Biochemical and Molecular Actions of Nutrients
Flavonoids from Almond Skins Are Bioavailable and Act Synergistically
with Vitamins C and E to Enhance Hamster and Human LDL
Resistance to Oxidation1,2
Chung-Yen Chen, Paul E. Milbury, Karen Lapsley,* and Jeffrey B. Blumberg3
Antioxidants Research Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research
Center on Aging, Tufts University and *The Almond Board of California, Modesto, CA
disease. Flavonoids, found predominantly in the skin of almonds, may contribute to their putative health benefit, but
their bioactivity and bioavailability have not previously been studied. Almond skin flavonoids (ASF) were extracted
with HCl:H2O:methanol (1:19:80) and their content of catechins and flavonols identified by HPLC with electro-
chemical detection. ASF bioactivity was assessed in vitro by their capacity to increase the resistance of human LDL
to oxidation induced by 10 ?mol/L Cu2?. ASF from 0.18 to 1.44 ?mol gallic acid equivalent (GAE)/L increased the
lag time to LDL oxidation in a dose-dependent manner (P ? 0.0001). Combining ASF with vitamin E or ascorbic
acid extended the lag time ?200% of the expected additive value (P ? 0.05). The bioavailability and in vivo
antioxidant activity of 40 ?mol ASF were examined in BioF1B hamsters. Peak plasma concentrations of catechin,
epicatechin, and flavonols (quercetin, kaempferol, and isorhamnetin) occurred at 60, 120, and 180 min, respec-
tively. The concentration of isorhamnetin was significantly elevated in liver at 180 min. Absorbed ASF enhanced the
ex vivo resistance of hamster LDL collected at 60 min to oxidation by 18.0% (P ? 0.028), and the in vitro addition
of 5.5 ?mol/L vitamin E synergistically extended the lag time of the 60-min sample by 52.5% (P ? 0.05). Thus, ASF
possess antioxidant capacity in vitro; they are bioavailable and act in synergy with vitamins C and E to protect LDL
against oxidation in hamsters. J. Nutr. 135: 1366–1373, 2005.
Consumption of tree nuts such as almonds has been associated with a reduced risk of coronary heart
almonds ● antioxidants ● bioavailability ● flavonoids ● synergy
Flavonoids are natural constituents of plant foods and have
been carefully studied in fruits and vegetables, but less atten-
tion has been paid to their presence in whole grains and tree
nuts (1,2). Flavonoids appear to possess a variety of biological
activities, including antioxidant, anti-inflammatory, and vaso-
dilatory actions (3). Putative health benefits of flavonoids were
suggested by epidemiologic studies showing an inverse associ-
ation between intake and risk of cardiovascular disease (4,5).
A similar relation was observed with tree nuts, leading to the
recent approval by the FDA of a qualified health claim for tree
nuts, including almonds, that eating 42.6 g/d (1.5 oz/d) as part
of a diet low in saturated fat and cholesterol may reduce the
risk of heart disease (6). It is worth noting here the randomized
clinical trial conducted by Jenkins et al. (7) demonstrating
that almond consumption can reduce total and LDL choles-
terol and increase the resistance of LDL to oxidation.
Almonds (Prunus amygdalus Batsch) rank highest among
tree nuts, such as hazelnut, pecan, pistachio, and walnut, in
total annual crop production (8), and are a good source of
nutrients associated with heart health such as vitamin E,
monounsaturated fatty acids, PUFA, arginine, and potassium
(9). Almond skins, removed from the nut by hot water blanch-
ing during preparation of almond meat, constitute ?4% of the
total almond weight and are generally treated as a waste
product. However, an array of flavonoids, including catechins,
flavonols, and flavanones in their aglycone and glycoside
forms, were identified in almond skin (10,11). These com-
pounds may contribute to the health benefits associated with
almond consumption. The bioavailability and pharmacokinet-
ics of individual flavonoids were demonstrated in several stud-
ies (12–14). Catechins and flavonols from food were also
found to be bioavailable in rodent and human studies with the
food, although its matrix and preparation may have a signifi-
cant effect on their bioavailabilities (15–20). However, no
studies have explored the bioavailability of flavonoids from
almond skins and their effect on antioxidant activity. There-
fore, we examined almond skin flavonoids (ASF)4for their in
vitro action with and without vitamins C and E on the resistance
1Presented in part at Experimental Biology 03, April 2003, San Diego, CA
[Chen, C.-Y., Shen, J., Li, T., Milbury, P. & Blumberg, J.
