Cannabis metabolites are gaining increasing interest over recent years due to the medical benefits associated with components such as terpenes. In response, research has attempted to refine analytical methods to improve plant breeding methods and extraction procedures to enhance medical treatments.
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The growing interest in the medicinal uses of cannabis and its metabolites
Cannabis is a genus of dioecious plants within the family Cannabaceae that has a rich and complex phylogeny of pharmacologically relevant phytochemicals as well as a longstanding history throughout human history. The earliest documented uses of cannabis included the development of hemp fabric for rope and sails in ancient times.
However, under the Single Convention on Narcotic Drugs of 1961, cannabis was deemed a plant without medicinal purpose and practically prohibited by its status as an illegal narcotic worldwide. This conclusion was based on limited scientific evidence or clinical trial data and cannabis chemistry and biology were largely limited to law enforcement and forensics applications as a result.
Nevertheless, over recent decades there has been a shift in attitude towards cannabis across various societies and countries. This has been linked to mounting evidence for the benefits of cannabinoids in the treatment of otherwise intractable medical conditions that have accelerated recent legislative changes, particularly in North America.
Cannabis research has largely focused on cannabis cultivation as well as the chemical extraction and purification of major cannabinoids including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These are now used for the treatment of a range of debilitating illnesses, such as Dravet’s Syndrome, and investigated for uses in treating many other physical and mental disorders.
However, when cannabis extracts are processed and separated, individual purified elements such as CBD do not carry the full range of beneficial effects desired, thus giving rise to the concept of the “entourage effect”, as the characteristic effects are generated by all components working together. This entourage effect describes how the modulating or synergistic effects of minor and secondary phytochemical compounds provide the recognized effects desired from cannabis administration when combining CBD, THC, and its metabolites, with little effects from isolated elements alone.
Despite being less recognized, cannabis metabolites such as terpenes and terpenoids also represent important secondary compounds. These play key roles in the desired results of medicinal cannabis and are gaining increasing interest among the research community.
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.
Therefore, research has aimed to improve experimental protocols to better understand and extract secondary metabolites.
Towards a validated and standardized analysis of terpenes
The analysis and extraction of metabolites such as terpenes has improved rapidly in recent years. New efforts are now focusing on the validation of a standardized method of analysis that would provide an overarching methodology across research and industry.
Standardizing the analytical procedure of terpenes would ensure adequate quality of extraction as well as a method for comparative analysis and provide a consistent way of selecting plant strains.
This was the focus of a new study led by Elsayed A. Ibrahim from the National Centre for Natural Products Research, University of Mississippi, USA, who worked with a team of Egyptian and American scientists to develop the first validated method of quantification of cannabinoids.
Researchers designed a simple and precise analytical method based on gas chromatography (GC) and mass spectrophotometry (MS) for the accurate and efficient determination of cannabis terpenes. Specifically, the method was able to quantify monoterpenes, α-pinene, β -pinene, β-myrcene, limonene, linalool, α-terpineol, and terpinolene and the sesquiterpenes, β-caryophyllene, α-humulene, and caryophyllene oxide, which represent the major classes of terpenes.
The method relies on extracting the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard before measuring the concentration-response relationship for all analyzed terpenes using GC and MS.
The scientists then measured repeatability and intermediate precisions as well as the detection limit of quantitation for all targeted terpenes. The method was tested with C. sativa produced by the facility at the University of Mississippi as well as confiscated marijuana samples to ensure consistent results. Findings showed that the method was accurate, precise, and showed highly selective responses to terpene groups.
The developed method, therefore, represents the first GC/MS method validated by the Association of Official Analytical Chemists (AOAC) which is the official body validating chemical analyses for food and produce analysis in the US, providing a valuable standardized method for terpene analysis.
Challenges, limitations, and alternatives to current analytical methods of terpenes
Despite the progressive move towards more standardized methods of metabolite analysis, several challenges have been highlighted in recent reviews that require consideration. This is particularly important when validating an overarching methodology.
Specifically, a study by Christian Krill and colleagues from the Centre for AgriBioscience of Bundoora in Australia discussed how current methodology to quantify terpenes in cannabis biomass mostly relies on large quantities of biomass, long extraction protocols, and long GC gradient times, often exceeding 60 min. These limitations could hinder standardized methods including the development of high-throughput cannabis breeding programs.
In response, the researchers developed an alternative method based on a small quantity of hexane extract and a gradient of less than 30 min. The method can detect 48 individual terpenes and terpenoids and was validated for selectivity, linearity, precision, intermediate precision, and accuracy (recovery) for 22 terpenes and terpenoids.
The method, therefore, covers a large cross-section of commonly detected cannabis volatiles, is validated for a large proportion of compounds it covers and offers significant improvement in terms of sample preparation and sample throughput over previously published studies.
Ultimately, despite the validation by institutions for a standardized method for cannabis breeding, metabolite extraction, and analysis, new methods are being continuously developed. Such progress could be taken into account to update existing procedures to further improve the standards of practice in the aims of refining medical treatments.
Sources:
- Aizpurua-Olaizola, O., Soydaner, U., ÖZtürk, E., Schibano, D., Simsir, Y., Navarro, P., Etxebarria, N., & Usobiaga, A. (2016). Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes. Journal of Natural Products, 79(2), 324–331. doi:10.1021/acs.jnatprod.5b00949
- Ibrahim, E., Wang, M., Radwan, M., Wanas, A., Majumdar, C., Avula, B., Wang, Y. H., Khan, I., Chandra, S., Lata, H., Hadad, G., Abdel Salam, R., Ibrahim, A., Ahmed, S., & ElSohly, M. (2019). Analysis of Terpenes in Cannabis sativa L. Using GC/MS: Method Development, Validation, and Application. Planta Medica, 85(05), 431–438. doi:10.1055/a-0828-8387
- 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
- Leghissa, A., Hildenbrand, Z. L., & Schug, K. A. (2019). The imperatives and challenges of analyzing Cannabis edibles. Current Opinion in Food Science, 28, 18–24. doi:10.1016/j.cofs.2019.02.010
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