DOI: 10.1007/s11130-006-0017-5 Increased Anti-oxidative Potency of Garlic by Spontaneous Short-term Fermentation EMIKO SATO,1 MASAHIRO KOHNO,1 HAMASUKE HAMANO2& YOSHIMI NIWANO1,∗ 1New In
Trang 12006 Springer Science+Business Media, Inc.
DOI: 10.1007/s11130-006-0017-5
Increased Anti-oxidative Potency of Garlic by Spontaneous Short-term Fermentation
EMIKO SATO,1 MASAHIRO KOHNO,1 HAMASUKE HAMANO2& YOSHIMI NIWANO1,∗
1New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan;2Genki 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,
Polyphe-nols
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
associ-ated with oxygen radical species and lipid peroxidation
The intrinsic antioxidant activities of garlic [7], garlic
ex-tract [8] and some garlic constituents [9] have been widely
documented Among the many commercial garlic
prod-ucts, aged garlic extract is known to contain unique and
bioactive organic sulfur compounds such as S-allylcystein
and S-allylmercaptocystein which show anti-oxidative
ef-fects [10–12] In addition to organic sulfur compounds, it
has been reported that aged garlic contains tetrahydro-
β-carboline derivatives which possess potent hydrogen
per-oxide 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 sponta-neous fermentation for 40 days at 60–70◦C, 85–95% rel-ative humidity without any additives (described as black garlic throughout the paper) Figure 1 shows representa-tive 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 pulver-ized in 80 % ethanol solution followed by filtration through
No 2 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 fol-lows; field sweep: 330.50–340.50 mT, field modulation frequency: 100 kHz, field modulation width: 0.07 mT, am-plitude: 200, sweep time: 2 min., time constant: 0.1 sec, microwave freq.: 9.420 GHz, microwave power: 4 mW
Trang 2Figure 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, 50µl of 2
mM HPX, 30µl 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
(DA-64) as a coloring agent suggested by the manufacture (Wako
Pure Chemical Industries, Tokyo, Japan) In brief, 50µl of
each sample (garlic extract or 80 % EtOH as a solvent) was
added to 150µl of hydrogen peroxide (final 21.3 µM) and
mixed Then, 100µl 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
peroxi-dase (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 Figure2
[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 400µl of saturated sodium carbonate solution
were mixed The absorbance was determined at 760 nm
after standing for 30 min A freshly prepared gallic acid was used as the standard
Results and Discussion
Figures3and4summarize the data of SOD-like activity, hydrogen peroxide concentrations as indices of hydrogen peroxide scavenging activity and the total polyphenol con-tent
When DMPO was added to a solution of the HPX-XOD reaction system, the spin adduct, DMPO-OOH, was formed The representative spectra of DMPO-OOH ob-tained from the solvent control, the control garlic extract and the black garlic extract are shown in Figure3, and the calcu-lated SOD-like activity is summarized in Figure4 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 de-rived from superoxide generated by the HPX-XOD reaction system [19] Figure4shows that SOD-like activity of the black garlic extract was about 13 times higher than that of the control garlic extract
As shown in Figure4, 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 gar-lic 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, hy-drogen 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 SOD-like 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-
β-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
Trang 3H 2 O 2 + 2H + 2H 2 O
N
N
N
C
CH3
H3C
CH3
CH3
HN CH2COONa
O DA-64
N+
N
N
CH3
H 3 C
CH3
CH 3
Cl-Bindschedler’s Green
Figure 2 The schematic figure of coloring reaction by DA-64 and hydrogen peroxide
Magnetic field/mT
Control garlic extract
Black garlic extract Solvent(80% EtOH)
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%
Trang 4N H
NH COOH
HOOC
5
6
7
8
4 3
2 1 9
Figure 5 Chemical structure of one of the tetrahydro- β-carboline
deriva-tives
hydrogen peroxide scavenging activity The proposed
mechanism by which tetrahydro-β-carboline derivatives
scavenge hydrogen peroxide has been reported [13] For
instance, in the case of one of the derivatives as shown
in Figure5, 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
anti-oxidative properties of the spontaneously fermented garlic,
quantification of organic sulfur compounds and
tetrahydro-β-carboline derivatives will be required as the next
step
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