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Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb. to control food-borne pathogenic and spoilage bacteria
International Journal of Food Microbiology 125 (2008) 117–122
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb. to control food-borne pathogenic and spoilage bacteria Vivek K. Bajpai, Atiqur Rahman, Sun Chul Kang ⁎ Department of Biotechnology, College of Engineering, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea
A R T I C L E
I N F O
Article history: Received 4 February 2008 Received in revised form 15 March 2008 Accepted 20 March 2008 Keywords: Nandina domestica Thunb. Essential oil Mono phenol 3,5-dimethylpyrazole Food-borne and spoiling bacteria Antibacterial activity
A B S T R A C T The aim of this study was to examine the chemical composition of the essential oil isolated from the floral parts of Nandina domestica Thunb. by hydrodistillation, and to test the efficacy of essential oil and various organic extracts against a panel of food-borne pathogenic and spoilage bacteria such as Bacillus subtilis ATCC6633, Listeria monocytogenes ATCC19166, Staphylococcus aureus KCTC1916, S. aureus ATCC6538, Pseudomonas aeruginosa KCTC2004, Salmonella typhimurium KCTC2515, Salmonella enteridis KCCM12021, Escherichia coli 0157-Human, E. coli ATCC8739, E. coli 057:H7 ATCC43888 and Enterobacter aerognes KCTC2190. The chemical composition of essential oil was analysed by GC–MS. It was determined that 79 compounds, which represented 87.06% of total oil, were present in the oil. The oil contained mainly 1-indolizino carbazole (19.65%), 2-pentanone (16.4%), mono phenol (12.1%), aziridine (9.01%), methylcarbinol (4.6%), ethanone (3.3%), furfural (2.96%), 3,5-dimethylpyrazole (1.29%) and 2(5H)-furanone (1.32%). The oil (1000 ppm/disc), and various organic extracts of hexane, chloroform, ethyl acetate and methanol (1500 ppm/disc) exhibited promising antibacterial effect as a diameter of zones of inhibition (9–18 and 7–13 mm) and MIC values (62.5 to 1000 and 250 to 2000 μg/ml), respectively against the tested bacteria. Also the oil had strong detrimental effect on the viable count of the tested bacteria. These results indicate the potential efficacy of plant-based natural products such as essential oil and organic extracts of N. domestica to control food-borne pathogenic and spoilage bacteria. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The presence and growth of microorganisms in food may cause spoilage and result in a reduction in quality and quantity (Soliman and Badeaa, 2002). Food-borne illnesses associated with Listeria monocytogenes, Staphylococcus aureus, Escherichia coli 0157:H7 and Salmonella enteritidis present a major public health concern throughout the world (Hall, 1997). One of the two mechanisms determining how foodborne diseases are primarily caused, is by infection as a consequence of consuming foods contaminated with the growth of pathogenic microorganisms, such as bacteria, mould, viruses and parasites (Vattem et al., 2004). In addition to passive transfer of pathogens to food, active growth of a pathogen may also occur in foods, for instance because of improper storage, which leads to marked increases in microbial load (Madigan et al., 1997). For these reasons, microbial contamination of food still poses important public health and economic concerns for the human society. Plant secondary metabolites, such as essential oils and plant extracts (Tepe et al., 2004), are studied for their antimicrobial activities and most essential oils derived from plants are known to possess antibacterial, insecticidal, antifungal, acaricidal and cytotoxic activities ⁎ Corresponding author. Tel.: +82 53 850 6553; fax: +82 53 850 6559. E-mail address: [email protected] (S.C. Kang). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.011
(Faleiro et al., 1999). Therefore, they are intensely screened and applied in the fields of food microbiology, pharmacology, pharmaceutical botany, medical and clinical microbiology, phytopathology and food preservation (Daferera et al., 2000). Since ancient times the crude herbal extracts of aromatic plants have been in use for different purposes, such as food, drugs and perfumery (Heath, 1981). The essential oils are considered among the most important antimicrobial agents present in the plants, and may also have antioxidant and antiinflammatory activities. Volatile oils are a complex mixture of compounds, mainly monoterpenes, sesquiterpenes, and their oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols and oxides). Other volatile compounds include phenylpropenes and specific sulphur- or nitrogen-containing substances. Generally, the oil composition is a balance of various compounds, although in many species one constituent may prevail over all others (Cowan, 1999). In the recent decades, antimicrobial plant products have gained special interest because of the resistance to antibiotics that some microorganisms have acquired (Essawi and Srour, 2000), the increasing popular concern about the safety of food and the potential impact of synthetic additives on health (Reische et al., 1998). Nandina domestica (Heavenly bamboo or Sacred bamboo), is a suckering shrub in the Barberry family, Berberidaceae, it is a monotypic genus, with this species as its only member. It is native to eastern Asia from the Himalaya east to Japan. Despite the common name, it is not a
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bamboo at all. It is an erect shrub growing to 2 m tall, with numerous, usually unbranched stems growing from the roots. The leaves are evergreen (sometimes deciduous in colder areas). The young leaves in spring are brightly coloured pink to red before turning green; old leaves turn red or purple again before falling. The flowers are white, borne in early summer in conical clusters held well above the foliage. It is widely grown in gardens as an ornamental plant; over 60 cultivars have been named. It has become naturalised in parts of eastern North America. In the Southeastern United States it is considered by many as a pest due to its invasive nature. Some even refer to it as Nandina Megalomania or Hitler Bamboo for its unbridled aggression toward other plants, its propensity to conquer the entire yard without provocation, and its seeming immortality (Dirr, 1990). To the best of our knowledge and literary survey, there is no report available on chemical composition analysis of essential oil derived from N. domestica, and its antibacterial properties. Hence, efforts have been made, to investigate the role of essential oil and various organic extracts of N. domestica as an antibacterial potential. In the present investigation, we examined the chemical compositions of the essential oil isolated from the floral parts of N. domestica Thunb., and tested the efficacy of essential oil and various leaf extracts against a diverse range of food-borne pathogens and food spoilage bacteria. 2. Materials and methods 2.1. Plant material Two types of samples, flowers and leaves of N. domestica were collected from the local area of Kyoungsan, Republic of Korea, in May– June 2007 and initially identified by morphological features and the data base present in the library at the Department of Biotechnology by Prof. Man Kyu Huh. A voucher specimen number (NDU-0099) has been deposited in the herbarium of College of Engineering, Department of Biotechnology, Daegu University, Republic of Korea. 2.2. Isolation of the essential oil The floral parts of N. domestica were air-dried for two weeks. The air-dried plant material (200 g) was subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus. The oil was dried over anhydrous Na2SO4 and preserved in a sealed vial at 4 °C prior to further analysis. 2.3. Preparation of leaf extracts The air-dried leaves of N. domestica 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). The extraction process yielded in methanol (7.1 g), hexane (3.1 g), chloroform (4.6 g) and ethyl acetate (4.3 g) extracts. 2.4. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the essential oil was performed using a Shimadzu GC–MS (GC-17A) equipped with a ZB-1 MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm). For GC– MS detection, an electron ionization system with 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 °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 finally raised to 250 °C at 10 °C/min. Diluted samples (1/ 100, v/v, in methanol) of 1.0 μl were injected manually in the split less
