Plant Foods for Human Nutrition 61: 157160, 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 2 & YOSHIMI NIWANO 1,
1 New

Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan; 2 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, 8595% 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

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 garlics color was rendered black by spontaneous fermentation for 40 days at 6070 C, 8595% relative humidity without any additives (described as black garlic throughout the paper). Figure 1 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 ltration through No. 2 lter paper. The obtained ltrate 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 & Ciocalteus 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; eld sweep: 330.50340.50 mT, eld modulation frequency: 100 kHz, eld modulation width: 0.07 mT, amplitude: 200, sweep time: 2 min., time constant: 0.1 sec, microwave freq.: 9.420 GHz, microwave power: 4 mW.

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 benet 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 [1012]. In addition to organic sulfur compounds, it has been reported that aged garlic contains tetrahydro-carboline derivatives which possess potent hydrogen peroxide scavenging properties [13, 14]. In this study, we have examined anti-oxidative properties of unique fermented-garlic. The garlics color is rendered black by spontaneous fermentation for 40 days without any additives. The black color is likely to be derived from antho-

158 after standing for 30 min. A freshly prepared gallic acid was used as the standard.

Results and Discussion Figures 3 and 4 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 4. 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 4 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 5 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 5 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 [912]. In adddition, tetrahydro--carboline derivatives which possess hydrogen peroxide scavenging activity have recently been identied 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-carboline derivatives, it is concluded that spontaneous fermentation of garlic for the relatively short period gives desirable anti-oxidative properties such as SOD-like and

Figure 1. 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 l of 2 mM HPX, 30 l of dimethyl sulfoxide (DMSO), and 50 l of the garlic extract dissolved in 80 % EtOH, or solvent (80 % EtOH) alone, 20 l of 4.5 M DMPO, and 50 l 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 quantied 90 sec after the addition of XOD. Signal intensities were evaluated from the peak height of the rst 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 l of each sample (garlic extract or 80 % EtOH as a solvent) was added to 150 l of hydrogen peroxide (nal 21.3 M) and mixed. Then, 100 l of the mixture was added to 900 l 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 min 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 2 [17]. Assay for Polyphenols Total polyphenol content was determined by Folin-Denis method [18]. In brief, 3.2 ml of pure water, 200 l of each garlic extract, 200 l of Folin & Ciocalteus Phenol Reagent and 400 l of saturated sodium carbonate solution were mixed. The absorbance was determined at 760 nm

159
H2O2 + 2H + 2H 2O

CH 3 N H3C N C O CH2COONa

CH 3 N CH 3

HRPOx

HRPRed

CH 3 N+ H3C ClN

CH 3 N CH 3

DA-64

HN

Bindschedlers Green

Figure 2. The schematic gure 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 3. 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 4. SOD-like activity, hydrogen peroxide scavenging activity and total polyphenol content of garlic extract. Hydrogen peroxide scavenging activity is expressed as the % of remaining hydrogen peroxide concentration in which the concentration of the solvent control (80% EtOH alone) is regarded as 100%

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5 6 7 8 4b 4a 2 1 4 3 NH COOH
6. Moriguchi T, Takashina K, Chu P, Saito H, Nishiyama N (1994) Prolongation of life span and improved learning in the senescence accelerated mouse produced by aged garlic extract. Biol Pharm Bull 17: 15891594. 7. Rietz B, Isensee H, Strobach H, Makdessi S, Jacob R (1993) Cardioprotective actions of wild garlic (Allium ursinum) in ischemia and reperfusion. Mol Cell Biochem 119: 143150. 8. Numagami Y, Suto S, Ohnishi ST (1996) Attenuation of rat ischemic brain damage by aged garlic extract: a possible protecting mechanism as antioxidants. Neurochemistry Int 29: 135143. 9. Ide N, Matsuura H, Itakura Y (1996) Scavenging effect of aged garlic extract and its constituents on active oxygen species. Phytotherapy Res 10: 340341. 10. Imai J, Ide N, Nagae T, Moriguchi H, Matsuura H, Itakura Y (1994) Antioxidant and radical scavenging effects of aged garlic extract and its constituents. Planta Med 60: 417420 11. Ide N, Lau BHS (1997) Garlic compounds protect vascular endothelial cells from oxidized low density lipoprotein-induced injury. J Pharm Pharmacol 49: 908911. 12. Ide N, Lau BHS (1999) Aged garlic extract attenuates intracellular oxidative stress. Phytomed 6: 125131. 13. Ichikawa M, Ryu K, Yoshida J, Ide N, Yoshida S, Sasaoka T, Sumi SI (2002) Antioxidant effects of tetrahydro--carboline derivatives identied in aged garlic extract. Biofactors 16: 5772. 14. Ichikawa M, Yoshida J, Ide N, Sasaoka T, Yamaguchi H, Ono K (2006) Tetrahydro-{beta}-Carboline Derivatives in Aged Garlic Extract Show Antioxidant Properties. J Nutr 136 :726S731S. 15. Kabuto H, Nishizawa M, Tada M, Higashio C, Shishibori T, Kohno M (2005) Zingerone [4-(4-hydroxy-3-methoxyphenyl)-2-butanone] prevents 6-hydroxydopamine-induced depression in mouse striatum and increases superoxide scavenging activity in serum. Neurochem Res 30: 325332. 16. Noda Y, Kohno M, Mori A, Packer L (1999) Automated ESR free radical detector assays for antioxidant activity in natural extracts. In: Abelson JN, Simon MI (eds), Methods in Enzymology. Pasadena, CA: Division of Biology California Institute of Technology 299, 2834 17. Cheng KL, Ueno K, Imamura T (1982) In: Cheng KL, Ueno K, Imamura T (eds), CRC Handbook of Organic Analytical Reagents. Boca Raton, FL: CRC Press, pp 452. 18. Schanderl SH (1970) Tannins and related phenolics. In: Joslyn MA (ed), Methods in Food Analysis. New York: Academic Press, pp 701724. 19. Tanigawa T, Yoshikawa T, Takahashi S, Naito Y, Kondo M (1994) Spin trapping of superoxide in aqueous solutions of fresh and aged cigarette smoke. Free Radical Biol Med 17: 361365

8a

9 9a N H HOOC

CH3

Figure 5. Chemical structure of one of the tetrahydro--carboline derivatives

hydrogen peroxide scavenging activity. The proposed mechanism by which tetrahydro--carboline derivatives scavenge hydrogen peroxide has been reported [13]. For instance, in the case of one of the derivatives as shown in Figure 5, the compound is decarboxylated at 1 position and dehydrated between 1 and 2 position, suggesting that the compound functions as an electron donor and scavenge hydrogen peroxide. To further characterize the antioxidative properties of the spontaneously fermented garlic, quantication of organic sulfur compounds and tetrahydro-carboline derivatives will be required as the next step. References
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