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(Last modification: 16. November 2010)
Sorry: It will be much work to get a good German version
Cannabinoids in Hemp (Cannabis sativa)
You might want to look at some Wikipedia pages which will provide you with lots of interesting information (note: often the English and German versions are quite different: if you can, look at both): Cannabis (English - German); Cannabinoids (English - German); Tetrahydrocannabinol (THC) (English - German);
A few pictures, taken from Wikipedia
Some explanation for the picture to the left (from
Franz Eugen Köhler's "Medizinal-Pflantzen"), text in German
(unfortunately, it is a bit difficult in the orginal to see the numbers and
therefore I enlarged them a bit):
Some Reviews Pertwee, 2010; Gertsch et al., 2010; Pertwee, 2005; Taura et al., 2007b, Flores-Sanchez and Verpoorte, 2008b; Clarke and Watson, 2007; Mechoulam et al., 2007; Ross, 2005; Turner et al., 1980.
Content of this page
The legal Situation in the U.S.A.
Introduction paragraph from an Internet article of
the National Institute of Drug Abuse (NIDA) (further
reading).
It is an illegal drug in most countries,
including the U.S.A. In view of this, I found it a bit unexpected that there is in the U.S.A. a University dedicated to teaching how to grow
and market Marihuana: the Oaksterdam University (a mixture of Oakland and Amsterdam)
that was founded in 2007, with the main location in downtown Oakland, California
(there are also campuses in Los Angeles, Sebastopol, and Michigan). The official mission
is: "to legitimize the business and work to change the law to make cannabis
legal". More details are in the
Wikipedia page.
Very briefly: on November 2 (2010) there will be a statewide ballot. The vote on Proposition 19 (also known as "Regulate, Control and Tax Cannabis Act of 2010") will possibly make important changes: it wants to legalize various marijuana-related activities, allow local governments to regulate these activities, permit local governments to impose and collect marijuana-related fees and taxes, and authorize various criminal and civil penalties. In March 2010 it qualified to be on the November statewide ballot. It requires a simple majority in order to pass, and would take effect the day after the election.Yes on 19 is the official advocacy group for the initiative. Update 03.11.2010: proposition 19 was refuted: 57% of the people participating in the vote did not want to legalize Cannabis.
In this context, you might also want to have a look at the Drug Policy of the Netherlands: more... There is really no need to add details here: the website is very detailed, and you find leads to everything you want (well, almost!).
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Cannabis sativa (Indian hemp) is an annual herb indigenous to Central and Western Asia; it is cultivated now in many other parts of the world. Main interests are: the fibre (hemp), the seed (seed oil), and the use as psychoactive compound, which is probably for many people the most well-known application (Marijuana, Hashish). The possession and consumption of the drug is illegal in most countries of the world, although there has been a lot of debate whether it might be less harmful than other drugs, e.g. alcohol or cigarettes. The Wikipedia sites give you more information, e.g. in English (Tetrahydrocannabinol) or German (Tetrahydrocannabinol). The contents of the two sites are not identical, and therefore you might look at both. More than 60 cannabinoids have been isolated from the leaves, and their pharmacological properties were extensively investigated. Fig. 1. shows the major compounds. It is important to note here that the carboxylated forms are predominant in the living plant; the removal of the carboxyl group occurs non-enzymatically in vitro during storage and processing of the drug (Yamauchi et al., 1967; Kimura and Okamoto, 1970). However, many people routinely use the names of the decarboxylated forms: it is easier, and that is what you get after the standard preparations / processing.
Major cannabinoids in
Cannabis sativa. Note: The predominant forms
in the plant are carboxylated (R =
COOH).
The carboxyl group is non-enzymatically removed during preparation of the drugs,
or by heating ("smoking"). Careful: the numbering of the atoms in THC
cannot be used with the other molecules: it is totally different, see the
Wikipedia page. It
doesn't get easier by the fact that various authors sometimes used different numbering
systems (e.g.
