Influence of Whisky Congeners on Health - The Institute of Brewing ...

<strong>Influence</strong> <strong>of</strong> <strong>Whisky</strong> <strong>Congeners</strong> on Health
Yoshihide Suwa*, Asuka Nemoto, Minako Koike, Eri Tsutsumi, Kayo Numaga, Seiichi
Koshimizu, Kazuo Nakatani ( Suntory Ltd., Osaka,Japan ), Kenji Oguchi (Gifu
International Institute <strong>of</strong> Biotechnology, Gifu, Japan ), Haruo Nukaya ( University <strong>of</strong>
Shizuoka, Shizuoka, Japan ), Hiroyuki Haraguchi (Fukuyama University, Hiroshima,
Japan ), Noriaki Shirasaka, Hajime Yoshizumi (Kinki University, Osaka, Japan ),
Chihiro Yabe (Kyoto Prefectural University <strong>of</strong> Medicine, Kyoto, Japan ), Kusuki
Nishioka (St. Marianna University, School <strong>of</strong> Medicine, Tokyo, Japan )
Abstract
Experimental or clinical data on whisky congener showed different effects to purine and
carbohydrate metabolism and melanin synthesis from other types <strong>of</strong> alcoholic beverages.
Moderate drinking <strong>of</strong> whisky did not enhance serum uric acid level, blood glucose, and
insulin level in healthy male subjects. <strong>Whisky</strong> congener showed the eliminative
properties <strong>of</strong> serum uric aid by excretion from blood to urine.
Secondly, whisky congener suppresses the activity <strong>of</strong> aldose reductase (AR). AR
inhibitors have been developed to prevent or to be effective for mild to moderate
symptoms <strong>of</strong> diabetic complication. No AR inhibition was observed in beer, wine, and
Shochu; a Japanese distilled liquor that does not involve any aging process in oak casks.
In whisky congeners, the longer it is aged in oak casks, the stronger the AR inhibition.
The active fraction from the congener consists <strong>of</strong> the compounds that have
1-formylpylogallol structure in their molecule. Especially, ellagic acid, which is one <strong>of</strong>
the major components in whisky congener, has an inhibitory effect on the production <strong>of</strong>
sorbitol in rat erythrocyte. Urolithin, a metabolite <strong>of</strong> ellagic acid, was shown to be
effective in the reduction <strong>of</strong> sorbitol accumulation. <strong>Whisky</strong> congener also eliminated the
absorption <strong>of</strong> sucrose from intestine.
Thirdly, whisky congener has antioxidant activity in proportion to the period <strong>of</strong>
maturation in white oak barrels. Lyoniresinol was isolated from whisky and identified
as a strong antioxidant. It suppresses the synthesis <strong>of</strong> melanin in vitro and in vivo by the
tyrosinase inhibition.
Key Words; <strong>Whisky</strong>, <strong>Congeners</strong>, Diabetes, Gout, Melanin, Antioxidant

Introduction
Studies on the effects <strong>of</strong> polyphenols contained in daily beverages on human health
have focused mainly on flavonoids, including catechin and epigallocatechin gallate in
tea, proanthocyanidin in red wine, naringenin and quercetin in citrus, or on their
condensed tannins (polymers). Stilbene, such as resveratrol in red wine and chlorogenic
acid and caffeic acid in c<strong>of</strong>fee, have also been vigorously investigated from the
perspective <strong>of</strong> preventative medicine.
Main polyphenols present in whisky are different from those described above. <strong>Whisky</strong>
polyphenols are composed <strong>of</strong> ellagic acid derived from ellagitannin (which is
hydrolyzable tannin), phenylpropanoids such as coniferyl aldehyde, vanillin, and lignan.
The effects <strong>of</strong> those ingredients on health have not been studied as much as those <strong>of</strong>
polyphenols such as the above-named flavonoids (Fig 1).
Number <strong>of</strong> literatures
3500
3000
2500
2000
1500
1000
500
0
catechin epicatechin resveratrol ellagic acid
Figure 1 Comparison <strong>of</strong> the number <strong>of</strong> studies on catechin, epicatechin,
resveratrol, and ellagic acid.
The number <strong>of</strong> literatures obtained from PubMed
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). “catechin, ”
“epicatechin,” “resveratrol,” or “ellagic acid” is used as a search term.

