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Springer-Verlag 1985
ABSTRACT
S u s p e n s i o n c u l t u r e s of B e r b e r i s w i l s o n a e
produce 4 berberine-type alkaloids: berberine, p a l m a t i n e , c o l u m b a m i n e
and J a t r o r rhizine. In particular the f o r m a t i o n of the
phenolic alkaloids c o l u m b a m i n e and Jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. W i t h h i g h e r a e r a t i o n rates, b e r b e r i n e
and J a t r o r r h i z i n e y i e l d s can be i n c r e a s e d
c o n s i d e r a b l y . T h u s we r e a c h e d an a l k a l o i d
y i e l d of m o r e t h a n 3 g x l- w i t h 50% d i s solved oxygen tension in the medium. As far
as we k n o w this is one of the b e s t r e s u l t s
in f e r m e n t i n g of a l k a l o i d - p r o d u c i n g
cell
cultures.
ABBREVIATIONS
P02, d i s s o l v e d o x y g e n c o n c e n t r a t i o n in %
s a t u r a t i o n ( u s i n g air); HPLC, h i g h - p e r f o r mance liquid chromatography; vvm, v o l u m e air
x v o l u m e m e d i u m -I x m i n u t e - l ; rpm, r e v o l u tions per minute; IAA, indole-3-acetic acid;
2,4-D, 2,4-dichlorophenoxy acetic acid.
INTRODUCTION
Culture conditions
Protoberberine-type
i s o q u i n o l i n e alkaloids
are c h a r a c t e r i s t i c
of the B e r b e r i d a c e a e .
M e m b e r s of this family are used in medicine,
e s p e c i a l l y in the F a r - E a s t , b e c a u s e of the
anti-bacterial and a n t i - i n f l a m m a t o r y properties of t h e i r a l k a l o i d s (Kondo 1976). In
addition, berberine-type alkaloids are k n o w n
to be c a p a b l e of i n d u c i n g c y t o s t a t i c a c t i v i t y in m a c r o p h a g e s in v i t r o ( K u m a z a w a et
al. 1984).
Berberis
s p e c i e s s y n t h e s i z e 4 m a i n berberine-type a l k a l o i d s : b e r b e r i n e , c o l u m b a m i n e , j a t r o r r h i z i n e and p a l m a t i n e (fig. I,
I k r a m 1975).
R10~
R1.R2=GH2 berberine
R20.,.,I.~ ~.1
~:CH3,R~CH
3 polmatine
~~I#=CH3,R'H
columbomine
F i g u r e I. The 4 m a i n a l k a l o i d s
wilsonae.
of B e r b e r i s
221
col, m o l w e i g h t 2025, E. M e r c k , D a r m s t a d t ,
FRG, x I - water). B e c a u s e of the g r a d u a l
d e p l e t i o n of the c a r b o n s o u r c e , a 30% g l u cose solution (w/v) was fed to the reaction
mixture
f r o m day 6 on. F e r m e n t a t i o n
was
carried out w i t h a constant aeration rate or
a constant dissolved oxygen concentration
(P02). The pO 2 w a s m e a s u r e d in s i t u w i t h a
p o l a r o g r a p h i c o x y g e n e l e c t r o d e (Dr. W. Ingold, U r d o r f , S w i t z e r l a n d ) and r e g u l a t e d
u s i n g an o x y g e n c o n t r o l u n i t f r o m B. B r a u n
(Melsungen, FRG).
A n a l y t i c a l procedures
i00 mg of the dried and p o w d e r e d tissue were
extracted under reflux with i0 ml methanol.
A f t e r c o o l i n g the e x t r a c t was f i l t e r e d and
diluted with m e t h a n o l 1:20.
The a l k a l o i d c o n t e n t of the s o l u t i o n was
a n a l y z e d by H P L C ( B e c k m a n G r a d i e n t L i q u i d
Chromatograph
M o d e l 332) u s i n g a 250 x 4.6
mm stainless steel column with Nucleosil
5 C 1 8 ( M a c h e r e y & Nagel, D ~ r e n , FRG) as the
stationary phase. The liquid phase was water
a d j u s t e d to pH3 w i t h H 3 P O 4 and 84% a c e t o nitrile redistilled in w a t e r adjusted to pH B
w i t h H 3 P O 4. We u s e d a m o d i f i c a t i o n of RHf ~
fer,s gradient (personal c o m m u n i c a t i o n 1984)
as s h o w n in fig. 2.
The flow rate was 1.5 ml x min. -I, m e a s u r i n g
w a v e length 228 rim, injection volume 20 ul.
0 % B (Acetonitrite,84%, pH 3)
96-
4431+"
27"
6 ......
' ....
1'0 ....
' -
2'0"
of pO 2 on cell g r o w t h
aeration rate
dissolved oxygen
spec growth rate
doubling time
dry weight at day1&
(vvm)
0.1-0.3 0.1-0./+ 0+1-0.5
{%p02)
20
40
50
(days-1)
Q15
0.15
0.13
(days)
4.6
4.6
5.5
(gxl-l)
25.5
25.1
24.8
T a b l e I. G r o w t h d a t a at v a r i o u s
oxygen concentrations.
dissolved
Effect of pO 2 on alkaloid f o r m a t i o n
r""
"
....