Δ9-THC
is
Δ1-THC
in another numbering system which seems to be obsolete now). According to the presence / absence of Δ9-THC (Δ9-Tetrahydrocannabinol), basically two chemical phenotypes can be distinguished:
CBCA, Cannabichromenic acid, the precursor of CBC (Cannabichromene) is a special case. It is apparently present in all plants and was detected very early (Gaoni and Mechoulam, 1966; Shoyama et al., 1968). Its expression is differently regulated, it seems to be mostly developmental specific: a fairly high content is found in young plants ("juvenile expression"), regardless of whether the plant is THC- or non-THC-type, and its percentage of the total cannabinoid content declines with maturation of the plants (see for example Rowan and Fairbairn, 1977; Vogelmann et al. 1988; Morimoto et al. 1997, 1998. There are also controls by light (Valle et al., 1978; Mahlberg and Hemphill, 1983). It has interesting biological activities (Turner et al., 1981)
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The Biosynthesis
My initial interest in tetrahydrocannabinol (THC) was the polyketide synthase that is the starting point of the biosynthesis. It seems quite clear that it should be by a polyketide synthase (PKS) reaction that involves hexanoyl-CoA as starter, three condensations with malonyl-CoA, and a stilbenecarboxylate-type (STCS) ring-folding to the pentylresorcylic acid (olivetolic acid). Stilbene synthase (STS) type ring-folding with retention of the terminal carboxyl group (STCS) is known from other plants, see in this website: Hydrangea macrophylla and Marchantia polymorpha, and they are postulated in the biosynthesis of anacardic acid and urushiols (more...). However, there is one important difference: In those cases the reaction sequence to the carboxylated end product contains a reduction step. That is not the case in the biosynthesis of olivetolic acid; this is potentially important because it has been argued that reduction and retention of carboxylic group may be linked: more.... Fig. 2 summarizes the proposed reaction. The available evidence for the reaction in Cannabis sativa is discussed here: In summary, it is pretty miserable, so far. To the best of my knowledge, not even the enzyme reaction has been demonstrated yet in vitro (October 2010).
Proposed type III PKS reaction in the biosynthesis of olivetolic acid in Cannabis sativa. The polyketide synthase reaction uses hexanoyl-CoA as starter and performs three condensations which are followed by a stilbenecarboxylate (STCS)-type ring-closure, i.e. with retention of the terminal carboxyl group. The colours indicate the carbon atoms introduced by the three condensation reactions.
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The next step is the prenylation of olivetolic acid (Fig. 3). The enzyme was characterized in Zenk's group (Fellermeier and Zenk, 1998), and has some interesting properties. Unlike many other prenyltransferases in secondary metabolism it is a soluble enzyme, not membrane-bound. The enzyme accepted not only geranyl-pyrophosphate (GPP), but also neryl-pyrophosphate (NPP). The activity with GPP was higher, consistent with a report that CBGA is more abundant than CBNA (Taura et al., 1995a). It was tempting to assume that CBNA would be the preferred precursor for cannabidiolic acid (CBDA, Fig. 1), but that was not the case, as discussed below with the cannabinoid synthases. The carboxyl group was essential for the prenyltransferase activity; olivetol was not a substrate for the enzyme. It is interesting to note that the terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the deoxyxylulose phosphate pathway (Fellermeier and Zenk, 2001).
Prenylation of Olivetolic acid. GPP = Geranylpyrophosphate, NPP = Nerylpyrophosphate
This turned out be a very interesting story: the three main cannabinoids are actually predominantly synthesized from a single precursor, CBGA, and the fourth, cannabigerol, is a simple decarboxylation product of CBGA. The reactions are summarized in Fig. 4. They are really quite interesting! If you get lost or confused by the use of the abbreviations (cannot easily be avoided): you are not the only one. This is what the figure should be good for: you can always have another look!
