Glycosylation of daidzein by the Eucalyptus cell cultures

Article abstract of DOI:10.1016/j.phytochem.2008.05.024

The sequential glycosylation of a soybean isoflavone, daidzein, with cultured suspension cells of Eucalyptus perriniana and cyclodextrin glucanotransferase was studied. Daidzein was converted into two glycosylation products, daidzein 7-O-β-d-glucopyranoside (39%) and daidzein 7-O-[6-O-(β-d-glucopyranosyl)]-β-d-glucopyranoside (β-gentiobioside, 6%), by cultured E. perriniana cells. Further glycosylation of daidzein 7-O-β-glucoside with cyclodextrin glucanotransferase gave daidzein 7-O-[4-O-(α-d-glucopyranosyl)]-β-d-glucopyranoside (β-maltoside, 26%), daidzein 7-O-β-maltotrioside (15%), and daidzein 7-O-β-maltotetraoside (7%).

Full text of DOI:10.1016/j.phytochem.2008.05.024

Phytochemistry 69 (2008) 2303–2306
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Glycosylation of daidzein by the Eucalyptus cell cultures
Kei Shimoda a, Noriaki Sato b, Tatsunari Kobayashi b, Hatsuyuki Hamada c, Hiroki Hamada d,
*
a Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
b Department of Applied Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
c National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan
d Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2007
Received in revised form 12 April 2008
Available online 5 July 2008
The sequential glycosylation of a soybean isoflavone, daidzein, with cultured suspension cells of Eucalyp-
tus perriniana and cyclodextrin glucanotransferase was studied. Daidzein was converted into two
glycosylation products, daidzein 7-O-b-
osyl)]-b-
daidzein 7-O-b-glucoside with cyclodextrin glucanotransferase gave daidzein 7-O-[4-O-(
osyl)]-b-
maltotetraoside (7%).
D
-glucopyranoside (39%) and daidzein 7-O-[6-O-(b-
D
-glucopyran-
D
-glucopyranoside (b-gentiobioside, 6%), by cultured E. perriniana cells. Further glycosylation of
a
-D
-glucopyran-
Keywords:
D
-glucopyranoside (b-maltoside, 26%), daidzein 7-O-b-maltotrioside (15%), and daidzein 7-O-b-
Eucalyptus perriniana
Cultured plant cells
Cyclodextrin glucanotransferase
Daidzein
Ó 2008 Elsevier Ltd. All rights reserved.
b-Gentiobioside
b-Maltooligosaccharide
1. Introduction
(6) by cultured cells of Eucalyptus perriniana and Cyclodextrin
glucanotransferase.
Soybean is one of the most important legumes in the human
diet. Daidzein (1) is a principal soy isoflavone and has been widely
studied for its anti-inflammatory activity such as inhibitory effect
on histamine release from mast cells (Dijsselbloem et al., 2004;
Marotta et al., 2006; Chacko et al., 2007). However, pharmacolog-
ical exploitation of daidzein (1) is limited due to its insolubility
in aqueous solution.
Many kinds of secondary metabolites, such as saponins, are pro-
duced in the form of glycosides in plant cells (Biswas et al., 2005;
Voutquenne et al., 2005; Eskander et al., 2006; Barile et al., 2007;
Melek et al., 2007; Shao et al., 2007; Vincken et al., 2007; Zhang
and Li, 2007). Glycosylation of organic compounds improves their
bioavailability and pharmacological properties. Thus, daidzein gly-
cosides are of pharmacological interest from the viewpoint of drug
development of isoflavone. Glycosylation using cultured plant cells
is useful for preparing water-soluble and stable glycosides from
water-insoluble and unstable compounds. In addition, cyclodextrin
glucanotransferase (Cyclodextrin glucanotransferase) is a conve-
nient biocatalyst to synthesize glucooligosaccharides (Jeang et al.,
2005).
2. Results and discussion
2.1. Glycosylation of daidzein (1) by cultured cells of E. perriniana
Cultured cells of E. perriniana have been reported to have high
potential to convert exogenous phenolic compounds into their gly-
coconjugates, and were used in this study (Shimoda et al., 2006).