mode. The relative percentage of the oil constituents was expressed as percentages by peak area normalization. Identification of components of the essential oil was based on GC retention time on a ZB-1 capillary column relative to 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). 2.5. Microorganisms In all, eleven bacterial pathogens including food spoilage bacteria, such as Pseudomonas aeruginosa KCTC2004 and Bacillus subtilis ATCC6633, and food-borne pathogenic bacteria, namely Enterobacter aerogenes KCTC2190, Salmonella typhimurium KCTC2515, E. coli ATCC8739, E. coli O157:H7 ATCC43888, E. coli O157:H7 (Human), L. monocytogenes ATCC19166, S. enteritidis KCCM 2021, S. aureus ATCC6538 and S. aureus KCTC1916, were obtained from the Korea Food and Drug Administration (KFDA), Daegu, Republic of Korea. Cultures of each bacterial strain were maintained on Luria broth (LB) agar medium at 4 °C. 2.6. 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 LB 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 away and the inoculum was allowed to dry for 5 min. To prepare the stock solution of the samples, the oil was dissolved in 5% dichloromethane, whereas the extracts (hexane, chloroform, ethyl acetate and methanol) were dissolved in their respective solvents. Then a Whatman No. 1 sterile filter paper disk (6 mm diameter) was impregnated with 5 μl essential oil, corresponding to 1000 ppm/disc, and 7.5 μl leaf extracts, corresponding to 1500 ppm/ disc, using a capillary micro-pipette. Negative controls were prepared using the same solvents employed to dissolve the samples. Standard reference antibiotics, streptomycin and tetracycline (10 μg/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 zones of inhibition using a vernier caliper against the tested bacteria. Each assay in this experiment was replicated three times. 2.7. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentrations (MICs) of essential oil and various leaf extracts of methanol, ethyl acetate, chloroform and hexane, were tested by a two-fold serial dilution method (Chandrasekaran and Venkatesalu, 2004). The tests of oil and leaf extracts of hexane, chloroform, ethyl acetate and methanol were dissolved in their respective solvents (5%), the solvents used for isolation of essential oil and for obtaining the various extracts, and were added to LB broth medium to get a final concentration of 4000 μg/ml, which were further serially diluted to achieve 2000, 1000, 500, 250 125, 62.5 and 31.25 μg/ml, respectively, where the final concentration of the solvent was 0.5%. 10 μl of standardized suspension of each tested organism (108 CFU/ml approximately) was transferred to each tube. The control tubes, containing only bacterial suspension, were incubated at 37 °C for 24 h. The lowest concentrations of the test samples, which did not show any growth of test organisms after macroscopic evaluation, were determined as MICs, which were expressed in μg/ml. 2.8. Effect of essential oil on viable counts of bacteria For viable counts, each of the tubes containing bacterial suspension (approximately 107 CFU/ml) of S. aureus KCTC 1916, B. subtilis ATCC 6633, S. aureus ATCC 6538, P. aeruginosa KCTC 2004 and L. mono-
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cytogenes ATCC19166 in LB broth medium was inoculated with the minimum inhibitory concentration of the essential oil in 10 ml LB broth, and kept at 37 °C (Shin et al., 2007). Samples for viable cell counts were taken out at 0, 20, 40, 60, 80, 100 and 140 min time intervals. Enumeration of viable counts on LB plates was monitored as followings: 0.1 ml sample of each treatment was diluted into buffer peptone water, there by diluting it 10-fold 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 same experimental condition as mentioned above. 2.9. Statistical analysis The data obtained for antibacterial activity of essential oil and various extracts were statistical analysed and mean values were calculated. A Student’s t-test was computed for the statistical significance of the results. 3. Results 3.1. Chemical composition of the essential oil GC–MS analyses of the oil led to the identification of 79 different components, representing 87.06% 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 contained a complex mixture consisting of mainly oxygenated mono- and sesqueterpenes, and mono- and sesqueterpene hydrocarbons. The major components in the oil detected were 1-indolizino carbazole (19.65%), 2-pentanone (16.4%), mono phenol (12.1%), azridine (9.01%), methylcarbinol (4.6%), ethanone (3.30%), furfural (2.96%), 1-hydroxy-4-methylbenzene (1.53%), 2(5H)furanone (1.32%) and 3,5-dimethylpyrazole (1.29%). In the present study, isovaleric acid (0.23%), α-toluenol (0.05%), suberon (0.45%), dihydrochavicol (0.16%), veltol (0.53%), brenzcatechin (0.74%) and fourrine (0.32%) were also found to be the minor components of N. domestica oil. 3.2. Antibacterial activity assay The in vitro antibacterial activities of essential oil and various leaf extracts (hexane, chloroform, ethyl acetate and methanol) of N. domestica, against the employed bacteria, were qualitatively and quantitatively assessed by the presence or absence of inhibition zones. According to the results given in Table 2, in all, eleven food-borne pathogens and spoilage bacteria, including four Gram-positive and seven Gram-negative bacteria, were tested for the evaluation of antibacterial potential of essential oil and various leaf extracts of methanol, ethyl acetate, chloroform and hexane. As shown in Table 2, the essential oil at 5 μl (1000 ppm/disc) exhibited potent inhibitory effect against all four Gram-positive (S. aureus KCTC1916, B. subtilis ATCC6633, S. aureus ATCC6538 and L. monocytogenes ATCC19166) and six Gram-negative bacteria (P. aeruginosa KCTC2004, E. coli ATCC8739, E. coli 0157 (human) E. aerogenes KCTC2190, S. typhimurium KCTC2515 and S. typhimurium KCTC12021), with their respective diameter zones of inhibition of 18, 11, 14, 12, 17, 12. 10, 12, 12 and 11 mm. Only one of the bacteria E. coli 0157:H7 ATCC43888 was found slight resistant to the oil at the used concentration with 9 mm diameter zone of inhibition. S. aureus KCTC1916 was found most inhibited bacterial pathogen by the essential oil. Also the methanol, ethyl acetate and chloroform extracts significantly inhibited the growth of some of the bacterial strains tested. However, chloroform and ethyl acetate extracts exhibited potential antibacterial effect against some of the Gram-positive and Gramnegative bacteria. The diameter zones of inhibition of chloroform and ethyl acetate extracts against the tested bacteria were found in the range from 7 to 13 and 9 to 13 mm, respectively (Table 2). Methanol
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Table 1 Chemical composition of volatile oil isolated by hydrodistillation from Nandina domestica Thunb. No.
Rt
Compound
Composition (%)
Method
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 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
0.9 1.0 1.1 1.4 1.7 1.8 1.9 2.0 2.12 2.19 2.3 2.4 2.5 2.7 2.9 3.0 3.07 3.1 3.4 3.5 3.7 3.9 3.98 4.0 4.1 4.4 4.46 4.6 4.8 4.9 5.0 5.2 5.3 5.4 5.6 5.9 6.0 6.1 6.17 6.4 6.5 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.7 8.0 8.1 8.3 8.5 8.8 9.1 9.3 9.4 9.5 9.7 9.9 10.1 10.3 10.4 10.6 10.7 10.9 11.4 11.6 11.8 11.9 12.2
Methylcarbinol 2-Pentanone 1-Indolizino carbazole 2-Butanone Propylene carbonate Propionic acid Tetrahydropyran Glyoxylic acid Imidazole 1.2-D2-Imidazole Aziridine Formic acid 2(3H)-Furanone 3-Pentanone Vinylcarbinol Propanoyl chloride 2-Aminooxazole 2,3-Dihydropyran 1-Methyltetrazole Furfural 3,5-Dimethylpyrazole Sorbaldehyde 2,4-Hexadien-1-o1 4-Pentenenitrile Pyrrolidine 2,4-Dimethylfuran Ethylidene-cyclopropane 4-Nonyne Octylazide Isovaleric acid Pyrazole Ketoisophrone Acetic acid 1,2-Propadiene Mono phenol 1-Nitrobutane 4-Chlorooctane Suberon Cresylic acid α-Toluenol 2-Methylphenol p-Oxytoluene p-Toluol 1-Hydoxy-4-methylbenzene Pyroguaiac acid Guaiacol Ethanone Leucine p-Hydoxyanisole 2,6-Dimethyl-1,7-octadiene-3-ol 2(5H)-Furanone Ortho-ethylphenol 3,4-Xylenol Dihydrochavicol Veltol Brenzcatechin Fourrine Phiaquin Benzohydroquinone Quinol dimethyl ether Orcinol 4-Methyl catechol Nicotinic acid 1-Furyl-1-ethoxy-ethanol Toluene Pyrazine Phenetole Trycarbonyl-1,3-cyclooctadiene Thynol Benzofuran 4-Ethylresorcinol Naphthalene 1,2,3,3,4-Pentamethyl cyclopentene
4.60 16.40 19.65 0.45 0.03 0.24 0.03 0.27 0.03 0.07 9.01 0.87 0.05 0.61 0.02 0.13 0.40 0.07 0.27 2.96 1.29 0.09 0.03 0.13 0.07 0.96 0.29 0.46 0.36 0.23 0.16 0.18 0.04 0.23 12.10 0.31 0.17 0.45 0.77 0.05 0.33 0.08 0.09 1.53 0.04 0.13 3.30 0.05 0.04 0.04 1.32 0.39 0.11 0.16 0.53 0.74 0.32 0.06 0.21 0.06 0.09 0.04 0.07 0.17 0.07 0.15 0.22 0.23 0.15 0.42 0.45 0.06 0.40
MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS
(continued (continued on next page)
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Table 1 (continued) (continued) No.
