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TRANSCRIPT
FOODMICROBIOLOGY
Food Microbiology 22 (2005) 139–144
ARTICLE IN PRESS
*Correspondi
2572.
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violettuna@simm
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doi:10.1016/j.fm
www.elsevier.nl/locate/jnlabr/yfmic
Short Communication
Volatile constituents from the leaves of Polygonum cuspidatumS. et Z. and their anti-bacterial activities
Yong-Suk Kima, Cheol-Seung Hwangb, Dong-Hwa Shinb,*aResearch Center for Industrial Development of BioFood Materials, Chonbuk National University, Republic of Korea
bFaculty 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 andthe growth of foodborne pathogens, and to increase theshelf-lives of foods. Currently, many food additives, i.e.,benzoic acid and sorbic acid are commercially producedfor the food industry (Cherry, 1999). Although thesesynthetic preservatives are effective, they can be detri-mental to human health (Cho et al., 1995), andconsequently an increasing number of consumersrequired food products, which are preservative free orcontain only trace amounts.
Volatile plant extracts commonly used for anti-bacterial tests are usually produced by simultaneoussteam distillation and solvent extraction (SDE). More-over, it has been found that the essential oils of Picea
ng author. Tel.: +82-63-270-2570; fax: +82-63-270-
sses: [email protected] (Y.-S. Kim),
ani.com (C.-S. Hwang),
honbuk.ac.kr (D.-H. Shin).
front matter r 2004 Elsevier Ltd. All rights reserved.
.2004.01.016
excelsa (Canillac and Mourey, 2001), oregano (Skanda-mis et al., 2002), and mustard seeds (Kim et al., 2001)have anti-bacterial activities upon putrefactive andpathogenic bacteria. The bactericidal effect of allylisothiocyanate on Listeria monocytogenes has also beeninvestigated (Ahn et al., 2001).
Polygonum cuspidatum S. et Z. belongs to thePolygonaceae perennial herb group found in Korea,Japan, Taiwan and China. In its dried form, it iscommonly known as knotweed root (Park and Lee,2000). The roots of the P. cuspidatum are used as alaxative, a diuretic, and for the treatment of suppurativedermatitis in the oriental medicine (Chi et al., 1982). Inaddition, the powder of the dried roots has been used inAsia to treat atherosclerosis (Kimura et al., 1985), andother medical ailments, such as coughs, asthma,hypertension, and cancer (Su et al., 1995). And, thecytotoxicity of P. cuspidatum have been tested on HL-60cells (Yeh et al., 1988).
The active compounds of P. cuspidatum havebeen identified as 2-methoxy-6-acetyl-7-methyljuglone(Kimura et al., 1983), emodin (Jayasuriya et al., 1992),
ARTICLE IN PRESSY.-S. Kim et al. / Food Microbiology 22 (2005) 139–144140
polygonin, emodinmono ethylether, and chrysophano(Park and Lee, 2000). Glycosides, reynoutrin, avicularinand 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 isknown to inhibit the growth of Staphylococcus aureus, a,and b-Streptococcus, Escherichia coli and Pseudomonas
aeruginosa. In addition, the taste of P. cuspidatum hasbeen reported to be bitter and its toxicity to be low (Parkand Lee, 2000).
The roots of the P. cuspidatum have been used as anagent for medicinal purposes for centuries, but studieson the anti-bacterial effects of the essential oil from itsleaves are scarce. As part of an ongoing investigationinto the potential medical uses of traditional remedies,we undertook this study to extract the essential oil of P.
cuspidatum leaves under different conditions, to identifythe volatile present, and to conduct tests upon their anti-bacterial activities, with a view towards increasing theshelf-life of foods without artificial preservatives.
