Biotransformation of naringin and naringenin by cultured Eucalyptus perriniana cells

Article abstract of DOI:10.1016/j.phytochem.2009.09.035

The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus perriniana. Naringin (1) was converted into naringenin 7-O-β-d-glucopyranoside (2, 15%), naringenin (3, 1%), naringenin 5,7-O-β-d-diglucopyranoside (4, 15

Full text of DOI:10.1016/j.phytochem.2009.09.035

Phytochemistry 71 (2010) 201–205
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Phytochemistry
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Biotransformation of naringin and naringenin by cultured Eucalyptus perriniana cells
Kei Shimoda a, Naoji Kubota a, Koji Taniuchi b, Daisuke Sato b, Nobuyoshi Nakajima c,
Hatsuyuki Hamada d, Hiroki Hamada b,
*
a Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
b Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
c Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization, Okayama Prefectural University, Soja, Okayama 719-1197, Japan
d National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus per-
Received 26 December 2008
Received in revised form 18 May 2009
Accepted 28 September 2009
riniana. Naringin (1) was converted into naringenin 7-O-b-
naringenin 5,7-O-b-
-diglucopyranoside (4, 15%), naringenin 40,7-O-b-
naringenin 7-O-[6-O-(b- -glucopyranosyl)]-b- -glucopyranoside (6, b-gentiobioside, 5%), naringenin 7-
O-[6-O-( -rhamnopyranosyl)]-b- -glucopyranoside (7, b-rutinoside, 3%), and 7-O-b-
-gentiobiosyl-40-
O-b- -glucopyranosylnaringenin (8, 1%) by cultured cells of E. perriniana. On the other hand, 2 (14%), 4
(7%), 5 (13%), 6 (2%), 7 (1%), naringenin 40-O-b-
-glucopyranoside (9, 4%), naringenin 5-O-b- -glucopyran-
oside (10, 2%), and naringenin 40,5-O-b-
-diglucopyranoside (11, 5%) were isolated from cultured
E. perriniana cells, that had been treated with naringenin (3). Products, 7-O-b- D-
-gentiobiosyl-40-O-b-
D
-glucopyranoside (2, 15%), naringenin (3, 1%),
D
D
-diglucopyranoside (5, 26%),
D
D
a
-L
D
D
D
Keywords:
Eucalyptus perriniana
Mystaceae
D
D
D
D
Eucalyptus
glucopyranosylnaringenin (8) and naringenin 40,5-O-b-
D-diglucopyranoside (11), were hitherto unknown.
Cultured plant cells
Biotransformation
Glycosylation
Naringin
Ó 2009 Elsevier Ltd. All rights reserved.
Naringenin
b-Glycosides
1. Introduction
et al., 1987; Shaw et al., 1991). These glycosides as well as their
aglycones have been studied in terms of their pharmaceutical prop-
erties, including anticarcinogenic, antiviral, anti-inflammatory, and
anticancer activities (Borradaile et al., 1999; Zhang et al., 2000;
Garg et al., 2001; Chiba et al., 2003; Miller et al., 2008; Nafisi
et al., 2008; Wu et al., 2008). Earlier, Lewinsohn et al. reported that
cultured cells of Citrus paradisi converted hesperetin into its 7-O-
glucoside and hesperidin (Lewinsohn et al., 1986, 1989). However,
little attention has been given to the biotransformation of naturally
occurring glycosides such as naringin (1) and hesperidin by
cultured plant cells. We report herein the biotransformation of
naringin (1) with plant cell cultures of E. perriniana to naringenin
7-O-glucoside (2), naringenin (3), naringenin 40,7-O-diglucoside
(5), naringenin 5,7-O-diglucoside (4), naringenin 7-O-gentiobioside
(6), naringenin 7-O-rutinoside (7), and 7-O-gentiobiosyl-40-O-glu-
cosylnaringenin (8), and biotransformation of naringenin (3) to its
40-O-glucoside (9), 5-O-glucoside (10), 7-O-glucoside (2), 5,7-O-dig-
lucoside (4), 40,7-O-diglucoside (5), 40,5-O-diglucoside (11), 7-O-
gentiobioside (6), and 7-O-rutinoside (7), respectively.