Rt
Compound
Composition (%)
Method
74 75 76 77 78 79
12.5 12.6 12.7 12.9 13.1 13.3
Mandelic acid Abiol, Paridol Solbrol Unimol 3-Amino-1,2,4-Triazole Furandione Total Not identified
0.05 0.03 0.21 0.03 0.07 0.11 98.10 1.9
MS MS MS MS MS MS MS MS
extract also exhibited antibacterial effect against all four Gram-positive bacteria (S. aureus KCTC1916, B. subtilis ATCC6633 and S. aureus ATCC6538 and L. monocytogenes ACTC19166) and one Gram-negative bacterium namely P. aeruginosa KCTC2004, with their respective diameter zones of inhibition of 11, 10, 10, 10 and 11 mm. Also, chloroform and ethyl acetate extracts exhibited moderate antibacterial effect against some of the bacterial pathogens tested (Table 2). Hexane extract exhibited low to moderate antibacterial effect against only three Gram-positive bacteria such as S. aureus KCTC1916, B. subtilis ATCC6633 and S. aureus ATCC6538, with their respective diameter zones of inhibition of 9, 8 and 8 mm. No antibacterial effect of hexane extract was observed against any of the Gram-negative bacteria tested. The solvents did not inhibit the growth of any of the bacteria tested at the used concentration. In this study, in some cases, the essential oil exhibited significantly higher antibacterial activity than that of standard streptomycin in regard of Gram-positive bacteria, while, tetracycline showed higher antibacterial effect, in some other cases, than those of essential oil and leaf extracts (Table 2). However, hexane extract did not show the significant results against all the bacterial strains tested as compared to standard antibiotics. 3.3. Determination of minimum inhibitory concentration (MIC) As shown in Table 3, the minimum inhibitory concentration values of essential oil against the employed bacterial strains were found more susceptible as compared to the leaf extracts. The essential oil displayed significantly remarkable antibacterial activity against all four Gram-positive bacteria such as S. aureus KCTC1916, B. subtilis ATCC6633, S. aureus ATCC6538, L. monocytogenes ATCC19166 and two Gram-negative bacteria such as E. coli
Table 3 Minimum inhibition concentration of essential oil and various leaf extracts of Nandina domestica Thunb. against the growth of food-borne and spoiling bacteria Minimum inhibitory concentration (MIC)a
Microorganism
Essential oilb
S. aureus (KCTC 1916) B. subtilis (ATCC 6633) S. aureus (ATCC 6538) P. aeruginosa (KCTC 2004) L. monocytogenes (ATCC 19166) E. coli 0157:H7 (ATCC 43888) E. coli (ATCC 8739) E. coli 0157 (Human) E. aerogenes (KCTC 2190) S. typhimurium (KCTC 2515) S. enteritidis (KCTC 12021)
62.5 125 125 125 125 1000 1000 1000 1000 500 1000
Leaf extractsc MeOH extract
Hexane
CHCl3
EtOAc
250 500 250 250 500 1000 1000 2000 2000 2000 2000
2000 2000 2000 ndd nd nd nd nd nd nd nd
250 250 500 250 250 nd 1000 2000 nd nd nd
250 250 500 250 500 nd nd nd nd nd nd
a
Minimum inhibitory concentration (MIC). MIC of essential oil (values in µg/ml). c MIC of various leaf extracts of MeOH (methanol), Hexane, CHCl3 (chloroform), EtOAc (ethyl acetate) (values in µg/ml). d nd: not detected. b
ATCC8739 and S. typhimurium KCTC2515, with their respective MIC values of 62.5, 125, 125, 125, 125 and 500 μg/ml. The essential oil also exhibited potential antibacterial effect against rest of the Gramnegative bacterial strains with MIC value 1000 μg/ml for each bacterium. Also, methanol, ethyl acetate and chloroform extracts exhibited strong antibacterial effect as compared to hexane extract. The solvents did not inhibit the growth of any of the bacteria tested at the used concentration. However, methanol, chloroform and ethyl acetate extracts exhibited similar antibacterial effect against S. aureus KCTC1916 and P. aeruginosa KCTC2004 with their respective MIC value of 250 for each bacterium. The MIC values of methanol, ethyl acetate and chloroform extracts against the employed bacteria were found in the range of 250–2000, 250–2000 and 250–500 μg/ ml, respectively. Comparatively, chloroform and ethyl acetate extracts exerted higher antibacterial susceptibility as compared to methanol extract. Moderate antibacterial effect of the hexane extract was observed as a minimum inhibitory concentration (2000 μg/ml for each bacterium) against only three Gram-positive bacterial strain tested (Table 3).
Table 2 Antibacterial activity of essential oil and various leaf extracts of Nandina domestica Thunb. against food-borne and spoiling bacteria Microorganism
Zone of inhibition Essential oila
S. aureus (KCTC 1916) B. subtilis (ATCC 6633) S. aureus (ATCC 6538) P. aeruginosa (KCTC 2004) L. monocytogenes (ATCC 19166) E. coli 0157:H7 (ATCC 43888) E. coli (ATCC 8739) E. coli 0157 (Human) E. aerogenes (KCTC 2190) S. typhimurium (KCTC 2515) S. enteritidis (KCTC 12021)
18.0 ± 2.1 11.0 ± 0.7 14.0 ± 0.9 17.0 ± 2.1 12.0 ± 1.7 9.0 ± 0.9 12.0 ± 1.2 10.0 ± 1.2 12.0 ± 1.7 12.0 ± 1.2 11.0 ± 0.9
Leaf extractsb
Standardc
MeOH
Hexane
CHCl3
EtOAc
SM
TC
11.0 ± 1.6 10.0 ± 1.2 10.0 ± 1.9 11.0 ± 1.5 10.0 ± 0.7 7.0 ± 1.3 9.0 ± 0.5 9.0 ± 0.7 7.0 ± 1.3 8.0 ± 1.7 7.0 ± 1.2
9.0 ± 1.4 8.0 ± 0.5 8.0 ± 0.5 ndd nd nd nd nd nd nd nd
13.0 ± 1.1 10.0 ± 0.5 10.0 ± 1.9 12.0 ± 0.7 11.0 ± 1.2 7.0 ± 0.3 9.0 ± 0.5 nd 9.0 ± 1.4 nd nd
13.0 ± 1.3 10.0 ± 1.1 10.0 ± 1.8 10.0 ± 1.2 10.0 ± 0.9 9.0 ± 0.2 9.0 ± 0.3 nd nd nd nd
14 ± 0.9 14 ± 0.2 14 ± 0.6 19 ± 0.4 14 ± 0.6 15 ± 0.9 24 ± 0.7 15 ± 0.7 13 ± 0.2 13 ± 0.2 14 ± 0.6
18 ± 0.5 18 ± 0.5 19 ± 0.6 20 ± 1.0 19 ± 0.5 20 ± 0.5 17 ± 1.1 19 ± 1.2 20 ± 0.6 21 ± 0.6 21 ± 1.0
Values are given as mean ± S.D. of triplicate experiment. a Diameter of inhibition zones of essential oil including diameter of disc 6 mm (tested volume 5 µl corresponding to 1000 ppm/disc). b Diameter of inhibition zones of various leaf extracts of MeOH (methanol), Hexane, CHCl3 (Chloroform) EtOAc (ethyl acetate) including diameter of disc 6 mm (tested volume 7.5 µl/ml corresponding to 1500 ppm/disc). c Standard antibiotics-SM: Streptomycin, TC: Tetracycline (10 µg/disc). d nd: not detected.
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Fig. 1. Effect of Nandina domestica Thunb. essential oil (MIC concentration) on viability of the tested bacterial pathogens. CT: control without treatment.
