Identification of antioxidants in dark soy sauce

IDENTIFICATION OF ANTIOXIDANTS IN DARK SOY SAUCE WANG HUANSONG A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCES DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements This journey is not possible without the help of so many people. First and foremost, I would especially like to express my most sincere and profound appreciation to my supervisor, Professor Barry Halliwell, for his constant guidance, invaluable suggestion and critical comments throughout this work. I am also grateful to all my colleagues and friends inside/outside Professor Halliwell’s group, who help me in this way or that way, especially to Dr Tang Soon Yew for his useful discussions and many technical support in cell culture, Dr Andrew Jenner and Dr Lee Chung-Yung for their useful suggestions, Dr Shui Guanghou, Professor Markus Wenk’s group, for his help in using LC-TOF-MS, Dr Koh Hwee Ling, Department of Pharmacy, for her help in using FT-IR, Dr Mark Richards, Agilent Technologies Singapore, for his help in LC-APCI-Ion trap -MS/MS measurement, and Professor Yong Eu Leong, Department of Obstetrics & Gynecology, for his help in using triple quadrupole LC-MS/MS. My heartfelt thanks also go to the Department of Biochemistry, the Centre of Life Sciences, the National University of Singapore for holding so many exciting talks and creating a wonderful academic atmosphere. Last but not least, I thank my wife, Shen Ping, for her patience and consistent support. I would like to dedicate this thesis to my son, Bo Qian. i    Table of Contents Acknowledgements i Table of Contents ii Summary iv List of Tables v List of Figures and Chart vi List of Symbols ix Chapter 1 Introduction 1 1.1 A brief history of soy sauce 1 1.2 The methodology of preparation of soy sauce 3 1.3 The functional components of soy sauce 5 1.4 The balance of free radicals/reactive species and antioxidants 6 Chapter 2 Materials and Methods 8 2.1 Chemicals 8 2.2 ABTS assay 8 2.3 Isolation of low molecular mass components from ethyl acetate extract 9 2.4 Fraction of colored components 9 2.5 HPLC determination of maltol in dark soy sauce 11 2.6 Mass spectrometry 12 2.7 Fourier transfer infrared spectrometry (FTIR) 13 2.8 Nuclear Magnetic resonance spectrometry (NMR) 13 2.9 Detection and determination of maltol metabolites in human urine samples 13 2.9.1 Standard preparation 13 2.9.2 Sample preparation 14 ii    2.9.3 HPLC-DAD detection of maltol metabolites and determination of total maltol content in urine 14 2.9.4 HPLC-MS/MS detection of maltol metabolites 15 2.10 GC-MS analysis of DNA base modification 16 2.10.1 Sample preparation 16 2.10.2 GC-MS analysis 16 2.11 Cell culture 17 2.12 Assessment of cell viability 17 2.13 Western blot analysis 18 2.14 Statistical analysis 18 Chapter 4 Results and Discussion 19 4.1 Separation and characterization of low molecular mass components 19 4.2 Content of maltol and its contribution to the total antioxidant activity of dark soy sauce 26 4.3 Maltol excretion in urine 32 4.4 Fractionation and characterization of the colored components 42 4.5 Protection against HOCl-induced DNA damage 49 4.6 Cytotoxicity on HT-29 cells 51 4.7 Inhibition of COX-2 protein expression in LPS-induced HT-29 cells 54 4.8 Discussion 56 Chapter 5 Conclusion 61 Bibliography 62 Appendices 70 iii      Summary Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS. In vitro, maltol, CP1 and CP2 can protect against hypochlorous acid (HOCl)-mediated DNA damage dosedependently. Furthermore, dark soy sauce potentially inhibits the growth of colon cancer HT 29 cells at high concentrations, while it decreases the up-regulation of cyclooxygenase-2 (COX-2) expression in LPS (lipopolysaccharide) -induced HT 29 cells at low concentrations. iv    List of Tables Table I. 1H- and 13C-NMR data of Compound 1. 25 Table II. Within- and between-assay precision and recoveries of the assay used to measure maltol. 27 Table III. The observed ions in TOF-MS spectrum of CP1. Three series were observed. Within each series, m/zs of the doubly-charged or the singly-charged ions consistently increase by 81 or 162, respectively. 48 Table IV. Inhibition of HOCl-induced DNA damage by maltol, the colored product 1 (CP1) and the colored product 2 (CP2). 50 v List of Figures and Chart Figure 1. Trolox equivalent antioxidant capacity (TEAC) values per g/ml of dark soy sauce (DSS) and three fractions: methanol extract residue (MeOH-R), ethyl acetate extract (EtOAc-extract) and ethyl acetate extract residue (EtoAc-R). Values are mean ± SD, n=3. 21 Figure 2. (A) Typical HPLC chromatogram of ethyl acetate extract. The absorbance was monitored at 270 nm. (B) Trolox equivalent antioxidant capacity (TEAC) values of 10 fractions of ethyl acetate extract per μg/mL. Results are mean ± SD, n=3. 22 Figure 3. EI-MS spectrum of compound 1 and the proposed mechanism for the formation of fragment ions. 23 Figure 4. The HPLC chromatograms of (a) ethyl acetate extract of dark soy sauce, (b) compound1 and (c) authentic maltol. (d) The overlaid spectra of maltol and compound 1; the match factor is 999.967 (It is generally considered to be matched well, if the match factor is no less than 990.). 24 Figure 5. Structure of 3-hydroxy-2-methyl-4H-pyran-4-one (maltol). 25 Figure 6. Standard curve of maltol at five concentrations, 0.25, 0.5 1.0, 1.5 and 2.0 mM . 28 Figure 7. (a) limit of detection (LOD): a typical chromatogram of maltol of 8 μM;.(b) limit of quantification (LOQ): a typical chromatogram of maltol of 25 μM. 29 Figure 8. Typical chromatograms of (a) dark soy sauce extract and (b) maltol standard (1.0mM) under the following HPLC conditions, Mobile phase: A: 0.1% formic acid; B: methanol. 10% of B for 15min, 10%-90% of B in 6min, 90% of B for another 5 min; Flow rate: 1.0 ml/min; Column: Agilent ZORBAX SB-C18 (4.6 mm i.d. × 250mm); Injection volume: 10 μl; Detection wavelength: 270nm. 30 Figure 9. The scavenging effects of dark soy sauce (a), maltol and trolox (b), on ABTS•+. Results are mean±SD, n≥3. 31 Figure 10. Typical HPLC chromatograms of (a) urine sample collected at 1 hour after the subject orally taken 70 mg maltol, and (b) the same urine sample as in (a) digested with β-glucuronidase. And the UV spectrum of (c) peak 1, whose retention time (RT) at 5.12 min, agrees well with that of (d) synthesized maltol sulphate. 34 Figure 11. (A) The total ion chromatogram of MS/MS scanning of maltol glucuronide from urine sample. (B) The mass spectrum of the peak with vi retention time at 3.12 min in total ion chromatogram A: The ion with m/z 303 is the protonated ion of maltol glucuronide; whereas the product ion with m/z 127 is protonated ion of maltol. 35 Figure 12. (A).Total ion chromatogram of MS/MS scanning of maltol sulfate in urine sample. (B). MS spectrum of the peak with retention time at 5.2 min in total ion chromatogram A. (C). MS spectrum of the peak with retention time at 1.7 min in total ion chromatogram A: The ion with m/z 207 is the protonated ion of maltol sulfate; whereas the product ion with m/z 127 is protonated ion of maltol. 36 Figure 13. (A). Total ion chromatogram of multiple reaction monitoring (MRM) scan of maltol glucuronide (303/127) and maltol sulfate (207/127) in urine sample.(B). Extract ion chromatogram of MRM scan of maltol glucuronide (303/127). (C). Extract ion chromatogram of MRM scan of maltol sulfate (207/127). 37 Figure 14. The typical HPLC chromatograms of (a) urine sample with subject taking 30 ml dark soy sauce and (b) that urine sample digested with enzyme: inset is the UV spectrum of peak with RT at 14.8 min which agrees with that of maltol. 38 Figure 15. Time course of digestion of urine samples with 5000U β-glucuronidase at 37°C. 39 Figure 16. (A). The maltol amount excreted in urine after one subject took 6 mg maltol or 30 ml dark soy sauce mixed with plain boiled rice. The accumulated maltol amounts excreted in urine for such two cases are also shown in (B). 40 Figure 17. The average total maltol (standardized with creatinine) measured in the different time point urine samples of 24 young healthy subjects who orally took 30 ml of dark soy sauce mixed with 200 gram of plain boiled rice. Data are mean±SD, n=24 (**P < 0.01 vs 0 h and 3 h). 41 Figure 18. The absorbance and ABTS•+ scavenging activity of MeOH-R fraction under acidic and basic conditions. The MeOH-R samples were incubated in 6 M HCl or 4.2 M NaOH in vacuo at 110°C for 18 hours. The colored components became insoluble in acidic condition, while in alkaline condition they were stable. Removal of insoluble colored components dramatically decreased the antioxidant activity of the acidic hydrolysate, indicating that the colored components could greatly contribute to the total antioxidant activity (TAA) of dark soy sauce. Values are mean ± SD, n=3. ***Comparision between the ABTS•+ scavenging activity of sample + 6 M HCl and that of sample + H2O (*** p < 0.001). 43 Figure 19. (A) Overlain Sephadex G-75 gel filtration chromatograms of EtOAc-R and MeOH-R of dark soy sauce. Fraction 26 to 34 of EtOAc-R were combined as Colored Product 1 (CP1), and fraction 3 to 9 of MeOH-R vii were combined as Colored Product 2 (CP2). The fragmentation range of Sephadex G75 gel filtration chromatography is 1000 ~ 50 000 Dalton. (B) The correlation of ABTS•+ scavenging activity and absorbance of CP1 and CP2 at 470 nm. Values are mean ± SD, n=3. 44 Figure 20. (a) 1H-NMR spectrum of CP1, (b) 1H-NMR spectrum of CP2, and (c) 13C-NMR spectrum of CP1. 45 Figure 21. TOF-MS spectrum of CP1. The inset shows a typical doubly charged ion. The peaks observed at around 1001.3 show the isotopic pattern at 0.5 Dalton distance. 47 Figure 22. Decrease of cell viability of HT 29 cells 24, 48 and 72 hours after incubation with various concentrations of dark soy sauce (DSS) (A), and DSS Nondialysable fraction (B). Results are mean±SD, n=3. 52 Figure 23. Cell viability tested on dark soy sauce only or plus catalase (1000 units/ml). 53 Figure 24. Time course of COX-2 expression and its inhibition by dark soy sauce in lipopolysaccharide (LPS)-induced HT-29 cells. Cells were pretreated with dark soy sauce (5 μl/ml) for half of an hour and then induced with LPS (100ng/ml) for various time indicated. Protein levels were estimated by Western Blot analysis as described in “Materials and Methods”. Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells treated with LPS (100 ng/ml) only); and lane 3, HT-29 cells simultaneously treated with dark soy sauce (5 μl/ml) and LPS (100 ng/ml). 54 Figure 25. Inhibitory effect of dark soy on COX-2 expression in LPSinduced HT-29 cells . Lane 1, untreated HT-29 cells; Lane 2, HT-29 cells treated with 1 μl/ml dark soy sauce(DSS); Lane 3, with 5 μl/ml DSS; Lane 4, with LPS (100 ng/ml); Lane 5, with DSS (1 μl/ml) and LPS (100 ng/ml); Lane 6, with DSS (5 μl/ml) and LPS (100 ng/ml). The western blot is from a single experiment, but is representative of 3 independent experiments. 55 Figure 26. The effect of maltol on COX-2 expression in LPS-induced HT-29 cells.Lane 1 is untrated HT-29 cells; lane 2, HT-29 cells treated with maltol (100 μM); lane 3, maltol (500 μM); lane 4; treated with LPS (100 ng/ml); lane 5, maltol (100 μM) and LPS (100 ng/ml); lane 6, maltol (500 μM) and LPS (100 ng/ml). 55 Chart 1. The flow chart of fractionation and isolation of maltol, CP1 and CP2 from dark soy sauce. 