International Journal of Food Microbiology 117 (2007) 112 119 www.elsevier.

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The in vitro antibacterial activity of dietary spice and medicinal herb extracts
Bin Shan a , Yi-Zhong Cai a , John D. Brooks b , Harold Corke a,
b

School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Institute of Food Nutrition and Human Health, Massey University, Palmerston North, New Zealand Received 25 August 2006; received in revised form 18 January 2007; accepted 7 March 2007

Abstract The in vitro antibacterial activities of a total of 46 extracts from dietary spices and medicinal herbs were investigated by agar-well diffusion method against five foodborne bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella anatum). Their total phenolic contents were also evaluated. Many herb and spice extracts contained high levels of phenolics and exhibited antibacterial activity against foodborne pathogens. Gram-positive bacteria were generally more sensitive to the tested extracts than Gram-negative ones. S. aureus was the most sensitive, while E. coli was the most resistant. There were highly positive relationships (R2 = 0.730.93) between antibacterial activities and phenolic content of the tested extracts against each bacterium. This suggested that the antibacterial activity of the tested extracts was closely associated with their phenolic constituents. 2007 Elsevier B.V. All rights reserved.
Keywords: Antibacterial activity; Spices; Herbs; Extracts; Phenolic compounds; Antioxidant activity

1. Introduction Food poisoning is still a concern for both consumers and the food industry despite the use of various preservation methods. Food processors, food safety researchers and regulatory agencies are continuously concerned with the high and growing number of illness outbreaks caused by some pathogenic and spoilage microorganisms in foods. The increasing antibiotic resistance of some pathogens that are associated with foodborne illness is another concern (Meng et al., 1998; Perreten et al., 1998; Stermitz et al., 2000). Consumers are also concerned about the safety of foods containing synthetic preservatives. Therefore, there has been increasing interest in the development of new types of effective and nontoxic antimicrobial compounds. There is growing interest in using natural antibacterial compounds, such as extracts of spices and herbs, for food preservation (Smid and Gorris, 1999). Spices and herbs have been added to foods since ancient times, not only as flavoring agents, but also as folk medicine and food preservatives (Beuchat, 1994; Nakatani, 1994; Cutler, 1995). In addition to imparting characteristic flavors, certain spices and
Corresponding author. Tel.: +852 22990314; fax: +852 28583477. E-mail address: hcorke@yahoo.com (H. Corke). 0168-1605/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.03.003

herbs prolong the storage life of foods by preventing rancidity through their antioxidant activity or through bacteriostatic or bactericidal activity (Beuchat and Golden, 1989). Spices and herbs and their constituents are generally recognized to be safe, either because of their traditional use without any documented detrimental impact or because of dedicated toxicological studies (Smid and Gorris, 1999). Being natural foodstuffs, spices and herbs appeal to many consumers who question the safety of synthetic food additives. Some spices and herbs used today are valued for their antimicrobial activities and medicinal effects in addition to their flavor and fragrance qualities. The extracts of many plant species have become popular in recent years and attempts to characterize their bioactive principles have gained momentum for varied pharmaceutical and food processing applications. The antimicrobial activities of plant extracts form the basis for many applications, including raw and processed food preservation, pharmaceuticals, alternative medicines and natural therapies (Lis-Balchin and Deans, 1997). Numerous studies have been published on the antimicrobial activities of plant extracts against different types of microbes, including foodborne pathogens (Beuchat, 1994; Lis-Balchin and Deans, 1997; Smith-Palmer et al., 1998; Hara-Kudo et al., 2004). However, the results reported for these different studies are difficult to compare directly, usually because of the low number