2. Materials and methods
2.1. Micro-organisms and cultures
Six different types of foodborne micro-organismswere used in this research. Bacillus cereus (ATCC 11778)and Salmonella Typhimurium (ATCC 14028) strainswere 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 SoyBroth or Tryptic Soy Agar (Difco, Detroit, MI, USA).Vibrio parahaemolyticus (ATCC 33844) strain wasgrown at 37�C in Tryptic Soy Broth or Tryptic SoyAgar supplemented with 3% (w/v) NaCl. The bacteriawere grown for 24 h in sterilized broth medium. Aportion of each culture (0.1ml) was transferred into newbroth medium 9.9ml and grown for 18 h for the anti-bacterial experiments.
2.2. Extraction of volatile components
The leaves of P. cuspidatum S. et Z. were collectedfrom Jeonju Arboretum (Jeonju, Korea) in September,2001, and washed and stored at �20�C. Extracts of thevolatile compounds in the leaves were obtained bysimultaneous steam distillation and solvent extraction(SDE) method using the ‘improved’ Likens–Nickersonunit (Parliment, 1997). After circulating 50ml of theextracting solvent (redistilled diethyl ether) through theapparatus at 36�C, 100 g of the leaves ground using aWaring blender (Waring, New Hartford, Connecticut,USA) and added to 1000ml of distilled water in a
round-flask. The mixture was heated at 100�C underdifferent conditions, as follows. The extraction condi-tion used for P. cuspidatum tree leaves were varied byusing 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. Anhydroussodium sulfate (about 5 g) was added, and incubated for12 h at �20�C. The mix was then concentrated to 1mlunder a nitrogen flow. The concentrates obtained wereused to identify the volatile components and to test foranti-bacterial activity. As internal standard, 10 ml of1-pentanol (n-amyl alcohol) was added to the extracts asan internal gas chromatography (GC) standard.
2.3. Analysis and identification of the volatile constituents
GC (GC-17A V3) and GC-MS (QP5050, ShimadzuCo., Kyoto, Japan); both using a Supelcowax 10 fusedsilica capillary column (60m� 0.25mm; 0.25 mm filmthickness); were used for this purpose. Helium was usedas a carrier gas at a flow rate of 1ml/min. The oventemperature was maintained at 50�C for 5min and thenincreased to 230�C at rate of 2�C/min, and then held for10min. The temperature of the injector was 250�C andof the flame ionization detector, 260�C. The split ratioused was 1:60 and a volume of 0.5 ml of extracts wasinjected for each run. The mass spectra ranged from 28to 400 m=e; and the ionizing voltage was 70 eV. Detectedcomponents were identified by comparing the spectraobtained with a mass spectrum library (Wiley NBS 139),and by comparing GC retention indices versus those ofusing known standards.
2.4. Anti-bacterial activities of SDE extracts
P. cuspidatum leaf extract (5.74 ml from 100 g ofleaves) of was obtained after completely removing thediethyl ether portion under a nitrogen atmosphere, and10% (v/v) Tween 80 (Showa Chemical Co. Ltd., Tokyo,Japan) in water was added up to a volume of 0.5ml. Theextract was then sterilized by passing it through amembrane filter (0.2 mm) (Kim et al., 1995; Naigre et al.,1996). To determine its anti-bacterial activity, 5.82ml ofmedia was supplemented with 0.12ml of the sterilizedleaf extract or with 10% Tween 80 as a control, and theninoculated with 0.06ml (105–106 cfu/ml) of each bacterialstrain. The concentration of the dispersion was adjustedto 2%, 4%, 8% or 10% (v/v) of the extract strength with10% Tween 80 to produce 6.00ml of total mixture.Aliquots of these cultures (0.3ml) were dispensed intothe Bioscreen C (Labsystem, Helsinki, Finland) wellsand incubated as described above for the respectivebacterial strains. Optical densities (600 nm) were mea-sured every 12 h for 3 days against the Tween 80 control.
