By Kevin She
21 Mar 2016
All plants, as an evolutionary legacy, produce and accumulate a unique variety of chemical small molecules. These small molecule chemicals are produced as secondary metabolites that contribute to the survival of the plant either directly or indirectly, and are called phytochemicals (literally, plant chemicals). Humans have been able to take advantage of many properties conferred by these small molecules from herbs and foods that have a pleasant taste or aroma such as mint, garlic, or onions to medicinal qualities such as the analgesic properties of salicylic acid from willow bark and codeine/morphine from poppies or the antimalarial effects of quinine from the cinchona tree.
Through selective breeding, many species of plants have been domesticated and selected for desirable traits such as increased accumulation of osmoprotectants – small molecules that accumulate inside vulnerable plant cells that protects against drought stress – or for larger and sweeter fruits (sugars can be considered a type of small molecule). Conversely many domesticated breeds were also selected to accumulate less undesirable small molecules that impart unpleasant flavors such as the reduction of bitter tasting glucosinolates in Brassica vegetables such as Brussels sprouts, kale, and broccoli (Heaney & Fenwick 1980).
Likewise with Cannabis, human intervention has shaped the genetics of this species. Two general categories of domesticated Cannabis are evident – one kind selected for high quality stem fibre and seeds, and the other kind selected for narcotic content. The narcotic and medical qualities of Cannabis are primarily derived by its production and accumulation of cannabinoids, of which over 100 has been described (Andre &al., 2016) although not all cannabinoids have apparent biological activity in people. Also, some cannabinoids have been shown to change the effects of other cannabinoids such as the ability of cannabidiol (CBD) to modulate the effects of tetrahydrocannabinol (THC) (Russo & Guy 2006). Cannabis plants also produce and accumulate a prodigious number of terpenes and phenolics, other classes of small molecules, some of which may have health benefits of their own and/or modulates the function of the active cannabinoids.
Figure 1: Chemical structures of a few of the major cannabinoids found in Cannabis
This entourage effect, where certain small molecules can affect the effect of the active cannabinoids, is incredibly complex and as yet incompletely understood. However, it is likely that these complex interactions contribute to the differing effects and efficacy of the various narcotic and medicinal strains of Cannabis on different conditions. However, with increasing legalization of medical and recreational Cannabis, progress is now being increasingly made in understanding the effects of various mixtures of cannabinoids on the human endocannabinoid system.
Adapted from Velasco &al., 2012
Figure 2: Chemical structures of plant-derived cannabinoid (THC) and human endocannabinoids (AEA, 2-AG)
Endocannabinoids are small molecules in humans and some other animals that are used as a messenger. The receptors for these chemical messengers are abundant in the brain and the nervous system and also in a subset of immune cells, both white blood cells and in some epithelial cells of the skin (which, yes, are part of the innate immune system) and some other tissues (Stander &al., 2005). Similar to some other signaling systems, phyto-cannabinoids were discovered before and ultimately led to the discovery of the signaling system that cannabinoids affect. In fact, the first human endocannabinoid discovered was named anandamide derived from the Sanskrit word ananda – meaning joy/bliss/delight – and amide refrring to an aspect of its chemical structure (Devane &al., 1992). The scientific name more commonly used now is N-arachidonoylethannolamine (abbreviated as AEA) is somewhat less romantic. The other major endocannabinoids so far discovered include 2-arachidonoylglycerol (2-AG), 2-arachidonoyl glyceryl ether (noladin ether), O-arachidonoylethanolamine (virodhamine), and N-arachidnoyldopamine (NADA).
Despite having superficially different shapes, the main cannabinoid receptors, of which two – CB1 and CB2 – are considered canonical, that recognize endocannabinoids are responsible for mediating the effects of phyto-cannabinoids. However, some endocannabinoids appear to also be recognized by other receptors which may not play a large role in recognizing phyto-cannabinoids (Pertwee 1997) such as the transient receptor potential vanilloid type-1 (TRPV1) receptors. Different endocannabinoids and phyto-cannabinoids bind to CB1 and CB2 receptors differently and ultimately produces different effects, which is further complicated by the entourage effect.
In broad terms, THC binds with and partially activates primarily CB1 receptors and can also activate CB2 receptors, albeit less strongly, and is responsible for producing the euphoria or “high” associated with the psychotropic effects of recreational Cannabis. CBD on the other hand binds to CB1 and CB2 receptors and temporarily turns them off and can temporarily displace THC molecules already bound to the receptor. Immune cells, when expressing cannabinoid receptors, mainly express CB2 receptors and antagonism by CBD is likely a major contributor to the anti-inflammatory effects of cannabinoids. CBD alone does not produce any euphoric or psychotropic effects but can differentially alter the quality of the psychotropic effects of THC depending on how much CBD is present relative to THC and other cannabinoids and when it is administered relative to THC (Pertwee 2008).