and antioxidant activity of polyphenolics from almond skins in hamsters. FASEB
J. 17: A377 (abs.)].
2Supported by the USDA Agricultural Research Service under Cooperative
Agreement No. 58-1950-4-401 and the Almond Board of California. The contents
of this publication do not necessarily reflect the views or policies of the USDA nor
does mention of trade names, commercial products, or organizations imply
endorsement by the U.S. government.
3To whom correspondence should be addressed.
4Abbreviations used: Cmax, maximal concentration; ECD, electrochemical
detection; GAE, gallic acid equivalents, Tmax, time to maximal concentration.
0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences.
Manuscript received 13 January 2005. Initial review completed 27 February 2005. Revision accepted 23 March 2005.
of human LDL to oxidation as well as their bioavailability,
pharmacokinetics, and in vivo antioxidant actions in hamsters.
METHODS AND MATERIALS
Chemicals and reagents. The following reagents were obtained
from Sigma Chemical: copper sulfate, vitamin E (?-tocopherol),
sodium chloride, quercetin, catechin, (-)-epicatechin, kaempferol,
isorhamnetin, sodium phosphate monobasic, sodium phosphate diba-
sic, BHT, Folin Ciocalteu phenol reagent, and ?-glucuronidase type
H-2 (containing sulfatase from Helix pomatia). All organic solvents,
glacial acetic acid, food-grade ascorbic acid, and potassium bromide
were purchased from Fisher.
Flavonoid profile of almond skin. Pulverized almond skin powder
provided by the Almond Board of California was used for ASF
extraction. The powder (0.5 g) was extracted twice with 10 mL
acidified methanol solution (HCl:H2O:methanol, 1:19:80) over 16 h
at 4°C. The sample was centrifuged at 1000 ? g for 15 min at 4°C and
methanol evaporated with nitrogen gas. The residue was reconsti-
tuted with the aqueous mobile phase and the ASF characterized by
HPLC with electrochemical detection (ECD) according to a slightly
modified method of Milbury (21). The residue was also reconstituted
with phosphate buffer (7.79 mmol/L Na2HPO4, 2.59 mmol/L
NaH2PO4, and 150 mmol/L NaCl, pH 7.4) for the in vitro LDL
oxidation assay. All HPLC procedures were performed with ESA
instruments, including 2 pumps (model 582), autosampler (model
542), and Coularray 5600 A detector. Analyte separation was
achieved using a Zorbax ODS C18 column (4.6 ? 150 mm, 3.5 ?m)
with a 0.6 mL/min flow rate and mobile phase gradient from mobile
phase A (75 mmol/L citric acid and 25 mmol/L ammonium acetate in
90% H2O and 10% acetonitrile) to mobile phase B (75 mmol/L citric
acid and 25 mmol/L ammonium acetate in 50% H2O and 50%
acetonitrile) for 68 min. The following mobile phase gradient profile
was used (% solvent B): 1% (0–15 min), 1–10% (15–25 min),
10–80% (25–60 min), 80–10% (60–65 min), and 10–1% (65–68
min). Detection was achieved with potentials applied from 60 to 720
mV with 60-mV increments. The identification of individual ASF
was achieved by comparing its retention time and electrochemical
response with purified standards obtained from Sigma Chemical. The
quantity of individual almond flavonoids was calculated according to
concentration curves constructed with purified standards. Total al-
mond skin phenolics were assessed via the Folin-Ciocalteu reaction
according to Singleton et al. (22) and expressed as ?mol/L gallic acid
Antioxidant capacity of ASF in human LDL. The antioxidant
capacity of ASF was assessed in vitro with human LDL according to
a slight modification of the method described by Esterbauer et al.
