The socioeconomic interest in cannabis has surged due to the increased recognition of medical benefits, and analytical methods are being continuously refined.
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The rising medical and economic interest in cannabis
Cannabis sativa is rapidly becoming one of the most studied plants in the world due to the increasing interest in its multifunctional benefits. In the US alone, the legal marijuana economy represented nearly 17 billion USD in 2016 and is estimated to reach up to 70 billion USD in 2021.
Scientific research has focused on the use of cannabinoids for a wide range of medical conditions including epilepsy, multiple sclerosis, diabetes, cancer, and post-traumatic stress disorder, with several findings indicating favorable benefits of cannabis treatments.
The most used elements of cannabis are generally cannabidiol (CBD) and cannabigerol (CBG), as well as the psychoactive compound tetrahydrocannabinol (THC). CBD represents the most valuable non-psychoactive pharmaceutical compound as it possesses antioxidative, anti-inflammatory, neuroprotective and antibiotic benefits. CBG also possesses anti‐inflammatory, antimicrobial and analgesic properties, making cannabis a promising medical solution for many medical conditions, both physical and mental.
Such benefits have subsequently generated many in vitro and in situ analytical methods that have many applications.
The range of modern analytical techniques
In a 2020 study by Lithuanian scientists published in the journal of Phytochemical Analysis, researchers were able to compare the effects of extraction methods and conditions on the yields of CBD and CBG in Cannabis sativa. To date, no other study has yet compared how methods and conditions can affect overall yield, or how sensitive each technique is relative to one another.
This study compared the use of the most common extraction methods of the past decade including ultrasound‐assisted (UAE), microwave‐assisted (MAE), and heated‐reflux extraction (HRE). Researchers concluded that the use of ultrasonication was most beneficial for extraction as it requires less time, energy, and cost.
The scientists were also able to design a compound-sensitive technique to quantify compounds post-extraction using gas chromatography (GC) combined with both a flame ionization detector (FID) and mass spectrometry (MS). This novel technique provides a reliable and effective tool to identify and sort through plants with higher content of CBD and CBG.
Such analytical methods are particularly valuable when selecting strains with desirable medicinal properties, but scientists are also focusing on other elements of cannabis plants too.
New frontiers for cannabis analysis: secondary metabolites.
Other analytical methods have also focused on other components such as cannabis terpenes and terpenoids. When cannabis extracts are administered, it is increasingly recognized that purified CBD does not carry the full range of beneficial effects, thus giving rise to the concept of the “entourage effect”. This process describes how the modulating or synergistic effects of minor and secondary phytochemical compounds provide the recognized effects desired from cannabis administration when combined with CBD and THC, with little effects from these isolated elements alone.
Metabolites such as terpenes and terpenoids represent such secondary compounds, which play key roles in the desired results of medicinal cannabis. Specifically, terpenes are a class of hydrocarbons synthesized from 5-carbon units and can be further classified based on the number of C5 units. Some of these units are responsible for the smell of the cannabis plant and its end-products.
These metabolites are proven to contribute to the medicinal effects of cannabis, and current methods to quantify terpenes in cannabis biomass mostly rely on large quantities of biomass, long extraction protocols, and long gradient times during gas chromatography. These limitations were put to the test by a study led by Krill et al. from 2020, which attempted to improve existing methods.
Indeed, such procedural limitations make it difficult to apply in the high-throughput environment of strain selection and breeding. In this study, the researchers developed a new methodology requiring as little as 40 mg of plant biomass, with a reduced amount of necessary dodecane, and a gradient half as long as standard methods.
Moreover, when tested this novel method detected up to 48 secondary metabolites comparable in accuracy to traditional extraction protocols currently being applied to medicinal cannabis precision breeding programs. The refinement of such methods is particularly promising for the future of the medicinal cannabis industry, contributing to the improvement of extraction and quantification methods of secondary metabolites.
Additional analytical methods being developed
Although the direct approach of studying key products such as CBG, CBD, and THC may be particularly valuable, researchers are also implementing alternative strategies to understanding and harnessing the medicinal properties of cannabis.
On a chromosomal level, a recent study by French scientists was able to map the sex chromosomes of cannabis plants to better understand the potency of cannabinoid elements in a 2020 study. The researchers identified over 500 sex-linked genes and found that the X-chromosome-specific region is particularly large compared to other plant systems. Further analysis of the sex-linked genes revealed that C. sativa has a strongly degenerated Y Chromosome and may represent the oldest plant sex chromosome system documented so far.
This is particularly important as THC reaches the highest concentrations in female inflorescences (referring to the cluster of branches on the stem), which are therefore the most economically viable and providing key indications as to how to enhance THC concentrations in cannabis plants by understanding and potentially manipulating key chromosomal regions.
Within such regions, expression patterns of genes may be also of key value. Further molecular work was done by Russian researchers in the International Journal of Molecular Sciences published in 2021, using high-throughput gene expression profiling data to detect the anti-inflammatory properties of the various cannabis extracts. The effects of various gene profiles were run on a range of human tissues, allowing researchers to calculate a cannabis drug efficiency index (CDEI) across a range of diseases.
These gene expression models were run across 241 different signaling pathways containing genes responding to the cannabis drug and ranked across CDEIs, resulting in a comprehensive understanding of which tissues are more or less affected by each extract.
Cannabis analysis looking forward
Cannabis and its multifunctional purposes have provided a number of biotechnological developments and methods that will be improved looking forward. Current applications are also being refined consistently, with multifaceted approaches yielding key insights into the medicinal properties of cannabis and how best to harvest such elements.
- Baranauskaite, J., Marksa, M., Ivanauskas, L., Vitkevicius, K., Liaudanskas, M., Skyrius, V., & Baranauskas, A. (2020). Development of extraction technique and GC/FID method for the analysis of cannabinoids in Cannabis sativa L. spp. santicha (hemp). Phytochemical Analysis, 31(4), 516–521. doi:10.1002/pca.2915
- Borisov, N., Ilnytskyy, Y., Byeon, B., Kovalchuk, O., & Kovalchuk, I. (2020). System, Method, and Software for Calculation of a Cannabis Drug Efficiency Index for the Reduction of Inflammation. International Journal of Molecular Sciences, 22(1), 388. doi:10.3390/ijms22010388
- Krill, C., Rochfort, S., & Spangenberg, G. (2020). A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass. Metabolites, 10(7), 276. doi:10.3390/metabo10070276
- Prentout, D., Razumova, O., Rhoné, B., Badouin, H., Henri, H., Feng, C., Käfer, J., Karlov, G., & Marais, G. A. (2020). An efficient RNA-seq-based segregation analysis identifies the sex chromosomes of Cannabis sativa. Genome Research, 30(2), 164–172. doi:10.1101/gr.251207.119
- Rana, S. S., & Gupta, M. K. (2020). Isolation of nanocellulose from hemp (Cannabis sativa) fibers by chemo‐mechanical method and its characterization. Polymer Composites, 41(12), 5257–5268. doi:10.1002/pc.25791