Biosynthesis of the major cannabinoids
by different enzymes, but from the same substrate.
These three enzymes represent a very nice example where most likely the product specificities of closely related proteins can be traced to specific aminoacid residues. And how about cannabigerol, the fourth compound? It appears that it is a decarboxylation product from cannabigerolic acid, in absence of the three other reactions.
Genetic work De Meijer et al., 2005, 2009a, 2009b; Mandolino et al., 2003.
There are some very interesting studies on the genes controlling these
conversions. Apparently, the formation of THC (Locus BT)
and Cannabidiol (Locus BT) are controlled
by two alleles of a single gene (called B). The conversion into CBG reflects the
inactivity of both alleles (Locus B0).
CBG accumulating plants have so far been
found in European fibre hemp populations that are generally composed of BD/BD
plants, and the observation that the B0 allele
possesses a residual ability to convert small amounts of CBG into CBD, make it
plausible that this B0 is a mutation of normally functional BD.
Therefore, B0 is considered as a member of the BD allelic series
encoding a CBD synthase isoform with greatly weakened substrate affinity and/or
catalytic capacity. The formation of the "juvenile cannabinoid" CBC is
apparently controlled by a different gene (although the protein may be similar).
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This is an interesting line that only recently really came to my attention: there are a number of natural cannabinoids that do not contain an isopentenyl but an isopropyl side chain. The most important compounds are show in Fig. 5. It is an easy suggestion that the biosynthesis should start with butyryl-CoA rather than with hexanoyl-CoA, but that all the subsequent steps are the same as with the isopentyl type cannabinoids; most likely the reactions are catalyzed by the same enzymes (reviewed in Clarke and Watson, 2007). These compounds were identified and characterized already in the early 70' of the last century (Gill, 1971; Merkus 1971; De Zeeuw et al., 1972) and reviews were established already several decades ago (e.g. Turner et al., 1980). It appears that high levels of these cannabinoids are specific to certain varieties (e.g. Cannabis indica) (Hillig and Mahlberg, 2004). By the way, the systematics of Cannabis and its varieties is seemingly not trivial, and a two-species origin of Cannabis was considered (C. sativa and C. indica) (Hillig and Mahlberg, 2004). What makes these cannabinoids so interesting? Without going into the complex details: the pharmacological properties are very different. THCV, for example, blocks the effects of THC (see for example: Thomas et al., 2005; Pertwee et al., 2007). This by itself of course makes them potentially very important for medical applications. There is also a very interesting overview comparing the pharmacology of THCV with cannabidiol and THC: Pertwee, 2008.
Fig. 5. Isopropyl Cannabinoids: the initial reaction starts with butyryl-CoA, but all the subsequent reactions seem to be the same as with hexanoyl-CoA as starter.
An interesting question, at least for those fascinated by polyketide synthases, is: what is the reason for the preference for isopropyl or isopentenyl cannabinoids? One or two enzymes? Considering the promiscuity of type III PKS (more...), one would think that both reactions could be very well done by a single enzyme. The apparent starter preference could be due to substrate channeling in a metabolic network. Actually, something like that must be postulated for almost any complex pathway, in particular in those cases where specialized organs like the trichomes in Cannabis are involved. One example, in this case with specialized root hairs, is the type III PKS in sorgoleone biosynthesis in Hordeum vulgare which is discussed in this website: the substrate preferences in vitro do not correspond to those in vivo (more...) Substrate channeling is likely the reason why in vitro experiments in most cases do not reflect the substrate use in vivo. One well-known example is flavonoid biosynthesis: although all chalcone synthases will accept many different substrates in vitro, the substrate use in vivo is exceptionally specific: it appears that the enzyme has only access to 4-coumaroyl-CoA, and this is a well-known example for pathway channeling (reviewed in Winkel-Shirley, 1999). As to the situation in Cannabis: as noted above, it has not even be possible to demonstrate the enzyme activity in vitro, and the genetic basis in C. sativa needs further investigation (De Meijer et al., 2009b). Interestingly, the situation in the closely related hop (Humulus lupulus) appears to be quite similar. There are two types of bitter acids, humulone and lupulone (more...). The initial reaction is by a type III PKS that uses either isovaleryl-CoA (-> humulone) or isobutyryl-CoA (-> lupulone), and I am not aware of any claim that two different enzymes are responsible. Actually, the problem was even more confounding: the reactions in humulone and lupulone biosynthesis are the same as performed by chalcone synthase, and there was for a long time considerable confusion on the question: which enzyme is responsible for what pathway? Several of the cloned proteins had both activities in vitro, and there are even ideas that a single enzyme serves both pathways (Okada et al., 2001).