ARTICLE IN PRESSY.-S. Kim et al. / Food Microbiology 22 (2005) 139–144 141
3. Results and discussion
3.1. Extraction times and anti-bacterial activities
The extracts of P. cuspidatum leaves strongly inhibitedthe growth of B. cereus and S. Typhimurium and theireffects increased with extraction time (Table 1). Thisresult concurs with a report by Seo et al. (1996), in whichthe anti-bacterial effect of hydrolysed mustard extractsalso increased with extraction time. However, theinhibitory effects of the extracts were weak on V.
parahaemolyticus and the other strains, though thegrowth of these strains also tended to decrease withextraction time. The anti-bacterial activity of extractsobtained using extraction times of 1.5 and 2.0 h weresimilar, therefore, in latter experiments an extractiontime of 1.5 h was selected. Previous reports indicate thata longer heat treatment can cause a chemical breakdownof the effective volatile components (Sides et al., 2000).
3.2. Changes in volatile components with extraction time
A total of 18 volatile compounds were identified in theextract of P. cuspidatum leaves at pH 4.5 (Table 2). Thenumbers and the types of the compounds were affectedby the extraction time, in which 13, 17, 17 and 18compounds were observed after 0.5, 1.0, 1.5 and 2.0 h.In addition, the concentrations of these compoundswere calculated to be 4.03, 5.45, 5.74 and 8.89 ml/100 g inextract, respectively. The concentration of the totalvolatiles after 2.0 h of extraction was nearly twice thatobtained at 0.5 h, and consequently, its anti-bacterialactivity also nearly doubled (Table 1). This finding is inagreement with previous work, in which the anti-bacterial effect of hydrolysed mustard extracts increasedwith extraction time (Seo et al., 1996) and the SDEextraction efficacy increased with time (Schultz et al.,1977).
The major volatile compound found in the extract ofP. cuspidatum leaves was 2-hexenal, which accounted for
Table 1
Anti-bacterial activity of volatile compounds from P. cuspidatum S. et Z. wi
Micro-organisms Extraction time
0.5 h
Bacillus cereus ATCC 11778 28.5a
Salmonella Typhimurium ATCC 14028 46.2
Vibrio parahaemolyticus ATCC 33844 11.7
Listeria monocytogenes ATCC 19111 16.4
Staphylococcus aureus ATCC 25923 11.0
Escherichia coli O157:H7 ATCC 43894 11.0
aGrowth inhibition rate (%)=100–(B=A� 100).
A: total area of growth curve of sample with only 10% Tween 80 by Bio
B: total area of growth curve of treated sample by Bioscreen C for 72 h
Values represent the mean of three replicates.
69.50–74.27% of the total peak areas. Other volatilecompounds identified were 3-hexen-1-ol (6.73–8.11%),n-hexanal (2.41–3.01%), 1-penten-3-ol (2.55–2.81%),and 2-penten-1-ol (2.21–2.91%). The level of 2-hexenalincreased with extraction time, in which 2.95, 4.05, 4.21and 6.18 ml/100 g were recorded when the leaves wereextracted for 0.5, 1.0, 1.5 and 2.0 h, respectively. Theantifungal activity of n-hexanal is well known (Hamilton-Kemp et al., 1996; Gardini et al., 1997), but it was foundin 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 bythe extracts, and this effect increased on reducing theextraction pH. However, this pH had no affect onbacterial growths of V. parahaemolyticus, L. monocyto-
genes, and S. aureus.
3.4. The effect of extraction pH on volatile compounds
Different extractions pHs produced extracts withdifferent numbers of compounds as shown in Table 4.A total of 18, 17, 15 and 17 compounds were found inthe extracts obtained at pHs of 3.5, 4.5 (control), 5.5 and6.5, respectively, and the concentrations of thosecompounds were 5.30, 5.74, 3.63 and 3.27 ml/100 g.Therefore, as the alkalinity of the extraction wasincreased, the total quantity extracted decreased.