Plant cells can conjugate sugar residues not only to endogenous
metabolic intermediates but also to xenobiotics. Glycosylation
reactions catalyzed by plant glycosyltransferases have diverse func-
tions in plants, i.e., increasing the solubility and stability of the agly-
cones, decreasing the toxicity of xenobiotics, and activating natural
compounds such as saponins. On the other hand, plant cultured
suspension cells are ideal systems for studying biotransformation
of exogenous organic compounds. Particularly, glycosylation is a
characteristic biotransformation reaction by cultured plant cells.
Recently, several papers on the biotransformation of phenolic com-
pounds by cultured suspension cells of Eucalyptus perriniana re-
ported that E. perriniana cell cultures had high potential to
produce their b-glycosides such as b-glucoside, b-gentiobioside,
and b-rutinoside of tocopherols, hesperetin, and daidzein (Shimoda
et al., 2007c, 2008a,b).
Citrus flavanones, naringenin and hesperetin, occur naturally as
the glycosides, naringenin 7-O-[2-O-(
glucopyranoside (naringin 1) and hesperetin 7-O-[6-O-(
rhamnopyranosyl)]-b-
a
-
L
-rhamnopyranosyl)]-b-
D
-
-
a-L
D
-glucopyranoside (hesperidin) (Rousseff
2. Results and discussion
Naringin (1) was subjected to biotransformation by cultured
cells of E. perriniana. Incubation of 1 with cultured E. perriniana
* Corresponding author. Tel.: +81 86 256 9473; fax: +81 86 256 8468.
E-mail address: hamada@dls.ous.ac.jp (H. Hamada).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.09.035

202
K. Shimoda et al. / Phytochemistry 71 (2010) 201–205
cells for 5 days yielded seven products 2–8. There were no glyco-
side products in the medium. The control cell cultures, which
had not been treated with naringin (1), were subjected to the same
extraction procedures and HPLC analyses, and neither naringin (1)
nor derivatives thereof were detected in the cells and in the med-
ium despite careful HPLC analyses. The structures of the products
were elucidated through use of HRFABMS, 1H and 13C NMR (Ta-
ble 1), H–H COSY, C–H COSY, HMBC spectroscopic analyses, and
Table 1
13C chemical shifts of glycosylation products 2 and 4–11 in DMSO-d6.
the products were identified as naringenin 7-O-b-
side (2, 15%) (Hai et al., 2004), naringenin (3, 1%), naringenin 5,7-
O-b- -diglucopyranoside (4, 15%) (Hai et al., 2004), naringenin
40,7-O-b-
-diglucopyranoside (5, 26%) (Liu et al., 2006), naringenin
7-O-[6-O-(b- -glucopyranosyl)]-b- -glucopyranoside (b-gentiobio-
side, 6, 5%) (Miura et al., 1986), naringenin 7-O-[6-O-( -rhamno-
pyranosyl)]-b- -glucopyranoside (b-rutinoside (narirutin), 7,
3%) (Shaw et al., 1991), and 7-O-b- -glu-
-gentiobiosyl-40-O-b-
D-glucopyrano-
Product
2
4
5
6
7
8
Aglycone
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
78.5
42.1
78.4
42.0
78.5
41.9
78.3
42.0
78.5
42.0
78.5
42.1
D
D
196.7
163.4
96.2
165.0
95.2
162.5
103.2
128.3
128.1
115.1
157.0
115.1
128.1
196.7
163.6
96.0
164.9
95.2
162.6
103.1
128.3
128.2
115.1
157.0
115.1
128.2
196.6
163.5
96.0
165.0
95.1
162.5
103.2
128.3
128.3
115.1
158.3
115.1
128.3
196.7
163.3
96.0
164.9
95.1
162.5
103.1
128.3
128.1
115.0
157.0
115.0
128.1
196.7
163.5
96.0
164.9
95.1
162.5
103.2
128.2
128.2
115.1
157.0
115.1
128.2
196.7
163.4
96.0
164.9
95.2
162.5
103.2
128.3
128.2
115.1
157.2
115.1
128.2
D
D
a
-L
D
D
D
copyranosylnaringenin (8, 1%), which has not been identified
before.
The HRFABMS spectrum of 8, which showed a [M+Na]+ peak at
m/z 781.2170, suggested the formula of C33H42O20 (calcd.
