Glycosylation of vanillin and 8-nordihydrocapsaicin by cultured Eucalyptus perriniana cells

Article abstract of DOI:10.3390/molecules17055013

Glycosylation of vanilloids such as vanillin and 8-nordihydrocapsaicin by cultured plant cells of Eucalyptus perriniana was studied. Vanillin was converted into vanillin 4-O-γ-D-glucopyranoside, vanillyl alcohol, and 4-O-γ-D-glucopyranosylvanillyl alcohol

Full text of DOI:10.3390/molecules17055013

Molecules 2012, 17, 5013-5020; doi:10.3390/molecules17055013
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Glycosylation of Vanillin and 8-Nordihydrocapsaicin by
Cultured Eucalyptus perriniana Cells
Daisuke Sato 1, Yuki Eshita 2, Hisashi Katsuragi 3, Hiroki Hamada 4,*, Kei Shimoda 1,* and
Naoji Kubota 1
1
Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593,
Japan
2
Department of Infectious Disease Control, Faculty of Medicine, Oita University, 1-1 Hasama-machi,
Oita 879-5593, Japan
3
Sunny Health Co. Ltd., Yaesu k Bilg., 2-1-6 Yaesu, Chuo-ku, Tokyo 104-0028, Japan
4
Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005,
Japan
* Authors to whom correspondence should be addressed; E-Mails: hamada@dls.ous.ac.jp (H.H.);
shimoda@med.oita-u.ac.jp (K.S.); Tel.: +81-86-256-9473 (H.H.); Fax: +81-86-256-8468 (H.H.);
Tel.: +81-97-586-5606 (K.S.); Fax: +81-97-586-5619 (K.S.).
Received: 29 March 2012; in revised form: 17 April 2012 / Accepted: 23 April 2012 /
Published: 2 May 2012
Abstract: Glycosylation of vanilloids such as vanillin and 8-nordihydrocapsaicin by
cultured plant cells of Eucalyptus perriniana was studied. Vanillin was converted into
vanillin 4-O--D-glucopyranoside, vanillyl alcohol, and 4-O--D-glucopyranosylvanillyl
alcohol by E. perriniana cells. Incubation of cultured E. perriniana cells with 8-nor-
dihydrocapsaicin gave 8-nordihydrocapsaicin 4-O--D-glucopyranoside and 8-nordihydro-
capsaicin 4-O--D-gentiobioside.
Keywords: biotransformation; glycosylation; vanilloid; plant cultured cells; Eucalyptus
perriniana
1. Introduction
Biotransformations are considered to be an important method for converting inexpensive and
plentiful organic compounds into costly and scarce ones [1–5]. In recent years, plant cell cultures have

Molecules 2012, 17
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been studied as potential agents for biotransformation reactions according to the biochemical potential
of plant enzymes to produce specific secondary metabolites [1]. Particularly, glycosylation by cultured
plant cells has attracted much attention, because one-step enzymatic glycosylation by cultured plant
cells is more convenient than chemical glycosylation which requires tedious protection-deprotection
procedures [6–13].
Vanilloids including capsaicinoids have been widely used as food additives and have been studied
for their broad range of physiological properties such as antioxidative, antimicrobial, analgesic,
antigenotoxic, antimutagenic, and anticarcinogenic effects [14–19]. Irrespective of such biological and
pharmacological activities, vanilloids are water-insoluble and poorly absorbable after oral
administration. Furthermore, capsaicinoids are pungent principles of hot peppers, exhibiting direct skin
and mucous membrane irritant effects [20]. These shortcomings have limited their use as food
additives and medicines. Glycosylation allows water-insoluble and pungent organic compounds to be
converted into the corresponding water-soluble and non-pungent compounds to improve their
bioavailability. Here we report the glycosylation of vanillin and 8-nordihydrocapsaicin to their
glucoconjugates, with greater water-solubility, by cultured plant cells of Eucalyptus perriniana.
2. Results and Discussion
2.1. Biotransformation of Vanillin
The biotransformation of vanilloids, i.e., vanillin and 8-nordihydrocapsaicin, was individually
investigated using cultured cells of E. perriniana. After addition of substrates to the cultured
suspension cells in Murashige and Skoog’s (MS) medium, the incubation was continued for five days,
after which products in each medium and MeOH extracts of cells were detected by HPLC. No products
were obtained in the control experiments undertaken without substrate.
Incubation of vanillin (1) with the cultured E. perriniana cells for five days yielded compounds 2–4
which were identified as vanillyl alcohol (2%), vanillin 4-O--D-glucopyranoside (20%), and 4-O--D-
glucopyranosylvanillyl alcohol (70%), respectively, by comparison of spectroscopic data, such as
1
HRFABMS, H- and 13C-NMR (Table 1), H-H COSY, C-H COSY, and HMBC-spectra, with those
reported previously [21,22]. A time course experiment (Figure 1) revealed that vanillin 4-O--D-
glucopyranoside (3) was formed, followed by formation of 4-O--D-glucopyranosylvanillyl alcohol (4)
(Scheme 1) because the amount of vanillin 4-O--D-glucopyranoside (3) decreased, accompanied by
an increase of that of 4-O--D-glucopyranosylvanillyl alcohol (4). These findings suggest that vanillin
4-O--D-glucopyranoside (3) was converted into 4-O--D-glucopyranosylvanillyl alcohol (4) by
E. perriniana cells.
Table 1. 13C chemical shifts of the glycosides 3, 4, 6, and 7 in CD3OD.
Compound
3
4
6
7
Aglycone
1
2
3
4
5
130.2
110.1
151.4
148.9
114.2
136.0
110.7
148.4
144.9
114.8
134.8
113.0
150.5
146.8
117.9
135.1
113.1
150.8
146.7
118.3

