Plant Foods for Human Nutrition 61: 157–160, 2006 c 2006 Springer Science+Business Media, Inc DOI: 10.1007/s11130-006-0017-5 157 Increased Anti-oxidative Potency of Garlic by Spontaneous Short-term Fermentation EMIKO SATO,1 MASAHIRO KOHNO,1 HAMASUKE HAMANO & YOSHIMI NIWANO 1, ∗ New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan; Genki Hamano Shokuhin Kogyo Co, Ltd., Sunrise Sangyo, 253 Yanohama, Owase, Mie 519-3672, Japan (∗ author for correspondence Research Center for Functional Food Materials, Sunny Health Co., Ltd., Saito Biotechnology Incubator, 7-7-15, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; e-mail: y.niwano@sunnyhealth.co.jp) Published online: 31 October 2006 Abstract Fundamental anti-oxidative properties of 80% ethanol extract from garlic fermented for the relatively short period of time (40 days at 60– 70◦ C, 85–95% relative humidity) were examined Superoxide dismutase (SOD)-like activity, scavenging activity against hydrogen peroxide and the polyphenol content of the garlic extract were increased 13-folds, more than 10-folds, and 7-folds, respectively, as compared with those of the control garlic extract The results indicate that relatively short-term spontaneous fermentation potentiates anti-oxidative properties of garlic in fresh form, which is, at least in part, attributable to the increased level of polyphenols Since superoxide is the primary upstream radical of the chain reaction with reactive oxygen species and hydrogen peroxide is generated from the scavenging reaction by SOD, the fermented- garlic is suggested to possess desirable anti-oxidative properties Keywords: Garlic, Anti-oxidative potency, Radical scavenging, Polyphenols Introduction Garlic (Allium sativum L.) has been considered a valuable healing agent by people of different cultures for thousands of years, and has long been used as a folk remedy for a variety of ailments Even today, it is commonly used for its medicinal benefit through the world, especially Eastern Europe and Asia Recently, it has also been suggested that garlic preparations including aged garlic prevented tumor promotion [2], cardiovascular diseases [3], liver damage [4, 5], and aging [6] which are considered to be associated with oxygen radical species and lipid peroxidation The intrinsic antioxidant activities of garlic [7], garlic extract [8] and some garlic constituents [9] have been widely documented Among the many commercial garlic products, aged garlic extract is known to contain unique and bioactive organic sulfur compounds such as S-allylcystein and S-allylmercaptocystein which show anti-oxidative effects [10–12] In addition to organic sulfur compounds, it has been reported that aged garlic contains tetrahydro-βcarboline derivatives which possess potent hydrogen peroxide scavenging properties [13, 14] In this study, we have examined anti-oxidative properties of unique fermented-garlic The garlic’s color is rendered black by spontaneous fermentation for 40 days without any additives The black color is likely to be derived from antho- cyanins so that a hypothesis is also raised that polyphenols are increased Materials and Methods Extraction of Garlic Garlic in fresh form has been harvested in August from Aomori prefecture of Japan and was stored in dry and dark depots The garlic’s color was rendered black by spontaneous fermentation for 40 days at 60–70◦ C, 85–95% relative humidity without any additives (described as black garlic throughout the paper) Figure shows representative examples of the black garlic As a control, the garlic in fresh form without spontaneous fermentation was used (described as control garlic throughout the paper) Both of the control and black garlic were freeze-dried and pulverized in 80 % ethanol solution followed by filtration through No filter paper The obtained filtrate was used as garlic extract Reagents Hypoxanthine (HPX) and superoxide dismutase (SOD from bovine erythrocytes) were purchased from Sigma-Aldrich Corp (St Louis, MO) Xanthine oxidase (XOD from cow milk) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were purchased from Labotec Co., Ltd (Tokyo, Japan) Folin & Ciocalteu’s Phenol Reagent was purchased from MP Biomedicals, LLC (Aurora, Ohio) All the other reagents used were of analytical grade Electron Spin Resonance (ESR) Analysis Measurement conditions of ESR (JES-FA-100, JEOL, Tokyo, Japan) throughout the experiment were as follows; field sweep: 330.50–340.50 mT, field modulation frequency: 100 kHz, field modulation width: 0.07 mT, amplitude: 200, sweep time: min., time constant: 0.1 sec, microwave freq.: 9.420 GHz, microwave power: mW 158 after standing for 30 A freshly prepared gallic acid was used as the standard Results and Discussion Figure The representative examples of the black garlic Assay for Superoxide Dismutase (SOD) Like Activity Assay for SOD-like activity was essentially identical to that described in the previous paper [15, 16] In brief, 50 µl of mM HPX, 30 µl of dimethyl