CA1293702C - Process for obtaining diterpenes of the labdane type, in particular forskolin, from coleus forskohlii - Google Patents
Process for obtaining diterpenes of the labdane type, in particular forskolin, from coleus forskohliiInfo
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- CA1293702C CA1293702C CA000550082A CA550082A CA1293702C CA 1293702 C CA1293702 C CA 1293702C CA 000550082 A CA000550082 A CA 000550082A CA 550082 A CA550082 A CA 550082A CA 1293702 C CA1293702 C CA 1293702C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
Abstract of the disclosure:
The present invention relates to a process for obtaining diterpenes of the labdane type, in particular forskolin, from cell cultures of Coleus forskohlii. The process comprises transferring the cell cultures from a mainten-ance medium into an induction medium which differs from the maintenance medium in respect of, in particular, the phytohormone content, and the diterpenes being obtained from it, after a specific time period, by extraction with organic solvents.
The present invention relates to a process for obtaining diterpenes of the labdane type, in particular forskolin, from cell cultures of Coleus forskohlii. The process comprises transferring the cell cultures from a mainten-ance medium into an induction medium which differs from the maintenance medium in respect of, in particular, the phytohormone content, and the diterpenes being obtained from it, after a specific time period, by extraction with organic solvents.
Description
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
(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
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
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-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 Light: continuous dark Temperature: 24C
Shaking speed: 100 rpm for further details, see Table 1 which follows:
~, 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~ _, 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
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~
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
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
(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
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
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-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 Light: continuous dark Temperature: 24C
Shaking speed: 100 rpm for further details, see Table 1 which follows:
~, 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~ _, 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
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~
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
Claims (12)
1. A process for obtaining diterpenes of the labdane type, which comprises cultivation of suspension cultures of Coleus forskohlii cells on a plant cell culture medium as the maintenance medium in the presence of phytohormones which are one or both of auxins in a concentration of 0. 2 - 5 mg/l, and cytokinins in a concentration of 0. 05 - 2 mg/l, then induction of diterpene formation in the cell cultures by transfer to an induction medium which differs from the maintenance medium in that the phytohormones are replaced by those having a less dedifferentiating action, or are entirely omitted, and isolation of the diterpenes.
2. The process as claimed in claim 1, wherein auxins, if included, are added in a concentration of 0.5-2 mg/l, and cytokinins, if included, are added in a concentration of 0.1-0.5 mg/l, to the maintenance medium.
3. The process as claimed in claim 1, wherein the auxins used in the maintenance medium are one or both of 2,4-dichlorophenoxyacetic acid and naphtylacetic acid, and these are replaced in the induction medium by indole-3-butyric acid or other auxins having a less dedifferentiating action, or are entirely omitted.
4. The process as claimed in claim 3, wherein the induction medium contains no phytohormones.
5. The process as claimed in claim 1, 2 or 3, wherein the induction medium which is used is a simplified medium, which may omit vitamins or amino acids, or pure carbohydrate solutions.
6. The process as claimed in claim 1, 2 or 3, wherein submerged cultures are used.
7. The process as claimed in claim 1, 2 or 3, wherein the process is carried out in the light, in the dark or in a light/dark rhythm.
8. The process as claimed in claim 1, 2 or 3, wherein the process is carried out at temperatures of 18 to 38°C.
9. The process as claimed in claim 1, 2 or 3, wherein an induction period lasts 7 to 30 days.
10. The process as claimed in claim 1, 2 or 3, wherein several induction periods take place successively by a part of the cells which have been stimulated to produce diterpenes being, in each case, further cultivated on fresh induction medium (semicontinuous process).
11. The process as claimed in claim 1, 2 or 3, wherein the process is carried out in glass vessels, (aeration by orbital shaking) or in fermenters (aeration by passing in gases).
12. The process as claimed in claim 1, 2 or 3, wherein forskolin is obtained.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863636397 DE3636397A1 (en) | 1986-10-25 | 1986-10-25 | METHOD FOR OBTAINING DITERPENS OF THE LABDAN TYPE, IN PARTICULAR FROM FORSKOLIN, FROM COLEUS FORSKOHLII |
| DEP3636397.9 | 1986-10-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1293702C true CA1293702C (en) | 1991-12-31 |
Family
ID=6312490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000550082A Expired - Fee Related CA1293702C (en) | 1986-10-25 | 1987-10-23 | Process for obtaining diterpenes of the labdane type, in particular forskolin, from coleus forskohlii |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0265810A3 (en) |
| JP (1) | JPS63123391A (en) |
| CN (1) | CN87107118A (en) |
| CA (1) | CA1293702C (en) |
| DE (1) | DE3636397A1 (en) |
| DK (1) | DK555787A (en) |
| FI (1) | FI874652A (en) |
| IE (1) | IE872858L (en) |
| IL (1) | IL84253A0 (en) |
| PT (1) | PT85973B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349206B (en) * | 2016-08-29 | 2018-09-21 | 珀莱雅化妆品股份有限公司 | A kind of preparation method for extracting Forskolin from Coleus forskohlii |
| CN117586217B (en) * | 2024-01-17 | 2024-03-19 | 云南省药物研究所 | Preparation method of forskolin based on domestic coleus forskohlii |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2717777A (en) * | 1976-07-21 | 1979-01-25 | Hoechst Ag | Pharmacologically effective substance from plants belonging to the family of labiatae |
-
1986
- 1986-10-25 DE DE19863636397 patent/DE3636397A1/en not_active Withdrawn
-
1987
- 1987-10-20 EP EP87115302A patent/EP0265810A3/en not_active Withdrawn
- 1987-10-22 FI FI874652A patent/FI874652A/en not_active Application Discontinuation
- 1987-10-22 PT PT85973A patent/PT85973B/en not_active IP Right Cessation
- 1987-10-23 CA CA000550082A patent/CA1293702C/en not_active Expired - Fee Related
- 1987-10-23 JP JP62266668A patent/JPS63123391A/en active Pending
- 1987-10-23 DK DK555787A patent/DK555787A/en not_active Application Discontinuation
- 1987-10-23 IE IE872858A patent/IE872858L/en unknown
- 1987-10-23 IL IL84253A patent/IL84253A0/en unknown
- 1987-10-24 CN CN198787107118A patent/CN87107118A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DK555787A (en) | 1988-04-26 |
| PT85973A (en) | 1987-11-01 |
| PT85973B (en) | 1990-07-31 |
| FI874652A (en) | 1988-04-26 |
| DE3636397A1 (en) | 1988-05-05 |
| EP0265810A2 (en) | 1988-05-04 |
| FI874652A0 (en) | 1987-10-22 |
| IE872858L (en) | 1988-04-26 |
| JPS63123391A (en) | 1988-05-27 |
| CN87107118A (en) | 1988-06-15 |
| EP0265810A3 (en) | 1989-06-07 |
| IL84253A0 (en) | 1988-03-31 |
| DK555787D0 (en) | 1987-10-23 |
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