Urolithins, intestinal microbial metabolites of pomegranate ~ellagitannins, exhibit potent antioxidant activity in a cell-based assay

Article abstract of DOI:10.1021/jf9025794

Many health benefits of pomegranate products have been attributed to the potent antioxidant action of their tannin components, mainly punicalagins and ellagic acid. While moving through the intestines, ellagitannins are metabolized by gut bacteria into urolithins that readily enter systemic circulation. In this study, the antioxidant properties of seven urolithin derivatives were evaluated in a cell-based assay. This method is biologically more relevant because it reflects bioavailability of the test compound to the cells, and the antioxidant action Is determined in the cellular environment. Our results showed that the antioxidant activity of urolithins was correlated with the number of hydroxy groups as well as the lipophilicity of the molecule. The most potent antioxidants are urolithins C and D with IC₅₀ values of 0.16 and 0.33 μM, respectively, when compared to IC₅₀ values of 1.1 and 1.4 μM of the parent ellagic acid and punicalagins, respectively. The dihydroxylated urolithin A showed weaker antioxidant activity, with an IC ₅₀ value 13.6 μM, however, the potency was within the range of urolithin A plasma concentrations. Therefore, products of the intestinal microbial transformation of pomegranate ellagitannins may account for systemic antioxidant effects.

Full text of DOI:10.1021/jf9025794

J. Agric. Food Chem. 2009, 57, 10181–10186 10181
DOI:10.1021/jf9025794
Urolithins, Intestinal Microbial Metabolites of Pomegranate
Ellagitannins, Exhibit Potent Antioxidant Activity in a
Cell-Based Assay
‡
DOBROSLAWA
B
IALONSKA
,
†,§ SASHI G. KASIMSETTY,† SHABANA I. KHAN
,†,‡
,
AND
DANEEL
F
ERREIRA
*
†Department of Pharmacognosy, School of Pharmacy, ‡National Center for Natural Products Research,
Research Institute of Pharmaceutical Sciences, The University of Mississippi, Oxford, Mississippi 38655,
and §Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krakow,
Poland
Many health benefits of pomegranate products have been attributed to the potent antioxidant action
of their tannin components, mainly punicalagins and ellagic acid. While moving through the
intestines, ellagitannins are metabolized by gut bacteria into urolithins that readily enter systemic
circulation. In this study, the antioxidant properties of seven urolithin derivatives were evaluated in a
cell-based assay. This method is biologically more relevant because it reflects bioavailability of the
test compound to the cells, and the antioxidant action is determined in the cellular environment. Our
results showed that the antioxidant activity of urolithins was correlated with the number of hydroxy
groups as well as the lipophilicity of the molecule. The most potent antioxidants are urolithins C and
D with IC50 values of 0.16 and 0.33 μM, respectively, when compared to IC50 values of 1.1 and 1.4
μM of the parent ellagic acid and punicalagins, respectively. The dihydroxylated urolithin A showed
weaker antioxidant activity, with an IC50 value 13.6 μM, however, the potency was within the range
of urolithin A plasma concentrations. Therefore, products of the intestinal microbial transformation of
pomegranate ellagitannins may account for systemic antioxidant effects.
KEYWORDS: Punica granatum L.; punicalagins; ellagic acid; urolithins; antioxidant activity; gut
bacterial metabolites of polyphenols
INTRODUCTION
The contribution of human gut microbiota toward health
improvement and genesis of various diseases has been widely
recognized (9, 10). One important function of intestinal bacteria
is the fermentation of undigested food components leading to the
production of metabolites of different physiological significance.
The products of microbial modifications can be potentially harm-
ful, like in the case of metabolites produced by Bacteroides and
Clostridia sp. (11). Other metabolites formed by gut bacteria may
exhibit significant health beneficial actions, for example, the short
chain fatty acids released by probiotic lactic bacteria (9). Human
intestinal bacteria are able to metabolize dietary polyphenolic
flavonoids by cleavage of the C-ring, hydroxylation, dehydroxyla-
tion, reduction of carbon-carbon double bonds, and lengthening
or shortening of aliphatic chains (12). Therefore, by the formation
of different phenolic acids and other aromatic derivatives, gut
microbiota can modify the bioactivity of the original compounds.
