Quinone is a class of bioactive compound that may be components of anti-cancer chemotherapy drugs [Dandawate et al., 2010]. 2,6-Dimethoxy-1,4-benzoquinone (2,6- DMBQ) exerts significant in vitro cytotoxicity against human tumor cell lines and also exhibits antibacterial activities (Fig. 1) [Tömösközi-Farkas and Daood, 2004; Lana et al, 2006; Kim et al., 2010]. 2,6-DMBQ has been identified in a variety of plant families, including wheat (Triticum vulgaris) and other food crops [Chen et al., 1994; Harasawa and Tagashira, 1994; Kwon et al., 2001]. 2,6-DMBQ and 2-methoxy-benzoquinone (2-MBQ) are especially abundant in wheat germ as the glycone type [Tömösközi-Farkas and Daood, 2004]. Lana et al. [2006] suggested that Eucalyptus tar could be a prominent substrate for 2,6-DMBQ production, because wood tar is inexpensive and contains abundant 2,6-dimethoxyphenol, which can be oxidized into 2,6-DMBQ. FWGE ® is the trade-name of a standardized, natural nutrient compound that has been extensively studied for the treatment of cancer and autoimmune diseases [Boros et al., 2005; Illmer et al., 2005; Telekes et al., 2009]. FWGE ® is made by fermenting wheat germ with baker’s yeast (Saccharomyces cerevisiae), which harbors βglucosidase activity, and of which aglycone-type 2,6- DMBQ and 2-MBQ are important components [Tömösközi-Farkas and Daood, 2004; Telekes et al., 2009].

Quinone is a class of bioactive compound that may be components of anti-cancer chemotherapy drugs [Dandawate et al., 2010]. 2,6-Dimethoxy-1,4-benzoquinone (2,6- DMBQ) exerts significant in vitro cytotoxicity against human tumor cell lines and also exhibits antibacterial activities (Fig. 1) [Tömösközi-Farkas and Daood, 2004; Lana et al, 2006; Kim et al., 2010]. 2,6-DMBQ has been identified in a variety of plant families, including wheat (Triticum vulgaris) and other food crops [Chen et al., 1994; Harasawa and Tagashira, 1994; Kwon et al., 2001]. 2,6-DMBQ and 2-methoxy-benzoquinone (2-MBQ) are especially abundant in wheat germ as the glycone type [Tömösközi-Farkas and Daood, 2004]. Lana et al. [2006] suggested that Eucalyptus tar could be a prominent substrate for 2,6-DMBQ production, because wood tar is inexpensive and contains abundant 2,6-dimethoxyphenol, which can be oxidized into 2,6-DMBQ. FWGE ® is the trade-name of a standardized, natural nutrient compound that has been extensively studied for the treatment of cancer and autoimmune diseases [Boros et al., 2005; Illmer et al., 2005; Telekes et al., 2009]. FWGE ® is made by fermenting wheat germ with baker’s yeast (Saccharomyces cerevisiae), which harbors βglucosidase activity, and of which aglycone-type 2,6- DMBQ and 2-MBQ are important components [Tömösközi-Farkas and Daood, 2004; Telekes et al., 2009].

 Nuruk, a traditional Korean fermentation starter, is produced by the natural proliferation of fungi and other microorganisms on crushed or whole wheat kernels [Yu et al., 1998]. Recent studies have demonstrated the biological activities of nuruk [Kim et al., 2008; Kwak et al., 2008; Lee et al., 2009]. Extracts of nuruk were associated with inhibitory effects on hypertension, platelet aggregation, migration, and angiogenesis of cancer cells [Lee et al., 2009]. Nuruk extract is also associated with decreases in lipopolysaccharide (LPS)- induced nitrite and interleukin (IL)-6 in RAW 264.7 cells by inhibiting the activation of p38 mitogen-activated protein kinase [Kim et al., 2008]. Four sterol compounds, ergosterol peroxide, stigmast-5-en-3β,7β-diol, ergosta7,22-dien-3β,5α,6β,9α-tetraol, and daucosterol, have been isolated from nuruk [Lee et al., 2009]. Accordingly, renewed evaluation of nuruk is warranted to explore its apparent health-enhancing biological activities. In the present study, the presence of 2,6-DMBQ in nuruk and the 2,6-DMBQ contents of five different nuruks are reported. Materials and Methods Nuruk and chemicals. Nuruk was purchased from five local markets (located in Andong, Busan, Hwaseong, Jinju, and Sangju) in Korea. Commercially available 2,6- DMBQ (TCI Chemicals, Tokyo, Japan) was used as the standard. Formic acid and methanol were purchased from Wako Pure Chemicals (Osaka, Japan). Extraction of 2,6-DMBQ. Nuruk (20 g) was added to a 2-L Erlenmeyer flask containing 300 mL sterile water. 

