of the dispersion was adjusted to 2%, 4%, 8% or 10% (v/v) of the extract strength with 10% Tween 80 to produce 6.00 ml of total mixture. Aliquots of these cultures (0.3 ml) were dispensed into the Bioscreen C (Labsystem, Helsinki, Finland) wells and incubated as described above for the respective bacterial strains. Optical densities (600 nm) were measured every 12 h for 3 days against the Tween 80 control.

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3. Results and discussion 3.1. Extraction times and anti-bacterial activities The extracts of P. cuspidatum leaves strongly inhibited the growth of B. cereus and S. Typhimurium and their effects increased with extraction time (Table 1). This result concurs with a report by Seo et al. (1996), in which the anti-bacterial effect of hydrolysed mustard extracts also increased with extraction time. However, the inhibitory effects of the extracts were weak on V. parahaemolyticus and the other strains, though the growth of these strains also tended to decrease with extraction time. The anti-bacterial activity of extracts obtained using extraction times of 1.5 and 2.0 h were similar, therefore, in latter experiments an extraction time of 1.5 h was selected. Previous reports indicate that a longer heat treatment can cause a chemical breakdown of the effective volatile components (Sides et al., 2000). 3.2. Changes in volatile components with extraction time

69.5074.27% of the total peak areas. Other volatile compounds identied were 3-hexen-1-ol (6.738.11%), n-hexanal (2.413.01%), 1-penten-3-ol (2.552.81%), and 2-penten-1-ol (2.212.91%). The level of 2-hexenal increased with extraction time, in which 2.95, 4.05, 4.21 and 6.18 ml/100 g were recorded when the leaves were extracted for 0.5, 1.0, 1.5 and 2.0 h, respectively. The antifungal activity of n-hexanal is well known (HamiltonKemp et al., 1996; Gardini et al., 1997), but it was found in small amounts (Table 2) in P. cuspidatum. 3.3. The effect of extraction pH on anti-bacterial activity As shown in Table 3, the bacterial growths of B. cereus and S. Typhimurium were strongly inhibited by the extracts, and this effect increased on reducing the extraction pH. However, this pH had no affect on bacterial growths of V. parahaemolyticus, L. monocytogenes, and S. aureus. 3.4. The effect of extraction pH on volatile compounds

A total of 18 volatile compounds were identied in the extract of P. cuspidatum leaves at pH 4.5 (Table 2). The numbers and the types of the compounds were affected by the extraction time, in which 13, 17, 17 and 18 compounds were observed after 0.5, 1.0, 1.5 and 2.0 h. In addition, the concentrations of these compounds were calculated to be 4.03, 5.45, 5.74 and 8.89 ml/100 g in extract, respectively. The concentration of the total volatiles after 2.0 h of extraction was nearly twice that obtained at 0.5 h, and consequently, its anti-bacterial activity also nearly doubled (Table 1). This nding is in agreement with previous work, in which the antibacterial effect of hydrolysed mustard extracts increased with extraction time (Seo et al., 1996) and the SDE extraction efcacy increased with time (Schultz et al., 1977). The major volatile compound found in the extract of P. cuspidatum leaves was 2-hexenal, which accounted for

Different extractions pHs produced extracts with different numbers of compounds as shown in Table 4. A total of 18, 17, 15 and 17 compounds were found in the extracts obtained at pHs of 3.5, 4.5 (control), 5.5 and 6.5, respectively, and the concentrations of those compounds were 5.30, 5.74, 3.63 and 3.27 ml/100 g. Therefore, as the alkalinity of the extraction was increased, the total quantity extracted decreased. The maximal area of the main volatile compound, 2hexenal, was 73.36% for P. cuspidatum leaves extracted at pH 4.5, and 71.78%, 72.25%, and 65.41% for leaves extracted at pH 3.5, 5.5 and 6.5, respectively. However, the levels of n-hexanal, trans-2-heptenal, 2-penten-1-ol and 2-hexen-1-ol were unaffected by the extraction pH, whereas, the level of 3-hexen-1-ol tended to increase with increasing pH, thus the extraction efcacy of each compound appears to be affected by pH.

