Antidiabetic ellagitannins from pomegranate flowers: Inhibition of α-glucosidase and lipogenic gene expression

Article abstract of DOI:10.1021/ol302548c

Two new ellagitannins containing a rare 3-oxo-1,3,3a,8b-tetrahydrofuro[3,4- b]benzofuran moiety, namely punicatannins A (1) and B (2), were isolated from pomegranate (Punica granatum) flowers. Their structures with absolute configuration were determined by detailed analysis of spectroscopic data, electronic circular dichroism (ECD) calculation, and chemical hydrolysis. A plausible biogenetic route involving a key enzymatic 1,4-Michael addition is proposed. Punicatannin A showed potent inhibition of α-glucosidase and lipogenic gene expression.

Full text of DOI:10.1021/ol302548c

ORGANIC
LETTERS
2012
Vol. 14, No. 20
5358–5361
Antidiabetic Ellagitannins from
Pomegranate Flowers: Inhibition of
r‑Glucosidase and Lipogenic Gene
Expression
Tao Yuan,† Yuanqing Ding,‡ Chunpeng Wan,† Liya Li,† Jialin Xu,† Ke Liu,§
Angela Slitt,† Daneel Ferreira,*,‡ Ikhlas A. Khan,*,‡ and Navindra P. Seeram*,†
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,
University of Rhode Island, Kingston, Rhode Island 02881, United States,
National Center for Natural Products Research and Department of Pharmacognosy,
School of Pharmacy, University of Mississippi, University, Mississippi 38677,
United States, and Department of Medicinal Chemistry, University of Kansas,
Lawrence, Kansas 66045, United States
dferreir@olemiss.edu; ikhan@olemiss.edu; nseeram@uri.edu
Received September 15, 2012
ABSTRACT
Two new ellagitannins containing a rare 3-oxo-1,3,3a,8b-tetrahydrofuro[3,4-b]benzofuran moiety, namely punicatannins A (1) and B (2), were
isolated from pomegranate (Punica granatum) flowers. Their structures with absolute configuration were determined by detailed analysis of
spectroscopic data, electronic circular dichroism (ECD) calculation, and chemical hydrolysis. A plausible biogenetic route involving a key
enzymatic 1,4-Michael addition is proposed. Punicatannin A showed potent inhibition of R-glucosidase and lipogenic gene expression.
The prevalence of diabetes is increasing rapidly world-
wide. The International Diabetes Federation predicts that
over 435 million people worldwide will be struggling with
this disease by 2030 if no effective prevention and control
programs are implemented. This global pandemic is driven
by type 2 diabetes which contributes to the vast majority of
all cases of diabetes.1 While several synthetic drugs includ-
ing sulfonylureas, biguanides, thiazolidinediones, and R-
glucosidase inhibitors are available to treat type 2 diabetes,
many have limited efficacy with significant associated side
effects. This has led to the search for more effective com-
pounds with less side effects, particularly natural products
and dietary agents, for the management of type 2 diabetes.
Punica granatum L. (Punicaceae), commonly known as
pomegranate, is commercially cultivated for its edible fruit
throughout the drier parts of Southeast Asia, the Medi-
terranean region, and the UnitedStates. The flowers of this
plant were used as a remedy for diabetes in the Unani and
Ayurvedic medicinal systems.2 Moreover, a pomegranate
flower extract was reported to markedly lower blood
† University of Rhode Island.
(2) Huang, T. H. W.; Peng, G.; Kota, B. P.; Li, G. Q.; Yamahara, J.;
Roufogalis, B. D.; Li, Y. Toxicol. Appl. Pharmacol. 2005, 207, 160–169.
(3) Li, Y.; Wen, S.; Kota, B. P.; Peng, G.; Li, G. Q.; Yamahara, J.;
Roufogalis, B. D. J. Ethnopharmacol. 2005, 99, 239–244.
‡ University of Mississippi.
§ University of Kansas.
(1) Zimmet, P.; Alberti, K. G.; Shaw, J. Nature 2001, 414, 782–787.
r
10.1021/ol302548c
Published on Web 10/10/2012
2012 American Chemical Society