3.4. Effect of essential oil on viable counts of bacteria Based on the susceptibility, further, elaborative study carried out on S. aureus KCTC 1916, B. subtilis ATCC 6633, S. aureus ATCC 6538, P. aeruginosa KCTC 2004 and L. monocytogenes ATCC19166, displayed different sensitivities of the essential oil. The effects of essential oil on the growth of all the tested bacterial strains demonstrated the reduced viability of the tested bacteria at MIC concentration of the essential oil. At 20 min exposure, complete inhibition of both the strains of S. aureus was observed at MIC concentration of the essential oil. Also the steep decline in CFU numbers was observed at 40 min exposure against B. subtilis ATCC 6633, L. monocytogenes ATCC19166 and P. aeruginosa KCTC 2004. Exposure of 80 min of the essential oil MIC concentration revealed complete inhibition of CFU numbers against all the bacterial strains tested (Fig. 1). 4. Discussion The traditional use of plants provides the basis for indicating the type of essential oils and plant extracts useful for specific food purposes. Historically, many plant oils and extracts have been reported to have antimicrobial properties (Hoffman, 1987). Also, the renewal of interest in the food industry, and increasing consumer demand for effective, safe, natural products, means that quantitative data on plant oils and extracts are required. Various publications have documented the antimicrobial activity of essential oil constituents and plant extracts (Morris et al., 1979). In recent years, several researchers have also 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. Actually, phenolics 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 function (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 bacteria. Also, the results of the antibacterial screening showed that essential oil and various leaf extracts of methanol, ethyl acetate and chloroform have potential antibacterial activity against S. aureus KCTC1916, S. aureus ATCC6538, B. subtilis ATCC6633, L. monocytogenes ATCC19166, P. aeruginosa KCTC2004, E. coli ATCC8739, S. typhimurium KCTC2515, E. coli 0157:H7 ATCC42888 and E. coli ACTC8739. This might be the result of the 1-indolizino carbazole, 2-pentanone, mono phenol, azridine, methylcarbinol, ethanone, furfural and 1-hydroxy-4-methyl-
benzene, present in the essential oil of N. domestica as these findings are strongly supported by the earlier work (Mevy et al., 2007). Essential oils, which are odorous and volatile products of plant secondary metabolism, have a wide application in the food flavouring and preservation industries (Smith-Palmer et al., 2001). N. domestica mediated oil also contained 2-pentanone, mono phenol, methylcarbinol, ethanone, furfural, 1-hydroxy-4-methylbenzene, 2(5H)-furanone, 3,5-dimethylpyrazole and isovaleric acid and, as earlier reported, the major/minor components of the various essential oils, have enormous potential against food spoilage and food-borne pathogenic bacteria (Maria-Rose et al., 2004). These findings were also confirmed in the present investigation. In addition, it is also possible that the minor components, such as α-toluenol, suberon, dihydrochavicol, veltol, brenzcatechin and fourrine might be involved in some type of antibacterial synergism with other active components of essential oil, as was also evident by the work of others (Marino et al., 2001). When comparing the data obtained in different studies, most publications provide generalization about whether or not a plant oil or extract possesses activity against Gram-positive and Gram-negative bacteria. However, few provide details about the extent or spectrum of this activity. Also, the results from viable count assay revealed that exposure of the MIC concentration of the essential oil had a severe effect on the cell viability of the tested bacteria. Both the strains of S. aureus were found more sensitive to the essential oil. Besides that, essential oil also exerted its maximum bactericidal activity against B. subtilis ATCC 6633, L. monocytogenes ATCC19166 and P. aeruginosa KCTC2004, as evident by the significant reduction in microbial counts at 40 min exposure and complete inhibition of cell viability at 80 min exposure of essential oil. Similar to our findings, one of n-6 essential oils also exerted an inhibitory effect against P. aeruginosa (Giamarellos-Bourboulis et al., 1998). Direct effect of n-6 essential oils on bacterial cells is prone to peroxidation ending in free radicals, capable of attacking bacterial outer membrane and facilitating the action of antimicrobials (Giamarellos-Bourboulis et al., 1998). However, similar mechanism can not be ruled out in case of essential oil action on P. aeruginosa KCTC2004. Deans et al. (1995) observed that the susceptibility of Grampositive and Gram-negative bacteria to plant volatile oils had little influence on growth inhibition (Deans and Ritchie, 1987). However, some oils appeared more specific, exerting a greater inhibitory activity against Gram-positive bacteria. It is often reported that Gram-negative 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). This is the reason that Gram-positive bacteria were found to be more sensitive to
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the essential oil and various leaf extracts of methanol, chloroform and ethyl acetate derived from N. domestica than were Gram-negative bacteria. However, in the present study, essential oil, ethyl acetate and chloroform extracts also severely inhibited the growth of Gramnegative bacteria in some cases (Table 2). In this research, we found that essential oil and various leaf extracts of N. domestica severely inhibited the growth of food-borne and spoiling bacteria. Therefore, essential oils and plant extracts are being considered as potential alternatives to synthetic bactericides or as a lead compounds for new classes of natural bactericides. In conclusion, the results of this study suggest that N. domestica mediated oil and organic extracts may act as an alternative to synthetic bactericides for using in the food industries, where bacterial pathogens cause severe destruction. Besides, the use of essential oils and plant extracts 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 (Tuley de Silva, 1996), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials. However, if plant oils and extracts are to be used for food preservation or medicinal purposes, issues of safety and toxicity will always need to be addressed. Acknowledgement This research was supported by the RIC program of MOCIE, Republic of Korea. References Adam, R.P., 2001. Identification of essential oil components by Gas chromatography/ Quadrupole mass spectroscopy. Allured Publishing Corporation, Carol stream, Illinois. Bezic, N., Skocibusic, M., Dinkic, V., Radonic, A., 2003. Composition and antimicrobial activity of Achillea clavennae L. essential oil. Phytotherapy Research 17, 1037–1040. 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 and Fragrance Journal 19 (1), 62–68. Chandrasekaran, M., Venkatesalu, V., 2004. Antibacterial and antifungal activity of Syzygium jambolanum seeds. Journal of Ethnopharmacology 91, 105–108. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Review 12, 564–582. Daferera, D.J., Ziogas, B.N., Polissiou, M.G., 2000. GC–MS Analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum. Journal of Agricultural Food Chemistry 48, 2576–2581. Deans, S.G., Noble, R.C., Hiltunen, R., Wuryani, W., Penzes, L.G., 1995. Antimicrobial and antioxidant properties of Syzygium aromaticum (L) Merr Perry: impact upon bacteria, fungi and fatty acid levels in ageing mice. Flavour and Fragrance Journal 10, 323–328.