For instance, the isoflavone daidzein is transformed into equol, a
bioavailable metabolite with potent estrogenic activity (13). In vitro
fermentation of punicalaganis and ellagic acid by human gut
bacteria resulted in the formation of a dibenzopyranone urolithin
A (Figure 2) (14). Urolithin A and related analogs (Figure 2) were
also confirmed as intestinal microbial metabolites of dietary
ellagitannins in animal studies (15,16). Common bacterial metabo-
lites of various ellagitannins indicate that ellagic acid, the product
Pomegranate products, including juice, tea, wine, and extracts,
are widely consumed and recognized for their health benefits,
including prevention of cancer and vascular diseases (1). In recent
years, various health advantages of pomegranate have been
attributed to the potent antioxidant activity of ellagitannins,
mainly punicalagins, punicalins (we prefer to use the plurals
punicalins and punicalagins, because these compounds exist in
solution as the R- and β-anomers as well as the acyclic hydro-
xyaldehyde analog), and ellagic acid (Figure 1) (2, 3). In fact,
increased antioxidant properties of plasma were found in human
volunteers after consumption of pomegranate products (4).
Although the punicalagins showed high antioxidant activity in
in vitro assays (5), they were not detected in human systemic
circulation after consumption of large amounts of pomegranate
products (4, 6), and only trace amounts of ellagic acid have been
found in human blood (7). Punicalagins undergo partial hydro-
lysis at the pH levels of the small intestine and under physiological
conditions of human cell lines (8). The remaining ellagitannins
and ellagic acid are retained unabsorbed in the gut lumen where
they can interact with complex intestinal bacteria.
*Corresponding author. Tel.: 001 662 915-7026. Fax: 001 662 915-
6975. E-mail: dferreir@olemiss.edu.
© 2009 American Chemical Society
Published on Web 10/13/2009
pubs.acs.org/JAFC

10182 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bialonska et al.
Figure 1. Chemical structures of pomegranate polyphenols.
volunteers after consumption of pomegranate products (4). How-
ever, previous investigations reported urolithins as insignificant
antioxidants compared to the original ellagitannins (15, 16).
In the present study, a cellular assay was applied for the first
time to comprehensively evaluate the antioxidant potency of
seven urolithins. This method reflects bioavailability of the test
compounds to the cell and employs cellular enzymes to reveal the
antioxidant action, hence better characterizing biological systems
in action. Six of the urolithins were earlier described as bacterial
metabolites of dietary ellagitannins (4, 6, 7, 16, 17). We also
included an O-methylated model derivative, 8,9-di-O-methylur-
olithin D for a comprehensive evaluation of the role of phenolic
hydroxy groups in the antioxidant activity.
MATERIALS AND METHODS
Chemicals. Resorcinol, 2-bromobenzoic acid, 2-bromo-4,5-dimethoxy-
benzoic acid, and chlorobenzene were purchased from Sigma Aldrich, St.
Louis, MO, 2-bromo-5-methoxybenzoic acid, from Alfa Aesar, WardHill,
MA, pyrogallol from Acros Organics, and copper sulfate, sodium hydro-
xide, and aluminum chloride from Fisher Scientific, Pittsburgh, PA. HL-
60 cells were from ATCC Manassas, VA. DCFH-DA was from Molecular
Probes, Eugene, OR.
Figure 2. Chemical structure of urolithins.
Purification of Compounds. The HPLC system consisted of a Waters
Delta 600, Waters 600 controller, Waters 996 Photodiode Array Detector,
and 10 ꢀ 250 mm column (Phenomenex, Prodigy ODS 10 μm C18). The
purification was performed in the gradient system A, 2.5% HOAc, B, 2.5%
HOAc in MeOH, starting from 100% A for 5 min, 0-60% B for 15 min,
and 60-100% B for 15 min. The flow rate was 1 mL/min and the pressure
600-800 mmHg. The elution of metabolites was monitored at 254 nm.
Identification of Compounds. The molecular mass of compounds
was confirmed using an LC-MS system consisting of a Waters Micromass
ZQ mass spectrometer, Waters 2695 Separation Module, and Waters
996 Photodiode Array Detector. Mass spectra were recorded in the
of hydrolysis of ellagitannins, may serve as substrate in the
formation of urolithins. A comprehensive investigation of the
production and bioavailability of urolithins was performed using
the Iberian Pig model (17).
