Natural antimicrobials have been developed to cure bacterial foodborne illnesses and to control the increasing bacterial resistance to antibiotics currently used in therapeutics [11, 14, 15, 18, 27, 28]. As such, antimicrobial research with natural products represents a new field. In addition, there is an increased need for natural preservatives in food, beverages, cosmetics, and food packaging. More than 2,000 naturally occurring quinones, such as anthraquinones, naphthoquinones, and benzoquinones, are now known and have been found to be widely distributed in nature as intermediates in cellular respiration and photosynthesis [2, 4, 20]. Hydro- and benzoquinones are members of the naturally occurring quinones, which have an interesting biological mode of action. Most quinones, including ubiquinones and menaquinones, are often involved in electron transport [16]. They provide a role in defense as a result of their effectiveness in inhibiting the growth of bacteria, fungi, and parasites [8]; therefore, a number of them have various physiological activities as antimicrobial and anticancer compounds [21]. Specifically, some benzoquinones such as 2,6-dimethoxy-1,4-benzoquinone (DMBQ) exhibit cytotoxic effects in Ehrlich ascites tumor cells (EATC) [22, 23], and thereby inhibit tumor propagation. DMBQ derived from different plant species is of special interest. Recent studies have reported that DMBQs from the roots of Gunnera perpensa [5] and Eucalyptus tar [13] are strongly antibacterial. Furthermore, it has been shown that benzoquinone-producing plants possess specific therapeutic properties that may be responsible for their beneficial effect on human health [10]. 

Natural antimicrobials have been developed to cure bacterial foodborne illnesses and to control the increasing bacterial resistance to antibiotics currently used in therapeutics [11, 14, 15, 18, 27, 28]. As such, antimicrobial research with natural products represents a new field. In addition, there is an increased need for natural preservatives in food, beverages, cosmetics, and food packaging. More than 2,000 naturally occurring quinones, such as anthraquinones, naphthoquinones, and benzoquinones, are now known and have been found to be widely distributed in nature as intermediates in cellular respiration and photosynthesis [2, 4, 20]. Hydro- and benzoquinones are members of the naturally occurring quinones, which have an interesting biological mode of action. Most quinones, including ubiquinones and menaquinones, are often involved in electron transport [16]. They provide a role in defense as a result of their effectiveness in inhibiting the growth of bacteria, fungi, and parasites [8]; therefore, a number of them have various physiological activities as antimicrobial and anticancer compounds [21]. Specifically, some benzoquinones such as 2,6-dimethoxy-1,4-benzoquinone (DMBQ) exhibit cytotoxic effects in Ehrlich ascites tumor cells (EATC) [22, 23], and thereby inhibit tumor propagation. DMBQ derived from different plant species is of special interest. Recent studies have reported that DMBQs from the roots of Gunnera perpensa [5] and Eucalyptus tar [13] are strongly antibacterial. Furthermore, it has been shown that benzoquinone-producing plants possess specific therapeutic properties that may be responsible for their beneficial effect on human health [10]. 

In our preliminary screening of several plant seeds previously characterized as antibacterial agents against Gram-negative and -positive bacteria, we discovered that wheat germ extract (WGE) from Triticum vulgaris contains DMBQ and is highly inhibitory to Staphylococcus aureus KCTC1927 and Bacillus cereus KCTC1014. Wheat germ is the nutrient-rich embryo of the wheat kernel that is removed during the processing of whole wheat grains to white flour. It makes up about 2% to 3% percent of the entire wheat kernel. The antibacterial activity of wheat germ has rarely been reported. We report herein the content of a major bioactive compound DMBQ in WGE by high-performance liquid chromatography (HPLC). We also evaluate the antibacterial activity of the standard benzoquinone derivatives 1,4- benzoquinone (BQ), hydroquinone (HQ), methoxybenzoquinone (MBQ), and DMBQ, using the well-diffusion assay against standard strains of S. aureus, Escherichia coli, Salmonella typhimurium KCTC2054, and B. cereus. To evaluate the relationship between molecular structure and antibacterial activity, we determined the minimum inhibitory concentration (MIC) and time-kill curve of the active compounds. MATERIALS AND METHODS Materials Wheat germ was purchased from DongA One Corporation (Seoul, Korea). BQ (98% purity), HQ (97%), MBQ (98%), DMBQ (97%), and chloramphenicol (CM, 98%) standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The test strains, S. aureus KCTC1927, E. coli KCTC2593, S. typhimurium KCTC2054, and B. cereus KCTC1014, were purchased from the Korean Collection for Type Cultures (Biological Resource Center, Daejeon, Korea). Nutrient broth (0.03% beef extract, 0.05% peptone) for the antibacterial assay was obtained from BBL Microbiology System (Cockeysville, MD, USA). 

