Glycosylation of trans-resveratrol by plant-cultured cells

Article abstract of DOI:10.1271/bbb.120126

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-β-D-glucopyranoside, 40-O-β-D-glucopyranoside, 3-O-(6-O-β-D-xylopyranosyl)-β-Dglucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-β- D-glucopyranoside. The 3-O-(6-O-β-D-x

Full text of DOI:10.1271/bbb.120126

Biosci. Biotechnol. Biochem., 76 (8), 1552–1554, 2012
Note
Glycosylation of trans-Resveratrol by Plant-Cultured Cells
Hiroya IMAI,1 Megumi KITAGAWA,1 Kohji ISHIHARA,1 Noriyoshi MASUOKA,1
y
y
1;
Kei SHIMODA,2 Nobuyoshi NAKAJIMA,3; and Hiroki HAMADA
1Department of Life Science, Faculty of Science, Okayama University of Science,
1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan
2Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
3Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization,
Okayama Prefectural University, Soja, Okayama 719-1197, Japan
Received February 23, 2012; Accepted April 27, 2012; Online Publication, August 7, 2012
[doi:10.1271/bbb.120126]
Plant-cultured cells of Catharanthus roseus converted
trans-resveratrol into its 3-O-ꢀ-D-glucopyranoside, 40-O-
ꢀ-D-glucopyranoside, 3-O-(6-O-ꢀ-D-xylopyranosyl)-ꢀ-D-
glucopyranoside, and 3-O-(6-O-ꢁ-L-arabinopyranosyl)-ꢀ-
D-glucopyranoside. The 3-O-(6-O-ꢀ-D-xylopyranosyl)-ꢀ-
D-glucopyranoside and 3-O-(6-O-ꢁ-L-arabinopyranosyl)-
ꢀ-D-glucopyranoside compounds of trans-resveratrol are
both new. Incubation of plant-cultured cells of Ipomoea
batatas and Strophanthus gratus with trans-resveratrol
gave trans-resveratrol 3-O-ꢀ-D-glucopyranoside and
trans-resveratrol 40-O-ꢀ-D-glucopyranoside.
with suction. The cells were extracted (ꢁ3) by homog-
enization with MeOH, and the resulting extract was
concentrated. The residue was partitioned between H2O
and EtOAc. The H2O layer was applied to a Diaion HP-
20 column, and the column was washed with H2O and
then eluted with MeOH. The MeOH eluate was subjected
to HPLC in a 150 ꢁ 20 mm column to give the products.
No products were apparent in the medium. A control
experiment using cells which had not been treated with
the substrate, resulted in no detection of the substrate or
products. The yield of the products was determined on
the basis of the peak area from HPLC, and is expressed as
a percentage relative to the total amount of whole
reaction products extracted and the substrate. Large-scale
biotransformation of a total of 500 mg of the substrate in
ten 1-L flasks each containing 500 mL of the medium
prepared the products for NMR analyses.
After a 5 d incubation period, products 2 (37%), 3
(15%), 4 (2%), and 5 (1%) were obtained from the
MeOH extract of the C. roseus cells treated with 1. The
structures of products 2 and 3 were determined to be
those of trans-resveratrol 3-O-ꢀ-D-glucopyranoside and
trans-resveratrol 40-O-ꢀ-D-glucopyranoside on the basis
of their HRFABMS, 1H and 13C NMR, H-H COSY,