Polyphenols exude from the oak cask into whisky during years <strong>of</strong> aging. Some <strong>of</strong>
them go through degradative and oxidizing reactions to turn into a variety <strong>of</strong> substances.
Concerning the relationship to health, whisky was reported to have the same level <strong>of</strong>
antioxidant activity in blood as wine (3) . However, the effects <strong>of</strong> polyphenols in whisky
on health remain unclear.
We first started determining the biochemical pr<strong>of</strong>iles <strong>of</strong> whisky congeners and
compared the pharmacological actions estimated from the pr<strong>of</strong>iles with those <strong>of</strong> other
alcohol-derived congeners. We then isolated and purified active ingredients responsible
for respective pharmacological actions to determine their chemical structures and
examine the correlation between structure and activity by using chemically synthesized
analogs. To be more specific, we assessed the effects <strong>of</strong> whisky constituents on purine
metabolism (hyperuricemia, gout), glucose metabolism (obesity, diabetes), and melanin
biosynthesis in the skin.
First, in relation to purine metabolism, the question <strong>of</strong> whether alcohol drinking
triggers hyperuricemia or gout had long been debated in the medical community until
multiple epidemiological studies conducted in Western countries and Japan recently
demonstrated that excessive intake <strong>of</strong> alcohol increases the risk <strong>of</strong> gout. In order to
prevent those lifestyle-related diseases and improve consumers’ quality <strong>of</strong> life, the
following two questions about drinking-related risks need to be answered (9) .
1) Does moderate drinking also raise the serum uric acid level?
2) Is there any difference in the serum uric acid level by the type <strong>of</strong> alcohol?
Regarding (2), the effects <strong>of</strong> moderate ingestion <strong>of</strong> distilled liquor on living
organisms were not fully examined. In addition, it is unknown whether a difference
exists in uric acid metabolism between when drinking distilled liquor such as whisky
and brandy that undergoes the process <strong>of</strong> aging in the oak cask and when drinking white
liquor.
Second, we studied the relationship <strong>of</strong> whisky constituents with diabetes and obesity.
Drinking alcohol had long been considered to exacerbate type II diabetes. These days,
however, a number <strong>of</strong> epidemiological studies (1) demonstrated that moderate drinking
rather helps prevent the onset <strong>of</strong> type II diabetes. Some studies (5) indicated a J-curve
relationship between beneficial effects and risks, showing that moderate-to-minimum
drinking helps prevent type II diabetes while excessive intake can induce the onset <strong>of</strong>
the disease (a study each in the U.S.A., Europe, and Japan).
The condition <strong>of</strong> diabetes worsens when complications (diabetic retinopathy,
nephropathy, and/or neuropathy) are present. Aldose reductase (AR) is an enzyme

esponsible for the exacerbation <strong>of</strong> these complications. This enzyme is activated when
the polyol metabolic pathway is highly activated in the cell. Accumulation <strong>of</strong> sorbitol
increased by activated AR is associated with the process <strong>of</strong> inducing diabetic
neuropathy and other complications (Fig 2). This is why AR inhibitors are used to treat
diabetic neuropathy (4, 8).
normal blood glucose level
glucose glucose-6-P CO2
high blood glucose level
aldose reductase
suppression
whisky components
sorbitol fructose
Figure 2 Pathogenesis <strong>of</strong> diabetic complications
diabetic complications
glycation reaction
oxidative stress
[neuropathy, retinopathy, nephropathy]
We investigated whether non-volatile ingredients in whisky represses AR inhibiting
activity and sorbitol accumulation under a hyperglycemic condition, as well as whether
whisky congeners inhibit absorption <strong>of</strong> glucose.
Third, we evaluated the effects <strong>of</strong> whisky constituents on melanin synthesis in the
skin. Being a dominant polyphenol member in whisky, ellagic acid is already available
as a whitening agent approved by the Ministry <strong>of</strong> Health, Labour and Welfare in Japan.
This substance inhibits the activity <strong>of</strong> tyrosinase responsible for melanin synthesis.
Studies conducted so far on ellagic acids have focused on those derived from berries,
not from whisky. In the present study, we compared the ability <strong>of</strong> whiskey congeners
and other alcohol congeners to hamper the activity <strong>of</strong> tyrosinase and then tried to isolate
and purify those active ingredients and determine their structures.

Experimental
Purine and carbohydrate metabolism
We (9) have shown the influences <strong>of</strong> moderate intake level <strong>of</strong> three types <strong>of</strong> alcoholic
beverages which were beer, whisky, and Shochu, Japanese distilled liquor, on purine
and carbohydrate metabolism <strong>of</strong> healthy male volunteers (7) . In addition to it, the
difference between two types <strong>of</strong> distilled spirits with (whisky) and without (Shochu) a
process with oak wood-barreling storage, on uric acid metabolism and excretion in
volunteers, have been found.
Uric acid in blood
Purine base concentration in beer was 64.4 mg/l, whereas the concentration was much
less in whisky and Shochu, being 0.20 mg/l and 0.07 mg/l, respectively.
Serum uric acid level did not increase after drinking Shochu (Japanese distilled liquor),
and it slightly decreased after drinking water or whisky. Thus, the serum uric acid
level before and one hour after drinking was 5.0±0.3 mg/dl and 4.8±0.3 mg/dl,
respectively, for water, 5.4±0.2 mg/dl and 5.2±0.2 mg/dl for whisky, demonstrating
decrease in concentration (p < 0.001) in both cases. The serum uric acid level before
and after drinking Shochu was 5.4±0.2 mg/dl and 5.2±0.2 mg/dl, respectively, with no
significant difference being observed. In contrast, drinking beer increased the serum
uric acid level from the basal level <strong>of</strong> 5.1±0.3 mg/dl to 5.8±0.3 mg/dl, an increase <strong>of</strong>
0.7±0.1 mg/dl (p < 0.001) which corresponded to 13.6% increase from the basal level
( Fig. 3). The pattern <strong>of</strong> urine production showed difference among three types <strong>of</strong>
alcoholic beverages. Thus, urine production started earlier in the beer group than in
the distilled liquor groups, reaching the peak urine excretion 1 to 2 hours after drinking
beer, compared to 2 to 5 hours after drinking whisky ( p < 0.05). The urine volume
tended to be smaller in the Shochu group than in the beer and whisky groups.

