Biotransformation of resveratrol to piceid by Bacillus cereus

Article abstract of DOI:10.1021/np980139b

Microbial transformation of resveratrol (1), trans-3,4',5- trihydroxystilbene, was studied. Preparative scale biotransformation of 1 with whole-cell suspensions of Bacillus cereus UI 1477 resulted in the production of metabolite 2 which was identical in a

Full text of DOI:10.1021/np980139b

J . Nat. Prod. 1998, 61, 1313-1314
1313
Biotr a n sfor m a tion of Resver a tr ol to P iceid by Ba cillu s cer eu s
Robert H. Cichewicz and Samir A. Kouzi*
Division of Basic Pharmaceutical Sciences, School of Pharmacy, Northeast Louisiana University, Monroe, Louisiana 71209
Received April 10, 1998
Microbial transformation of resveratrol (1), trans-3,4′,5-trihydroxystilbene, was studied. Preparative scale
biotransformation of 1 with whole-cell suspensions of Bacillus cereus UI 1477 resulted in the production
of metabolite 2 which was identical in all respects to an authentic sample of piceid, resveratrol 3-O-â-
D-glucoside.
Resveratrol (1) (trans-3,4′,5-trihydroxystilbene), a con-
B. cereus UI 1477 was the only organism capable of
stituent of several food products and medicinal plants, is
a potent phytoalexin which displays an array of pharma-
biotransforming 1 to a more polar metabolite. A prepara-
tive scale biotransformation of 1 using whole-cell suspen-
sions of B. cereus afforded metabolite 2 as a pure compound
in 11% yield. Metabolite 2 was isolated and purified by
preparative TLC. The FAB mass spectrum of metabolite
1
2
3
cological and antimicrobial activities. It exhibits strong
4
in vitro antioxidant activity; however, its in vivo role is
questionable.5 In addition, studies have demonstrated that
resveratrol inhibits platelet aggregation in vitro and in
+
2, MH at m/z 391, indicated that it was a glucoside
6
vivo which may contribute to its potential cardioprotective
conjugate of 1. The physical and spectral data of 2 were
consistent with the reported data for piceid.1
4-17
In addi-
properties. Other investigations have suggested that res-
veratrol is capable of inhibiting certain steps in arachido-
nate metabolism7 and may possess antiinflammatory ac-
tivity. Further studies indicate that 1 is an inhibitor of
tion, metabolite 2 was identical in all respects to an
authentic sample of piceid confirming that 2 is the 3-O-â-
D-glucoside conjugate of 1.
8
+
+
9
monoamine oxidase A and gastric H , K -ATPase. Res-
veratrol (1) has also demonstrated cancer chemopreventive
properties.10
Piceid (2) has previously been isolated from a variety of
14
18
plant species and synthesized.
Piceid has also been
4,7,8,14,18
reported to exhibit a number of biological activities.
Recently, a dehydrodimer has been reported as a trans-
formation product of 1 by the grapevine pathogen Botrytis
cinerea.19 This is the first report of preparing piceid (2)
from 1 using a microorganism as a biocatalyst. Further