Urolithins appear in human systemic circulation within a few
hours of consumption of pomegranate products, reaching max-
imium concentrations between 24 and 48 h. They are present in
the plasma and urine for up to 72 h, in free and conjugated
forms (1, 4, 14). Therefore, microbial metabolites could account
for the increased antioxidant properties of plasma in human

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10183
negative mode. The capillary voltage was 4000/3500 V, gas temperature
Table 1. Antioxidant Activity of Pomegranate Tannins and Microbial Meta-
bolitesa
300 °C, and a 3.0 ꢀ 150 mm analytical column (Phenomenex, Luna 5 μ
˚
C18 100 A) was used. The analyses were performed in the gradient system
antioxidant activity
(IC50 μg/mL)
cytotoxicity HL-60
(IC50 μg/mL)
A, 1% formic acid, B, 1% formic acid in MeOH, starting from 100% A for
5 min, 0-60% B for 15 min, and 60-100% B for 15 min. The flow rate was
0.3 mL/min and the pressure 900-1500 mmHg. The elution of metabolites
was monitored at 254 nm.
compound
urolithin A
urolithin B
3.1 (13.6 μM)
NA
>31.25
31.25
NC
Synthesis of Urolithins. The urolithins were sythesized by the
condensation of either resorcinol or pyrogallol with the appropriately
substituted benzoic acids, via modification of literature protocols (16,18).
The identity of urolithins was confirmed based on their molecular mass
and 1H NMR spectra.
urolithin C
urolithin D
0.04 (0.16 μM)
0.085 (0.33 μM)
NC
8-O-methylurolithin A
8,9-di-O-methylurolithin C
8,9-di-O-methylurolithin D
NA
NA
NA
>31.25
NA
NC
Urolithin B (3-Hydroxy-6H-dibenzo[b,d]pyran-6-one). 2-
Bromobenzoic acid (1 g), resorcinol (1.1 g), and NaOH (0.4 g) in water
(5 mL) were heated under reflux for 30 min. After the addition of aqueous
CuSO4 (5%, 2 mL) the mixture was refluxed again for 10 min, during
which time urolithin B (0.62 g) precipitated as a white powder. The
precipitate was filtered and subjected to HPLC for purification.
8-O-Methylurolithin A (3-Hydroxy-8-methoxy-6H-dibenzo-
[b,d]pyran-6-one). 2-Bromo-5-methoxybenzoic acid (1 g), resorcinol
(1 g), and NaOH (0.375 g) in water (4.5 mL) were heated under reflux for
30 min. After the addition of aqueous CuSO4 (5%, 1.8 mL) the mixture
was refluxed for a further 10 min, during which time 8-O-methylurolithin
A (0.72 g) precipitated as a white powder. The precipitate was filtered and
subjected to HPLC for purification.
punicalagins
punicalins
ellagic acid
gallic acid
1.6 (1.4 μM)
1.8 (2.3 μM)
0.33 (1.1 μM)
2.1 (3.2 μM)
NC
NC
NC
NC
vitamin C
doxorubicin
0.35 (1.9 μM)
NT
NT
0.25
a Inhibition of intracellular generation of ROS was determined in PMA-induced
cells. IC50 values were obtained from the dose response curves of % inhibition
versus test concentrations. NA: not active up to the highest test concentration of
31.25 μg/mL; NC: not cytotoxic up to the highest test concentration of 31.25 μg/mL;
NT: not tested; IC50: the concentration of compound that causes a 50% inhibition of
intracellular ROS generation.
Urolithin A (3,8-Dihydroxy-6H-dibenzo[b,d]pyran-6-one).
A solution of 2-bromo-5-methoxybenzoic acid (2.5 g) and AlCl3 (7.5 g) in
chlorobenzene (75 mL) was refluxed for 2.5 h. After cooling, the mixture
was added to ice and extracted with diethyl ether (3 ꢀ 125 mL). The ether
was evaporated to yield 2-bromo-5-hydroxybenzoic acid (2 g). The 2-bro-
mo-5-hydroxybenzoic acid (1.53 g), resorcinol (1.6 g), and NaOH (0.58 g)
in water (8 mL) were heated under reflux for 30 min. After the addition of
aqueous CuSO4 (5%, 3 mL) the mixture was refluxed for a further 10 min,
during which time urolithin A (1 g) precipitated as a white powder. The
precipitate was filtered and subjected to HPLC for purification.