serum uric acid level (%)
115
110
105
100
95
90
0 1 2 3 4 5
hour
Figure 3 Time course <strong>of</strong> serum uric acid level.
beer
Shochu
whisky
water
Significant interactions were observed between each type <strong>of</strong> alcoholic beverage
and time course. [ p

time, and the samples were subjected to the measurement <strong>of</strong> 58 biochemical
markers related to uric acid metabolism, glucose metabolism, alcohol
metabolism and hepatic, pancreatic and renal functions. Alcohol and
acetaldehyde concentrations in blood were determined using a headspace gas
chromatography (type HS-101 8700), and oxypurine concentration in urine was
determined by colorimetry using a Hitachi type 736-15 automated analyzer.
Among the 13 subjects, seven healthy males[mean age: 31.9±1.9 (S.E), serum
uric acid: 5.0±0.3 mg/dL] who fulfilled the criteria specified in the study protocol
throughout the study period were used as the final evaluable subjects.
Alcohol intake at each drinking test (0.8 mL/kg body weight) was set at a level
slightly higher than the moderate amount <strong>of</strong> alcohol described in the NIAAA
review (2) (ca. 30.4 mL or less /male), taking into consideration the possible
individual difference in extrapolating the results <strong>of</strong> the study to healthy people
and patients in general. Except for low purine beer, each beverage used in the
study was commercially available and was purchased from a market.
Excretion <strong>of</strong> uric acid
Serum uric acid concentration tended to be consistently lower after drinking whisky
than after drinking Shochu, another distilled liquor, although the difference was not
significant (Fig 3). With the aim <strong>of</strong> clarifying whether components in whisky have
activity to decrease serum uric acid concentration, subjects were asked to drink whisky
and Shochu in turn with intervals <strong>of</strong> one week while eating high purine food
( oil-packed sardines, total purine intake: approx 50 mg/subject ).
As shown in Fig.4-a, serum uric acid concentration was consistently lower by
approximately 2 to 3% after drinking whisky relative to the concentration after drinking
Shochu or water. And the urinary excretion <strong>of</strong> uric acid, which was measured
simultaneously, increased by 27% after drinking whisky (Fig.4-b) ( p < 0.05 ).

serum uric acid level (%)
115
110
105
100
urinary excretion <strong>of</strong> uric acid (%)
high purine food
0 alcohol intake 1 2
120
100
80
hour
140 (n= 7)
Shochu whisky
Shochu
whisky
n = 7

Figure 4 <strong>Influence</strong> <strong>of</strong> whisky drinking on excretion <strong>of</strong> serum (a) and
urinary excretion (b) <strong>of</strong> uric acid
Eight subject [30-54 years old (35.2 ± 4.1 (standard deviation) years old; 7 <strong>of</strong>
whom remained eligible until the end <strong>of</strong> the study) ingested whisky or Shochu
under the same conditions as used in figure 3, and kinetics <strong>of</strong> uric acid
metabolism were compared between the two distilled liquors. During the 30 min
<strong>of</strong> drinking each beverage, the subjects were instructed to eat oil-packed
sardines as a high-purine food (total purin intake: approx 50 mg/subject).
Xanthine oxidase inhibition
In addition to the increase <strong>of</strong> urinary excretion <strong>of</strong> uric acid after drinking whisky, we
examined the inhibitory activity <strong>of</strong> whisky to xanthine oxidase, the enzyme responsible
for the biosynthesis <strong>of</strong> uric acid (Fig.5).
purine body
uric acid
oxidation crystallization
HN
O
O
N
H
Figure 5 Uric acid biosynthesis pathway
H
N
N
H
O
needle crystal

Whiskies showed the tendency to inhibit the xanthine oxidase activity, and the
inhibitory activity increased with the time whisky is stored in an oak cask (Fig. 6).
Sake A
Sake B
Shochu A
Shochu B
Brandy A
Brandy B
<strong>Whisky</strong> A (aged 10 years)
<strong>Whisky</strong> B (aged 10 years)
<strong>Whisky</strong> C (aged 12 years)
<strong>Whisky</strong> D (aged 18 years)
<strong>Whisky</strong> E (aged 25 years)
Activity at 250 μg / ml
0 20 40 60 80 1 00
XO inhibiting activity (%)
Figure 6 Xanthine oxidase (XO)-inhibiting activity <strong>of</strong> whisky
0.3 ml <strong>of</strong> the test substance was added to 2.6 ml <strong>of</strong> 15 μg/ml xanthine solution
and the mixture was preincubated, after which 0.1 ml <strong>of</strong> 0.044 unit/ml XO
solution was added and the mixture was incubated for 5 min. Absorbance at
290 nm was determined 5 min after the addition <strong>of</strong> XO, and XO-inhibiting activity
was calculated from the absorbance by using the following equation:
XO-inhibiting activity (%) = (absorbance in the presence <strong>of</strong> the test substance) /
(absorbance in the presence <strong>of</strong> equal concentration <strong>of</strong> alcohol) x 100
We isolated and purified this XOI active ingredient under the procedures described in
(figure 7) to identify the structure <strong>of</strong> ellagic acid. Regarding in vitro oxidase inhibiting
activity, the 50% inhibition was achieved with 260 mg/L <strong>of</strong> whisky congeners (single
malt, 12 years <strong>of</strong> storage) or with 4.9 mg/L <strong>of</strong> ellagic acid, in comparison with
allopurinol, a treatment for hyperuricemia and grout.

