Preventive oral treatment with resveratrol pro-prodrugs drastically reduce colon inflammation in rodents

Article abstract of DOI:10.1021/jm1007006

There is no pharmaceutical or definitive surgical cure for inflammatory bowel diseases (IBDs). The naturally occurring polyphenol resveratrol exerts anti-inflammatory properties. However, its rapid metabolism diminishes its effectiveness in the colon. The design of prodrugs to targeting active molecules to the colon provides an opportunity for therapy of IBDs. Herein we explore the efficacy of different resveratrol prodrugs and pro-prodrugs to ameliorate colon inflammation in the murine dextran sulfate sodium (DSS) model. Mice fed with a very low dose (equivalent to 10 mg for a 70 kg-person) of either resveratrol-3-O-(6′-O-butanoyl)-β-d-glucopyranoside (6) or resveratrol-3-O-(6′-O-octanoyl)-β-d-glucopyranoside (7) did not develop colitis symptoms and improved 6-fold the disease activity index (DAI) compared to resveratrol. Our results indicate that these pro-prodrugs exerted a dual effect: (1) they prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon and (2) they reduced mucosal barrier imbalance and prevented diarrhea, which consequently facilitated the action of the delivered resveratrol in the colon mucosa.

Full text of DOI:10.1021/jm1007006

J. Med. Chem. 2010, 53, 7365–7376 7365
DOI: 10.1021/jm1007006
Preventive Oral Treatment with Resveratrol Pro-prodrugs Drastically Reduce Colon
Inflammation in Rodents
†
Mar Larrosa, Joao Tome-Carneiro, Marı
†
†
‡
†
†
ꢀ
ꢀ~ ꢀ ꢀ
ꢀ
a J. Yanez-Gascon, David Alcantara, Marıa V. Selma, David Beltran,
´
´
Marıa T. Garcı
´ ´
Juan Carlos Espı
a-Conesa, Cristina Urban, Ricardo Lucas, Francisco Tomas-Barberan, Juan C. Morales,*,‡ and
†
†
‡
†
ꢀ
ꢀ
ꢀ
´
n*,†
†Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC,
‡
30100 Campus de Espinardo, Murcia, Spain, and Department of Bioorganic Chemistry, Instituto de Investigaciones Quımicas (IIQ),
´
CSIC-Universidad de Sevilla, 41092 Sevilla, Spain
Received June 11, 2010
There is no pharmaceutical or definitive surgical cure for inflammatory bowel diseases (IBDs). The
naturally occurring polyphenol resveratrol exerts anti-inflammatory properties. However, its rapid
metabolism diminishes its effectiveness in the colon. The design of prodrugs to targeting active molecules
to the colon provides an opportunity for therapy of IBDs. Herein we explore the efficacy of different
resveratrol prodrugs and pro-prodrugs to ameliorate colon inflammation in the murine dextran sulfate
sodium (DSS) model. Mice fed with a very low dose (equivalent to 10 mg for a 70 kg-person) of either
resveratrol-3-O-(60-O-butanoyl)-β-D-glucopyranoside (6) or resveratrol-3-O-(60-O-octanoyl)-β-D-gluco-
pyranoside (7) did not develop colitis symptoms and improved 6-fold the disease activity index (DAI)
compared to resveratrol. Our results indicate that these pro-prodrugs exerted a dual effect: (1) they
prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon
and (2) they reduced mucosal barrier imbalance and prevented diarrhea, which consequently facilitated
the action of the delivered resveratrol in the colon mucosa.
Introduction
etiology and pathogenesis of IBDs are poorly understood, the
most acceptedtheoryincludesthe loss of balancebetween host
susceptibility, enteric microbiota, and mucosal immunity. This
results in an exacerbated immune system response with an
imbalance of pro-inflammatory cytokines, adhesion molecules,
and reactive oxygen metabolites provoking tissue injury.1
Treatments for IBDs are restricted to controlling symp-
toms, maintaining remission, and preventing relapse. Acute
treatment uses antibiotics and anti-inflammatory drugs
such as corticosteroids and aminosalicylates. Sulfasalazine
and 5-aminosalicylates are used in the management of mild
to moderate disease, whereas the glucocorticoids remain the
primary therapy for patients suffering from moderate to severe
disease. However, prolonged use of corticosteroids has signi-
ficant side effects preventing their use for long-term treatments.2
At the present time, there is no pharmaceutical or definitive
surgical cure for IBDs.2 Therefore, the search for new drug
treatments remains absolutely necessary.
A limitation to the use of orally administered drugs in the
treatment of IBDs is that they undergo absorption from
the upper parts of the gastrointestinal tract before reaching
the colon. This has two major drawbacks, the absorption,
conjugation, and further excretion of a high proportion of the
drug without reaching the colon as well as the potential non-
desirable systemic effects of some drugs. In this context, the
design of pro-drugs targeting active molecules to the colon
provides an opportunity to improve the therapeutic potential
of drugs intended for topical therapy of the intestinal mucosa.3
Polyphenols, plant secondary metabolites abundant in
our diet through the intake of fruits and vegetables, have been
The term “inflammatory bowel diseases” (IBDsa) groups a
wide set of clinical manifestations and presentations whose
main characteristic is the chronic inflammation of the gastro-
intestinal tract. The major IBD forms, Crohn’s disease and
ulcerative colitis, affect millions of individuals. Although the
*To whom correspondence should be addressed. For J.C.M.: phone,
þ34-954-489561; fax, þ34-954-460565; E-mail, jcmorales@iiq.csic.es.
For J.C.E.: phone, þ34-968-396344; fax, þ34-968-396213; E-mail,
jcespin@cebas.csic.es.
a Abbreviations: anh, anhydrous, AUC, area under the curve; BUT,
mice group fed with trans-resveratrol-3-O-(60-O-butanoyl)-β-D-gluco-
pyranoside (6); t-BuOH, tert-butanol, Cmax, maximum concentration;
COX-2, cyclooxygenase-2; DAI, disease activity index; DCM, dichlor-
omethane; DIGLUC, mice group fed with trans-resveratrol-3,40-di-β-D-
glucopyranoside (4); DMF, dimethylformamide; DSS, dextran sodium
sulfate; GM-CSF, granulocyte macrophage colony-stimulating factor;
HED, human equivalent dose; HRMS, high resolution mass spectro-
metry; HSP70, 70 kDa heat shock protein; IBDs, inflammatory bowel
diseases; IGF-1, insulin growth factor type 1; IL-3, interleukin-3; IL-6,
interleukin-6; IL-10, interleukin 10; MeOH, methanol; MIG, IFNγ-
inducible T cell chemoattractant monokine; MIP-1γ, macrophage in-
flammatory protein-1-γ (CCL9); MPO, myeloperoxidase; MS, molec-
ular sieves, MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NF-κB, nuclear factor κ-B; MW, molecular weight; NMR,
nuclear magnetic resonance spectroscopy; OCT, mice group fed with
trans-resveratrol-3-O-(60-O-octanoyl)-β-D-glucopyranoside (7); PGE2,
prostaglandin E2; PIC, mice group fed with trans-piceid (compound 2);
PON2, paraoxonase-2, PTGES, prostaglandin E synthase; RES, mice
group fed with free resveratrol (3,5,40-trihydroxy-trans-stilbene, com-
pound 1); rt, room temperature; SIRT1, silent mating type information
regulation 2 homologue 1; sTNF RI, p55 subunit of the TNFR receptor
(TNFRp55, CD120a); TBDMS, tert-butyldimethylsilyl; THF, tetrahydro-
furan; TLC, thin layer chromatography; VCAM, vascular cell adhesion
molecule.
r
2010 American Chemical Society
Published on Web 09/24/2010
pubs.acs.org/jmc

7366 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
Figure 1. Resveratrol (1), piceid (2), and the different resveratrol pro-drugs (3-10) synthesized and assayed in the present study.
reported to exert a number of health-beneficial effects, including
the prevention and/or amelioration of cardiovascular diseases
and cancer.4 Among polyphenols, trans-resveratrol (1) (Figure 1),
naturally occurring in grapes and wine, has shown antiplatelet,
antioxidant, antitumor, and anti-inflammatory activities.5
Resveratrol has been reported to ameliorate the inflammation
status in mice and rat IBD models.6-8 Recently, our group
demonstrated that a human equivalent dose (HED) of 10 mg
resveratrol per day for a 70 kg person for 20 days was able to
ameliorate colon inflammation in an IBD rat model.7 How-
ever, like other phenolics, resveratrol is rapidly absorbed and
conjugated by phase II enzymes to yield mostly sulfate and
glucuronate derivatives,9 which reduces resveratrol delivery
to the colon and decreases its topical effectiveness in the colon
mucosa.