Biotransformation of daidzein (1) by cultured suspension cells of
E. perriniana resulted in production of glycosides 2 (39%) and 3
(6%), the structures of which were determined to be daidzein 7-
O-b-
D-glucopyranoside (2) and daidzein 7-O-[6-O-(b-D-glucopyr-
anosyl)]-b-
D
-glucopyranoside (b-gentiobioside, 3) based on their
HRFABMS, 1H and 13C NMR (Table 1), H–H COSY, C–H COSY, NOE,
and HMBC spectroscopic data. The b-gentiobioside product 3 has
not been identified previously.
The molecular formula of 3 was established as C27H30O14 based
on its HRFABMS spectrum, which included a pseudomolecular ion
[M + Na]+ peak at m/z 601.1537 (calcd. 601.1533 for C27H30O14Na).
HRFABMS suggested that 3 was composed of one molecule of 1 and
two hexoses. Its 1H NMR spectrum showed two anomeric proton
signals at d 4.20 (1H, d, J = 8.0 Hz) and 5.11 (1H, d, J = 7.6 Hz). This
suggested the presence of two b-anomers. The 13C NMR spectrum
included two anomeric carbon signals at d 99.5 and 103.4. The su-
We report here the sequential glycosylation of daidzein into the
corresponding 7-O-b-glucoside (2), 7-O-b-gentiobioside (3), 7-O-b-
maltoside (4), 7-O-b-maltotrioside (5), and 7-O-b-maltotetraoside
gar component of 3 was determined to be b-
D-glucopyranose based
* Corresponding author. Tel.: +81 86 256 9473; fax: +81 86 256 8468.
E-mail address: hamada@das.ous.ac.jp (H. Hamada).
on the chemical shifts of the carbon signals. The 13C resonance of
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.05.024

2304
K. Shimoda et al. / Phytochemistry 69 (2008) 2303–2306
Table 1
100
13C chemical shifts of the glycosylation products 2-6 in DMSO-d6
Daidzein (1)
Product
2
3
4
5
6
Aglycone
2
3
4
5
6
7
8
9
153.0
122.1
174.6
127.0
115.5
161.3
103.3
157.2
118.4
123.6
130.0
114.9
156.9
114.9
130.0
153.1
122.1
174.6
127.0
115.5
161.2
103.3
157.1
118.4
123.6
130.0
114.9
156.9
114.9
130.0
153.1
122.1
174.6
126.9
115.4
161.2
103.3
157.2
118.4
123.6
130.0
114.9
156.9
114.9
130.0
153.0
122.2
174.7
127.0
115.5
161.2
103.3
157.2
118.4
123.6
130.0
114.9
156.9
114.9
130.0
153.1
122.1
174.6
127.0
115.4
161.2
103.3
157.1
118.4
123.6
130.0
114.9
156.9
114.9
130.0
50
2
3
10
11
12
13
14
15
16
Glc
10
48
96
Time/h
Fig. 1. Time-course of the glycosylation of daidzein (1) by cultured cells of E.
perriniana. aYield is expressed as a percentage relative to the total amount of all
reaction products.
99.9
73.0
77.1
69.5
76.4
60.5
99.5
73.3
77.0
69.8
76.1
68.7
103.4
72.9
76.5
69.2
75.9
60.8
98.9
73.3
76.0
78.9
75.3
61.0
100.6
72.0
73.2
69.6
72.6
60.2
99.5
73.4
75.9
79.4
75.3
60.7
100.5
71.9
73.2
79.0
71.7
60.2
100.7
72.4
73.1
69.8
72.6
60.2
98.8
73.1
76.0
79.3
75.3
60.7
100.5
72.0
73.1
79.3
72.0
60.2
100.6
72.0
73.1
78.9
72.0
60.2
100.7
72.6
73.1
69.7
72.6
60.2
20
30
D-glucopyranosyl)]-b-D-glucopyranoside (b-maltoside, 4), daidzein
40
7-O-b-maltotrioside (5), and daidzein 7-O-b-maltotetraoside (6).
The di- and tetrasaccharide products 4 and 6 have not been iden-
tified previously.
