Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
02
HOECHST AKTIENGESELLSCHAFT HOE 86/F 263 Dr.LA/mu
Description:
A process for obtaining diterpenes of the labdane type,
in particular forskolin, from Coleus forskohlii
s
The plant Coleus forskohlii Briq., which belongs to the
Lamiaceae family and has its habitat in India, contains
several diterpenes of the labdane type. Those present
in the greatest amounts are 1,9-dideoxyforskolin ~DDF)
and forskolin (BHAT et al., Tetrahedron Letters 19 (1977)
1669-1672). The content of forskolin, based on dry
weight, is about 0.05% for the whole plant and about 0.1%
for the root (SHAH et al., Planta Medica 39 (1980), 183-
185).
Forskolin (= 73-acetoxy-8,13-epoxy-1~,6B,9~-trihydroxy-
labd-14-en-11-one) has the following struc-
tural formula:
1~
H~;) qj~ ~ls
~--8--CH3
The interest in the plant Coleus forskohlii is determined
by the pronounced pharmacological effects of forskolin.
It has a positive inotropic effect, increases the heart
rate and lowers the blood pressure (LINDNER et al.,
Arzneim.-Forsch.lDrug Research 28 (I) No. 2 (1978), 284-
289). This and many other actions are mediated by acti-
vation of the adenylate cycLase of the membranes, which
results in an increase in the intracellular concentration
of cyclic adenosine monophosphate (cAMP) (METZGER H.,
LINDNER E., Arzneim.-Forsch./Drug Research 31 (II), No. 8
7~)Z
-- 2
(1981) 1248-1250; SEAMON K.B., DALY J.W., J. Cycl. Nucl.
Res. 7 (4) (1981), 201-224; DE SOUZA N. et al., Med.
Res. Rev. 3 (1983), 201-219). Thus the importance of
forskolin is two-fold: on the one hand as a medicament,
and on the other hand as an excellent aid to the investi-
gation of hormone actions which are triggered via cAMP as
the second messenger.
Hitherto, forskolin has been obtained mainly from wild-
growing stands, as well as from cultivated stands in some
cases, of Coleus forskohlii by extraction of the dried
roots.
The process of the present invention which is described
hereinafter now represents a new method of obtaining the
various diterpenes, but especially forskolin.
For this purpose, Coleus forskohlii cell cultures are
maintained as suspension cultures. The formation of for-
skolin derivatives taking place on what is called themaintenance medium is zero or only slight. In order to
stimulate production, the cell aggregates are transferred
into an "induction medium" which differs from the main-
tenance medium in respect of, in particular, the phyto-
hormone contents. The diterpene formation reaches itsmaximum in a period of 2-6 weeks, and concentrations of
0.005-0.06~ forskolin, based on dried cell material,
result.
The advantage of this new production method is the inde-
pendence of the preparation of the substance from seasonal
and climatic factors; nor is it bound to the habitat in
India, and it also reduces economic and political risks.
The process according to the invention is described in
detail hereinafter. Initially, callus cultures of Coleus
forskohlii plants are set up by known methods. After
some passages (= culture per ods) on nutrient medium
3;~()Z
-- 3 --
solidified with agar, it is possible to transfer the cell
aggregates into liquid medium and maintain them there as
suspension cultures.
Suitable culture conditions for the maintenance culture
are:
storage in glass vessels, for example Erlenmeyer flasks
aeration by shaking on an orbital shaker at 80-15û rpm
(revolutions per minute)
temperature 18-38C
continuous light, continuous dark or light/dark rhythm.
It is possible to use customary plant-cell culture media
as maintenance media, for example Gamborg's B5 medium
(GAMBORG, D.L., Plant Physiol. 45, 372 (1970)) or MS
medium (MURASHlGE, T. and SKOOG, F., Plant Physiol. 28,
473-497 (1962)) with the addition of phytohormones, for
example auxins such as 2,4-dichlorophenoxyacetic acid
(2,4-D), indole-3-butyric acid (IBA) or naphthylacetic
acid (NAA) in a suitable concentration, for example of
0.2-5 mg/l, preferably û.5-2 mg/l, and/or cytokinins such
as kinetin or 6-benzylaminopurine (BAP) in a suitable
concentration, for example of 0.05-2 mg/l, preferably
0.1-0.5 mg/l.
The crucial step for stimulating the production of for-
skolin comprises transferring the cells into a "working
or induction medium". This medium differs from the main-
tenance medium in that it either
a) contains no phytohormones, or
b) in place of auxins having a strong dedifferentiating
action, such as, for example, 2,4-D, contains one or
more other auxins such as, for example, IBA or NAA, or
c) contains no auxins but only cytokinins, or
d) contains auxins
and/or cytokinins in lower concentrations than in the
maintenance medium.
OZ
-- 4
The induction medium is renewed at intervals ot 7-30 days,
and, after each passage, part of the cell material is harvested
in order to obtain forskolin.
