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Identification of Bioactive Metabolites Dihydrocanadensolide, Kojic Acid, and Vanillic Acid in Soy Sauce Using GC-MS, NMR Spectroscopy, and Single-Crystal X‑ray Diffraction Ying Li,† Zi Teng,† Kirk L. Parkin,# Qin Wang,† Qingli Zhang,⊥ Wei Luo,○ Deyun Ma,△ and Mouming Zhao*,§ †

Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, People’s Republic of China # Department of Food Science, University of WisconsinMadison, 1605 Linden Drive, Madison, Wisconsin 53705, United States ⊥ Department of Animal and Food Sciences, Texas Tech University, Lubbock, Texas 79409, United States ○ Analysis and Test Center of South China University of Technology, Guangzhou 510640, People’s Republic of China △ School of Chemistry and Chemical Engineering, Zhaoqing University, Zhaoqing 526061, People’s Republic of China §

ABSTRACT: Microbial transformations of intrinsic substrates offer immense potential for generating new bioactive compounds in fermented food products. The aim of this work was to characterize the secondary metabolites in soy sauce, one of the oldest fermented condiments. Ethyl acetate extract (EAE) of soy sauce was separated using flash column chromatography, crystallized, and analyzed by nuclear magnetic resonance (NMR), single-crystal X-ray diffraction (SC-XRD), and mass spectroscopy. Dihydrocanadensolide (DHC), an antiulcer agent, was identified in a food for the first time. The natural stereostructure of DHC, which remained controversial for several decades, was determined as (3S,3aS,6R,6aR)-6-butyl-3-methyltetrahydrofuro[3,4b]furan-2,4-dione using SC-XRD analysis. Kojic acid (KA) and vanillic acid (VA) were also identified from EAE as bioactive metabolic products of fungi and yeasts. Moreover, a new polymorphic form of KA was determined by SC-XRD. KEYWORDS: bioactive metabolites, soy sauce, dihydrocanadensolide (DHC), kojic acid, polymorphism, single-crystal X-ray diffraction (SC-XRD), NMR, GC-MS

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INTRODUCTION Fermented foods have fascinated humans since antiquity. Over the past few decades, extensive research has been done on the secondary metabolites in fermented foods and their potential health-promoting benefits. In fermented dairy foods, besides the notable B vitamins (over)produced by lactic acid bacteria (LAB), the essential biogenic peptides generated by LAB also drew attention because of their antihypertensive, antimicrobial, and immunomodulatory properties.1−3 Monascus speciesfermented “red mold rice” contains bioactive metabolites, such as monacolins, monascin, and monascopyridines; this red mold rice is a popular functional food regarded in East Asia as conferring antioxidant, cancer chemopreventive, hypolipidemic, hypotensive, and hypoglycemic benefits.4,5 Kojic acid (KA), first isolated from cultures of Aspergillus oryzae in steamed rice, is widely used now as an antibrowning food additive and a skinlightening agent in the cosmetics industry.6 Discovery of the beneficial activities of secondary metabolites produced by microorganisms has resulted in renewed interest and exploration of fermentation processes. Soy sauce, one of the world’s oldest fermented soy products, is documented for being effective in suppressing inflammation and clearing toxins according to ancient Chinese literature.7,8 Since the 1990s, there have been increasing studies on the chemopreventive, antioxidant, and antimicrobial activities of soy sauce.9 Many aroma compounds in soy sauce, such as Furaneol, homofuraneol, norfuraneol, 4-ethylphenol, 4-ethyl© XXXX American Chemical Society

guaiacol, and 2-phenylethanol, exhibit antioxidant functions. These compounds were identified as byproducts of a salttolerant yeast Zygosaccharomyces rouxii.10−13 The newly found phase II enzyme inducers in soy sauce, flazin and perlolyrin, are metabolic products of Pediococcus halophilus, and they are candidates for further evaluation as cancer preventive agents.14,15 Owing to the complexity of the raw materials and the fact that mixed microbes collectively contribute to the whole fermentation process, soy sauce could be a rich reservoir of a diversity of food-derived microbial products that remain to be identified.16 In the present study, we report the isolation and identification of bioactive microbial metabolites in traditional fermented soy sauce based on solvent extraction, column chromatography, nuclear magnetic resonance (NMR), gas chromatography−mass spectrometry (GC-MS), and singlecrystal X-ray diffraction (SC-XRD) analyses.

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MATERIALS AND METHODS

Materials. KA, vanillic acid (VA), formic acid, ethyl acetate, hexane, and acetone were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Silica gel for column chromatography Received: May 7, 2014 Revised: August 1, 2014 Accepted: August 4, 2014

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dx.doi.org/10.1021/jf502159m | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 1. GC-MS total ion chromatograms of ethyl acetate extract of raw soy sauce (A) and dihydrocanadensolide (B). Peaks in (A): 1, maltol; 2, pyranone; 3, methylmaltol; 4, benzeneacetic acid; 5, 2-methoxy-4-vinylphenol; 6, 4-hydroxybenzoic acid; 7, vanillic acid; 8, syringic acid; 9, phthalic acid, isobutyl octyl ester; 10, palmitic acid; 11, oleic acid; 12, stearic acid; 13, oleylamide; 14, cyclo(phe-pro); 15, erucylamide. Flash Column Chromatography. One and a half liters of pasteurized soy sauce was extracted with ethyl acetate (1:1, v/v) three times by continuous shaking in a separatory funnel for 10 min each. The combined ethyl acetate phases were dried over anhydrous Na2SO4 and concentrated using a rotary evaporator under vacuum (Buchi rotavapor RII, Buchi Labortechnik AG, Flawil, Switzerland) to yield ∼3 g of solids from the ethyl acetate extract (EAE). Two grams of EAE solids was redissolved in minimum volume of ethyl acetate and mixed with 10 g of silica gel. Then the mixture was dried in a vacuum desiccator at room temperature (∼25 °C) for 4 h. The extract-laden silica gel was then loaded onto a normal phase silica gel flash column (4.5 cm i.d. × 12.2 cm). The column was eluted successively with a gradient of hexane/acetone (9:1, 8:2, ..., 2:8, 1:9; 500 mL for each step). Portions (120 mL) of eluate collected were subject to solvent evaporation, and a small sample (∼10 μL) of each portion was applied to a 20 cm × 10 cm precoated TLC plate (silica gel 60 A F254). Spots were detected by ultraviolet illumination (254