glucose levels in the Zucker diabetic fatty rat model.3
However, there is limited knowledge on pomegranate
flower constituents4 and the bioactive antidiabetic com-
pounds present therein remain unknown.
small coupling constants of the protons of the glucopyr-
anosyl unit indicated its 1C4 conformation with the anome-
ric acyl group in an R-equatorial orientation consistent
with isocorilagin (3). Analysis of HMBC data (Figure 1)
permitted assignment of the galloyl and HHDP groups to
C-1 and C-3, 6, respectively, by the correlations from H-1
to C-70, from H-3 to C-7000, and from H2-6 to C-70000. The
latter correlations further supported the 1C4 conformation
of the glucopyranosyl moiety carrying an HHDP unit at its
C-3 and C-6 oxygen functions.7
In continuation of our characterization of new antidia-
betic compounds from natural sources,5 this study sought to
identify the bioactive compounds present in pomegranate
flowers. Preliminary screening of a pomegranate flower
extract showed promising R-glucosidase inhibitory activity
(IC50 = 3.2 μg/mL). Subsequent bioassay-guided isolation
afforded two unprecedented ellagitannins, named punica-
tannins A (1) and B (2), both containing a rare 3-oxo-
1,3,3a,8b-tetrahydrofuro[3,4-b]benzofuran moiety attached
to a (1C4)-glucopyranose core, along with a biosynthetically
related compound, isocorilagin (3).6 Herein we present the
details of the isolation and structure elucidation of 1 and 2,
the evaluation of their antidiabetic activities, and a plausible
biosynthetic pathway for these two compounds.
1
Figure 1. 1Hꢀ H COSY (bold line) and key HMBC correlations
(HfC) of 1.
The remaining part of the molecule was established by
analysis of 1D and 2D NMR data. Apart from the above
functionalities, carbon resonancesforsixaromaticcarbons
(δC 112.7, 118.6, 119.1, 136.4, 147.8, 148.6), four ester
carbonyls (δC 165.4, 169.9, 171.1, 175.0), two methines
(δC 51.5, 80.9), an oxgenated quaternary (δC 89.6), one
methylene (δC 39.3), and two O-methyls (δC 52.8, 53.0)
were observed in the 13C NMR spectrum. The chemical
shifts of the aromatic carbon signals implied the presence
ofa pyrogallol ring. The HSQCspectrum permitted assign-
ment of the protons to their bonding carbons, and the
Compound 1 was isolated as a tan amorphous powder.
The standardcolor reaction withferric chloride(darkblue)
and sodium nitriteꢀacetic acid (reddish brown) suggested
that compound 1 was most likely an ellagitannin. Its
molecular formula was established as C43H34O28 with an
index of hydrogen deficiency of 27 based on the HR-ESI-
MS exhibiting an [MꢀH]ꢀ ion at m/z 997.1151 (calcd for
1
1Hꢀ H COSY and HMBC spectra (Figure 1) were applied
1
C43H33O28, 997.1158). The H and 13C NMR spectra
to construct the structure of the remaining part of the
[Supporting Information (SI) Table S2] of 1 showed
signals due to a highly acylated hexopyranosyl moiety
along with aromatic and carboxylic carbon resonances,
strongly indicative of compound 1 being a hydrolyzable
tannin. The presence of galloyl and hexahydroxydiphenoyl
(HHDP) groups was readily determined by the 1H NMR
signals at δH = 7.08 (2H, s), δH = 6.72 (s), 6.83 (s) and
characteristic13C NMR signals in thearomatic region. The
1
1
molecule. The Hꢀ H COSY correlation between H-8b00
and H-100 revealed the linkage of C-8b00 and C-100. In the
13
HMBC spectrum, the mutual 1Hꢀ C correlation between
an aromatic singlet (δH = 7.12, H-700) and the C-1200 ester
carbonyl carbon (δC 165.4), C-500, and C-8a00, indicated
that the C-1200 ester carbonyl was linked at C-800 of a
pyrogallol-type ring. The HMBC correlations from H-8b00
to C-4a00, C-800, and C-8a00, suggested that the CH-8b00
methine group was attached at C-8a00 of the pyrogallol-
type ring. The HMBC correlations from H2-1000 to C-300
(δC 175.0), C-3a00 (δC 89.6), and C-8b00 (δC 51.5) indicated
the contiguous linkage of C-300, C-3a00, and C-8b00 and also
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68, 959–964. (b) Wan, C.; Yuan, T.; Li, L.; Kandhi, V.; Cech, N. B.; Xie,
M.; Seeram, N. P. Bioorg. Med. Chem. Lett. 2012, 22, 597–600.
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Org. Lett., Vol. 14, No. 20, 2012
5359