whisky
whisky congener
liquid-liquid distribution
(AcOEt、n-BuOH、H 2 2O) O)
active fraction (Kd=3.0-3.4)
Figure 7 Isolation and structural determination <strong>of</strong> the XOI active
component from whisky
Effect on diabetic complications
Glucose and insulin in blood
Being a distilled liquor, whisky does not contain carbohydrates, and its calories are
virtually 60% <strong>of</strong> those <strong>of</strong> beer, a fermented liquor, when the same amount <strong>of</strong> alcohol is
consumed.
Evaporation and lyophilization
AcOEt and n-BuOH fraction
Sephadex LH-20
column chromatography
Elution : MeOH
active component
HO
HO
Regarding the blood glucose level and blood insulin level after drinking alcoholic
beverages, distinct differences were observed in these levels between beer and the two
types <strong>of</strong> distilled liquor. Thus, blood glucose level and insulin level increased on
average by 26.7% and 5.1-fold, respectively, after drinking beer, whereas there were no
changes in these parameters observed after drinking whisky or Shochu(Fig 8-a&b).
O
O
O
O
Ellagic acid
OH
OH

whisky
Evaporation and lyophilization
whisky congener
liquid-liquid distribution
(AcOEt、n-BuOH、H 2 2O) O)
AcOEt and n-BuOH fraction
Sephadex LH-20
column chromatography
Elution : MeOH
active fraction (Kd=3.0-3.4)
active component
serum insulin insulin (μ (μ U/mL)
30
20
10
HO
HO
Figure 8 Plasma glucose and serum insulin levels after alcohol intake
The experiment was conducted as described in Fig 3.
The level <strong>of</strong> ellagic acid contained in the whisky used in this study was approximately 7
O
O
O
O
Ellagic acid
beer
OH
whisky
OH
0
0 1 2 3 4 5
alcohol intake
hour

ppm. This level and its specific activity explained that ellagic acid was responsible for
about 33.6% <strong>of</strong> whisky’s in vitro XOI activity.
Aldose reductase inhibition
We assessed the effect <strong>of</strong> respective alcoholic beverages on AR activity. When 5 μL <strong>of</strong>
whisky, Sake, beer, or Shochu was added to the 1 mL <strong>of</strong> AR enzyme reaction reagent,
AR’s activity was inhibited by 94%, 8.4%, 0%, or 0%, respectively, showing that
whisky was more potent in inhibiting the activity <strong>of</strong> the enzyme than any other
beveragel (Fig 9). Adding 40% ethanol solution instead <strong>of</strong> an alcoholic beverage
produced no inhibitory activity.
low low inhibiting inhibiting activity activity High High
whisky
red wine
Shochu
beer
Sake
0 0.1 0.2 0.3 0.4
<strong>Whisky</strong> products
aged 21 years
aged 17 years
aged 12 years
aged 10 years
aged short period
0 0.2 0.4 0.6
1/ IC 50 ( L / mg )
Figure 9 Aldose reductose (AR) inhibiting activitiy <strong>of</strong> whisky
The reaction mixture contained 0.1 M sodium phosphate buffer (pH 6.2), 150 μM
NADPH, 10 mM DL-glyceraldehyde, enzyme (6) and test sample in a total
volume <strong>of</strong> 0.1 ml. Test samples were dissolved in DMSO and added to the
reaction mixture resulting the final concentration <strong>of</strong> DMSO <strong>of</strong> 1 %. The reaction
was initiated by the addition <strong>of</strong> glyceraldehyde and incubated for 30 min. The
decrease <strong>of</strong> absorbance was measured at 340 nm.

When oolong tea or green tea was added as a non-alcoholic,
plant-compound-containing beverage, the enzyme activity was reduced by 55% or 54%,
respectively. Those findings indicated that whisky was more potent in inhibitory
activity than non-alcoholic beverages as well (data not shown).
When 10 μL <strong>of</strong> malt whisky <strong>of</strong> 5, 10, 15, 20, or 30 years <strong>of</strong> age was added to 1 mL <strong>of</strong>
the AR enzyme reaction reagent, AR’s activity was suppressed by 20%, 32%, 33%,
42%, or 56%, respectively, demonstrating that the inhibitory activity increased in
proportion to the age <strong>of</strong> the whisky (Fig 9). These findings suggested that the AR
inhibiting substances in whisky were oak compounds derived from the cask.
To evaluate the AR inhibitory compounds from whisky congener, a systematic
fractionation was carried out and their effects on hrAR were tested. The n-BuOH
fraction was found to exhibit the strongest AR inhibitory activity, its IC50 being 1.5
mg/L. An active principle as a potent AR inhibitor was isolated from this active
n-BuOH fraction and its chemical structure was identified as ellagic acid by spectral
analysis and comparing it with published data in the literature. Ellagic acid exhibited a
significant inhibition <strong>of</strong> AR in concentration-dependent manner, its IC50 value being
0.067 mg/L. The potency <strong>of</strong> this compound appeared equal to that <strong>of</strong> the known ARI,
ONO-2235 (Epalrestat) (data not shown).
Sorbitol was accumulated in erythrocytes when they were incubated in the medium
containing 30mM glucose. In the presence <strong>of</strong> ellagic acid, sorbitol accumulation was
inhibited in a dose-dependent manner (Fig. 10). At 100 mg/L, sorbitol accumulation
was inhibited by 70 %. In comparison with ONO-2235, which exhibits an IC50 <strong>of</strong>
1.5×10 -6 M, ellagic acid was less potent.
Although less potent than drugs, the specific activity <strong>of</strong> whisky congeners against AR
belongs to the most potent class for foods and beverages including alcoholic drinks. We
are evaluating the AR inhibiting activity <strong>of</strong> ellagic acid metabolites now.