Herein we explore the efficacy of different resveratrol pro-
drugs and pro-prodrugs to ameliorate colon inflammation in
a colitis-induced mouse model. We hypothesized that increas-
ing the delivery of resveratrol to the colon could improve its
anti-inflammatory effects in the colon mucosa. We have
prepared two series of resveratrol derivatives: the first one
with glucosyl modifications, inspired by the natural molecule
trans-piceid (2) (Figure 1), and the second one with glucosyl-
and acyl-modifications, by attaching fatty acid groups of different
length to the piceid. Three candidates, trans-resveratrol-3,5-
di-O-β-D-glucopyranoside (4), trans-resveratrol-3-O-(60-O-
butanoyl)-β-D-glucopyranoside (6), and trans-resveratrol-3-
O-(60-O-octanoyl)-β-D-glucopyranoside (7), (Figure 1) were
selected for in vivo studies after preliminary in vitro cell
assays. We reasoned that a preventive oral treatment with
low doses of the prodrugs would protect the mucosal barrier
from inflammation, avoiding disease development. Mice were
fed with 2.1 mg/kg day (HED of 10 mg for a 70 kg person) of
each derivative for 3 weeks prior to the induction of colitis for
8 days. The protective effects observed for both 6 and 7 were
very remarkable despite the very low dose assayed. Mice fed
with compounds 6 or 7 hardly developed any symptoms of
colitis, asevidencedby various markers: diseaseactivityindex,
serum haptoglobin level, colon length and tissue damage,
colonic microbiota counts, myeloperoxidase activity, prosta-
glandin PGE2 production, and levels of several cytokines in
the colon tissue. Our results suggest that compounds 6 and 7
exerted a dual effect: (1) higher delivery of RES quantities to
the colon and (2) reduction of the mucosal barrier imbalance
which prevented diarrhea and, consequently, improved the
action of the delivered RES in the colon mucosa.
Results
Synthesis. Glucosylated resveratrol derivatives 3-5 were
prepared following the strategy reported by Zhang et al10
with modifications (Scheme 1). Random TBDMS-protection
of resveratrol 1 and subsequent chromatographic separation
afforded the four possible phenolic alcohol precursors 11, 12,
13, 14, and fully silylated resveratrol 15 (24, 18, 6, 22, and 7%
yields, respectively). The amount of fully protected 15 was
reduced from 27 to 7% just by two additions (during 15 min
each) of 1þ0.5 equiv of TBDMSCl (3 h interval) with respect
to the previously reported 1þ1 equiv of TBDMSCl added in
two shots (also 3 h interval). Glycosylation was carried out
using 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl trichloroa-
cetimidate 16 as the glycosyl donor,11 much more common
and simpler to prepare than the previously used trifluoro-
acetimidate derivative.10 However, the yields of resveratrol deri-
vativesmonoglucosylated (19) and diglucosylated (17 and 18)
compounds were slightly lower (60, 40, and 64%, respectively)
than those reported with the trifluoroacetimidate donor.
One-step deprotection with aqueous 3N NaOH in MeOH-
THF afforded compounds 3, 4, and 5 (50, 83, and 84% yields,
respectively).
Piceid acyl derivatives 6-10 were prepared in one-step by
enzymatic acylation of piceid 2 using Thermomyces lanugi-
nosus lipase immobilized on granulated silica (Lipozyme TL
IM) (Scheme 2). The reactions were carried out in tert-butyl
alcohol, and the acylating agents were the corresponding
fatty acid vinyl esters. Short column chromatography of the
reaction mixtures produced pro-prodrugs 6-10 in very high
yields (88-97%).
In Vitro Anti-inflammatory and Cell Metabolism Assays.
To test whether these structural modifications of the resver-
atrol molecule had any effect on the capacity of resveratrol to
reduce prostaglandin E2 (PGE2) synthesis under inflammatory
conditions, we measured the production of PGE2 by human
colon CCD18-Co cells after costimulation with 1 ng/mL of
IL-1β and the synthesized resveratrol derivatives for 18 h
(Supporting Information Figure S1). The inflammatory cytokine
IL-1β caused a significant increase (100-fold) in the PGE2
levels (P<0.01). Co-treatment with 1 μM of resveratrol (1)
partially prevented the production of PGE2, which was

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7367
Scheme 1. Synthetic Route to Resveratrol Prodrugs 3-5a
a
ꢀ
Conditions: (a) TBDMSCl, imidazole, anh. DMF, rt; (b) TMSOTf, DCM, 4 A MS; (c) 3N NaOH, MeOH-THF (5:1), 0 °C to rt.
Scheme 2. Synthesis of Piceid Fatty Acid Esters 6-10a
a Conditions: (a) lipozyme TL IM, t-BuOH, 60 °C
completely eliminated by resveratrol concentrations ranging
between 2.5 and 25 μM. Co-treatment with 25 μM of com-
pound 3 partially reduced PGE2 production, whereas com-
pounds 2, 4, and 5 did not prevent PGE2 induction (Support-
ing Information Figure S1A).
All the acyl-glucosyl derivatives, at concentrations in the
range from 1 to 7.5 μM, showed anti-inflammatory activity
by reducing PGE2 production (Supporting Information
Figure S1B). Concentrations higher than 10 μM (results
not shown) of the compounds 6-10, in particular of the com-
pounds 8, 9, and 10, were cytotoxic to these cells (as mea-
sured by the MTT method) upon 8 h of incubation and were
not further considered.
The chemical stability and metabolism of resveratrol
derivatives were evaluated after incubation with human
colon cancer Caco-2 cells. This cancer cell line was chosen
because its metabolism is faster than that of normal cells such
as CCD18-Co. Cell media samples were analyzed by LC-
MS-MS. Representative chromatograms of cell media sam-
ples after incubation of Caco-2 cells with resveratrol glucosyl
derivatives for 6 h are shown in Supporting Information
Figure S2. Overall, the glucosyl modifications conferred
protection to 1 by retarding its metabolism because no direct
phase II conjugation (glucuronidation, sulphation, methylation,
etc.) of the glucosyl derivatives was detected. The only
conjugated metabolites detected were those of the resveratrol
(mostly glucuronides), which were formed after the hydro-
lysis of the glucosyl derivatives and subsequent conjugation
of resveratrol by phase II enzymes. From all the glucosyl
derivatives tested, compound 4 was the most resistant to
hydrolysis because a high proportion of this compound re-
mainedintact in the cell medium even after 24 h of incubation
(results not shown), providing a slow release and metabolism
of the active molecule resveratrol. Although compound 4 did
not show anti-inflammatory activity in the cell model, its
resistance to be hydrolyzed and conjugated in the cell culture
suggested that it might be a good candidate as a pro-prodrug
to increase the delivery of resveratrol to the colon.
Regarding the cell metabolism of glucosyl-acyl derivatives
(Supporting Information Figure S3), all the compounds
presented a similar trend. The glucosyl-acyl modification
also conferred protection to resveratrol by slowing down
their metabolism. Conjugated resveratrol metabolites were
only detected upon hydrolysis of glucosyl-acyl derivatives
and subsequent resveratrol conjugation by phase II enzymes.
Compounds 6 and 7 were selected for the in vivo inflammatory
assays due to their higher stability compared to compounds
8, 9, and 10 and also to their direct in vitro anti-inflammatory
activity.