(23). Collection of venous blood, approved by the Institutional
Review Board of the Tufts-New England Medical Center, was per-
formed between 1400 and 1430 h in 6 healthy Caucasian women who
were not fasting; the women were 28–64 y old with a mean body
weight of 63 ? 15 kg. LDL was separated from plasma according to
Chung et al. (24) using a Beckman NVT-90 rotor in a Beckman
L8-mol/L centrifuge (329,271 ? g for 90 min). KBr and EDTA were
removed from the sample using a PD-10 column (Amersham Phar-
macia Biotech). LDL protein was determined using a bicinchoninic
acid protein assay kit (Pierce). LDL samples from the first 3 subjects
were used to assess the dose-response relation of ASF, and from the
last 3 subjects for experiments on the interaction between ASF with
vitamins C and E. All LDL experiments were performed in duplicate
in each of 3 subjects. LDL (182 nmol/L) were oxidized by 10 ?mol/L
CuSO4in a total volume of 1.0 mL phosphate buffer (7.79 mmol/L
Na2HPO4, 2.59 mmol/L NaH2PO4, and 150 mmol/L NaCl, pH 7.4).
Formation of conjugated dienes was monitored by absorbance at 234
nm at 37°C over 6 h using a Shimadzu UV1601 spectrophotometer
(Japan) equipped with a 6-position automated sample changer. The
results of the LDL oxidation are expressed as lag time (defined as the
intercept at the abscissa in the diene-time plot) (25). An aliquot of
ASF in acidified methanol was dried under nitrogen and redissolved
in an equal volume of phosphate buffer (pH 7.4) for testing in the
assay. Concentrations ofASF from 0.18 to 1.44 ?mol/L were selected
to reflect amounts that can be obtained from dietary intakes (26).
Concentrations of vitamin E and C employed in these experiments
were 9–18% and 3–6% of plasma concentrations, respectively, levels
generally found in healthy people (27). Vitamin E (?-tocopherol)
was dissolved in methanol and subsequently diluted with phosphate
buffer to obtain the selected concentrations; the final concentration
of methanol was 0.5%. ASF and vitamin E were incubated with LDL
at 37°C for 30 min before initiation of oxidation, and ascorbic acid
was dissolved in PBS and added to the reaction immediately before
initiation of oxidation. The effect of ASF plus vitamin C or E on the
resistance of LDL to oxidation was expressed as the lag time increase
compared with the lag time of LDL without the addition of ASF or
vitamin C or E.
Preparation of ASF for hamster gavage.
with acidic aqueous methanol as described above with the substitu-
tion of white vinegar for HCl. Methanol was removed using a
Rotavapor 134 (Buchi, Brinkmann Instrument) and the resulting
slurry used for the hamster gavage.
Animals. BioF1B strain Golden Syrian Hamsters (n ? 22; Bio-
Breeders) were used in the bioavailability experiments due to the
similarity of their lipoprotein metabolism to that of humans (28). The
1-y-old hamsters, with a body weight of 136.0 ? 1.2 g (mean ? SE),
were housed individually in cages with a 10:14 h light:dark cycle. To
increase lipoprotein production for subsequent collection, hamsters
consumed ad libitum a nonpurified diet (Harlan) enriched with 10 g
coconut oil and 0.5 g cholesterol/100 g for 2 wk before the acute
administration of the ASF slurry (29).