10 viii List of Symbols ABTS 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) APCI-ITMS atmospheric pressure chemical ionization-ion trap mass spectrometry COX-2 cyclooxygenase-2 CP1/2 the colored product 1/2 DAD diode array detection DSS dark soy sauce EI-MS electron impact – mass spectrometry ESI-MS electrospray-ionization mass spectrometry EtOAc-R ethyl acetate extract residue FTIR Fourier transform infrared GIT gastrointestinal tract HEMF 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone HPLC high performance liquid chromatography LC-MS/MS liquid chromatography-mass spectrometry/mass spectrometry LOD limit of detection LOQ limit of quantification LPS lipopolysaccharide MeOH-R methanol extract – residue MRM multiple reaction monitoring NMR nuclear magnetic resonance RP reverse phase RT retention time ix SPE solid phase extraction TAA total antioxidant activity TEAC trolox equivalent antioxidant capacity TOF time-of-flight UV ultraviolet x   Chapter 1 Introduction Pour it into soup, and watch the artful dark tongue mix its own remedy. It is the same as looking at hard men cry as they watch cream weave through coffee. (From Tina Chang’s poem, Ode to Soy Sauce [1]) Soy sauce is a traditional fermented seasoning of East Asian countries and is currently used in cooking worldwide. Not only do the flavor components of soy sauce improve the taste of many types of foods, but its coloring ingredients can enhance the appearance of the dipped food or the mixed soup. Moreover, recent studies indicate that some ingredients in soy sauce are potentially beneficial to human health, showing effects such as anticarcinogenesis, antihypertension and antihyperlipidemia [2-4]. 1.1 A brief history of soy sauce The history of soy sauce goes back over three thousand years. The origin of soy sauce is generally considered to be in China, where soy sauce is called jiang-you, the extract of jiang. Jiang was first recorded in the books of Zhou dynasty (1121-256 B.C.), while Jiang-you was first mentioned in a book of ‘Qimenyaoshu’, Jia Sixie, Bei-Wei dynasty (220-265) [5]. It is speculated that, to preserve foodstuffs against periods of scarcity, the ancient Chinese accidently discovered Jiang when they mixed salt with meat, fish and vegetables, and innocently incorporated some harmless aeroborne fungi, which fermented the raw material after a long period of culture. With the introduction of Buddism to China, meat was excluded from the diet of the Buddist monks. Plant1      origin materials, such as soy bean and wheat, then began to be widely used to make sauce [6]. Buddist monks are thought to have played an important role in spreading soy sauce from China to Japan. Soy sauce was first introduced into Japan by a Buddist monk, Jian Zhen, in Tang Dynasty (618-907) [7]. But some consider that Japanese soy sauce originated from that brought back by a Japanese Buddist monk, Kakushin, from China in Song Dynasty (960-1279) [8-10]. It is in Japan that the making of soy sauce was modernized and exported to Europe and North America. Between 17th and 18th century, a large quantity of soy sauce was exported from Japan by a Dutch company to Europe [8]. As early as in 1867 Japanese soy sauce was taken along by immigrants to Hawaii [11]. In 1972, the Kikkoman Company opened a modern soy sauce plant at Walworth, Wisconsin [8]. Soy sauce was brought to East Asian countries by the Chinese immigrants. In Singapore, it is said that the small-scale manufacturing of soy sauce started by a small number of Xin-hui Cantonese, a sub-dialect group from Guangdong Province of South China, just a couple of years after the first arrival of Chinese immigrants in the early nineteenth century [12]. After being successful, many of them shifted their business to other more profitable fields. Only a few survived. For example, Chuen Cheong Food Industries, which produces Tiger brand sauce, was established in 1930, earlier than other local famous soy sauce manufacturers, such as Yeo Hiap Seng (1938) and Tai Hua Food Industries (1949). The business and technologies of soy sauce fermentation have been passed from father to son. Now the fourth generation is in the charge of the business [13, 14]. 2      1.2 The methodology of preparation of soy sauce The ratios of starting raw materials, soy bean and wheat, are different for Chinese style and Japanese style: around 50:50 for Japanese style, while approximately 60:40 for Chinese style [15]. For both styles of traditionally fermented soy sauce, the basic procedure of production is very similar. The method of soy sauce production practiced locally is based on the traditional Chinese fermentation process, the main steps of which are as follows [16]: Raw material preparation: Soy beans or defatted soy flakes are soaked and then cooked, while wheat is roasted The Koji fermentation process The raw materials are mixed and inoculated with mold (seed Koji), and then cultured for 2-3 days with controlled temperature and moisture. The mold grows enough to provide the enzymes necessary to hydrolyze the raw materials, thus, then, also called sauce Koji. The mash fermentation The sauce Koji is poured into fermentation tanks and mixed with saline water, aging for 3 months to 2 years, upon the culture temperature. During the fermentation period, the enzymes from Koji mold hydrolyze most of the protein to amino acids and lowmolecular-weight peptides, and some polysaccharides into simple sugars. Refinement The aged mash is filtered. The filtrate is called raw soy sauce. The raw soy sauce is further pasteurized and finally bottled. To make dark soy sauce, the aging process is 3      further extended for another 2-4 months, and caramel is added to make the final product thicker and the color deeper. Traditionally, the making of soy sauce is time-consuming and labor-consuming. The mash is usually fermented in earthenware, and sunlight provides the thermal energy for aging. So in the North of China or Japan, the process of manufacturing can last as long as 2 years. To save time, in some modern soy sauce factories, thermal-controlled fermentation tanks are used. The making process of soy sauce can be shortened to three months [17]. After World War II, when the supplies of soy beans were scarce, many manufacturers applied chemical soy sauce, acid hydrolyzed vegetable proteins. But the flavor of such products is poor. To keep some flavor as of fermented soy sauce, semi-chemical and blended soy sauce were produced. Semi-chemical soy sauce is produced by further fermentation of acid hydrolyzed products; while blended soy sauce is produced by mixing fermented soy sauce with chemical soy sauce and/or enzymatically hydrolyzed vegetable protein. As Asian economies have grown, fermented soy sauce becomes the main product in Asian markets. The United States is left to be the largest chemical soy sauce producer in the world. Japanese producers once proposed the world-trade regulations that the manufacturing methods should be included in the label. But the US suggested that individual countries should make their own decisions on labeling [18]. In Singapore, soy sauce can be made from soy beans with or without other foodstuffs, by “either enzymic reaction or acid hydrolysis or by both methods” [19], although local products are traditionally fermented. But there is no labeling requirement on the manufacturing process. 4      1.3 The Functional components of soy sauce In China, soy sauce has been traditionally used for treatment of anorexia, ulcers post thermal burn etc, recorded in Traditional Chinese medicinal books (Su Jing, Xinxiubencao, Tang Dynasty, 659; Sun Simiao, Qianjinfang, Tang Dynasty, 625) [7]. Modern scientific research has provided some supporting evidence for such usage: e.g. soy sauce can promote gastric juice secretion in humans; soy sauce has antimicrobial activity, due to the synergistic effects of NaCl, ethanol, pH, and preservatives [20]. Soy sauce has a variety of biologically active effects, such as hypotensive, anticarcinogenic, anticataract, and antiplatelet . Nicotianamine was found to be the major bioactive components inhibiting angiotensin I-converting enzyme. 4-hydroxy3(2H)-furanone derivatives, namely, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)furanone, 4-hydroxy-5-methyl-3(2H)-furanone and 4-hydroxy-2,5-dimethyl-3(2H)furanone, are antioxidants, also having anticarcinogenic and anticataract activities. Shoyu-flavones, derivatives of daidzein, genistein, and 8-hydroxygenistein, are antioxidants and histidine decarboxylase inhibitors. 1-methyl-1,2,3,4,-tetrahydro-βcarboline and 1-methyl-β-carboline are found to be the active antiplatelet components [20]. During the fermentation process, the soybean and wheat proteins are degraded into peptides and amino acids. Thus the IgE-mediated hypersensitive response to wheat is markedly reduced in soy sauce. Furthermore, the soy sauce polysaccharides that cannot be hydrolyzed during the fermentation process can inhibit hyaluronidase activity and histamine release. In vitro, these polysaccharides can increase the production of IgA from Peyer’s patch cells. In vivo, soy sauce polysaccharides can 5      increase the concentration of IgA in the intestines of mice, suppress passive cutaneous anaphylaxis reaction in the ears of allergy model mice, and regulate the balance of Th1/Th2 cell response in mice, potentially enhancing host defenses [21-23]. Clinically, oral administration of soy sauce polysaccharides can improve allergic symptoms of patients with perennial allergic rhinitis or seasonal allergic rhinitis [23]. Soy sauce polysaccharides can also inhibit pancreatic lipase activity and reduce the absorption of lipid in mice and humans [4]. 1.4 The balance of free radicals/reactive species and antioxidants A free radical is any species capable of independent existence containing one or more unpaired electrons [24]. Free radicals can be formed by homolytic or heterolytic fission of a covalent bond. For example, UV-induced homolytic fission of the O-O bond in H2O2 can produce hydroxyl radical, OH•. Reactive oxygen species (ROS) includes not only the oxygen radicals (O2• − and OH•) but also non-radical derivatives of O2 (H2O2, HOCl and O3). Univalent reduction of molecular oxygen can form a variety of ROS. Mitochondrial electron transport chain is the main cellular source of ROS. Various oxidases in cell membranes, cytosol, and other organelles can transfer single electrons onto dioxygen,e.g. Cytochrome P450, a monooxygenase in the endoplasmic reticulum, is able to reduce dioxygen to superoxide radicals. Transition metals, e.g. iron, having the capability of donating and/or receiving electrons, play an important role in oxygen radical formation. Inflammation can also result in excessive production of free radicals. Free radicals and ROS are not only formed endogenously, but also introduced outside [25]. Our body has evolved antioxidant defense systems. Small molecules and antioxidant enzymes are two major categories. Antioxidant enzymes, such as superoxide 6      dismutase, catalase, and glutathione reductase, are important for antioxidant defense [24]. Imbalance between formation of free radicals/reactive oxygen species and levels of antioxidants in vivo has been suggested to play a role in the development of various diseases, such as atherosclerosis, diabetes, rheumatoid arthritis, cancer and neurodegenerative diseases [24, 26]. Some (but not all) studies show that nutritional antioxidants can decrease oxidative damage in the human body and may have beneficial effects on disease prevention [26 – 29]. This has led to a growing interest in antioxidants from natural products [26 – 31]. Several papers have alluded to the presence of antioxidants in soy sauce [32 – 36]. Tiger brand soy sauce is a local brand sauce with an 80-years history [13, 14]. Our group’s studies showed that dark soy sauce, especially Tiger brand products, had extremely high total antioxidant activity (TAA) in vitro [36] as judged by the ability to scavenge the nitrogen-centred ABTS•+ radical, an assay that is frequently used to assess the antioxidant activity of beverages, food extracts and body fluids [37]. In vivo Dark soy sauce of this brand also decreased lipid peroxidation in human volunteers [38]. In this study, using Tiger brand products, we attempted to identify the major components that contribute to the high antioxidant activity of dark soy sauce. 