(230−400 mesh, 60 Å) and thin-layer chromatography (TLC) glass plates (precoated with silica gel 60 F254) were purchased from Merck (Darmstadt, Germany). Soy sauce produced by the traditional high-salt dilute-state fermentation method was obtained from a local manufacturer (Guangdong, China). The fermentation process was conducted as follows: Raw whole soybeans were thoroughly cooked by steam at 125 °C for 15 min and mixed with wheat flour. Spore suspensions (Aspergillus oryzae) were inoculated (0.05%, w/w) into the mixtures of soybean and flour, and the medium was kept at 35−40 °C, relative humidity 70−90% for 2−3 days (koji process). After turning yellowishgreen, the molded materials were mixed with NaCl solution (16−18%, w/v) to make wet mash (moromi). Finally, raw soy sauce was obtained by collecting liquid dripping from the wet mash after 3 months of fermentation. Raw soy sauce was batch-pasteurized at 80 °C for 30 min before analysis. B



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Figure 2. Mass spectra (EI/MS) fragmentation and cleavage sites of dihydrocanadensolide. and 365 nm) and iodine vapors. The consecutive portions of eluate showing single spots with the same Rf on TLC developed by the same developing solvents (mixtures of hexane and ethyl acetate) were combined into fractions and concentrated. Fraction 1 comprised material migrating to an Rf of 0.26 on a TLC plate developed with hexane/ethyl acetate/formic acid (80:20:1, v/v/v). This fraction was concentrated under vacuum to ∼2 mL and sealed in vials held at room temperature (∼25 °C) to allow slow crystallization. The resulting colorless rectangular prismatic crystals (∼40 mg) were washed carefully with methanol and recrystallized with ethyl acetate for further analysis. Fraction 2 was recovered as material migrating to an Rf of 0.52 on TLC plates that were developed by hexane/ethyl acetate/ formic acid (60:40:1, v/v/v). Upon evaporation of desorbing solvent, 15 mg of white powder was recovered. Fraction 3 was composed of material migrating to an Rf of 0.37 on TLC plates developed by hexane/ethyl acetate/formic acid = 70:30:1 (v/v/v). The recovered material afforded colorless needle-shaped crystals (∼18 mg) after slow solvent evaporation in sealed vials. Gas Chromatographic Analyses. GC-MS analyses of EAE and dihydrocanadensolide (DHC) were performed on a 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a mass spectrometer (MS 5975, Agilent Technologies). Helium was used as a carrier gas with a flow rate of 1 mL/min. Diluted EAE (0.2 μL) was injected into a splitless injector (270 °C). An HP-5MS (5% phenyl methyl siloxane) capillary GC column (30 m length, 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies) was used. The GC oven temperature was held at 80 °C for 3 min, then increased to 280 °C at 10 °C/min, and finally held for 5 min at 280 °C. The mass selective detector was operated in positive electron ionization (EI) mode (70 eV) with a mass scan range from m/z 33 to 450. The database NIST.05 was used to determine compound structures. Quantitative GC analysis was carried out using an Agilent 5890 II instrument with a flame ionization detector (FID). The capillary column and temperature program were the same as those of the GCMS analysis. Nuclear Magnetic Resonance (NMR) Analysis. 1H NMR, 13C NMR, and gradient correlation spectroscopy (gCOSY) NMR spectra were recorded with a Bruker AVANCE spectrometer (Bruker DRX400, Bruker Biospin Co., Karlsruhe, Germany) using solvent signals as an internal standard. Chemical shifts (δ) and coupling constants (J) are expressed in parts per million (ppm) and hertz (Hz), respectively. MestReNova V5.3 was used to visualize and process NMR data (Mestrelab, Escondido, CA, USA). Signal multiplicities were described as follows: s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublets of doublets), dt (doublet of

triplets), dq (doublet of quartets), t (triplet), m (multiplet), and b (broad). Single-Crystal X-Ray Crystallography (SC-XRD) Analysis. Well-shaped single crystals of fractions 1 and 3 were selected for the X-ray diffraction experiment. The crystals were mounted on glass fibers and positioned on the goniometer head. SC-XRD data of both fractions were collected on an R-axis Spider diffractometer (Rigaku, Japan) at 293(2) K using monochromatic Mo Kα radiation (λ = 0.71073 Å) at the Analytical and Testing Center of South China University of Technology. The programs SHELXS-97 and SHELXL97 were used for structure determination and refinement. All H atoms were placed at calculated positions. Before the last cycle of refinement, all H atoms were fixed and were allowed to ride on their parent atoms.

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RESULTS AND DISCUSSION GC-MS Analysis of EAE. Previous studies of GC-MS analysis of volatile compounds in soy sauce were usually conducted on polar GC capillary columns, such as DB-FFAP/ HP-FFAP,17,18 DB-WAX,19 and HP-Innowax.20 In this study, a nonpolar HP-5MS column was adopted, in the hope of searching for a different polarity category of metabolites in soy sauce. A total ion chromatogram of volatile compounds in EAE of soy sauce separated by the HP-5MS column was obtained (Figure 1A). Among the tentatively identified compounds, maltol, VA, and syringic acid were reported to possess antioxidant activities, nitric oxide scavenging effects, and cytoprotective ability in the prevention of diabetic neuropathy complications.21−24 Other principal peaks included oleic acid, palmitic acid, and stearic acid, which were likely derived from soybean lipids.25 However, no match was found for the mass spectrum of the major peak (retention time = 14.57 min) in the commercial mass spectra libraries or online mass spectra database. For structure identification, this unknown compound was isolated from EAE by silica gel column chromatography. Identification of DHC in Soy Sauce and Its Absolute Configuration. Eluates of silica gel chromatography were pooled according to their TLC patterns. Fraction 1 formed colorless crystals after slow solvent evaporation. After washing and recrystallizing, the crystal emanated a strong characteristic odor of soy sauce. In GC-MS TIC spectra, the peak retention time and mass spectrum of this compound (Figure 1B) were consistent with those of the major peak in Figure1A. The C



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Figure 3. (A)1H NMR spectrum of DHC in CDCl3; (B) gCOSY spectrum of DHC in CDCl3 For the convenience of interpretation, atomic numbers of C, H, and O were designated as shown in Figure 4.