Mice Weight and Disease Activity Index (DAI). Mice were
fed for 21 days with standard mouse chow supplemented
with 2.1 mg/kg day of the different compounds. The abbre-
viations RES, PIC, DIGLUC, BUT, and OCT refer to the
mice groups whose diets were supplemented with com-
pounds 1, 2, 4, 6, or 7, respectively. Afterward, 1% DSS
was included in the drinking water for 8 days. Mice were still
fed with each of the compound for six more days in the
absence of DSS (post-treatment phase) (Supporting Infor-
mation Scheme S1A). Food and water intake as well as stool
blood, stool form, and body weight were monitored daily for
the whole experimental period (35 days). There were no signi-
ficant differences in the food and water intake among the
different groups (data not shown). The body weight increased
at a similar rate in all the groups during the 21 days prefeeding

7368 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
Figure 2. (a) DAI values, (b) colon length, (c) myeloperoxidase activity (MPO), and (d) prostaglandin E2 (PGE2) levels in mice. Deter-
minations of (c) and (d) were carried out in colon mucosa samples. Results shown in (b), (c), and (d) were determined in samples taken after 1%
DSS consumption for 8 days. RES group, fed with 1; PIC group, fed with 2; DIGLUC group, fed with 4; BUT group, fed with 6; OCT group,
fed with 7. Different letters inside each graph indicates significant differences (P < 0.05). a, different from control; b, different from DSS; a,b,
different from both control and DSS; c, different from other treatments (comparison within test compounds).
period (Supporting Information Figure S4). Upon DSS admini-
stration, mouse weight decreased very rapidly in the DSS,
RES, PIC, and DIGLUC groups, however, DSS had no
effect on the weight of the animals prefed with compounds 6
or 7 (BUT and OCT groups, respectively), which behaved
like the control group. During the recovery phase, the weight
slowly increased in the affected groups, the weight recovery
being faster in the DIGLUC group.
The DAI value (which combines stool blood, stool con-
sistency, and weight loss) increased in all the groups after
DSS administration although much lower DAI values were
clearly obtained in the last four days of DSS consumption for
BUT and OCT groups (Figure 2a). During the recovery
phase, both BUT and OCT groups reached DAI values close
to those of the control untreated group, whereas the rest of
the treated groups still exhibited higher DAI values than the
control animals (Figure 2a).
Mice from the DSS-treated group (animals fed with chow
diet only) showed ataxia, hair brightness loss, alopecia,
leanness, and severe rectal bleeding (Supporting Information
Figure S5). However, the animals fed with the resveratrol
prodrugs for 3 weeks prior to DSS consumption showed a
substantial improvement in their appearance in the following
order: 7 ≈ 6 . 4 ≈ 2 ≈ 1>DSS. The improvement was most
remarkable in the case of mice fed with compounds 6 or 7 at
day 29 (Supporting Information Figure S5), which, based on
their external appearance, seemed not to be affected by DSS
consumption.
(Supporting Information Scheme S1B), no differences in the
DAI index were observed among the groups (Supporting
Information Figure S6). These results clearly indicated a
preventive effect rather than a therapeutic effect of the low
doses of the compounds 6 and 7 tested (2.1 mg/kg day).
Colon Length. Colon shortening is a well-known feature of
colon inflammation in the murine DSS model. Mice in the
control group had an average colon length of 8.9 ( 0.4 cm,
which was reduced to 6.3 ( 0.2 cm in the DSS group (P<0.01)
(Figure 2b). The colon length shortening caused by DSS was
only significantly prevented in the groups of animals prefed
with compounds 6 or 7, which had colon lengths of 7.7 ( 0.5
and 8.2 ( 0.5 cm, respectively, (P < 0.004) (Figure 2b).
Myeloperoxidase (MPO) Activity. MPO activity is usually
increased in the colon mucosa of IBD patients as a result of
leukocyte infiltration. As expected, MPO activity was in-
duced 30-fold (P<0.01) in the mice colon mucosa upon DSS
administration for 8 days. This increase was only significantly
reduced upon pretreatments with compounds 6 (3.7-fold)
and 7 (7.2-fold) (P < 0.01) (Figure 2c).
Prostaglandin (PGE2) Levels. PGE2 is an important pro-
inflammatory lipid mediator that is produced in excess in the
colon mucosa of IBD patients. PGE2 levels increased 15-fold
(P < 0.01) in mouse colon mucosa after DSS treatment.
Prefeeding with 6 or 7 attenuated the induced levels of PGE2
by 2.5- and 3.5-fold (P< 0.01), respectively (Figure 2d),
whereas the rest of the compounds were not able to reduce
PGE2 levels after DSS administration.
In a separate set of experiments in which mice were not
prefed with any of the compounds prior to DSS administration
Histological Evaluation. Microscopically, colon samples
from the control group (Figure 3a) showed the normal histology

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7369
in time and space modulate the development, recurrence, and
exacerbation of the inflammatory process in IBD. Forty cyto-
kines (Supporting Information Figure S7) were analyzed in
the colon mucosa of mice at the end of the DSS administra-
tion. Proteins exhibiting significant changes (P<0.05) are
shown in Figure 4 and Supporting Information Figure S8.
DSS increased the levels of soluble TNFR receptor type I
(sTNF RI, also known as TNFRp55; Figure 4a) by 2-fold
(P<0.05), macrophage inflammatory protein-1-γ (MIP-1γ,
also known as CCL9; Figure 4b) by 3-fold (P < 0.05),
interferon-γ-inducible T cell chemoattractant monokine
(MIG; Figure 4c) by 2-fold (P < 0.05) and interleukin-6
(IL-6; Figure 4d) by 3-fold (P<0.05). Preadministration of
compounds 1, 2, 4, 6, and 7 decreased the expression of these
cytokines induced by DSS. The compounds 6 and 7 showed a
stronger inhibition of the expression of sTNF RI (Figure 4a)
and MIP-1γ (Figure 4b) than the rest of the resveratrol
derivatives, reaching protein levels similar to those of the
control animals. The levels of MIP-1γ were also detected and
measured in serum but were not affected by DSS treatment,
and no differences were found among the groups (results not
shown). Other proteins such as the granulocyte macrophage
colony-stimulating factor (GM-CSF; Figure 4e) and inter-
leukin-10 (IL-10; Figure 4f) were not significantly affected by
DSS, and only compounds 6 and 7 significantly decreased
the levels of these two cytokines, even below those deter-
mined for control animals. Interleukin-3 (IL-3; Supporting
Information Figure S8) was not induced by DSS. However,
all the resveratrol derivatives significantly increased the
levels of this cytokine. IL-3 was not detected in serum.
Acute Phase Proteins. Both haptoglobin and fibrinogen
are acute phase proteins that determine systemic inflamma-
tion and are usually elevated in IBD patients. DSS increased
the levels of serum haptoglobin (Figure 5a) and fibrinogen
(Figure 5b) by 9-fold and 2-fold (P<0.05), respectively.
Prefeeding with 1, and more markedly, with 6 or 7, reduced
haptoglobin values to those of the control animals (Figure 5a).
The compounds 4, 6, and 7 also prevented the increase of
fibrinogen serum level induced by DSS (Figure 5b).
Fecal Microbiota. An altered gut microbial composition
could be involved in the pathogenesis of IBD. On the other
hand, high counts of some specific microbial groups such as
bifidobacteria, lactobacilli, and clostridia have been corre-
lated with an improvement of the intestinal health. Groups
pretreated with the resveratrol prodrugs showed a significant
increase (P < 0.001) of fecal lactobacilli and bifidobacteria
counts after 21 days of dietary supplementation with each of
the compounds (Figure 6a,b), whereas animals fed on un-
supplemented chow had constant numbers of these bacteria.
The DSS induced a marked decrease of the counts of these
two groups of bacteria in the untreated group as well as in the
groups prefed with 1, 2, and 4 (Figure 6a,b). However, lacto-
bacilli and bifidobacteria counts continue rising in the groups
treated with 6 or 7 (Figure 6a,b).
Regarding clostridia, enterobacteria, and Escherichia coli,
the compounds 2 and 4 were not evaluated as these experi-
ments were focused on the most effective compounds (6 and 7)
and using different controls including the unmodified
resveratrol molecule (1). Clostridia counts increased slightly
in the RES (compound 1), BUT (compound 6), and OCT
(compound 7) groups after 21 days of prefeeding but were
only statistically significant in the case of the OCT group
(Figure 6c). As in the case of lactobacilli and bifidobacteria
counts, DSS administration drastically reduced the levels of
Figure 3. Histology of mouse colonic samples stained with hematoxylin
and eosin. (a) Control group showing a normal histological section
of mouse colon. (b) Mucosal injury induced by DSS administration
for 8 days with loss of crypts and epithelial integrity as well as severe
inflammatory cell infiltration. (c-g) Colonic samples from mice
pretreated for 21 days with compounds 1, 2, 4, 6, or 7 (2.1 mg/kg day)
before DSS administration. Prodrugs administration protected
the colonic structure, especially in the case of the BUT (f) and
OCT (g) groups. Epithelium, crypts, and cell infiltration are indi-
cated by arrow, square, and circle, respectively. The scale bar in the
first microphotograph represents 50 μm and applies to all images.
