Cannabis and Cannabinoid Research Volume 2.1, 2017 DOI: 10.1089/can.2016.0040

Cannabis and Cannabinoid Research

ORIGINAL RESEARCH

Open Access

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

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

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 Excelsior Analytical Laboratory, Inc., Union City, California.

*Address correspondence to: Justin T. Fischedick, PhD, Excelsior Analytical Laboratory, Inc., 30099 Ahern Avenue, Union City, CA 94587, E-mail: jtfi[email protected]

ª Justin T. Fischedick 2017; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

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Fischedick; Cannabis and Cannabinoid Research 2017, 2.1 http://online.liebertpub.com/doi/10.1089/can.2016.0040

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 (0.3%) and high CBD (>0.5%), and type III high CBD (>0.5%) and low THC (10%) as determined by high performance liquid chromatography (HPLC) (data not shown). Cultivar names, which were analyzed at least five times (n ‡ 5) in our laboratory over the data collection period, were selected to develop the classification scheme. This resulted in a data set containing 233 samples with 30 different cultivar names. The weight loss upon drying was between 6% and 14% indicating that the samples were not excessively dry and had similar moisture content. Typically, samples were submitted for testing a few days before they were made available to patients and usually represented samples from different batches purchased by the dispensary from various producers (personal communication with dispensary owners). Cannabinol (CBN), the primary degradation product of THC was trans-ocimene a-Pinene: b-pinene *2:1 trans-Ocimene >limonene a-Pinene >limonene

Cultivars in italics were added to PLS-DA model 3. PLS-DA, partial least squares discriminant analysis.

Gelato Bubba Kush, Master Kush

Mr. Nice

Mr. Nice

Purple

Grape Ape, Purple Cream, Purple Princess, Purple Urkle, Blue Mazaar, Granddaddy Purple, Purple Max, Watermelon Blue Dream Strawberry Haze, Strawberry Cough Godfather, AK-47

Blue Dream Strawberry High myrcene

Fischedick; Cannabis and Cannabinoid Research 2017, 2.1 http://online.liebertpub.com/doi/10.1089/can.2016.0040

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Table 4. Partial Least Squares Discriminant Analysis Model Classification Inputs and Q2 (Cross-Validated R2) Value from Five Component Model PLS-DA class assignments Cultivar name as classes Class names from Table 3 classes Cultivars with three to four samples added to model 2 Classes with >15 samples

Model number

Samples in model

Classes in model

Q2

1 2

217 217

30 13

0.29750 0.54896

3

274

13

0.57430

4

208

6

0.81654

five-component PLS-DA model was built between the terpenoid data and permuted class labels. Histograms show permutation test scores, and compare it to the performance based on the defined classifications (red arrow) (Supplementary Figs. S5–S8, bottom). The further the separation distance, based on a sum of squares between/sum of squares (B/W) ratio, between the observed statistic and the distribution resulting from permuted data, the more significant the discrimination.44 The separation difference increased from model 1 to 2 from 2 to 3 and from 3 to 4 (Supplementary Figs. S5–S8, bottom). The PLSDA results confirm that classification based on terpenoid classes outperforms classification based on cultivar name. When new samples were added to the sample set from models 1 and 2, a slightly improved model 3 resulted. However, the highest performing model was achieved with classes that contain ‡15 samples (model 4). These results suggest that better predictive models are constructed from terpenoid profiles with more representative samples. Conclusion This study has demonstrated an approach to discriminating terpenoid chemotypes among cannabis cultivars, despite obscure nomenclature. Overall, a hierarchy of chemotype was observed that could initially be broken down into five major terpenoid groups based on dominant terpenoid and relative levels of hydroxylated terpenoids. These five major groups could be broken down into 13 classes. The Cookie, Og Kush/Limonene Og Kush, Purple, and Terpinolene classes were clearly distinguishable chemotypes comprised of many representative cultivar names. Blue Dream represented a chemotype with only one cultivar name. The remaining classes could represent either new chemotypes pending confirmation from more representative samples, or rather a continuum of variation within a larger chemo-