sulfoxide (DMSO), and 50 µl of the garlic extract dissolved in 80 % EtOH, or solvent (80 % EtOH) alone, 20 µl of 4.5 M DMPO, and 50 µl of 0.4 U/ml XOD were placed in a test tube and mixed The mixture was transferred to the ESR spectrometry cell, and the DMPO-OOH spin adduct was quantified 90 sec after the addition of XOD Signal intensities were evaluated from the peak height of the first signal of the DMPO-OOH spin adduct The SOD-like activity was determined by the calibration curve of the enzyme activity of authentic SOD Assay for Hydrogen Peroxide Scavenging Activity The protocol used for hydrogen peroxide assay was the procedure using N-(carboxymethylaminocarbonyl)4,4 -bis(dimethylamino)-diphenylamine sodium salt (DA64) as a coloring agent suggested by the manufacture (Wako Pure Chemical Industries, Tokyo, Japan) In brief, 50 µl of each sample (garlic extract or 80 % EtOH as a solvent) was added to 150 µl of hydrogen peroxide (final 21.3 µM) and mixed Then, 100 µl of the mixture was added to 900 µl of reaction solution consisting of 0.1 mM DA-64, 0.1 M PIPES buffer (pH7.0), 0.5% Triton X-100 and horse radish peroxidase (1 unit/ml), and the optical density at 727 nm was read 10 after the onset of the reaction The concentrations of hydrogen peroxide were determined by a calibration curve in which known concentrations of hydrogen peroxide were used as the standard The principle of the coloring reaction of DA-64 and hydrogen peroxide is illustrated in Figure [17] Assay for Polyphenols Total polyphenol content was determined by Folin-Denis method [18] In brief, 3.2 ml of pure water, 200 µl of each garlic extract, 200 µl of Folin & Ciocalteu’s Phenol Reagent and 400 µl of saturated sodium carbonate solution were mixed The absorbance was determined at 760 nm Figures and summarize the data of SOD-like activity, hydrogen peroxide concentrations as indices of hydrogen peroxide scavenging activity and the total polyphenol content When DMPO was added to a solution of the HPXXOD reaction system, the spin adduct, DMPO-OOH, was formed The representative spectra of DMPO-OOH obtained from the solvent control, the control garlic extract and the black garlic extract are shown in Figure 3, and the calculated SOD-like activity is summarized in Figure Since it has been reported that the addition of superoxide dismutase (a scavenger for superoxide) resulted in the disappearance of the ESR spectrum, DMPO-OOH was indicated to be derived from superoxide generated by the HPX-XOD reaction system [19] Figure shows that SOD-like activity of the black garlic extract was about 13 times higher than that of the control garlic extract As shown in Figure 4, the control garlic extract equivalent to mg garlic showed slight hydrogen peroxide scavenging activity (about 15% of hydrogen peroxide was scavenged), while the black garlic extract equivalent to 1.25 mg garlic scavenged about 63% of hydrogen peroxide, indicating that the hydrogen peroxide scavenging activity of the black garlic extract was at least 10 times higher than that of the control garlic extract When mg garlic equivalent of the black garlic extract was added to the reaction mixture, hydrogen peroxide was scavenged to a trace level As shown in Figure 4, it is indicated that polyphenols were increased about 7-fold in the black garlic extract as compared with those in the control garlic extract Aged garlic extract manufactured by a more than 10 months natural aging process is well known to contain bioactive organic sulfur compounds such as S-allylcysteine and S-allylmercaptocysteine both of which show a variety of biological activities including anti-oxidative properties [9–12] In adddition, tetrahydro-β-carboline derivatives which possess hydrogen peroxide scavenging activity have recently been identified in aged garlic extract [13, 14] In this study, as is the case with the aged garlic, the SOD-like activity and hydrogen peroxide scavenging activity are greatly increased in garlic fermented spontaneously for only 40 days without any additives The increased SODlike activity is, at least in part, attributable to the increased amount of polyphenols Althogh we have not determined the levels of organosulfur compounds and tetrahydro-βcarboline derivatives, it is concluded that spontaneous fermentation of garlic for the relatively short period gives desirable anti-oxidative properties such as SOD-like and 159 H2O2 + 2H + CH CH N N H3C HRPOx CH 2H 2O HRPRed N+ H3C Cl- N C DA-64 HN CH CH O N CH N CH2COONa Bindschedler’s Green Figure The schematic figure of coloring reaction by DA-64 and hydrogen peroxide Solvent(80% EtOH) Control garlic extract Black garlic extract 330.5 335.5 Magnetic field/mT 340.5 Figure The representative ESR spectra of DMPO-OOH (for superoxide determination) from the solvent control (80% EtOH alone), the control garlic extract and the black garlic extract Figure SOD-like activity, hydrogen peroxide scavenging activity and 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