characteristic splitting patterns in the 1H NMR spectra. The signals of protons on C3a and C6a were dd because they were affected not only by each other (J = 6 Hz) but also by H3 and H6, respectively (J = 1.4 and 4 Hz). For H3, the quartet intensity 1:3:3:1 (J = 8 Hz) suggested that a methyl group was connected to C3, and the pattern of quartet peaks splitting to doublets occurred owing to the influence of H3a at the adjacent stereocenter (J = 1.4 Hz). It is not surprising that H6 showed a similar pattern of ddd (J = 8, 6, 4 Hz), which was also observed by Sharma.26 Attention should be paid to the interpretation of the ddd effect of H6, evoked by vicinal coupling from H6a, and individual diastereotopic protons H7(1) and H7(2). This could be easily confused with a doublet of triplets, which was the case interpreted by Birch and led to stereostructure III in Table 1.27 Third, an n-butyl residue on C6 could be deduced from the downfield 1H NMR spectra. The chiral center C6 may cause the restricted rotation of diastereotopic protons on C7, resulting in geminal coupling of these two protons. Similar geminal coupling effects were also recorded by Isaka in the

molecular weight of the compound was m/z 212 according to the EI mass spectrum (EI/MS) (Figure 2). Confirmation of the structure and assignment of this compound were conducted from detailed analyses of the 13C NMR spectra, 1H NMR spectra (Figure 3A), gCOSY (Figure 3B), and EI/MS (Figure 2). First, the chemical shifts observed by 13C NMR analysis (in CDCl3) were δ 13.98, 17.31, 22.59, 27.68, 28.72, 38.57, 49.22, 78.49, 82.61, 174.83, and 176.90. The chemical shifts in high field permitted the assignments of two R−COO−R′ groups. From the 1H NMR spectra, no signal was evident in the range of δ 6−14 ppm, indicating no carboxylic acid, benzene ring, or double bond present in the molecule. The integration of two peaks at δ 4.4−5.2 in 1H NMR were both 1, suggesting two α hydrogen within two −COO−CH− fragments. The 13C NMR signals at δ 78.49 and 82.61 imply two carbons are connected to oxygen atoms. Therefore, the two ester groups appeared to be confirmed. Second, the four chiral centers (C3, C3a, C6, and C6a in Figure 4A) in the cis-fused dilactonic ester structures could explain the D



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Figure 4. (A) Oak Ridge thermal ellipsoid plot (ORTEP) diagram for crystal structures of DHC; (B) ORTEP of KA; (C) packing diagram of KA. Molecules of equivalent symmetry are shown in the same color. Lengths of O−H···O hydrogen bond: 2.465, 2.702, 2.740, and 2.760 Å.

identification of a furofurandione.28 Also considering the influence from H8, splitting patterns of H7 appeared to be more complex than a triplet of doublets (1.7−2.0 ppm in Figure 3A), consistent with locating the n-butyl group on C6 (−CH3). These effects were confirmed by gCOSY spectra, which reflect the interactions between the coupling protons H3a−H6a, H6−H6a, H6−H7, H3−CH3, H7(1)−H8, H7(2)− H8, and H7(1)−H7(2) (Figure 3B). In addition, analysis of EI/ MS spectra is consistent with the speculated structure of DHC (Figure 2). The parent peak indicated that m/z of the molecule was 212. The base peak at m/z 98 probably results from loss of the n-butyl connected B ring. The appearance of the second major molecular ion peak at m/z 69 can be ascribed to fragmentation by ring opening of ring A. The differences of 13, 15, 16, 18, and 29 among other minor ions were postulated to arise from the loss of the groups −CH, −CH3, −O, H2O (or CO), and −CH−O, respectively (Figure 2). Although mass

fragments of DHC were previously reported by Birch,27 the detailed EI/MS spectra provided here have the advantage of indicating the relative abundance of fragments. Because of the complexity brought about by the four chiral centers, there has been debate about the natural configuration of DHC since its discovery (Table 1). When McCorkindale first isolated DHC from synthetic medium of Penicillum canadense, the structure was suggested as III.29 In the same year, Birch identified DHC, also as III, from Aspergillus indicus.27 However, in their studies, the absence of NMR data of protons H7 precluded a discussion of the possibility of stereoconfiguration reflected by long-range couplings from H6a, if III was the correct assignment of structure. More recently, Yoshikoshi reported the protons on the B ring had been interpreted as trans-positioned by McCorkindale, and corrected the structure III to II by evidence obtained from stereoselective synthesis of (+)-canadensolide from hex-1E



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uous.41 The critical issue for SC-XRD is obtaining a single crystal with suitable size.42 In this study, single crystals weighing up to 15 mg of DHC were obtained for SC-XRD analysis for the first time. Information on crystallographic data collection and structure refinement is summarized in Table 2. The

Table 1. Dissension on Molecular Configuration of DHC

Table 2. Crystal Data and Structure Refinements for DHC and KA parameter

DHC

KA

diffraction radiation wavelength (Å) diffraction radiation type temperature (K) crystal system chemical formula formula weight unit cell dimensions a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) Z F (000) density (mg/cm3) absorption coefficient (mm−1) GOF (goodness of fit) θ range for data collection (deg) value of Rint residual factor R for all

0.71073 Mo Kα 293(2) monoclinic C11H16O4 212.24

0.71073 Mo Kα 293(2) monoclinic C6H6O4 142.11

10.008 (2) 8.6505(17) 13.474(3) 90 103.03(3) 90 1136.4(4) 4 520 1.428 0.117 1.036 3.10−25.19 0.0202 0.0447

3.8334(7) 36.876(7) 8.512(3) 90 96.66(3) 90 1195.1(5) 8 592 1.580 0.136 1.085 3.27−25.19 0.0212 0.0387

absolute configuration of DHC isolated from soy sauce was identified as (3S,3aS,6R,6aR)-6-butyl-3-methyltetrahydrofuro[3,4-b]furan-2,4-dione (I in Table 1). A search of the Cambridge Structural Database and online databases showed no match with crystallographic data reported here for DHC. Detailed chemical bond lengths and bond angles of DHC can be found in the CIF format data deposited with the Cambridge Crystallographic Data Centre (CCDC) under reference no. 982491. Previous studies on bioactivities of DHC were mostly focused on the antiulcer activities on gastric or duodenal ulcers. Animal experiments revealed that DHC produced by fermentation method showed better healing properties in gastric ulceration induced by betamethasone and fenclozic acid, compared to the commonly used drug carbenoxolone. In addition, DHC significantly reduced duodenal ulceration in rats induced by acetic acid, whereas carbenoxolone did not show this property.38,43 The reported fermentation method for producing DHC involved 10 days of agar fermentation and 33 days of submerged fermentation, which was similar to the koji (solid) and moromi (liquid) fermentation process of soy sauce, respectively.38 DHC was discovered in the metabolites of Penicillium canadense and Aspergillus indicus.29 Penicillium and Aspergillus species were also found in soy sauce. A. oryzae and A. sojae are two important starter fungi for soy sauce fermentation.44,45 In our study, ∼40 mg of DHC crystals was obtained in 2 g of EAE (i.e., ∼40 mg/L soy sauce). Considering the loss during extraction, isolation, and crystallization processes, the concentration of DHC in soy sauce should be