7    Chapter 2 Materials and Methods 2.1 Chemicals All chemicals were obtained from Sigma-Aldrich, Singapore unless otherwise stated. Ethyl acetate, formic acid 37% [Guaranted Reagent (GR) for analysis], sodium hydroxide (GR for analysis), were from Merck, Germany; methanol (HPLC grade) was from Labscan Analytical Science, Thailand. Dark soy sauce (Tiger brand, Chuen Cheong Food Industries, Singapore) was purchased from a local supermarket. 2.2 ABTS assay This was carried out as described in Ref. [36, 37]. 2,2ʹ-Azino-bis[3ethylbenzothiazoline-6-sulfonate] (ABTS) in water (7 mM final concentration) was oxidized using potassium persulfate (2.45 mM final concentration) for at least 12 h in the dark. The ABTS•+ solution was diluted to an absorbance of 0.70 ± 0.02 at 734 nm (Beckman UV – VIS spectrophotometer, Model DU640B, UK) with phosphate buffered saline (PBS 10 mM, pH 7.4). Extracts of dark soy sauce (10 μl) or trolox standard (10 μl) were added to 1 ml of ABTS•+ solution. Absorbance was measured 1 min after initial mixing. Antioxidant properties of fractions of dark soy sauce extracts were expressed as Trolox equivalent antioxidant capacity (TEAC), calculated from at least three different concentrations of extract tested in the assay and giving a linear response. 8    2.3 Isolation of low molecular mass components from ethyl acetate extract Dark soy sauce (6.4 L) was extracted three times with a four-fold volume of methanol. The methanol extracts were combined, filtered by filter paper and evaporated to dryness under vacuum at 40ºC (MeOH-extract, yield 2.6 kg). No significant retention of antioxidant components from soy sauce by the filter paper was detected. The residue after methanol extraction (MeOH-R) yielded 1.4 kg. The MeOH-extract was suspended in water and partitioned with ethyl acetate three times. The ethyl acetate fractions were combined and evaporated to dryness under vacuum at 30ºC (EtOAc-extract, yield 11.6 g). The remaining aqueous fraction (EtOAc-R) yielded 2.6 kg (Chart 1.). As the EtOAc-extract exhibited strong ABTS radical scavenging activity, it was subjected to flash chromatographic separation with a silica gel RP18 (particle size 40 – 63 µm, Merck KGaA, Darmsadt, Germany) packed column (6 × 42 cm) eluting with methanol and water. Fractions eluted with 10% methanol had the highest ABTS•+ radical scavenging activity and were further purified by a prep-HPLC (Agilent 1100 Series, equipped with a fraction collector) using a ZORBAX SB-C18 PreP HT column (21.2 × 250 mm, 7 µm) (Agilent, USA) at 20 ml/min with methanol—0.1% formic acid in MilliQ water (10:90, v/v) as mobile phase. Ten fractions showing antioxidant activity in the ABTS assay, Fr.1 to 10, were obtained. Fraction 9 was found to have the most ABTS scavenging activity and yielded Compound 1 (41mg). 2.4 Fractionation of colored components Approximately, 1 gm of ethyl acetate extract residue (EtOAc-R) was resuspended in 25 ml distilled water and dialyzed against distilled water for seven days, using a 9    Dark soy sauce (1) extracted with 4 fold methanol (MeOH) 3 times MeOH-R MeOH-extract (1) dialyzed against distilled water (2) fractionated with gel filtration chromatography (1) filtered by filter paper, evaporated to dryness under vacuum at 40 ºC (2) and suspended in water; (3) partitioned with ethyl acetate (EtOAc) three times       Colored Product 2 EtOAc-extract (1) Fractionated with RP18 column chromatography (2) Further fractionated with prep HPLC. Eluted with methanol – 0.1% formic acid in milli Q water (10:90) Fr.1 to Fr.10 (Fr. 9 Æ maltol) EtOAc-R (1) dialyzed against distilled water (2) fractionated with gel filtration chromatography Colored Product 1 (CP1) Chart 1. The flow chart of fractionation and isolation of maltol, CP1 and CP2 from dark soy sauce.   10    cellulose dialysis tubing (Pierce, Rockford, USA; molecular weight cutoff 3500). Initially, we investigated the influence of dialysis time and temperature (room temperature, ~25ºC, and cold-room temperature, ~4ºC), on the antioxidant capacity of the colored products as measured by the ABTS assay and found no significant effect. For convenience, the dialysis experiments were carried out at room temperature. The non-dialyzable fraction was freeze-dried. Approximately, 85 mg of the freeze-dried product was dissolved in 5 ml of water and loaded onto a fine Sephadex G-75 gel filtration chromatography column (2 × 100 cm). The colored fractions (absorbance at 470 nm), 4 ml per tube, were collected. The fractions from No. 26 to 34 possessed significantly higher TEAC values and consequently were combined as Colored Product 1 (CP1) (yield 61 mg). The MeOH-R fraction (approximately 2 g) was dialyzed against distilled water. The non-dialyzable fraction (85mg) was fractionated with gel chromatography in the same way as EtOAc-R. The fractions from No. 3 to 9 were combined as Colored Product 2 (CP2) (yield 47mg) (Chart 1.). 2.5 HPLC determination of maltol in dark soy sauce Dark soy sauce (10 ml) was extracted with 40 ml methanol on an orbital shaker (SLOS-20, Seoulin Bioscience, Seoul, Korea) at a speed of 150 rpm for 24 h, and then centrifuged at 3000g for 30 min. This procedure was repeated three times. The supernatants were pooled and dried using a rotary evaporator under vacuum at 40ºC. The residue was dissolved in 20 ml water and extracted three times with 20 ml ethyl acetate. The ethyl acetate extracts were combined and evaporated to dryness at 30ºC. Prior to HPLC analysis, the dried samples were dissolved in 10 ml methanol-0.1% 11    formic acid (1:9) and then filtered through 0.45 µm disposable nylon filters (Agilent Technology, USA). Analysis was performed using an Agilent 1100 HPLC with an Agilent ZORBAX SBC18 column (4.6 × 250 mm, 5 µm), maintained at 35ºC. The mobile phase was formic acid in MilliQ water (0.1%, v/v) (A) and methanol (B) with a gradient program as follows: 10% of B for 15 min, 10 – 90% of B in 6 min, 90% of B for another 5 min, with flow rate at 1 ml/min. The injection volume for all samples was 10 μl and absorbance was monitored at 270 nm. Spectra were recorded from 190 to 400 nm. 2.6 Mass spectrometry An Agilent XCT Plus ion trap mass spectrometer (ITMS) (Agilent Technology, US) was used to analyze the fractions, Fr. 1 – 10, obtained from ethyl acetate extracts. Atmospheric pressure chemical ionization (APCI) MS was performed in the positive mode. The dry gas and vaporizer temperatures were 350 and 400ºC, respectively. Compound 1 was also analyzed by an electron impact (EI) MS spectrometer (Agilent Technologies), sample dissolved in methanol and introduced by a gas chromatography (GC) interface (Agilent Technologies). For the analysis of CP1, Electrospray-Ionization Mass Spectrometry (ESI-MS) was performed using a Waters Micromass Q-Tof micro mass spectrometer (Waters, USA). For acquiring mass spectra, sample was directly infused at a speed of 10 µl/min. The capillary and sample cone voltages were maintained at 3.0 kV and 50 V, respectively. The source and desolvation temperatures were 80 and 250ºC, respectively. The mass spectra were acquired from m/z 100 to 5000 in the positive ion mode. 12    2.7 Fourier transfer infrared spectrometry (FTIR) IR spectra (KBr disc) were recorded on a JASCO FT/IR-430 spectrometer (Japan). 2.8 Nuclear magnetic resonance spectrometry (NMR) The NMR spectra of CP1 and CP2 and compound 1 were recorded on a Bruker Advance AMX500 NMR spectrometer (Rheinstetten, Germany) at 500.13 MHz (1H) and 125.75 MHz (13C), respectively. Compound 1 was dissolved in methanol-d4; CP1 and CP2 were dissolved in D2O. 2.9 Detection and determination of maltol metabolites in human urine samples 2.9.1 Standard preparation Maltol glucuronide was isolated and puried from the urine samples of a healthy volunteer using solid phase extraction (SPE), reverse-phase (RP) C-18 flash column chromatography and preparative HPLC. The purity of the isolated compound was checked on an analytical HPLC system. Its identity was confirmed based on NMR data. Maltol glucuronide, 1 H-NMR, 2.39 (s), 3.56 (m), 3.86 (d), 4.95 (d,dd), 6.48 (d), 7.98 (d) 13 C-NMR, 15.12, 71.24, 73.11, 75.23, 102.48, 115.98, 141.39, 156.70, 164.73, 172.53, 176.62 Maltol sulfate was synthesized based on a published method [39]. To the solution of 200 mg maltol in pyridine, 300 mg pyridine-sulfur trioxide complex was added and the mixture was stirred at 4 °C for 60 hours. The precipitating solid in the reaction mixture was filtered off and washed with CHCl3, then dried in vacuo to give 390 mg 13    solid. The solid was then dissolved in water and further purified using a preparative HPLC (Agilent Technologies). A series of different concentrations (0.05 mM - 1 mM) of maltol standard solutions were prepared in 10% methanol/0.1% formic acid- MilliQ water. 2.9.2 Sample preparation To test how fast maltol is metabolized, the urine samples at different time points (0.5, 1, 2, 3, 4, 5 and 6h) were collected from a human volunteer who had consumed 6 mg maltol or 30 ml dark soy sauce mixed with 200 gram of plain boiled rice. The washtime between these two experiments were more than one week. The urine samples were also collected from 24 young health volunteers in an observer-blinded, randomized, placebo controlled, crossover clinical study [38]. The urine samples were deproteinized with three volumes of methanol, then centrifuged at 1500 × g for 10 min. For detection of maltol metabolites, the supernatants were directly injected onto HPLC column. For determination of total maltol content in urine, these supernatants were dried under N2 flow, digested with 200 μl 11.1 mg/ml β-D-galactonidase in 0.5 mM acetate buffer at 37°C overnight, and then centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were collected and analyzed immediately using HPLC. 2.9.3 HPLC-DAD detection of maltol metabolites and determination of total maltol content in urine HPLC analysis was performed on an agilent 1100 HPLC system, equipped with a diode array detector. The samples were introduced by an autosampler. A Phenomenex column (4.6 × 250 mm, 5.0 μm) was used, with temperature kept at 14    35 °C in a thermostat column compartment. UV spectra (190-400 nm) were recorded. The mobile phase was composed of 10% methanol in 0.1% formic acid, with a running rate of 1ml/min. 2.9.4 HPLC-MS/MS detection of maltol metabolites LC-MS/MS analysis of maltol metabolites in urine was performed on an Agilent 1200 LC system (Palo Alto, CA, USA) coupled to an API3200 Triple-Quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, USA). A Turbo V source with ESI prober was used for the analysis. Data acquisition was performed with Analyst 1.4.2 software (AB MDS Sciex). The chromatographic separation was performed on a 150 × 2.0 (i.d.) mm Phenomenex Synergi 4µ Polar-RP 80 Å column (Phenomenex, CA, USA) with a column oven temperature of 35°C. The mobile phase was premixed 10% methanol in 0.1% formic acid. Flow rate was 0.4 ml/min. Standards or samples were introduced into the LC using an Agilent 1200 G1367B autosampler and injection volume was 10.0 μl. The ion source was operated in the positive mode. For survey scan (Q1 MS scan mode), ion-spray voltage and temperature were set at 5000 V and 600oC. Ion source gas 1(GS1) and ion source gas 2 (GS2) were set at 40 and 45 psi. Interface heater (ihe) was set as on. The curtain gas (CUR), declustering potential (CE) and entrance energy (EP) settings were at 10 psi, 50 V and 10 V, respectively. In product ion scan (MS2) mode, for maltol glucuronide, parent ion 303 was used, and scan range was set at 50 – 400; whereas for maltol sulphate, parent ion 207, scan range was 50 -300. For both chemicals, collision energy (CE) was set at 5 V. In multiple reaction monitoring (MRM) mode, the ion pairs monitored for maltol glucuronide and maltol sulphate were 303/127 and 207/127 respectively. 