sorbitol in erythrocytes
(nmol/mLRBC)
glucose
5.5mM
glucose
30mM
glucose
ellagic
acid
1 mg/L
glucose
glucose
glucose
30mM 30mM 30mM 30mM
+ + + +
ellagic
acid
3 mg/L
ellagic
acid
10 mg/L
Epalrestat
3 mg/L
Figure 10 Ellagic acid inhibits the accumulation <strong>of</strong> sorbitol in
rerythrocytes
Blood was collected into a heparinized tube from the abdominal aorta <strong>of</strong> the rats.
Erythrocytes were obtained by centrifugation at 2000 xg and washed with cold
saline twice. 1 ml <strong>of</strong> erythrocytes was added into 3 ml <strong>of</strong> Kreb’s-Ringer
bicarbonate buffer, containing 30 mM glucose and each compound dissolved in
30 μL <strong>of</strong> dimethyl sulfoxide (DMSO), and was incubated at 37 ℃ in 5 % CO2 in
air for 3 h. At the end <strong>of</strong> incubation periods, red blood cells were separated by
centrifugation, washed, and precipitated with cold 6 % perchloric acid. The
supernatant was neutralized with a same volume <strong>of</strong> 0.5 M Na3PO4 (pH 11).
The sorbitol contents were measured enzymatically by the sorbitol
dehydrogenase reaction. The amount <strong>of</strong> sorbitol is proportional to the formation
<strong>of</strong> NADPH as measured by the increase in fluorescence under the excitation and
emission at 355 and 460 nm, respectively.

Suppressive effect to melanogenesis
Inhibition <strong>of</strong> tyrosinase activity
A process <strong>of</strong> aging in the oak cask allows whisky and brandy to contain substances
exuded from oak during aging in the cask as well as products made from their
interactive or degenerating reactions such as lignin-derived aromatic components and
polyphenols. Researchers have reported that some plant elements with a phenol
structure inhibit melanin synthesis.
The inhibitory effect <strong>of</strong> respective test substances was examined on the enzyme
activity <strong>of</strong> tyrosinase prepared from mouse B16 melanoma cells (9) . <strong>Whisky</strong> and brandy
had a more potent tyrosinase inhibiting activity than other alcohols (Sake, Shochu, wine,
beer) (Fig 11) This inhibitory activity was comparable to one observed in extremely
potent plant extracts and increased in proportion to the length <strong>of</strong> storage in the cask.
Non-volatile fractions were more active than volatile fractions. (data not shown)
brown spirits
<strong>Whisky</strong> A
<strong>Whisky</strong> B
<strong>Whisky</strong> C
Brandy A
Brandy B
Brandy C
Shochu A
Shochu B
Shochu C
low
Inhibiting activity
High
kojic acid
(
positive control)
0.00 0.01 0.02 0.03 0.04 0.05
1 / IC50 (ml / μg)
Figure 11 Inhibitory effects <strong>of</strong> distilled liquors to tyrosinase activity
Tyrosinase source was prepared from murin B16 melanoma cells (Riken Cell
Bank, Tsukuba, Japan). The cells were lysed by incubating at 4°C for 1hr in
RIPA buffer (10 mM Tris-HCl, pH 7.5, 1 % NP-40, 0.1 % sodium deoxycholate,

0.1 % SDS, 150 mM NaCl, 1 mM EDTA, 0.5 mM AEBSF, and 10 μg/mL
leupeptin). The lysate were centrifuged at 10,000g for 30 min to obtain the
supernatant as the crude tyrosinase extract for the activity assay. The reaction
mixture contained 50 mM phosphate buffer, pH 6.8, 0.05 % L-dopa and the
supernatant (tyrosinase source). After incubation at 37°C for 20 min,
dopachrome formation was monitored by measuring absorbance at wavelength
492 nm in a microplate reader.
Effect <strong>of</strong> anti-melanogenesis
Inhibitory effect <strong>of</strong> whiskey congener on melanin synthesis was determined in B16
melanoma cell line (Fig 12). We isolated and identified several constituents from
whisky congeners and assessed the ability to inhibit melanin synthesis with an
ultraviolet-induced pigmentation test system in the skin <strong>of</strong> the colored guinea pig. As a
result, lyoniresinol, a polyphenol, was found to be effective in reducing pigmentation.
Its activity was approximately 7 times as potent as that <strong>of</strong> arbutin, which was used as a
positive control. The test result demonstrated that lyoniresinol did not directly act
against the activity <strong>of</strong> tyrosinase (Fig 13).
whisky (-) whisky (+)
Figure 12 <strong>Whisky</strong> effect <strong>of</strong> anti-melanogenesis in B16 melanoma cell line.
Mouse B16 melanoma cells were purchased from Riken Cell Bank (Tsukuba,
Japan). B16 melanoma cells were cultured in Dulbecco’s modified eagle’s
medium (DMEM) that was supplemented with 10 % (vol/vol) fetal bovine serum,
100 unit/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified
CO2-controlled (5 %) incubator. Induction <strong>of</strong> melanogenesis was initiated by
addition <strong>of</strong> 1 μM [Nle 4 , D-Phe 7 ] α-MSH. 0-200 μg/mL <strong>of</strong> whiskey congener was
applied.