Molecules 2012, 17
5015
Table 1. Cont.
Compound
3
4
6
7
6
7
8
9
125.1
191.2
118.2
62.5
121.1
43.6
175.7
37.0
27.0
30.3
30.3
30.3
32.9
23.6
14.4
56.6
102.7
74.7
77.9
71.2
77.6
62.4
121.4
43.8
176.0
37.1
27.1
30.3
30.3
30.3
32.9
23.7
14.4
56.7
102.5
75.1
77.8
71.5
77.6
69.4
104.3
74.8
77.6
71.3
77.8
62.6
10
11
12
13
14
15
16
OCH3
1'
2'
3'
4'
5'
55.4
99.1
72.8
76.9
69.3
76.6
60.3
55.3
99.9
73.0
76.7
69.4
76.6
60.4
Glc
6'
1''
2''
3''
4''
5''
6''
Figure 1. Time course of the biotransformation of vanillin (1) by cultured cells of
E. perriniana. Yields of 1 (■), 2 (○), 3 (●), and 4 (▲) are plotted.
100
50
48
96
Time/h

Molecules 2012, 17
Scheme 1. Biotransformation pathway of vanillin (1) by plant cultured cells of E. perriniana.
5016
HO
H CO
3
CH OH
2
5
HO
4
3
6
Vanillyl alcohol (2)
7
1
H CO
3
CHO
2
GlcO
Vanillin (1)
GlcO
CH OH
H CO
3
2
CHO
H CO
3
4
3
2.2. Biotransformation of 8-Nordihydrocapsaicin
After cultured cells of E. perriniana were incubated with 8-nordihydrocapsaicin (5) for five days,
compounds 6 and 7 were obtained as the products. They were identified as 8-nordihydrocapsaicin 4-O-
-D-glucopyranoside (62%) [23] and 8-nordihydrocapsaicin 4-O--D-gentiobioside (Glc--1,6-Glc)
(1%), respectively. The time-course experiment results (Figure 2) indicated that 8-nordihydrocapsaicin
4-O--D-glucopyranoside was produced first and further glucosylation gave 8-nordihydrocapsaicin 4-
O--D-gentiobioside, as shown in Scheme 2. The HRFABMS spectrum of cmpound 7 included a
pseudomolecular ion [M+Na]+ peak at m/z 640.2920, consistent with a molecular formula of
C29H47NO13 (calcd. 640.2903 for C29H47NO13Na).
Figure 2. Time course of the biotransformation of 8-nordihydrocapsaicin (5) by cultured
cells of E. perriniana. Yields of 5 (■), 6 (▲), and 7 (○) are plotted.
100
50
48
96
Time/h
1
The H NMR spectrum of compound 7 included proton signals at  4.35 (1H, d, J = 7.2 Hz) and
1
4.92 (1H, d, J = 7.6 Hz), indicating the presence of two -anomers in the sugar moiety. The H- and
13C-NMR spectra of compound 7 indicated that it was a -gentiobiosyl analogue of
8-nordihydrocapsaicin. Thus, the structure of compound 7 was determined as 8-nordihydrocapsaicin
4-O--D-gentiobioside, which has not been reported before.