15    2.10 GC-MS analysis of DNA base modification 2.10.1 Sample preparation Salmon testis DNA was dissolved in 100mM phosphate buffer (final concentration 1 mg/ml). 1 ml of DNA sample was pretreated with or without various concentrations of soy sauce extracts before hypochlorous acid (HOCl) exposure. HOCl concentration was quantified spectrophotometrically (290 nm, pH 12, ∈ = 350 M-1cm-1) [40]. HOCl dilutions were prepared in Krebs buffer (mmol/l: NaCl 130, KCl 5.7, NaHCO3 25, NaH2PO4 10, D-glucose 5, CaCl2 1, and MgCl2 0.5, pH 7.4) immediately before use. Here we use the hypochlorous acid (pKa 7.46) to refer to an ≈50% ionized mixture of HOCl and OCl‾ (hypochlorite). After incubation with HOCl (500 μM) at 37 °C for 1 hour, the DNA samples were dialyzed against distilled water for 24 hours in a cold room. The concentration of DNA was measured spectrophotometrically at 260 nm (E260 = 50 μg/ml). Aliquots of 100 μg DNA were hydrolyzed by adding 0.5 ml of 60% (v/v) formic acid and heating at 140 °C for 45 min in an evacuated, sealed hydrolysis tube. The internal standards were added to the cooled hydrolyzed samples and freezedried. Derivatization was carried out for 2 hour at 25 °C using BSTFA (+1% TMCS)/acetonitrile/ethanethiol (16:3:1(v/v)) mixture. 2.10.2 GC-MS analysis The GC-MS analysis was performed as previously reported [41, 42]. Derivatized samples were analyzed by GC-MS (Agilent gas chromatography 6890 interfaced with Agilent 5973 mass selective detector). The injection port and the GC-MS interface were kept at 250 and 290 °C, respectively. Separations were carried out on a fused silica gel capillary column (12 m × 0.2 mm i.d.) coated with cross-linked 5% phenylmethylsiloxane (film thickness 0.33 μm) (Agilent). Helium was the carrier gas 16    with a flow of 1.0 ml/min. Derivatized samples (1.0 μl) were injected into the GC port using a split ratio of 8:1. Column temperature was increased from 125 to 175 °C at 8 °C/min after 2 min at 125 °C, then increased from 175 to 220 °C at 30 °C/min, held at 220 °C for 1 min, and finally increased from 220 to 290 °C at 40 °C/min and held at 290 °C for 2 min. Selected-ion monitoring was performed using the electronionization mode with the ion source maintained at 189 °C. 2.11 Cell culture Human colorectal adenocarcinoma cells (HT29), obtained from the American Type Culture Collection, were cultured in McCoy’s 5A media with 1% (v/v) penicillin, 10% (v/v) fetal bovine serum (FBS), 5% CO2 at 37 °C. The cells were maintained in the logarithmic growth phase by routine passage every 2-3 days. 2.12 Assessment of cell viability Viability was assessed by the MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) method as previously reported [43, 44]. Cells in McCoy’s 5A full media were seeded overnight at a density around of 1.5 × 104 cells per well in 96-well plates. After treatment with various concentration of dark soy sauce in full medium, 200 μl of MTT (0.5mg/ml final concentration) dissolved in McCoy’s 5A only (with FBS). Cells were incubated at 37 °C in darkness for 1 hour and then MTT was removed. 200 μl of dimethyl sulfoxide (DMSO) was added to solubilize the formazan formed. After shaking in the dark for 15 min, absorbance at 550 nm was measured using a microplate reader (SpectraMax190, Molecular Device). 17    2.13 Western blot analysis Experiments were performed as describe in [43, 45]. Control and treated cells were washed twice with cold PBS buffer, incubated in lysis buffer on ice for 30 min, and then scraped into Eppendorf tubes. Cells were centrifuged at 13800 rpm at 4 °C for 15 min using a desktop centrifuge (Eppendorf) to remove unbroken cells, nuclei and other organelles. The protein concentration of the resulting supernatant was measured with the DC protein assay Kit (Bio-Rad), and 20 μg of protein was boiled for 10 min, and then loaded in a 12.5% (v/v) SDS – PAGE gel. The separated proteins were then transferred to nitrocellulose membranes and probed with antibodies against COX-2, β-actin, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies. 2.14 Statistical analysis The mean values were calculated from data taken from at least three separate experiments. Where significance testing was performed, a Student’s t-test was used; P-values of, 0.05 were considered to be statistically significant. 18    Chapter 4 Results and Discussion 4.1 Separation and characterization of low molecular mass components Dark soy sauce was extracted with methanol, and the methanol extract was further partitioned with ethyl acetate as described in Experimental section (Chart 1). The three fractions of dark soy sauce, methanol extract residue (MeOH-R), ethyl acetate extract (EtOAc-extract), and ethyl acetate extract residue (EtOAc-R), were subjected to the ABTS assay. The MeOH-R fraction contributed about 60% of the total antioxidant activity (TAA) of dark soy sauce. However, the EtOAc-extract showed the strongest antioxidant activity, approximately 12-fold higher than that of the dark soy sauce (Figure 1). After pretreatment with a reverse phase (RP)-C18 column (mainly to remove benzoic acid, which has no antioxidant activity in the ABTS assay), the EtOAc-extract was separated with preparative high performance liquid chromatography (HPLC) resulting in 10 fractions (Figure 2A). Among them, the ABTS assay (Figure 2B) showed that Fr.9 has by far the strongest activity. Fr.9 was further purified to yield compound 1. The positive atmospheric pressure chemical ionization - ion trap mass spectrometry (APCI-ITMS) spectrum of Compound 1 showed a protonated molecular ion peak at m/z 127 [M + H]+. The electron impact – mass spectrometry (EI-MS) spectrum also shows its molecular ion at m/z 126 (Figure 3). The major fragment ions (m/z 97, 71, 55) were observed. The molecular ion m/z 126 can be decomposed to a furan ion (m/z 97) by losing one H and CO. The furan fragment ion (m/z 97) can be further decomposed to ion m/z 55 by losing CH3CO. Via another pathway, the molecular ion 19    (m/z 126) can give the fragment ion m/z 71 by losing CH2CH2 and CO. The mechanism for these fragment ion formation is proposed in Figure 3. The ion fragment pattern agrees with that previously reported [46]. The proton and carbon 13 nuclear magnetic resonance (1H- and 13C-NMR) spectral data of compound 1 are listed in Table I. The 1H-NMR spectrum of compound 1 (500MHz, methanol-d4) showed two cis-olefinic proton signals at δH 7.94 (1H, d, J = 5.5 Hz) and 6.39 (1H, d, J = 5.5 Hz). In addition, the signal of one methyl group directly attached to an olefinic carbon at δH 2.35 (3H, s) was observed. The 13C-NMR spectrum showed one carbonyl carbon signal at δC175.3, and four olefinic carbon signals at δc 156.3, 152.2, 144.6 and 114.4, indicating the presence of a pyranone ring. The signal at δc 14.2 corresponded to the carbon of the methyl group. The Fourier transform infrared (FTIR) spectrum of compound 1 shows the major absorbance bands (cm-1): 3258, 3069, 1655, 1617, 1560, 1459, 1257, 1199. Compound 1 was also analyzed by high performance liquid chromatography – diode array detection (HPLC-DAD). Its retention time and ultraviolet (UV) spectra (absorption maxima at 210 nm and 275 nm) agreed very closely with those obtained from authentic maltol (Figure 4). So the MS spectrum, NMR data, FTIR spectrum, the retention time and UV spectrum support the identity of compound 1 as 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) (Figure 5). Fractions 1-8 and 10 were also analyzed by APCI-ITMS, suggesting these fractions contain molecules with molecular weight ranging from 124-214 (Appendix: Table A1).     20          1800 1580 1600 TEAC (mM) 1400 1200 1000 800 600 400 200 309 122 51 0 D SS MeO H -R EtoA cextract EtoA c-R Figure 1. Trolox equivalent antioxidant capacity (TEAC) values per g/ml of dark soy sauce (DSS) and three fractions: methanol extract residue (MeOH-R), ethyl acetate extract (EtOAc-extract) and ethyl acetate extract residue (EtoAc-R). Values are mean ± SD, n=3. 21      mA U 250 9 5 (A) 200 150 1 3 100 10 7 50 2 8 6 4 0 0 5 10 15 20 25 30 35 40 min Retention Time    25.00 21.21 (B)  T EA C (mM)  20.00 15.00 10.00 5.00   0.00 1.85 1.81 1.27 0.39 0.34 0.58 2 3 4 0.22 0.43 5 6   1   0.88 7 Fraction Number 8 9   10   Figure 2. (A) Typical HPLC chromatogram of ethyl acetate extract. The absorbance was monitored at 270 nm. (B) Trolox equivalent antioxidant capacity (TEAC) values of 10 fractions of ethyl acetate extract per μg/mL. Results are mean ± SD, n=3. 22    +. O OH + O _ H. _ O CH 3 CH 3 O + + _ CH CO 2 CO O O OH O CH 3 m/e = 55 m/e = 97 m/e = 126 O O O O + _C H 2 2 C O CH 3 O + + O CH 3 O O O _ CO CH 3 O CH 3 + m/e = 71 A bundance  2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000  600000  400000  200000  0 126 71 55  50  97 60 70 80 90 100 m/z 110 120 130 140 Figure 3. EI-MS spectrum of compound 1 and the proposed mechanism for the formation of fragment ions. 23    N orm 160 *Fr.2a9 *maltol (d) Match factor: 999.967 120 80 40 0 200 mA U  700 250 300 350 nm (a)  600 500 400 (b)  300 200 100 (c)   0 2.5 5 7.5 10 12.5 15 17.5 min  Figure 4. The HPLC chromatograms of (a) ethyl acetate extract of dark soy sauce, (b) compound1 and (c) authentic maltol. (d) The overlaid spectra of maltol and compound 1; the match factor is 999.967 (It is generally considered to be matched well, if the match factor is no less than 990.). 24    Figure 5. Structure of 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) Table I. 1H- and 13C-NMR data of Compound 1. C/H number δC (ppm) δH (ppm) 2 152.2 3 144.6 4 175.3 5 114.4 6.39 (1H, d, J = 5.5 Hz) 6 156.3 7.94 (1H, d, J = 5.5 Hz) 7 14.2 2.35 (3H, s) 25    4.2 Content of maltol and its contribution to the TAA of dark soy sauce A considerable part of the TAA of the dark soy sauce was contained in MeOH-R and EtOAc-R fractions and in fractions 1-8 and 10 (Figure 1, 2). Although maltol was identified as the active fraction of Fr. 9, we needed to determine its overall contribution to TAA. Thus an HPLC method was developed for determination of maltol in dark soy sauce. Maltol standard was dissolved in methanol-0.1% formic acid (10:90, v/v) yielding concentrations of 0.25, 0.5, 1.0, 1.5, 2.0 mM. Three different sets of standard solutions were prepared and analyzed each day and in three continuous days. Calibration curves for the quantification of maltol were obtained by plotting amount (mM) against peak area. The calibration curve (Figure 6) was linear within the investigated concentration range (0.25 – 2.0 mM) with the following regression equation: A = 4869.4C – 43.532 (R2 = 0.9999, n=9) where A is the peak area (mAU*S), and C is the concentration of maltol solution (mM). The within- and between-assay precision were established by injecting three sets of samples at three spike concentration levels (0.25, 1.0 and 2.0 mM) within one and three days, respectively. The within-assay and between-assay was in the range 0.3-1.8% and 1.4-3.4%, respectively (Table II). The limit of detection (LOD) was determined as the amount that resulted in a peak with a height two or three times that of the baseline noise. The limit of quantification (LOQ) was determined as the amount that resulted in a peak with a height ten times that of the baseline noise. The LOD and LOQ were 8 26    and 25 μM respectively. Figure 7 shows a typical chromatogram at such concentration. The accuracy was evaluated through recovery studies by adding known amounts of maltol at different levels (0.25, 0.5, 1.0, 1.5, 2.0 mM) to the sample. The unspiked samples and each of the spiked samples were analyzed in triplicate. The recoveries at the different concentration levels were in the range 89.8 to 94.2% (Table II). Using this validated method, the average concentration of maltol in dark soy sauce was determined to 1.15 ± 0.04 mM (n=5). One typical HPLC chromatogram of ethyl acetate extract of dark soy sauce is shown in Figure 8. Based on the ABTS assay, the total antioxidant activity of dark soy sauce and matol was evaluated. The antioxidant activity of maltol (TEAC value) was 2.67 ± 0.05 mM (n=5) (Figure 9). This concentration of maltol was calculated to contribute around 2.42% of the TAA of dark soy sauce. Table II. Within- and between-assay precision and recoveries of the assay used to measure maltol. Concentration level/mM 0.25 Within-assay precision (% RSD, n=3) 1.8 Between-assay precision (% RSD, n=3) 3.4 0.5 1.0 89.8 89.9 0.4 2.4 1.5 2.0 Recovery (%) 92.9 94.2 0.3 1.4 93.9 27    12000 y = 4869.4x - 43.532 R 2 = 0.9999 (n=9) UV absorbance (mAU) 10000 8000 6000 4000 2000 0 0 0.5 1 1.5 2 2.5 Concentration (mM)   Figure 6. Standard curve of maltol at five concentrations, 0.25, 0.5 1.0, 1.5 and 2.0 mM, with UV detection at 270nm.       28      mA U   0.16  0.14  (a) Maltol 8μM; injection 10μl; Signal/N oise ≈ 3   0.12  0.1  0.08  0.06  5 7.5 10 12.5 15  17.5 min   mA U   0.35  0.3  (b) Maltol 25μM; injection 10μl; Signal/N oise ≈ 10   0.25  0.2  0.15  0.1  5 7.5 10 12.5 15 17.5 min   Figure 7. (a) limit of detection (LOD): a typical chromatogram of maltol of 8 μM;.