Beef extract and peptone were purchased from Difco Laboratories (Detroit, MI, USA). Unless otherwise noted, all chemicals were purchased from Sigma. Sample Preparation Ground wheat germ (10 g) was dissolved in 250 ml of double distilled water and then extracted three times by shaking with 100 ml of chloroform (CHCl3). The CHCl3 layers were pooled, washed twice with distilled water, and dried over anhydrous Na2SO4. The filtrate was evaporated to dryness in a vacuum evaporator at a maximum temperature of 40o C. The dry material (600 mg) was finally redissolved in CHCl3 and filtered through a 0.45-µm PTFE filter aid. Twenty µl of the final filtrate was injected into the HPLC column. Antibacterial Assay Antibacterial activity was detected by the modified disk diffusion method [1]. S. aureus, E. coli, S. typhimurium, and B. cereus were subcultured in nutrient medium and incubated for 18 h at 37o C (30o C for B. cereus), and then the bacterial cells were suspended in saline solution according to the McFarland protocol to produce a suspension of about 105 CFU/ml. A volume of 0.5 ml of this suspension was mixed with 150 ml of nutrient agar at 40o C and poured onto an agar plate (23×23 cm) in a laminar flow cabinet. Finally, agar wells were cut from the seeded agar medium using a hollow tube (7 mm diameter) and applying slight negative pressure to remove the plug of agar. Each test compound was dissolved in CHCl3, and 150 µl was added to wells containing bacterial cells. Wells containing CM (100, 500, and 1,000 ppm) and CHCl3 only were used as positive and negative controls, respectively. 

The susceptibility of the bacteria to the test compounds was determined by the formation of an inhibition zone after 18 h of incubation at 37o C. Experiments were run in triplicate, and the results are presented as mean values of the three measurements. The MIC was evaluated by the macrodilution test using the method of Christoph et al. [3] with slight modifications. Briefly, serial 2-fold dilutions of the test compounds were prepared in dimethylsulfoxide, and 0.5 ml of each dilution was added to 9.0 ml of nutrient broth. These solutions were inoculated with 0.5 ml of an overnight culture of S. aureus, E. coli, S. typhimurium, or B. cereus. After incubation of the cultures at 37o C (30o C for B. cereus) for 48 h, the MIC was determined as the lowest concentration of the test compound that demonstrated no visible growth. HPLC Analysis of 1,4-Benzoquinones The 1,4-benzoquinones in WGE were analyzed by an HPLC method [29] with minor modifications. The extract was prepared as described above. The HPLC system consisted of a Tosoh 8010 series (Tosoh Corporation, Japan) equipped with a UV 8010 diodearray UV-vis detector (Tosoh) at 275 nm, and an RP-Amide C16 (250×4.6 mm) column (Supelco, Bellefonte, PA, USA). The mobile phase used a water:acetonitrile [80:20 (v/v)] mixture containing 0.0025 M KH2PO4, where the flow rate and sample injection volume were fixed at 0.7 ml/min and 20 µl, respectively. As the reference ingredient, DMBQ in 100% CHCl3 was used to calibrate the standard curve and retention times. Time-Kill Study Bacteria were cultivated with each compound as described above for the determination of MIC. At selected time points, samples were withdrawn and serially diluted in sterile saline, plated on nutrient agar.

Thus, the chloroform extract of wheat germ, which has a high DMBQ content, has potential as a food-based source for the safe production and enhancement of antimicrobial components. Antibacterial Spectrum of 1,4-Benzoquinones Since WGE showed strong antibacterial activity against the selected foodborne pathogens (Table 1) and had a high DMBQ content, we compared its activity with that of various concentrations (100, 500, 1,000 ppm) of the 1,4- benzoquinone standards BQ, MBQ, DMBQ, and HQ (Table 2). Pure DMBQ had the highest anti-S. aureus activity (27.7 mm) followed by CM (24.2 mm), MBQ (14.2 mm), HQ (12.6 mm), and BQ (11.4 mm). These results reveal an interesting structure-dependent activity for these compounds. Among them, BQ is a 1,4-benzoquinone, whereas MBQ and DMBQ are methoxylated 1,4-benzoquinones. BQ was active against all four bacteria tested, whereas DMBQ and MBQ showed especially potent antibacterial activity against the Gram-positive bacterium S. aureus with an inhibition zone of 27.7 and 14.2 mm in diameter, respectively. In the case of DMBQ, the anti-S. aureus (27.7 mm) and anti-S. typhimurium (23.5) activities were higher than that of the reference antibiotic CM (24.2 and 18.3 mm, respectively) at a concentration of 1,000 ppm. Many of the benzoquinone derivatives tested showed a comparable and selective inhibition of the foodborne pathogens. These results suggest that DMBQ, the major benzoquinone component of WGE, may play an important role in the antibacterial activity of wheat germ. MICs of 1,4-Benzoquinones The MICs of the active 1,4-benzoquinones were determined and are shown in Table 3. BQ was active against S. aureus up to a dose of 8 µg/ml, the same MIC found for DMBQ.