' ....
....
'tm+n]
columbamine
J: J a t r o r r h i z l n e
p: p a l m a t i n e
b: berberine
F i g u r e 2.a: P e r c e n t a g e of a c e t o n i t r i l e in
the l i q u i d phase. R i n s i n g
cycle between
m i n u t e 25 and 35. b: H P L C - c h r o m a t o g r a m
of
the 4 p r o t o b e r b e r i n e alkaloids.
c:
Fig. 3 shows that increasing the oxygen tension and a e r a t i o n rate enhances the a c c u m u lation of berberine, c o l u m b a m i n e and jatrorrhizine, w h e r e a s p a l m a t i n e is only affected
to a v e r y m i n o r e x t e n t . T h i s r e f l e c t s the
close b i o s y n t h e t i c i n t e r r e l a t i o n s h i p of the
f i r s t t h r e e a l k a l o i d s ; the f i n a l s t e p s in
p a l m a t i n e b i o s y n t h e s i s may be s o m e w h a t divergent. W h e r e a s fig. 3 only shows the m a x i mal a m o u n t of alkaloids, fig. 5 d e m o n s t r a t e s
the k i n e t i c s of a c c u m u l a t i o n
for the f o u r
a l k a l o i d s d u r i n g the g r o w t h c y c l e of the
cells. M a x i m a l berberine a c c u m u l a t i o n occurs
v e r y e a r l y (days 2-4) d u r i n g the lag p h a s e
in the g r o w t h cycle, d i m i n i s h i n g
w h e n columbamine
and j a t r o r r h i z l n e a c c u m u l a t i o n
i n c r e a s e d u r i n g the l o g a r i t h m i c and e a r l y
222
mg g-1
mg g.-1
a, O~ P2 :
1 (3 20%
~81
132=
40'/,,/~
132:
50%
I 1I
s t a t i o n a r y g r o w t h phase. This, too, d e m o n strates the intermediate function of berberine in columbamine and Jatrorrhizine biosynthesis, as was found in Beecher and Kelleher,s enzymatic studies (1983).
The maximum
Jatrorrhizine
accumulation
(10.1%) in percent of dry m a t t e r can be
o b s e r v e d on day 15. A l k a l o i d b i o s y n t h e s i s ,
however, continues until day 20, as is shown
in fig. 4.
ao
20t ~r~
-5~I0
o~
bpc j
bpc j
vvm
wm=
00~
vvm= ,,1
bpc j
bpc j
bpc j
I\
mg g-1
80
ill
"
60~
N
l..
~o ~,
b)c j
of alkaloid
rates.
forma-
20
g1-1
gl-1
4
&O
I
0
3I
30
-
C@
E
2~
o
20
L.
x
0
o
0
10
12
16 20
deys
12
16 20
doys
Figure 5. The kinetics of alkaloid accumulation d u r i n g the g r o w t h cycle and the yield
in dry w e i g h t at P02=50% in the culture
broth.
Because of the significant increase in cell
dry w e i g h t there is still an i n c r e a s e in the
to{al a l k a l o i d yield up to m o r e than 3 g x
I- . To our knowledge, these are the highest
alkaloid yields from plant cell cultures in
bioreactors.
Although the oxygen demand of plant cells is
quite low when compared with microorganisms
the e x p e r i m e n t s p r e s e n t e d here show very
clearly that it may be very i m p o r t a n t to
control the oxygen tension in the bioreactor
in order to obtain optimal secondary product
yields f r o m plant cell cultures. S i m i l a r
results were o b t a i n e d by S p i e l e r et ai.
(1985). M o r e o v e r , it can be seen that by
p r o v i d i n g the a p p r o p r i a t e c u l t u r a l conditions it is possible to improve the product
yields in bioreactors substantially as compared with the results in shake flasks.
ACKNOWLEDGEMENTS
We w o u l d like to thank Dr. M. R G f f e r (Munich, FRG) for her kind advice and v a l u a b l e
information on alkaloid separation by HPLC.
223
This w o r k was s u p p o r t e d by the B u n d e s m i n i sterium fGr Forschung und Technologie.
REFERENCES
531
Beecher
letters
Fowler
CWW, Kelleher
24: 469-472
WJ
(1983) Tetrahedron
Tanaka H (1982)
442
F u k u i H, N a k a g a w a
K, T s u d a S, T a b a t a M
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Hinz H, Zenk MH (1981) N a t u r w i s s e n s c h a f t e n
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Ikram M (1975) Planta Med 2 8 : 3 5 3 - 3 5 8
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K u m a z a w a Y, Hagaki A, F u k u m o t o M, F u j i s a w a
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F (1962)
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S (1982) U n t e r s u c h u n g e n
zur
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