(b) limit of quantification (LOQ): a typical chromatogram of maltol of 25 μM.   29    22.856 13.932 8.007 mA U   (a) 300 50  15.618 10.068  100 6.213 6.846 2.591 150 8.597 200 11.955  250 0 5  0 10 15 20  min   13.974 mA U   (b) 250 200 150 100 50  0 0 5  10 15 20  min   Figure 8. Typical chromatograms of (a) dark soy sauce extract and (b) maltol standard (1.0mM) under the following HPLC conditions, Mobile phase: A: 0.1% formic acid; B: methanol. 10% of B for 15min, 10%-90% of B in 6min, 90% of B for another 5 min; Flow rate: 1.0 ml/min; Column: Agilent ZORBAX SB-C18 (4.6 mm i.d. × 250mm); Injection volume: 10 μl; Detection wavelength: 270nm. 30    60 y = 423.12x 2 R = 0.9947 (n=9) (a) 50 %inhibition 40 30 20 10 0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 dark soy sauce con (mg)   (b) 70 y = 10.944x 2 R = 0.9991 60 y = 4.1053x 2 R = 0.9992 %inhibition 50 40 maltol std trolox 30 20 10 0 0 5 10 15 20 con μM     Figure 9. The scavenging effects of dark soy sauce (a), maltol and trolox (b), on ABTS•+. Results are mean±SD, n≥3.      31    4.3 Maltol excretion in urine To test how fast maltol is metabolized, we measured the content of maltol in urine samples after a human volunteer had consumed dark soy sauce. The total volume of urine at different time points (0.5, 1, 2, 3, 4, 5, 6 h) were collected. No free form of maltol was found in urine samples, but after the urine sample was digested with β-Dgalactonidase, the HPLC chromatogram shows two main peaks (RT at 5.12, 8.13 min respectively) disappeared, while the peak of maltol (RT at 14.75 min) was observable (Figure 10 a, b). Peak 1 was tentatively identified as maltol sulfate by comparing this peak with that of synthesized maltol sulfate. Its RT and UV spectrum agreed well with those of the synthesized maltol sulfate (Figure 10 c, d). Peak 2 was isolated and purified using solid phase extraction, RP C-18 flash chromatography and identified as maltol glucuronide based on NMR data (Appendix Figure A-2). The presence of these two major metabolites of maltol was further confirmed with LC-MS/MS analysis. The total ion chromatogram of MS/MS scanning of maltol glucuronide of the urine sample shows a peak with RT at 3.12 min, whose mass spectrum presents two typical ions with m/z 303 and 270, the protonated parent ion maltol glucuronide and fragment ion after losing glucuronide chain, respectively (Figure 11). Although the total ion chromatogram of MS/MS scanning of maltol sulfate shows two peaks with RTs at 1.7 and 5.2 min, only the peak with RT at 1.7 min presents the desirable mass fragment pattern with protonated parent ion (m/z 207) and fragment ion after losing sulfate (m/z 127) (Figure 12). The multiple reaction monitoring (MRM) scan of maltol glucuronide (303/127) and maltol sulfate (207/127) also confirmed the presence of these two components in the urine samples (Figure 13). 32    The total amount of maltol excreted in urine was estimated by enzyme digestion of the urine samples. The HPLC method for determination of maltol in dark soy sauce was modified here for this purpose. Figure 14 shows the typical HPLC chromatograms of the urine sample with or without enzyme digestion. The urine samples were extracted with methanol and then dried up and digested with βglucuronidase. The time course of digestion was investigated. After 12 hours of digestion, the concentration of free form of maltol in the samples reached a plateau (Figure 15). After a subject took 6 mg of maltol only or 30 ml of dark soy sauce mixed with 200 gram of plain boiled rice, the amount of maltol excreted in urine at different time points was estimated. The maltol was excreted faster after taking maltol only than taking dark soy sauce mixed with plain boiled rice. When 6 mg maltol only was taken, around 0.9 mg maltol as its derivatives (sulfate and glucuronide) was found in the urine during 0.5 to 1.0 hour interval; whereas, when 30 ml of dark soy sauce mixed with 200 gram of plain boiled rice was taken, similar amount of maltol was excreted in the urine during 1 to 2 hour interval (Figure 16 A). The excretion of maltol is fast. Within 3 hour the accumulated maltol excreted in urine almost reached a plateau (Figure 16 B). In an observer-blinded, randomized, placebo controlled, crossover clinical study [38], the average amount of total maltol excreted in 24 young health volunteers was measured for different time points. Standardized with creatinine, the concentrations of total maltol in urine samples collected at 1 hour and 2 hour were significantly higher than those collected at 0 hour and 3 hour (Figure 17). 33    1200 Æpeak 1  800 600 400 200 Æpeak 2 (a)  7.656 2.749 3.164 3.517 4.001 4.507 4.815 1000 8.131 m AU 1400 5.122 D AD 1 D , S ig=258,4 R ef=600,100 (WAN G H S \MAU C 7050.D ) 0 0 5 10 15 D AD 1 C , S ig=274,4 R ef=600,100 (WAN G H S \MAU C 7064.D ) 14.754 4.514 m AU 1400 20 1200 1000 25 m in 25 m in  (b)  400 200 7.680 2.756 3.525 4.008 600 5.188 5.580 800 0 0 5 10 15 20     *D AD 1, 5.124 (1634 m AU ,A   m AU         *D AD 1, 5.224 (245 m AU ,Ap m AU 1400 1200 1000 800 600 400 200 0 (c)  200 (d)  150 100 50 0 200 250 300 350 nm 200 250 300 350 nm   Figure 10. Typical HPLC chromatograms of (a) urine sample collected at 1 hour after the subject orally taken 70 mg maltol, and (b) the same urine sample as in (a) digested with β-glucuronidase. And the UV spectrum of (c) peak 1, whose retention time (RT) at 5.12 min, agrees well with that of (d) synthesized maltol sulphate. 34    (A)    ( (B)      Figure 11. (A) ( The totaal ion chrom matogram of o MS/MS scanning of m maltol gluccuronide from urine sample. (B)) The mass spectrum of the peak with w retentioon time at 3.12 3 min in total ion chromatogrram A: Thee ion with m/z m 303 is thhe protonateed ion of maaltol glucuronidee; whereas the t product ion with m/z m 127 is prrotonated ioon of maltol.     35    (A)    (B)    (C)      Figure 12. (A).Total ion chromatogram of MS/MS scanning of maltol sulfate in urine sample. (B). MS spectrum of the peak with retention time at 5.2 min in total ion chromatogram A. (C). MS spectrum of the peak with retention time at 1.7 min in total ion chromatogram A: The ion with m/z 207 is the protonated ion of maltol sulfate; whereas the product ion with m/z 127 is protonated ion of maltol. 36    (A)    (B)    (C)      Figure 13. (A). Total ion chromatogram of multiple reaction monitoring (MRM) scan of maltol glucuronide (303/127) and maltol sulfate (207/127) in urine sample.(B). Extract ion chromatogram of MRM scan of maltol glucuronide (303/127). (C). Extract ion chromatogram of MRM scan of maltol sulfate (207/127).   37        35 6.633 2.732 3.522 3.973 4.494 5.202 m AU 9.457 D AD 1 D , S ig=258,4 R ef=600,100 (WAN G H S \MAU C 7018.D ) 30 21.747 11.720 7.661 9.052 10 10.054 10.687 15 5.992 6.347 7.190 7.855 8.115 2.932 3.017 3.316 3.732 3.880 4.323 20 4.813 3.183 25 5 0 0 5 10 15 D AD 1 C , S ig=274,4 R ef=600,100 (WAN G H S \MAU C 7030.D ) 2.730 3.521 3.967 4.496 m AU 20 25 m in *D AD 1, 14.787 (10.2 m AU , 35 m AU 8 5.255 30 6 25 14.784 0 11.727 6.616 9.443 9.717 10.061 10 2 7.852 7.661 15 6.008 2.926 3.046 3.120 3.316 3.730 3.876 20 4.815 5.360 4 200 250 300 350 nm 5 0 0 5 10 15 20 25 m in  Figure 14. The typical HPLC chromatograms of (a) urine sample with subject taking 30 ml of dark soy sauce and (b) that urine sample digested with enzyme: inset is the UV spectrum of peak with RT at 14.8 min which agrees with that of maltol.     38    0.6 0.5 Conc (mM) 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 Time (hr)   Figure 15. Time course of digestion of urine samples with 5000U β-glucuronidase at 37°C. 39    (A)  1.20 maltol amount (mg) 1.00 0.80 Maltol (6mg) 0.60 D SS (30mL) 0.40 0.20 0.00 0~0.5 0.5~1 1~2 2~3 3~4 4~6 Time (h) (B)  Maltol amount secreted (mg) 2.50 2.00 1.50 maltol (6mg) 1.00 0.50 0.00 0 1 2 3 4 Time (h) 5 6 7 Figure 16. (A). The maltol amount excreted in urine after one subject took 6 mg of maltol or 30 ml of dark soy sauce mixed with plain boiled rice. The accumulated maltol amounts excreted in urine for such two cases are also shown in (B). 40    maltol concentration (mmol/mol creatinine) 30.0 25.0 ** ** 20.0 15.0 10.0 5.0 0.0 0 1 2 3 4 24 Time point (hour) Figure 17. The average total maltol (standardized with creatinine) measured in the different time point urine samples of 24 young healthy subjects who orally took 30 ml of dark soy sauce mixed with 200 gram of plain boiled rice. Data are mean±SD, n=24 (**p < 0.01 vs 0 h and 3 h). 41    4.4 Fractionation and characterization of the colored components When the MeOH-R fractions of dark soy sauce were incubated in 6 M HCl in vacuo at 110°C for 18 h, the colored components became insoluble (the solvent became transparent, and colored precipitate was present at the tube bottom), while in 4.2 M NaOH they were stable. Removal of colored components dramatically decreased the antioxidant activity of the hydrolysate (Figure 18), suggesting that the colored components could greatly contribute to the TAA of dark soy sauce. To further investigate the colored components, we used gel filtration chromatography to fractionate the dark soy sauce. The MeOH-R and EtOAc-R fractions were separately dialyzed against distilled water and further fractionated with gel filtration chromatography as described in Experimental section. Figure 19A shows the gel filtration chromatograms of these two fractions. We obtained a high-molecular-mass fraction (CP2) from the MeOH-R, and a lower-molecular-mass fraction (CP1) from the EtOAc-R. Indeed, the absorbance at 470 nm and ABTS•+ scavenging activity of CP1 and CP2 were highly correlated, r = 0.9945 for CP1 and 0.9999 for CP2 (Figure 19B). However, the ratios of scavenging activity to absorbance for the two fractions were not equal, 1629.5 for CP1 and 1125.9 for CP2, suggesting that different compositions are present. The 1H-NMR and 13C-NMR spectra of CP1 (Figure 20a and 20c) are very complex. Despite this, it was possible to observe the resonance signals related to carbonyl groups (δ 166.8). The spectra also indicated the presence of carbohydrates: the proton signals at δ 5.36-5.33 and carbon signals at δ 99.9-98.5 were due to the anomeric hydrogens and carbons, and a complex of multiplet between δ 3.93 and δ 3.37 and the 42    carbon signals at the range of δ 77.6 to δ 60.5 corresponded to other sugar backbone protons and carbons, respectively. Inhibition 2.5 50 45 40 35 30 25 20 15 10 5 0 Abs at 470 nm 2 1.5 1 *** 0.5 0 Sa mp le+ Sa 6M HC l mp le + Sa 4 .2 M mp Na le + %Inhibition A bs H2 O OH Figure 18. The absorbance and ABTS•+ scavenging activity of MeOH-R fraction under acidic and basic conditions. The MeOH-R samples were incubated in 6 M HCl or 4.2 M NaOH in vacuo at 110°C for 18 hours. The colored components became insoluble in acidic condition, while in alkaline condition they were stable. Removal of insoluble colored components dramatically decreased the antioxidant activity of the acidic hydrolysate, indicating that the colored components could greatly contribute to the total antioxidant activity (TAA) of dark soy sauce. Values are mean ± SD, n=3. ***Comparision between the ABTS•+ scavenging activity of sample + 6 M HCl and that of sample + H2O (*** p < 0.001).     