Deans, S.G., Ritchie, G., 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5, 165–180. Dirr, M.A., 1990. Manual of Woody Landscape Plants: their Identification, Ornamental Characteristics, Culture, Propagation, and Uses, 4th Ed. Stipes Publishing Co., Champaign, IL, p. 1007. Essawi, T., Srour, M., 2000. Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology 70, 343–349. Faleiro, L., Miguel, G.M., Guerrero, C.A., Brito, J.M.C., 1999. Antimicrobial activity of essential oils of Rosmarinus offıcinalis L., Thymus mastichina (L) L. ssp. mastichina and Thymus albicans. Proceedings of the II WOCMAP Congress on Medicinal and Aromatic Plants, Part 2: Pharmacognosy, Pharmacology, Phytomedicine, Toxicology. 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. Prostaglandins Leukotrienes and Essential Fatty Acids 58, 283–287. Hall, R.L., 1997. Food-borne illness: implications for the future. Emerging Infectious Diseases 3, 555–559. Heath, H.B., 1981. Source Book of Flavours. Avi, Westport, p. 890. Hoffman, D.L., 1987. The herb user’s guide. Thorsons Publishing Group, Wellingborough, UK. Madigan, M.T., Martinko, J.M., Parker, J., 1997. Biology of microorganisms, 8th Ed. Prentice-Hall International Inc., New Jersey. 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. International Journal of Food Microbiology 67, 187–195. 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 Chemistry 101, 682–685. Morris, J.A., Khettry, A., Seitz, E.W., 1979. Antimicrobial activity of aroma chemicals and essential oils. Journal of American Oil Chemical Society 56, 595–603. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H., 1995. Manual of clinical microbiology, 6th Ed. ASM, Washington. Reische, D.W., Lillard, D.A., Eintenmiller, R.R., 1998. Antioxidants in food lipids. In: Ahoh, C.C., Min, D.B. (Eds.), Chemistry, Nutrition and Biotechnology. New York, pp. 423–448. Reynolds, J.E.F., 1996. Martindale the extra harmacopoeia, 31st Eds. Royal Pharmaceutical Society of Great Britain, London. Shin, S.Y., Bajpai, V.K., Kim, H.R., Kang, S.C., 2007. Antibacterial activity of eicosapentaenoic acid (EPA) against foodborne and food spoilage microorganisms. LWT 40, 1515–1519. Smith-Palmer, A., Stewart, J., Fyfe, L., 2001. The potential application of plant essential oils as natural food preservatives in soft cheese. Food Microbiology 18, 463–470. Soliman, K.M., Badeaa, R.I., 2002. Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food Chemical Toxicology 40, 1669–1675. Tepe, B., Donmez, E., Unlu, M., Candan, F., Daferera, D., Vardar-Unlu, G., 2004. Antimicrobial and antioxidative activities of the essential oils and methanol extracts of Salvia cryptantha (montbret et aucher ex benth.) and Salvia multicaulis (vahl). Food Chemistry 84, 519–525. Tuley de Silva, K., 1996. A manual on the essential oil industry. United Nations Industrial Development Organization, Vienna. Vattem, D.A., Lin, Y.T., Labbe, R.G., Shetty, K., 2004. Phenolic antioxidant mobilization in cranberry pomace by solid-state bioprocessing using food grade fungus Lentinus edodes and effect on antimicrobial activity against select food-borne pathogens. Innovative Food Science and Emerging Technology 5, 81–91.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb. to control food-borne pathogenic and spoilage bacteria Vivek K. Bajpai, Atiqur Rahman, Sun Chul Kang ⁎ Department of Biotechnology, College of Engineering, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea
A R T I C L E
I N F O
Article history: Received 4 February 2008 Received in revised form 15 March 2008 Accepted 20 March 2008 Keywords: Nandina domestica Thunb. Essential oil Mono phenol 3,5-dimethylpyrazole Food-borne and spoiling bacteria Antibacterial activity
A B S T R A C T The aim of this study was to examine the chemical composition of the essential oil isolated from the floral parts of Nandina domestica Thunb. by hydrodistillation, and to test the efficacy of essential oil and various organic extracts against a panel of food-borne pathogenic and spoilage bacteria such as Bacillus subtilis ATCC6633, Listeria monocytogenes ATCC19166, Staphylococcus aureus KCTC1916, S. aureus ATCC6538, Pseudomonas aeruginosa KCTC2004, Salmonella typhimurium KCTC2515, Salmonella enteridis KCCM12021, Escherichia coli 0157-Human, E. coli ATCC8739, E. coli 057:H7 ATCC43888 and Enterobacter aerognes KCTC2190. The chemical composition of essential oil was analysed by GC–MS. It was determined that 79 compounds, which represented 87.06% of total oil, were present in the oil. The oil contained mainly 1-indolizino carbazole (19.65%), 2-pentanone (16.4%), mono phenol (12.1%), aziridine (9.01%), methylcarbinol (4.6%), ethanone (3.3%), furfural (2.96%), 3,5-dimethylpyrazole (1.29%) and 2(5H)-furanone (1.32%). The oil (1000 ppm/disc), and various organic extracts of hexane, chloroform, ethyl acetate and methanol (1500 ppm/disc) exhibited promising antibacterial effect as a diameter of zones of inhibition (9–18 and 7–13 mm) and MIC values (62.5 to 1000 and 250 to 2000 μg/ml), respectively against the tested bacteria. Also the oil had strong detrimental effect on the viable count of the tested bacteria. These results indicate the potential efficacy of plant-based natural products such as essential oil and organic extracts of N. domestica to control food-borne pathogenic and spoilage bacteria. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The presence and growth of microorganisms in food may cause spoilage and result in a reduction in quality and quantity (Soliman and Badeaa, 2002). Food-borne illnesses associated with Listeria monocytogenes, Staphylococcus aureus, Escherichia coli 0157:H7 and Salmonella enteritidis present a major public health concern throughout the world (Hall, 1997). One of the two mechanisms determining how foodborne diseases are primarily caused, is by infection as a consequence of consuming foods contaminated with the growth of pathogenic microorganisms, such as bacteria, mould, viruses and parasites (Vattem et al., 2004). In addition to passive transfer of pathogens to food, active growth of a pathogen may also occur in foods, for instance because of improper storage, which leads to marked increases in microbial load (Madigan et al., 1997). For these reasons, microbial contamination of food still poses important public health and economic concerns for the human society. Plant secondary metabolites, such as essential oils and plant extracts (Tepe et al., 2004), are studied for their antimicrobial activities and most essential oils derived from plants are known to possess antibacterial, insecticidal, antifungal, acaricidal and cytotoxic activities ⁎ Corresponding author. Tel.: +82 53 850 6553; fax: +82 53 850 6559. E-mail address: [email protected] (S.C. Kang). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.011
(Faleiro et al., 1999). Therefore, they are intensely screened and applied in the fields of food microbiology, pharmacology, pharmaceutical botany, medical and clinical microbiology, phytopathology and food preservation (Daferera et al., 2000). Since ancient times the crude herbal extracts of aromatic plants have been in use for different purposes, such as food, drugs and perfumery (Heath, 1981). The essential oils are considered among the most important antimicrobial agents present in the plants, and may also have antioxidant and antiinflammatory activities. Volatile oils are a complex mixture of compounds, mainly monoterpenes, sesquiterpenes, and their oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols and oxides). Other volatile compounds include phenylpropenes and specific sulphur- or nitrogen-containing substances. Generally, the oil composition is a balance of various compounds, although in many species one constituent may prevail over all others (Cowan, 1999). In the recent decades, antimicrobial plant products have gained special interest because of the resistance to antibiotics that some microorganisms have acquired (Essawi and Srour, 2000), the increasing popular concern about the safety of food and the potential impact of synthetic additives on health (Reische et al., 1998). Nandina domestica (Heavenly bamboo or Sacred bamboo), is a suckering shrub in the Barberry family, Berberidaceae, it is a monotypic genus, with this species as its only member. It is native to eastern Asia from the Himalaya east to Japan. Despite the common name, it is not a