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Receptors, endocannabinoids, and a bit more...
See also the page in Wikipedia: more... One of the interesting questions after identification of the cannabinoids, e.g. THC, was of course whether they acted in some way nonspecifically or whether there were true receptors. That was resolved in the eighties/nineties of the last century: receptors were functionally identified in membranes (Devane et al., 1988) and it was shown that they likely operated by modulation of cAMP concentrations (Bidaut-Russell et al., 1990); this was suggestive of G protein coupled receptors. The receptors were called cannabinoid receptors (CB) because endogenous effectors were unknown. The cloning of the first receptor (CB1, cannabinoid receptor 1) was reported already in 1990; it was characterized as G protein-coupled receptor found in rat brain and neural cell lines (Matsuda et al., 1990). However, this strict specificity had to be relaxed later, as expression was also detected in lung, liver, and kidneys. A year later followed the description of human CB1 (Gerard et al., 1991); the rat and human proteins were 97% identical.
A
few years later a second receptor named CB2 was cloned; it was clearly different
from CB1 (48% protein identity), and most importantly, the expression pattern
was really different: it was predominant in peripheral tissues (e.g. the immune
system), and notably absent from most normal nervous tissues (Munro
et al., 1993). The next fascinating question was whether there were natural substances binding to these receptors: indeed there are. They are now summarily described as endocannabinoids, and the International Union of Pharmacology in 2002 stuck to this definition (Howlett et al., 2002) because at least at that time it was not clear whether the two or three eicosanoid molecules most analyzed (see below, e.g. anandamide) are the primary endogenous receptor agonists. Arachidonylethanolamide (named: Anandamide, apparently from "ananda", the Sanskrit word for "bliss") was identified in a screen of membrane fractions for endogenous ligands for the cannabinoid receptor (Devane et al., 1992). The function was confirmed by many experiments, e.g. Vogel et al., 1993; Felder et al., 1993. A second major compound binding to the cannabinoid receptors, 2-Arachidonoylglycerol, was identified and characterized a few years later (Mechoulam et al., 1995; Sugiura et al., 1995; Stella et al., 1997). However, there are several more arachidonic acid derivatives that are less characterized. Figure 6 shows a simple summary of the structures of the main molecules and the likely routes of synthesis and degradation. If you look in the Internet for the structure, you’ll find different ways of drawing them: I used the proposal which suggested that anandamide and its congeners adopt tightly curved U/J-shaped conformations at CB1 (Barnett-Norris et al., 2002).
Fig. 6. A simplified overview of two endocannabinoids based on arachidonic acid (Anandamide, 2-Arachidonoylglycerol), and the most plausible biosynthesis and degradation: see below for a brief discussion of the complexities. Abbreviations: NAPE-PLD (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D); FAAH, Fatty acid amide hydrolase; MAGL, monoacylglycerol lipase.
A very short overview on endocannabinoid metabolism
Beyond that grand overview, nothing appears to be simple
I will not even try to go into the pharmacology; the topic is much too complicated. If you want to study the details: here are some recent reviews; some of them also summarize the complex pharmacology (which is impossible to cover here): more...
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A bit on Prescription Drugs based on cannabinoids
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