Identification of Terpenoid Chemotypes Among High (-)-trans-D9- Tetrahydrocannabinol-Producing Cannabis sativa L. Cultivars

Justin T. Fischedick

Cannabis and Cannabinoid Research, 2017, 2, 1, 34-47

DOI: 10.1089/can.2016.0040

 

Abstract

Introduction : With laws changing around the world regarding the legal status of Cannabis sativa (cannabis) it is important to develop objective classification systems that help explain the chemical variation found among various cultivars. Currently cannabis cultivars are named using obscure and inconsistent nomenclature. Terpenoids, responsible for the aroma of cannabis, are a useful group of compounds for distinguishing cannabis cultivars with similar cannabinoid content.

Methods : In this study we analyzed terpenoid content of cannabis samples obtained from a single medical cannabis dispensary in California over the course of a year. Terpenoids were quantified by gas chromatography with flame ionization detection and peak identification was confirmed with gas chromatography mass spectrometry. Quantitative data from 16 major terpenoids were analyzed using hierarchical clustering analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).

Results : A total of 233 samples representing 30 cultivars were used to develop a classification scheme based on quantitative data, HCA, PCA, and OPLS-DA. Initially cultivars were divided into five major groups, which were subdivided into 13 classes based on differences in terpenoid profile. Different classification models were compared with PLS-DA and found to perform best when many representative samples of a particular class were included.

Conclusion : A hierarchy of terpenoid chemotypes was observed in the data set. Some cultivars fit into distinct chemotypes, whereas others seemed to represent a continuum of chemotypes. This study has demonstrated an approach to classifying cannabis cultivars based on terpenoid profile.

Keywords : chemotype; hierarchical clustering analysis; orthogonal partial least squares discriminant analysis; partial least squares discriminant analysis; principal component analysis; terpenes

 

Introduction

Cannabis sativa L. (cannabis) is an annual diecious member of the Cannabaceae family. Since ancient
times cannabis has been used by humans for its fiber, seed, as well as its psychoactive and medicinal resin.1,2 Despite a long history of use, the legal status of cannabis in modern times often depends on its
intended use. Cannabis grown for its fiber or seed, commonly known as hemp, is legally cultivated in many nations. Cannabis used for its psychoactive properties, in North American commonly known as ‘‘marijuana,’’ has been illegal in most nations worldwide since the 1961 United Nations Single Convention on Narcotic Drugs.3 Recently however, laws concerning the legal status of cannabis are changing around the world. In the United States of America, many states have legalized cannabis for medical use, whereas some have even legalized cannabis for adult consumption.4 Uruguay recently legalized cannabis and laws in various countries within the European Union (EU) are also changing regarding cannabis.5,6 Due to its many and controversial uses, the taxonomic classification of cannabis has been the subject of both legal and scientific debate.

From a morphological perspective, three main types of cannabis have been described sativa, indica, and
ruderalis. Generally sativa plants are described as taller and loosely branched, whereas indica is typically
shorter, more densely branched, and conical in shape. Ruderalis is described as short (£2 feet) at maturity and sparsely if at all branched.7 Whether the genus Cannabis is monotypic and composed of just a single species (C. sativa) or polytypic and composed of multiple species is an old taxonomic debate.8,9 A more recent taxonomic classification dividing cannabis into seven putative taxa based on morphological, geographical, and genetic traits has been proposed.1,10

Cannabinoids are a group of terpenophenolic compounds found in cannabis. Today over 100 cannabinoids from cannabis have been characterized.11–14 ()-Trans-D9-tetrahydrocannabinol (THC) is considered the primary active ingredient responsible for the intoxicating and medical effects attributed to cannabis. THC has antiemetic, neuroprotectant, and anti-inflammatory properties as well as the ability to reduce certain forms of neuropathic and chronic pain.15–17 Another important cannabinoid, cannabidiol (CBD), has neuroprotective, anti-inflammatory, antipsychotic, and antiseizure properties without the intoxicating effects of THC.18–20 Other minor cannabinoids, such as cannabigerol (CBG), cannabichromene (CBC), and tetrahydrocannabivarin (THCV), also exhibit interesting pharmacological properties.17,21

Since cannabinoids are the major active ingredients found in cannabis, it makes sense to classify cannabis from a chemotaxonomic perspective according to cannabinoid levels for both medical and legal purposes. Early studies noted that cannabis used for fiber tended to have higher levels of CBD, whereas cannabis used for drug purposes had higher levels of THC.22 Small and Beckstead identified three chemical types (chemotypes) based on ratios of THC and CBD: type I, which contained high THC (>0.3%) and low CBD (<0.5%), type II high THC (>0.3%) and high CBD (>0.5%), and type III high CBD (>0.5%) and low THC (<0.3%).23 The three chemotype concepts were confirmed by Hillig and Mahlberg among cultivars originating from different geographic locations in addition to noting other minor cannabinoids that were characteristic of certain cultivars.24 Studies on the inheritance of cannabinoid phenotypes have demonstrated that chemotype can be independent from the plants morphology. 25 In recent decades drug type I cultivars have increased in potency containing upward of about 15– 20% THC,26,27 as have type II and type III cultivars.28–30 Clinical research has demonstrated that the combination of THC and CBD can alter their effects31–33 indicating the importance of knowing active compound ratios when using cannabis for medical purposes.

Terpenoids represent another interesting group of biologically active compounds found in cannabis. Due to their volatile nature, the mono- and sesquiterpenoids found in cannabis contribute to the plants’ aroma and flavor. About 100 terpenoids have been identified in cannabis, many of which are found in
other plants.11,34 Both cannabinoids and terpenoids are produced in the trichomes of cannabis, which are found at highest density on female flower buds.35–37 Terpenoids are usually present in cannabis flower buds in the 0.5–3.5% range28 and are found at significant levels in cannabis smoke and vapor.38 As biologically active compounds, terpenoids may play a role in the overall effects of herbal cannabis.17 The popularly understood distinctions between indica and sativa may have more to do with aroma and subjective effects than plant morphology. Recent studies have shown that terpenoids are useful in distinguishing cannabis cultivars that have similar cannabinoid content.28,39 A study of cannabinoid and terpenoid profiles among medical cannabis samples analyzed by a cannabis testing laboratory in California found a continuum of terpenoid profiles among the wide variety of sample names.29 Another study found that cannabis samples described as indica contained more myrcene and hydroxylated terpenoids, whereas those described as sativa tended to contain more terpinolene, 3-carene, and a few specified sesquiterpenes.30

However, in the aforementioned studies, it was difficult to define specific terpenoid chemotypes (or ‘‘chemovars’’ as described by Hazekamp)30,39 associated with commonly used cultivar names. This was likely due to the wide degree of quantitative variation in the sample sets as well as the lack of any formally agreedupon nomenclature for cannabis cultivars. Confusing and obscure nomenclature makes it difficult for doctors and patients to decide which cultivars they should use for various medical conditions. Furthermore, given recent advances in cannabis legalization, describing this chemical variation more systematically has never been more pertinent from both an agricultural and industrial
perspective. Therefore, the purpose of this study was to assess the variation of terpenoid chemotypes
among high THC-producing cannabis cultivars available to medical cannabis patients in the state of California. We chose to analyze a sample set obtained by monitoring the terpenoid content of samples submitted by a single medical cannabis dispensary over the course of a year. The single source was chosen with the assumption that some pattern or consistency in nomenclature would be used by the dispensary most likely based on smell or some knowledge of the source plant material.

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can.2016.0040(1)