Irwin Cheah,a Jim Kelly,b Steven J. Langforda and Gordon Troupc
Three single malt Scotch and two USA whiskies were investigated
by EPR spectrometry and their antioxidant activity evaluated.
The EPR spectra obtained for the five samples showed the presence
of a Cu2+ species that was unique to each whisky as well as an
expected free radical signal. The results of the antioxidant assay
show that whisky (either neat or as its extract) confers excellent
antioxidant activity and that these properties relate to the "non-phenolic"
constituents of the extract.
The antioxidant action of Scotch Whiskies was first reported in
1996 . It was measured by an EPR technique observing the decay
of superoxide radicals: however, no EPR spectra of the whiskies
were reported. Since the antioxidant action was presumably due
to gallates and other similar (phenolic) compounds extracted from
toasted hard oak barrels previously used to age sherry, a stable
free radical signal should be detectable by EPR spectrometry.
The research work described in this paper aimed at characterising
five whisky samples by EPR spectrometry and also to evaluate quantitatively
the antioxidant activity reported by McPhail et al. .
As part of a research programme aimed at finding preventative
methods for diseases relating to neurodegenerative conditions,
we developed a simple assay to determine the efficiency of our
drug candidates based on previous literature.[2,3] This assay
has been used here to determine the antioxidant efficacy of the
whiskies against the know antioxidants, vitamin C (ascorbic acid)
and vitamin E (atocopherol).
For the EPR work, each sample of 150 ml was concentrated to 10
ml on a rotary evaporator (25-30°C). Samples containing the solid
and supernatant were placed in standard quartz tubes and frozen
to avoid microwave polar water losses. A Varian E-12 EPR spectrometer,
operating at 9.1 GHz was used to collect the spectra. There was
little difference between the shape spectra of the liquid and
solid portions of the heterogenous samples.
A convenient test to determine the efficiency of antioxidants
in aqueous systems was setup using 2,2-azobis(2-amidinopropane).2HCl
(AAPH) as a free radical initiator (Scheme 1). The production
of conjugated diene hydroperoxide (LOOH) generated through the
oxidation of linoleic acid in an aqueous system at 37ºC is monitored
at 234nm for 15 minutes using a Cary 100 UV-Vis spectrophotometer.
The efficiency of the antioxidant is measured by its ability to
quench free radicals and hence slow or stop oxidation of linoleic
acid. Prior to testing the efficiency of the five whisky samples
(supplied by Angoves Pty Ltd) at a volume of 10 mL, a number of well known antioxidants including
vitamin E and ascorbic acid were tested (0.01 M solutions), giving
similar results to those presented in literature.[2,3]
The efficiency is determined according to a standard, that is
the generation of free radicals in the absence of an antioxidant.
Efficiency is then calculated using the following equation:
Efficiency (%) = 1 [K2 /K1] x 100
K1 = rate of oxidation of standard (no antioxidant) = [difference
in absorbance]/ [time (sec)]
K2 = rate of oxidation with antioxidant = [difference in absorbance]/
Lipid oxidation can usually be prevented by the addition free
radical scavengers (FRS), more commonly known as antioxidants.
Their role is to turn very reactive and harmful molecules such
as hydroxy, nitroxy and superoxide radicals into innocuous molecules
that can be passed or converted by biological processes.
One of the main pathways that lead to apoptosis in cells is oxidative
stress. This event is a product of the action of these harmful
radicals on the cell walls and membranes, leading to their breakdown.
In terms of the antioxidant evaluation (Scheme 1), AAPH was utilised
as a free radical initiator generating superoxide, and the efficiency
was calculated based on the formation of a conjugated diene hydroperoxide
generated through the oxidation of linoleic acid in the presence
and absence of a free radical scavenger. The conjugated diene
hydroperoxide mimics the products of cellular oxidative stress
imposed on the cell wall. The antioxidant can act at any stage
of the reaction to stop or slow down the formation of the conjugated
diene by reacting with the appropriate radicals.Quite clearly,
all five whiskies conferred excellent antioxidant action (77-95%)
Alcoholic solutions (40% v/v ethanol) were ineffective suggesting
that the antioxidant action is a result of the extract. Interestingly,
the removal of phenolic compounds by passage of the whisky through
polyvinylpolypyrrolidone (PVPP) still conferred excellent activity.
This contradicts the proposal of McPhail et al.  and suggests that other components are responsible for the
Fig.1 Schematic representation of the spectrophotometric results at
234 nm obtained upon the addition of antioxidant (10 mL) to linoleic
acid in the presence of AAPH.
The spectra of the highest quality Scotch Whisky and the lowest
quality USA are shown in Figure 1. Note the free radical signals
and the differing Cu2+ spectra. In fact, the observed Cu2+ spectrum
is unique for each whisky and is presumably a result of leaching
from the copper within the stills, the different Cu2+spectra a
result of different ligation which is most likely a result of
the different fermentation methods employed. Whisky samples treated
with polyvinylpolypyrrolidone (PPVP) to remove phenolics prior
to cold evaporation showed reduced free radical signals, but the
reduction was not as dramatic as in red wines (90%). The antioxidant
efficiency achieved by the treated whiskies was approx. 10% of
that of the untreated, showing that the removable phenolics did
not contribute significantly.
Fig. 2. EPR spectra of concentrated samples of (a) USA Whiskey 1 and
(b) Malt Whisky 1 at 77K click to enlarge
The results indicate that whisky acts as an antioxidant with a
similar efficiency to a-tocopherol and greater efficiency than
ascorbic acid. Given these results, a shot of whisky will give
the equivalent "antioxidant potential" to the recommended daily
intake of vitamin C! We are at present investigating the source
of the antioxidant action with a view to utilizing it for neurodegenerative
 D.B. McPhail, P.T. Gardner, G.G. Duthie and G.M. Steele, in
International Society for Free Radical Research - VIII Biennial Meeting, Barcelona, Spain, 5.163, p. 125-1 (1996).
 W.A. Pryor, J.A. Cornicelli, L.J. Devall, B. Tait, B.K. Trivedi,
D.T. Witiak and M. Wu, J. Org. Chem. 58, 3521 (1993).
 C. Liegeois, G. Lermusieau and S. Collin J. Agric. Food. Chem. 48, 1129 (2000).
Irwin Cheah,a Jim Kelly,b Steven J. Langforda and Gordon Troupca School of Chemistry, Monash University, Victoria 3800, bAngoves
Pty Ltd, and cSchool of Physics and Material Science, Monash University,