After overnight food deprivation, hamsters were randomly as-
signed on the basis of their body weight to 6 time point groups: 0
(baseline), 60, 90, 120, 180, and 300 min (n ? 4, 4, 4, 4, 4, and 2,
respectively). Because Ku ¨hnau (30) estimated daily intake of fla-
vonoids and other polyphenolics by a person weighing 70 kg at 14
mg/kg, we chose a test dose of 50 mg/kg body weight or 40 ?mol
GAE/hamster because rodents consume 5–6 times more food-based
energy per unit of body weight than humans (31). Thus, ASF with 40
?mol GAE (6.8 mg) was delivered in 1.0 mL via stomach gavage to
hamsters anesthetized with Aerrane™ (Baxter); 1.0 mL saline was
administered to the baseline group. Blood from each hamster was
collected from the orbital sinus via micro-hematocrit tubes coated
with heparin and stored in microtubes containing ETDA. Plasma
samples were collected after the whole blood was centrifuged at 1000
? g for 15 min at 4°C. An aliquot of plasma was stored at ?80°C for
determination of flavonoid status with the remainder used immedi-
ately for analysis of LDL oxidation. Whole liver was removed, rinsed
with saline, and snap-frozen in liquid nitrogen for additional fla-
vonoid analysis. This study was approved by the Animal Care and
Use Committee of the Jean Mayer USDA Human Nutrition Re-
search Center on Aging at Tufts University.
Analysis of plasma and liver flavonoids. ASF in hamster plasma
and liver were assessed by the HPLC-ECD as described above. Briefly,
20 ?L vitamin C-EDTA (200 mg ascorbic acid plus 1 mg EDTA in
1.0 mL of 0.4 mol/L NaH2PO4,pH 3.6) and 20 ?L ?-glucuronidase
(98,000 kU/L ?-glucuronidase and 2400 kU/L sulfatase) were added
to 200 ?L plasma and the mixture was incubated at 37°C for 45 min.
ASF were extracted with 500 ?L acetonitrile; 500 ?L of supernatant
was removed following a vigorous vortex for 30 s and centrifugation
at 14,000 ? g for 5 min, dried under purified nitrogen, and reconsti-
tuted in 200 ?L of the aqueous HPLC mobile phase. After centrifu-
gation at 14,000 ? g for 5 min, 100 ?L of supernatant was injected
into the HPLC.
Liver samples (0.5 g) frozen in liquid nitrogen were pulverized and
extracted twice with 5 mL acetonitrile containing 0.01% BHT. The
supernatant was removed after centrifugation at 14,000 ? g for 5 min,
combined, dried under nitrogen, and reconstituted in 500 ?L of 1
mol/L sodium acetate buffer, pH 5.5. After incubation with 10 ?L
?-glucuronidase (98,000 kU/L ?-glucuronidase and 2400 kU/L sulfa-
tase) at 37°C overnight, the mixture was processed in the same
manner as plasma samples. Due to a lower volume of ?-glucuronidase
and different buffer system, deconjugation of flavonoid metabolites
took longer in liver than in plasma samples. Spiked, purified stan-
ASF were extracted
ALMOND SKIN FLAVONOID BIOAVAILABILITY
dards, rather than an internal standard, were added at the beginning
of sample processing to construct standard curves and account for
extraction losses and quantify concentrations (2). The recovery rates
of spiked standards ranged from 75 to 80%.
Ex vivo antioxidant capacity of ASF. The ex vivo resistance of
hamster LDL to Cu2?-induced oxidation was performed as described
above for the in vitro experiments to test antioxidant activity of
flavonoids in ASF-gavaged hamsters. Because hamster LDL obtained
through ultracentrifugation was lower than human LDL (determined
by protein content), 91 nmol/L LDL was oxidized by 5 ?mol/L
CuSO4in a total volume of 1.0 mL phosphate buffer (pH 7.4). Based
on the synergistic results obtained with ASF plus vitamin E in the in
vitro experiments, vitamin E was added in vitro to hamster LDL to a
final concentration of 5.5 ?mol/L before initiation of oxidation.