781.2167 for C33H42O20Na), indicating the incorporation of three
hexoses in the molecule. The 1H NMR spectrum of compound 8
showed signals for anomeric protons at d 4.25 (1H, d, J = 8.0 Hz),
4.90 (1H, d, J = 8.0 Hz), and 5.06 (1H, d, J = 7.6 Hz), suggesting the
presence of three b-anomers. The 13C NMR spectrum of 8 included
anomeric carbon signals at d 99.6, 99.8, and 103.9. On the basis of
the chemical shifts of the carbon resonances due to the sugar moi-
Glc
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
100.2
72.9
77.6
69.5
76.7
60.7
100.4
72.8
77.0
69.3
76.8
60.5
103.7
72.9
77.5
69.5
77.5
60.8
100.5
72.9
76.9
69.5
76.7
60.6
99.7
72.8
77.6
69.4
77.6
60.3
100.0
73.2
76.7
69.9
76.3
68.5
104.1
72.9
76.3
69.2
76.0
60.7
99.5
72.8
77.0
69.5
76.6
66.0
100.6
70.2
70.5
71.6
68.2
17.8
99.8
73.1
77.0
69.8
76.6
68.3
103.9
73.0
76.5
69.3
76.1
60.8
99.6
ety, the sugar component of 8 was determined as b-D-glucopyra-
nose. HMBC correlations between proton and carbon resonances,
d 5.06 (H-10000 )/d 157.2 (C-40), d 4.90 (H-100)/d 164.9 (C-7), and d
4.25 (H-1000)/d 68.3 (C-600), established that glucosyl residues were
attached at the 40- and 7-positions of naringenin (3), and that the
10000
20000
30000
40000
50000
60000
pair of b-D-glucopyranosyl residues at C-7 position was 1,6-linked.
Thus, compound 8 was 7-O-b-D D-glucopy-
-gentiobiosyl-40-O-b-
72.9
76.9
69.5
77.0
60.8
ranosylnaringenin.
Fig. 1 shows the time-course of the biotransformation of 1 with
cultured E. perriniana cells. It was found that formation of 7-O-b-
glucoside 2 first occurred, followed by production of di- and trisac-
charides 4–8. Very small amounts of naringenin (3) were found
during the incubation period. These findings suggested that
9
10
11
naringenin 7-O-b-D-glucopyranoside (2), which had been formed
by hydrolysis of 1 with E. perriniana cells, was glucosylated to af-
ford di- and trisaccharides 4–8, as shown in Scheme 1.
Next, naringenin (3) was transformed by the same biotransfor-
mation system with cultured E. perriniana cells as the case of narin-
gin (1). After incubation, products 2 (14%), 4 (7%), 5 (13%), 6 (2%), 7
Aglycone
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
78.5
42.2
196.6
163.5
96.0
78.4
42.1
78.4
42.1
196.7
163.7
96.0
164.8
95.0
162.5
103.2
128.2
128.1
115.2
157.0
115.2
128.1
196.6
163.6
96.0
164.8
95.1
162.6
103.1
128.2
128.1
115.2
157.2
115.2
128.1
164.9
95.0
162.6
103.1
128.3
128.3
115.1
157.5
115.1
128.3
100
50
Glc
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
99.5
72.8
77.3
69.5
77.5
60.6
103.5
72.9
77.6
69.7
77.4
60.4
99.2
72.7
77.6
69.3
77.5
60.4
103.7
72.7
77.3
69.6
77.3
60.5
10000
20000
30000
40000
50000
60000
48
Time (h)
96
Fig. 1. Time-course of biotransformation of naringin (1) by cultured cells of E.
perriniana. Yield is expressed as a percentage relative to the total amount of all
reaction products. Yields of 1 (j), 2 (ꢀ), 3 (s), 4 (d), 5 (e), 6 (ꢀ), 7 (N), and 8 (h) are
plotted.

K. Shimoda et al. / Phytochemistry 71 (2010) 201–205
203
OH
O
HO
OH
O
3
OH
GlcO
O
OGlcO
4
3'
OH
OH
OGlc
4'
5'
2'
1
8
GlcO
O
O
GlcO
O
Rham(1 2)GlcO
7
9
2
1'
E. perriniana
6'
3
4
6
10
5
OGlc
O
2
O
5
OH
O
1
OH
OH
O
Glc(1 6)GlcO
OH
O
OH
8
O
O
Glc(1 6)GlcO
OH
6
OH
O
O
Rham(1 6)GlcO
OH
7
Scheme 1. Biotransformation of naringin (1) by cultured cells of E. perriniana.