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of plant samples tested, different test methods and diverse bacterial strains and sources of antimicrobial samples used. Many studies have reported that phenolic compounds in spices and herbs significantly contributed to their antioxidant and pharmaceutical properties (Cai et al., 2004; Shan et al., 2005; Wu et al., 2006). Some studies claim that the phenolic compounds present in spices and herbs might also play a major role in their antimicrobial effects (Hara-Kudo et al., 2004). There has been no large scale systematic investigation of the relationship between bacterial inhibition and total phenolic content of spices and herbs. Previous studies (Cai et al., 2004; Shan et al., 2005) showed that a highly positive linear relationship exists between antioxidant activity and total phenolic content in some spices and herbs. However, there is no reported data on the relationship between antibacterial activity and antioxidant capacity of spices and herbs. The objectives of this study were: (1) to evaluate and compare the in vitro antibacterial activity of 46 spice and herb extracts; (2) to establish the relationship between bacterial inhibition and total phenolic content to confirm whether the phenolic constituents are responsible for antibacterial activity; (3) to compare the antibacterial activity and antioxidant capacity of the extracts and to determine any correlation. 2. Materials and methods 2.1. Plant materials Twenty-one Chinese medicinal herbs were purchased and collected from a well-known market for Chinese herbal medicines in Qichun, Hubei, China. Twenty fresh or dried spice materials, originally from seven Asian countries/regions and four Western countries, were purchased from local Hong Kong supermarkets and drugstores. Five traditional Indian herbs were obtained from traditional medicine stores in Madras, India. These medicinal or spice plants were harvested and processed and naturally dried, according to traditional procedures. The scientific names and tested parts of the 46 plant materials (20 dietary spices and 26 medicinal herbs) are detailed in Table 1. 2.2. Microorganisms and culture A total of five foodborne bacteria were kindly provided by the Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong. They are Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella anatum. The strains were cultured at 37 C on plate count agar (PCA) medium. 2.3. Chemicals and reagents 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin-Ciocalteu reagent was from BDH (Dorset, England). Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was from Fluka Chemie AG (Buchs, Switzerland). Potassium persulfate, potassium phosphate and sodium carbonate

were purchased from Sigma/Aldrich (St. Louis, MO), and sodium dihydrogen phosphate monohydrate from Merck (Darmstadt, Germany). Authentic standards, such as penicillin G (16 mg/mL) and gentamicin solution (10 mg/mL), were purchased from Sigma/ Aldrich. PCA medium was from BD (Sparks, NA). 2.4. Preparation of methanolic extracts Fresh plant samples were cleaned, freeze-dried in a Heto FD3 freeze dryer (Heto-Holten A/S, Denmark), and ground into a fine powder by a Kenwood Multi-Mill (Kenwood, Havant, UK) and passed through a sieve (24-mesh). Dried plant samples were further air-dried in a ventilated oven at 40 C for 24 h, then ground into a fine powder and passed through a sieve as above. Powdered sample (2 g) was extracted with 50 mL 80% methanol in water at room temperature ( 23 C) for 24 h in a shaking water bath (Shaking Bath 5B-16, Techne, Cambridge, UK). The extract was filtered by a Millipore filter with a 0.45-m nylon membrane under vacuum at 23 C. The filtrates were concentrated by Rotavapor (R-114) (Buchi, Flawil, Switzerland) and then freeze-dried by a Heto FD3 freeze-dryer (Heto-Holten A/S, Allerod, Denmark). The samples were stored at 4 C until use. 2.5. Determination of antibacterial activity An agar-well diffusion method was employed for determination of antibacterial activities (NCCLS, 1999). The freezedried extract samples of spices and herbs were dissolved in phosphate buffered saline (PBS, pH 7.07.2) to the final concentration of 100 mg/mL and sterilized by filtration through 0.22 m sterilizing Millipore express filter (Millex-GP, Bedford, OH). All bacteria were suspended in sterile water and diluted to 106 CFU/mL. The suspension (100 L) was spread onto the surface of PCA medium. Wells (4.6 mm in diameter) were cut from the agar with a sterile borer and 60 L extract solutions were delivered into them. Negative controls were prepared using PBS solution. Penicillin G (960 g/well) and gentamicin (600 g/well) were used as positive reference standards to determine the sensitivity of each microbial species tested. The inoculated plates were incubated at 35 C for 24 h. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (DIZ) of the tested bacteria. DIZ was expressed in millimeters. All tests were performed in triplicate. 2.6. Determination of total antioxidant capacity A total antioxidant capacity assay was carried out by the improved ABTS method as described in the previous study (Shan et al., 2005). ABTS+ radical cation was generated by reacting 7 mM ABTS and 2.45 mM potassium persulfate after incubation at room temperature (23 C) in the dark for 16 h The ABTS + solution was diluted with 80% ethanol to an absorbance of 0.700 0.005 at 734 nm. The blank was 3.9 mL of ABTS+ solution with 0.1 mL of 80% ethanol. The extract sample was diluted with 80% ethanol to give 2080% inhibition of the blank absorbance with 0.1 mL of sample (Cai