Statistics. All results are reported as means ? SE. The Tukey-
Kramer honestly significant difference test was used in experiments
on ASF in hamster plasma, and in vitro hamster and human LDL
oxidation after significant differences were obtained by one-way
ANOVA. The equal variance assumption was assessed by Hartley’s
test (32) and data, including epicatechin and isorhamnetin in plasma
and catechin, epicatechin, quercetin, and kaempferol in liver, were
square root-transformed before ANOVA analysis (2). The difference
in lag time of ex vivo hamster LDL oxidation between baseline and
ASF administration was determined by a simple t test. A paired t test
was performed to determine the significance of the synergy between
ASF and vitamins C or E in the in vitro human LDL oxidation by
comparing the observed lag time during co-incubation with the
expected (calculated) sums of values observed for ASF and vitamin C
or E treatment alone. Data from all time points was included in
correlation tests. Differences with P ? 0.05 were considered signifi-
cant. The JMP IN 4 statistical software package (SAS Institute) was
used to perform all statistical analyses.
The total phenolic content in the almond skins as deter-
mined by the Folin-Ciocalteu method was 8.2 ?mol GAE/g.
HPLC-ECD chromatography resolved ?30 peaks with detect-
able redox potential, indicating the presence of numerous
potential antioxidant compounds in almond skin (Fig. 1). Five
aglycone flavonoids were identified and quantified as follows
nanoamp (nA). Each trace reflects the electrochemical response of a
specific applied potential (as mV) in the ECD. Labeled peaks are: (1)
catechin, (2) epicatechin, (3) quercetin, (4) kaempferol, (5) isorhamnetin.
HPLC-ECD profile of ASF. The unit of the ordinate is
human LDL in vitro. Lag time of control (no added ASF) ? 49.3 ? 3.7
min. Values are means ? SE, n ? 3. Means without a common letter
differ, P ? 0.0001.
Effect of ASF on lag time to Cu2?-induced oxidation of
VE) on the lag time of human LDL oxidation in vitro. LDL (182 ?mol/L)
was oxidized by 10 ?mol/L Cu2?with the addition of ASF, VC or VE, or
ASF ? VC or VE. Values are means ? SE, n ? 3. Lag time of control (no
added ASF, VC, or VE) ? (A) 45.5 ? 0.7 min and (B) 47.7 ? 1.5 min.
Bars are stacked to illustrate the calculated additive effect of the 2
treatments. The percentage value above the solid bar indicates the
observed synergy greater than the calculated sums of the individual
treatments. Symbols indicate different from the calculated sums: *P
? 0.05,†P ? 0.01,‡P ? 0.005.
The synergistic effect of ASF and vitamin C or E (VC or
CHEN ET AL.
(nmol/g): catechin (186.9), epicatechin (77.5), isorhamnetin
(20.8), quercetin (6.0), and kaempferol (3.9). These 5 fla-
vonoids represent 3.6% of the total phenolic content of al-
In vitro, ASF increased the resistance of human LDL
against Cu2?-induced oxidation in a dose-dependent manner
(P ? 0.0001) (Fig. 2). Ascorbic acid at 2.5 and 5.0 ?mol/L
increased lag time by 12.9 ? 1.6 and 34 ? 2.7 min, respec-
tively; vitamin E at 2.75 and 5.5 ?mol/L increased lag time by
19.1 ? 1.4 and 24.3 ? 0.7 min, respectively. A 1-fold synergy
(i.e., an observed value twice the calculated additive value)
was observed with 0.18 and 0.36 ?mol/L of ASF in combina-
tion with 5.0 ?mol/L ascorbic acid (P ? 0.01); no synergy was
observed with the 2.5 ?mol/L dose of vitamin C (Fig. 3A).
The lag time was 289% longer than the expected additive
value with the combination of 0.36 ?mol/L ASF and 5.5
?mol/L vitamin E (P ? 0.001) (Fig. 3B).