6
white
5
⊿ L*
4
3
2
1
0
-1
-2
Figure 13 Effect <strong>of</strong> anti-melanogenesis in vivo
Chemosynthesized compounds <strong>of</strong> respective whisky-derived substances were
applied to the UVB-irradiated skin <strong>of</strong> the colored guinea pig to evaluate their
effects on pigmentation.
Discussion
UV application
**
*
**
-3
black
-4
-7 0 7 14 21 28 35 42 49 56 63
time (date)
Polyphenols contained in whisky, including precursors <strong>of</strong> reaction products during the
aging process, derive from the white oak cask. The effects <strong>of</strong> whisky congeners on
health may therefore be similar to those <strong>of</strong> polyphenols inherent in white oak. The most
abundant polyphenol present in white oak is ellagic acid. And tannin present in oak is
ellagitannin, or a hydrolysable polymer <strong>of</strong> ellagic acid. The pattern <strong>of</strong> this polymer
differs from that <strong>of</strong> condensed type tannin present in tea.
**
N=12-25
*; p

<strong>Whisky</strong> and brandy congeners composed <strong>of</strong> those substances have distinctly different
properties from other daily foods and beverages.
Containing a variety <strong>of</strong> characteristic polyphenols, whisky congeners probably have
a greater affinity for oxidoreductases than other foods and drinks do, and therefore are
distinctively potent in inhibiting enzyme activities. <strong>Whisky</strong> congeners effectively
suppressed the activities <strong>of</strong> different types <strong>of</strong> oxidoreductases, i.e., xathine oxidase for
urid acid production, aldose reductase in the polyol pathway, and tyrosinase for melanin
production.
The potency <strong>of</strong> in vitro enzyme inhibiting activity, however, does not necessarily
correlate with that <strong>of</strong> in vivo activity. Ellagic acid plays a major role in vitro, while
other constituents may account for the inhibitory effect in cell or in vivo. For instance,
lyoniresinol in vitro showed a weaker ability than ellagic acid against melanin synthesis
but a stronger ability in melanoma cell lines and the skin <strong>of</strong> the guinea pig.
Exploration <strong>of</strong> the effects <strong>of</strong> whisky congeners on health has just taken <strong>of</strong>f.
Acknowledgment
Many thanks are due to Dr. Tetsuya Yamamoto, Department <strong>of</strong> Internal Medicine III,
Hyogo College <strong>of</strong> Medicine, Pr<strong>of</strong>essor Takayuki Sumida, School <strong>of</strong> Medicine,
University <strong>of</strong> Tsukuba, Dr. Masako Iwatani, Institute <strong>of</strong> Rheumatology, Tokyo
Women’s Medical College for their valuable advice. The authors also thank Dr.
Hir<strong>of</strong>umi Koda, Ms. Aki Kusumoto, Mr. Yoshiyuki Ishikura, Research Center, Suntory
Limited, for their kind collaborations.
References
1. Ajani, U.A., Hennekens, C.H., Spelsberg, A., Manson, J.E., Alcohol consumption
and risk <strong>of</strong> type 2 diabetes mellitus among US male physicians. Arch. Intern. Med.,
2000, 160(7), 1025-1030.
2. Dufour, M.C., What is moderate drinking? Defining “Drink” and gouty humans.
Alcohol Res. Health, 1999, 23, 5-14.
3. Duthie, G.G., Pederson, M.W., Gardner, P.T., Morrice, P.C., Jenkinson, A.McE.,
McPhail, D.B., Steele, G.M., The effect <strong>of</strong> whisky and wine consumption on total
phenol content and antioxidant capacity <strong>of</strong> plasma from healthy volunteers. Eur. J. Clin.
Nutr., 1998, 52, 733-736.

4. Yabe-Nishimura, C., Aldose reductase in glucose toxicity: A potential target for the
prevention <strong>of</strong> diabetic complications. Pharmacol. Rev., 1998, 50(1), 21-33.
5. Nakanishi, N., Suzuki, K., Tatara, K., Alcohol consumption and risk for development
<strong>of</strong> impaired fasting glucose or type 2 diabetes in middle-aged Japanese men. Diabetes
Care, 2003, 26(1), 48-54.
6. Nishimura, C., Yamaoka, T., Mizutani, M., Yamashita, K., Akera, T., Tanimoto, T.,
Purification and characterization <strong>of</strong> the recombinant human aldose reductase expressed
in baculovirus system. Biochim. Biophys. Acta, 1991, 1078, 171-178.
7. Nishioka, K., Sumida, T., Iwatani, M., Kusumoto, A., Ishikura, Y., Hatanaka, H.,
Yomo, H., Kohda, H., Ashikari, T., Shibano, Y., Suwa, Y., <strong>Influence</strong> <strong>of</strong> moderate
drinking on purine and carbohydrate metabolism. Alc. Clin. Exp. Res., 2002, 26(8),
20S-25S.
8. Haraguchi, H., Ohmi, I., Kubo, I., Inhibition <strong>of</strong> aldose reductase by maesanin and
related p-benzoquinone derivatives and effects on other enzymes. Bioorg. Med. Chem.,
1996. 4(1), 49-53.
9. Ohguchi, K., Banno Y., Akao Y., Nozawa Y., Involvement <strong>of</strong> Phospholipase D1 in
Melanogenesis <strong>of</strong> Mouse B16 Melanoma Cells. J. Biol. Chem., 2004. 279(5),
3408-3412
10. Suwa, Y., Purine metabolism and the influence <strong>of</strong> alcoholic beverages. In:
Comprehensive Handbook <strong>of</strong> Alcohol Related Pathology Volume 2, V.Preedy and
R.Watson,Eds., Academic Press / Elsevier, 2005, 1069-1081.