yne.30,31 However, this correction still mistakenly adopted the structure of a synthetic epimer as the natural configuration. After comparing samples from both McCorkindale and Yoshikoshi, Anderson and Fraser-Reid suggested a dehydro-I as the natural structure of canadensolide in 1978.32 In 1975, Aldridge32 patented a formula containing fermented DHC as a gastric and duodenal ulcer-healing agent and acknowledged II as the structure corrected by Yoshikoshi.30,31 Over the past two decades, chemical synthesis of DHC in its natural configuration has been gaining increasing attention. Although extensive studies have been done by exploring the synthetic processes for I, II, and the epimers III and IV, the natural configuration of DHC has not been conclusively established.26,30,33−37 Molecular chirality is essential to activities of biologically/ pharmacologically active compounds, as many drug components exhibit significant differences in their pharmacologic activities compared with their enantiomers.39 Therefore, knowledge of the absolute configuration of chemicals that are potential therapeutic agents is extremely important. By far, the most powerful structural method for determination of the absolute configuration of molecules is single-crystal X-ray crystallography.40 Because the anomalous dispersion effect of heavy atoms can be measured very accurately, the absolute stereostructure obtained by SC-XRD is clear and unambigF



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preventing the secondary complications of diabetes64 by VA have also gained increasing attention. Identification of KA. Fraction 3 was separated by normal phase column chromatography and obtained as colorless needle-like crystals. 1H NMR spectra (δ 4.35 (2H, s, H6), 6.47 (1H, s, H2), 8.16 (1H, s, H5), 5.75 (1H, b, −O(2)H), 9.13 (1H, b, −O(4)H), 400 MHz, DMSO-d6) and data obtained from an authentic compound confirmed this compound as 5hydroxy-2-hydroxymethyl-4-pyrone (KA).65 As the physical appearance of KA crystal obtained in this study was different from that described in the literature (0.1 × 0.15 × 0.15 mm, KA obtained from Aspergillus tamaril strain),66 a further analysis oby SC-XRD was carried out to gain more insight into the crystal polymorphs, because SC-XRD was considered to outweigh other methodologies on providing detailed and undisputable crystal packing information.67 A crystal of KA (1 × 0.8 × 3 mm) was mounted on a glass fiber and positioned on the goniometer head for SC-XRD analysis. Our results showed different unit cell dimensions from reports in the literature,66 mainly in the observation that Z = 8 molecules instead of 4 molecules (Table 1; Figure 4B,C). Another report showed that a crystal of KA (0.48 × 0.5 × 0.08 mm) obtained from the strain of Aspergillus oryzae WP-D-1 had the formula (C6H6O4)2.68 The crystal packing of KA in our study was more complex than those previously reported. Whereas all KA molecules were connected by hydrogen bonding with three other molecules instead of two, they were aligned along the baxis facing mainly two directions (Figure 4C). More specifically, the planes of KA were tilted ∼23.4°, ∼26.7°, ∼153.3°, and ∼156.6° off the bc plane and approximately normal to the ab plane. The difference in packing motif provides evidence for the polymorphism of KA, and our report added an additional polymorphic variety. Because of its superior activities of antioxidant and inhibition of tyrosinase, KA has been used as an antibrowning food additive and a skin-lightening agent in the cosmetics industry.6 Other health beneficial effects such as antimelasma and antineoplastic activities have also been reported.69,70 Literature on the presence of kojic acid in soy sauce is the subject of debate. Nakadai claimed that KA was not detected in commercial soy sauce.71 Shinshi studied 84 commercially used koji-mold strains for their capabilities of producing KA. Most of them generate 18 mg/L in the original sample. Kojic acid is produced by numerous Aspergillus species. Carbon sources for KA fermentation include reducing sugars, sugar acids, and sugar alcohols. The chain length of these compounds could be 2−7 carbons or 12 or 18 carbons. Polysaccharides can also serve as a carbon source for KA.74 Although the studies on the biosynthesis of KA started six decades ago, the pathway remains speculative.6 One important theory suggesting that KA can be synthesized from glucose via direct conversion was supported by isotope-traced D-glucose experiments.75 Typically, oxidation of glucose to 3-ketoglucose by glucose 3-dehydrogenases was suggested as the initial step, followed by dehydration at the 4- and 5-positions of 3ketoglucose to form KA. For carbon sources with fewer than six carbon atoms, it was difficult to determine the exact route,