allowed the attachment of CH2-1000 to C-3a00. A methox-
ycarbonyl group was attached to both C-1000 and C-100
based on the HMBC correlations from H2-1000 and the O-
methyl proton signals (δH = 3.79) to C-1100 and from
H-8b00, H-100, and the O-methyl proton signals (δH = 3.31)
to C-900. The linkage of C-100 and C-300 via oxygen was
determined by the HMBC correlation between H-100 and
C-300. The index of hydrogen deficiency of 26 in compound
1 was accounted for by the isocorilagin moiety (17), four
carbonyl groups (4), a pyrogallol ring (4), and a furan ring
(1). The remaining index of hydrogen deficiency required
the presence of an additional ring. Analysis of the 13C
NMR data suggested the presence of an ether bridge
between oxygenated quaternary carbon C-3a00 and aro-
matic carbon C-4a00 to furnish a ring. Thus, the molecular
structure of the remaining part was determined as the rare
5,6-dihydroxy-3a-(2-methoxy-2-oxoethyl)-1-(methoxycar-
bonyl)-3-oxo-1,3,3a,8b-tetrahydrofuro[3,4-b]benzofuran-
8-carboxylic acid moiety.8 Finally, the HMBC correlation
between H-4 and C-1200 facilitated the placement of this
structural unit at C-4 of the glucopyranosyl core via an
ester bridge.
issue.11 The mandatory assigned (100R, 3a00R, 8b00R)-con-
figuration was selected for the conformational search of 1a
by using the OPLS2005 force field in the Schrodinger 9.2
MacroModel software package. After the detailed calcu-
lated ECD analysis (SI S4.1), the (100R, 3a00R, 8b00R)
absolute configuration of partial structure 1a was unequi-
vocally confirmed.
Compound 2 was isolated from the same fraction as 1
and had the same molecular formula (C43H34O28) and
similar UV, IR, 1H, and 13C NMR spectra as compound 1.
1
A combined analysis of H, 13C NMR (SI Table S2),
1
1Hꢀ H COSY, HSQC, and HMBC data of 2 indicated
that it possessed the same molecular structure as 1. The
glucopyranosyl core also had the same conformation and
D-configuration. The experimental ECD spectrum exhib-
ited a negative Cotton effect at 233 nm indicative of an
HHDP moiety with an (R)-configured biphenyl bond. The
anomeric galloyl group was assigned an R-equatorial
orientation based on the ca. 1.0 Hz coupling constant of
theanomericproton. The 5,6-dihydroxy-3a-(2-methoxy-2-
oxoethyl)-1-(methoxycarbonyl)-3-oxo-1,3,3a,8b-tetrahy-
drofuro[3,4-b]benzofuran-8-carboxylic acid moiety also
showed the same relative configuration as 1. Thus, 1 and 2
only differ as far as the absolute configuration of this rare
structural moiety is concerned. Compound 2 is, thus, a
(C-100, C-3a00, C-8b00) quasi-enantiomer of compound 1
and accordingly named punicatannin B.
The relative configuration of the 5,6-dihydroxy-3a-(2-
methoxy-2-oxoethyl)-1-(methoxycarbonyl)-3-oxo-1,3,3a,
8b-tetrahydrofuro[3,4-b]benzofuran-8-carboxylic acid moi-
ety was determined by the ROESY spectrum and the
H-100ꢀH-8b00 coupling constant (9.6 Hz) reminiscent of
the cis-configuration of these protons.9 The ROESY corre-
lation between H-8b00 and H-1000 indicated that H-8b00 and
the C-3a00 2-methoxy-2-oxoethyl group were cofacial.
A combination of electronic circular dichroism (ECD)
analyses, chemical hydrolysis, and calculated ECD spectra
were used to elucidate the absolute configuration of com-
pound 1. First, the HHDP moiety was assigned an R-
configuration by the negative Cotton effect at 234 nm.
Notably, a Cotton effect at 235 nm is a diagnostic criterion
in determining the absolute configuration of HHDP units
in ellagitannins, which is unaffected by the presence or
absence of galloyl groups and the conformation of the
glucose core.10 Acid hydrolysis of 1 afforded D-glucose,
which was identified by direct comparison with an authen-
tic sample (SI S1). However, the challenging issue to
determine the absolute configuration of the acyl group
linked at C-4 in compound 1 still remained. Hydrolysis of 1
Scheme 1. Postulated Biosynthesis of the 4-Acyl Group of
Punicatannins A (1) and B (2)
1
in hot water yields 1a, which was confirmed by the H
A possible biosynthetic pathway to the 5,6-dihydroxy-
3a-(2-methoxy-2-oxoethyl)-1-(methoxycarbonyl)-3-oxo-
1,3,3a,8b-tetrahydrofuro[3,4-b]benzofuran-8-carboxylic
acid moiety of compounds 1and 2is postulated in Scheme 1.
Its biosynthetic precursor is proposed to be an HHDP
group, similar to that in, e.g., compound 1, which is
converted into A by oxidation and ketoꢀenol tautomer-
ism. Intermediate A would be cleaved into ketene B by
hydrolysis.12 This may be hydrolytically transformed into
the enantiomeric intermediates C1 and C2 that may under-
go a 1,4-Michael addition reaction to yield intermediates
D1 and D2. These may be converted into E1 and E2 by
NMR and mass spectra (SI S20 and S21). As the absolute
configuration of 1a could not be resolved directly by the
analysis of its ECD curve, comparison between experi-
mental and calculated ECD spectra using the time-depen-
dent DFT method was successfully applied to resolve this
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Biology of Ellagitannins, An Underestimated Class of Bioactive Plant
Polyphenols; Quideau, S., Ed.; World Scientific Publishing: Singapore,
2009; Vol. 1, pp 1ꢀ54. (b) Ding, Y.; Li, X. C.; Ferreira, D. J. Nat. Prod.
2009, 72, 327–335.
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Org. Lett., Vol. 14, No. 20, 2012