Table 1 Anti-bacterial activity of volatile compounds from P. cuspidatum S. et Z. with the extraction time of SDE Micro-organisms Extraction time (pH 4.5) 0.5 h Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028 Vibrio parahaemolyticus ATCC 33844 Listeria monocytogenes ATCC 19111 Staphylococcus aureus ATCC 25923 Escherichia coli O157:H7 ATCC 43894
a

1.0 h 35.6 50.2 12.4 19.0 13.9 11.2

1.5 h 49.8 69.1 12.0 21.8 16.1 15.0

2.0 h 53.7 68.7 16.2 24.8 24.2 16.6

28.5 46.2 11.7 16.4 11.0 11.0

Growth inhibition rate (%)=100(B=A 100). A: total area of growth curve of sample with only 10% Tween 80 by Bioscreen C for 72 h incubation. B: total area of growth curve of treated sample by Bioscreen C for 72 h incubation. Values represent the mean of three replicates.

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142 Y.-S. Kim et al. / Food Microbiology 22 (2005) 139144 Table 2 Volatile components of P. cuspidatum S. et Z. with the extraction time of the SDE at pH 4.5 Peak no. Component RIa Peak area (%)b 0.5 h 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Acetaldehyde Ethyl acetate Ethyl alcohol 2-ethyl furan n-Valeraldehyde Ethyl vinyl ketone Toluene n-Hexanal 2-pentenal 2-methyl-4-pentenal 1-penten-3-ol trans-2-Heptenal 2-hexenal 2-penten-1-ol n-Hexanol 3-hexen-1-ol 2-hexen-1-ol Durenol Total peak area (%) Total amounts (ml/100 g)
a b

IMc 1.0 h 0.75 0.31 0.64 0.68 0.51 1.25 0.28 3.01 0.51 0.26 2.73 3.10 74.27 2.27 0.95 6.73 1.74 0 99.99 5.45 1.5 h 0.80 0.33 0.92 0.79 0.77 1.13 0.28 2.81 0.51 0 2.55 3.05 73.36 2.21 0.94 6.97 1.71 0.59 99.72 5.74 2.0 h 0.49 0.26 0.63 0.70 0.66 1.16 0.22 2.93 0.51 0.22 2.81 2.95 69.50 2.91 0.87 7.54 1.47 0.64 96.47 8.89 A, A, A, A, A, A, A, A, A, A A, A A, A, A, A, A, A B B B B B B B B B B B B B B B

444 619 702 758 830 959 1038 1208 1447 1491 1586 1873 1983 2695 2935 3186 3357 8974

0.81 0.30 0 0 0.60 0.37 0 2.41 0.30 0 2.70 3.35 73.04 2.50 1.31 8.11 2.41 0 98.21 4.03

RI: retention index. Peak area (%) on the gas chromatogram. c IM: identication mode. Components identied by gas chromatography-MS are designated as A, and by retention index of authentic compounds are designated as B. Values represent the mean of three replicates.

Table 3 Anti-bacterial activity of volatile compounds from P. cuspidatum S. et Z. with the SDE pH Micro-organisms Extraction pH (1.5 h) pH 3.5 Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028 Vibrio parahaemolyticus ATCC 33844 Listeria monocytogenes ATCC 19111 Staphylococcus aureus ATCC 25923 E. coli O157:H7 ATCC 43894 Values represent the mean of three replicates. a See footnotes in Table 1. 58.6 49.3 10.8 13.6 13.6 17.8
a

pH 4.5 49.8 69.1 12.0 21.8 16.1 15.0

pH 5.5 30.7 35.0 8.5 15.8 14.5 10.1

pH 6.5 32.6 26.1 7.3 12.1 15.8 10.3

In previous reports the yield of volatiles was found to be affected by the dispersion media type (Ebeler et al., 1988) and extraction pH (Bredie et al., 2002). In addition, in common with our results, Schultz et al. (1977) reported that the extraction pH for SDE did not affect the extraction efcacy of the most volatile compounds without linalool and citonellal at pH 3.4 were 73% and 59%, respectively, and 100% at pH. 6.5. 3.5. The effect of extract concentration on anti-bacterial activity The growth of B. cereus was slightly inhibited by 2% of the extract of P. cuspidatum, and the addition of 4%

of the extract inhibited its growth for up to 36 h. It growth was inhibited completely by 10% of the extract (Fig. 1). The growth of S. Typhimurium was inhibited by increasing the extract dose from 2%, 4% to 8%, but was only completely inhibited by 10% for up to 60 h. The growth of V. parahaemolyticus was slightly inhibited at 2% and 4%, and the 10% extract inhibited growth for up to 72 h. However, the growth of E. coli O157:H7 was not inhibited even by 10% of the extract, demonstrating that its resistance to P. cuspidatum leaf extract is high. When purchased 2-hexenal was treated at 1000 ppm, which corresponded to same quantity of P. cuspidatum extract, the growths of all tested microorganisms were inhibited by more than 97%. Thus,