higher than this value. Quantification of DHC in the ether extract of raw soy sauce was performed by GC-FID under the same conditions as GC-MS described under Materials and Methods, using purified DHC (>99% judged by GC-MS) as an external standard. The results showed that the concentration of DHC in raw soy sauce was approximately 83 mg/L. Although chemically diverse, all secondary metabolites are produced via a few common biosynthetic pathways.46 Studies with [2-14C]acetic acid suggested that DHC might arise from the condensation of two chains, octanoic acid and pyruvic acid (or oxaloacetic acid with subsequent decarboxylation).27 Oxaloacetic acid is an intermediate in the citric acid cycle, a major metabolic pathway of primary metabolism found in living systems. On the basis of these facts, it is likely that DHC is derived from the citric acid cycle of fungi. Ma et al. reported that a nucleophile-promoted, biscyclization of keto acids could spontaneously happen, suggesting this mechanism might be involved in the biosynthesis of natural bicyclic lactones.47 Similar biosynthetic routes have been defined for canadensolide, indicating that this compound was mainly formed by condensation of α-methylene of a fatty acid and the carbonyl group of oxalacetic acid.48 1-14C- and 2-13C-labeling during the evolution of another bislactone, avenaciolide, gave more evidence for this biosynthetic pathway.48 Identification of VA. Identification of fraction 2 was conducted by GC-MS and 1H NMR. The EI/MS profile of fraction 2 matched that of VA in the NIST 0.05 database. Its retention time (tR of 12.78 min, Figure 1A) on an HP-5MS column, as well as 1H NMR profile [δ 7.59 (1H, dd, J = 8.0 Hz, 2.0 Hz, H4), 7.56 (1H, d, J = 2.0 Hz, H6), 6.92 (1H, d, J = 8.0 Hz, H3), 3.90 (3H, s, −OCH3), 400 MHz, acetone-d4)] corresponded with that of the authentic compound. VA is a flavoring compound in soy sauce.49 It might be derived directly from soybeans, which contain VA as a major phenolic acid.50 Another phenolic compound, ferulic acid in soybeans, can be converted to VA through the metabolism of Aspergillus niger,51 Debaryomyces hansenii,52 and Pseudomonas putida.53 Moreover, Zygosaccharomyces rouxii, a salt-tolerant yeast commonly found in soy sauce, is capable of producing VA as a metabolite.54 The concentration of VA in soy sauce recovered in this study was ∼15 mg/L, which was within the range of 1.9−294 mg/L reported by others.49,55,56 Considering the dry contents in soy sauce (25−58 g/100 mL),57 the concentration of VA relative to total solids of soy sauce was comparable to that in the root of the medicinal plant Angelica sinensis (or Dang Gui), which contains the highest known amount of VA among all plants (6.1−94.4 mg/kg).58,59 Hence, soy sauce may be a rich source for VA. VA has been reported to exhibit potential antiulcer activity in ulcerative colitis.60 It reduced the severity of the clinical signs of dextran sulfate sodium-induced colitis in mice, including weight loss and shortening of colon length, and the disease activity index. A hepatoprotective effect of VA was also reported.61 VA significantly decreased the transaminase activity and suppressed the disorganization of the hepatic sinusoids in concanavalin Atreated mice. VA was capable of reducing plasma interleukin-6 in both ulcerative colitis-healing and hepatoprotective effects. Moreover, VA has been associated with antihypertensive and antioxidant activities demonstrated in rats.62 VA restored systolic and diastolic blood pressure to normal levels, preserved the plasma nitric oxide metabolites concentration, and reduced plasma lipid peroxidation products and cellular defensing enzymes. The potential of inhibiting carcinogenesis63 and G



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this study is the first to offer SC-XRD data of DHC and identify DHC in a food. This is an advance over earlier attempts to resolve the long-lasting debate on natural configuration of DHC. In addition, polymorphism of KA was discovered by SCXRD analysis. The polymorphic variety observed in this study had the crystal packing of eight molecules in a unit cell, and each molecule was connected with three other molecules via hydrogen bonding. These data, as well as the detailed information on EI fragmentation in EI/MS spectra of DHC, can extend our knowledge of the structural identification of similar microbial metabolites in a wide range of fungusfermented foods. On the basis of the results from this study, future works can be done on the quantification of these bioactive compounds in various fermented foods, improvement of fermentation processes to enrich these metabolites, and development of new food products bearing potential ulcer protection activities.

because the presence of a C6 precursor has not been definitely identified, and 14C positions in the KA product were more extensively redistributed than direct conversion, by evidence obtained with [2-14C]-dihydroxyacetone and [1-14C]-D-ribose.6,76 Fermentation is the major production technology for soy sauce by far. Typically, the fermentation process consists of two stages, namely, koji (aerobic growth of the starter fungi on soyand wheat-based materials for a few days) and moromi (anaerobic incubation of molded materials in salt solutions for months).77 Depending on the temperature and the ratio of solid to brine solution, the producing methods are mainly categorized into high-salt dilute-state (HSDS) and low-salt solid-state (LSSS) fermentation. HSDS fermentation is widely adopted among Asian countries, although there are differences in raw materials (whole soybean, defatted soy flake, roast wheat, flour, and wheat bran), starter fungal species, and fermentation time for moromi (3−9 months). On the other hand, LSSS fermentation requires less time for both koji (1 day) and moromi (3 weeks) processes, a smaller volume of moromi brine solution (half that of HSDS), and a higher moromi temperature (40−45 °C).78 It has been known that the amounts and compositions of amino acids and the aroma compounds in soy sauce can be regulated by controlling the fermentation conditions.78,79 Similarly, it can be inferred that bioactive compounds can also be modulated. Considering the possible biosynthesis routes of KA, this compound may arise from heterocyclic compounds derived from the primary intermediates in the citric acid cycle of fungal metabolism during fermentation. The primary intermediates, particularly oxalacetic acid in the citric acid cycle, can be converted to secondary intermediates, which may be responsible for the formation of DHC in soy sauce. Secondary metabolites are usually not produced during the phase of rapid growth (trophophase); instead, they are synthesized during the subsequent production state (idiophase), especially when microbial growth is limited by the exhaustion of one key nutrient.80 Therefore, the production of microbial secondary metabolites could be enhanced by (1) allowing sufficient microbial growth and exhaustive utilization of certain culturing ingredients in the koji process, followed by (2) proceeding to the moromi stage at which the brine solution could further inhibit the rapid growth of the microbes. This was supported by reports that solid fermentation followed by liquid fermentation resulted in a higher yield of KA, compared to adopting liquid fermentation alone.6,81,82 The relatively short time (1 day) of koji and high temperature of moromi processes of the LSSS method are designed to achieve highest protease activities, which may compromise the adequate conditions for microbial trophophase growth and idiophase metabolite production, compared to HSDS fermentation. In addition, VA in soy sauce is generated from various sources, including the soy materials and enzymatic modification of ferulic acid by fungi, yeasts, or bacteria. Utilization of soy materials with high VA and/or ferulic acid contents may be beneficial for elevating the levels of VA in soy sauce. Until now, the key fermentation conditions that affect microbial survival status, metabolic pathways, and microbeculturing material interaction are still largely unknown. Further studies need to be done on the effects of various fermentation factors on the contents of bioactive compounds in soy sauce. The findings of the present study illustrate the isolation and structural determination of three bioactive microbial secondary metabolites, namely, DHC, VA, and KA. To our knowledge,