Molecules 2012, 17
Scheme 2. Biotransformation pathway of 8-nordihydrocapsaicin (5) by plant cultured cells
5017
of E. perriniana.
HO
GlcO
H
N
H
N
H3CO
H3CO
O
O
8-Nordihydrocapsaicin (5)
6
GlcGlcO
H
N
H3CO
O
7
3. Experimental
3.1. Analysis of the Products
The structures of the products were determined on the basis of analysis of their HRFABMS, 1H- and
1
13C-NMR, H-H COSY, C-H COSY, and HMBC spectra. The H- and 13C-NMR, H-H COSY, C-H
COSY, and HMBC spectra were recorded using a Varian XL-400 spectrometer in CD3OD solution and
the chemical shifts are expressed in  (ppm) referring to TMS. The HRFABMS spectra were measured
using a JEOL MStation JMS-700 spectrometer.
3.2. Cell Line and Culture Conditions
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 [24]. A suspension
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).
3.3. Biotransformation and Purification of Products
To the 500 mL flask containing 200 ml of MS medium and the suspension cultured cells (100 g) of
E. perriniana was added 15 mg of substrate. The cultures were incubated at 25 °C for 5 days on a
rotary shaker (120 rpm) in the dark. After the incubation period, the cells and medium were separated
by filtration with suction. Extraction and purification procedures of biotransformation products were
performed according to the previously reported methods [9–13]. The yield of the products was determined
on the basis of the peak area from HPLC [column: YMC-Pack R&D ODS column (150 × 30 mm);
solvent: MeOH-H2O (9:11, v/v); detection: UV (280 nm); flow rate: 1.0 mL/min] and expressed as a
relative percentage to the total amount of the whole reaction products extracted.

Molecules 2012, 17
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8-Nordihydrocapsaicin 4-O--D-gentiobioside (2 mg): HRFABMS: m/z 640.2920 [M+Na]+ (calcd
1
640.2903 for C29H47NO13Na); H-NMR (CD3OD):  0.89 (3H, t, J = 6.8 Hz, H-16), 1.31 (10H, m,
H-11, 12, 13, 14, 15), 1.63 (2H, q, J = 7.6 Hz, H-10), 2.24 (2H, t, J = 7.6 Hz, H-9), 3.10–4.11 (12H, m,
H-2', 2'', 3', 3'', 4', 4'', 5', 5'', 6', 6''), 3.85 (3H, s, OCH3), 4.31 (2H, d, J = 8.0 Hz, H-7), 4.35 (1H, d,
J = 7.2 Hz, H-1''), 4.92 (1H, d, J = 7.6 Hz, H-1'), 6.85 (1H, dd, J = 8.0, 1.6 Hz, H-6), 6.94 (1H, s, H-2),
7.15 (1H, d, J = 8.4 Hz, H-5); 13C-NMR (CD3OD), see Table 1.
4. Conclusions
The results of this experiment revealed that the cultured suspension cells of E. perriniana are able to
convert vanilloids, including capsaicinoid, into the corresponding -glucosides and -gentiobioside.
Recently, several attempts have been made to glycosylate vanillin by cultured plant cells that resulted
in the production of only vanillin 4-O--D-glucopyranoside [21,22]. We have already reported that
cultured plant cells of E. perriniana had high potential to glycosylate exogenous compounds to their
glycosides including gentiobiosides [10,12,13]. However, there have been no reports on the
glycosylations of vanillin to the glycoside of vanillyl alcohol and of 8-nordihydrocapsaicin to its
gentiobioside derivative, and the biotransformation pathway of vanilloids in cultured plant cells has
not yet been elucidated. This paper reports, for the first time, the biological production by cultured
plant cells of vanilloid glycosides, such as -glucoside of vanillyl alcohol and 8-nordihydrocapsaicin
-gentiobioside, which was more soluble [25]. The biotransformation pathway of vanillin to -
glucoside of vanillyl alcohol has not been reported so far. Recently, a few studies were reported on the
biocatalytic synthesis of capsaicin primeveroside and vicianoside using Catharanthus roseus cells [26],
and on chemo-enzymatic synthesis of dihydronorcapsaicin β-D-glucopyranoside [27]. Cultured
C. roseus cells are more active rather than E. perriniana cells in the capsaicin glycosylations. Further
studies on the pharmacological activities of vanilloid glycosides are now in progress.
Supplementary Materials
Supplementary materials can be accessed at http://www.mdpi.com/1420-3049/17/5/5013/s1.
Conflict of Interest
The authors declare no conflict of interest.
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