(1%), naringenin 40-O-b-
and naringenin 5-O-b- -glucopyranoside (10, 2%) (Ogundaini et al.,
D
-glucopyranoside (9, 4%) (Ko et al., 2006),
1H NMR spectrum of 11 displayed two anomeric proton signals
at d 4.97 (1H, d, J = 7.6 Hz) and 5.05 (1H, d, J = 8.0 Hz). The sugar
D
1996), and 11 (5%) were isolated from the MeOH-extracts of the
component in product 11 was determined to be b-D-glucopyra-
cells. Product 11 was identified as naringenin 40,5-O-b-
D-digluco-
nose, according to the chemical shifts of the sugar carbon reso-
nances. The 13C NMR spectrum of 11 showed two anomeric
carbon resonances at d 99.4 and 103.7, as the HMBC spectrum of
11 exhibited interactions between the anomeric proton at the 100-
position (d 5.05) and the carbon at the 40-position (d 157.2), as well
as between the anomeric proton at 1000-position (d 4.97) and the
carbon at 5-position (d 163.6). These findings established that the
glucopyranosyl residues were attached to the phenolic hydroxyl
group at the C-40 and C-5 positions of naringenin (3). Thus, struc-
ture of compound 11 was determined to be naringenin 40,5-O-b-
pyranoside, which has not been identified before.
The HRFABMS spectrum of product 11 showed a pseudomolec-
ular ion [M+Na]+ peak at m/z 619.1641 consistent with a molecular
formula of C27H32O15 (calcd. 619.1639 for C27H32O15Na), suggest-
ing that two new hexoses were introduced to the substrate. The
100
D-diglucopyranoside.
A time-course experiment was carried out to investigate the
ability of cultured cells of E. perriniana to convert 3 (Fig. 2). The bio-
transformation pathway of naringenin (3) is shown in Scheme 2.
50
3. Conclusion
The results of this study established that naringin (1) was con-
verted into six glycosides of naringenin and into a small amount of
naringenin (3) by cultured E. perriniana cells. It is postulated that
five glycosides, i.e., naringenin 40,7-O-b-
naringenin 5,7-O-b-
gentiobioside, naringenin 7-O-b-
biosyl-40-O-b-
-glucopyranosylnaringenin (8), were generated
from naringenin 7-O-b- -glucoside (2), which had been produced
D
-diglucopyranoside (5),
-diglucopyranoside (4), naringenin 7-O-b-
-rutinoside, and 7-O-b- -gentio-
D
D-
48
Time (h)
96
D
D
D
D
Fig. 2. Time-course of biotransformation of naringenin (3) by cultured cells of E.
perriniana. Yield is expressed as a percentage relative to the total amount of all
reaction products. Yields of 2 (ꢀ), 3 (j), 4 (ꢀ), 5 (h), 6 (x), 7 (N), 9 (d), 10 (e), and 11
(s) are plotted.
by hydrolysis of naringin, not from naringenin.
On the other hand, naringenin (3) was converted into eight
glycosides, i.e., naringenin 40-O-b-
D-glucopyranoside (9), naringe-

204
K. Shimoda et al. / Phytochemistry 71 (2010) 201–205
OGlc
OH
HO
O
O
GlcO
GlcO
HO
OH
O
O
OGlc
9
4
OGlc
OGlc
O
OH
OH
O
HO
O
O
HO
E. perriniana
O
OH
5
OH
O
OGlc
3
10
O
OH
O
OGlc
GlcO
O
11
OH
OH
O
O
OH
Glc(1 6)GlcO
2
O
OH
OH
6
O
Rham(1 6)GlcO
O
7
Scheme 2. Biotransformation of naringenin (3) by cultured cells of E. perriniana.
nin 5-O-b-
D
-glucopyranoside (10), naringenin 7-O-b-
-diglucopyranoside (11),
-diglucopyranoside (5), naringenin 5,7-O-b-
-diglucopyranoside (4), naringenin 7-O-b- -gentiobioside (6),
and naringenin 7-O-b- -rutinoside (7). This is the first descrip-
tion of production of naringenin 7-O-b- -rutinoside by biotrans-
formation of naringin (1) and naringenin (3) with cultured E.