microbial transformation studies of 1 are in progress.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined in open capillary tubes with a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Opti-
cal rotations were measured with a Perkin-Elmer 241 digital
polarimeter. IR spectra were recorded in KBr using a Nicolet
Impact 400D FT-IR spectrophotometer. UV spectra were
recorded in MeOH using a Gilford spectrophotometer. Cen-
trifugation of whole-cell suspensions and microbial cultures
was carried out using a Heraeus Megafuge 2.0R centrifuge at
4 °C and 2724g. The term in vacuo refers to removal of solvent
with a rotary evaporator under water aspirator vacuum (15-
30 mmHg). 1H and C NMR spectra were obtained in acetone-
Despite the widespread human exposure to 1, there have
been no reports on the mammalian metabolism of this
bioactive stilbene. A study showed that 1 is likely to
undergo metabolic transformation as it accumulates in the
livers of rats following oral administration.11 To facilitate
the mammalian metabolism studies of 1, microorganisms
are being utilized in our laboratory as in vitro models to
mimic and predict the metabolic fate of resveratrol in
mammalian systems. Furthermore, we are interested in
utilizing microbial cultures as in vitro biocatalysts to
prepare new and potentially active analogues of 1 since
13
d
4
6
on a J EOL-Eclipse 400 FT-NMR spectrometer operating at
00 and 100 MHz, respectively. Standard pulse sequences
were used for COSY, HETCOR, DEPT, and APT experiments.
Low- and high-resolution FAB mass spectra were obtained
using a VG 70-SEQ mass spectrometer (VG Analytical,
Manchester, England) at the Mass Spectrometry Facility,
Department of Medicinal Chemistry, University of Washing-
ton, Seattle, WA 98195.
many of its known analogues have demonstrated equal or
greater bioactivity.1
2,13
In the present study we describe
the isolation and structure elucidation of piceid (2), res-
veratrol 3-O-â-D-glucoside, as a microbial metabolite of
resveratrol (1) from whole-cell suspensions of Bacillus
cereus UI 1477.
A total of 12 microbial cultures were screened for their
ability to biotransform resveratrol (1). On the basis of
comparative TLC analyses of test cultures and controls,
Ch r om a togr a p h ic Con d ition s. TLC analyses were car-
ried out on precoated Silica G-25 UV254 plates (E. Merck,
Darmstadt). Visualization of TLC plates was performed using
1
0% phosphomolybdic acid in absolute ethanol as a spray
reagent. Spots were visualized by spraying the plates and
then heating them at 110 °C for 3 min in an oven. The solvent
system used for developing TLC plates was a solution of
EtOAc-hexane-acetic acid (8:1:1 v/v). Preparative TLC
separations were carried out on 20 × 20 cm precoated Silica
Gel 60 F254 plates with 2 mm layer thickness (E. Merck,
*
To whom correspondence should be addressed. Phone: (318)342-1693.
Fax: (318)342-3286. E-mail: pykouzi@alpha.nlu.edu.
1
0.1021/np980139b CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/19/1998