8,9-Di-O-methylurolithin C (3-Hydroxy-7,8-dimethoxy-
6H-dibenzo[b,d]pyran-6-one). 2-Bromo-4,5-dimethoxybenzoic acid
(1.3 g), resorcinol (1.1 g), and NaOH (0.4 g) in water (5 mL) were heated
under reflux for 30 min. After the addition of aqueous CuSO4 (5%, 2 mL)
the mixture was heated again for 10 min, during which time 8,9-di-O-
methylurolithin C (0.82 g) precipitated as a white powder. The precipitate
was filtered and subjected to HPLC for purification.
12-myristate-13-acetate (PMA, 100 ng/mL) for 30 min. DCFA-DA (5 μg/
mL) was added and cells were further incubated for 15 min. Vitamin C (0.05
μg/mL-12.5 mg/mL) was used as positive control. Levels of fluorescent
DCF (produced by reactive oxygen species (ROS) catalyzed oxidation of
DCFH) were measured on a PolarStar with excitation wavelength of 485
nm and emission of 530 nm. DCFH-DA is a nonfluorescent probe that
diffuses into the cells, where cytoplasmic esterases hydrolyze it to the
nonfluorescent 20,70-dichlorodihydrofluorescein (DCFH). ROS generated
within HL-60 cells oxidize DCFH to the fluorescent dye 20,70-dichloro-
fluorescein (DCF). The ability of the test compounds to inhibit ROS
mediated oxidation of DCFH in PMA-treated HL-60 cells is measured in
comparison to the vehicle control. The cytotoxicity to HL-60 cells was also
determined after incubation of cells (2 ꢀ 104 cells/well in 225 μL) with test
samples for 48 h by the XTT method as described earlier (5, 19).
RESULTS AND DISCUSSION
Urolithin C (3,7,8-Trihydroxy-6H-dibenzo[b,d]pyran-6-one).
A
The cellular injury caused by oxidative stress and excess of free
radicals has been associated with aging and linked to over 200
clinical disorders, including cancer, heart disease, liver damage,
and other degenerative diseases related to inflammation (20). A
growing body of evidence shows that dietary antioxidants offer
effective protection from peroxidative damage caused by ROS
and other free radicals (21). Plant-derived polyphenols are known
for their strong antioxidant potency, acting as ROS scavengers,
peroxide decomposers, quenchers of singlet oxygen, electron
donor, labilehydrogendonor, and inhibitorsoflipoxygenase(22).
In the previous report, the cell-based assay revealed a potent
antioxidant activity of pomegranate extracts and individual
tannin components (punicalagins, punicalins, ellagic acid, and
gallic acid) (5). The antioxidant potency of the compounds has
been attributed to multiple phenolic hydroxy groups in the
hexahydroxydiphenyl (HHDP) and gallagyl moieties with poten-
tial to form o- or p-quinones. In the present study, antioxidant
properties of urolithins, the gut bacterial metabolites of pome-
granate ellagitanins, were evaluated in terms of inhibition of
intracellular generation of ROS. In contrast to earlier reports (15,
16), our results showed that urolithins exhibited a significant
antioxidant action correlated with the number of hydroxy groups
as well as lipophilicity of the molecules. The highest antioxidant
activity was detected for urolithin C, carrying hydroxy groups at
C-3, C-4, and C-8 (IC50 0.16 μM), and urolithin D, with one
additional hydroxy group at C-9 (IC50 0.33 μM; Table 1). The
solution of 8,9-O-dimethylurolithin C (0.563 g) and AlCl3 (1.69 g) in
chlorobenzene (16.9 mL) was refluxed for 2.5 h. After cooling, the mixture
was added to ice and extracted with diethyl ether (3ꢀ25 mL). The ether was
evaporated and subjected to chromatography over a 43ꢀ1.1 cm Sephadex
LH-20 column developed with EtOH f EtOH/MeOH (1:1) f MeOH, to
yield urolithin C (0.211 g) from eluates of EtOH and EtOH/MeOH.