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bamboo at all. It is an erect shrub growing to 2 m tall, with numerous, usually unbranched stems growing from the roots. The leaves are evergreen (sometimes deciduous in colder areas). The young leaves in spring are brightly coloured pink to red before turning green; old leaves turn red or purple again before falling. The flowers are white, borne in early summer in conical clusters held well above the foliage. It is widely grown in gardens as an ornamental plant; over 60 cultivars have been named. It has become naturalised in parts of eastern North America. In the Southeastern United States it is considered by many as a pest due to its invasive nature. Some even refer to it as Nandina Megalomania or Hitler Bamboo for its unbridled aggression toward other plants, its propensity to conquer the entire yard without provocation, and its seeming immortality (Dirr, 1990). To the best of our knowledge and literary survey, there is no report available on chemical composition analysis of essential oil derived from N. domestica, and its antibacterial properties. Hence, efforts have been made, to investigate the role of essential oil and various organic extracts of N. domestica as an antibacterial potential. In the present investigation, we examined the chemical compositions of the essential oil isolated from the floral parts of N. domestica Thunb., and tested the efficacy of essential oil and various leaf extracts against a diverse range of food-borne pathogens and food spoilage bacteria. 2. Materials and methods 2.1. Plant material Two types of samples, flowers and leaves of N. domestica were collected from the local area of Kyoungsan, Republic of Korea, in May– June 2007 and initially identified by morphological features and the data base present in the library at the Department of Biotechnology by Prof. Man Kyu Huh. A voucher specimen number (NDU-0099) has been deposited in the herbarium of College of Engineering, Department of Biotechnology, Daegu University, Republic of Korea. 2.2. Isolation of the essential oil The floral parts of N. domestica were air-dried for two weeks. The air-dried plant material (200 g) was subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus. The oil was dried over anhydrous Na2SO4 and preserved in a sealed vial at 4 °C prior to further analysis. 2.3. Preparation of leaf extracts The air-dried leaves of N. domestica 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). The extraction process yielded in methanol (7.1 g), hexane (3.1 g), chloroform (4.6 g) and ethyl acetate (4.3 g) extracts. 2.4. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the essential oil was performed using a Shimadzu GC–MS (GC-17A) equipped with a ZB-1 MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm). For GC– MS detection, an electron ionization system with 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 °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 finally raised to 250 °C at 10 °C/min. Diluted samples (1/ 100, v/v, in methanol) of 1.0 μl were injected manually in the split less
mode. The relative percentage of the oil constituents was expressed as percentages by peak area normalization. Identification of components of the essential oil was based on GC retention time on a ZB-1 capillary column relative to 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). 2.5. Microorganisms In all, eleven bacterial pathogens including food spoilage bacteria, such as Pseudomonas aeruginosa KCTC2004 and Bacillus subtilis ATCC6633, and food-borne pathogenic bacteria, namely Enterobacter aerogenes KCTC2190, Salmonella typhimurium KCTC2515, E. coli ATCC8739, E. coli O157:H7 ATCC43888, E. coli O157:H7 (Human), L. monocytogenes ATCC19166, S. enteritidis KCCM 2021, S. aureus ATCC6538 and S. aureus KCTC1916, were obtained from the Korea Food and Drug Administration (KFDA), Daegu, Republic of Korea. Cultures of each bacterial strain were maintained on Luria broth (LB) agar medium at 4 °C. 2.6. 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 LB 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 away and the inoculum was allowed to dry for 5 min. To prepare the stock solution of the samples, the oil was dissolved in 5% dichloromethane, whereas the extracts (hexane, chloroform, ethyl acetate and methanol) were dissolved in their respective solvents. Then a Whatman No. 1 sterile filter paper disk (6 mm diameter) was impregnated with 5 μl essential oil, corresponding to 1000 ppm/disc, and 7.5 μl leaf extracts, corresponding to 1500 ppm/ disc, using a capillary micro-pipette. Negative controls were prepared using the same solvents employed to dissolve the samples. Standard reference antibiotics, streptomycin and tetracycline (10 μg/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 zones of inhibition using a vernier caliper against the tested bacteria. Each assay in this experiment was replicated three times. 2.7. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentrations (MICs) of essential oil and various leaf extracts of methanol, ethyl acetate, chloroform and hexane, were tested by a two-fold serial dilution method (Chandrasekaran and Venkatesalu, 2004). The tests of oil and leaf extracts of hexane, chloroform, ethyl acetate and methanol were dissolved in their respective solvents (5%), the solvents used for isolation of essential oil and for obtaining the various extracts, and were added to LB broth medium to get a final concentration of 4000 μg/ml, which were further serially diluted to achieve 2000, 1000, 500, 250 125, 62.5 and 31.25 μg/ml, respectively, where the final concentration of the solvent was 0.5%. 10 μl of standardized suspension of each tested organism (108 CFU/ml approximately) was transferred to each tube. The control tubes, containing only bacterial suspension, were incubated at 37 °C for 24 h. The lowest concentrations of the test samples, which did not show any growth of test organisms after macroscopic evaluation, were determined as MICs, which were expressed in μg/ml. 2.8. Effect of essential oil on viable counts of bacteria For viable counts, each of the tubes containing bacterial suspension (approximately 107 CFU/ml) of S. aureus KCTC 1916, B. subtilis ATCC 6633, S. aureus ATCC 6538, P. aeruginosa KCTC 2004 and L. mono-
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cytogenes ATCC19166 in LB broth medium was inoculated with the minimum inhibitory concentration of the essential oil in 10 ml LB broth, and kept at 37 °C (Shin et al., 2007). Samples for viable cell counts were taken out at 0, 20, 40, 60, 80, 100 and 140 min time intervals. Enumeration of viable counts on LB plates was monitored as followings: 0.1 ml sample of each treatment was diluted into buffer peptone water, there by diluting it 10-fold 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 same experimental condition as mentioned above. 2.9. Statistical analysis The data obtained for antibacterial activity of essential oil and various extracts were statistical analysed and mean values were calculated. A Student’s t-test was computed for the statistical significance of the results. 3. Results 3.1. Chemical composition of the essential oil GC–MS analyses of the oil led to the identification of 79 different components, representing 87.06% 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 contained a complex mixture consisting of mainly oxygenated mono- and sesqueterpenes, and mono- and sesqueterpene hydrocarbons. The major components in the oil detected were 1-indolizino carbazole (19.65%), 2-pentanone (16.4%), mono phenol (12.1%), azridine (9.01%), methylcarbinol (4.6%), ethanone (3.30%), furfural (2.96%), 1-hydroxy-4-methylbenzene (1.53%), 2(5H)furanone (1.32%) and 3,5-dimethylpyrazole (1.29%). In the present study, isovaleric acid (0.23%), α-toluenol (0.05%), suberon (0.45%), dihydrochavicol (0.16%), veltol (0.53%), brenzcatechin (0.74%) and fourrine (0.32%) were also found to be the minor components of N. domestica oil. 3.2. Antibacterial activity assay The in vitro antibacterial activities of essential oil and various leaf extracts (hexane, chloroform, ethyl acetate and methanol) of N. domestica, against the employed bacteria, were qualitatively and quantitatively assessed by the presence or absence of inhibition zones. According to the results given in Table 2, in all, eleven food-borne pathogens and spoilage bacteria, including four Gram-positive and seven Gram-negative bacteria, were tested for the evaluation of antibacterial potential of essential oil and various leaf extracts of methanol, ethyl acetate, chloroform and hexane. As shown in Table 2, the essential oil at 5 μl (1000 ppm/disc) exhibited potent inhibitory effect against all four Gram-positive (S. aureus KCTC1916, B. subtilis ATCC6633, S. aureus ATCC6538 and L. monocytogenes ATCC19166) and six Gram-negative bacteria (P. aeruginosa KCTC2004, E. coli ATCC8739, E. coli 0157 (human) E. aerogenes KCTC2190, S. typhimurium KCTC2515 and S. typhimurium KCTC12021), with their respective diameter zones of inhibition of 18, 11, 14, 12, 17, 12. 10, 12, 12 and 11 mm. Only one of the bacteria E. coli 0157:H7 ATCC43888 was found slight resistant to the oil at the used concentration with 9 mm diameter zone of inhibition. S. aureus KCTC1916 was found most inhibited bacterial pathogen by the essential oil. Also the methanol, ethyl acetate and chloroform extracts significantly inhibited the growth of some of the bacterial strains tested. However, chloroform and ethyl acetate extracts exhibited potential antibacterial effect against some of the Gram-positive and Gramnegative bacteria. The diameter zones of inhibition of chloroform and ethyl acetate extracts against the tested bacteria were found in the range from 7 to 13 and 9 to 13 mm, respectively (Table 2). Methanol
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Table 1 Chemical composition of volatile oil isolated by hydrodistillation from Nandina domestica Thunb. No.