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AUTHOR INFORMATION

Corresponding Author

*(M.Z.) Phone/fax: 86-20-87113914. E-mail: femmzhao@scut. edu.cn. Funding

This work was supported by the National Science Technology Supporting Project for the 12th Five-Year Plan (Nos. 2012BAD34B03 and 2012BAK17B11) and the Key Technology R&D Program of Guangdong Province (No. 2012cxy004). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GC, gas chromatography; GC-MS, gas chromatography−mass spectrometry; GC-FID, GC with flame ionization detector; NMR, nuclear magnetic resonance; SC-XRD, single-crystal Xray diffraction; KA, kojic acid; VA, vanillic acid; TLC, thin-layer chromatography; EAE, ethyl acetate extract; DHC, dihydrocanadensolide; EI, electron ionization; MS, mass spectra; gCOSY, gradient correlation spectroscopy; ORTEP, Oak Ridge thermal ellipsoid plot

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REFERENCES

(1) Hayes, M.; Stanton, C.; Fitzgerald, G. F.; Ross, R. P. Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions. Biotechnol. J. 2007, 2, 435−449. (2) Stanton, C.; Ross, R. P.; Fitzgerald, G. F.; Sinderen, D. V. Fermented functional foods based on probiotics and their biogenic metabolites. Curr. Opin. Biotechnol. 2005, 16, 198−203. (3) Hugenholtz, J.; Smid, E. J. Nutraceutical production with foodgrade microorganisms. Curr. Opin. Biotechnol. 2002, 13, 497−507. (4) Loret, M.-O.; Morel, S. Isolation and structural characterization of two new metabolites from Monascus. J. Agric. Food Chem. 2009, 58, 1800−1803. (5) Wild, D.; Tóth, G.; Humpf, H.-U. New Monascus metabolite isolated from red yeast rice (angkak, red koji). J. Agric. Food Chem. 2002, 50, 3999−4002. (6) Bentley, R. From miso, sake and shoyu to cosmetics: a century of science for kojic acid. Nat. Prod. Rep. 2006, 23, 1046−1062. (7) Huang, G. Truth-seeking in materia medica (Ben Cao Qiu Zhen, 1769). In Classical Ten Books of Materia Medica (Ben Cao Jing Dian Lun Zhu Shi Ren Shu); Academy Press (Xue Yuan): Beijing, China, 2011; Vol. 7, p 324. (8) Tao, Z., Other records by famous doctors (Ming Yi Bie Lu, between 220−450 A.D). In Compilation of Ancient Books of Traditional

H



Article

Chinese Medicin Series (Zhong Yi Gu Ji Zheng Li Cong Shu); Shang, Z., Ed.; People’s Medical Publishing House: Beijing, China, 1986; Vol. 3, p 314. (9) Kataoka, S. Functional effects of Japanese style fermented soy sauce (shoyu) and its components. J. Biosci. Bioeng. 2005, 100, 227− 234. (10) Hecquet, L.; Sancelme, M.; Bolte, J.; Demuynck, C. Biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone by Zygosaccharomyces rouxii. J. Agric. Food Chem. 1996, 44, 1357−1360. (11) Sasaki, M.; Nunomura, N.; Matsudo, T. Biosynthesis of 4hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone by yeasts. J. Agric. Food Chem. 1991, 39, 934−938. (12) Suezawa, Y.; Suzuke, M. Bioconversion of ferulic acid to 4vinylguaiacol and 4-ethylguaiacol and of 4-vinylguaiacol to 4ethylguaiacol by halotolerant yeasts belonging to the genus Candida. Biosci., Biotechnol., Biochem. 2007, 71, 1058−1062. (13) van der Sluis, C.; Tramper, J.; Wijffels, R. H. Enhancing and accelerating flavour formation by salt-tolerant yeasts in Japanese soysauce processes. Trends Food Sci. Technol. 2001, 12, 322−327. (14) Li, Y.; Zhao, M.; Parkin, K. L. β-Carboline derivatives and diphenols from soy sauce are in vitro quinone reductase (QR) inducers. J. Agric. Food Chem. 2011, 59, 2332−2340. (15) Kihara, K. Elucidation of shoyu pigment. 1. Formation of shoyu pigment from flazin and the end product. Nippon Shoyu Kenkyusho Zasshi 1995, 21, 187−196. (16) O’Toole, D. K. The role of microorganisms in soy sauce production. In Advances in Applied Microbiology; Saul, L. N., Allen, I. L., Eds.; Academic Press: San Diego, CA, USA, 1997; Vol. 45, pp 87− 152. (17) Wanakhachornkrai, P.; Lertsiri, S. Comparison of determination method for volatile compounds in Thai soy sauce. Food Chem. 2003, 83, 619−629. (18) Steinhaus, P.; Schieberle, P. Characterization of the key aroma compounds in soy sauce using approaches of molecular sensory science. J. Agric. Food Chem. 2007, 55, 6262−6269. (19) Hayashida, Y.; Nishimura, K.; Slaughter, J. C. The influence of mash pre-aging on the development of the flavour-active compound, 4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF), during soy sauce fermentation. Int. J. Food Sci. Technol. 1997, 32, 11−14. (20) Lee, S. M.; Seo, B. C.; Kim, Y.-S. Volatile compounds in fermented and acid-hydrolyzed soy sauces. J. Food Sci. 2006, 71, C146−C156. (21) Kim, Y. B.; Oh, S. H.; Sok, D. E.; Kim, M. R. Neuroprotective effect of maltol against oxidative stress in brain of mice challenged with kainic acid. Nutr. Neurosci. 2004, 7, 33−39. (22) Kang, K. S.; Yokozawa, T.; Kim, H. Y.; Park, J. H. Study on the nitric oxide scavenging effects of ginseng and its compounds. J. Agric. Food Chem. 2006, 54, 2558−2562. (23) Zhao, H. F.; Chen, W. F.; Lu, J.; Zhao, M. M. Phenolic profiles and antioxidant activities of commercial beers. Food Chem. 2010, 119, 1150−1158. (24) Huang, S.-M.; Chuang, H.-C.; Wu, C.-H.; Yen, G.-C. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Mol. Nutr. Food Res. 2008, 52, 940−949. (25) Namgung, H. J.; Park, H. J.; Cho, I. H.; Choi, H. K.; Kwon, D. Y.; Shim, S. M.; Kim, Y. S. Metabolite profiling of doenjang, fermented soybean paste, during fermentation. J. Sci. Food Agric. 2010, 90, 1926− 1935. (26) Sharma, G. V. M; Gopinath, T. Radical-mediated stereoselective synthesis of (+)-dihydrocanadensolide and (−)-3-epi-dihydrocanadensolide from D-xylose. Tetrahedron Lett. 2001, 42, 6183−6186. (27) Birch, A. J.; Qureshi, A. A.; Rickards, R. W. Metabolites of Aspergillus indicus − structure and some aspects of biosynthesis of dihydrocanadensolide. Aust. J. Chem. 1968, 21, 2775−2784. (28) Isaka, M.; Chinthanom, P.; Boonruangprapa, T.; Rungjindamai, N.; Pinruan, U. Eremophilane-type sesquiterpenes from the fungus Xylaria sp BCC 21097. J. Nat. Prod. 2010, 73, 683−687.