All 5 identified flavonoids from almond skins were bioavail-
able in hamster plasma (Fig. 4). Based on their pharmacoki-
netic profile in the plasma, the maximum concentrations
(Cmax) of catechin, epicatechin, kaempferol, quercetin, and
isorhamnetin were 376, 133, 72, 222, and 761 nmol/L, respec-
tively (Fig. 5). The time to reach the Cmax(Tmax) was 60 and
120 min for catechin and epicatechin, respectively, and 180
ples obtained at 60 min (A) and 180 min (B) after administration of 40
?mol GAE ASF and immediately after gavage with saline (baseline). (A)
210 mV ECD trace. (B) 70 mV ECD trace. Labeled peaks are: (1)
catechin, (2) epicatechin, (3) quercetin, (4) kaempferol, (5) isorhamnetin.
HPLC-ECD chromatographs of hamster plasma sam-
kaempferol, and isorhamnetin in the plasma of hamsters administered
40 ?mol GAE ASF. Values are means ? SE, n ? 4. Means in each panel
without a common letter differ, P ? 0.05.
Time course of catechin, epicatechin, quercetin,
ALMOND SKIN FLAVONOID BIOAVAILABILITY
min for the 3 flavonols. At 300 min, the plasma concentra-
tions of these flavonoids were less than half the Cmaxand their
concentrations (with the exception of quercetin and isorham-
netin) did not differ from the baseline value. Correlation
coefficients between the 3 plasma flavonols ranged from 0.66
to 0.78 although no relation was noted between the catechins
Pharmacokinetic patterns of the flavonoids in liver, except
for isorhamnetin, differed from those in the plasma (Fig. 6).
Concentrations of catechin, quercetin, and kaempferol in liver
were not affected by acute ASF gavage. The epicatechin
concentration was significantly elevated at 300 min. The Cmax
of liver quercetin and kaempferol at 73 and 130 nmol/g did not
differ from baseline and their Tmaxwas 90 min, in contrast to
the 180 min observed in plasma. The Cmaxof isorhamnetin in
the liver was 698 nmol/g at 180 min after administration;
isorhamnetin remained higher than baseline levels after 300
min (P ? 0.05). Concentrations of quercetin and kaempferol
were correlated (Table 1). Isorhamnetin pharmacokinetics
paralleled those of kaempferol, but not quercetin. Isorhamne-
tin showed a similar pharmacokinetic pattern in both plasma
The lag time of ex vivo hamster Cu2?-induced LDL oxi-
dation was 30.8 ? 0.8, 36.3 ? 1.8, 34.5 ? 1.4, 38.1 ? 1.7, and
38.9 ? 3.9 min at the baseline, 60, 90, 120, and 180 min time
points, respectively. Absorbed ASF appeared to induce a small
increase of 18.0 and 24.0% in the ex vivo resistance of LDL to
oxidation at 60 and 120 min (P ? 0.028 and 0.008, respec-
tively). When 5.5 ?mol/L vitamin E was added in vitro to the
reaction, the lag time of LDL oxidation was 119.9 ? 2.4, 182.9
? 13.5, 152.3 ? 17.0, 160.1 ? 8.0, and 160.0 ? 6.7 min,
respectively. However, only LDL collected at 60 min showed
a synergistic increase with a 52.5% longer lag time than that
collected at baseline (P ? 0.05).
The putative health benefits of flavonoids have been attrib-
uted in part to their antioxidant activity (33,34). However,
most of the supportive evidence has been based only on in
vitro experiments or in vivo feeding studies with a single
flavonoid aglycone or glycoside (26). In contrast, we examined
the behavior of a complex array of polyphenolics in almond
skins while measuring several specific flavonoid constituents.
Almond skins were previously reported to contain 3 classes of
flavonoids, i.e., catechins, flavanones, and flavonols (10,11).
Using HPLC-ECD, we quantified 5 aglycones (catechin, epi-
catechin, quercetin, kaempferol, and isorhamnetin) and mea-
sured ?25 other redox compounds (Fig. 1), likely flavonoids or
related polyphenolics, such as glycosides of quercetin, kaemp-
ferol, isorhamnetin, and naringenin as well as protocatechuic
acid, vanillic acid, and p-hydroxybenzoic acid (10,11).