type. More sensitive methods for terpenoid analysis in cannabis samples such as a recently published method by Giese et al.45 as well as the unequivocal identification of difficult-to-resolve sesquiterpenoids in cannabis would aid classification efforts. Information about terpenoid chemotypes can allow doctors and clinical researchers to design studies to assess whether they have different medicinal or subjective effects, despite similar cannabinoid content. Since it is unlikely that the popularly used cultivar names (‘‘strain’’ names as they are commonly referred to in the cannabis industry) will go away, the chemotype approach allows a more objective way of understanding cannabis chemical diversity for the newly emerging cannabis industry. Combining chemotaxonomic data, with morphological and genetic data, would provide a more complete picture of cannabis taxonomy. Acknowledgments The author would like to thank Dr. Andrea Lubbe for assistance with multivariate data analysis and article review. The author would also like to acknowledge the Garden of Eden Cooperative for allowing them to use their sample data in this study. This study was selffunded by Excelsior Analytical Laboratory, Inc. Author Disclosure Statement No competing financial interests exist. References 1. Clarke R, Merlin M. Cannabis: evolution and ethnobotany. University of California Press: Berkeley, CA, 2013. 2. Russo EB. History of cannabis and its preparations in saga, science, and sobriquet. Chem Biodivers. 2007;4:1614–1648. 3. Mead AP. International control of cannabis. In: Handbook of Cannabis (Pertwee R, ed.). Oxford University Press: Oxford, United Kingdom, 2014, pp. 44–64. 4. Maxwell JC, Mendelson B. What do we know now about the impact of the laws related to marijuana? J Addict Med. 2016;10:3–12. 5. Chatwin C. UNGASS 2016: insights from Europe on the development of global cannabis policy and the need for reform of the global drug policy regime. Int J Drug Policy. 2015 [Epub ahead of print]; DOI: 10.1016/i.drugpo.2015.12.17. 6. Room R. Legalizing a market for cannabis for pleasure: Colorado, Washington, Uruguay and beyond. Addiction 2014;109:345–351. 7. Schultes RE, Klein WM, Plowman T, et al. Cannabis: an example of taxonomic neglect. Botanical Museum Leaflets, Harvard University 1974;23:337–367. 8. Erkelens JL, Hazekamp A. An essay on the history of the term Indica and the taxonomical conflict between the monotypic and polytypic views of Cannabis. Cannabinoids. 2014;9:9–15. 9. Piomelli D, Russo EB. The Cannabis sativa versus Cannabis indica debate: an interview with Ethan Russo, MD. Cannabis Cannabinoid Res. 2016;1:44–46. 10. Hillig, KW. Genetic evidence for speciation in Cannabis (Cannabaceae). Genet Resour Crop Evol. 2005;52:161–180. 11. Turner CE, Elsohly MA, Boeren EG. Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J Nat Prod. 1980;43: 169–234.

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Cite this article as: Fischedick JT (2017) Identification of terpenoid chemotypes among high ()-trans-D9-tetrahydrocannabinol-producing Cannabis sativa L. cultivars, Cannabis and Cannabinoid Research 2:1, 34–47, DOI: 10.1089/can.2016.0040.

Abbreviations Used CBC ¼ cannabichromene CBD ¼ cannabidiol CBG ¼ cannabigerol CBN ¼ Cannabinol EU ¼ European Union FID ¼ flame ionization detector GC-MS ¼ gas chromatography–mass spectrometry HCA ¼ hierarchical clustering MeOH ¼ methanol OPLS-DA ¼ orthogonal partial least squares discriminant analysis PC1 ¼ Principal component 1 PCA ¼ principal component analysis PLS-DA ¼ partial least squares discriminant analysis THC ¼ tetrahydrocannabinol THCV ¼ tetrahydrocannabivarin RRT ¼ relative retention time

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