The maximal area of the main volatile compound, 2-hexenal, was 73.36% for P. cuspidatum leaves extractedat pH 4.5, and 71.78%, 72.25%, and 65.41% for leavesextracted at pH 3.5, 5.5 and 6.5, respectively. However,the levels of n-hexanal, trans-2-heptenal, 2-penten-1-oland 2-hexen-1-ol were unaffected by the extraction pH,whereas, the level of 3-hexen-1-ol tended to increasewith increasing pH, thus the extraction efficacy of eachcompound appears to be affected by pH.
th the extraction time of SDE
(pH 4.5)
1.0 h 1.5 h 2.0 h
35.6 49.8 53.7
50.2 69.1 68.7
12.4 12.0 16.2
19.0 21.8 24.8
13.9 16.1 24.2
11.2 15.0 16.6
screen C for 72 h incubation.
incubation.
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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 IMc
0.5 h 1.0 h 1.5 h 2.0 h
1 Acetaldehyde 444 0.81 0.75 0.80 0.49 A, B
2 Ethyl acetate 619 0.30 0.31 0.33 0.26 A, B
3 Ethyl alcohol 702 0 0.64 0.92 0.63 A, B
4 2-ethyl furan 758 0 0.68 0.79 0.70 A, B
5 n-Valeraldehyde 830 0.60 0.51 0.77 0.66 A, B
6 Ethyl vinyl ketone 959 0.37 1.25 1.13 1.16 A, B
7 Toluene 1038 0 0.28 0.28 0.22 A, B
8 n-Hexanal 1208 2.41 3.01 2.81 2.93 A, B
9 2-pentenal 1447 0.30 0.51 0.51 0.51 A, B
10 2-methyl-4-pentenal 1491 0 0.26 0 0.22 A
11 1-penten-3-ol 1586 2.70 2.73 2.55 2.81 A, B
12 trans-2-Heptenal 1873 3.35 3.10 3.05 2.95 A
13 2-hexenal 1983 73.04 74.27 73.36 69.50 A, B
14 2-penten-1-ol 2695 2.50 2.27 2.21 2.91 A, B
15 n-Hexanol 2935 1.31 0.95 0.94 0.87 A, B
16 3-hexen-1-ol 3186 8.11 6.73 6.97 7.54 A, B
17 2-hexen-1-ol 3357 2.41 1.74 1.71 1.47 A, B
18 Durenol 8974 0 0 0.59 0.64 A
Total peak area (%) 98.21 99.99 99.72 96.47
Total amounts (ml/100 g) 4.03 5.45 5.74 8.89
aRI: retention index.bPeak area (%) on the gas chromatogram.c IM: identification mode. Components identified 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 pH 4.5 pH 5.5 pH 6.5
Bacillus cereus ATCC 11778 58.6a 49.8 30.7 32.6
Salmonella Typhimurium ATCC 14028 49.3 69.1 35.0 26.1
Vibrio parahaemolyticus ATCC 33844 10.8 12.0 8.5 7.3
Listeria monocytogenes ATCC 19111 13.6 21.8 15.8 12.1
Staphylococcus aureus ATCC 25923 13.6 16.1 14.5 15.8
E. coli O157:H7 ATCC 43894 17.8 15.0 10.1 10.3
Values represent the mean of three replicates.aSee footnotes in Table 1.
Y.-S. Kim et al. / Food Microbiology 22 (2005) 139–144142
In previous reports the yield of volatiles was found tobe affected by the dispersion media type (Ebeler et al.,1988) and extraction pH (Bredie et al., 2002). Inaddition, in common with our results, Schultz et al.(1977) reported that the extraction pH for SDE did notaffect the extraction efficacy of the most volatilecompounds without linalool and citonellal at pH 3.4were 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. Itgrowth was inhibited completely by 10% of the extract(Fig. 1). The growth of S. Typhimurium was inhibitedby increasing the extract dose from 2%, 4% to 8%, butwas only completely inhibited by 10% for up to 60 h.The growth of V. parahaemolyticus was slightly inhib-ited at 2% and 4%, and the 10% extract inhibitedgrowth 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 leafextract is high. When purchased 2-hexenal was treatedat 1000 ppm, which corresponded to same quantity of P.