perriniana cells. No formation of naringenin 40-O-b-
-glucopyran-
oside (9), naringenin 5-O-b- -glucopyranoside (10), and naringe-
nin 40,5-O-b-
-diglucopyranoside (11) was found in the case of
the biotransformation of naringin (1) by E. perriniana cells. These
findings also indicated that naringenin 7-O-b- -glucopyranoside
D-glucopy-
4. Experimental
ranoside (2), naringenin 40,5-O-b-
D
naringenin 40,7-O-b-
D
D
4.1. General
D
D
Naringin (1) and naringenin (3) were purchased from Aldrich
Chemical Co. 1H and 13C NMR, H–H COSY, C–H COSY, and HMBC
spectra were measured using a Varian XL-400 spectrometer in
DMSO-d6 solution, whereas HRFABMS was performed using a JEOL
MStation JMS-700 spectrometer. HPLC utilised a YMC-Pack R&D
ODS column (150 ꢁ 30 mm) [solvent: CH3CN–H2O (3:17, v/v);
detection: UV (280 nm); flow rate: 1.0 ml/min].
D
D
D
D
D
(2) was converted into other five glycosides 4–8 in the biotrans-
formation of naringin (1). Several studies on the biotransforma-
tion of flavanones by plant cell cultures have been reported so
far (for example, Lewinsohn et al., 1986, 1989; Shimoda et al.,
2008a). However, there are no reports on the biotransformation
of both flavanone glycosides and their aglycone flavanones to
clarify the biotransformation pathways of flavanone glycosides.
It would be useful for investigation of metabolism of flavanone
glycosides by cultured plant cells to compare the biotransforma-
tion pathway of flavanone glycosides with that of aglycone
flavanones.
4.2. Plant material and culture conditions
Cultured cells of E. perriniana have been cultivated for over
20 years in our laboratory (Furuya et al., 1987), and were used in
this study. Cultured E. perriniana cells were cultivated at 4-week
intervals on solid medium (MS medium; 100 ml in a 300-ml coni-
cal flask) containing 10 mM 2,4-dichlorophenoxyacetic acid and 1%
agar (adjusted to pH 5.7) in the dark. A solid culture was changed
to a suspension culture by transferring the solid cells to liquid
medium (100 ml) in a 300-ml conical flask.
Recently, we reported the glycosylation of hesperetin to
hesperidin, i.e., hesperetin 7-O-b-rutinoside, by cultured cells
of E. perriniana (Shimoda et al., 2008a). The results obtained
here suggested that cultured cells of E. perriniana can glycosy-
late flavanones such as hesperetin and naringenin (3) to the
corresponding 7-O-b-rutinosides. The present biotransformation
system is useful to convert flavanone glycosides to other glu-
cosyl conjugates of flavanones. Further studies on the physio-
logical properties of flavanone glycosides are now in progress.
4.3. Biotransformation of naringin (1) and naringenin (3) by cultured
E. perriniana cells and isolation of products
Substrate (a total amount of 1.5 mmol), naringin (1) or naringe-
nin (3), was individually administered to the 10 flasks (0.15 mmol/
flask) containing the cultured suspension cells and the flasks were
incubated at 25 °C on a rotary shaker (120 rpm) in the dark accord-
ing to the previously reported methods (Shimoda et al., 2007a,b,c,

K. Shimoda et al. / Phytochemistry 71 (2010) 201–205
205
2008a,b). After a 5-day incubation period, the cells and medium
References
were separated by filtration with suction. The filtered medium
was extracted with EtOAc. The medium was further extracted with
n-BuOH. The n-BuOH fraction was analyzed by HPLC. The cells
were extracted with MeOH for 12 h and then sonicated for 5 min.
The MeOH fraction was concentrated, and partitioned between
H2O and EtOAc. The EtOAc fractions were combined, concentrated,
and analyzed by HPLC. The H2O fraction was applied to a Diaion
HP-20 column, which was washed with H2O and then eluted with
MeOH. The MeOH eluate was subjected to preparative HPLC to iso-
late products.
Borradaile, N.M., Carroll, K.K., Kurowska, E.M., 1999. Regulation of HepG2 cell
apolipoprotein metabolism by the citrus flavanones hesperetin and
B
naringenin. Lipids 34, 591–598.
Chiba, H., Uehara, M., Wu, J., Wang, X., Masuyama, R., Suzuki, K., Kanazawa, K.,
Ishimi, Y., 2003. Hesperidin, a citrus flavonoid, inhibits bone loss and decreases
serum and hepatic lipids in ovariectomized mice. J. Nutr. 133, 1892–1897.
Furuya, T., Orihara, Y., Hayashi, C., 1987. Triterpenoids from Eucalyptus perriniana
cultured cells. Phytochemistry 26, 715–719.