Number
<strong>of</strong> literatur
e s
3500
3000
2500
2000
1500
1000
500
0
catechin epicatechin resveratrol ellagic acid
Figure 1 Comparison <strong>of</strong> the number <strong>of</strong> studies on catechin, epicatechin,
resveratrol, and ellagic acid.
The number <strong>of</strong> literatures obtained from PubMed
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). “catechin, ”
“epicatechin,” “resveratrol,” or “ellagic acid” is used as a search term.

normal blood glucose level
glucose glucose-6-P CO2
high blood glucose level
aldose reductase
suppression
whisky components
Figure 2 Pathogenesis <strong>of</strong> diabetic complications
sorbitol fructose
diabetic complications
glycation reaction
oxidative stress
[neuropathy, retinopathy, nephropathy]

serum uric acid level (%) ( %)
115
110
105
100
95
90
0 1 2 3 4 5
hour
Figure 3 Time course <strong>of</strong> serum uric acid level.
beer
Shochu
whisky
water
Significant interactions were observed between each type <strong>of</strong> alcoholic beverage
and time course. [ p

markers related to uric acid metabolism, glucose metabolism, alcohol
metabolism and hepatic, pancreatic and renal functions. Alcohol and
acetaldehyde concentrations in blood were determined using a headspace gas
chromatography (type HS-101 8700), and oxypurine concentration in urine was
determined by colorimetry using a Hitachi type 736-15 automated analyzer.
Among the 13 subjects, seven healthy males[mean age: 31.9±1.9 (S.E), serum
uric acid: 5.0±0.3 mg/dL] who fulfilled the criteria specified in the study protocol
throughout the study period were used as the final evaluable subjects.
Alcohol intake at each drinking test (0.8 mL/kg body weight) was set at a level
slightly higher than the moderate amount <strong>of</strong> alcohol described in the NIAAA
review (2) (ca. 30.4 mL or less /male), taking into consideration the possible
individual difference in extrapolating the results <strong>of</strong> the study to healthy people
and patients in general. Except for low purine beer, each beverage used in the
study was commercially available and was purchased from a market.

a
serum uric acid level (%)
b
115
110
105
100
urinary excretion <strong>of</strong> uric acid (%)
high purine food
0 alcohol intake 1 2
120
100
80
hour
140 (n= 7)
Shochu whisky
Shochu
whisky
n = 7
Figure 4 <strong>Influence</strong> <strong>of</strong> whisky drinking on excretion <strong>of</strong> serum (a) and

urinary excretion (b) <strong>of</strong> uric acid
Eight subject [30-54 years old (35.2 ± 4.1 (standard deviation) years old; 7 <strong>of</strong>
whom remained eligible until the end <strong>of</strong> the study) ingested whisky or Shochu
under the same conditions as used in figure 3, and kinetics <strong>of</strong> uric acid
metabolism were compared between the two distilled liquors. During the 30 min
<strong>of</strong> drinking each beverage, the subjects were instructed to eat oil-packed
sardines as a high-purine food (total purin intake: approx 50 mg/subject).

purine body
xanthine oxydase
uric acid
oxidation crystallization
HN
O
O
N
H
Figure 5 Uric acid biosynthesis pathway
H
N
N
H
O
needle crystal

Sake A
Sake B
Shochu A
Shochu B
Brandy A
Brandy B
<strong>Whisky</strong> A (aged 10 years)
<strong>Whisky</strong> B (aged 10 years)
<strong>Whisky</strong> C (aged 12 years)
<strong>Whisky</strong> D (aged 18 years)
<strong>Whisky</strong> E (aged 25 years)
Activity at 250 μg / ml
0 20 40 60 80 1 00
XO inhibiting activity (%)
Figure 6 Xanthine oxidase (XO)-inhibiting activity <strong>of</strong> whisky
0.3 ml <strong>of</strong> the test substance was added to 2.6 ml <strong>of</strong> 15 μg/ml xanthine solution
and the mixture was preincubated, after which 0.1 ml <strong>of</strong> 0.044 unit/ml XO
solution was added and the mixture was incubated for 5 min. Absorbance at
290 nm was determined 5 min after the addition <strong>of</strong> XO, and XO-inhibiting activity
was calculated from the absorbance by using the following equation:
XO-inhibiting activity (%) = (absorbance in the presence <strong>of</strong> the test substance) /
(absorbance in the presence <strong>of</strong> equal concentration <strong>of</strong> alcohol) x 100

whisky
Evaporation and lyophilization
whisky congener
liquid-liquid distribution
(AcOEt、n-BuOH、H 2 2O) O)
AcOEt and n-BuOH fraction
Sephadex LH-20
column chromatography
Elution : MeOH
active fraction (Kd=3.0-3.4)
active component
Figure 7 Isolation and structural determination <strong>of</strong> the XOI active
component from whisky
HO
HO
O
O
O
O
Ellagic acid
OH
OH

a
b
plasma glucose (mg/dL)
serum insulin (μ U/mL)
140
130
120
110
100
90
beer
whisky
80
0 1 2 3 4 5
alcohol intake
30
20
10
hour
beer
whisky
0
0 1 2 3 4 5
alcohol intake
hour
Figure 8 Plasma glucose and serum insulin levels after alcohol intake
The experiment was conducted as described in Fig 3.