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Bacillus cereus ATCC 11778 Optical density (600 nm) Optical density (600 nm) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 60 Incubation time (h) 72 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 60 Incubation time (h) 72 Salmonella Typhimurium ATCC 14028

143

Vibrio parahaemolyticus ATCC 33844 Optical density (600 nm) 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 60 Incubation time (h) 72 Optical density (600 nm) 1.2 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

Listeria monocytogenes ATCC 19111

12

24 36 48 60 Incubation time (h)

72

Staphylococcus aureus ATCC 25923 Optical density (600 nm) Optical density (600 nm) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 60 Incubation time (h) 72 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Escherichia coli O157:H7 ATCC 43894

12

24 36 48 60 Incubation time (h)

72

Fig. 1. Anti-bacterial activity of compounds obtained by SDE method for 1.5 h at pH 4.5 from P. cuspidatum S. et Z. against several foodborne micro-organisms. Table 4 Volatile components of P. cuspidatum S. et Z. with the extraction pH in the SDE for 1.5 h Peak no. Component RI Peak area (%) pH 3.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Acetaldehyde Ethyl acetate Ethyl alcohol 2-ethyl furan n-Valeraldehyde Ethyl vinyl ketone Toluene n-Hexanal 2-pentenal 2-methyl-4-pentenal 1-penten-3-ol trans-2-Heptenal 2-hexenal 2-penten-1-ol n-Hexanol 3-hexen-1-ol 2-hexen-1-ol Durenol Total peak area (%) Total amounts (ml/100 g) See footnotes in Table 2. Values represent the mean of three replicates. 444 619 702 758 830 959 1038 1208 1447 1491 1586 1873 1983 2695 2935 3186 3357 8974 0.38 0.26 1.76 0.51 0.60 1.51 0.32 2.80 0.76 0.25 2.66 3.34 71.78 2.55 1.13 6.44 1.95 1.00 100 5.30 pH 4.5 0.80 0.33 0.92 0.79 0.77 1.13 0.28 2.81 0.51 0 2.55 3.05 73.36 2.21 0.94 6.97 1.71 0.59 99.72 5.74 pH 5.5 0.80 0.41 0.96 1.13 0 0.41 0 3.17 0.41 0 2.92 3.66 72.25 2.61 0.99 7.71 1.65 0.36 99.44 3.63 pH 6.5 0.83 0.46 3.03 1.19 1.62 1.10 0.46 2.66 0.40 0 3.09 3.06 65.41 2.57 1.22 10.18 2.08 0.64 100 3.27 A, A, A, A, A, A, A, A, A, A A, A A, A, A, A, A, A B B B B B B B B B B B B B B B IM