Statistical analysis of significant differences is included in Support-
ing Information Table S1.
of the mouse distal colon. Damage to the colonic mucosa
assessed by total histological score was significantly higher
(P<0.005) in the DSS group than in the other groups (Sup-
porting Information Table S1). In the DSS group, colonic
sections showed typical inflammatory changes in colonic
architecture such as severe crypt and surface epithelial loss as
well as strong infiltration of inflammatory cells (mononuclear
cells, neutrophils, and eosinophils) (Figure 3b). A complete
disruption of the epithelial architecture was observed in some
areas. In general, pretreatment with the resveratrol prodrugs
reduced the morphological signs of cell damage and preserved
the mucosal architecture (Figure 3c-g). In some areas, the
epithelium remained almost intact. These protective effects
were most remarkable in animals prefed with the compounds
6 or 7 (Figure 3f,g), particularly in the case of the OCT group
fed with 7 (Figure 3g, Supporting Information Table S1).
Cytokine Array of Colon Samples. Imbalance of cytokines
is contributing to the pathogenesis of IBD. Cytokines levels

7370 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
Figure 4. Effect of pretreatment with compounds 1, 2, 4, 6, and 7 after consumption of 1% DSS on different cytokines levels in colon samples.
(a) sTNF RI, p55 subunit of the TNFR receptor (TNFRp55, CD120a); (b) MIP-1γ, macrophage inflammatory protein-1-γ (CCL9); (c) MIG,
IFNγ-inducible T cell chemoattractant monokine; (d) IL-6, interleukin-6; (e) GM-CSF, granulocyte macrophage colony-stimulating factor
and (f) IL-10, interleukin-10. Different letters inside each graph indicates significant differences (P<0.05). a, different from control; b,
different from DSS; a,b, different from both control and DSS; c, different from other treatments (comparison within test compounds).
clostridia in the DSS group (no-pretreatment) and in the
RES group (Figure 6c) but not in the BUT and OCT groups
(Figure 6c).
A significant growth induction of Escherichia coli and
enterobacteria (P<0.01) was observed upon DSS adminis-
tration (Figure 6d). E. coli induction was significantly di-
minished (P<0.01) by prefeeding with compounds 1, 6, or 7.
Enterobacteria were only significantly decreased (P < 0.01)
by 6 and 7 (Figure 6d).
Absorption, Metabolism and Gastro-Intestinal Transit Kine-
tics of Resveratrol Derivatives. For the metabolic studies,
healthy mice (Supporting Information Scheme S2A) were
intragastrically administered with 1, 2, 4, 6, or 7 at a much
higher dose (84 mg/kg) than that used for the intestinal
inflammatory assays (2.1 mg/kg). This enabled us to detect
the parent compounds as well as the different resulting meta-
bolites in the gastrointestinal tract and in plasma. Stomach,
small intestine, colon, and plasma samples were taken at nine
time-points after DSS administration and were monitored by
HPLC-MS-MS.
Supporting Information Figure S9 shows the evolution
of the concentration of the initially administered parent
compounds (1, 2, 4, 6, and 7) (Supporting Information
Figure S9A) and their hydrolysis products piceid (2) and
free resveratrol (compound 1) (Supporting Information
Figure S9B and S9C, respectively) in the stomach. The corres-
ponding AUC (area under the curve) and Cmax (maximum
concentration) values are shown in Supporting Information
Table S2. Resveratrol (1) remained intact in the stomach 8 h
after intragastric administration (Supporting Information
Figure S9A) and showed very high AUC and Cmax values
that diminished upon the intestinal transit and absorption.
The rest of the dosed compounds (2, 4, 6, and 7) were
partially hydrolyzed in the stomach to release different
quantities of the intermediate piceid (2) (Supporting Infor-
mation Figure S9B) and of the final hydrolysis product, free

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7371
Figure 5. Serum levels of acute phase proteins after DSS treatment. (a) Haptoglobin and (b) fibrinogen. Different letters inside each graph
indicates significant differences (P < 0.05). a, different from control; b, different from DSS; a,b, different from both control and DSS.
Figure 6. Microbial counts in mouse feces before and after DSS treatment. (a) Lactobacilli: before DSS, the prodrug group was different from
control and, after DSS, only 6 and 7 showed differences from DSS. (b) Bifidobacteria: before DSS, the prodrug group was different from
control and, after DSS, only OCT and BUT showed differences from DSS. (c) Clostridia: before DSS, the OCT was different from control and,
after DSS, OCT and BUT showed differences from DSS. (d) E. coli and enterobacteria: different letters inside graph indicates significant
differences. a, different from control; b, different from DSS. Significance level (P<0.05).
resveratrol (1) (Supporting Information Figure S9C). The hydro-
lysis process continued in the small intestine (Supporting
Information Figure S10). Glucuronide and sulfate resveratrol
derivatives were also detected in the small intestine (results
not shown). Regarding the levels of circulating resveratrol
metabolites (mainly resveratrol-glucuronide) in the systemic
bloodstream after the ingestion of the glucosyl and acyl-
glucosyl prodrugs, it was found that the AUC and Cmax values
were approximately 10-fold lower and between 3- to 30-fold
lower, respectively, than those determined after ingestion of com-
pound 1 (Supporting Information Figure S11 and Table S2).
These results clearly indicated that all the glucosyl and
acyl-glucosyl modifications diminished resveratrol absorp-
tion and conjugation. Consequently, the use of these pro-
drugs should increase the delivery of resveratrol to the colon,
a fact that was confirmed by higher AUC and Cmax values of
free resveratrol detected in the colon (Figure 7a, Supporting
Information Table S2). Only 4.5% of resveratrol ingested as
such (1) reached the colon, whereas the highest Cmax values
were observed for 2, 6, and 7. None of the initially adminis-
tered parent compounds were detected in the colon.
The differences between the results obtained with 1, 6, and
7 were accentuated in mice with DSS-induced colitis (Sup-
porting Information Scheme S2B). In this second experiment,
we focused only on the prodrugs with the highest delivery of
resveratrol to the colon (6 and 7) and compared them to
resveratrol itself (1). One time-point (4 h postingestion) was
analyzed (Figure 7b). It should be noted that in healthy mice
the amount of resveratrol delivered to the colon increased
by 2.5- and 3.3-fold in the 6 and 7 dosed animals, respectively,

7372 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
this molecule to the colon. Other anti-inflammatory drugs
(naproxen, flurbiprofen, sulindac, 5-aminosalicylic acid, etc.)
present similar problems, and a prodrug approach has been
quite successful to improve drug delivery to the colon. For
example, azo derivatives as prodrugs of 5-aminosalicylic acid
have shown potent anticolitic effect in a rat model.13 In addi-
tion, colon targeting is very important in the case of steroidal
drugs to reduce side effects. In this context, a cyclization-
activation prodrug strategy was validated in a murine DSS
model to depress steroid absorption and consequently to
reduce side effects.14
Our preliminary in vitro inflammation assay revealed that
resveratrol was the most effective anti-inflammatory molecule
in a model with direct cell-molecule interaction (Supporting
Information Figure S1). However, as resveratrol (1) is rapidly
metabolized and excreted in vivo, a more efficient delivery of 1
to the colon should involve both protection from rapid 1
metabolism and further prodrug hydrolysis to deliver 1 to the
colon. According to the in vitro inflammation and cell meta-
bolism assays, the compounds 2, 4, 6, and 7 could potentially
fulfill the above requirements.