50
60
100
200
The HRFABMS spectrum of 4 with a pseudomolecular ion
[M + Na]+ peak at m/z 601.1538 indicated a molecular formula of
300
400
C
27H30O14 (calcd. 601.1533 for C27H30O14Na). The 1H NMR spec-
500
trum of 4 included two anomeric proton signals at d 5.07 (1H, d,
J = 3.4 Hz) and 5.17 (1H, d, J = 7.6 Hz), indicating the presence of
600
1000
2000
3000
4000
5000
6000
10000
20000
30000
40000
50000
60000
both the a
- and b-anomers in the sugar moiety. The 13C NMR spec-
troscopic data of 4 showed two anomeric carbon signals at d 98.9
and 100.6. HMBC correlations were observed between the anomer-
ic proton signal at d 5.17 (H-10) and the carbon resonance at d 161.2
(C-7), and between the anomeric proton signal at d 5.07 (H-100) and
the carbon resonance at d 78.9 (C-40). These data confirm that the
inner b-
of daidzein (1), and that the second
and the inner b- -glucopyranosyl residue were 1,4-linked. Thus,
compound 4 was identified as daidzein 7-O-[4-O-( -glucopyran-
osyl)]-b- -glucopyranoside (b-maltoside).
D
-glucopyranosyl residue was attached to the C-7 position
a-D-glucopyranosyl residue
D
a
-D
D
The HRFABMS spectrum of 6, which included a pseudomolecu-
lar ion [M + Na]+ peak at m/z 925.2602, established that the molec-
ular formula of 6 was C39H50O24 (calcd. 925.2590 for C39H50O24Na).
The 1H NMR spectrum of 6 showed four anomeric proton signals at
d 5.00 (2H, d, J = 3.4 Hz), 5.09 (1H, d, J = 3.0 Hz), and 5.17 (1H, d,
C-60 was shifted downfield to d 68.7. Correlations were observed
between the anomeric proton signal at d 5.11 (H-10) and the carbon
resonance at d 161.2 (C-7), and between the anomeric proton sig-
nal at d 4.20 (H-100) and the carbon resonance at d 68.7 (C-60) in the
HMBC spectrum. These findings confirmed that the inner glucopyr-
anosyl residue was attached to the phenolic hydroxyl group at C-7
J = 7.6 Hz), suggesting the presence of three
a-anomers and one
b-anomer in the sugar moiety. The 13C NMR chemical shifts of
C-40 (d 79.3), C-400 (d 79.3), and C-4000 (d 78.9) were shifted relatively
downfield. The HMBC experiment established connections
between H-10 (d 5.17) and C-7 (d 161.2), between H-100 (d 5.09)
and C-40 (d 79.3), between H-1000 (d 5.00) and C-400 (d 79.3), and
between H-10000 (d 5.00) and C-4000 (d 78.9). Thus, compound 6 was
identified as daidzein 7-O-b-maltotetraoside.
of daidzein (1), and that the pair of b-
was 1,6-linked. Thus, 3 was identified as daidzein 7-O-[6-O-(b-
glucopyranosyl)]-b- -glucopyranoside.
D-glucopyranosyl residues
D
-
D
A time-course experiment was carried out to investigate the
ability of cultured E. perriniana cells to biotransform daidzein (1).
As shown in Fig. 1, 1 was readily glucosylated to 2 at an early stage
of incubation, whereas the yield of product 3 was not so high
throughout the incubation period. E. perriniana cell culture was a
good biocatalyst for producing daidzein 7-O-b-glucoside (2) rather
than daidzein 7-O-b-gentiobioside (3).
3. Conclusion
The results obtained with cultured cells of E. perriniana showed
that E. perriniana can glycosylate daidzein (1) to give the corre-
sponding 7-O-b-glucoside (2) and 7-O-b-gentiobioside (3) (Scheme
1). This is the first description of daidzein 7-O-b-gentiobioside (3)
formation using cultured plant cells. Recently, it has been reported
that cultured E. perriniana cells converted exogenously added
thymol, carvacrol, and eugenol into their mono-glucosides and
gentiobiosides (Shimoda et al., 2006). The yield of daidzein 7-O-
b-gentiobioside (3) obtained here is relatively low, probably due
to the substrate specificity of glycosyltransferases which produce
gentiobiosides.
