PlantCell Reports
Plant Cell Reports (1987) 6:150-152
© Springer-Verlag1987
Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L. R. Braemer and M. Paris Laboratoire de Pharmacognosie, Facult6 de Pharmacie, Rue J. B. C16ment, F-92296 Chatenay Malabry C6dex, France Received September 16, 1986 / Revised version received December 15, 1986 - Communicated by A. M. Boudet
ABSTRACT
Conversion of CBD and THC with cell suspensions: Regarding to the toxicity of these products (5), 4 mg
A cell suspension culture of Cannabis sativa L. is able to convert cannabidiol to bound cannabielsains and delta-9 tetrahydrocannabinol to cannabicoumaronon. The localization and the mechanism of the bioconversian are discussed.
of substrate (12,74 # moles) in 0.2 ml of dimethylformamide were added to nine-day-old cell suspensions. Five flasks were used for each substrate. CBD or THC and cell suspensions were incubated for 6 days in the same conditions as the suspension culture. The control consisted of sub~'ate added to
INTRODUCTION
fresh medium. At the end of the incubation time, the cells
Until the work of Heitrich and Binder (I) became known,
were ground in EtOAc (twice) and the medium was extracted
callus cultures of Cannabis sativa L. appeared to be unable
with EtOAc (twice). The organic extracts were collected and
to synthesize cannabinoids. Cell suspension culture of
analysed by GLC (6) and TLC. With CBD as substrate, the
Cannabis, without endogenous cannabinoids, have the capa-
organic extract was dried in vacuo and treated t~ 2N HCI,
bility to convert cannabidiol (CBD) to cannabielsoins (CBE)
1 hour at 100°C under reflux. The acid phase was extracted
(2). We have recently reported that a callus culture of
with CH2CI2.
Cannabis transforms CBD to CBE in bound forms only (3). A cell suspension culture was established from this callus culture. We compare here the capabilities of cells to bioconvert CBD and delta-9 tetrahydrocannabinol (THC).
Conversion of CBD, THC and hexahydrocannabinol epoxide with filtered medium: Nine-day-old cell suspensions were filtered on mesh filters of 48#m diameter. Four mg of substrate (CBD, THC or hexahydrocannabinol epoxide (EHHC)) were added to the medium
MATERIAL AND METHODS
in the same conditions as before. For each substrate 5 flasks
A callus culture was established from a leaf of Cannabis
were used. The substrate and the filtered medium were incu-
sativa L. containing delta-9 tetrahydrocannabivarol as the
bated for 24 hours in the conditions used for the cell suspen-
major cannabinoid (South Africa origin) in 1979. The cells
sion culture. At the end of the incubation time, the medium
were grown on B5 medium (4) with added kinetin (5.10-4g l-l),
was extracted with CH2CI2 (twice). The organic extracts
2,4-dichlorophenoxyacetic acid
(lO-3g l-l), glucose (30 g 1-1)
and agar (7 g l-l). The pH was adjusted to 5.5 before sterilisation. For the establishment of the cell suspension culture,
were collected and analysed by GLC and TLC. The organic extracts were chromatographed on silicagel plates (Merck). The solvent was hexane-dioxane (4-i, V/V).
i g (fresh weight) of callus was transferred into i00 ml of
Visualization of cannabinoids was achieved by spraying the
fresh liquid medium of the same composition but wihtout
plates with a 0.5 % aqueous solution of Fast Blue B salt (FBS)
agar. Subcultures were made every week by inoculation of 1 g
or with an anisaldehyde solution (7). With FBS, (R) CBE was
of cells into 100 ml of fresh nutritive medium contained in
red (Rf: 0.37) and the 1,8 cineol derivative of (S) CBE was
250 ml flasks. Suspensions were maintained on a rotary shaker
violet (Rf: 0.53). With anisaldehyde, cannabicoumaronon
(120 rpm) at 25°C in total darkness. Bioconversian tests were
(CBon) gave a blue spot (Rf: 0.51).
made one year after the transfer into the liquid medium.
Offprint requests to: R. Braemer
151 RESULTS AND DISCUSSION
The identification of the bioconversion products was accomplished using Varian Mat III gas chromatograph~mass
With the cell suspension cultures, after 6 days of contact,
spectrometer (70 eV).
no more cannabidiol and no other free cannabinoids are de-
1,8 cineol derivative of (S) CBE: m/z: 330 (50), 205 (100),
tected. (R) cannabielsoin and (S) cannabielsoin-l,8 cineol
204 (0.8), 148 (0.4), 147 (0.3).
derivative appear after HCI treatment of the organic extract.
