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and various
Triticum
species other than
T. aestivum
, whereas low levels (40-100 mg/g)
are found in barley (
Hordeum vulgare
). In
cereals, AR are located in the outer layers of
the kernel (Tluscik, 1978) and more than
99% of the AR content is present in the
outer cuticle of testa/inner cuticle of peri-
carp (Fig. 10.4b) (Landberg
et al.
, 2008).
This situation makes it possible to use ARs
as biomarkers of cereal intake. In the past
decade 5-n-ARs have been found in seed-
lings and plants of rice, sorghum and rye,
reinforcing the hypothetical phytoanticipin
function of these molecules. Interestingly,
ARs were not found in grains of rice and
sorghum, and further research may provide
more evidence of the presence of ARs in
seedlings and later phenological stages of
cereal plants.
Several 5-n-ARs have been identified
in strains of soil bacteria from
Azotobacter
and
Pseudomonas
families as well as in
Streptomyces
(Tsuge
et al.,
1992),
Arthrobacter
and
Micrococcus
genus. The ARs occur in
both vegetative and cyst forms (Kozubek
et al
., 1996). An interesting feature of micro-
bial 5-n-ARs is that the alkyl side chains are
always saturated. Moreover, resorcinolic
lipids were also found in fungal species
from
Basidiomycetes
(Gianetti
et al
., 1978)
and
Hyphomycetes
(Stodola
et al
., 1973), as
well as in
Fusarium culmorum
(Zarnowski
et al
., 2000a).
Extractions with solvents more polar than
acetone, such as methanol or ethanol,
should be avoided because they co-extract
other compounds that may interfere with
later colorimetric and chromatographic
analysis. Selectivity is in part achieved
when whole grains are extracted, because
ARs are only accumulated in the outer
layers of the kernels. In our laboratory,
extractions with acetone followed by
flash micro-filtration on silica gel using
hexane:chloroform:ethyl acetate (2:1:1,
v/v/v) as mobile phase allow us to elimi-
nate higher polar co-extracted metabolites,
increasing the selectivity of the extracting
method.
Extracting methods developed for ARs
in cereal grains often have a low per-
formance when applied to food products,
where ARs sometimes may form inclusion
complexes with other components, such
as the starch in the bread, as occurs with
other polar lipids (Ross
et al
., 2004a).
In such a situation ARs require more
drastic extracting conditions. For example,
complete recovery of AR from bread only
was achieved using hot propanol:water
(3:1, v/v), a method previously used for total
lipid extraction from starch (Ross
et al
.,
2003b). This extraction method, however,
is time consuming. Sometimes, the main
purpose of AR analysis is to know the homo-
logue composition more than the total
content. In such cases, extracts from food
products (i.e. pasta samples) obtained after
continous stirring in ethyl acetate had the
same composition of ARs as those origi-
nated from hot propanol:water extraction
(Knödler
et al
., 2009).
10.4
Extraction of ARs
As previously mentioned, ARs are insoluble
in water, but are soluble in more hydropho-
bic solvents such as methanol, ethanol, ace-
tone, ethyl acetate, diethyl ether, chloroform,
cyclohexane and
n
-hexane. ARs are often
extracted from cereal grains. Extraction is
performed at room temperature from 1 g of
cereal grains with 40 ml of acetone or ethyl
acetate for 16-24 h, or from 25 g of grains
extracted three times with 25 ml of acetone
for 24 h each, and then filtered to remove
solid particles (Kozubek and Tyman, 1999).
Soxleth extractions for 2 h with acetone or
cyclohexane were indicated as efficient as
the last one (Zarnowski and Suzuki, 2004).
10.5 Rapid and Easy Methods
for Detection/Preliminary
Analysis of ARs
Several reasons have been presented on the
need for efficient, fast and sensitive meth-
ods to analyse total content of ARs in cereal
grains, including the use of ARs as biomar-
kers of whole grain or measure of ARs
in breeding programmes of cereal crops.
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