The complex of ASF effectively increased the resistance of
human LDL to oxidation in vitro in a dose-dependent fashion
within physiologically relevant concentrations (Fig. 2) (26).
These results are consistent with several reports of the anti-
oxidant activity of flavonoids such as catechin and quercetin
with concentrations administered ranging from 0.25 to 10
?mol/L (35–37). In contrast, Filipe et al. (38) reported a
prooxidant activity of low concentrations of quercetin (?2
?mol/L) indicated by increased malondialdehyde formation
during Cu2?-induced LDL oxidation. If a prooxidant action of
quercetin occurred in our study, it may have been masked by
other constituents of almond skin. It is possible that the
potential prooxidant actions of single flavonoids do not occur
in natural mixtures of plant polyphenols in which opportuni-
ties for recycling oxidized compounds may exist, such as the
regeneration of oxidized malvidin 3-glucoside by catechin
(39). Such interactions (14,40,41) may also partly account for
the synergy between the ASF and vitamins C and E observed
here (Fig. 3). Similar synergies were observed between
genistein and ascorbic acid (40), oat phenolics and ascorbic
acid (2), and quercetin and urate (42). In addition to recycling
mechanisms, Hwang et al. (40) suggested that polyphenolics
may stabilize the LDL particle structure via an interaction with
the apoprotein-B domain. Further, vitamin C may contribute
to the synergy by inhibiting the decomposition of lipid perox-
ides and/or preventing Cu2?from binding to LDL (40).
Extrapolations about flavonoids from in vitro results are
limited because of their relatively poor general bioavailability
and extensive biotransformation in vivo (14). The bioavail-
abilities of selected single catechin and flavonol compounds
were reported (14,16,19,43,44), but little information is avail-
able regarding the concurrent absorption profile of mixtures.
Similar to our finding in hamsters, others found a faster plasma
Tmaxfor catechin than quercetin when single compounds were
fed to rats (16,44), suggesting that catechin is rapidly absorbed
Correlation coefficients among flavonoids in the plasma and liver of hamsters administered 40 ?mol GAE ASF1
Plasma and liver
1Based on one observation in 22 hamsters.
CHEN ET AL.
in the upper gastrointestinal tract and cleared from plasma
within several hours. In contrast to a Tmaxof 120 min for
epicatechin in hamsters (Fig. 5), Baba et al. (44) reported a
60-min value in rats. However, caution is warranted when
interpreting these values due to pharmacokinetic differences
between species as well to other confounding variables such as
differing ingredients in the diet fed and potential competition
between polyphenolics for absorption (44). In addition, gly-
coside moieties appear to have a substantial influence on
pharmacokinetics, e.g., quercetin-4?-glycoside and rutin (quer-
cetin rutinoside) have Tmaxof 0.5 and 7 h, respectively, in
humans (26). We observed a Tmaxat 180 min for the 3 almond
skin flavonol aglycones after deconjugation by glucuronidase
and sulfatase in hamsters (Fig. 5), suggesting the potential
contribution to this value from unidentified flavonol glycosides
in almond skins, such as quercetin, isorhamnetin, and
kaempferol glycosides (10,11). Similar to the relations sug-
gested by Manach et al. (13) for humans, we found a shorter
half-life in plasma for the catechins compared with the fla-
vonols in hamsters. The half-life of flavonols in the hamsters
(5 h) was shorter than that in rats (11–28 h), although such
differences could be accounted for by differences in their
The plasma pharmacokinetics of the almond flavonols were
similar with high correlation coefficients (Table 1). Although
isorhamnetin is a metabolite of quercetin (45), the association
between their plasma concentrations was low, possibly due to
direct contribution of isorhamnetin from almond skins. No
association was noted between the plasma concentrations of
the 2 catechins.