1
314 J ournal of Natural Products, 1998, Vol. 61, No. 10
Notes
Darmstadt). Visualization of preparative TLC plates was
performed under UV light (254 nm).
Micr oor ga n ism . The B. cereus UI 1477 culture was
obtained from the Department of Medicinal and Natural
Products Chemistry, College of Pharmacy, University of Iowa,
Iowa City, IA.
vacuo yielding 662 mg of a yellow residue. The residue was
dissolved in a minimal amount of EtOAc:MeOH (3:1) and
chromatographed on 2 preparative TLC plates. Metabolite 2
was visualized under UV light (254 nm) as an intense violet
f
band (R 0.15), and the silica from this region was removed
from the plate. The silica (8 g) was crushed into a fine powder
and extracted with EtOAC:MeOH (3:1) (8 × 4 mL/1 g silica).
The solvent was then aspirated, filtered, and evaporated in
vacuo. The resulting 454 mg of light yellow residue was again
chromatographed on 2 preparative TLC plates under identical
conditions, yielding 298 mg of off-white foamy powder. Crys-
Med ia . All preliminary screening experiments were carried
out in a medium consisting of dextrose, 20 g; yeast extract, 5
g; peptone, 5 g; NaCl, 5 g; K
2 4 2
HPO , 5 g; and distilled H O,
1
000 mL. Stock cultures of fungi and bacteria were stored on
slants of Mycophil and Eugon (Difco, Detroit, Michigan) agar,
respectively, at 4 °C. The 0.1 M phosphate buffer (pH 7.2)
2 2
tallization from H O:Me CO (9:1) yielded 121 mg of 2 as white
1
4
used for whole-cell suspensions of B. cereus consists of K
0.6 g; KH PO , 4.08 g; dextrose, 20 g; and distilled H O, 1000
mL.
2
HPO
4
,
needles (11% yield): mp 136-138 °C (lit. mp 135-137 °C);
16 25
1
2
4
2
[R]25
D
-51.8°(c 0.5 g/100 mL, MeOH) (lit. [R]
D
-60°);
+
FABMS (positive) m/z 391 [MH] ; HR FABMS (positive) m/z
F er m en ta tion P r oced u r es. Microbial metabolism studies
were carried out by incubating the cultures with shaking on
an Innova 5000 Gyrotory shaker (New Brunswick Scientific
Co., NJ ), operating at 150 rpm and 25 °C. Preliminary
screening experiments were carried out in 125-mL stainless
steel capped DeLong culture flasks containing 25 mL of
medium. The media were sterilized at 121 °C and 18 psi for
391.1403 (calcd for C20
H
23
O
8
, 391.1392); spectral data (IR, UV,
1
13
H and C NMR) of 2 were in agreement with those reported
in the literature for piceid.1
4,15
Metabolite 2 was identical in
all respects to an authentic sample of piceid.
Ack n ow led gm en t. Financial support from the National
Cancer Institute is greatly appreciated. The purchase of the
4
00 MHz FT-NMR spectrometer was funded by the Louisiana
1
5 min. Fermentations were carried out according to a
Education Quality Support Fund and Northeast Louisiana
University. The authors wish to thank Professor J ean-Michel
M e´ rillon, Facult e´ des Sciences Pharmaceutiques, Universit e´
de Bordeaux 2, Bordeaux, France, for kindly providing us with
the authentic sample of piceid.
standard two-stage protocol. In general, the substrate was
added in DMF (1 mg/10 µL) to the incubation media 24 h after
inoculation of stage II cultures at a concentration of 0.2 mg/
mL. The fermentations were sampled at 24 h intervals by
extracting 3 mL of the culture broth with 3 × 3 mL of EtOAc.
The extracts were concentrated and chromatographed on TLC
plates. Substrate controls were composed of sterile media to
which the substrate was added and incubated without micro-
organisms. Culture controls consisted of fermentation blanks
in which the microorganisms were grown under identical
conditions without the addition of the substrate. Substrate-
autoclaved culture controls consisted of microbial cultures that
were grown under the usual conditions to maturity (usually
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chased from Sigma Chemical Co. (St. Louis, MO) and Phar-
mascience Inc. (Montreal, QC). The physical and spectral data
1
39.
of 1 have been widely reported in the literature.1
4,17
A
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39.
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bioconversion of 1 to 2. Twenty 2-L flasks, each containing
(
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00 mL of sterile medium, were inoculated with 3 mL of 72 h
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stage I B. cereus culture and incubated on a shaker for 72 h.
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centrifuged at 2724g for 18 min at 4 °C. The supernatant
portions were decanted and discarded. Each pellet from the
aliquots was gently rinsed with 10 mL of sterile phosphate
buffer before being resuspended in 200 mL of dextrose-
enriched phosphate buffer (filter-sterilized, 0.22 µm). Whole-
cell suspensions of B. cereus from each aliquot were then placed
separately into 16 1-L flasks. Forty milligram portions (in 400
µL DMF) of resveratrol were added to each flask before
incubating on the shaker. All incubations and extraction
procedures were conducted in covered vessels in order to avoid
UV-induced isomerization of 1 and 2.
2
20.
(
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Following a 72 h incubation, 500 mL aliquots of the whole-
cell suspensions were centrifuged under the previously out-
lined conditions. The pellets were each rinsed twice with 50
mL of EtOAc. One-liter portions of the supernatant (the
buffer) were extracted with 3 × 330 mL of EtOAc. The EtOAc
8
13-816.
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(
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extract was dried over Na
2
SO
4
, filtered, and evaporated in
NP980139B