43      (A)    50 000                                                                                    1000   Fractionation Range (M Mr) (Dextrans)   (B B)  %Inhibition 40 35 C P1 30 C P2 y = 1629.5x r = 0.9945 p < 0.001 25 20 25.9x y = 112 r = 0.9 9999 p < 0.001 15 10 5 0 0 0 0.005 0.01 0.015 0.02 0.025 Abs orban ce at 470nm   Figure 19. (A) ( Overlaiin Sephadexx G-75 gel filtration f chromatogram ms of EtOAc-R and MeOH-R of o dark soy sauce. s Fracttions 26 to 34 3 of EtOA Ac-R were coombined as Colored Prooduct 1 (CP P1), and fracctions 3 to 9 of MeOH--R were com mbined as Colored C Product 2 (CP2). The fragmentati f on range off Sephadex G75 gel filttration chromatogrraphy is 10000 ~ 50 0000 Dalton. (B B) The correelation of AB BTS•+ scaveenging activity andd absorbancce of CP1 annd CP2 at 470 nm. Valuues are meaan ± SD, n= =3. 44      Figure 20. (a) 1H-NM MR spectrum m of CP1, (b b) 1H-NMR spectrum of CP2, and (c) 13CNMR specttrum of CP11. 45    1 H-NMR spectrum of CP2 (Figure 20b) showed similar signals to those of CP1 (Figure 20a). However, the ratios of integral of the signal peaks at δ 8.25 to that at δ 5.36-5.33 are different for these two fractions; around 1:2 for CP1 and 1:1 for CP2. This difference suggested that their compositions are somewhat different. The ESI-TOF mass spectrum of CP1 (Figure 21) showed that most of the observed ions were doubly-charged (as shown in the inset of Figure 21, the signal was observed at 1001.3 with the peaks of the isotopic pattern at 0.5 Dalton distance), and the m/z range was from 200 to 2200, centered at 1000, indicating that the average molecular weight was about 2000. As shown in Table 3, the observed ions can be assigned to several series. Interestingly, within each series, m/zs of the doubly-charged or the singly-charged ions consistently increase by 81 or 162, respectively. This moiety with a mass of 162 could be a hexose losing one molecule of water (180-18). These results were consistent with our NMR data, suggesting that CP1 is a sugar-containing fraction. Using TOF-MS, we also investigated the effect of adding the ABTS•+ free radical on the structure of the colored components. We compared the mass spectra of the CP1 solution spiked with ABTS•+ stock solution with those without spiking. Interestingly, we found the intensities of doubly-charged ions, especially those in Series 2 (Table III), were markedly decreased, while the singly-charged ions have no or little change. These findings suggest that the sugars contribute little to the antioxidant activity; while possibly chromophore units are present, contributing to both the color and the antioxidant activity. 46    1001.3344 100 1001.3344 839.2879 1001.8270 920.2736 100 1001.8270 1002.3366 1082.3546 1175.4012 758.2728 % 1002.7953 1337.4618 0 596.1904 1176.3951 344.1183 425.1577 1662.7813 400 600 m 1003 1661.7953 1338.5027 1500.6201 263.0956 0 200 1002 1499.5386 677.2410 % 1001 1003.3053 800 1000 1200 1400 1600 1824.1770 1986.4733 1800 2000 2200 m/z Figure 21. TOF-MS spectrum of CP1. The inset shows a typical doubly charged ion. The peaks observed at around 1001.3 show the isotopic pattern at 0.5 Dalton distance.       47      Table III. The observed ions in TOF-MS spectrum of CP1. Three series were observed. Within each series, m/zs of the doubly-charged or the singly-charged ions consistently increase by 81 or 162, respectively. Series 2† Series 1 ions Charge M.W. states (Da) 263.0956 2 528.2 344.1483 2 425.1577 ions Series 3 Charge M.W. states (Da) 353.1367 2 708.3 690.3 434.1462 2 2 852.3 515.1589 506.1745 2 1014.3 587.2086 2 668.2075 ions Charge M.W states (Da) 1191.4094 1 1192.4 870.3 1353.4515 1 1354.5 2 1032.3 1515.6155 1 1516.6 596.1904 2 1194.4 1677.6451 1 1678.6 1176.4 * 677.2410 2 1356.5 1839.7515 1 1840.8 2 1338.4* 758.2728 2 1518.6 749.2477 2 1500.5* 839.2879 2 1680.6 1175.4012 1 1176.4* 920.2736 2 1842.5 1337.4618 1 1338.5* 1001.3334 2 2004.7 1499.5386 1 1500.5* 1082.3546 2 2166.7 1661.6138 1 1662.6 1163.4160 2 2328.8 1823.7094 1 1824.7 1244.4283 2 2490.9 1985.7924 1 1986.8 1325.4880 2 2653.0 1406.5306 2 2815.1 1487.5840 2 2977.2   * Both singly-charged and doubly-charged ions were observed. Intensities of peaks which were markedly decreased after reaction of CP1 with ABTS•+.    † 48    4.5 Protection against HOCl-induced DNA damage Hypochlorous acid (HOCl), a reactive oxidative agent, can be generated by myeloperoidase that is released by activated phagocytes at sites of inflammation [40]. HOCl can oxidize various biological molecules, such as lipids, amino acids, proteins, nucleotides and DNA repair enzymes. Chronic inflammatory diseases in GI tracts, e.g. ulcerative colitis and Crohn’s disease, are thought to cause tissue damage and be associated with the increased risk of tumorigenesis. Thus we are to tests whether the components of dark soy sauce can protect against HOCl induced DNA damage. Treatment of DNA with HOCl led to various detectable base modification products. Significant increases were observed for 5-Cl uracil, 5-OH uracil, and 5-OH cytosine, but not for 5-(OH, Me) hydantoin, FAPy adenine, FAPy guanine and 8-OH guanine (Table IV). The formation of HOCl-induced base modification products was inhibited dose-dependently by pretreatment of DNA with maltol, CP1 or CP2 (Table IV).   49    Table IV. Inhibition of HOCl-induced DNA damage by maltol, the colored product 1 (CP1) or the colored product 2 (CP2) #. Oxidized base Control DNA+HOCl DNA+HOCl+maltol DNA+HOCl+CP1 DNA+HOCl+CP2 50 μM 250 μM 500 μM 25 μg/ml 125 μg/ml 250 μg/ml 25 μg/ml 125 μg/ml 250 μg/ml 5-Cl uracil 0.002±0.002 0.986±0.308 0.111±0.015 0.068±0.005 0.048±0.008 1.317±0.239 0.770±0.062 0.528±0.013 1.321±0.191 0.632±0.040 0.521±0.038 5-(OH,Me) hydantoin 0.079±0.002 0.096±0.013 0.084±0.000 0.079±0.002 0.081±0.011 0.097±0.002 0.092±0.004 0.088±0.005 0.098±0.004 0.093±0.007 0.094±0.006 5-OH uracil 0.004±0.001 0.912±0.325 0.144±0.005 0.074±0.007 0.048±0.006 1.190±0.072 0.673±0.070 0.456±0.036 1.021±0.083 0.547±0.032 0.448±0.014 5-(OH, Me) uracil 0.027±0.015 0.095±0.063 0.085±0.039 0.065±0.029 0.052±0.029 0.109±0.034 0.097±0.043 0.095±0.042 0.120±0.032 0.116±.022 0.107±0.028 5-OH cytosine 0.019±0.009 1.517±0.552 0.201±0.021 0.110±0.020 0.093±0.025 1.224±0.127 0.822±0.055 0.558±0.078 1.217±0.094 0.673±0.030 0.533±0.021 FAPy adenine 0.037±0.007 0.046±0.011 0.060±0.006 0.054±0.006 0.043±0.007 0.054±0.008 0.052±0.006 0.056±0.005 0.059±0.004 0.052±0.002 0.049±0.007 8-OH adenine 0.075±0.012 0.466±0.180 0.123±0.042 0.104±0.019 0.166±0.138 0.475±0.056 0.337±0.051 0.269±0.060 0.457±0.040 0.280±0.018 0.250±0.029 2-OH adenine 0.057±0.012 0.224±0.054 0.146±0.091 0.104±0.046 0.123±0.074 0.286±0.126 0.243±0.135 0.215±0.127 0.304±0.132 0.185±0.054 0.159±0.034 FAPy guanine 0.107±0.025 0.101±0.032 0.175±0.023 0.267±0.217 0.115±0.014 0.176±0.073 0.302±0.221 0.190±0.078 0.135±0.004 0.125±0.009 0.122±0.016 8-OH guanine 0.415±0.067 0.239±0.068 0.394±0.126 0.349±0.054 0.307±0.188 0.389±0.134 0.381±0.128 0.386±0.119 0.290±0.034 0.276±0.053 0.336±0.046 # Data are mean±SD, n=3. 50    4.6 Cytotoxicity on HT-29 cells To explore the potent effects of dark soy sauce and its components on GI tract, using MTT assay, the cytotoxicity of dark soy sauce was tested on HT-29 cells, a human colon adenocarcinoma cell line. Figure 22A shows that dark soy sauce did not result in significant cytoxicity at low concentrations, i.e. 1, 2.5, 5 μl/ml, of dark soy sauce, but at higher concentrations, such as 10 μl/ml of dark soy sauce for 72 hr, only around 30% cells survived. When the concentration of dark soy sauce increased to 20 μl/ml, the viability was reduced to around 15% after 48 hr exposure. Dark soy sauce has high concentration of salts, e.g. sodium chloride. In case the salts influenced the cytotoxicity results, the cell viability was also tested using non-dialysable part of dark soy sauce. Their cytotoxic effects were dose- and time-dependent: the higher the dose and the longer exposure used, the lower the cell viability observed (Figure 22B). In cell culture, it is commonly observed that the cytotoxic effects of adding compounds are possibly caused by the artificially formed H2O2 in the medium [47]. When 5 μl/ml of dark soy sauce was spiked into the culture medium, around 50 μM H2O2 was detected after 60 min, while 100 μM H2O2 was produced when the concentration of spiked dark soy sauce increased to 20 μl/ml. (Figure 23A). But when exposed to 100 μM H2O2 for 72 hr, around more than 60% of HT-29 cells still survived (Figure 23B). Furthermore, using catalase to neutralize the effects of H2O2, the cell viability did not change when exposed to 20 μl/ml of dark soy sauce with or without catalase (Figure 23C), suggesting that the cytotoxic effects observed was not caused by H2O2 . 51      .       (A)  140 % Viable Cells 120 100 24hr 80 48hr 60 72hr 40 20 0 0 1 2.5 5 10 20 DSS Concentration (μl/ml)     (B)  120 Cell viability (%) 100 80 24hr 60 48hr 40 72hr 20 0 0 1 2.5 5 10 20 DSS Nondialysable (mg/ml)   Figure 22. Decrease of cell viability of HT 29 cells 24, 48 and 72 hours after incubation with various concentrations of dark soy sauce (DSS) (A), and DSS Nondialysable fraction (B). Results are mean±SD, n=3. 52       D SS D SS+Catalase 120 % Viable Cells 100 80 60 40 20 0 0 5 20 DSS concentration ( ul/ml)   Figure 23. Cell viability tested on dark soy sauce only or plus catalase (1000 units/ml).    53    4.7 Inhibition of COX-2 protein expression in LPS-induced HT-29 cells HT-29 cells expressed a low basal level of cyclooxygenase-2 (COX-2) protein [48], which can be obviously up-regulated by lipopolysacchride (LPS) induction at 100 ng/ml. The time course in Figure 24 shows that after 2 hours, the levels of COX-2 protein expression had not much difference among the control, treatment with LPS only, or treatment with both dark soy sauce and LPS. After 24 hours induction, the up-regulation of COX-2 expression in HT-29 cells was observable, but such effect was inhibited by treatment with 5 μl/ml dark soy sauce. The inhibitory effects of dark soy sauce on the expression of COX-2 protein in LPS-induced HT-29 cells were further observed at the concentrations of 1 μl/ml and 5 μl/ml (Figure 25), at which concentrations, the dark soy sauce has no significant cytotoxic effects (Figure 22). But at the concentrations of 100 μM and 500 μM, maltol did not exhibit remarkable inhibitory effects (Figure 26).           