The in vivo assay described in the present study implied the
long-term administration of low doses of the test compounds
mixed with the diet. This approach takes into account three
important points: (1) oral administration, because it is a challenge
to develop effective orally administered colon anti-inflammatory
drugs,2 (2) use of a very low dose (HED: 10 mg/day for a
70 kg-person). Although no significant acute toxicity has been
reported for resveratrol, treatments with high resveratrol
concentrations for long periods have not been evaluated
so far, (3) because it is widely known that many dietary com-
pounds can interfere with the absorption, metabolism and
action of drugs,15 the prodrugs were administered mixed with
the diet to consider the potential neutralizing effect of food
matrix.
The mice fed with the resveratrol pro-prodrugs 6 and 7
hardly developed any intestinal inflammation symptoms,
which was evidenced by the overall appearance of mice as
well as by the different parameters and molecular markers
evaluated. In general, the lack of colitis symptoms in mice fed
with 6 and 7 was confirmed by the scarce colon tissue damage
(Figure 3f,g), which matched with low myeloperoxidase activ-
ity (Figure 2c) and low levels of leukocyte-derived cytokines
(Figure 4a-e) determined in these groups. In addition, the
levels of the pro-inflammatory mediator PGE2 in the colon
mucosa of these two groups were significantly lower than in
the other groups (Figure 2d). PGE2 is crucial for monocyte-
derived dendritic cells to acquire potent T-helper cell stimula-
tory capacity and chemotactic responsiveness to lymph node-
derived chemokines.16 PGE2 is a key mediator in intestinal
inflammation and colorectal cancer.17 The role of PGE2 in
IBDs appears to have a dual effect. Whereas a low PGE2
synthesis seems to be related to the recovery of inflammatory-
induced damage,18 high PGE2 levels can exacerbate inflam-
matory processes.19 Therefore, the low PGE2 values detected
in mice fed with 6 and 7 (BUT and OCT groups, respectively,
Figure 2d) also indicated that these groups hardly developed
colitis. In addition, as part of the healing mechanisms upon
intestinal inflammation, PGE2 has been reported to induce
IL-10, which is a known anti-inflammatory cytokine.20 In
agreement with this, our results showed no IL-10 induction after
DSS treatment in both BUT and OCT groups (Figure 4f).
Cytokine arrays analyses revealed that the induction, under
inflammatory conditions, of a number of cytokines such as
Figure 7. Kinetics of resveratrol delivered in the colon upon adminis-
tration of resveratrol (1), piceid(2), and the resveratrol pro-prodrugs 4,
6, and 7. (a) Healthy mice (no DSS and no prodrugs pretreatment)
and (b) animals pretreated with 2.1 mg/kg of compounds 1, 6, or 7
for 3 weeks followed by DSS-induced colitis.
as compared to the 1 dosed group (Figure 7a). However, in
mice pretreated for 21 days with 6, 7, or 1 and with DSS-
induced colitis, the amount of resveratrol delivered to the
colon increased by 12.6- and 28-fold in BUT (compound 6)
and OCT (compound 7) groups, respectively, compared to
the RES (compound 1) group (Figure 7b).
Discussion
The intestinal inflammatory model based on oral DSS
administration is widely accepted, and it has been reported
to be relevant for the translation of data from mice to humans
to identify and validate new therapies for IBDs.12 Orally
administered DSS disrupts the epithelial barrier causing acute
clinical symptoms (diarrhea and bloody stools), epithelial
crypt loss, and subsequently inflammation. These inflammatory
features are found in ulcerative colitis and Crohn’s disease,
as the increased colonic permeability leads to an increased
interaction between microbiota and immune system, one of
the important factors on initiation of IBD.
Resveratrol (1) exerts a plethora of different biological
activities.5 Previous studies have reported the amelioration
of DSS-induced colitis in mouse and rat models using high6,8
and low7 resveratrol doses through the downregulation of COX-2
(cyclooxygenase-2) and PTGES (prostaglandin E synthase)6,7
as well as the inhibition of NF-κB activation mediated by
SIRT1 (silent mating type information regulation 2 homo-
logue 1) induction.8 Larrosa et al.7 reported the regulation of
genes implicated on different pathways such as interleukin 6
(IL-6) NetPath 18, HSP70, mitochondrial fatty acid oxidation,
Wnt signaling, and T-cell-receptor NetPath 11, as revealed by
functional analysis of differentially expressed genes from
colon mucosa samples of rats treated with DSS and 1 mg/kg
day resveratrol.7 In addition, the low dose of 1treatment counter-
acted the deleterious effect of DSS treatment by regulating the
expression of other important genes including IGF-1, leptin,
VCAM, PON-2, adiponectin, SIRT3, and SIRT7.7 Because 1
is rapidly absorbed, metabolized, and excreted,9 which limits
its potential use as a colon anti-inflammatory drug, it is of great
interest to design resveratrol prodrugs to improve delivery of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7373
MIG, IL-6, and especially, TNF-Rp55 (Figure 4a), MIP-γ
(Figure 4b), and GM-CSF (Figure 4e) was prevented by 6 and 7.
TNF-Rp55 is the major soluble TNF-R receptor (sTNFR)
in the lamina propria and submucosal regions of the colon.
Mice lacking TNF-Rp55 and treated with DSS have been
reported to show reduced mucosal damage, reduced infiltra-
tion of macrophages and neutrophils, and attenuated subse-
quent tumor formation.21 The precise role of the granulocyte
macrophage colony-stimulating factor (GM-CSF) in IBD
remains to be elucidated. Whereas GM-CSF can be induced
upon DSS administration22 it has been also reported to elicit
bone marrow-derived cells that promote efficient colonic
mucosal healing.23 The role of the murine MIP-1γ (CCL9)
in experimental colitis has not been previously reported. Our
study shows, for the first time, the induction of this chemokine
in the DSS-induced experimental colitis model. CCL9 is secreted
by the murine follicle-associated epithelium of Peyer’s patches
and recruits CD11bþ dendritic cells, which seem to be a critical
mechanism to capture antigens and present them to CD4þ T
cells.24 Subsequently, T cells mount an adaptative immune
response against potentially pathogenic organisms. There-
fore, induction of CCL9 and consequent impairment of this
process can contribute to an exacerbated inflammatory re-
sponse. This induction was completely prevented by both 6
and 7 (Figure 4b). Although the role of sTNFR, PGE2, GM-
CSF, and CCL9 in inflammatory bowel diseases is not fully
understood, it is reasonable to argue, in accordance with our
results, that keeping their values similar to those of the control
(healthy) group resembles a normal intestinal homeostasis.
The mechanism by which 6 and 7 prevented DSS-induced
colitis could not be simply explained by the observed increase
in bifidobacteria, lactobacilli, or clostridia because the other
resveratrol prodrugs tested also caused a similar or even
higher increase of bacteria counts before DSS administration
(Figure 6). However, after DSS administration, the bacterial
population was maintained or even increased in the case of 6
and 7, while the bacterial counts dramatically decreased in the
rest of the groups. Therefore, the maintenance of bacterial
counts in the BUT and OCT groups after DSS administration
seemed to be a consequence of the normal mucosal barrier
balance promoted by 6 and 7. In this context, Larrosa et al.7
hypothesized a potential synergistic effect of the induced colonic
microbiota with resveratrol in the final anti-inflammatory
effects observed. According to the present study, the resver-
atrol prodrugs-induced microbiota did not seem to be critical
in the prevention of colon inflammation symptoms because 1, 2,
and 4 increased microbiota counts before DSS administration
(Figure 6) but were not as effective as 6 and 7 in preventing the
colitis.
mice previously fed with 6 and 7 because these animals did not
suffer from diarrhea, whereas animals fed with 1 did have
diarrhea. Resveratrol delivery to the colon was also much
higher in the DSS-treated mice in BUT and OCT groups
compared to the RES group (Figure 7b). Therefore, the
prevention of diarrhea by 6 and 7 improved colonic resvera-
trol delivery in the BUT and OCT groups. Both prevention of
diarrhea and increased delivery of resveratrol to the colon
seemed to be relevant in the prevention of inflammation
symptoms in DSS-treated mice fed with 6 and 7. This is even
more relevant when taking into account that the same pro-
prodrug dose administered (2.1 mg/kg day) implies different
quantities of initial resveratrol. This is important if resveratrol
is the active core of the prodrugs. Considering the molecular
weight of resveratrol (MW=228), the dose administered of 1
(unmodified resveratrol) was 2.1 mg/kg day (i.e., ratio = 1).