Rt
Compound
Composition (%)
Method
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 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
0.9 1.0 1.1 1.4 1.7 1.8 1.9 2.0 2.12 2.19 2.3 2.4 2.5 2.7 2.9 3.0 3.07 3.1 3.4 3.5 3.7 3.9 3.98 4.0 4.1 4.4 4.46 4.6 4.8 4.9 5.0 5.2 5.3 5.4 5.6 5.9 6.0 6.1 6.17 6.4 6.5 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.7 8.0 8.1 8.3 8.5 8.8 9.1 9.3 9.4 9.5 9.7 9.9 10.1 10.3 10.4 10.6 10.7 10.9 11.4 11.6 11.8 11.9 12.2
Methylcarbinol 2-Pentanone 1-Indolizino carbazole 2-Butanone Propylene carbonate Propionic acid Tetrahydropyran Glyoxylic acid Imidazole 1.2-D2-Imidazole Aziridine Formic acid 2(3H)-Furanone 3-Pentanone Vinylcarbinol Propanoyl chloride 2-Aminooxazole 2,3-Dihydropyran 1-Methyltetrazole Furfural 3,5-Dimethylpyrazole Sorbaldehyde 2,4-Hexadien-1-o1 4-Pentenenitrile Pyrrolidine 2,4-Dimethylfuran Ethylidene-cyclopropane 4-Nonyne Octylazide Isovaleric acid Pyrazole Ketoisophrone Acetic acid 1,2-Propadiene Mono phenol 1-Nitrobutane 4-Chlorooctane Suberon Cresylic acid α-Toluenol 2-Methylphenol p-Oxytoluene p-Toluol 1-Hydoxy-4-methylbenzene Pyroguaiac acid Guaiacol Ethanone Leucine p-Hydoxyanisole 2,6-Dimethyl-1,7-octadiene-3-ol 2(5H)-Furanone Ortho-ethylphenol 3,4-Xylenol Dihydrochavicol Veltol Brenzcatechin Fourrine Phiaquin Benzohydroquinone Quinol dimethyl ether Orcinol 4-Methyl catechol Nicotinic acid 1-Furyl-1-ethoxy-ethanol Toluene Pyrazine Phenetole Trycarbonyl-1,3-cyclooctadiene Thynol Benzofuran 4-Ethylresorcinol Naphthalene 1,2,3,3,4-Pentamethyl cyclopentene
4.60 16.40 19.65 0.45 0.03 0.24 0.03 0.27 0.03 0.07 9.01 0.87 0.05 0.61 0.02 0.13 0.40 0.07 0.27 2.96 1.29 0.09 0.03 0.13 0.07 0.96 0.29 0.46 0.36 0.23 0.16 0.18 0.04 0.23 12.10 0.31 0.17 0.45 0.77 0.05 0.33 0.08 0.09 1.53 0.04 0.13 3.30 0.05 0.04 0.04 1.32 0.39 0.11 0.16 0.53 0.74 0.32 0.06 0.21 0.06 0.09 0.04 0.07 0.17 0.07 0.15 0.22 0.23 0.15 0.42 0.45 0.06 0.40
MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS
(continued (continued on next page)
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Table 1 (continued) (continued) No.
Rt
Compound
Composition (%)
Method
74 75 76 77 78 79
12.5 12.6 12.7 12.9 13.1 13.3
Mandelic acid Abiol, Paridol Solbrol Unimol 3-Amino-1,2,4-Triazole Furandione Total Not identified
0.05 0.03 0.21 0.03 0.07 0.11 98.10 1.9
MS MS MS MS MS MS MS MS
extract also exhibited antibacterial effect against all four Gram-positive bacteria (S. aureus KCTC1916, B. subtilis ATCC6633 and S. aureus ATCC6538 and L. monocytogenes ACTC19166) and one Gram-negative bacterium namely P. aeruginosa KCTC2004, with their respective diameter zones of inhibition of 11, 10, 10, 10 and 11 mm. Also, chloroform and ethyl acetate extracts exhibited moderate antibacterial effect against some of the bacterial pathogens tested (Table 2). Hexane extract exhibited low to moderate antibacterial effect against only three Gram-positive bacteria such as S. aureus KCTC1916, B. subtilis ATCC6633 and S. aureus ATCC6538, with their respective diameter zones of inhibition of 9, 8 and 8 mm. No antibacterial effect of hexane extract was observed against any of the Gram-negative bacteria tested. The solvents did not inhibit the growth of any of the bacteria tested at the used concentration. In this study, in some cases, the essential oil exhibited significantly higher antibacterial activity than that of standard streptomycin in regard of Gram-positive bacteria, while, tetracycline showed higher antibacterial effect, in some other cases, than those of essential oil and leaf extracts (Table 2). However, hexane extract did not show the significant results against all the bacterial strains tested as compared to standard antibiotics. 3.3. Determination of minimum inhibitory concentration (MIC) As shown in Table 3, the minimum inhibitory concentration values of essential oil against the employed bacterial strains were found more susceptible as compared to the leaf extracts. The essential oil displayed significantly remarkable antibacterial activity against all four Gram-positive bacteria such as S. aureus KCTC1916, B. subtilis ATCC6633, S. aureus ATCC6538, L. monocytogenes ATCC19166 and two Gram-negative bacteria such as E. coli
Table 3 Minimum inhibition concentration of essential oil and various leaf extracts of Nandina domestica Thunb. against the growth of food-borne and spoiling bacteria Minimum inhibitory concentration (MIC)a
Microorganism
Essential oilb
S. aureus (KCTC 1916) B. subtilis (ATCC 6633) S. aureus (ATCC 6538) P. aeruginosa (KCTC 2004) L. monocytogenes (ATCC 19166) E. coli 0157:H7 (ATCC 43888) E. coli (ATCC 8739) E. coli 0157 (Human) E. aerogenes (KCTC 2190) S. typhimurium (KCTC 2515) S. enteritidis (KCTC 12021)
62.5 125 125 125 125 1000 1000 1000 1000 500 1000
Leaf extractsc MeOH extract
Hexane
CHCl3
EtOAc
250 500 250 250 500 1000 1000 2000 2000 2000 2000
2000 2000 2000 ndd nd nd nd nd nd nd nd
250 250 500 250 250 nd 1000 2000 nd nd nd
250 250 500 250 500 nd nd nd nd nd nd
a
Minimum inhibitory concentration (MIC). MIC of essential oil (values in µg/ml). c MIC of various leaf extracts of MeOH (methanol), Hexane, CHCl3 (chloroform), EtOAc (ethyl acetate) (values in µg/ml). d nd: not detected. b
ATCC8739 and S. typhimurium KCTC2515, with their respective MIC values of 62.5, 125, 125, 125, 125 and 500 μg/ml. The essential oil also exhibited potential antibacterial effect against rest of the Gramnegative bacterial strains with MIC value 1000 μg/ml for each bacterium. Also, methanol, ethyl acetate and chloroform extracts exhibited strong antibacterial effect as compared to hexane extract. The solvents did not inhibit the growth of any of the bacteria tested at the used concentration. However, methanol, chloroform and ethyl acetate extracts exhibited similar antibacterial effect against S. aureus KCTC1916 and P. aeruginosa KCTC2004 with their respective MIC value of 250 for each bacterium. The MIC values of methanol, ethyl acetate and chloroform extracts against the employed bacteria were found in the range of 250–2000, 250–2000 and 250–500 μg/ ml, respectively. Comparatively, chloroform and ethyl acetate extracts exerted higher antibacterial susceptibility as compared to methanol extract. Moderate antibacterial effect of the hexane extract was observed as a minimum inhibitory concentration (2000 μg/ml for each bacterium) against only three Gram-positive bacterial strain tested (Table 3).
Table 2 Antibacterial activity of essential oil and various leaf extracts of Nandina domestica Thunb. against food-borne and spoiling bacteria Microorganism
Zone of inhibition Essential oila
S. aureus (KCTC 1916) B. subtilis (ATCC 6633) S. aureus (ATCC 6538) P. aeruginosa (KCTC 2004) L. monocytogenes (ATCC 19166) E. coli 0157:H7 (ATCC 43888) E. coli (ATCC 8739) E. coli 0157 (Human) E. aerogenes (KCTC 2190) S. typhimurium (KCTC 2515) S. enteritidis (KCTC 12021)
18.0 ± 2.1 11.0 ± 0.7 14.0 ± 0.9 17.0 ± 2.1 12.0 ± 1.7 9.0 ± 0.9 12.0 ± 1.2 10.0 ± 1.2 12.0 ± 1.7 12.0 ± 1.2 11.0 ± 0.9
Leaf extractsb
Standardc
MeOH
Hexane
CHCl3
EtOAc
SM
TC
11.0 ± 1.6 10.0 ± 1.2 10.0 ± 1.9 11.0 ± 1.5 10.0 ± 0.7 7.0 ± 1.3 9.0 ± 0.5 9.0 ± 0.7 7.0 ± 1.3 8.0 ± 1.7 7.0 ± 1.2
9.0 ± 1.4 8.0 ± 0.5 8.0 ± 0.5 ndd nd nd nd nd nd nd nd
13.0 ± 1.1 10.0 ± 0.5 10.0 ± 1.9 12.0 ± 0.7 11.0 ± 1.2 7.0 ± 0.3 9.0 ± 0.5 nd 9.0 ± 1.4 nd nd
13.0 ± 1.3 10.0 ± 1.1 10.0 ± 1.8 10.0 ± 1.2 10.0 ± 0.9 9.0 ± 0.2 9.0 ± 0.3 nd nd nd nd
14 ± 0.9 14 ± 0.2 14 ± 0.6 19 ± 0.4 14 ± 0.6 15 ± 0.9 24 ± 0.7 15 ± 0.7 13 ± 0.2 13 ± 0.2 14 ± 0.6
18 ± 0.5 18 ± 0.5 19 ± 0.6 20 ± 1.0 19 ± 0.5 20 ± 0.5 17 ± 1.1 19 ± 1.2 20 ± 0.6 21 ± 0.6 21 ± 1.0
Values are given as mean ± S.D. of triplicate experiment. a Diameter of inhibition zones of essential oil including diameter of disc 6 mm (tested volume 5 µl corresponding to 1000 ppm/disc). b Diameter of inhibition zones of various leaf extracts of MeOH (methanol), Hexane, CHCl3 (Chloroform) EtOAc (ethyl acetate) including diameter of disc 6 mm (tested volume 7.5 µl/ml corresponding to 1500 ppm/disc). c Standard antibiotics-SM: Streptomycin, TC: Tetracycline (10 µg/disc). d nd: not detected.