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Table 1 Antibacterial activity, antioxidant capacity, and total phenolic content of 46 extracts from dietary spices and medicinal herbs Family and scientific name Common name (English or Chinese) Edible parts tested TEAC TPC Antibacterial activity (DIZ) (mm) c (mmol trolox/ (g GAE/ B. L. S. E. 100 g DW) a 100 g DW) b cereus monocytogenes aureus coli 5.5 61.8 107.7 7.0 6.6 168.7 20.3 34.3 33.8 20.0 29.6 100.7 6.3 11.2 4.6 9.0 37.8 51.9 38.1 36.9 39.6 0.6 6.3 11.9 0.9 0.2 14.4 2.0 4.2 5.2 1.6 3.6 10.2 1.0 0.4 0.3 0.8 5.1 5.3 4.5 3.1 4.1 4.6 10.3 15.4 4.6 4.6 14.0 4.7 6.8 8.0 4.6 6.3 11.0 4.6 4.6 4.6 4.6 7.7 7.9 9.0 5.8 7.2 4.6 8.9 11.5 4.6 4.6 13.7 4.6 5.3 6.6 4.6 4.6 13.7 4.6 4.6 4.6 4.6 6.5 6.8 7.7 4.6 6.6 4.6 12.1 15.7 4.6 4.6 21.3 4.6 8.1 9.8 4.6 7.2 24.2 4.6 4.6 4.6 4.6 8.9 9.1 8.8 7.8 8.7

S. Mean anatum 4.6 8.6 12.6 4.6 4.6 15.3 4.7 5.9 7.0 4.6 5.5 13.3 4.6 4.6 4.6 4.6 6.7 6.9 7.3 5.7 6.8

20 dietary spices Carum carvi L. Cinnamomum cassia Presl Cinnamomum burmannii B. Coriandrum sativum L. Cuminum cyminum L. Eugenia caryophylata Thunb. Illicium verum Hook. f. Laurus nobilis L. Mentha canadensis L. Myristica fragrans Houtt. Ocimum basilicum L. Origanum vulgare L. Petroselinum crispum L. Piper nigrum L. Piper nigrum L. Piper nigrum L. Rosmarinus officinalis L. Salvia officinalis L. Thymus vulgaris L. Zanthoxylum bungeanum Maxim. Mean of 20 spices 26 medicinal herbs Areca catechu L. Artemisia capillaris Thunb. Artemisia caruifolia Buch. Ham. Astragalus mongholicus Bge. Bupleurum scorzonerifolium Willd. Campsis radicans (L.) Seem. Cassia auriculata L. Chrysanthemum morifolium Ramat. Cornus officinalis Sieb. et Zucc. Fagopyrum cymosum (Trev.) Meisn. Houttuynia cordata Thunb. Inula britannica L. Lonicera japonica Thunb. Matteuccia struthiopteris (L.) Todaro Mucuna pruriens (L.) DC. Myrica nagi Thunb. Phellodendron amurense Rupr. Polygonum cuspidatum Sieb. et Zucc. Polygonum multiflorum Thunb. Prunella vulgaris L. Punica granatum L. Rhus succedanea L. Sanguisorba officinalis L. Scutellaria baicalensis Georgi Terminalia bellirica Roxb. Viola yedoensis Makino Mean of 26 herbs Overall mean
a b

Caraway Cinnamon Cinnamon stick Coriander Cumin Clove Star anise Bay Mint Nutmeg Sweet basil Oregano Parsley Green peppercorn Black pepper White pepper Rosemary Sage Thyme Chinese prickly ash

Fruit Cortex/bark Cortex/bark Whole plant Fruit Bud Fruit Leaf Leaf and branch Fruit Leaf Leaf Leaf Fruit Fruit Fruit Leaf and branch Leaf and branch Leaf and branch Fruit coat

4.6 4.6 5.0 7.0 8.7 12.1 4.6 4.6 4.6 4.6 10.1 17.5 4.6 4.7 4.6 4.6 4.6 5.9 4.6 4.6 4.6 4.6 7.3 10.5 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 5.8 4.6 6.0 4.6 6.6 4.6 5.6 5.2 6.4