The reaction mixture was incubated at 30o C for 24 h with vigorous shaking and centrifuged at 6,000 g for 10 min. After measurement of β-glucosidase enzyme activity as described by Hong et al. [2009], the supernatant was sterilized at 121o C for 15 min, filtered through a 0.45-µm filter (Whatman, Maidstone, England), and dried using a freeze-dryer (Ilshin Engineering Co., Seoul, Korea). The lyophilizate was harvested and stored at −80o C until further analysis. High-performance liquid chromatography (HPLC) analysis. The operation conditions for HPLC analysis described previously were implemented with slight modification [Tömösközi-Farkas and Daood, 2004]. One gram of the lyophilizate was dissolved in 50 mL sterile water and extracted three times with 100 mL chloroform. The chloroform layers were pooled and evaporated to dryness using a vacuum evaporator (EYELA, Tokyo, Japan). The resulting dry pellet was dissolved in 20 mL mobile phase [methanol (24%, v/v) in water containing 1 mM formic acid, pH 4.2]. Analysis of 2,6-DMBQ was performed on a Shimadzu Prominence LC-20A HPLC system equipped with an UV/VIS SPD-20A detector (Shimadzu Co., Kyoto, Japan) and an inertsil ODS-3 C18 column (GL Sciences, Tokyo, Japan). The wavelength of the UV detector was set at 290 nm, and the flow rate of the mobile phase was 0.7 mL/min. Column temperature was maintained at 40o C. LC-MS and LC-MS/MS analyses. Liquid chromatography-mass spectrophotometry (LC-MS) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) experiments were performed using the Quantum Ultra Triple-Stage Quadrupole system (Thermo Fisher Scientific, Pittsburgh, PA). 

A C18 Hydrosphere column (GL Sciences, Tokyo, Japan) was used for separation. The mobile phase flowed at a rate of 200 µL/ min. The ionization was achieved using an electrospray ionization source in the positive selected reaction monitoring (SRM) mode. The spray voltage, capillary temperature, sheath gas pressure, auxiliary gas pressure, and capillary temperature were 4.2 kV, 320o C, 50 psi, and 20 psi, and 320o C, respectively. Results and Discussion The HPLC chromatograms of the samples prepared from nuruk were similar to that of the commercially available 2,6-DMBQ (standard) (Fig. 3). In addition, the m/z (mass-to-charge ratio) values of both the standard 2,6-DMBQ and nuruk extract were 168.91, and MS2 fragmentation induced by a collision energy of 54 eV were the same for the samples and standard. The results of HPLC and LC-MS/MS analyses showed that nuruk contains 2,6-DMBQ. Among the five samples, the highest 2,6-DMBQ content was found in nuruk purchased in Hwaseong, which contains 2,6-DMBQ at 1.16±0.07 mg/50 g. Nuruk purchased in Jinju showed the lowest content (0.38±0.01 mg/50 g). However, 2-MBQ, which is found in fermented wheat germ, was not detected in nuruk. Contents of 2,6-DMBQ in nuruk from the five different local markets are shown in Table 1. In the present study, 2,6-DMBQ was hypothesized to be produced by the actions of microbial β-glucosidases in nuruk. Nuruk contained much lower 2,6-DMBQ content than that of the commercial product FWGE ®, which may be explained by the fact that nuruk is usually not made of wheat germ, but with wheat bran or wheat kernel [Lee et al., 1994] in which the intrinsic content of the glycone type of 2,6-DMBQ is low compared to that of wheat germ [Tömösközi-Farkas and Daood, 2004]. 

The specific microorganisms present in different nuruk and their enzymes may play important roles in converting the glycone form of 2,6-DMBQ to its aglycone form. Nuruk is naturally inoculated by airborne microorganisms, and thus contains a variety of fungi, yeast, and bacteria [Yu et al., 1998]. Accordingly, nuruk from different local areas may vary in microbial flora that produce βglucosidase. Nuruk purchased in Hwaseong exhibited the highest β-glucosidase enzyme activity among the five nuruk samples (Table 1), which suggests that the enzyme activity of b-glucosidase is critical to the production of the aglycone form of 2,6-DMBQ in nuruk. Because the pivotal microorganisms involved in production of 2,6- DMBQ in nuruk are unknown, further study on enumeration and characterization of the microorganisms harboring β-glucosidase activity could be necessary. In conclusion, to the best of our knowledge, this is the first study to demonstrate the presence of the bioactive compound 2,6-DMBQ in nuruk, which may help explain the health-promoting functions of traditional Korean fermented alcoholic beverages that employ nuruk as a Acknowledgments. This work was supported by the Korea Small & Medium Business Administration (C1007722-01-01) and the Regional Partnership Development Project of the Ministry of Knowledge Economy (70011259).