During the production period the cultivation is carried out preferably
in continuous dark.
In the first induction passage, initially the culture
growth is found to be still hardly changed, and the for-
mation of forskolin derivatives is usually confined to
small amounts of 1,9-dideoxyforskolin (DDF) and traces
of the other diterpenes. The maximum content of for-
skoLin and its derivatives is then reached as early as
the second or third induction passage. The entire spec-
trum of diterpenes which is found in the root of the plant
is formed.
The production phase can be continued in Erlenmeyer
flasks for a period of up to several months. However,
the forskolin content and culture growth greatly depend
on whether the process is carried out on hormone-free
medium (--~ production period about 3 passages) or in the
presence of phytohormones in the form of an auxin/cyto-
kinin combination. A medium containing 1.0 mg/l IBA and
0.2 my/l BAP is, for example, suitable for long-term pro-
duction.
To obtain diterpene-containing cell material on a larger
scale, the culture is cultivated on the induction medium
in commercially available fermenters. The preculture
(i.e. the passage(s) before the start of an induction
experiment) for an experiment of this type is preferably
carried out in an Erlenmeyer flask, but can also be moved
into a fermenter from which the maintenance medium can be
removed in a suitable manner.
After completion of a production experiment, the cells
and nutrient medium are separated, and the latter is dis-
carded because it contains only minimal amounts of di-
terpene. The cells are dried and, in analogy to the
roots, are extracted with organic solvents (toluene, di-
02
-- 5
chloromethane, methanol, inter alia) (see, for example,
German Patent No. 2,557,784). Finally, forskolin and,
where appropriate, other labdane derivatives, for example
1,9-dideoxyforskolin and 1-deoxyforskolin, are isolated
from the crude extract by chromatography, for example on
silica gel columns.
Quantitative determination is carried out by, for example,
GLC, HPLC (INAMDAR et al., J. Pharm. Sci. 69 (1980), 1449-
19 1451; Planta Medica 50 (1984), 30-34) or TLC scanning.
Example 1
Induction of a light suspension
A suspension culture of Coleus forskohlii maintained under
continuous light is used.
Preculture
Initial weight: 16 g of cell material (fresh
weight) in a 1 l Erlenmeyer
flask containing 300 ml of
medium
Medium: B5 (plant cell culture medium
of GAMBORG, Plant Physiol.,
45 (1970), 372) containing
1.0 mg/l 2,4-D and 0.2 mg/l
kinetin
Temperature: 24C
Shaking speed: 100 rpm
Light: continuous light
Duration of passages: 14 days
Multiplication: 12
Induction and production periods
Medium: B5 medium containing 0.4 mg/l
IBA
;3'7~)2
-- 6
Light: continuous dark
Temperature: 24C
Shaking speed: 100 rpm
for further details, see Table 1 which follows:
02
~,
o U)
~o ~ I >~ o~ o~
~c ~ ~ o
~~ 1/1 ~1 ~ ~J N
c ~n ~OO O
J~ ~~ ~I . .
~~ ~ ~ ~ O O
J
c
O V)
O ._ 1/)
u~ ~ ~ ~ ~o r~ ~
C ~ (O 0 ~ ~ ~)
u~ ~:n ~ u~ u~
Q C V~ O O O ~
as ~o ~ . . . ~J
Q ~ ~ ~O O O
V~ ~ ~
N
~,OQ
~ CQ ' C O
O ~
tn ~IJ
oo
~ C ~ V) ~7) ~ ~-- ~ D~Jl O C V) . ~ O
~n u~ O +
J Q ~ ~ ~ ~ O
~n D
C .C
-
O
_ _ ~ ~ O
v) a) ~ E u~
~ ~ ~ ~ ~ ~ , ~
O) ~ 3 ' C~ ~ ~:5) 0
_ C O - ~U) ~CJ
Q ~ tl) E ~r, ~ 3 C
E ~ ~ a~ ID C ~ O
10 C ~ L ~ Q O .,>~
x o ~r) ~ o ., ~, ,_
LLI L,~ ., ~ ~ ~ 3 ~ 1'0
J O ~
3 E ~ C ~~ ,
J 11~ 0 O _ _ ~ O
t~ ~ O., Q O ;~ ~
~ ., ~ )~ .,_ ~ ~,
D ., ~ LL _ L
~o C ~ ~~ ~O ;~
E Ql C~ c-L~ _,
02
-- 8 -
Working up and quantitative determination
Freeze-drying of the freshly harvested cells after re-
moval of nutrient medium
Powdering of the cell material in a porcelain mortar
Extraction of the diterpenes with toluene (2 hours, 50C)
while rotating in a non-evacuated rotary evaporator
Filtration and concentration of the filtrate to dryness
in a rotary evaporator (T = 50C)
Crude extract taken up in methanol in an ultrasonic bath
The forskolin derivatives are quantitatively determined
by development of thin-layer chromatograms and detection
by means of TLC scanning:
Mobile phase = toluene/ethyl acetate 85:15
Development = 2 x 15 cm
Plates = precoated silica gel 60 F2s4 plates
Detection = STAHL anisaldehyde/sulfuric acid reagent,
2û immersion bath; subsequent heating at
130-140C for about 5 min
Scanner = Shimadzu Dual CS-930 from Desaga
wavelength 500 nm
Example 2
Fermentation ~ith light suspension, production on hormone-
free med;um
This example shows that cultivation of Coleus forskohlii
suspension cultures under induction conditions is also
possible on a larger scale. The culture container used
is a 20 l airlift fermenter (WAHL J., Dissertation,
Tubingen, 1977) with an automatic facility for maintaining
the content of dissolved oxygen in the medium (P02) con-
stant.