Betelnut Yinchenhao Qinghao Huangqi Chaihu Lingxiaohua Cassia Juhua Shanzhuyu Jinqiaomai Yuxingcao Xuanfuhua Jinyinhua Guanzhong Cow-itch plant Box myrtle Huangbo Huzhang Heshouwu Xiakucao Shiliupi Diyu Huangqin Belliric myrobalan Diding

Seed Seed Aerial parts Root Root Flower Leaf and flower Inflorescence Fruit Rhizome Aerial parts Inflorescence Floral bud Rhizome Seed Bark Bark Rhizome Root Inflorescence Peel Gall Root Root Fruit Whole plant

107.0 15.8 23.3 4.8 3.0 17.9 118.6 29.3 26.1 65.2 14.4 14.3 23.6 87.1 90.8 153.8 13.4 74.0 72.5 36.4 249.6 224.8 177.9 52.9 132.5 25.7 71.3 57.5

11.8 2.6 3.5 0.8 0.5 2.2 9.5 4.8 2.3 6.7 2.2 2.4 3.6 10.1 6.2 15.0 1.7 8.1 5.7 4.7 22.6 12.1 15.9 8.2 9.3 2.4 6.7 5.6

18.3 4.8 6.2 4.6 4.6 4.6 12.7 7.4 5.6 9.3 5.0 4.6 6.3 17.5 8.8 17.6 4.6 16.4 8.3 7.4 24.6 15.5 16.5 10.9 12.1 4.6 10.0 8.8

17.9 4.6 4.6 4.6 4.6 4.6 13.2 6.0 4.6 8.4 4.6 4.6 4.6 16.2 7.7 19.5 4.6 19.9 8.0 6.0 15.2 18.9 13.8 11.0 11.9 4.6 9.4 8.2

21.1 5.5 7.0 4.6 4.6 4.6 17.4 9.2 10.9 12.4 6.0 4.6 7.2 19.9 11.6 26.9 4.6 20.4 11.6 9.1 32.3 22.0 20.9 15.1 17.3 4.6 12.8 11.0

8.6 4.6 4.6 4.6 4.6 4.6 7.1 4.6 4.6 5.2 4.6 4.6 4.6 7.5 4.9 9.5 4.6 6.4 4.6 4.6 14.5 8.8 11.3 6.2 7.3 4.6 6.2 5.8

12.0 4.6 4.6 4.6 4.6 4.6 10.6 5.5 4.6 7.3 4.6 4.6 4.6 7.7 6.8 15.9 4.6 12.8 6.3 7.5 19.2 8.6 12.4 8.6 12.2 4.6 7.9 7.2

15.6 4.8 5.4 4.6 4.6 4.6 12.2 6.5 6.1 8.5 5.0 4.6 5.5 13.7 8.0 17.9 4.6 15.2 7.8 6.9 21.2 14.8 15.0 10.4 12.2 4.6 9.2 8.2

TEAC (trolox equivalent antioxidant capacity) expressed as millimoles of trolox equivalent per 100 g dry weight (DW). TPC (total phenolic content) expressed as grams of gallic acid equivalents (GAE) per 100 g dry weight (DW). c The zone diameter of wells cut in PCA medium is 4.60 mm and the diameter of inhibition zone (DIZ) of negative control for each bacterium is also 4.6 mm. If the DIZ value is 4.6 mm (), that means the extract has no inhibitory activity against this bacterium.

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et al., 2004). ABTS+ solution (3.9 mL; absorbance of 0.700 0.005) was added to 0.1 mL of the test samples and mixed thoroughly. The reactive mixture was allowed to stand at room temperature for 6 min and the absorbance was immediately recorded at 734 nm using a Spectronic Genesys 5 spectrophotometer (Milton Roy, NY). Trolox standard solution (final concentration 015 m) in 80% ethanol was prepared and assayed under the same conditions. The absorbance of the resulting oxidized solution was compared with that of the calibrated trolox standard. Results were expressed in terms of trolox equivalent antioxidant capacity (TEAC, mmol trolox equivalents per 100 g dry weight of spice/herb powder). All tests were performed in triplicate. 2.7. Determination of total phenolic content Total phenolic content was estimated using the FolinCiocalteu colorimetric method described previously (Shan et al., 2005). The appropriate dilutions of the extract samples