C-H COSY, HMBC, and NOE spectra. The 1H- and
13C-NMR, H-H COSY, C-H COSY, and HMBC spectra
were recorded in a DMSO-d6 solution by using a Varian
XL-400 spectrometer and the chemical shifts are ex-
pressed in ꢂ (ppm), referring to TMS. Products 4 and 5
were identified as trans-resveratrol 3-O-(6-O-ꢀ-D-xylo-
pyranosyl)-ꢀ-D-glucopyranoside and trans-resveratrol
Key words: biotransformation; trans-resveratrol; plant-
cultured cell
trans-Resveratrol is one of the most important plant
polyphenols and has attracted considerable pharmaceut-
ical interest because of its diverse biological activities.1)
However, the water-insoluble nature of trans-resveratrol
limits its further pharmacological exploitation. How-
ever, its water-soluble derivatives, i.e., trans-resveratrol
3-O-ꢀ-D-glucoside and trans-resveratrol 40-O-ꢀ-D-glu-
coside, have been reported to show such pharmaceutical
properties as cancer prevention,2,3) anti-oxidative activ-
ity,4,5) and estrogenic activity.6,7) Several attempts have
recently been made to synthesize resveratrol glycosides
by chemical methods involving tedious protection-
deprotection procedures, and resulted in low yields.8,9)
Plant cell cultures would be useful for the practical
preparation of glycosides, due to the high potential of
plant glycosyltransferases to diastereoselectively pro-
duce glycosides by one-step enzymatic glycosylation.
We report here the biotransformation of trans-
resveratrol by plant-cultured cells to trans-resveratrol
3-O-(6-O-ꢀ-D-xylopyranosyl)-ꢀ-D-glucopyranoside and
trans-resveratrol 3-O-(6-O-ꢁ-L-arabinopyranosyl)-ꢀ-D-
glucopyranoside which are both new compounds.
3-O-(6-O-ꢁ-L-arabinopyranosyl)-ꢀ-D-glucopyranoside.
These disaccharide products 4 and 5 have not been
identified before. The yield of products 4 and 5 was
lower than that of previously reported 3-O-(6-O-ꢀ-D-
xylopyranosyl)-ꢀ-D-glucopyranosides and 3-O-(6-O-ꢁ-
L-arabinopyranosyl)-ꢀ-D-glucopyranosides of capsaici-
noids, which had been produced by incubating of
C. roseus cells with capsaicinoids,10) probably due to
the substrate specificity of the enzymes participating in
the formation of the 3-O-(6-O-ꢀ-D-xylopyranosyl)-ꢀ-D-
glucopyranosides and 3-O-(6-O-ꢁ-L-arabinopyranosyl)-
ꢀ-D-glucopyranosides. The spectral data for products 4
and 5 follow.
The substrate, trans-resveratrol (1), was biotrans-
formed by using plant-cultured cells as biocatalysts. To
a 500-mL flask containing 200 mL of the culture medium
and suspension-cultured cells (100 g) was added 15 mg
of the substrate. The culture was incubated at 25 ꢀC for
5 d on a rotary shaker (120 rpm). After the incubation
period, the cells and medium were separated by filtration
y
To whom correspondence should be addressed. Nobuyoshi NAKAJIMA, Tel/Fax: +81-866-94-2157; E-mail: nkmt-nakajima@fhw.oka-pu.ac.jp;
Hiroki HAMADA, Tel: +81-86-256-9473; Fax: +81-86-256-8468; E-mail: hamada@dls.ous.ac.jp

