**2. Terpene Biosynthesis**

Terpenes originate from the 5-carbon precursor isopentenyl diphosphate (IPP), which is biosynthesized from either pyruvate and glyceraldehyde (via the methylerythritol phosphate (MEP) pathway in plastids) [27] or from acetyl-coA (via the mevalonic acid (MEV) pathway in the cytoplasm) [28] (Figure 1). One or more IPPs condense with dimethylallyl diphosphate (DMAPP) in a 1–4 fashion to form geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), or geranylgeranyl diphosphate (GGPP, C20). GPP and FPP serve as substrates for a multitude of synthetic reactions, condensing together to form the precursors of carotenoids and steroids, or cyclizing to form a myriad of terpene natural products (e.g., monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20)) [12,29]. GPP also condenses with a diphenol with an alkyl chain (e.g., olivetolic acid) to form the cannabinoids [30]. In cannabis, over 200 terpenes have been published to date [31].

Terpenoid biosynthesis is governed by a family of homologous enzymes, the terpene synthases (TPS) [29,32], which catalyze the formation of different types of terpenes, including monoterpenes, diterpenes, hemiterpenes, and sesquiterpenes. These essential enzymes are encoded in large gene families that have been broken down into seven subfamilies based on phylogenetic analyses rendering, TPS-a, -b, -c, -d, -e/-f, -g, and -h, each based on amino acid length and location of emergence, such as angiosperms or gymnosperms [29,33]. In angiosperms, the TPS-a subfamily contains sesquiterpene synthases (sesqui-TPSs); the TPS-b subfamily contains monoterpenes synthases (mono-TPSs) and hemiterpene synthases [34].

**Figure 1.** General scheme of terpene synthesis pathway in *Cannabis sativa* L.

Booth et al. analyzed the genome and transciptome of Purple Kush cannabis to identify more than 30 cannabis terpene synthases (CsTPS genes) [35], which has been expanded to over 14 cultivars, representing chemotypes I, II, and III [34,36,37]. The characterized TPS genes of cannabis are documented as being a part of the TPS-a and TPS-b subfamilies [29]. Only nine of the 30 CsTPS genes have been fully characterized with respect to their catalytic functions, eight of which are multi-product enzymes that can generate different terpene structures from either GPP or FPP substrates [35,38]. Interestingly, genetic variation in these CsTPS has been associated with differences in the Sativa-Indica scale of cannabis labeling. Genotyping 100 cannabis samples for >100,000 single nucleotide polymorphisms revealed that Sativa- and Indica-labelled samples were indistinguishable from a genome perspective; however, variation in CsTPS genes translated to shifts in the terpene profile and was correlated with the current dichotomous label system, suggesting terpenes (and genetic markers associated with terpene biosynthesis) could have a large role in governing the strain classification [39]. This biosynthetic plasticity could be one explanation for the diversity of terpenes found in cannabis; however, it is important to keep in mind that the CsTPS responsible for many cannabis terpenes remain unexplored. When considering the incredible diversity of cannabis terpenes, it is unknown how the expression levels of different CsTPS could vary with plant development stage, plant organ and cell-type, and environmental factors. In addition, non-enzymatic modifications of terpenes, such as cyclization and oxidation, can increase structural diversity independent of enzymatic biochemical reactions. Even post-harvest considerations can change the terpene profile, especially the smaller, more volatile hemiterpenes and monoterpenes [40]. More qualitative and quantitative studies are needed to comprehensively profile the terpenes found in cannabis and how those concentrations relate to expression levels and functionality of the CsTPS.

#### **3. Terpene Diversity in Cannabis**

Over 20,000 terpenes have been identified in the Plantae kingdom, making these highly volatile compounds one of the most structurally and functionally diverse groups of natural products [41]. Cannabis is widely known for its assorted terpene profiles. To date, 200 terpenes/terpenoids have been detected in cannabis [42]. However, the complete identification and quantification of the vast majority of terpenes/terpenoids remains undetermined, blunting our knowledge of the impact of cannabis terpenes on plant and human health [43]. Thus, the complete identification of terpenes in cannabis may sugges<sup>t</sup> a substantial assortment of cannabis terpenes unknown to current breeders and researchers.

With the tremendous diversity of compounds in cannabis, researchers seek to categorize the main chemical constituents of cannabis cultivars or 'strains' by establishing five classes of chemotypes based on cannabinoid ratios. These are classified as Chemotypes (I): high THCA:CBDA ratio; (II) intermediate ratios of THCA:CBDA; (III) low THCA:CBDA ratio; (IV) high CBGA content/low ratio of THCA:CBDA; and (V) containing almost no cannabinoids [44]. This classification has drawn researchers to further categorize cannabis chemical profiles by associating cannabinoid content with bioactive metabolites such as terpenes. Table 1 illustrates the concentration range (mg/g) of terpenes and terpene derivatives reported in published research articles investigating the terpene content of specific cannabis chemotypes. Chemical profiles of common cannabis cultivars continue to show that myrcene, β-caryophyllene, limonene, α-terpinene, and α-pinene are the most prominent terpenes that can be found in the first three chemotype varieties [34,45–48]. Terpene profiles of the remaining chemotypes are limited or have ye<sup>t</sup> to be investigated. Conversely, the classification of secondary terpenes (terpenes found in lower concentrations) in cannabis chemotypes is limited, as they are often disregarded or unreported due to a lack of reference material. More studies on cannabis terpene chemotypes are required to identify the relationships between specific terpenes and cannabinoid content.