0.2 mL were oxidized 4 min with 1 mL of 0.5 M Folin-Ciocalteu reagents and then the reaction was neutralized with saturated sodium carbonate (75 g/L) 1 mL. The absorbance of the resulting blue color was measured at 760 nm with a spectrophotometer after incubation for 2 h at room temperature. Quantification was done based on a standard curve of gallic acid. Results were expressed as gram of gallic acid equivalent (GAE) per 100 g of dry weight (DW). All tests were performed in triplicate. 3. Results 3.1. Antibacterial activities Three of the five bacteria used (B. cereus, L. monocytogenes and S. aureus) were Gram-positive and two (E. coli and S. anatum) were Gram-negative. There was a significant variation in the antibacterial activities (DIZ values) of 46 extracts (Table 1). For B. cereus, the DIZ values of 14 extracts (accounting for 30% of the 46 tested extracts) were between 10.3 mm and

Fig. 1. Relationship between diameter of inhibition zone (mm) and total phenolic content (g gallic acid equivalents/100 g DW) of 46 extracts from spices and herbs for five foodborne bacteria.

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24.6 mm and those of 18 extracts (39%) were between 4.7 mm and 9.3 mm. However, 14 extracts (30%) had no inhibitory activity. Punica granatum (shiliupi) exhibited the strongest antibacterial activity (DIZ = 24.6 mm), followed by Areca catechu (betelnut) (18.3 mm) and Myrica nagi (box myrtle) (17.6 mm). For S. aureus, 18 extracts (39%) exhibited high inhibitory activity (DIZ = 10.932.3 mm) and 13 extracts (28%) had low activity (5.59.8 mm). The remaining 15 extracts (33%) showed no inhibitory activity. The sample with the strongest antibacterial activity was P. granatum (DIZ = 32.3 mm). For L. monocytogenes, the DIZ values of 13 extracts (28%) ranged from 11.0 mm to 19.9 mm and those of 11 extracts (24%) were between 5.3 mm and 8.9 mm. The other 22 extracts (48%) showed no inhibitory activity. Polygonum cuspidatum (huzhang) showed the strongest inhibitory activity (DIZ = 19.9 mm). Of the two Gram-negative bacteria tested, for S. anatum, the DIZ values of 10 extracts (22%) varied from 10.5 mm to 19.2 mm. Fifteen extracts (33%) had low inhibitory activity (DIZ ranging between 4.7 mm and 8.6 mm). The other 21 plant

extracts (46%) showed no inhibitory activity. P. granatum exhibited the strongest antibacterial activity (DIZ = 19.2 mm), followed by Eugenia caryophylata (clove) (17.5 mm), and M. nagi (15.9 mm). For E. coli, 3 extracts (7%) exhibited high inhibitory activity (DIZ = 10.114.5 mm) and 13 extracts (28%) possessed less inhibitory activity (DIZ = 4.99.5 mm) while the other 30 extracts (65%) had no inhibitory activity. P. granatum also showed the strongest inhibitory activity (DIZ = 14.5 mm), followed by Sanguisorba officinalis (diyu) (11.3 mm) and E. caryophylata (10.1 mm). In general, a total of 12 spices and herbs showed relatively high inhibitory activities against the five foodborne pathogenic bacteria tested, i.e. P. granatum, M. nagi, S. officinalis, A. catechu, E. caryophylata, P. cuspidatum, Rhus succedanea, Matteuccia struthiopteris, Origanum vulgare, Cinnamomum burmannii B., Terminalia belliric, and Cassia auriculata. Comparison of the average values of antibacterial activities between 20 spices (mean DIZ = 6.8 mm) and 26 herbs (9.2 mm) (Table 1) showed that medicinal herbs exhibited significantly stronger average activity than dietary spices.

Fig. 2. Relationship between diameter of inhibition zone (mm) and total antioxidant capacity (mmol trolox/100 g DW) of 46 extracts from spices and herbs for five foodborne bacteria.