 Interestingly, the MICs observed for DMBQ, which bears two methoxy groups, were as high as those obtained for BQ, except when tested against B. cereus, in which case the value was twice as high. This result is due to an interesting effect of the methoxy identity (presence of methoxy groups) of 1,4-benzoquinones. As shown in Table 3, the anti-S. typhimurium activity (MIC) of MBQ was significantly lower than that of DMBQ (>512 vs. 32 µg/ml, respectively). The difference in the number of methoxy groups in the 1,4-benzoquinones significantly affected their antibacterial activities against the Gram-negative bacteria S. typhimurium and E. coli; the introduction of one methoxy group in the nucleus resulted in decreased activity. Furthermore, HQ, a reduced form of BQ, had significantly lower antibacterial activity than BQ (Table 3). This result also indicates that reduction of BQ could decrease its antibacterial activity. These data clearly demonstrate that the number of methoxy groups and reduction of BQ may play an important role in antibacterial activity, an observation that is in agreement with the previous study by Lana et al. [13]. Similar results have been reported by Koyama [10], who demonstrated that the dimethoxylated structure is crucial for potent antibacterial activity. Time-Kill Curves of Selected 1,4-Benzoquinones The bactericidal activities of BQ against S. typhimurium and S. aureus were confirmed by the time-kill curve experiment, as shown in Fig. 2 and 3. The result verifies that the MIC and minimal bactericidal concentration (MBC) of BQ against S. typhimurium were the same. The MIC of BQ (32 µg/ml) significantly reduced the growth rate of S. typhimurium (Fig. 2A).

 It should be noted that lethality occurred quickly, within the first hour after the addition of BQ, suggesting that the antibacterial activity of BQ against S. typhimurium was associated with membrane disruption, similar to its effect on S. aureus (Fig. 3A). The bactericidal effect of DMBQ was also confirmed by the time-kill curve experiment, as shown in Fig. 2B and 3B. The effect of DMBQ against S. typhimurium cells was bacteriostatic for the first 5 h of incubation after addition of the compound, but its bactericidal effect was expressed after 12 h of incubation. Lethality against S. aureus occurred more slowly than with BQ, 12 h after adding DMBQ (Fig. 3A and 3B). Thus, doubling the MIC of DMBQ to 16 µg/ml reduced the growth rate of S. aureus but did not have a siginificant effect on the final cell count (Fig. 3B). of antibacterial action of BQ and DMBQ against S. typhimurium and S. aureus differ to some extent. Previous researchers have demonstrated that BQ is a potent sulfhydryl arylator that is highly cytotoxic [26]. BQ may have undergone arylation, binding to glutathione or protein thiols [26]. In contrast, DMBQ, while capable of strongly inducing oxidative stress by redox cycling, is only slightly cytotoxic [6]. Quinones are important biological molecules that are active against a variety of cancer cells [2, 25], viruses [12], and fungi [17]. Some semisynthetic analogs of substituted 1,4-benzoquinones have in vitro cytotoxic and antioxidant activities [19]. Quinones also play a critical role in energy metabolism and even in chemotherapy where redox cycling drugs are utilized. However, the molecular mechanisms involved in quinone cytotoxicity are still mostly unknown [26]. 

Since quinones are widely used as antibiotics and antitumor agents and for a variety of other purposes, it is critical that we understand their effects on cellular function. The present study revealed the selective antibacterial activity of 1,4-benzoquinone derivatives against microorganisms that cause food poisoning [7, 9, 24] and once again points out the importance of structure in the antibacterial activity of these compounds. The antibacterial mechanism of 1,4- benzoquinone compounds from wheat germ against Grampositive bacteria seems to be an interesting subject for further studies. The simple preparation of active 1,4- benzoquinone derivatives from wheat germ, reported herein, should be of interest to food industries, where such economically useful compounds could be extracted from waste materials. The toxicity of benzoquinone derivatives and structurally similar compounds is not well defined, but seems to be low enough to permit the development of new antimicrobials for human use or as agents to prevent bacterial food spoilage [13]. The results of the present study provide further insight into the molecular basis of the antibacterial action of these compounds, and a foundation for further studies regarding the production and enhancement of bioactive antimicrobial compounds using grains with high DMBQ content. In conclusion, WGE could serve as a safe and inexpensive natural antimicrobial against foodborne pathogens for use in food and cosmetic applications. 

Wheat germ, a by-product generated in large quantities by the flour milling industry, could thus be utilized as a source of this antimicrobial agent. Acknowledgments This research was financially supported by the Ministry of Education, Science and Technology (MEST) and the Korea Institute for Advancement of Technology (KIAT) through the Human Resource Training Project for Regional Innovation, and a research grant from Hannam University, Daejeon, Korea in 2010.