Initially, a growth passage on normal maintenance medium
(cf. EXAMPLE 1) is carried out in the fermenter, and then
B * denote s trade -mark
12~;~702
the cell suspension is removed, apart from 4 l which are
left, and the fermenter is made up to the working volume
of 19 l again with hormone-free B5 medium.
In the first induction passage which now follows, the
culture growth continues to be very strong; a packed cell
volume (PCV) of 95% is reached after only about 13 days.
Thereafter, small amounts of DDF, but no forskolin, are
detectab.e in the cells.
To continue the experiment, the cell suspension is again
removed, apart from 3.5 l which are left, and is replaced
by fresh hormone-free B5 medium. In this second induction
passage the formation of substantial amounts of diterpenes
starts, and it reaches its maximum of 0.0053% forskolin
and 0.0116% DDF on day 9, which is thus the optimal time
for harvesting cell material. If the fermentation is
continued, the diterpene content decreases continuously,
owing to part of the culture dying.
Data on the fermentation
Culture: light suspension of Coleus for-
skohlii
Preculture in flasks: 15 days, initial weight 16-17 9
fresh weight in 300 ml of B5
maintenance medium (fresh weight =
weight of the wet cells after
drainage of the medium)
Temperature: 24C
Working volume: 19 l
Sucrose/glucose: a residual glucose content > 0.5%
is maintained by addition of
sucrose solution (30%).
Growth passage (diffuse daylight)
Medium: B5 (1.0 mg/l 2,4-D and 0.2 mg/l
kinetin)
'7~Z
- 10 -
Inoculum: 2.4 l = 8 preculture flasks
(amount inoculated)
Duration: 16 days
P02: 35%, increasing to 50~ from day 14
1st induction passage (continuous dark)
Medium: B5, hormone-free
Inoculum: 4 l of suspension from the growth
passage
Duration: 13 days
P02: 35%
Foam suppression: addition of a total of about
250 ml of polypropylene glycol
solution
2nd induction passage (continuous dark)
Mediuw: B5, hormone-free
20 Inoculum: 3.5 l of suspension from the
1st induction passage
Duration: 26 days
P02: decreasing from 70~ to 40%
Aeration rate: vvm = 0.06-0.10 (= 70-110 l of
air per hour and per 19 l of
~orking volume)
Diterpene content (% by weight) Forskolin DDF
Day 7 0.00350.0092
Day 8 0.00320.0093
Day 9 0.00530.0116
Day 11 0.00160.0102
Example 3
Induction of a light suspension, isolation of diterpenes
A suspension culture of Coleus forskohlii maintained under
continuous light is used.
)Z
Preculture: as in Examp(e 1
Induction and production periods: as in Example 1
uith the follouing differences
Medium: ~5, hormone-free
In;tial weight: 22.5 9 and 29.5 9 fresh ueight/
300 ml of medium
(2nd induction passage)0 Duration sf passages: 1st induction passage 15 days
2nd induction passage 11 days
At the end of the second induction passage, the cells are
harvested for isolation of diterpenes.
Extraction: 2.26 9 of freeze-dried cells uith
toluene (cf. Example 1)
Crude extract: 68.6 mg - 3.0% - only partly
soluble in methanol
The forskolin derivatives are prepared pure by column
chromatography.
Column: Labomati~, about 50 ml, packed
uith 30 ~m silica gel
Mobile phase: dichloromethane/ethyl acetate 8:1
Extract solution: methanol is replaced as solvent
by the mobile phase
Amount of substance
30 for separation: about 25 mg (i.e. about 50% of
the original soluble crude extract)
The total amount of substance uhich can be eluted, 12.8 mg,
yields, inter alia:
0.7 mg of forskolin
1.0 mg of 1,9-dideoxyforskolin (DDF)
0.4 mg of 1-deoxyforskolin.
* denotes trade-mark
B~ ~
0~
- 12 -
The identity of the individual fractions is checked by
GLC, HPLC (INAMDAR et aL., 1980 and 1984) or by two-
dimensional thin-layer chromatography.
TLC plates: HPTLC, silica gel 60 F254
10 x 10 cm
Mobile phase: I. dichloromethane/ethyl ace~ate
8:1
II. diisopropyl ether/chloroform
7:3