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3.2. Sensitivity of five foodborne bacteria B. cereus, L. monocytogenes, S. aureus, E. coli, and S. anatum strains exhibited varying sensitivities to the 46 tested extracts (Table 1). Of the three Gram-positive bacteria tested, S. aureus was the most sensitive. Thirty-one extracts had inhibitory activity against this bacterium (mean DIZ = 11.0 mm; ranging from 5.5 to 32.3 mm) and 15 extracts were without activity. With respect to B. cereus thirty-two extracts showed inhibitory activity (mean = 8.8 mm; 4.724.6 mm) and 14 extracts had no activity. L. monocytogenes was the most resistant of the tested organisms. Twenty-four extracts exhibited inhibitory activity (mean = 8.2 mm; 5.319.9 mm) and 22 extracts had no activity. Of the two Gram-negative bacteria, S. anatum was more sensitive than E. coli. Twenty-five extracts showed inhibitory activity against S. anatum (mean = 7.2 mm; 4.719.2 mm) and only 16 extracts exhibited inhibitory activity against E. coli (mean = 5.8 mm; 4.914.5 mm). Based on the mean values of inhibitory activity, Gram-positive bacteria demonstrated more sensitivity to the extracts than Gram-negative bacteria. 3.3. Relationships between antibacterial activity, total phenolic content, and antioxidant capacity The correlations between antibacterial activity and total phenolic content are shown (Fig. 1). The R2 values were between 0.93 and 0.73, and decreased in the following order: S. aureus N B. cereus N E. coli N S. anatum N L. monocytogenes. The relationship between antibacterial activity and antioxidant capacity of the 46 extracts was also computed (Fig. 2). The R2 values were between 0.84 and 0.70, and decreased in the following order: E. coli N S. aureus N B. cereus N S. anatum N L. monocytogenes. 4. Discussion Of the 46 spice and herb extracts tested in this study, twelve exhibited high antibacterial activities against the five foodborne bacteria. To some extent, these results were similar to those of previous studies. Methanolic and ethanolic extracts of P. granatum were effective against B. cereus, E. coli, and S. aureus (Negi and Jayaprakasha, 2003; Voravuthikunchai et al., 2004; Voravuthikunchai and Kitpipit, 2005). The extracts and essential oils of E. caryophylata, O. vulgare, and C. burmannii B. had significant inhibitory properties against S. aureus, E. coli, and L. monocytogenes (Shelef, 1984; Zaika, 1988; Smith-Palmer et al., 1998; Kalemba and Kunicka, 2003; Ceylan and Fung, 2004; Lopez et al., 2005). Kokoska et al. (2002) reported that the ethanolic extracts of S. officinalis had strong antimicrobial activity against B. cereus, E. coli, and S. aureus. Kim et al. (2005) found that the extracts of P. cuspidatum strongly inhibited the growth of B. cereus, S. aureus, and E. coli. Samy and Ignacimuthu (2000) reported that C. auriculata exhibited significant activity against E. coli and S. aureus. Further, this study has shown a link between the concentration of phenolic compounds in the extracts and their antibacterial activity.

In the present study, among the five bacteria tested, S. aureus was the most sensitive to the 46 extracts, while E. coli was the most resistant. The highest sensitivity of S. aureus may be due to its cell wall structure and outer membrane (Zaika, 1988). Our results suggest that Gram-positive bacteria are generally more sensitive to the spice and herb extracts than Gram-negative bacteria. This was consistent with the previous studies on other spices and herbs (Smith-Palmer et al., 1998; Zaika, 1988; Ceylan and Fung, 2004; Lopez et al., 2005). A possible explanation for these observations may lie in the significant differences in the outer layers of Gram-negative and Grampositive bacteria. Gram-negative bacteria possess an outer membrane and a unique periplasmic space not found in Grampositive bacteria (Nikaido, 1996; Duffy and Power, 2001). The resistance of Gram-negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier to the penetration of numerous antibiotic molecules and is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside (Russell, 1991; Nikaido, 1994; Gao et al., 1999). Gram-positive bacteria do not have such an outer membrane and cell wall structure. Antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane and result in a leakage of the cytoplasm and its coagulation (Kalemba and Kunicka, 2003). However, three extracts (E. caryophylata, T. bellirica, and P. vulgaris) tested in this study did not completely follow the trend described above. Although a Gram-positive bacterium (S. aureus) was more sensitive to these three extracts than a Gram-negative bacterium (E. coli), the inhibitory activities of these three extracts against S. anatum were greater than the effects on B. cereus and L. monocytogenes. This suggests that there might be some particular anti-Gram-negative substances in these three extracts. Many previous studies have reported the antibacterial activity, phenolic content or antioxidant activities of spices and herbs. But it was not easy to compare directly the results of different studies and to establish reasonable relationships between antibacterial activity, phenolic content and antioxidant activity because of the low number of spice and herb samples tested, different determination methods and different bacterial strains used. Previous results (Shan et al., 2005) found high correlations, based on the analysis of a large number of samples and the wide ranges in total phenolic content and antioxidant capacity. The current study investigated a total of 46 spice and herb extracts from different regions and resulted in good linear relationships between antibacterial activity and total phenolic content. The results emphasized the importance of phenolic compounds in the antibacterial activity of spice and herb extracts and also indicated that the phenolic compounds significantly contributed to their antibacterial activity. In addition, the present study shows that highly positive relationships also exist between the antibacterial activity and antioxidant capacity of the extracts. Previous work (Cai et al., 2004; Shan et al., 2005) showed that there were highly positive linear correlations between antioxidant capacity and total