Birenboim et al., 2022, were the first to demonstrate a highly accurate classification of medicinal cannabis chemovars based on their cannabinoid and terpene profiles. Using a partial least-square discriminant analysis multivariate (PLS-DA) technique, Birenboim et al. were able to differentiate terpene content between the inflorescences of three major chemovars (high-THCA, high-CBGA, and a hybrid). They concluded that the terpenes of the three major classes were significantly different in their concentrations of different terpenes [49], providing evidence of the high-THCA class having a higher abundance of limonene, β-caryophyllene, β-pinene, α-humulene, γ-elemene, and seychellene. Within the hybrid class, α-pinene and β-myrcene are more pronounced, followed by a high abundance of γ-eudesmol, α-bisabolol, and guaiol in the high-CBGA class. However, these results represent 14 different cannabis chemovars, including seven high THC chemovars, five hybrid chemovars, and only two high-CBG chemovars. The plant material used was from commercial breeding lines that could not be affiliated to a specific subspecies because of crossings between different cultivars over many generations. Moreover, several factors have been shown to influence terpene diversity, such as plant genetics, pest presence, overall plant health, soil composition, proper drying, curing, and microbiology [34,50–55].

Variations in terpene expression can also be dependent upon the stage of growth. In 2016, Aizpurua-Olaizola et al. analyzed the terpene and cannabinoid content of the leaves and flowers of cannabis chemotypes I, II, and III. For 23 weeks, a chemical profile was generated on a weekly basis, providing the researchers with a total content of cannabinoids and terpenes at different stages of growth. Researchers found that chemotypes II and III required more time to reach their peak production of monoterpenes compared to chemotype I. Major terpene differences were also observed between chemotypes I and III. The distinct terpenes of chemotype I included γ-selinene, β-selinene, α-gurjunene, γ-elemene, Selina-3.7 (11) diene, and β-curcumene, while chemotype III displayed βeudesmol, γ-eudesmol, guaiol, α-bisabolol, or eucalyptol. This suggests a chemotypedependent terpene distribution, as the investigators describe the more prominent terpenes in chemotype III as having a higher correlation coefficient with CBDA and chemotype I terpenes having a higher correlation coefficient with THCA [53]. Despite the differences in terpene content at different stages of growth, limitations of terpenes and cannabinoid expression may be observed based on light exposure and select spectra.

A high abundance of terpenes and cannabinoids can be found on the surface of cannabis inflorescence and leaves in the glandular appendages known as trichomes [56,57]. Trichomes are believed to be a defense mechanism against several different stresses, including light stress [58,59]. This has led to the proposed ecological function of cannabinoids and terpenes aiding in protection against high light exposure [58]. Additionally, research has shown the altering effects LED light can have on THC and terpene concentrations, but not CBD [57,58]. One study provided evidence of supplemental green light increasing THC and terpene content in comparison to controls. However, quantification of IPP and DMAPP were not conducted, leaving the mechanistic implications undetermined [52]. With the increasing application of LED lighting for indoor cultivation, the chemical profiles of the desired chemotype may be susceptible based on light application. Nonetheless, with the information surrounding the factors that influence terpene concentrations, terpene biosynthesis, and genetic expression, new cultivars with desired cannabinoid and terpene profiles may become attainable as the research surrounding terpenes in cannabis continues.

**Table 1.** Concentrations of terpenes found in cannabis. Concentration range is given by chemotype where available; Tr—trace (<level of quantitation).



**Table 1.** *Cont.*



#### **4. Potential Roles of Secondary Terpenes**

The biological activity of cannabis terpenes is a growing topic that been extensively covered in multiple reviews [13,23,66–71]. These reviews on the therapeutic properties of cannabis terpenes primarily cover the commonly encountered mono- and sesquiterpenes

(e.g., β-caryophyllene, β-myrcene, α- and β-pinene, α-humulene, limonene, terpenoline, and linalool). For this reason, this review aims to further investigate nine secondary terpenes of cannabis, based on their abundance in the plant and their therapeutic potential (Figure 2). Regardless of their minor presence, the significant therapeutic value could point towards stronger or novel synergistic effects. The following is a summary of the more uncommon but notable secondary terpenes/terpenoids in cannabis and their potential therapeutic value; it is impossible to describe all the pharmacological effects of terpenes/terpenoids in this paper, but we shall give some examples of how these compounds possess multi-functional bioactivity. It is worth noting that the compounds often have multiple potential activities, and there is overlap of activities between terpene compounds.

**Figure 2.** Structures of secondary terpenes present in *Cannabis sativa* L.
