Variable Accumulation of Bioactive Metabolites Revealed in Medicinal Cannabis

Despite more than half of American states approving of the medicinal prescription of cannabis to treat select conditions, and nine states approving recreational cannabis use, at the federal level cannabis remains a Schedule 1 drug under the Controlled Substance Act. As a result, there is not a large amount of historical scientific literature on cannabis science available due to strict government regulations on research in the field. With time it has become easier to obtain the necessary licenses needed to study cannabis in the United States, but the lack of historic literature means that it is incredibly challenging to trace the origins of cannabis strains.

Before the establishment of regulated medical cannabis farms, years of cultivation by illicit drug growers had facilitated the creation of many different cannabis strains and variants in the search to create strains with more desirable effects for drug users. More modern research has determined that these differing effects are linked the relative ratios of cannabinoid compounds present in a strain. 

The most important cannabinoids concerning the expression of beneficial medicinal properties in a cannabis sample are Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), and the Δ9-THC variant Δ9-tetrahydrocannabivarin (Δ9-THCV). In addition to the cannabinoids, cannabis plants also contain a number of terpenes that are responsible for the physical properties of the cannabis plant, such as odor and flavor. These terpenes are of particular interest to the recreational cannabis industry, as odor and flavor are important qualities to consumers.

A new method for metabolite quantification

There are a variety of preexisting methods by which the analysis of the cannabinoids and terpenes present in a sample can be carried out, but the creation of new methods is still a very active area of research. A novel gas chromatography (GC) method for the separation and quantification of these metabolites has been recently reported by a research group based in New Mexico, USA. 

In many current gas chromatography methods, it has been noted that it can be difficult to attain adequate baseline separation of some of the minor cannabinoids. In order to remedy this, the team altered the typical GC analysis conditions. 

The largest change to traditional procedures is that here the cannabis plant samples were extracted using acetone. Typically one would use an extraction solvent like methanol, methanol-acetonitrile, or methanol-chloroform. There is a large environmental benefit to limiting to the use of these solvents as they can be toxic to animal life and expensive to dispose of. Acetone was selected as a potential alternative due to the fact that it is an excellent solvent for Δ9-THC. Acetone is also a less polar solvent than the methanol-based alternatives, and as such extracts fewer sugars from the plant material than methanol does. This is important as a gradual build-up of these sugars in the GC column can affect the performance of the apparatus. 

In addition to changing the extraction solvent, the group also used a single chromatographic system, gas chromatography with flame ionization detection (GC-FID), where it is more commonplace to use systems such as gas chromatography with mass spectrometry (GC-MS), or high-performance liquid chromatography (HPLC). Finally, the group used nitrogen gas as the carrier gas, where helium would usually be used. This change was made as helium is becoming a more expensive gas to obtain and the team wanted to ensure that their analysis method could remain both simple and affordable. 

The method the team used, comprising of GC-FID, a nitrogen carrier gas, and acetone as an extraction solvent, reported a better limit of detection and limit of quantitation capability for their target metabolites than the more conventional HPLC analysis method.

Metabolite accumulation findings

In addition to developing this new analysis procedure, the researchers also used it to study the cannabinoid content across 16 common medicinal cannabis strains. It was found that just as how cannabinoid levels can vary from strain to strain, they can also vary between different locations on the cannabis plant. 

A comparison of Δ9-THC levels found that, in general, the Δ9-THC content of floral tissues were around ten-fold larger than the leaf tissues. Expressed as a percentage of the dry weight, the floral tissues varied between 3-21% Δ9-THC, whereas the leaf tissues had a range of 0.3 - 2.7%. Additional cannabinoids, CBG and Δ9-THCV, also exhibited a similar pattern of being present in greater levels in floral samples compared to leaf samples. 

To further study the behavior of cannabinoids in flower and leaf material, samples of both were taken for two strains, Sour Willie and Bohdi Tree, as the plants flowered and matured. As the plants matured, Δ9-THC content increased over time for the flower samples and decreased slightly in the leaf samples over the same time period. 

In the course of studying the leaf samples, the researchers also confirmed the anecdotal knowledge of medicinal cannabis cultivators who believe that the flowers from nearer the top of the cannabis plant are more potent. Across four distinct strains it was observed that the flower samples taken from the upper third of the plant had a higher Δ9-THC content than those at the base of the plant, and in two of the strains tested the difference was equivalent to the upper flowers being around twice as potent as the lower flowers. A larger sample size is needed before this can be a statistically significant observation, but it is certainly an interesting preliminary finding.

The final cannabinoid study that the researchers carried out with their novel analysis method was to establish whether future Δ9-THC and CBD content could be predicted by analyzing the vegetative cannabis leaves before the plant flowers. In plotting the levels of CBD in vegetative leaves versus the cannabinoid content of the mature flowers across 16 different cannabis strains, a positive correlation was observed. Where no CBD was detected in the vegetative leaves, the strain would go on to contain predominantly Δ9-THC in its flowers. Conversely, when appreciable levels of CBD were detected, the mature flowers would go on to contain at least 0.5% CBD by weight. 

The impact of this research

With cannabis science gaining traction as, the demand for an effective, cheap, and simple analysis method will only grow. The method presented by the research team in New Mexico is an accessible analysis method that can meet this demand. 

In addition to creating this novel methodology, the study in intra-plant variation in cannabinoid content could have important repercussions for the medicinal cannabis industry, who rely on compositional analysis in order to establish the optimal treatment method for health conditions.