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Fig. 1. Effect of Nandina domestica Thunb. essential oil (MIC concentration) on viability of the tested bacterial pathogens. CT: control without treatment.
3.4. Effect of essential oil on viable counts of bacteria Based on the susceptibility, further, elaborative study carried out on S. aureus KCTC 1916, B. subtilis ATCC 6633, S. aureus ATCC 6538, P. aeruginosa KCTC 2004 and L. monocytogenes ATCC19166, displayed different sensitivities of the essential oil. The effects of essential oil on the growth of all the tested bacterial strains demonstrated the reduced viability of the tested bacteria at MIC concentration of the essential oil. At 20 min exposure, complete inhibition of both the strains of S. aureus was observed at MIC concentration of the essential oil. Also the steep decline in CFU numbers was observed at 40 min exposure against B. subtilis ATCC 6633, L. monocytogenes ATCC19166 and P. aeruginosa KCTC 2004. Exposure of 80 min of the essential oil MIC concentration revealed complete inhibition of CFU numbers against all the bacterial strains tested (Fig. 1). 4. Discussion The traditional use of plants provides the basis for indicating the type of essential oils and plant extracts useful for specific food purposes. Historically, many plant oils and extracts have been reported to have antimicrobial properties (Hoffman, 1987). Also, the renewal of interest in the food industry, and increasing consumer demand for effective, safe, natural products, means that quantitative data on plant oils and extracts are required. Various publications have documented the antimicrobial activity of essential oil constituents and plant extracts (Morris et al., 1979). In recent years, several researchers have also 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. Actually, phenolics 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 function (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 bacteria. Also, the results of the antibacterial screening showed that essential oil and various leaf extracts of methanol, ethyl acetate and chloroform have potential antibacterial activity against S. aureus KCTC1916, S. aureus ATCC6538, B. subtilis ATCC6633, L. monocytogenes ATCC19166, P. aeruginosa KCTC2004, E. coli ATCC8739, S. typhimurium KCTC2515, E. coli 0157:H7 ATCC42888 and E. coli ACTC8739. This might be the result of the 1-indolizino carbazole, 2-pentanone, mono phenol, azridine, methylcarbinol, ethanone, furfural and 1-hydroxy-4-methyl-
benzene, present in the essential oil of N. domestica as these findings are strongly supported by the earlier work (Mevy et al., 2007). Essential oils, which are odorous and volatile products of plant secondary metabolism, have a wide application in the food flavouring and preservation industries (Smith-Palmer et al., 2001). N. domestica mediated oil also contained 2-pentanone, mono phenol, methylcarbinol, ethanone, furfural, 1-hydroxy-4-methylbenzene, 2(5H)-furanone, 3,5-dimethylpyrazole and isovaleric acid and, as earlier reported, the major/minor components of the various essential oils, have enormous potential against food spoilage and food-borne pathogenic bacteria (Maria-Rose et al., 2004). These findings were also confirmed in the present investigation. In addition, it is also possible that the minor components, such as α-toluenol, suberon, dihydrochavicol, veltol, brenzcatechin and fourrine might be involved in some type of antibacterial synergism with other active components of essential oil, as was also evident by the work of others (Marino et al., 2001). When comparing the data obtained in different studies, most publications provide generalization about whether or not a plant oil or extract possesses activity against Gram-positive and Gram-negative bacteria. However, few provide details about the extent or spectrum of this activity. Also, the results from viable count assay revealed that exposure of the MIC concentration of the essential oil had a severe effect on the cell viability of the tested bacteria. Both the strains of S. aureus were found more sensitive to the essential oil. Besides that, essential oil also exerted its maximum bactericidal activity against B. subtilis ATCC 6633, L. monocytogenes ATCC19166 and P. aeruginosa KCTC2004, as evident by the significant reduction in microbial counts at 40 min exposure and complete inhibition of cell viability at 80 min exposure of essential oil. Similar to our findings, one of n-6 essential oils also exerted an inhibitory effect against P. aeruginosa (Giamarellos-Bourboulis et al., 1998). Direct effect of n-6 essential oils on bacterial cells is prone to peroxidation ending in free radicals, capable of attacking bacterial outer membrane and facilitating the action of antimicrobials (Giamarellos-Bourboulis et al., 1998). However, similar mechanism can not be ruled out in case of essential oil action on P. aeruginosa KCTC2004. Deans et al. (1995) observed that the susceptibility of Grampositive and Gram-negative bacteria to plant volatile oils had little influence on growth inhibition (Deans and Ritchie, 1987). However, some oils appeared more specific, exerting a greater inhibitory activity against Gram-positive bacteria. It is often reported that Gram-negative 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). This is the reason that Gram-positive bacteria were found to be more sensitive to
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the essential oil and various leaf extracts of methanol, chloroform and ethyl acetate derived from N. domestica than were Gram-negative bacteria. However, in the present study, essential oil, ethyl acetate and chloroform extracts also severely inhibited the growth of Gramnegative bacteria in some cases (Table 2). In this research, we found that essential oil and various leaf extracts of N. domestica severely inhibited the growth of food-borne and spoiling bacteria. Therefore, essential oils and plant extracts are being considered as potential alternatives to synthetic bactericides or as a lead compounds for new classes of natural bactericides. In conclusion, the results of this study suggest that N. domestica mediated oil and organic extracts may act as an alternative to synthetic bactericides for using in the food industries, where bacterial pathogens cause severe destruction. Besides, the use of essential oils and plant extracts 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 (Tuley de Silva, 1996), as both consumers and regulatory agencies are more comfortable with the use of natural antimicrobials. However, if plant oils and extracts are to be used for food preservation or medicinal purposes, issues of safety and toxicity will always need to be addressed. Acknowledgement This research was supported by the RIC program of MOCIE, Republic of Korea. References Adam, R.P., 2001. Identification of essential oil components by Gas chromatography/ Quadrupole mass spectroscopy. Allured Publishing Corporation, Carol stream, Illinois. Bezic, N., Skocibusic, M., Dinkic, V., Radonic, A., 2003. Composition and antimicrobial activity of Achillea clavennae L. essential oil. Phytotherapy Research 17, 1037–1040. 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 and Fragrance Journal 19 (1), 62–68. Chandrasekaran, M., Venkatesalu, V., 2004. Antibacterial and antifungal activity of Syzygium jambolanum seeds. Journal of Ethnopharmacology 91, 105–108. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Review 12, 564–582. Daferera, D.J., Ziogas, B.N., Polissiou, M.G., 2000. GC–MS Analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum. Journal of Agricultural Food Chemistry 48, 2576–2581. Deans, S.G., Noble, R.C., Hiltunen, R., Wuryani, W., Penzes, L.G., 1995. Antimicrobial and antioxidant properties of Syzygium aromaticum (L) Merr Perry: impact upon bacteria, fungi and fatty acid levels in ageing mice. Flavour and Fragrance Journal 10, 323–328.
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