(29) McCorkindale, N. J.; Wright, J. L. C.; Brian, P. W.; Clarke, S. M.; Hutchinson, S. A. Canadensolide − an antifungal metabolite of penicillium canadense. Tetrahedron Lett. 1968, 9, 727−730. (30) Nubbemeyer, U. Diastereoselective zwitterionic aza-Claisen rearrangement: the synthesis of bicyclic tetrahydrofurans and a total synthesis of (+)-dihydrocanadensolide. J. Org. Chem. 1996, 61, 3677− 3686. (31) Kato, M.; Tanaka, R.; Yoshikoshi, A. Synthesis of (±)-canadensolide and its C-5 epimer. Revision of the stereochemistry. J. Chem. Soc. D 1971, 1561−1562. (32) Anderson, R. C.; Fraser-Reid, B. An asymmetric synthesis of naturally occurring canadensolide. Tetrahedron Lett. 1978, 19, 3233− 3236. (33) Sharma, G. V. M; Gopinath, T. Radical cyclisation approach for the synthesis of (+)dihydrocanadensolide, (+)dihydrosporothriolide and their C-3 epimers from d-xylose. Tetrahedron 2003, 59, 6521− 6530. (34) Aldridge, D. C.; Nicholson, S.; Taylor, P. J. Rates and mechanisms for the ring-opening, ring-closure and ring transformation reactions of the di-γ-lactone dihydrocanadensolide (DHC). J. Chem. Soc., Perkin Trans. 2 1995, 1929−1938. (35) Mulzer, J.; Kattner, L. Doubly stereodifferentiating hiyama addition with mismatched reactants; enantio- and diastereo-controlled synthesis of dihydrocanadensolide. Angew. Chem., Int. Ed. 1990, 29, 679−680. (36) Chen, M.-J.; Narkunan, K.; Liu, R.-S. Total synthesis of natural bicyclic lactones (+)-dihydrocanadensolide, (±)-avenociolide, and (±)-isoavenociolide via tungsten-π-allyl complexes. J. Org. Chem. 1999, 64, 8311−8318. (37) Adrio, J.; Carretero, J. C. Butenolide synthesis by molybdenummediated hetero-Pauson-Khand reaction of alkynyl aldehydes. J. Am. Chem. Soc. 2007, 129, 778−779. (38) Aldridge, D. C.; Borrow, A.; Gerring, E. E. L. Pharmaceutical compositions. U.S. 3,930,014, 1975. (39) Kasprzyk-Hordern, B. Pharmacologically active compounds in the environment and their chirality. Chem. Soc. Rev. 2010, 39, 4466− 4503. (40) Albright, A.; White, J. Determination of absolute configuration using single crystal X-ray diffraction. In Metabolomics Tools for Natural Product Discovery; Roessner, U., Dias, D. A., Eds.; Humana Press: Totowa, NJ, USA, 2013; Vol. 1055, pp 149−162. (41) Fun, H.-K.; Boonnak, N.; Chantrapromma, S. Single crystal xray structural determination of natural products. AIP Conf. Proc. 2012, 1455, 19−28. (42) Harada, N. Powerful chiral molecular tools for preparation of enantiopure alcohols and simultaneous determination of their absolute configurations by X-ray crystallography and/or 1H NMR anisotropy methods. In Chirality in Drug Research; Wiley-VCH Verlag: Weinheim, Germany, 2006; pp 283−321. (43) Aldridge, D. C.; Crawley, G. C.; Strawson, C. J. Pharmaceutical compositions. U.S. 4,103,023 1978. (44) Biesebeke, R. T.; Record, E. Scientific advances with Aspergillus species that are used for food and biotech applications. Microbes Environ. 2008, 23, 177−181. (45) Pitt, J. I.; Hocking, A. D. Fungi and Food Spoilage, 2nd ed.; Blackie Academic and Professional: London, UK, 1997; p 593. (46) Keller, N. P.; Turner, G.; Bennett, J. W. Fungal secondary metabolism − from biochemistry to genomics. Nat. Rev. Microbiol. 2005, 3, 937−947. (47) Ma, G.; Nguyen, H.; Romo, D. Concise total synthesis of (±)-salinosporamide A, (±)-cinnabaramide A, and derivatives via a bis-cyclization process: implications for a biosynthetic pathway? Org. Lett. 2007, 9, 2143−2146. (48) Hanson, J. R. Fungal Metabolites Derived from the Citric Acid Cycle; Royal Society of Chemistry: Cambridge, UK, 2008; pp 121− 124. (49) Kaneko, S.; Kumazawa, K.; Nishimura, O. Studies on the key aroma compounds in raw (unheated) and heated Japanese soy sauce. J. Agric. Food Chem. 2013, 61, 3396−3402. I