Garg, A., Garg, S., Zaneveld, L.J.D., Singla, A.K., 2001. Chemistry and pharmacology of
the citrus bioflavonoid hesperidin. Phytother. Res. 15, 655–669.
Hai, L., Zhang, Q., Zhao, Y., Liang, H., Du, N., 2004. Studies on chemical constituents
from root of Hedysarum polybotrys. Zhongg. Zhong. Zazhi 29, 432–434.
Ko, J.H., Kim, B.G., Ahn, J.-H., 2006. Glycosylation of flavonoids with
a
The time-course experiment was carried out according to the
previously reported procedures (Shimoda et al., 2007a,b,7c,
2008a,b).
glycosyltransferase from Bacillus cereus. FEMS Microb. Lett. 258, 263–268.
Lewinsohn, E., Berman, E., Mazur, Y., Gressel, J., 1986. Glucosylation of exogenous
flavanones by grapefruit (Citrus paradisi) cell cultures. Phytochemistry 25,
2531–2535.
Lewinsohn, E., Berman, E., Mazur, Y., Gressel, J., 1989. Glucosylation and (1–6)
rhamnosylation of exogenous flavanones by undifferentiated citrus cell
cultures. Plant Sci. 61, 23–28.
Liu, Y., Yang, J., Cai, Z., 2006. Chemical investigation on Sijunzi decoction and its two
major herbs Panax ginseng and Glycyrrhiza uralensis by LC/MS/MS. J. Pharm. Bio.
Anal. 41, 1642–1647.
Miller, E.G., Peacock, J.J., Bourland, T.C., Taylor, S.E., Wright, J.M., Patil, B.S., Miller,
E.G., 2008. Inhibition of oral carcinogenesis by citrus flavonoids. Nutr. Cancer
60, 69–74.
Miura, H., Kawashima, K., Kitamura, Y., Sugii, M., 1986. Studies on the tissue culture
of Swertia japonica Makino. III. Glycosylation of naringenin in cultured cells.
Shoyakugaku Zasshi 40, 40–43.
Nafisi, S., Hashemi, M., Rajabi, M., Tajimir-Riahi, H.A., 2008. DNA adducts with
antioxidant flavonoids: morin, apigenin, and naringin. DNA Cell Biol. 27, 433–
442.
Ogundaini, A., Farah, M., Perera, P., Samuelsson, G., Bohlin, L., 1996. Isolation of two
new antiinflammatory biflavanoids from Sarcophyte piriei. J. Nat. Prod. 59, 587–
590.
Rousseff, R.L., Martin, S.F., Youtsey, C.O., 1987. Quantitative survey of narirutin,
naringin, hesperidin and neohesperidin in citrus. J. Agri. Food Chem. 35, 1027–
1030.
Shaw, P.E., Calkins, C.O., McDonald, R.E., Greany, P.D., Webb, J.C., Nisperos-Carriedo,
M.O., Barros, S.M., 1991. Changes in limonin and naringin levels in grapefruit
albedo with maturity and the effects of gibberellic acid on these changes.
Phytochemistry 30, 3215–3219.
4.4. Identification of biotransformation products
Structures of products were elucidated on the basis of spectro-
scopic techniques including HRFABMS, 1H and 13C NMR, H–H
COSY, C–H COSY, and HMBC spectroscopic analyses.
Spectral data for new compounds are as follows.