However, depending on the structural modification, the initial
quantity of resveratrol administered was different, i.e. com-
pound 2 (MW=390), ratio prodrug:resveratrol=0.58, com-
pound 4 (MW=552), ratio=0.41, compound6(MW=460),
ratio = 0.5, and compound 7 (MW = 515), ratio = 0.44.
Therefore, despite the lower amount of resveratrol initially
administered, the colonic resveratrol delivery as well as the
overall preventive effect against DSS-induced ulcerative coli-
tis was higher for all the prodrugs than for free (unmodified)
resveratrol (1).
The small differences found in colonic resveratrol delivery
upon ingestion of 2, 6, and 7 seem to indicate that other
specific mechanisms are involved in the protective effects of 6
and 7 in this intestinal inflammatory model. A potential direct
action of the compounds 6 and 7 before hydrolysis might be
possible in the upper gastrointestinal tract but not in the colon
where only resveratrol was detected. Although DSS affects
mainly colon distal parts, an increase in the small intestinal
mucosal permeability is part of the early stages of the DSS-
induced colitis which precedes the histological detectable
changes.25 It has been recently reported that DSS can affect
the small intestine by producing changes in the mucosal
homeostasis that can influence the colonic response after
DSS treatment as well as the dimension of the inflammatory
response to heal intestinal damage.26
Butyric acid and octanoic acid, released upon hydrolysis
of 6 and 7, could also be involved in the preventive effect
observed because these molecules are known anti-inflamma-
tory short and medium fatty acids, respectively.27,28 This
scenario would involve a pro-double-drug mechanism, where
the fatty acid chain and the resveratrol molecule would be
playing a role side-by-side. However, the total amount of
butyrate releaseddailyupon hydrolysisof6 couldreachvalues
of only 0.4 mg/kg (1.9 mg for a 70 kg person). It would be
difficult to explain the remarkable protective effects observed
in our study with such a small amount of butyrate when
some studies in animals and humans with IBD reported
discrete anti-inflammatory responses using 2000-fold higher
In an attempt to prove our prodrug approach as a means to
achieve higher delivery of resveratrol to the target colon, the
gastrointestinal transit kinetics of all the resveratrol prodrugs
was evaluated. Despite the huge number of reports on resver-
atrol, our study also shows for the first time the monitoring
of resveratrol transit through the gastrointestinal tract. In
healthy mice, the maximum concentration of resveratrol was
detected in the colon after 4 h of oral administration. The
lowest concentration was detected in the case of mice fed with
resveratrol itself (compound 1), whereas the highest values of
resveratrol were found in mice fed with 6 and 7. These results
indicatedthatthe addition of butanoyl-glucose- andoctanoyl-
glucose- residues to the resveratrol molecule improved the
delivery of this bioactive compound to the colon. We then hypo-
thesized that resveratrol delivery might have been improved in
quantities of butyrate (HED 4 g for a 70 kg person).29,30
A
similarreasoning couldbeapplied tocompound7. Despitethe
above, the beneficial effects of long-term low-dose oral ad-
ministration of butyrate and octanoate in IBDs cannot be
ruled out.
Our results suggest that 6 and 7 act as resveratrol pro-
prodrugs with a dual activity, i.e., modulating the barrier mucosal
homeostasis and delivering effectively anti-inflammatory re-
sveratrol to the colon. This suggests that chronic consumption
of 6 and 7 could be very important in a potential maintenance

7374 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
or remission stage of IBD to prevent relapse. It is important to
remark that the low pro-prodrugs dose assayed effectively
prevented colitis symptoms upon repeated oral administra-
tion before colitis induction. In this context, the investigation
of potential therapeutic effects at higher prodrugs concentra-
tions is warranted.
(Carom), 125.4 (CHdCH), 115.1, 107.0, 105.2, (Carom), 100.5
(C-1), 76.5 (C-3), 73.9 (C-5), 73.4 (C-2), 70.6 (C-4), 63.4 (C-6),
33.6, 31.4, 28.9, 28.6, 24.6, 22.3, 22.2 (CH2), 13.0 (CH3). HRMS
m/z calculated for C28H36O9, [M þ Na]þ 539.2257, found
539.2269.
(E)-1-(3-(60-O-Lauroyl)-β-D-glucopyranosyloxy-5-hydroxy-
phenyl)-2-(4-hydroxyphenyl)ethene 8. The pure compound was
obtained using vinyl laurate as the acylating agent (138 mg, 94%).
TLC (ethyl acetate) Rf 0.36. [R]D20 -39.2 (c, 1.0 in MeOH). 1H
NMR (MeOD, 300 MHz), δ ppm: 7.38 (d, 2H, Jortho =8.4 Hz,
Experimental Section
Chemistry. TLC was carried out on precoated Silica-Gel
60 plates F254 and stained by heating with Mostain (500 mL
of 10% H2SO4, 25 g of (NH4)6Mo7O24 4H2O, 1 g Ce(SO4)2
H
arom), 7.03 (d, 1H, J=16.5 Hz, CHdCH), 6.86 (d, 1H, J=16.5
Hz, CHdCH), 6.79 (d, 2H, Jortho =8.4 Hz, Harom), 6.74 (s, 1H,
arom), 6.64 (s, 1H, Harom), 6.42 (s, 1H, Harom), 4.90 (s, 1H, H-1),
H
3
3
4H2O). Products were purified by flash chromatography with
silica gel60 (200-400 mesh). NMR spectra were recorded on
either a Bruker AVANCE 300 or ARX 400 MHz (300 or 400 MHz
(1H), 75 or 100 (13C)) spectrometer at room temperature for solu-
tions in CDCl3, D2O, or CD3OD. Chemical shifts are referred to
the solvent signal and are expressed in ppm. 2D NMR experi-
ments (COSY, TOCSY, ROESY, and HMQC) were carried out
when necessary to assign the corresponding signals of the new
compounds. High resolution FAB (þ) mass spectral analyses
were obtained on a Micromass AutoSpec-Q spectrometer. Com-
pound purities of test compounds 1-10 by reversed-phase HPLC
(integration of chromatograms at λ = 320 nm) were g95%.
General Procedure for the Synthesis of the Piceid Fatty Acid
Esters 6-10. Preparation of derivatives 6-10 was carried out by
regioselective enzymatic acylation of piceid 2 (from Sigma
Aldrich) by the following general procedure: lipozyme TL IM
(Novozymes) (100-150 mg) was added to a mixture of piceid 2
(100-150 mg, 1 equiv) and the corresponding acylating agent
(20 equiv) in 15-20 mL of tert-butyl alcohol. The mixture was
stirred in an orbital shaker at 60 °C for 16 h. The enzyme was
decanted and separated. The solvent was evaporated, and the
product was purified by flash column chromatography (ethyl
acetate/MeOH from 1:0 to 9:1) to yield the corresponding
acylated piceid derivatives 6-10 (88-97%).
4.45 (m, 1H, H-6), 4.28-4.21 (dd, 1H, J = 7.5 and 11.7 Hz,
H-60), 3.74-3.69 (m, 1H, H-5), 3.51-3.47 (m, 2H, H-3, H-2),
3.38-3.32 (m, 1H, H-4), 2.30 (t, 2H, J=7.5 Hz, CH2), 1.50 (m,
2H, CH2), 1.30-1.16 (m, 16H, 8 ꢀ CH2), 0.90 (t, 3H, J=6.9 Hz,
CH3). 13C NMR (MeOD, 75 MHz), δ ppm: 174.2 (CdO), 158.9,
158.2, 157.1, 139.9, 128.8 (Cqarom), 128.6 (CHdCH), 127.5 (Carom),
125.3 (CHdCH), 115.1 (Carom), 107.0, 105.3, 102.9 (Carom) 100.5
(C-1), 76.5 (C-3), 73.9 (C-5), 73.4 (C-2), 70.6 (C-4), 63.5 (C-6),
33.6, 31.7, 29.6, 29.4, 29.2, 29.1, 28.9, 28.7, 24.6, 22.3 (CH2) 13.1
(CH3). HRMS m/z calculated for C32H44NaO9, [M þ Na]þ
595.2883, found 595.2921.