cuspidatum extract, the growths of all tested micro-organisms were inhibited by more than 97%. Thus,
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0.00 12 24 36 48 60 72
0.2
0.4
0.6
0.8
1.0
1.2
Incubation time (h)
0 12 24 36 48 60 72Incubation time (h)
0 12 24 36 48 60 72Incubation time (h)
0 12 24 36 48 60 72Incubation time (h)
0 12 24 36 48 60 72Incubation time (h)
0 12 24 36 48 60 72Incubation time (h)
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Vibrio parahaemolyticus ATCC 33844
Bacillus cereus ATCC 11778 Salmonella Typhimurium ATCC 14028
Listeria monocytogenes ATCC 19111
Staphylococcus aureus ATCC 25923 Escherichia coli O157:H7 ATCC 43894
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 (%) IM
pH 3.5 pH 4.5 pH 5.5 pH 6.5
1 Acetaldehyde 444 0.38 0.80 0.80 0.83 A, B
2 Ethyl acetate 619 0.26 0.33 0.41 0.46 A, B
3 Ethyl alcohol 702 1.76 0.92 0.96 3.03 A, B
4 2-ethyl furan 758 0.51 0.79 1.13 1.19 A, B
5 n-Valeraldehyde 830 0.60 0.77 0 1.62 A, B
6 Ethyl vinyl ketone 959 1.51 1.13 0.41 1.10 A, B
7 Toluene 1038 0.32 0.28 0 0.46 A, B
8 n-Hexanal 1208 2.80 2.81 3.17 2.66 A, B
9 2-pentenal 1447 0.76 0.51 0.41 0.40 A, B
10 2-methyl-4-pentenal 1491 0.25 0 0 0 A
11 1-penten-3-ol 1586 2.66 2.55 2.92 3.09 A, B
12 trans-2-Heptenal 1873 3.34 3.05 3.66 3.06 A
13 2-hexenal 1983 71.78 73.36 72.25 65.41 A, B
14 2-penten-1-ol 2695 2.55 2.21 2.61 2.57 A, B
15 n-Hexanol 2935 1.13 0.94 0.99 1.22 A, B
16 3-hexen-1-ol 3186 6.44 6.97 7.71 10.18 A, B
17 2-hexen-1-ol 3357 1.95 1.71 1.65 2.08 A, B
18 Durenol 8974 1.00 0.59 0.36 0.64 A
Total peak area (%) 100 99.72 99.44 100
Total amounts (ml/100 g) 5.30 5.74 3.63 3.27
See footnotes in Table 2.
Values represent the mean of three replicates.
Y.-S. Kim et al. / Food Microbiology 22 (2005) 139–144 143
ARTICLE IN PRESSY.-S. Kim et al. / Food Microbiology 22 (2005) 139–144144
2-hexenal showed stronger anti-bacterial activity thanthat of the extract (data not shown). We presume thatthe anti-bacterial effect of the extract was interfered withby the other extract constituents.
When the essential oil of Melaleuca alternifolia wasadded to Candida, the lower concentration (o0.25%)were found to have higher anti-yeast effect, whereashigher concentrations did not show any effect (Hammeret al., 1998). Marino et al. (1999) reported that Gram-positive as S. aureus is more resistant to the essential oilfrom 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 effecton E. coli and on S. aureus in the presence of oreganoessential oil. These results indicate that the anti-bacterialactivity depends on the type of essential, which agreeswith our finding.
Acknowledgements
This research was supported by Research Center forIndustrial Development of Biofood Materials in Chon-buk National University, Chonju, Korea. The centeris designated as a Regional Research Center appointedby the Korea Science and Engineering Foundation(KOSEF), Jeollabuk-do Provincial Government andChonbuk National University.
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