Figure 24. Time course of COX-2 expression and its inhibition by dark soy sauce in lipopolysaccharide (LPS)-induced HT-29 cells. Cells were pretreated with dark soy sauce (5 μl/ml) for 30 min and then induced with LPS (100ng/ml) for various time indicated. Protein levels were estimated by Western Blot analysis as described in “Materials and Methods”. Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells treated with LPS (100 ng/ml) only); and lane 3, HT-29 cells simultaneously treated with dark soy sauce (5 μl/ml) and LPS (100 ng/ml). 54        Figure 25. Inhibitory effect of dark soy on COX-2 expression in LPS-induced HT-29 cells . Lane 1, untreated HT-29 cells; Lane 2, HT-29 cells treated with 1 μl/ml dark soy sauce(DSS); Lane 3, with 5 μl/ml DSS; Lane 4, with LPS (100 ng/ml); Lane 5, with DSS (1 μl/ml) and LPS (100 ng/ml); Lane 6, with DSS (5 μl/ml) and LPS (100 ng/ml). The western blot is from a single experiment, but is representative of 3 independent experiments.   Figure 26. The effect of maltol on COX-2 expression in LPS-induced HT-29 cells.Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells treated with maltol (100 μM); lane 3, maltol (500 μM); lane 4; treated with LPS (100 ng/ml); lane 5, maltol (100 μM) and LPS (100 ng/ml); lane 6, maltol (500 μM) and LPS (100 ng/ml).   55    4.8 Discussion Our data show that the ethyl acetate extract of dark soy sauce has the strongest antioxidant activity based on the ABTS assay which detects a wide range of antioxidants [35, 36]. Fractionation of that extract produced several components with antioxidant activity, but the most active was indentified as maltol. We also developed an HPLC method to determine the maltol level in dark soy sauce. Although maltol has been identified in many soy sauce products [49-51], to the authors’ knowledge, this is the first study to determine its concentration in soy sauce and its contribution to the TAA of dark soy sauce. Maltol can be formed from reducing sugars during food processing and has been identified in a wide variety of other heated materials such as bread crusts, coffee, cocoa beans, cereals, dried whey and chicory [51]. Therefore, the amount of the daily maltol intake can be considerable. Maltol has even been suggested to be capable of exerting neuroprotective activity by inhibiting oxidative damage [52-54], and its iron chelate has been used to treat anaemia in humans [55]. At present the pharmacokinetics of maltol after oral dosing have only been investigated in dogs and indicate rapid and extensive absorbtion, followed by conjugation and rapid excretion [56]. However, the oral bioavailability of maltol in humans has not yet been examined. In our study on healthy human subjects, maltol in conjugated form was at high concentrations in urines 2 hours after oral administration, consistent with rapid aborption, metabolism and excretion of the products in urine. The flavonoids, e.g. daidzein and genistein, have been identified in some soy sauce products and postulated to have antioxidant activities [57], but in our study, we found the levels in the dark soy sauce to be undetectable. This is not perhaps surprising, since soy sauce is generally regarded as a poor source of isoflavones [58]. 56    However, the major antioxidant activity of dark soy sauce appears to reside in high molecular weight colored components, some soluble in methanol and others not, containing carbohydrate residues. These are most probably melanoidins, brown polymers formed by Maillard reactions between reducing sugars and compounds possessing a free amino group, such as free amino acids and the amino groups of peptides [59]. Although soy sauce is a traditional cooking ingredient throughout Asia, the actual process and the composition of the starting ingredients for making the soy sauce is known to differ between countries. It is highly likely that differences in the raw materials, fermentation time and heating processes used during the manufacture of soy sauce may affect the composition and antioxidant activity of the final products. Indeed, variation in the antioxidant capacity of different soy sauce products has previously been demonstrated [36]. Conventional soy sauce making involves a solidstate aerobic fermentation process by Aspergillus species on a soybean and wheat mixture. Aspergillus produces extracellular enzymes, in particular proteases and amylases, which hydrolyze the proteins and polysaccharides of soybean and wheat. Subsequently, the sugars and amino acids produced are further digested during a brine fermentation process and, depending on the extent of fermentation process that takes place, the amount of Maillard reaction products can vary. Full details of commercial soy sauce manufacture tend to be carefully guarded by Asian food companies and the exact details of manufacture for the brand examined in this study are not known. The antioxidant characteristics of Maillard related products from fermented soy sauce have been studied [60] and melanoidins have previously been isolated from soy sauce [61]. Melanoidins have been characterized in many foods, such as beer [62], bread [63] and roasted coffee [64,65]. Many studies have attempted to elucidate the structure 57    characteristics of melanoidins [59, 66], using a variety of starting materials. Ando [32] investigated different soy sauce products, and found that the color of soy sauce was not proportional to its antioxidant capacity. In our study, even for one soy sauce product, the ratios of free radical scavenging ability to color density of two different fractions, CP1 and CP2, were different. This could be due to the variety of chemical structures of the colored components. In this study, NMR and MS were used to eludicate the structural characteristics of melanoidins. NMR and MS data of CP1 indicate the presence of carbohydrate residues, which agrees with the finding that sugar moieties could be part of the melanoidin backbone [66]. The carbonyl resonance and nonsaturated proton signals of CP1 suggest that amino acids may be involved in the formation of the chromophore unit. Based on our ESI-TOF-MS data, the molecular weights of CP1 are in the range of 400 – 4400, which is comparable to soy sauce pigments fractionated by Motai et al. [67]. Maillard reaction products add the flavor and brown color to soy sauce [68]. One of these antioxidant flavor components, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)furanone (HEMF) was reported to inhibit benzo[α]pyrene-induced forestomach neoplasia in mice and reduce the hydrogen peroxide concentration in human polymorphonuclear leucocytes [34]. The colored components of soy sauce have also been linked to the antioxidant capacity of soy sauce [32]. For example, soy sauce melanoidin strongly inhibited NO-induced DNA damage in a dose dependent manner, as determined by the comet assay [69]. In vivo, the formation of hypochlorous acid (HOCl) by activated netrophils in defense against microorganisms may cause DNA damage [70]. Our studies also suggest that, in vitro, maltol and the colored products of dark soy sauce can reduce HOCl-induced DNA base modification. In an in vitro study, melanoidin has been demonstrated to affect the growth of human colonic 58    bacteria [71]. Melanoidins extracted from soy sauce have been suggested to inhibit colon cancer cell growth [72]. Our data also indicate that dark soy sauce exibits some inhibitory effect on growth of the HT29 colon cancer cell line. As has been suggested for other nutritional antioxidants [73, 74], melanoidins may play a role in the antioxidant defence of the gastrointestinal tract (GIT), since they are unlikely to be absorbed as such. Maltol may well be absorbed, however, and could conceivably exert systemic antioxidant effects. Postprandial oxidative stress is thougth to be associated with increased risk for various metabolic syndromes, such as atherosclerosis, due to an increased susceptibility of the organism toward oxidative damage after consumption of a meal rich in lipids and/or carbohydrates [75]. Epidemiological studies suggest that food and diets rich in antioxidants could be benefitful to decrease the postprandial oxidative stress. However, clinical studies on the efficacy of antioxidants in the prevention of atherotic diseases have reported conflicting results. It has reported that the timing of administration of antioxidants has various effects on postprandial oxidative serum markers [76]. The failure of observation of antioxidant effects in some clinical studies could be due to the missed timing. Our data show that maltol can be found in urine at high concentration two hours after orally taken, indicating it may be rapidly absorbed. . In our in vivo study, dark soy sauce has a rapid (3-4 h) antioxidant effect [38]. Thus when dark soy sauce is used as seasoning for high fat diets, maltol and other small molecule components with similar structures could timely reduce the postprandial oxidative stress. Recent findings suggest that tumor microenvironment can be crucial for carcinogenesis [77]. Many molecular targets in the microenvironment might be 59    selected for chemoprevention, such as cyclooxygenase-2 (COX-2). COX-2 overexpression has been documented in more than 80% of human colon carcinomas [78]. Population-based studies suggested that long-term intake of non-steroidal antiinfammatory drugs, compounds that inhibit the enzymatic activity of cyclooxygenase (COX), reduces the relative risk for developing colorectal cancer [79]. COX-2 expression may protect cancer cells from apoptosis induced by a variety of stimuli and could enhance cell tumorigenic potential. So COX-2 inhibitors might be useful for colorectal cancer prevention and treatment. Our data suggest that dark soy sauce can decrease COX-2 protein levels in LPS-induced HT29 cells after 24 hour incubation.   60    Chapter 5 Conclusion   Our data show that 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) is one major active compounds in an ethyl acetate extract of dark soy sauce and present at millimolar concentrations in dark soy sauce. In human urine, maltol is excreted quickly and mainly in conjugated form. Our data of nuclear magnetic resonance (NMR) and electrospray-ionization time-offlight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydratecontaining pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS. In vitro, maltol and the colored products can protect against hypochlorous acid (HOCl)-mediated DNA damage dose-dependently. Furthermore, dark soy sauce potentially inhibits the growth of colon cancer HT 29 cells at high concentrations, while decreases the up-regulation of cyclooxygenase-2 (COX-2) expression in LPS (lipopolysaccharide) -induced HT 29 cells at low concentrations. Although maltol can scavenge ABTS free radicals and protect HOCl-induced DNA damage in vitro, it is still unknown whether it exerts such antioxidant effects in vivo. Since maltol might be readily well absorbed, it is worthwhile to test whether it can reduce the postprandial oxidative stress in model animals or human subjects. The potent chemopreventive efficacy of the colored products of dark soy sauce on colon cancer can be further tested using an inducible inflammation-driven colorectal carcinogenesis rat/mouse model. 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[79] Thun MJ, Namboodiri MM, Heath CWJ. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 1991; 325:1593-1596. [80] Wang HS, Jenner AM, Lee CYJ, Shui GH, Tang SY, Whiteman M, Wenk MR, Halliwell B. The identification of antioxidants in dark soy sauce. Free Radic Res 2007; 41:479-488. 69 Appendices     (A)     (B)   Figure A-1. (A) 1H-NMR and (B) 13C-NMR spectra of compound 1. 70    (A)       (B)     Figure A‐2. (A) 1H‐NMR and (B) 13C‐NMR spectra of maltol glucuronide.   