glucose (Figure 2). This suggests that compound 1 may
repress the induction of lipogenic gene expression by high
glucose exposure and could aid in the prevention of high
glucose induced de novo lipogenesis and subsequently
inhibit lipid accumulation.
Table 1. R-Glucosidase Inhibitory Activities of Compounds 1ꢀ3
no.
IC50 (μM)a
1
2
3
7.15 ( 0.27
12.39 ( 0.05
104.89 ( 7.73
208.60 ( 3.39
acarboseb
a IC50 values are shown as mean ( SD from three independent
experiments. b Positive control.
trans-esterification. Methyl esterification of E1 and E2
would lead to the structural moieties F1 and F2 of com-
pounds 1 and 2.
Given the ethnomedicinal uses of pomegranate flowers
for treating diabetes, we evaluated the isolated compounds
for biological activities. First, compounds 1ꢀ3 and the
clinical drug, acarbose, were tested for R-glucosidase
inhibitory activities. This enzyme is a carbohydrate hydro-
lase that breaks down starch and R-linked di-, oligo-, and
polysaccharides to glucose leading to elevated blood glu-
cose levels. Thus, inhibition of the enzyme decreases the
rate of digestion of carbohydrates and less glucose is
absorbed. As shown in Table 1, compounds 1ꢀ3 showed
potent and concentration-dependent R-glucosidase inhibi-
tory activities (SI Figure S3). Remarkably, punicatannin A
Figure 2. Compound 1 prevents elevation of Acc1, Scd1, and
CD36 gene expression in HepG2 cells exposed to high glucose.
(*) P < 0.05, group of high glucose compared to group of low
glucose treatments in absence of compound 1. (#) P < 0.05,
group of high glucose compared to group of low glucose
treatments in presence of compound 1 (50 μM). ($) P < 0.05,
group of high glucose compared to group of low glucose
treatments in presence of compound 1 (100 μM).
(1) was ca. 30 times more potent than acarbose (IC50
7.15 vs 208.60 μM, respectively).
=
Finally, based on compound availability, we selected
compound 1 to evaluate its ability to inhibit lipogenic gene
expression. It is known that obesity associated with insulin
resistance and type 2 diabetes increases the risk of hepatic
lipid accumulation. Lipid accumulation is also associated
with increased de novo lipogenesis, and high glucose con-
centrations have been shown to induce lipid accumulation
in HepG2 cells.13 This is known to occur through the
transcription factor, Srebp1c, which along with the lipo-
genic enzymes Acc-1, Fas, and Scd1 regulate lipogenesis.
Furthermore, loss of Scd1 protects against obesity induced
by decreased de novo lipogenesis,14 and forced CD36
expression increases fatty acid uptake in HepG2 cells.15
Here we showed that compound 1 prevents Acc1, Scd1,
and CD36 induction in HepG2 cells exposed to high
In summary, this work describes the discovery of two
new ellagitannins containing the rare 3-oxo-1,3,3a,8b-
tetrahydrofuro[3,4-b]benzofuran moiety. The abilities of
these compounds to inhibit R-glucosidase enzyme activity
and lipogenic gene induction could account for the re-
ported antidiabetic properties of pomegranate flowers.
Furthermore, the structure identified in these natural
compounds could serve as a scaffold for the synthesis of
structural analogs with enhanced antidiabetic activities.
Supporting Information Available. General experimen-
tal procedures, plant materials, extraction and isolation
of compounds, hydrolysis of compound 1 and sugar
analysis, R-glucosidase and lipogenic gene expression
inhibition assays, calculated ECD analysis, tabulated
NMR data and full NMR, MS spectra for compounds
1 and 2, and 1H NMR and MS spectra for compound 1a.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Herscovitz, H.; Brecher, P.; Ruderman, N. B.; Cohen, R. A. J. Biol.
Chem. 2004, 279, 47898–47905.
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1018–1024.
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J.; Ehehalt, R. Int. J. Med. Sci. 2011, 8, 599–614.
The authors declare no competing financial interest.
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