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phenolic content of a large number of spices and herbs (including most of the samples tested in this study). The present work shows that antibacterial activity is closely related to the concentration of phenolic compounds and thus to the antioxidant capacity of the extracts. Other researchers have also reported that phenolic compounds from different plant sources could inhibit various foodborne pathogens (Nychas, 1995; Smid and Gorris, 1999; Prashanth et al., 2001; Kim et al., 2005). Polyphenols, such as tannins and flavonoids, are important antibacterial substances. Ellagitannin (punicalagin) in pomegranate (P. granatum) is the active substance responsible for its antimicrobial activity (Machado et al., 2002). Many plant flavonoids (e.g., epigallocatechin, catechin, myricetin, quercetin) have antimicrobial activity (Cushnie and Lamb, 2005). The most active constituents (essential oils) of many spices having wide spectra of antimicrobial activity are aromatic phenolic compounds, such as thymol and carvacrol in oregano and thyme, eugenol in clove and cinnamon, and cinnamic aldehyde in cinnamon (Karapinar and Aktug, 1987; Beuchat and Golden, 1989). These bioactive principles in the related dietary spices and medicinal herbs were also identified in previous studies (Cai et al., 2004; Shan et al., 2005). The antimicrobial activities of phenolic compounds may involve multiple modes of action. For example, essential oils degrade the cell wall, interact with the composition and disrupt cytoplasmic membrane (Sikkema et al., 1994; Helander et al., 1998; Ultee et al., 1999; Lambert et al., 2001), damage membrane protein, interfere with membrane integrated enzymes (Raccach, 1984), cause leakage of cellular components, coagulate cytoplasm, deplete the proton motive force, change fatty acid and phospholipid constituents, impair enzymatic mechanisms for energy production and metabolism, alter nutrient uptake and electron transport (Taniguchi et al., 1988), influence the synthesis of DNA and RNA and destroy protein translocation and the function of the mitochondrion in eukaryotes (Raccach, 1984; Nychas, 1995). All of these mechanisms are not separate targets; some are affected as a consequence of another mechanism being targeted. The mode of action of antimicrobial agents also depends on the type of microorganisms and is mainly related to their cell wall structure and the outer membrane arrangement. Plants including spices and herbs contain complex phenolics (e.g., phenolic acids, flavonoids, tannins, lignans, coumarins, quinines). The phenolic compounds in the spices and herbs tested had not been identified thoroughly. The mechanisms of action of each phenolic compound against various bacteria are also very complicated (Kalemba and Kunicka, 2003; Burt, 2004). Therefore, it is necessary to investigate further and understand the relationship between antibacterial activity and chemical structure of each phenolic compound in the tested extracts. In conclusion, this is the first systematic study to report a highly positive relationship between antibacterial activity and total phenolic content in a large number of spice and herb extracts. This suggested that the phenolic compounds might significantly contribute to their antibacterial activity. The present study also demonstrated that many of the extracts (especially medicinal herbs) contained high levels of phenolics and possessed strong antibacterial activity. They could be a

potential source for inhibitory substances against some foodborne pathogens as well as antioxidant agents. Twelve extracts with high antibacterial activity selected in this study may be candidates for future studies of synergism, compatibility, and activity in foods or food-processing systems and mechanisms of activity against specific pathogens. Acknowledgements This work was supported by the Faculty of Science Research Collaboration Grant of The University of Hong Kong. We thank Mr. S. Surveswaran for supply of five Indian help samples. References
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