low low inhibiting inhibiting activity activity High High
whisky
red wine
Shochu
beer
Sake
0 0.1 0.2 0.3 0.4
<strong>Whisky</strong> products
aged 21 years
aged 17 years
aged 12 years
aged 10 years
aged short period
0 0.2 0.4 0.6
1/ IC IC50 50 ( L / mg )
Figure 9 Aldose reductose (AR) inhibiting activitiy <strong>of</strong> whisky
The reaction mixture contained 0.1 M sodium phosphate buffer (pH 6.2), 150 μM
NADPH, 10 mM DL-glyceraldehyde, enzyme (6) and test sample in a total
volume <strong>of</strong> 0.1 ml. Test samples were dissolved in DMSO and added to the
reaction mixture resulting the final concentration <strong>of</strong> DMSO <strong>of</strong> 1 %. The reaction
was initiated by the addition <strong>of</strong> glyceraldehyde and incubated for 30 min. The
decrease <strong>of</strong> absorbance was measured at 340 nm.

sorbitol in erythrocytes
(nmol/mLRBC)
glucose
5.5mM
glucose
30mM
glucose
ellagic
acid
1 mg/L
glucose
glucose
glucose
30mM 30mM 30mM 30mM
+ + + +
ellagic
acid
3 mg/L
ellagic
acid
10 mg/L
Epalrestat
3 mg/L
Figure 10 Ellagic acid inhibits the accumulation <strong>of</strong> sorbitol in
rerythrocytes
Blood was collected into a heparinized tube from the abdominal aorta <strong>of</strong> the rats.
Erythrocytes were obtained by centrifugation at 2000 xg and washed with cold
saline twice. 1 ml <strong>of</strong> erythrocytes was added into 3 ml <strong>of</strong> Kreb’s-Ringer
bicarbonate buffer, containing 30 mM glucose and each compound dissolved in
30 μL <strong>of</strong> dimethyl sulfoxide (DMSO), and was incubated at 37 ℃ in 5 % CO2 in
air for 3 h. At the end <strong>of</strong> incubation periods, red blood cells were separated by
centrifugation, washed, and precipitated with cold 6 % perchloric acid. The
supernatant was neutralized with a same volume <strong>of</strong> 0.5 M Na3PO4 (pH 11).
The sorbitol contents were measured enzymatically by the sorbitol
dehydrogenase reaction. The amount <strong>of</strong> sorbitol is proportional to the formation
<strong>of</strong> NADPH as measured by the increase in fluorescence under the excitation and
emission at 355 and 460 nm, respectively.

own spirits
<strong>Whisky</strong> A
<strong>Whisky</strong> B
<strong>Whisky</strong> C
Brandy A
Brandy B
Brandy C
Shochu A
Shochu B
Shochu C
low
Inhibiting activity
kojic acid
(
positive control)
0.00 0.01 0.02 0.03 0.04 0.05
1 / IC50 (ml / μg)
Figure 11 Inhibitory effects <strong>of</strong> distilled liquors to tyrosinase activity
High
Tyrosinase source was prepared from murin B16 melanoma cells (Riken Cell
Bank, Tsukuba, Japan). The cells were lysed by incubating at 4°C for 1hr in
RIPA buffer (10 mM Tris-HCl, pH 7.5, 1 % NP-40, 0.1 % sodium deoxycholate,
0.1 % SDS, 150 mM NaCl, 1 mM EDTA, 0.5 mM AEBSF, and 10 μg/mL
leupeptin). The lysate were centrifuged at 10,000g for 30 min to obtain the
supernatant as the crude tyrosinase extract for the activity assay. The reaction
mixture contained 50 mM phosphate buffer, pH 6.8, 0.05 % L-dopa and the
supernatant (tyrosinase source). After incubation at 37°C for 20 min,
dopachrome formation was monitored by measuring absorbance at wavelength
492 nm in a microplate reader.

whisky (-) whisky (+)
Figure 12 <strong>Whisky</strong> effect <strong>of</strong> anti-melanogenesis in B16 melanoma cell line.
Mouse B16 melanoma cells were purchased from Riken Cell Bank (Tsukuba,
Japan). B16 melanoma cells were cultured in Dulbecco’s modified eagle’s
medium (DMEM) that was supplemented with 10 % (vol/vol) fetal bovine serum,
100 unit/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified
CO2-controlled (5 %) incubator. Induction <strong>of</strong> melanogenesis was initiated by
addition <strong>of</strong> 1 μM [Nle 4 , D-Phe 7 ] α-MSH. 0-200 μg/mL <strong>of</strong> whiskey congener was
applied.

6
white
5
⊿ L*
4
3
2
1
0
-1
-2
UV application
**
*
-3
black
-4
-7 0 7 14 21 28 35 42 49 56 63
time (date)
Figure 13 Effect <strong>of</strong> anti-melanogenesis in vivo
Chemosynthesized compounds <strong>of</strong> respective whisky-derived substances were
applied to the UVB-irradiated skin <strong>of</strong> the colored guinea pig to evaluate their
effects on pigmentation.
**
**
**
**
*
vehicle
1%lyoniresinol
1% arbutin
7% arbutin
1% ellagic acid
N=12-25
*; p