Glycosylation of trans-Resveratrol
1553
trans-Resveratrol 3-O-(6-O-ꢀ-D-xylopyranosyl)-ꢀ-D-
100
glucopyranoside (4). HRFABMS m=z ðM þ NaÞþ: calcd.
1
for C25H30O12Na 545.1519; found, 545.1522. H-NMR
(400 MHz, DMSO-d6) ꢂH: 3.12–3.70 (11H, m, H-200, 2000,
300, 3000, 400, 4000, 500, 5000, 600), 4.80 (1H, d, J ¼ 8:0 Hz,
H-1000), 5.10 (1H, d, J ¼ 7:6 Hz, H-100), 6.33 (1H, m, H-4),
6.55 (1H, d, J ¼ 1:8 Hz, H-6), 6.73 (1H, d, J ¼ 1:8 Hz,
H-2), 6.76 (2H, d, J ¼ 8:8 Hz, H-30, 50), 6.86 (1H, d,
J ¼ 16:0 Hz, H-7), 7.01 (1H, d, J ¼ 16:0 Hz, H-8), 7.39
(2H, d, J ¼ 8:8 Hz, H-20, 60). 13C-NMR (100 MHz,
DMSO-d6) ꢂC: 60.6 (C-5000), 68.8 (C-600), 69.8, 69.9
(C-400, C-4000), 72.5 (C-2000), 73.3 (C-200), 76.4, 76.7 (C-300,
C-3000), 77.0 (C-500), 100.2 (C-100), 102.0 (C-1000), 102.7
(C-2), 104.7 (C-4), 107.0 (C-6), 115.5 (C-30, C-50), 125.1
(C-7), 127.9 (C-20, C-60), 128.5 (C-8), 129.9 (C-10), 139.3
(C-1), 157.2 (C-40), 158.2 (C-3), 158.8 (C-5).
trans-Resveratrol 3-O-(6-O-ꢁ-L-arabinopyranosyl)-ꢀ-
D-glucopyranoside (5). HRFABMS m=z ðM þ NaÞþ:
calcd. for C25H30O12Na, 545.1519; found, 545.1520.
1H-NMR (400 MHz, DMSO-d6) ꢂH: 3.09–3.78 (11H, m,
H-200, 2000, 300, 3000, 400, 4000, 500, 5000, 600), 4.77 (1H, d,
J ¼ 6:0 Hz, H-1000), 5.09 (1H, d, J ¼ 7:6 Hz, H-100), 6.32
(1H, m, H-4), 6.55 (1H, d, J ¼ 1:8 Hz, H-6), 6.73 (1H, d,
J ¼ 1:8 Hz, H-2), 6.76 (2H, d, J ¼ 8:8 Hz, H-30, 50), 6.86
(1H, d, J ¼ 16:0 Hz, H-7), 7.00 (1H, d, J ¼ 16:0 Hz, H-8),
7.39 (2H, d, J ¼ 8:8 Hz, H-20, 60). 13C-NMR (100MHz,
DMSO-d6) ꢂC: 59.5 (C-5000), 67.0 (C-4000), 68.5 (C-600),
69.8 (C-400), 72.5 (C-2000), 72.9 (C-3000), 73.2 (C-200), 76.7
(C-300), 77.0 (C-500), 100.1 (C-100), 101.5 (C-1000), 102.8
(C-2), 104.9 (C-4), 107.0 (C-6), 115.5 (C-30, C-50), 125.1
(C-7), 127.7 (C-20, C-60), 128.5 (C-8), 129.9 (C-10), 139.3
(C-1), 157.2 (C-40), 158.5 (C-3), 158.8 (C-5).
50
4
8
Time (d)
100
50
4
8
Time (d)
100
50
A time-course experiment was used to investigate the
biotransformation pathway for 1 by the cultured cells of
C. roseus. Figure 1A shows that mono-glucoside prod-
ucts 2 and 3 were produced at an early stage of
incubation, whereas disaccharides 4 and 5 were accu-
mulated after 3 d of incubation. These findings indicate
that the disaccharide products were formed from the
corresponding mono-glucosides, as shown in Fig. 2. The
entire experiments were conducted three times with
essentially the same result, and one representative data
set is shown in Fig. 1.
4
8
Time (d)
Fig. 1. Time-Course Characteristics for the Biotransformation of
trans-Resveratrol (1) by Cultured Cells of (A) C. roseus,
(B) I. batatas, and (C) S. gratus.
The substrate, trans-resveratrol (1, 15 mg), was incubated at 25 ꢀC
with 100 g of the suspension cell culture on a rotary shaker
(120 rpm). The relative peak areas by HPLC of substrate 1 ( ) and
product 2 ( ), 3 ( ), 4 ( ) and 5 ( ) are plotted.
On the other hand, the biotransformation of trans-
resveratrol by cultured cells of I. batatas and S. gratus
O
HO
HO
1''' O
6''
H
HO
HO
HO
O1''
O
3
4'
OH
H
HO
1
GlcO
C. roseus
HO
4
5
OH
OH
HO
O
HO
1''' O
6''
C. roseus
I. batatas
S. gratus
H
HO
O1''
2
HO
HO
HO
HO
O
3
4'
OH
OH
H
HO
1
HO
HO
HO
OGlc
trans-Resveratrol (1)
3
Fig. 2. Biotransformation Pathway for trans-Resveratrol (1) by Plant-Cultured Cells.
Arrows indicate the HMBC correlations.

1554
H. IMAI et al.
resulted in the formation of its 3-O-ꢀ-D-glucopyranoside
and 40-O-ꢀ-D-glucopyranoside (Fig. 1B and C).
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We thank Regional Innovation Creation R&D Pro-
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support.