2.2. Glycosylation of daidzein 7-O-b-D-glucoside (2) by Cyclodextrin
glucanotransferase
Daidzein 7-O-b-D-glucoside (2), which had been produced by
the biotransformation of daidzein (1) with cultured E. perriniana
cells, was subjected to further glycosylation with Cyclodextrin glu-
canotransferase. Incubation of 2 with Cyclodextrin glucanotrans-
ferase in the presence of starch gave three products, 4 (26%), 5
(15%), and 6 (7%), which were identified as daidzein 7-O-[4-O-(a-

K. Shimoda et al. / Phytochemistry 69 (2008) 2303–2306
2305
OH
O
HO
HO
1''
O
OH
HO
HO
OH
1
8
5
O
1'
O
O
7
9
HO
HO
1'
O
O
O
O
O
HO
O
2
OH
OH
3
12
15
11
16
6
13
14
OH
10
4
O
OH
OH
3
2
Daidzein (1)
Scheme 1. Glycosylation of daidzein (1) by cultured cells of E. perriniana.
OH
O
HO
HO
OH
OH
OH
O
O
O
n
HO
HO
HO
O
O
O
O
O
O
OH
OH
OH
OH
2
4:
n
=1; 5: n=2; 6: n=3
Scheme 2. Glycosylation of daidzein 7-O-b-D-glucopyranoside (2) by Cyclodextrin glucanotransferase.
Oligomerization of sugar at the C-7 position of daidzein gave
daidzein (1) 7-O-b-maltooligosaccharides, i.e., 7-O-b-maltoside
(4), 7-O-b-maltotrioside (5), and 7-O-b-maltotetraoside (6), by
the glycosylation of daidzein 7-O-b-glucoside (2) with Cyclodextrin
glucanotransferase in the presence of starch (Scheme 2). Daidzein
7-O-b-maltoside (4) and daidzein 7-O-b-maltotetraoside were
identified for the first time. Li et al. reported that daidzein 7-O-b-
glucoside was glycosylated to daidzein 7-O-b-maltotrioside, daidz-
ein 7-O-b-maltopentaoside, daidzein 7-O-b-maltoheptaoside, and
daidzein 7-O-b-maltononaoside by maltosyltransferase from
Thermotoga maritime, and daidzein 7-O-b-maltotrioside (5) was
isolated and subjected to structure determination using spectro-
scopic methods (Li et al., 2004). The glycosylation system in the
present study is useful for the practical preparation of a series of
daidzein 7-O-b -maltooligosaccharides which differ by a glucose
unit.
4.2. Plant cell culture
A cell culture of E. perriniana was induced in our laboratory, and
has been cultivated for over 20 years (Furuya et al., 1987). Cultured
E. perriniana cells were subcultured at 4-week intervals on solid
Murashige and Skoog (MS) medium (100 ml in a 300-ml conical
flask) containing 3% sucrose, 10 mM 2,4-dichlorophenoxyacetic
acid, and 1% agar (adjusted to pH 5.7) at 25 °C in the dark. A sus-
pension culture was started by transferring the cultured cells to
100 ml of liquid medium in a 300-ml conical flask, and incubated
on a rotary shaker (120 rpm) at 25 °C in the dark. Prior to use for
this work, part of the callus tissues (fr. wt 40 g) was transplanted
to freshly prepared MS medium (100 ml in a 300-ml conical flask)
and grown with continuous shaking for 2 weeks on a rotary shaker
(120 rpm).
Sequential glycosylation with E. perriniana cells and Cyclodex-
trin glucanotransferase may be useful for the production of isoflav-
one maltooligosaccharides as food additives. This method is simple
and environmentally friendly. Further studies on the physiological
activities of daidzein glycosides are now in progress.
4.3. Glycosylation by cultured cells of E. perriniana
The substrate daidzein (1) was added to each of ten 300-ml
flasks (1 mmol/l) containing 100 ml of MS medium and cultured
suspension cells of E. perriniana, and the cultures were incubated
at 25 °C for five days on a rotary shaker (120 rpm) in the dark. After
incubation, the cells and medium were separated by filtration with
suction. Extraction and purification of the biotransformation prod-
ucts were achieved as previously reported (Shimoda et al., 2006,
2007a, 2007b).
4. Experimental
4.1. General experimental details
Daidzein (1) was purchased from Aldrich Chemical Co.
(St. Louis, MO). Cyclodextrin glucanotransferase from Bacillus mac-
erans was purchased from Amano Pharmaceutical Co. Ltd. (Nagoya,
Japan). HPLC was performed using a YMC-Pack R&D ODS column
(150 ꢀ 30 mm, YMC Co. Ltd., Kyoto, Japan) (solvent: MeOH-H2O
(9:11, v/v); detection: UV (280 nm); flow rate: 1.0 ml/min).