(E)-1-(3-(60-O-Hexadecanoyl)-β-D-glucopyranosyloxy-5-hydroxy-
phenyl)-2-(4-hydroxyphenyl)ethene 9. The pure compound was
obtained using vinyl palmitate as the acylating agent (142 mg,
88%). TLC (ethyl acetate) Rf 0.42. [R]D20 -40.2 (c, 1.0 in MeOH).
1H NMR (MeOD, 300 MHz), δ ppm: 7.38 (d, 2H, Jortho=8.4 Hz,
H
arom), 7.03 (d, 1H, J=16.2 Hz, CHdCH), 6.86 (d, 1H, J=16.2
Hz, CHdCH), 6.79 (d, 2H, Jortho =8.7 Hz, Harom), 6.74 (s, 1H,
arom), 6.64 (s, 1H, Harom), 6.43 (s, 1H, Harom), 4.90 (m, 1H,
H
H-1), 4.45 (m, 1H, H-6), 4.28-4.21 (d, 1H, J = 7.6, 11.7 Hz,
H-60), 3.73-3.68 (m, 1H, H-5), 3.53-3.47 (m, 2H, H-3, H-2),
3.44-3.32 (m, 1H, H-4), 2.30 (t, 2H, J=7.5 Hz, CH2), 1.51 (m,
2H, CH2), 1.29-1.17 (m, 24H, 12 ꢀ CH2), 0.91 (t, 3H, J=6.9 Hz,
CH3). 13C NMR (MeOD, 75 MHz), δ ppm: 174.1 (CdO), 158.8,
158.2, 157.1, 139.9, 128.8 (Cqarom), 128.5 (CHdCH), 127.5 (Carom),
125.4 (CHdCH), 115.1 (Carom), 107.0, 105.3, 102.9 (Carom),
100.5 (C-1), 76.5 (C-3), 73.9 (C-5), 73.4 (C-2), 70.6 (C-4), 63.5
(C-6), 33.6, 31.7, 29.7, 29.4, 29.2, 29.1, 28.9, 28.7, 24.6, 22.3 (CH2),
13.0 (CH3). HRMS m/z calculated for C36H52NaO9, [MþNa]þ
651.3509, found 651.3549.
(E)-1-(3-(60-O-Octadecanoyl)-β-D-glucopyranosyloxy-5-hydroxy-
phenyl)-2-(4-hydroxyphenyl)ethene 10. The pure compound was
obtained using vinyl stearate as the acylating agent (155 mg,
92%). TLC (ethyl acetate) Rf 0.46. [R]D20 -46.5 (c, 1.0 in MeOH).
1H NMR (MeOD, 300 MHz), δ ppm: 7.38 (d, 2H, Jortho = 8.7
Hz, Harom), 7.03 (d, 1H, J=16.2 Hz, CHdCH), 6.86 (d, 1H, J=
16.2 Hz, CHdCH), 6.79 (d, 2H, Jortho=8.7 Hz, Harom); 6.74 (s,
1H, Harom), 6.64 (s, 1H, Harom), 6.43 (t, 1H, J=2.1, Harom), 4.91
(d, 1H, J=7.5 Hz, H-1), 4.45 (dd, 1H, J=1.8 and 12 Hz, H-6),
4.28-4.21 (dd, 1H, J = 7.6, 11.7 Hz, H-60), 3.73-3.68 (m, 1H,
H-5), 3.54-3.47 (m, 2H, H-3, H-2), 3.44-3.32 (m, 1H, H-4),
2.30 (t, 2H, J=7.5 Hz, CH2), 1.50 (m, 2H, CH2), 1.30-1.16 (m,
28H, 14 ꢀ CH2), 0.90 (t, 3H, J = 6.6 Hz, CH3). 13C NMR
(MeOD, 75 MHz), δ ppm:174.2 (CdO), 158.8, 158.2, 157.1,
139.9, 128.8 (Cqarom), 128.6 (CHdCH), 127.5 (Carom), 125.4
(CHdCH), 115.4 (Carom), 107.0, 105.3, 102.9 (Carom), 100.5 (C-1),
76.5 (C-3), 73.9 (C-5), 73.4 (C-2), 70.6 (C-4), 63.5 (C-6), 33.6,
31.7, 29.4, 29.3, 29.2, 29.1, 28.9, 28.7, 24.6, 22.3 (CH2), 13.1 (CH3).
HRMS m/z calculated for C38H56O9Na [M þ Na]þ 679.3822,
found 679.3857.
(E)-1-(3-(60-O-Butyryl)-β-D-glucopyranosyloxy-5-hydroxyphenyl)-
2-(4-hydroxyphenyl)ethene 6. The pure compound was obtained
using vinyl butanoate as the acylating agent (170 mg, 97%).
20
1
TLC (ethyl acetate) Rf 0.3. [R]D -55.0 (c, 1 in MeOH). H
NMR (MeOD, 300 MHz), δ ppm: 7.39 (d, 2H, J = 8.4 Hz,
Harom); 7.03 (d, 1H, J = 16.2 Hz, CHdCH)), 6.86 (d, 1H, J =
16.2 Hz, CHdCH)), 6.79 (d, 2H, Jortho=8.7 Hz, Harom), 6.74 (s,
1H, Harom), 6.64 (s, 1H, Harom), 6.43 (t, 1H, J=2.1 Hz, Harom),
4.92 (m, 1H, H-1), 4.45 (dd, 1H, J = 2.1 and 12 Hz, H-6),
4.28-4.21 (m, 1H, H-60), 3.73-3.67 (m, 1H, H-5), 3.53-3.47 (m,
2H, H-2, H-3), 3.37 (m, 1H, H-4), 2.30 (t, 2H, J=7.5 Hz, CH2),
1.59-1.52 (m, 2H, CH2), 0.83 (t, 3H, J = 7.2 Hz, CH3). 13C
NMR (MeOD, 75 MHz), δ ppm: 174.1 (CdO), 158.8, 158.2,
157.0, 139.9 (Cqarom), 128.8 (CHdCH), 128.6, 127.5 (Carom),
125.4 (CHdCH), 115.2 (Carom); 107.0, 105.5, 102.9 (Carom),
100.5 (C-1), 76.5 (C-3), 73.9 (C-5), 73.4 (C-2), 70.5 (C-4), 63.4
(C-6), 35.5 (CH2), 17.9 (CH2), 12.5 (CH3). HRMS m/z calcu-
lated for C24H28NaO9, [M þ Na]þ 483.1631, found 483.1648.
(E)-1-(3-(60-O-Octanoyl)-β-D-glucopyranosyloxy-5-hydroxy-
phenyl)-2-(4-hydroxyphenyl)ethene 7. The pure compound was
obtained using vinyl octanoate as the acylating agent (181 mg,
92%). TLC (ethyl acetate) Rf 0.35. [R]D20 -50.0 (c, 0.5 in MeOH).
1H NMR (MeOD, 300 MHz), δ ppm: 7.38 (d, 2H, Jortho = 8.7
Hz, Harom), 7.03 (d, 1H, J=16.2 Hz, CHdCH), 6.86 (d, 1H, J=
16.2 Hz, CHdCH), 6.78 (d, 2H, Jortho=8.7 Hz, Harom), 6.74 (s,
1H, Harom), 6.64 (s, 1H, Harom), 6.43 (t, 1H, J=2.1 Hz, Harom),
4.90(d, 1H, J=7.3 Hz, H-1), 4.45 (dd, 1H, J=1.8 and 11.7 Hz,
H-6), 4.28-4.21 (dd, 1H J=7.5 and 11.7 Hz, H-60), 3.73-3.68
(m, 1H, H-5), 3.51-3.45 (m, 2H, H-2, H-3), 3.45-3.33(m, 1H,
H-4); 2.30 (t, 2H, J = 7.5 Hz, CH2), 1.52-1.47 (m, 2H, CH2),
Animals and Experimental Design. C57BL/6J mice weighing
23 ( 2 g were provided by the Animal Centre of the University of
Murcia (Spain). All experiments were in accordance with the
recommendations of the European Union regarding animal
experimentation (Directive of the European Council 86/609/
EC) and approved by and performed according to the guidelines
of the Animal Ethics Committee of the University of Murcia.