8,9-Di-O-methylurolithin D (2,3-Dihydroxy-7,8-dimethoxy-
6H-dibenzo[b,d]pyran-6-one). 2-Bromo-4,5-dimethoxybenzoic acid
(1.3 g), pyrogallol (1.26 g), and NaOH (0.4 g) in water (5 mL) were heated
under reflux for 30 min. After the addition of aqueous CuSO4 (5%, 2 mL),
the mixture was heated again for 10 min, during which time 8,9-di-O-
methylurolithin D (0.9 g) precipitated as a yellow powder (0.656 g). The
precipitate was filtered and subjected to HPLC for purification.
Urolithin D (2,3,7,8-Tetrahydroxy-6H-dibenzo[b,d]pyran-
6-one). 8,9-Di-O-methylurolithin D (0.5 g) in a mixture of a glacial
HOAc (30 mL) and aqueous HBr (15 mL) was heated under reflux for 11
h. The starting material dissolved in 2 h and upon cooling the product
precipitated as darkbrown solid (0.295 g) that was filtered and subjected to
HPLC for purification.
Antioxidant Assay. Antioxidant activity was determined by the
DCFH-DA (20,70-dichlorodihydrofluorescein diacetate) method as de-
scribed earlier (5, 19). Myelomonocytic HL-60 cells (1 ꢀ 106 cells/mL,
ATTC; Manassas, VA) were suspended in RPMI-1640 medium containing
10% fetal bovine serum, penicillin (50 units/mL), and streptomycin
(50 units/mL). The cell suspension (125 μL) was added to wells of a 96-
well plate. After treatment with different concentrations (0.01-31.25 μg/
mL) of the test compounds for 30 min, cells were stimulated with phorbol

10184 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bialonska et al.
Figure 3. Proposed pathway of punicalagins transformation to urolithins by intestinal bacteria.
higher activity of the trihydroxydibenzopyranone, urolithin C,
over the tetrahydroxydibenzopyranone, urolithin D, can be
explained in terms of the superior lipophilicity, hence, increased
bioavailability of urolithin C (17). Both urolithins C and D had
higher antioxidant potency than ellagic acid and the punicalagins
with IC50 values of 1.1 and 1.4 μM, respectively (Table 1). They
also showed higher activity than vitamin C (Table 1). The
dihydroxydibenzopyranone, urolithin A, with hydroxy groups
at C-3 and C-8, exhibited less significant antioxidant activity
(IC50 13.6 μM). The monohydroxylated urolithin B, as well as
methylated compounds, 8,9-di-O-methylurolithin C, 8,9-di-O-
methylurolithin D, and 8-O-methylurolithin A, did not show
antioxidant activity (Table 1). The general decrease in the anti-
oxidant activity with the reduced number of phenolic hydroxy
groups, as well as the lack of activity of O-methyl urolithins,
reconfirms the importance of free hydroxy groups in antioxidant
mechanisms.
Previously, urolithin A was reported as a less potent antiox-
idant compared to the punicalagins, with a 42- and 3500-fold
lower activity in the DPPH (1,10-diphenyl-2-picrylhydrazyl) and
ABTS (2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as-
says (15). In our cell-based assay, urolithin A exhibited a 10-fold
lower activity than the punicalagins (Table 1). In another study,
the antioxidant properties of urolithins A and C were screened in
assays for SOD (super oxide dismutase)-like activity generated by
(1) the xanthine/XOD (xanthine oxidase system) and by (2) the
PMS (phenazine methyl sulfate)/NADH system. A weak anti-
oxidant activity was found for urolithin C, with an IC50 of 51 μM,
when compared to 1.3 μM of the precursor ellagitannin geraniin.
In addition, urolithin C showed a radical scavenging effect in the
DPPH assay with an IC50 of 1.9 μM, compared to 0.6 μM of
geraniin (16).
urolithins display considerable activity cognizant of the in-
creased plasma antioxidant properties demonstrated in human
volunteers after consumption of pomegranate products (4).