(R) CBE: m/z: 330 (40.5), 247 (100), 205 (83.1), 204 (25.7),
These products are identified from their mass spectrums
148 (37.6), 147 (27.7).
according to (10). (S) cannabielsoin submitted to the same
CBon: m/z: 328 (61.3), 313 (17.6), 285 (100), 271 (19.6),
acid treatment is completely transformed to its 1,8 cineol
258 (44.2), 257 (41.8), 243 (21.9), 214 (53.4).
derivative. (R) cannabielsoin, with an equatorial C-9 hydroxyl group, cannot be transformed to 1,8 cineol derivative.
(S) CBE was synthesized as described by Uliss et al. (8) and EHHC as described by Turner et al. (9) for its acetylated
It seems that (I{) cannabielsoin and (S) cannabielsoin are
derivative. The liberation of EHHC was obtained with a 10%
two products of biotransformation of cannabidiol. The cell
NaOH solution in H20-MeOH (I-I,V/V).
suspension culture is able to convert cannabidiol to bound forms of eannabielsoins. (I{) cannabielsoin is liberated by HCI; (S) cannabielsoin appears to be transformed into its 1,8 cineol derivative during its liberation. The yield of liberated products is too low to realize quantitative analyses
OH
with our experimental conditions. When delta-9 tetrahydrocannabinol is added to the suspension cultures, its concentration decreases and after 6 days
C5HII cannabidiol
~0~0
of contact no more tetrahydrocannabinol is detected. With-
0
out HCI treatment, the presence of a supplementary product
delta-9 tetrahydrocannabinol
is observed by GLC analysis compared to the control flask. Analysis by TLC does not permit the presence of cannabinoid to be visualized with FBS reagent but a blue spot appears with anisaldehyde. The mass spectrum of this product is identical to that of cannabicoumaronon according to (i i). The absence of any phenolic hydroxyl group explains the fact
c5~ii
that it is impossible to visualize cannabicoumaronon by FBS. Cannabicoumaronon represents 17 % of the initial substrate.
hexahydrocannabinolepoxide
Austin and Brown (12) have already reported that marine-
~..OH
HO~>~
Plant Cell Reports (1987) 6:150-152
© Springer-Verlag1987
Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L. R. Braemer and M. Paris Laboratoire de Pharmacognosie, Facult6 de Pharmacie, Rue J. B. C16ment, F-92296 Chatenay Malabry C6dex, France Received September 16, 1986 / Revised version received December 15, 1986 - Communicated by A. M. Boudet
ABSTRACT
Conversion of CBD and THC with cell suspensions: Regarding to the toxicity of these products (5), 4 mg
A cell suspension culture of Cannabis sativa L. is able to convert cannabidiol to bound cannabielsains and delta-9 tetrahydrocannabinol to cannabicoumaronon. The localization and the mechanism of the bioconversian are discussed.
of substrate (12,74 # moles) in 0.2 ml of dimethylformamide were added to nine-day-old cell suspensions. Five flasks were used for each substrate. CBD or THC and cell suspensions were incubated for 6 days in the same conditions as the suspension culture. The control consisted of sub~'ate added to
INTRODUCTION
fresh medium. At the end of the incubation time, the cells
Until the work of Heitrich and Binder (I) became known,
were ground in EtOAc (twice) and the medium was extracted
callus cultures of Cannabis sativa L. appeared to be unable
with EtOAc (twice). The organic extracts were collected and
to synthesize cannabinoids. Cell suspension culture of
analysed by GLC (6) and TLC. With CBD as substrate, the
Cannabis, without endogenous cannabinoids, have the capa-
organic extract was dried in vacuo and treated t~ 2N HCI,
bility to convert cannabidiol (CBD) to cannabielsoins (CBE)
1 hour at 100°C under reflux. The acid phase was extracted
(2). We have recently reported that a callus culture of
with CH2CI2.
Cannabis transforms CBD to CBE in bound forms only (3). A cell suspension culture was established from this callus culture. We compare here the capabilities of cells to bioconvert CBD and delta-9 tetrahydrocannabinol (THC).