71    (A)  %T   100  90 4000 3 3500 3000 2000 2 1750 1500 Wavenumber (1/cm) 509.23       708.87 691.51 846.79   918.16     1256.68 1250 1198.81   1459.21  1397.49 2500 1617.38 30   40 1655.00 50   3257.91   60 1560.48 70   3068.88   1073.43 1021.35   80 1000 0  750  500   (B) (C) ompound 1,, (B) CP1 annd (C) CP2. Figure A-3. Fourier infrared specttra of (A) co 72    Table A-1. Summary of LC-ITMS/MS parent ion and product ions and their possible identities of the 10 fractions from dark soy sauce. Fr No. Parent ion and product ions Possible identities 1 126, 108 182, 164, 136 3-Methoxy-2[1h]pyridone; 2-Amino-3-(4-hydroxyphenyl)-propanoic acid, tyrosine 2 215, 197, 179, 137 185, 167, 149 2-methyl-Dodecanoic acid; 4-Hydroxy-3,5-dimethoxybenzyl alcohol 3 155, 137 108 4 125, 107 88 2-acetyl-5-methylfuran 5 141, 127 109 199, 181 163 121 3-Hydroxy-4-methoxymandelic acid 6 127, 97 79 7 125‚ 107 2-acetyl-5-methylfuran 8 167, 149 125, 107 2-acetyl-5-methylfuran 9 127 maltol 10 155, 123 141, 123 95 81 2-pentylthiophene 3-methoxypyrocatechol 73    Intens. x10 8 2 3 2 1 3 4 5 1 0 0 1 2 Intens. 2. x10 8 3 4 5 6 7 8 T ime [min] +MS , 2.0-2.0min #136-#138 126.3 2 1 108.4 0 x10 6 2. 6 182.3 +MS 2(126.4), 2.0-2.1min #137-#139 108.4 4 2 0 50 100 150 Intens. 3. x10 7 200 250 300 350 400 450 m/z +MS , 2.1-2.2min #144-#146 126.3 182.3 3 2 108.4 1 96.4 140.3 170.3 212.3 0 x10 6 3. +MS 2(108.4), 2.1-2.2min #145-#147 126.3 1.0 0.5 0.0 50 100 Intens. 4. x10 7 150 200 250 300 350 400 450 m/z +MS , 2.1-2.3min #140-#154 126.3 6 182.3 4 108.4 2 96.4 0 x10 4 4. 3 140.3 +MS 2(182.4), 2.1-2.3min #141-#155 136.3 2 164.3 1 183.2 391.4 0 50 100 150 200 250 300 350 400 450 m/z Figure A-4. Total ion chromatogram (TIC), MS and MS2 [126], MS and MS2 [108] , MS and MS2 [182] of fraction 1. 74    Intens. x10 9 1.25 4 1.00 5 0.75 0.50 0.25 1 0.00 0 1 2 Intens. 4. x10 8 3 2 3 6 7 8 9 101112 4 5 6 T ime [min] +MS , 3.7-3.9min #261-#271 185.3 215.3 3 2 1 0 x10 7 4. 3 +MS 2(215.3), 3.8-3.9min #262-#272 197.3 2 179.3 137.3 1 0 100 150 Intens. 8. x10 8 200 250 300 350 400 450 m/z +MS , 3.8-4.3min #265-#299 185.3 215.3 1 123.3 0 x10 7 8. +MS 2(185.4), 3.8-4.3min #266-#300 167.3 1.0 0.5 0.0 149.3 107.4 100 150 Intens. 5. x10 8 200 250 300 350 400 450 m/z +MS , 3.9-3.9min #273-#275 185.3 4 215.3 2 123.3 0 x10 5 5. 1.0 +MS 2(123.4), 3.9-4.0min #274-#276 391.3 95.5 0.5 139.3 0.0 100 150 200 250 300 350 400 450 m/z Figure A-5. Total ion chromatogram (TIC), MS and MS2 [215], MS and MS2 [185] , MS and MS2 [123] of fraction 2. 75    Intens. x10 8 6 3 4 4 2 1 0 0 2 Intens. 4. x10 8 3 4 56 8 7 9 2 6 8 10 T ime [min] +MS , 5.9-6.3min #356-#384 155.3 2 1 137.3 0 x10 7 4. +MS 2(155.4), 5.9-6.3min #357-#385 137.3 2 1 0 100 Intens. 3. x10 8 150 200 250 300 350 400 450 m/z +MS , 5.8-6.2min #352-#380 155.3 3 2 137.3 1 0 x10 4 3. 6 +MS 2(137.2), 5.8-6.2min #353-#381 391.3 4 2 108.4 0 100 150 200 250 300 350 400 450 m/z Figure A-6. Total ion chromatogram (TIC), MS and MS2 [155], MS and MS2 [137] of fraction 3. 76    Intens. x10 8 3 1 2 2 1 3 4 56 7 0 0 2 Intens. 2. x10 8 4 6 8 10 T ime [min] +MS , 7.1-7.6min #371-#405 125.3 1.0 0.5 107.4 0.0 x10 6 2. +MS 2(125.5), 7.1-7.6min #372-#406 107.4 1.0 0.5 391.3 0.0 100 Intens. 1. x10 8 150 200 250 300 350 400 450 m/z +MS , 7.2-7.5min #375-#395 125.3 2 1 107.4 0 x10 4 1. +MS 2(107.4), 7.2-7.5min #376-#396 391.2 2 1 0 88.5 100 150 200 250 300 350 400 450 m/z Figure A-7. Total ion chromatogram (TIC), MS and MS2 [125], MS and MS2 [107] of fraction 4. 77    I n te n s. x1 0 8 4 5 3 1 .5 6 1 .0 7 0 .5 1 2 0 .0 0 2 I n te n s. 3 . x1 0 7 4 6 8 10 T i m e [m i n ] + M S , 7 .1 -7 .1 m i n # 3 8 9 -# 3 9 1 1 2 7 .3 1 0 9 .4 4 2 0 x1 0 5 3. + M S 2 (1 0 9 .4 ), 7 .1 -7 .1 m i n # 3 9 0 -# 3 9 2 3 8 7 .3 1 .0 8 1 .5 0 .5 2 9 5 .4 0 .0 100 I n te n s. 4 . x1 0 7 6 150 200 300 4 7 0 .3 350 400 450 m /z + M S , 7 .1 -7 .2 m i n # 3 9 3 -# 3 9 5 1 2 7 .3 1 0 9 .4 1 4 1 .3 4 2 0 x1 0 6 250 1 9 9 .3 4. + M S 2 (1 4 1 .4 ), 7 .1 -7 .2 m i n # 3 9 4 -# 3 9 6 1 0 9 .3 2 1 3 9 1 .4 0 100 150 200 I n te n s. 5 . x1 0 7 300 350 400 450 m /z + M S , 7 .0 -7 .3 m i n # 3 8 5 -# 4 0 7 1 9 9 .3 1 2 7 .3 1 0 9 .3 2 250 1 0 x1 0 6 1 .5 5. + M S 2 (1 2 7 .4 ), 7 .0 -7 .3 m i n # 3 8 6 -# 4 0 8 1 0 9 .3 1 .0 0 .5 3 9 1 .3 0 .0 100 150 200 In te n s. 7 . x1 0 7 2 1 250 300 350 400 450 m /z + M S , 7 .2 -7 .6 m i n # 3 9 7 -# 4 2 3 1 9 9 .3 1 2 7 .3 1 0 9 .3 0 x1 0 6 7 . + M S 2 (1 9 9 .4 ), 7 .2 -7 .6 m i n # 3 9 8 -# 4 2 4 1 8 1 .3 1 .0 1 6 3 .3 0 .5 1 2 1 .4 0 .0 100 150 200 250 300 350 400 450 m /z Figure A-8. Total ion chromatogram (TIC), MS and MS2 [109], MS and MS2 [141], MS and MS [127], MS and MS2 [199] of fraction 5. 78    Intens. x10 7 2 5 3 4 1 3 2 1 0 6.0 6.5 Intens. 2. x10 7 7.0 7.5 8.0 8.5 9.0 9.5 10.0 T ime [min] +MS , 7.9-8.0min #432-#434 127.3 1.0 0.5 126.3 0.0 x10 5 2. 1.00 0.75 157.3 177.3 205.3 271.3 +MS 2(126.6), 8.0-8.0min #433-#435 391.2 97.4 0.50 0.25 0.00 127.2 79.5 100 Intens. 3. x10 7 187.4 150 200 250 300 350 400 450 m/z +MS , 8.0-8.1min #436-#442 125.3 1.0 157.3 0.5 0.0 x10 4 3. 6 126.3 205.3 271.3 +MS 2(126.0), 8.0-8.1min #437-#443 97.4 4 403.1 2 79.5 0 100 150 200 250 300 350 400 450 m/z Figure A-9. Total ion chromatogram (TIC), MS and MS2 [127], MS and MS2 [126] of fraction 6. 79    Intens. x10 8 2 2.5 1 2.0 1.5 1.0 34 0.5 0.0 0 2 Intens. 2. x10 8 4 6 8 10 12 14 T ime [min] +MS , 9.1-9.6min #420-#454 125.3 1.5 1.0 0.5 0.0 x10 5 2. 4 +MS 2(125.2), 9.2-9.6min #421-#455 125.3 391.3 2 107.4 187.3 0 100 150 200 250 300 350 400 450 m/z Figure A-10. Total ion chromatogram (TIC), MS and MS2 [125] of fraction 7. 80    Intens. x10 7 2 3 5 4 3 1 2 1 0 7 Intens. 2. x10 7 8 9 10 11 12 T ime [min] +MS , 9.2-9.4min #437-#447 125.3 1.5 167.3 1.0 149.3 0.5 197.3 0.0 x10 6 2. +MS 2(167.7), 9.2-9.4min #438-#448 149.3 2 1 0 100 Intens. 3. x10 7 150 200 250 300 350 400 450 m/z +MS , 9.3-9.7min #441-#467 125.3 2 1 167.3 0 x10 4 3. 6 +MS 2(125.2), 9.3-9.7min #442-#468 125.4 391.3 4 2 107.4 187.3 0 100 150 200 250 300 350 400 450 m/z Figure A-11. Total ion chromatogram (TIC), MS and MS2 [167], MS and MS2 [125] of fraction 8. 81    Intens. x10 8 1 3 2 1 2 0 0.0 2.5 5.0 7.5 10.0 12.5 3 4 56 15.0 17.5 Intens. 1. x10 8 20.0 22.5 T ime [min] +MS , 12.1-12.6min #417-#453 3 127.3 2 1 0 x10 6 1. +MS 2(127.3), 12.1-12.6min #418-#454 391.3 1.0 0.5 0.0 50 100 150 200 250 300 350 400 450 m/z Figure A-12. Total ion chromatogram (TIC), MS and MS2 [127] of fraction 9. 82    Intens. x10 7 5 1 2 3 4 3 2 1 0 0 2 4 Intens. 1. x10 7 6 8 10 12 14 16 18 T ime [min] +MS , 14.2min #449 141.3 123.3 2 155.3 1 0 x10 6 1. 1.5 +MS 2(155.4), 14.2min #450 123.3 1.0 0.5 0.0 50 100 Intens. 2. x10 7 2 150 200 250 300 350 400 450 m/z +MS , 14.1-14.3min #443-#457 141.3 123.3 1 0 x10 4 2. +MS 2(123.4), 14.1-14.3min #444-#458 95.4 3 81.4 2 401.3 1 0 50 100 Intens. 3. x10 7 150 200 250 300 350 400 450 m/z +MS , 14.0-14.4min #439-#465 141.3 1.5 123.3 1.0 0.5 0.0 x10 6 3. +MS 2(141.4), 14.0-14.4min #440-#466 123.3 1.5 1.0 0.5 391.2 0.0 50 100 150 200 250 300 350 400 450 m/z Figure A-13. Total ion chromatogram (TIC), MS and MS2 [155], MS and MS2 [123], MS and MS2 [141] of fraction 10. 83    (A) (B) Figure A-14. UV spectra of (A) CP1 and (B) CP2. 84    [...]... countries by the Chinese immigrants In Singapore, it is said that the small-scale manufacturing of soy sauce started by a small number of Xin-hui Cantonese, a sub-dialect group from Guangdong Province of South China, just a couple of years after the first arrival of Chinese immigrants in the early nineteenth century [12] After being successful, many of them shifted their business to other more profitable fields... components of soy sauce improve the taste of many types of foods, but its coloring ingredients can enhance the appearance of the dipped food or the mixed soup Moreover, recent studies indicate that some ingredients in soy sauce are potentially beneficial to human health, showing effects such as anticarcinogenesis, antihypertension and antihyperlipidemia [2-4] 1.1 A brief history of soy sauce The history of soy. .. sauce [6] Buddist monks are thought to have played an important role in spreading soy sauce from China to Japan Soy sauce was first introduced into Japan by a Buddist monk, Jian Zhen, in Tang Dynasty (618-907) [7] But some consider that Japanese soy sauce originated from that brought back by a Japanese Buddist monk, Kakushin, from China in Song Dynasty (960-1279) [8-10] It is in Japan that the making... hydrolyzed vegetable proteins But the flavor of such products is poor To keep some flavor as of fermented soy sauce, semi-chemical and blended soy sauce were produced Semi-chemical soy sauce is produced by further fermentation of acid hydrolyzed products; while blended soy sauce is produced by mixing fermented soy sauce with chemical soy sauce and/or enzymatically hydrolyzed vegetable protein As Asian economies... polysaccharides can 5      increase the concentration of IgA in the intestines of mice, suppress passive cutaneous anaphylaxis reaction in the ears of allergy model mice, and regulate the balance of Th1/Th2 cell response in mice, potentially enhancing host defenses [21-23] Clinically, oral administration of soy sauce polysaccharides can improve allergic symptoms of patients with perennial allergic rhinitis or seasonal... were injected into the GC port using a split ratio of 8:1 Column temperature was increased from 125 to 175 °C at 8 °C/min after 2 min at 125 °C, then increased from 175 to 220 °C at 30 °C/min, held at 220 °C for 1 min, and finally increased from 220 to 290 °C at 40 °C/min and held at 290 °C for 2 min Selected-ion monitoring was performed using the electronionization mode with the ion source maintained... fermented soy sauce becomes the main product in Asian markets The United States is left to be the largest chemical soy sauce producer in the world Japanese producers once proposed the world-trade regulations that the manufacturing methods should be included in the label But the US suggested that individual countries should make their own decisions on labeling [18] In Singapore, soy sauce can be made from soy. .. labeling requirement on the manufacturing process 4      1.3 The Functional components of soy sauce In China, soy sauce has been traditionally used for treatment of anorexia, ulcers post thermal burn etc, recorded in Traditional Chinese medicinal books (Su Jing, Xinxiubencao, Tang Dynasty, 659; Sun Simiao, Qianjinfang, Tang Dynasty, 625) [7] Modern scientific research has provided some supporting evidence... thermal energy for aging So in the North of China or Japan, the process of manufacturing can last as long as 2 years To save time, in some modern soy sauce factories, thermal-controlled fermentation tanks are used The making process of soy sauce can be shortened to three months [17] After World War II, when the supplies of soy beans were scarce, many manufacturers applied chemical soy sauce, acid hydrolyzed... The filtrate is called raw soy sauce The raw soy sauce is further pasteurized and finally bottled To make dark soy sauce, the aging process is 3      further extended for another 2-4 months, and caramel is added to make the final product thicker and the color deeper Traditionally, the making of soy sauce is time-consuming and labor-consuming The mash is usually fermented in earthenware, and sunlight ... List of Figures and Chart vi List of Symbols ix Chapter Introduction 1.1 A brief history of soy sauce 1.2 The methodology of preparation of soy sauce 1.3 The functional components of soy sauce. ..   4.2 Content of maltol and its contribution to the TAA of dark soy sauce A considerable part of the TAA of the dark soy sauce was contained in MeOH-R and EtOAc-R fractions and in fractions 1-8... modern soy sauce plant at Walworth, Wisconsin [8] Soy sauce was brought to East Asian countries by the Chinese immigrants In Singapore, it is said that the small-scale manufacturing of soy sauce