Article

(71) Nakadai, T. Kojic acid. Nippon Shoyu Kenkyusho Zasshi 2004, 30, 55−59. (72) Eiichiro, S.; Masaru, M.; Tetshuhisha, G.; Yukiko, M.; Kenji, T.; Sinji, M. Studies on the fluorescent compounds in fermented foods. Part VII. The degradation of added kojic acid during soy sauce fermentation. Nippon Shoyu Kenkyusho Zasshi 1984, 10, 151−155. (73) Tanaka, K.; Kushiro, M.; Manabe, M. A review of studies and measures to improve the mycotoxicological safety of traditional Japanese mold-fermented foods. Mycotoxin Res. 2006, 22, 153−158. (74) Beélik, A. Kojic acid. In Advances in Carbohydrate Chemistry; Melville, L. W., Tipson, R. S., Eds.; Academic Press: New York, 1956; Vol. 11, pp 145−183. (75) Arnstein, H. R. V.; Bentley, R. Kojic acid biosynthesis from 1C14-glucose. Nature 1950, 166, 948−949. (76) Arnstein, H.; Bentley, R. The biosynthesis of kojic acid. 1. Production from [1-14C] and [3:4-14C2] glucose and [2-14C]-1:3dihydroxyacetone. Biochem. J. 1953, 54, 493. (77) Hui, Y. H. Handbook of Food Science, Technology, And Engineering; CRC Press: Boca Raton, FL, USA, 2006; Vol. 149. (78) Laskin, A. I.; Bennett, J. W.; Gadd, G. M. Advances in Applied Microbiology; Academic Press: San Diego, CA, USA, 2003; Vol. 45, pp 104−132. (79) Feng, Y.; Cui, C.; Zhao, H.; Gao, X.; Zhao, M.; Sun, W. Effect of koji fermentation on generation of volatile compounds in soy sauce production. Int. J. Food Sci. Technol. 2012, 48, 609−619. (80) Pandey, A., Soccol, C. R., Larroche, C., Eds. Current Developments in Solid-State Fermentation; Asiatech Publishers: New Delhi, India, 2007; pp 304−305. (81) Rosfarizan, M.; Ariff, A. B. Kinetics of kojic acid fermentation by Aspergillus f lavus link S44-1 using sucrose as a carbon source under different pH conditions. Biotechnol. Bioprocess. Eng. 2006, 11, 72−79. (82) Ogawa, A.; Wakisaka, Y.; Tanaka, T.; Sakiyama, T.; Nakanishi, K. Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484. J. Ferment. Bioeng. 1995, 80, 41−45.

(50) Chung, I.-M.; Seo, S.-H.; Ahn, J.-K.; Kim, S.-H. Effect of processing, fermentation, and aging treatment to content and profile of phenolic compounds in soybean seed, soy curd and soy paste. Food Chem. 2011, 127, 960−967. (51) Lesage-Meessen, L.; Delattre, M.; Haon, M.; Thibault, J.-F.; Ceccaldi, B. C.; Brunerie, P.; Asther, M. A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. J. Biotechnol. 1996, 50, 107−113. (52) Mathew, S.; Abraham, T. E.; Sudheesh, S. Rapid conversion of ferulic acid to 4-vinyl guaiacol and vanillin metabolites by Debaryomyces hansenii. J. Mol. Catal. B: Enzym. 2007, 44, 48−52. (53) Muheim, A.; Lerch, K. Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 1999, 51, 456−461. (54) Naylin, N.; Suganuma, T.; Taing, O. Antioxidant activities of compounds isolated from fermented broth of Zygosaccharomyces rouxii. Food Biotechnol. 2006, 20, 131−141. (55) Li, X.; Hiramoto, K.; Yoshida, M.; Kato, T.; Kikugawa, K. Identification of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) with DNA breaking activity in soy sauce. Food Chem. Toxicol. 1998, 36, 305−314. (56) Kihara, K. Determination of benzoic acid and vanillic acid in shoyu by micro high-performance liquid chromatography. Nippon Shoyu Kenkyusho Zasshi 1981, 7, 112−115. (57) Li, Y.; Zhao, H.; Zhao, M.; Cui, C. Relationships between antioxidant activity and quality indices of soy sauce: an application of multivariate analysis. Int. J. Food Sci. Technol. 2010, 45, 133−139. ́ R.; Piekut, J.; Lewandowski, W. The relationship (58) Swisłocka, between molecular structure and biological activity of alkali metal salts of vanillic acid: spectroscopic, theoretical and microbiological studies. Spectrochim. Acta, Part A 2013, 100, 31−40. (59) Lü, J.-L.; Zhao, J.; Duan, J.-A.; Yan, H.; Tang, Y.-P.; Zhang, L.-B. Quality evaluation of Angelica sinensis by simultaneous determination of ten compounds using LC-PDA. Chroma 2009, 70, 455−465. (60) Kim, S.-J.; Kim, M.-C.; Um, J.-Y.; Hong, S.-H. The beneficial effect of vanillic acid on ulcerative colitis. Molecules 2010, 15, 7208− 7217. (61) Itoh, A.; Isoda, K.; Kondoh, M.; Kawase, M.; Kobayashi, M.; Tamesada, M.; Yagi, K. Hepatoprotective effect of syringic acid and vanillic acid on concanavalin A-induced liver injury. Biol. Pharm. Bull. 2009, 32, 1215−1219. (62) Kumar, S.; Prahalathan, P.; Raja, B. Antihypertensive and antioxidant potential of vanillic acid, a phenolic compound in LNAME-induced hypertensive rats: a dose-dependence study. Redox Rep. 2011, 16, 208−215. (63) Erdem, M. G.; Cinkilic, N.; Vatan, O.; Yilmaz, D.; Bagdas, D.; Bilaloglu, R. Genotoxic and anti-genotoxic effects of vanillic acid against mitomycin C-induced genomic damage in human lymphocytes in vitro. Asian Pac. J. Cancer Prev. 2012, 13, 4993−4998. (64) Prabhakar, P. K.; Doble, M. Effect of natural products on commercial oral antidiabetic drugs in enhancing 2-deoxyglucose uptake by 3T3-L1 adipocytes. Ther. Adv. Endocrinol. Metab. 2011, 2, 103−114. (65) Saruno, R. Kojic acid, a tyrosinase inhibitor from Aspergillus albus. Agric. Biol. Chem. 1979, 43, 1337−1338. (66) Lokaj, J.; Kozisek, J.; Koren, B.; Uher, M.; Vrabel, V. Structure of kojic acid. Acta Crystallogr. C 1991, 47, 193−194. (67) Gavezzotti, A.; Flack, H. 21. Crystal Packing; University College Cardiff Press: Cardiff, Wales, 2001; pp 14−15. (68) Zhang, L.-R.; Yan, X.-F.; Liu, S.-S.; Yu, M.; Chen, J.-J.; Shen, Y.M. 5-Hydroxy-2-hydroxymethyl-4-pyrone, a major secondary metabolite isolated from the strain of Aspergillus oryzae WP-D-1. J. Life Sci. 2008, 2, 1−4. (69) Novotný, L.; Rauko, P.; Abdel-Hamid, M.; Vachalkova, A. Kojic acid − a new leading molecule for a preparation of compounds with an anti-neoplastic potential. Neoplasma 1998, 46, 89−92. (70) Lim, J. T. E. Treatment of melasma using kojic acid in a gel containing hydroquinone and glycolic acid. Dermatol. Surg. 1999, 25, 282−284. J


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