This also seems to be supported by the production and dis-
tribution of urolithins as described in the Iberian Pig experi-
ment (17). Based on data reported in this model (17) and on the
time course profile of in vitro ellagitannin transformations by
rat intestinal microbials (16), we propose the pathway in
Figure 3, to account for the formation of the urolithins in the
intestinal tract. The ingestion of ellagitannins involves abiotic
hydrolysis at the pH levels of the small intestines and sponta-
neous internal lactone formation to give ellagic acid, which
probably constitutes the substrate for the formation of the
urolithins in bacterial metabolism (15). Significant amounts of
urolithins D and C and trace amounts of urolithin A were
detected in the jejunum of Iberian pigs fed with an ellagitannin-
rich diet (17). Therefore, urolithin formation starts as early as in
the small intestines, and bacteria present in the small intestines
are able to metabolize ellagitannins to a number of degradation
metabolites (17). Because urolithin B was not detected in the
small intestines, it suggests that the bacterial metabolism of
ellagitannins continues in the colon and culminates with the
formation of urolithin B as the final product (17). The distribu-
tion of urolithins in the digestive tract also points to urolithin D
as the first product of microbial transformation of ellagic acid
and subsequent modifications lead to intermediates with a
decreasing number of phenolic hydroxy groups: urolithin
C (3,7,8-trihydroxydibenzopyranone), urolithin A (3,8-dihy-
droxydibenzopyranone), and finally urolithin B (3-hydroxy-
dibenzopyranone) (Figure 3). The alternative pathway, based
on in vitro transformation of the ellagitannin, geraniin by rat
colon bacteria, proposes a metabolite hydroxylated at posi-
tions C-3, C-8, C-9, and C-10, as alternative to urolithin D in
urolithin formation. Subsequent deoxygenations at positions C-
9 and C-10 lead to urolithin A (Figure 3). It should be noted that
metabolites M1-M4 as well as 8,9-di-O-methylurolithin C were
regarded as rat feasible intermediates but have not been de-
tected in human systemic circulation nor in tissues of the Iberian
pig (4, 6, 7, 16, 17).
The relatively weak antioxidant potency of the urolithins
reported in the earlier studies could be explained by the fact that
test methods utilized previously were based on physical and
enzymatic processes. In contrast, in the present assay, the
inhibition of intercellular generation of ROS was measured
and the antioxidant potential of test compounds was deter-
mined in the cellular environment. Our results showed that the

Article
Urolithins C and D, the first products of bacterial transforma-
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10185
human plasma and some persist in urine for up to 48 h. J. Nutr. 2006,
136, 2481–2485.
tions, are present in significant concentrations in the intestines.
The urolithins are present in plasma at trace concentrations
because of enteropathic circulation (17). However, they can still
provide health beneficial effects resulting form their extremely
high antioxidant potency (Table 1). Urolithin A is the major
metabolite of ellagitannins present in plasma, urine, tissues, and
digestive tract (4,6,17). Inthe present study, urolithin A exhibited
a relatively weak antioxidant activity compared to its precursors,
that is, punicalagins and ellagic acid (Table 1). Despite this, the
antioxidant IC50 value of 13 μM is still in the range of the plasma
concentrations of urolithin A, that is, 4-18 μM (23, 24). This
compound remains in systemic circulation for up to 72 h (14), and
although urolithin A is the major metabolite, urolithins C and D
with their much superior antioxidant capacity will, no doubt, also
contribute to the total antioxidant potency. Hence, the regular
consumption of pomegranate products should maintain an
efficient concentration of urolithins in the systemic circulation
to provide protection against oxidative stress. Urolithin B, the
final product of ellagitannin microbial transformation is present
in significant amounts in plasma and urine (14), but it did not
exhibit any antioxidant properties (Table 1). However, other
health protective properties, including anticarcinogenic and an-
ticancer activities, were demonstrated for this molecule (25). It is
also important that the urolithins did not exhibit toxicity against
mammalian cells at concentrations up to 31.25 μg/mL (Table 1).
In conclusion, urolithins, the bioavailable products of the
intestinal microbial transformation of pomegranate ellagitannins
may account for systemic antioxidant effects and protection
against oxidative stress. In addition, the original ellagitannins
as well as some of the urolithins retained in the gut may also
provide potential local gastrointestinal tract beneficial effects.
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ACKNOWLEDGMENT
We thank POM Wonderful, Los Angeles, for financial sup-
port. United States Department of Agriculture (USDA), agricul-
tural research service specific cooperative agreement, No. 58-
6408-2-0009, is also acknowledged for partial support of the
work. We also thank Katherine Martin for her excellent technical
support.
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Received for review July 24, 2009. Revised manuscript received October
1, 2009. Accepted October 5, 2009.