Like keys that happen to fit locks that they weren’t designed to open, the biologically active cannabinoids nonetheless exert their effects through cannabinoid receptors in the brain. Of course, those endocannabinoid receptors didn’t evolve millions of years ago just to wait around for someone to get high, but then, what are their normal roles?
1964: Gaoni: THC isolated: active ingredient in drug cannabis
1965: Mechoulam: total chemical synthesis of THC
1988: Devane: demonstrated the existence of a cannabinoid receptor
1990: Matsuda: cloning of receptor for THC (CB1)
1992: Devane: isolation and characterization of anandamide, first discovered endocannabinoid
1993: Munro: cloning of CB2 receptor
1995: Mechoulam: isolation and characterization of 2-arachidonyl glycerol, an endocannabinoid
Table 1 Early timeline of the discovery of cannabinoids/endocannabinoids.
Endocannabinoid signaling has been suspected in regulating the immune system and in regulating metabolism in fat cells. The most studied roles of endocannabinoids have been their functions in the brain and nervous system and some of these functions have been described and more are being actively investigated.
Neurotransmission, signaling in the brain, is extraordinarily complicated and very high controlled. The human brain has about 100 billion neurons and approximately 150 trillion synapses, the points of contact between neurons through which they communicate with one another. Cannabinoid receptors typically reside on the pre-synaptic side (the origin of forward neurotransmission) of the synapse and endocannabinoids are synthesized on demand by enzymes on the post-synaptic side and travel the very short distance to the pre-synaptic side to stimulate the cannabinoid receptors there (retrograde signaling). However, not all synapses contain cannabinoid receptors and may also contain different kinds and different amounts of these receptors. Endocannabinoids are rapidly degraded or otherwise removed from the synapse after they are released so the signaling is transient and highly regulated. One function of endocannabinoids appear to be as a mechanism for fine-tuning neurotransmission and is typically produced in response to over-signaling and act as a very locationally precise brake to decrease excessive signaling (Kreitzer 2005). However, there is evidence that endocannabinoids can also signal in a non-retrograde manner and synaptic endocannabinoid signaling is more complex and diverse than originally thought and work continues to further our understanding of this complex signaling system (Castillo &al., 2012).
Over 100 different phyto-cannabinoids have been described to date. Like other secondary metabolites in plants, they are made by various enzymes that break apart larger molecules, build new molecules from smaller ones, or modify an existing small molecule. Sometimes a single enzyme can turn two different precursors into two different small molecules; sometimes a single enzyme can only catalyze one specific reaction. Cannabinoids are no different; Cannabis has a large repertoire of enzymes that can create cannabinoid precursors in a step-by-step manner. Separate specific enzymes then turn the common precursor into the different cannabinoids. For example, olivetolic acid is one of the common cannabinoid precursors that is turned into cannabigerolic acid.
THC (Δ9-tetrahydrocannabinol), CBD (cannabidiol), and CBC (cannabichromene) are three abundant cannabinoids in drug-type Cannabis (to be precise, these cannabinoids are created in their acidic form in the plant; these acid forms undergo spontaneous decarboxylation while in the plant, upon drying/curing of the flowers, and upon exposure to heat during cooking, vapourizing, or smoking to become the active neutral forms; see Figure 3). THCA synthase is the specific enzyme that turns cannabigerolic acid into THCA, CBD synthase for CBDA, and CBC synthase for CBCA.
Figure 3: Part of the cannabinoid biosynthetic pathway leading to the end products THC, CBD, and CBC.
Initial analysis of the Cannabis genome as well as breeding studies done in the past suggest that there is one gene locus (loosely, a physical position on a chromosome) that can accommodate THCA synthase or CBDA synthase genes. Because Cannabis is normally a diploid organism (two of each chromosome) so there can be two THCA synthase genes, two CBDA synthase genes, or one of each. In the case of hemp, it appears that the frequency of the THCA synthase gene in the hemp population is virtually non-existent and so do not produce THC but produce primarily CBD. The fact that there are non-zero amounts of THC in hemp plants, which lack THCA synthase, suggests that the cannabinoid biosynthetic pathway may be “leaky” and THC can be produced in a non-specific manner at very low frequency by other cannabinoid biosynthetic enzymes.
However, the amounts and ratios of THC and CBD (and other cannabinoids) produced by a given strain is not only determined by which synthase gene repertoire that they possess. The control of how much precursor is produced, how much of each enzyme is made, and how rapidly they are removed and recycled contribute to how much end product is produced. As yet, the genetic control mechanisms are incompletely understood but there are many potential avenues to explore such as gene mutation, gene duplication, the existence of pseudogenes, differences in transcriptional regulation, and differences in the amounts and types of micro-RNAs that are produced in the plant. Of course, the conditions that the plant was grown in strongly affect the final phytochemical profile in the mature flowers, both the absolute amounts of individual cannabinoids as well as their relative abundance to one another.
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