7-O-b-D D-glucopyranosylnaringenin (8):
-Gentiobiosyl-40-O-b-
½
a 2D5
ꢂ
ꢃ59.2 (c 0.09, MeOH); HRFABMS: m/z 781.2170 [M+Na]+; 1H
NMR (400 MHz, DMSO-d6): d 2.72 (1H, dd, J = 17.3, 3.0 Hz, H-3a),
3.17 (1H0,000 m, H-3b), 3.25–3.81 (18H, m, H-200, 2000, 20000 , 300, 3000, 30000 ,
400, 4000, 4 , 500, 5000, 50000 , 600, 6000, 60000 ), 4.25 (1H, d, J = 8.0 Hz, H-1000),
4.90 (1H, d, J = 8.0 Hz, H-100), 5.06 (1H, d, J = 7.6 Hz, H-10000 ), 5.48
(1H, dd, J = 12.6, 3.0 Hz, H-2), 6.10 (2H, m, H-6, 8), 6.90 (2H, d,
J = 8.4 Hz, H-30, 50), 7.40 (2H, d, J = 8.4 Hz, H-20,000060); 13C NMR
(100 MHz, DMSO-d6): d 42.1 (C-3), 60.8 (C-6000, C-6 ), 68.3 (C-600),
69.3 (C-4000), 69.5 (C-40000 ), 69.8 (C-400), 72.9 (C-20000 ), 73.00000(C-2000),
73.1 (C-200), 76.1 (C-5000), 76.5 (C-3000), 76.6 (C-500), 76.9 (C-3 ), 77.0
(C-300, C-50000 ), 78.5 (C-2), 95.2 (C-8), 96.0 (C-6), 99.6 (C-10000 ), 99.8
(C-100), 103.2 (C-10), 103.9 (C-1000), 115.1 (C-30, C-50), 128.2 (C-20,
C-60), 128.3 (C-10), 157.2 (C-40), 162.5 (C-9), 163.4 (C-5), 164.9 (C-
7), 196.7 (C-4).
Shimoda, K., Harada, T., Hamada, H., Nakajima, N., Hamada, H., 2007a.
Biotransformation of raspberry ketone and zingerone by cultured cells of
Phytolacca americana. Phytochemistry 68, 487–492.
Shimoda, K., Kwon, S., Utsuki, A., Ohiwa, S., Katsuragi, H., Yonemoto, N., Hamada, H.,
Hamada, H., 2007b. Glycosylation of capsaicin and 8-nordihydrocapsaicin by
cultured cells of Catharanthus roseus. Phytochemistry 68, 1391–1396.
Shimoda, K., Kondo, Y., Akagi, M., Abe, K., Hamada, H., Hamada, H., 2007c.
Glycosylation of tocopherols by cultured cells of Eucalyptus perriniana.
Phytochemistry 68, 2678–2683.
Naringenin 40,5-O-b-
D
-diglucopyranoside (11): ½a D25
ꢃ35.1 (c
ꢂ
0.15, MeOH); HRFABMS: m/z 619.1641 [M+Na]+; 1H NMR
(400 MHz, DMSO-d6): d 2.73 (1H, dd, J = 17.8, 2.8 Hz, H-3a), 3.15
(1H, m, H-3b), 3.20–3.55 (12H, m, H-200, 2000, 300, 3000, 400, 4000, 500, 5000,
600, 6000), 4.97 (1H, d, J = 7.6 Hz, H-1000), 5.05 (1H, d, J = 8.0 Hz, H-100),
5.51 (1H, dd, J = 12.6, 2.8 Hz, H-2), 6.11 (2H, m, H-6, 8), 6.90 (2H,
d, J = 8.4 Hz, H-30, 50), 7.41 (2H, d, J = 8.4 Hz, H-20, 60); 13C NMR
(100 MHz, DMSO-d6): d 42.1 (C-3), 60.4 (C-600), 60.5 (C-6000), 69.3
(C-400), 69.6 (C-4000), 72.7 (C-200, C-2000), 77.3 (C-3000, C-5000), 77.5 (C-
500), 77.6 (C-300), 78.4 (C-2), 95.1 (C-8), 96.0 (C-6), 99.2 (C-100),
103.1 (C-10), 103.7 (C-1000), 115.2 (C-30, C-50), 128.1 (C-20, C-60),
128.2 (C-10), 157.2 (C-40), 162.6 (C-9), 163.6 (C-5), 164.8 (C-7),
196.6 (C-4).
Shimoda, K., Hamada, H., Hamada, H., 2008a. Glycosylation of hesperetin by plant
cell cultures. Phytochemistry 69, 1135–1140.
Shimoda, K., Sato, N., Kobayashi, T., Hamada, H., Hamada, H., 2008b. Glycosylation
of daidzein by Eucalyptus cell cultures. Phytochemistry 69, 2303–2306.
Wu, J.B., Fong, Y.C., Tsai, H.Y., Chen, Y.F., Tsuzuki, M., Tang, C.H., 2008. Naringin-
induced bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun
and AP-1 pathway in osteoblasts. Eur. J. Pharmacol. 588, 333–341.
Zhang, M., Zhang, J.P., Ji, H.T., Wang, J.S., Qian, D.H., 2000. Effect of six flavonoids on
proliferation of hepatic stellate cells in Vitro. Acta Pharmacol. Sin. 21, 253–
256.