Although the number of reports is limited, flavonoids were
detected in mouse and rat tissues with concentrations from 30
to 3000 ng aglycone equivalents/g (13). In hamsters, after ASF
gavage, the 2 catechins and 3 flavonols were detected in liver
although, with the exception of isorhamnetin, their pharma-
cokinetic patterns were quite different from those in plasma
(Fig. 6). Noticeable amounts of catechin, quercetin, and
kaempferol in hamster liver may be attributed to their pres-
ence in the basal nonpurified diet fed to hamsters. It is not
clear why ASF administration did not increase catechin, epi-
catechin, kaempferol, or quercetin in the liver, although it is
possible that these flavonoids were quickly redistributed to
other tissues. Manach et al. (14) reported that ?90% of
catechin and quercetin in rat liver was methylated. Similar to
their relation in plasma (Table 1), quercetin and kaempferol
concentrations in the liver were correlated. Although iso-
rhamnetin status in plasma was correlated with its concentra-
tion in liver, it was not associated with other hepatic flavonols.
The relatively high level of isorhamnetin in liver may reflect
a slow rate of clearance compared with other flavonoids or a
gradual contribution from methylation of aglycone and gly-
cone quercetin. The increase in hepatic catechins, especially
epicatechin, at 300 min may reflect an unusually slow distri-
bution to this tissue or result from the small sample size
employed at this one time point.
Consistent with the antioxidant capacity of almond skins
using the LDL oxidation assay, Halvorsen et al. (46) demon-
strated the antioxidant potency of whole almonds in vitro with
the ferric reducing antioxidant power assay, although the
vitamin E content of the nut may have contributed substan-
tially to this result. In vivo, a very modest antioxidant effect of
ASF was suggested by the small increase in the resistance of
LDL in hamsters treated with the almond skin extract com-
pared with saline. Although a higher dose may have produced
a greater effect, the weak direct response elicited in vivo may
be a result of a lower potency of glucuronidated, sulfated,
and/or methylated metabolites of the ASF. Indeed, Moon et
isorhamnetin in the liver of hamsters administered 40 ?mol GAE ASF.
Values are means ? SE, n ? 4. Means in each panel without a common
letter differ, P ? 0.05.
Catechin, epicatechin, quercetin, kaempferol, and
ALMOND SKIN FLAVONOID BIOAVAILABILITY
al. (47) and Cren-Olive et al. (48) both reported that conju-
gated derivatives of quercetin and catechin provided less an-
tioxidant protection than their parent compounds against LDL
oxidation in vitro. Further, many flavonoid metabolites appear
bound to plasma proteins in vivo; thus, they may be less
available to interact with LDL (16,20). Nonetheless, despite a
weak effect of ASF on the resistance of LDL to oxidation ex
vivo, its antioxidant action was strengthened by their synergy
with the vitamin E added in vitro in this experiment. A similar
synergistic relation was observed between oat phenolics and
vitamin C in the same hamster model (2). Interestingly, the
ASF-vitamin E synergy appeared in LDL collected at 60 min,
the Tmaxfor catechin, rather than at 180 min, when the
greatest concentration of flavonols was obtained. It may be
attributed to the more potent antioxidant activity of catechin
against copper-induced LDL oxidation than quercetin (49).
In summary, ASF possess antioxidant capacity and interact
with vitamins E and C in a synergistic manner to protect LDL
against oxidation in vitro. Of the 5 identified flavonoids in the
ASF, all appeared in plasma and liver after oral administration.
These flavonoids and/or related compounds in the ASF
slightly enhanced the resistance of hamster LDL against ex
vivo Cu2?-induced oxidation, and their antioxidant capacity
was amplified with the in vitro addition of vitamin E. Because
almonds represent one of the richest dietary sources of vitamin
E, further research is warranted examining the relations be-
tween the natural ingredients in this whole food and their
potential association with health benefits, such as a reduction
in the risk of cardiovascular disease.
We thank Donald Smith for his contributions to establishing the
hamster model and Ting Li and Jennifer O’Leary for their excellent
technical assistance in the laboratory.
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