1.25-1.16 (m, 8H, 4 ꢀ CH2); 0.86 (t, 3H, J=7.2 Hz, CH3). 13
C
NMR (MeOD, 75 MHz), δ ppm:174.1 (CdO), 158.8, 158.2,
157.1, 139.9 (Cqarom), 128.8 (CHdCH), 128.5, 127.8, 127.5

Article
Four types of assays were carried out (see more details in
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7375
of the experimental period described for inflammation assay-1
(Supporting Information Scheme S1).
Supporting Information Schemes S1, S2). “Inflammation assay-1”
(7 groups with n = 10 mice per group). In this study, animals
were fed with standard chow supplemented with each of the follow-
ing compounds: trans-resveratrol (1, RES group), trans-piceid
(2, PIC group), trans-resveratrol-3,5-di-O-β-D-glucopyranoside
(4, DIGLUC group), trans-resveratrol-3-O-(60-O-butanoyl)-β-
D-glucopyranoside (6, BUT group), and trans-resveratrol-3-
O-(60-O-butanoyl)-β-D-glucopyranoside (7, OCT group) for 29
days. Colitis was induced for the last 8 days by giving 1% DSS in
sterilized drinking tap water ad libitum. “Inflammation assay-2”
(7 groups, n =14 mice per group): each of the compounds was
coadministered with the DSS for 8 days (no pretreatment).
In both assays, the animal dose assayed for each compound
was 2.1 mg/kg day, which was equivalent to 10 mg of compound
for a 70 kg person according to the human equivalent dose
formula (HED31). Animals were randomly assigned to seven
groups, i.e., five groups with each of the compounds (1, 2, 4, 6, or 7
plus DSS), DSS group (only DSS, no compound), and control
group (only standard diet, no compound, no DSS).
The third type of experiment was designed to investigate the
bioavailability and metabolism of the different compounds in
healthy mice (metabolism assay-1). In this case, 84 mg of each
compound/kg was administered to the animals with an intra-
gastric probe using ethanol:water (5:95, v:v) as vehicle. Mice
were distributed in the same groups described above with n=24
animals per group. Three mice from each group were sacrificed
at each time-point after ingestion of the compounds (0.25, 0.5, 1,
2, 4, 8, 12, and 24 h), and plasma as well as the contents of
stomach, small intestine, and colon were analyzed by HPLC-
MS-MS to determine the presence and concentration of parent
compounds or derived metabolites.
The fourth and last experiment (metabolism assay-2) was
focused on the detection of resveratrol in the colon of mice
subjected to the treatment described for the inflammation assay-1
(SupportingInformationSchemeS1A).Afterprefeeding(2.1mg/kg
day) for three weeks with the compounds 1, 6, or 7 and colitis
induction with DSS for 8 days, animals were then given a high
dose (84 mg/kg) of the compounds 1, 6, or 7 with an intragastric
probe (n = 4 animals per group). Mice were sacrificed 4 h
postingestion of the compounds and their colon content ana-
lyzed by HPLC-MS-MS. Details for sampling procedure can be
found in the Supporting Information.
Disease Activity Index. A combinatorial index of disease, or
disease activity index (DAI), defined as stool blood, stool form,
and weight loss32 (see Supporting Information) was used to ana-
lyze the therapeutic benefit of the compounds 4, 6, and 7 as
compared to the standards 1 and 2.
Prostaglandin Assay. PGE2 levels were measured in distal
colon mucosa homogenates using an EIA immunoenzymatic
method (Cayman Chemicals, San Diego, CA,) according to
Larrosa et al.35 (see Supporting Information).
HPLC Analyses. Test compounds 1-10, cell media, plasma,
stomach, small intestine, and colon content samples were all
analyzed by HPLC (Agilent Technologies, Waldbronn, Germany),
equipped with a photodiode array detector and an HTC Ultra
ESI ion-trap mass detector in series (Bruker Daltonics, Bremen,
Germany). Chromatographic separations of compound-enriched
diet, cell media, blood, stomach, small intestine, and colon content
samples as well as the identification and quantification of resver-
atrol metabolites were carried out as reported by Larrosa et al.7
(see Supporting Information).
Antibody Arrays. Mucosa samples (40 mg) from distal colon
were homogenized in PBS containing proteases inhibitors
(Roche, Manhein, Germany). The mixtures were centrifuged
at 15000g for 30 min at 4 °C. Protein concentration was mea-
sured in the supernatant using the Bradford’s reagent. Colon
mucosa supernatants recovered from all the animals in each
group were pooled together to obtain 300 μg of protein and used
to measure the levels of the 40 cytokines represented in the
RayBio Mouse Inflammation Antibody Array 1 (RayBiotech,
Inc., Norcross, GA) according to the manufacturer’s protocol.
Measurements were carried out in duplicate. The cytokine pre-
sence was detected by chemiluminescence using the Chemidoc
XRS equipment (Biorad Laboratories, Barcelona, Spain). Arrays
were scanned with a densitometer and converted to densito-
metric units using the software ScanAlyze.37
Statistical Analysis. Differences for each parameter or marker
were evaluated using the ANOVA test followed by the Tukey
posthoc test. Mean values ( SD are shown. In the case of
histological scores, data are represented as mean ( SEM.
Statistical significance between differences in scores was calcu-
lated using the Kruskal-Wallis test and, when significant, the
Mann-Whitney’s U-test to test for differences between two
subscores. Statistic tests were carried out using the SPSS 15.0
software (SPSS Inc., Chicago, IL).
Acknowledgment. This work wassupportedbytheprojects
200670F0131 (CSIC), CICYT-BFU2007-60576, and CSD2007-
00063 (Fun-C-Food; Consolider Ingenio 2010). M.L. and R.L.
are holders of a JAE-DOC grant from CSIC and M.V.S of
ꢀ
a “Ramon y Cajal” grant from MCINN. J.T.C. is holder of
a predoctoral grant from MCINN.
Sampling Procedure. Cell media were processed as described
ꢀ
by Gonzalez-Sarrı
´
as et al.33 Blood, stomach, small intestine, and
colon samples were processed as described by Espı
Supporting Information)
´
n et al.34 (see
Supporting Information Available: In vitro anti-inflammatory
and cell metabolism assays; experimental design of inflamma-
tion and metabolism assays; evolution of mice weight; repre-
sentative pictures of C57Bl/6 mice after DSS treatment in the
absence or presence of the compounds 1, 6, or 7; evolution of
DAI values upon coadministration of DSS and each of the
compounds tested for 8 days without pretreatment with the
compounds for 21 days; histological scores for evaluating
colonic tissue damage; mouse inflammation antibody array;
IL-3 levels colon mucosa; absorption, metabolism and gastro-
intestinal transit kinetics of resveratrol derivatives in mice
and AUC and Cmax values. Supplementary Methods: In vitro
anti-inflammatory activity, cell uptake and metabolism, cell
viability, animals and experimental design, disease activity
index and animal appearance, sampling procedure, haptoglo-
bin and fibrinogen, prostaglandin assay, histological analysis
of colon samples, fecal microbiota analysis and HPLC-MS-
Haptoglobin and Fibrinogen. Serum concentrations of hapto-
globin and fibrinogen were determined spectrophotometrically
(see Supporting Information).
Histological Analyses. Tissue samples from the distal colon
were processed as reported elsewhere35 and evaluated under a
light microscope (see Supporting Information). The mucosal
damage scoring system of Araki et al.36 was used to evaluate the
degree of colitis. The samples were graded 0-4 for surface
epithelial loss, crypt destruction, and inflammatory cell infiltra-
tion into the mucosa (maximum score=12).
Fecal Microbiota Analysis. Microbial counts were determined
in fecal samples according to Larrosa et al.7 Total bifidobacteria,
lactobacilli, clostridia, enterobacteria, and E. coli were determined
in samples from the control group and DSS-treated group as
well as in the groups pretreated with 1, 6, or 7. In the groups
pretreated with 2 and 4 only bifidobacteria and lactobacilli were
determined. Microbial counts were determined on days 0, 21
(before DSS administration), and 29 (after DSS administration)
1
MS analysis. H and 13C NMR spectra of compounds 6-10.
This material is available free of charge via the Internet at
http://pubs.acs.org.

7376 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Larrosa et al.
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