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144 Y.-S. Kim et al. / Food Microbiology 22 (2005) 139144 tion of menthone and isoamyl acetate. J. Agric. Food Chem. 36, 791796. Gardini, F., Lanciotti, R., Caccioni, D.R.L., Guerzoni, M.E., 1997. Antifungal activity of hexanal as dependent on its pressure. J. Agric. Food Chem. 45, 42974302. Hamilton-Kemp, T.R., Archbold, D.D., Loughrin, J.H., Collins, R.W., Byers, M.E., 1996. Metabolism of natural volatile compounds by strawberry fruit. J. Agric. Food Chem. 44, 28022805. Hammer, K.A., Carson, C.F., Riley, T.V., 1998. In-vitro activity of essential oil, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J. Antimicrob. Chemother. 42, 591595. Huang, K.C., 1993. The Pharmacology of Chinese Herbs. CRC Press, Boca Raton, FL. USA, pp. 295296. Jayasuriya, H., Koonchanok, N.M., Geahlen, R.L., McLaughlin, J.L., Chang, C.J., 1992. Emodin, a protein tyrosine kinase inhibitor from Polygonum cuspidatum. J. Nat. Prod. 55, 696698. Kim, J.M., Marshall, M.R., Wei, C.I., 1995. Antibacterial activity of some essence oil component against ve food borne pathogens. J. Agric. Food Chem. 43, 28392845. Kim, Y.S., Park, S.B., Lee, J.Y., Kim, Y.H., Shin, D.H., 2001. Volatile compounds and antimicrobial effects of mustard seeds and leaf mustard seeds according to extraction method. Food Sci. Biotechnol. 10, 468474. Kimura, Y., Kozawa, M., Baba, K., Hata, K., 1983. New constituents of roots of Polygonum cuspidatum. Plant Med. 48, 164168. Kimura, Y., Okuda, H., Arachi, S., 1985. Effects of stilbene on arachidonate metabolism in leukocytes. Biochim. Biophys. Acta 834, 275278. Marino, M., Bersani, C., Comi, G., 1999. Antimicrobial activity of the essential oil of Thymus vulgaris L. measured using a bioimpedometric method. J. Food Prot. 62, 10171023. Naigre, R., Kalck, P., Roques, C., Roux, I., Michel, G., 1996. Comparison of antimicrobial properties of monoterpenes and their carbonylated products. Planta Med. 62, 275277. Park, J.H., Lee, J.K., 2000. The Encyclopedia of Medicinal Plants. Shinil Books Co., Ltd, Seoul, Korea. Parliment, T.H., 1997. Solvent extraction and distillation techniques. In: Marsili, R. (Ed.), Techniques for Analyzing Food Aroma. Marcel Dekker, Inc, New York, USA, pp. 126. Schultz, T.H., Flath, R.A., Mon, R., Eggling, S.B., Teranishi, R., 1977. Isolation of volatile components from a model system. J. Agric. Food Chem. 25, 446449. Seo, K.I., Park, S.K., Park, J.R., Kim, H.C., Choi, J.S., Shim, K.H., 1996. Changes in antimicrobial activity of hydrolyzate from mustard seed (Brassica juncea). J. Korean Soc. Food Nutr. 25, 129134. Sides, A., Robards, K., Helliwell, S., 2000. Developments in extraction techniques and their application to analysis of volatiles in foods. Trends Anal. Chem. 19, 322329. Skandamis, P., Tsigarida, E., Nychas, G.-J.E., 2002. The effect of oregano essential oil on survival/death of Salmonella typhimurium in meat stored at 5 C under aerobic, VP/MAP conditions. Food Microbiol. 19, 97103. Su, H.Y., Cherng, S.H., Chen, C.C., Lee, H., 1995. Emodin inhibits the mutagenicity and DNA adducts induced by 1-nitropyrene. Mutat. Res. 329, 205212. Vastano, B.C., Chen, Y., Zhu, N., Ho, C.T., Zhou, Z., Rosen, R.T., 2000. Isolation and identication of stilbenes in two varieties of Polygonum cuspidatum. J. Agric. Food Chem. 48, 253256. Yeh, S.F., Chou, T.C., Liu, T.S., 1988. Effects of anthraquinones of Polygonum cuspidatum on HL-60 cells. Planta Med. 54, 413414.

2-hexenal showed stronger anti-bacterial activity than that of the extract (data not shown). We presume that the anti-bacterial effect of the extract was interfered with by the other extract constituents. When the essential oil of Melaleuca alternifolia was added to Candida, the lower concentration (o0.25%) were found to have higher anti-yeast effect, whereas higher concentrations did not show any effect (Hammer et al., 1998). Marino et al. (1999) reported that Grampositive as S. aureus is more resistant to the essential oil from Thymus vulgaris L. than Gram-negative as E. coli O157:H7 or S. Typhimurium. Dorman and Deans (2000) reported that the essential oils of the pepper, clove or geranium showed a potent anti-bacterial effect on E. coli and on S. aureus in the presence of oregano essential oil. These results indicate that the anti-bacterial activity depends on the type of essential, which agrees with our nding.

Acknowledgements This research was supported by Research Center for Industrial Development of Biofood Materials in Chonbuk National University, Chonju, Korea. The center is designated as a Regional Research Center appointed by the Korea Science and Engineering Foundation (KOSEF), Jeollabuk-do Provincial Government and Chonbuk National University.

References
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