Synthesis of anti-tumor dimeric indole alkaloids in catharanthus roseus was promoted by irradiation with near-ultraviolet light at low temperature

Article abstract of DOI:10.1271/bbb.90545

We have found that coupling between catharanthine and vindoline occurs non-enzymatically in the presence of flavin mononucleotide and manganese ions with near- ultraviolet light irradiation in vitro. The present study found that the concentrations of cath

Full text of DOI:10.1271/bbb.90545

Biosci. Biotechnol. Biochem., 74 (2), 386–389, 2010
Note
Synthesis of Anti-Tumor Dimeric Indole Alkaloids in Catharanthus roseus
Was Promoted by Irradiation with Near-Ultraviolet Light at Low Temperature
Mamiko ASANO,1 Kazuo HARADA,1 Tomoaki YOSHIKAWA,1
y
1;
Takeshi BAMBA,2 and Kazumasa HIRATA
1Department of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Biotechnology, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Received July 27, 2009; Accepted November 15, 2009; Online Publication, February 7, 2010
[doi:10.1271/bbb.90545]
We have found that coupling between catharanthine
and vindoline occurs non-enzymatically in the presence
of flavin mononucleotide and manganese ions with near-
ultraviolet light irradiation in vitro. The present study
found that the concentrations of catharanthine and
vindoline in Catharanthus roseus decreased and those
of dimeric indole alkaloids increased under near-
ultraviolet light at 4 ꢀC. It indicates that this coupling
reaction at 4 ꢀC occurs non-enzymatically.
Freshly harvested leaf sections were used as the 0 d
control without any light treatment. After irradiation, the
leaf sections were frozen in liquid N2 and crushed. The
crushed material was added to 500 ml of methanol and
sonicated for 5 min. The mixture was centrifuged at
18;000 g at 4 ꢀC for 5 min, and the supernatant was
collected. These extraction steps were repeated at 3
times. Ten ml of the extract was subjected to HPLC as
outlined below. HPLC analyses were performed follow-
ing ref. 6 except for the following point: the mobile
phase was acetonitrile/triethylamine/water (60/0.006/
40). Detection of DIAs (anhydrovinblastine, leurosine,
and vinblastine) was by fluorescence emission excited at
305 nm. One section was irradiated with white light
(20 W/m2) as a control. The other section was irradiated
with NUV (5 W/m2). The temperatures for light
irradiation were set at 25 ꢀC, at which both the
enzymatic and non-enzymatic coupling reactions occur-
red, or at 4 ꢀC. Changes in the alkaloid concentrations of
C. roseus leaf sections irradiated with NUV at 4 ꢀC and
at 25 ꢀC are shown in Fig. 2. At both temperatures, even
at 4 ꢀC, the concentrations of catharanthine and vindo-
line decreased and the total DIAs content increased after
irradiation with NUV. Significant changes in the
alkaloid concentrations at 4 ꢀC were observed at 5 d
after irradiation, but those at 25 ꢀC were observed
immediately. The catharanthine and vindoline concen-
trations as irradiated with NUV decreased as compared
with white light after 7 d of irradiation at 4 ꢀC and 25 ꢀC
respectively, whereas the concentrations of DIAs
reached maximum at 5 and 7 d of irradiation at 4 ꢀC
and 25 ꢀC respectively. NUV, with one-fourth the light
intensity of white light, induced DIA synthesis.
Key words: FMN; near-ultraviolet light (NUV); Cathar-
anthus roseus; dimeric indole alkaloids
(DIAs)
Dimeric indole alkaloids (DIAs) produced by Cathar-
anthus roseus, including vinblastine, anhydrovinblas-
tine, and leurosine (Fig. 1), have antineoplastic activ-
ities,1) but these alkaloids are very expensive because of
their low concentrations in plants. Hence, the biosyn-
thetic pathway of DIAs has been investigated actively.
DIAs were synthesized by a coupling reaction between
catharanthine and vindoline with oxidization of cathar-
anthine as the first step. Kutney et al. have proposed that
this initial step is catalyzed by enzyme including
peroxidase.2) In contrast, we have found that the
coupling reaction occurred in vitro in the presence of
FMN and Mn2þ and in the absence of any enzymes after
irradiation by near-ultraviolet light (NUV), with a peak
at 370 nm.3,4) Moreover, we also found that the amount
of DIAs in the leaves of multiple shoot cultures and
intact plants specifically increased with NUV irradia-
tion.4,5) This study was the first step to determine
whether the coupling reaction occurs non-enzymatically
in vivo. We analyzed the concentrations of alkaloids in
plant leaves after NUV irradiation at 4 ꢀC, a temperature
at which the rate of enzymatic reactions was slow.
Catharanthus roseus was obtained following ref. 4
and grown outdoors in soil to 2 months of age. The
leaves were sliced along the center vein to obtain two
sections. Paired leaf sections were floated on water and
irradiated with NUV (FL15BA-37K, Matsushita Elec-
tric, Tokyo) as treatment and white light (FL10ENW,
Matsushita Electric) as the control for 1, 3, 5, and 7 d.
The important point is that the coupling reaction
occurred in vivo depending on the NUV at 4 ꢀC.
Additionally, two in vitro experiments based on the
coupling reaction were performed at 4 ꢀC. One was an
enzymatic reaction due to horseradish peroxidase which
is known as the coupling reaction in vitro, and the other
was a non-enzymatic reaction that we have reported
which occurred in the presence of FMN and Mn2þ under
NUV light irradiation. These reactions in vitro were
performed at the highest rate and yield. In vitro
y
To whom correspondence should be addressed. Tel/Fax: +81-6-6879-8235; E-mail: hirata@phs.osaka-u.ac.jp
Abbreviations: NUV, near-ultraviolet light; DIAs, dimeric indole alkaloids

