tetrehydrocannabinolic acid (THCA) synthase has recently been cloned fromC.
sativaplants and its heterologous expression in tobacco hairy root cultures has been
reported (Sirikantaramas et al. 2004 ; Taura 2009 ). This opens the way for
biotechnological production of pharmacologically active THC using wild type
transformed orrolABCtransgenic root cultures ofC. sativa.
We also studied the presence, in hemp root cultures, of metabolites in the
nitrogen compounds class ofC. sativaconstituents (i) atropine (tropane alkaloid)
and (ii) choline and muscarine (quaternary amines) (Wahby 2007 ; Wahby et al.
2006 ) (Fig.14.2), which have considerable pharmacological implications. Choline
is the precursor of the neurotransmitter acetylcholine as well as key membrane
(phosphatydil choline and sphingomieline) and signaling (platelet activating factor)
lipids and further it also serves a regulatory function of the peripheral and central
nervous systems (Blusztajn 1998 ; Zhang et al. 2004 ). Biological effects of mus-
carine resemble those of acetyl choline (Calabresi et al. 1998 ). Atropine is normally
used as a parasympatholytic, anticholinergic, spasmolytic and antiemetic drug
(Eeva et al. 1998 ; Ye et al. 2001 ).
Only few papers have reported the identification of choline inCannabistissues
including roots, root calli and leaves (Veliky and Genest 1972 ; Turner et al. 1980 ),
but quantitative data were never presented, while the occurrence of muscarine and
atropine-type substances on the other hand, have just been inferred from the
pharmacological effects of crude extracts of different plant tissues (Gill et al. 1970 ).
Further, intoxication with atropine-rich plant material was reported to cause hal-
lucinations, tachycardia, modification of secretionfluxes (Halpern 2004 ) and pupil
dilatation (Guharoy and Barajas 1991 ), the same symptoms observed after
Cannabisingestion (Stark et al. 2003 ).
Transformed roots and untransformed plant material (roots and leaves) were
collected, frozen in liquid nitrogen, lyophilized and stored desiccated until use.
Samples (1–3gfine powder) are extracted in a Soxhlet apparatus with 70%
aqueous methanol for 16 h. Afterfiltration, concentration and clarification (Wahby
2007 ), the extracts are kept at−20 °C before analysis. Extracts were analyzed for
the presence of choline, muscarine and atropine by an optimized capillary elec-
trophoresis coupled to electrospray ionization (ion trap) mass spectrometry
(CE-ESI-MS) method (collaboration with Dr. Antonio Segura Carretero,
Fig. 14.2 Chemical structure
of choline (a) atropine (b) and
muscarine (c)
308 I. Wahby et al.