Conversion of CBD, THC and hexahydrocannabinol epoxide with filtered medium: Nine-day-old cell suspensions were filtered on mesh filters of 48#m diameter. Four mg of substrate (CBD, THC or hexahydrocannabinol epoxide (EHHC)) were added to the medium
MATERIAL AND METHODS
in the same conditions as before. For each substrate 5 flasks
A callus culture was established from a leaf of Cannabis
were used. The substrate and the filtered medium were incu-
sativa L. containing delta-9 tetrahydrocannabivarol as the
bated for 24 hours in the conditions used for the cell suspen-
major cannabinoid (South Africa origin) in 1979. The cells
sion culture. At the end of the incubation time, the medium
were grown on B5 medium (4) with added kinetin (5.10-4g l-l),
was extracted with CH2CI2 (twice). The organic extracts
2,4-dichlorophenoxyacetic acid
(lO-3g l-l), glucose (30 g 1-1)
and agar (7 g l-l). The pH was adjusted to 5.5 before sterilisation. For the establishment of the cell suspension culture,
were collected and analysed by GLC and TLC. The organic extracts were chromatographed on silicagel plates (Merck). The solvent was hexane-dioxane (4-i, V/V).
i g (fresh weight) of callus was transferred into i00 ml of
Visualization of cannabinoids was achieved by spraying the
fresh liquid medium of the same composition but wihtout
plates with a 0.5 % aqueous solution of Fast Blue B salt (FBS)
agar. Subcultures were made every week by inoculation of 1 g
or with an anisaldehyde solution (7). With FBS, (R) CBE was
of cells into 100 ml of fresh nutritive medium contained in
red (Rf: 0.37) and the 1,8 cineol derivative of (S) CBE was
250 ml flasks. Suspensions were maintained on a rotary shaker
violet (Rf: 0.53). With anisaldehyde, cannabicoumaronon
(120 rpm) at 25°C in total darkness. Bioconversian tests were
(CBon) gave a blue spot (Rf: 0.51).
made one year after the transfer into the liquid medium.
Offprint requests to: R. Braemer
151 RESULTS AND DISCUSSION
The identification of the bioconversion products was accomplished using Varian Mat III gas chromatograph~mass
With the cell suspension cultures, after 6 days of contact,
spectrometer (70 eV).
no more cannabidiol and no other free cannabinoids are de-
1,8 cineol derivative of (S) CBE: m/z: 330 (50), 205 (100),
tected. (R) cannabielsoin and (S) cannabielsoin-l,8 cineol
204 (0.8), 148 (0.4), 147 (0.3).
derivative appear after HCI treatment of the organic extract.
(R) CBE: m/z: 330 (40.5), 247 (100), 205 (83.1), 204 (25.7),
These products are identified from their mass spectrums
148 (37.6), 147 (27.7).
according to (10). (S) cannabielsoin submitted to the same
CBon: m/z: 328 (61.3), 313 (17.6), 285 (100), 271 (19.6),
acid treatment is completely transformed to its 1,8 cineol
258 (44.2), 257 (41.8), 243 (21.9), 214 (53.4).
derivative. (R) cannabielsoin, with an equatorial C-9 hydroxyl group, cannot be transformed to 1,8 cineol derivative.
(S) CBE was synthesized as described by Uliss et al. (8) and EHHC as described by Turner et al. (9) for its acetylated
It seems that (I{) cannabielsoin and (S) cannabielsoin are
derivative. The liberation of EHHC was obtained with a 10%
two products of biotransformation of cannabidiol. The cell
NaOH solution in H20-MeOH (I-I,V/V).
suspension culture is able to convert cannabidiol to bound forms of eannabielsoins. (I{) cannabielsoin is liberated by HCI; (S) cannabielsoin appears to be transformed into its 1,8 cineol derivative during its liberation. The yield of liberated products is too low to realize quantitative analyses
OH
with our experimental conditions. When delta-9 tetrahydrocannabinol is added to the suspension cultures, its concentration decreases and after 6 days
C5HII cannabidiol
~0~0
of contact no more tetrahydrocannabinol is detected. With-
0
out HCI treatment, the presence of a supplementary product
delta-9 tetrahydrocannabinol
is observed by GLC analysis compared to the control flask. Analysis by TLC does not permit the presence of cannabinoid to be visualized with FBS reagent but a blue spot appears with anisaldehyde. The mass spectrum of this product is identical to that of cannabicoumaronon according to (i i). The absence of any phenolic hydroxyl group explains the fact
c5~ii
that it is impossible to visualize cannabicoumaronon by FBS. Cannabicoumaronon represents 17 % of the initial substrate.
hexahydrocannabinolepoxide
Austin and Brown (12) have already reported that marine-
~..OH
HO~>~