Synthesis of Dimeric Indole Alkaloids with Near-Ultraviolet
387
N
N
O
OH
N
O
O
N
H
O
H
O
O
O
Catharanthine
Vindoline
N
N
N
H
O
O
OH
O
N
O
H
O
O
O
Anhydrovinblastine (AVLB)
N
N
OH
O
N
OH
N
N
H
O
N
H
O
O
O
OH
O
O
N
N
O
O
H
O
H
O
O
O
O
O
Vinblastine
Leurosine
Fig. 1. Biosynthesis of DIAs (vinblastine, anhydrovinblastine, and leurosine) from Monomeric Precursors Catharanthine and Vindoline.
A
B
1200
800
1200
800
600
600
0
0
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
C
D
1200
1200
800
600
0
800
600
0
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Time (d)
Catharanthine
Vindoline
DIAs (vinblastine, anhydrovinblastine, leurosine)
Fig. 2. Changes in Alkaloid Concentrations in Plant Leaves at 4 ꢀC (A, C) and 25 ꢀC (B, D) under Irradiation by NUV.
Leaf sections were irradiated with NUV as the treatment (C, D) and with white light as the control (A, B) for 1, 3, 5, and 7 d. Freshly harvested
leaf sections were used as the 0 d control without light treatment.

388
M. ASANO et al.
A
B
C
D
30
20
10
0
30
20
10
0
30
20
10
0
30
20
10
0
0
20
40
60
0
20
40
60
0
20
40
60
0
1
2
3
4
5
6
7
Time (min)
Time (d)
E
F
G
30
20
10
0
30
20
10
0
30
20
10
0
0
20
40
60
0
20
40
60
0
20
40
60
Time (min)
H
I
J
30
30
20
10
0
30
20
10
0
20
10
0
0
20
40
60
0
20
40
60
0
20
40
60
Time (min)
K
L
M
30
30
20
10
0
30
20
10
0
20
10
0
0
20
40
60
0
20
40
60
0
20
40
60
Time (min)
Fig. 3. Effects of Temperature and Light on the Enzymatic and Non-Enzymatic Coupling Reaction of Anhydrovinblastine.
Enzymatic reaction, vindoline and catharanthine (150 mM) were reacted in 0.1 M Tris–HCl buffer (pH 7.0) in the presence of 18 U/ml
horseradish peroxidase, and 1 mM H2O2 under dark (A, D, E) and irradiation with white (B, F) or NUV light (C, G) at 4 ꢀC (A, B, C, D) and 25 ꢀC
(E, F, G). Non-enzymatic reaction, vindoline and catharanthine (150 mM) were reacted in 0.1 M Tris–HCl buffer (pH 7.0) in the presence of
100 mM FMN and 1 mM MnCl2 under dark (H, K) and irradiation with white (I, L) or NUV light (J, M) at 4 ꢀC (H, I, J) and 25 ꢀC (K, L, M).
enzymatic reactions were performed following ref. 6,
except for the following: the reaction was initiated by
adding 300 ml of 180 U/ml peroxidase (Amresco, Solon,
OH). The non-enzymatic reaction was carried out using
the reaction mixture containing 1 mM of MnCl2 and
excluding H2O2. The reaction was started by adding
30 ml of 10 mM FMN. The DIAs produced by in vitro
coupling reactions were converted to AVLB by reduc-
tion of NaBH4. Figure 3 shows the time-courses of
AVLB production by the in vitro enzymatic and non-
enzymatic coupling reactions. Catharanthine and vindo-
line of the enzymatic reaction at 25 ꢀC and the non-
enzymatic reaction at 4 ꢀC and 25 ꢀC did not occur at all
at 60 min. The catharanthine and vindoline of the
enzymatic reaction at 4 ꢀC were still present at 60 min
and reduced for 24 h, but AVLB was not synthesized.
Although the enzymatic reactions occurred at 25 ꢀC
under both lighting conditions, the enzymatic reactions
at 4 ꢀC were suppressed completely. In contrast, the non-
enzymatic reactions occurred at both temperatures under
NUV and the rate of the coupling reaction increased at
25 ꢀC, as did the yield of AVLB at 4 ꢀC under irradiation
of NUV. Our in vivo experiments indicated that NUV
was required for the increase in DIAs levels at low
temperature, whereas our in vitro experiments indicated
that the non-enzymatic coupling reaction at low temper-
ature proceeded by NUV irradiation while the enzymatic
coupling reaction at low temperature did not proceed.
It is apparent that the enzymatic reaction was sup-
pressed at 4 ꢀC and that the synthesis of DIAs might
have occurred non-enzymatically in vivo. To confirm
this possibility, we should perform experiments that to
relate in vitro and in vivo to determine whether this non-
enzymatic reaction occurs in vivo. For example, we
should verify that the peroxidase in Catharanthus roseus
suppress activity at 4 ꢀC as well as horseradish perox-
idase does. There is no report that peroxidases in vivo
increase the activity due to NUV at 4 ꢀC, but we need to
analyze the peroxidase activity in Catharanthus roseus.
Also, we could not compare the light intensity of NUV

Synthesis of Dimeric Indole Alkaloids with Near-Ultraviolet
389
in vitro and in vivo because there are various pigments
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there is a possibility that irradiation of NUV promotes
the redox state and the coupling reaction might not
depend on flavins. If the coupling reaction in vivo relates
to flavins, the flavins might eliminate the oxidation
energy generated by NUV in the cell by biosynthesis
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