HRFABMS was performed using a JEOL MStation JMS-700 spec-
trometer (JEOL Ltd., Tokyo, Japan). 1H and 13C NMR, H–H COSY,
C–H COSY, NOE, and HMBC spectra were measured using a Varian
XL-400 spectrometer (Varian Technologies Japan Ltd., Tokyo,
Japan) in DMSO-d6 solution and the chemical shifts were expressed
in d (ppm) with reference to TMS.
4.4. Glycosylation by Cyclodextrin glucanotransferase
Daidzein 7-O-b-D-glucoside (2, 50 mg) was incubated with 3 lk
at of Cyclodextrin glucanotransferase in 10 ml of 25 mM sodium
phosphate buffer (pH 7.0) containing 5 g of soluble starch at
40 °C for 24 h. After incubation, the reaction mixture was centri-
fuged at 3000 g for 10 min. The supernatant was applied to a
Sephadex G-25 column equilibrated with water. Fractions that
contained glycosides were lyophilized, re-dissolved with water,
and purified by preparative HPLC using a YMC-Pack R&D ODS col-
umn (150 ꢀ 30 mm).

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their HRFABMS, 1H and 13C NMR, H–H COSY, C–H COSY, NOE, and
HMBC spectra.
Daidzein 7-O-[6-O-(b-D-glucopyranosyl)]-b-D-glucopyranoside
(3): HRFABMS: m/z 601.1537 [M + Na]+; 1H NMR (400 MHz,
DMSO-d6): d 3.18-3.71 (12H, m, H-20, 200, 30, 300, 40, 400, 50, 500, 60,
600), 4.20 (1H, d, J = 8.0 Hz, H-100), 5.11 (1H, d, J = 7.6 Hz, H-10),
6.82 (2H, d, J = 6.4 Hz, H-13, 15), 7.13 (1H, dd, J = 8.6, 2.0 Hz, H-
6), 7.24 (1H, d, J = 2.0 Hz, H-8), 7.40 (2H, d, J = 6.4 Hz, H-12, 16),
8.04 (1H, d, J = 8.6 Hz, H-5), 8.39 (1H, s, H-2); for 13C NMR
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Daidzein 7-O-[4-O-(a-D-glucopyranosyl)]-b-D-glucopyranoside
(b-maltoside, 4): HRFABMS: m/z 601.1538 [M + Na]+; 1H NMR
(400 MHz, DMSO-d6): d 3.02–3.78 (12H, m, H-20, 200, 30, 300, 40, 400,
50, 500, 60, 600), 5.07 (1H, d, J = 3.4 Hz, H-100), 5.17 (1H, d, J = 7.6 Hz,
H-10), 6.81 (2H, d, J = 6.4 Hz, H-13, 15), 7.13 (1H, dd, J = 8.6,
2.0 Hz, H-6), 7.25 (1H, d, J = 2.0 Hz, H-8), 7.41 (2H, d, J = 6.4 Hz,
H-12, 16), 8.05 (1H, d, J = 8.6 Hz, H-5), 8.40 (1H, s, H-2); for 13C
NMR (100 MHz, DMSO-d6) spectroscopic data see Table 1.
Daidzein 7-O-b-maltotetraoside (6): HRFABMS: m/z 925.2602
[M + Na]+; 1H NMR (400 MHz, DMSO-d6): d 3.05-3.95 (24H, m, H-
20, 200, 2000, 20000, 3’, 300, 3000, 30000, 40, 400, 4000, 40000, 50, 500, 5000, 50000, 60, 600,
6000, 60000), 5.00 (2H, d, J = 3.4 Hz, H-1000, 10000), 5.09 (1H, d, J = 3.0 Hz,
H-100), 5.17 (1H, d, J = 7.6 Hz, H-10), 6.81 (2H, d, J = 6.4 Hz, H-13,
15), 7.14 (1H, dd, J = 8.6, 2.0 Hz, H-6), 7.24 (1H, d, J = 2.0 Hz, H-8),
7.40 (2H, d, J = 6.4 Hz, H-12, 16), 8.05 (1H, d, J = 8.6 Hz, H-5), 8.39
(1H, s, H-2); for 13C NMR (100 MHz, DMSO-d6) spectroscopic data
see Table 1.
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