1. IntroductionCannabis sativa L. is a dioecious plant of the Cannabaceae family and is perhaps most famous for its production of the psychedelic metabolite delta-9 tetrahydrocannabinol (D9-THC). Cannabis has been used in traditional medicine for millennia across several continents; cannabis has been used in traditional Chinese medicine therapies for the treatment of gout, pain, convulsions, insomnia, cough, headache, itching, and anemia [1], while in traditional Aryuvedic practices, cannabis has been reported to stimulate digestion, function as an analgesic and sedative, and have aphrodisiac, anti-parasitic, and anti-viral properties [2]. Review articles covering the chemistry, pharmacology, botany, genomics, and ethnology of cannabis are regularly published as the plant’s usage grows in prevalence [3,4,5,6,7]. In addition to THC, cannabis produces a number of other cannabinoid compounds with potent activities. Cannabidiol (CBD) is one non-psychedelic cannabinoid that has emerged as a popular botanical supplement ingredient [8]. A majority of Americans are aware of CBD, and ca. 18% have tried or are regular users of CBD products [9]. The US hemp-derived market in cannabidiol (CBD) topped $4.7 billion in 2021 and is expected to reach $12.0 billion by 2026 [10]. However, while many bioactivities can be ascribed to the presence of cannabinoids, cannabis is a prolific biosynthetic organism, producing over 750 known phytochemicals, including flavonoids and terpenoids, many of which possess putative medicinal properties [11], yet the majority of these phytochemical constituents and their mechanisms of action have not been fully explored.Terpenes (also termed isoprenoids) are the most diverse class of natural products and are the most abundant by mass [12]; in cannabis, terpenes account for 3–5% of the dry mass of the inflorescence [13]. Terpenes have incredible potential for bioactivity against both infectious and chronic health conditions [14,15,16] and have been employed for thousands of years for therapeutic purposes, including in anti-inflammatory, anti-microbial, antioxidant, antitumor, and antidiabetic capacities [17]. In addition, terpenes often provide the foundation for the flavor and aroma of numerous plants and food products [18,19,20], including cannabis [21], granting the plant earthy or herbal aromas that combine with hints of sweet, citrusy, or piney scents. The terpene profile and content of cannabis has been reviewed previously [13,22,23]; however, analytical profiling studies, as well as cannabis phytochemistry reviews, traditionally focus on the more prevalent, terpenes such as myrcene, α-pinene, limonene, β-caryophyllene, linalool, humulene, ocimene, bisabolol, and terpinolene. The presence of a vast array of terpenes highlights the additional complexity of cannabis, as well as the further potential for bioactivity within this complex plant.In botanical samples, mixtures of phytochemicals are often more effective than their individual constituents in isolation due to additive or synergistic interactions among compounds. Indeed, many chronic and infectious diseases are not regulated by a single cellular target, but often have multiple regulating pathways [24,25]. As organisms in a complex and dynamic ecological environment, plants have evolved to address this multifactorial disease etiology through the synthesis of structurally and functionally diverse phytochemicals. Thus, cannabis may also exert its bioactive effects via a combination of multiple constituents. Originally hypothesized in the late 20th century and termed the “entourage effect” [26], synergy between different cannabinoids has been documented in several studies. However, the potential for synergy between cannabinoids and other chemical classes, especially terpenes, has remained underreported.

This review aims to synthesize recent studies and information regarding the compositional diversity of terpenes, especially ‘minor’ terpenoid structures (compounds that are less prevalent in the plant on a by-mass basis) that have not been the focus of other reviews, yet are found in diverse cultivars of cannabis and have unique and varied bioactivities as well. This is a unique feature of this review. In addition, we will build on the body of knowledge regarding how terpenes can potentially work in concert with cannabinoids to enhance bioactivity, as this is a timely topic given the upswing in interest in cannabis and potential synergy/entourage effects.

2. Terpene BiosynthesisTerpenes 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].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 CannabisOver 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 suggest 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 [34,45,46,47,48]. Terpene profiles of the remaining chemotypes are limited or have yet 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 [34,50,51,52,53,54,55].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 β-eudesmol, γ-eudesmol, guaiol, α-bisabolol, or eucalyptol. This suggests a chemotype-dependent 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. Concentrations of terpenes found in cannabis. Concentration range is given by chemotype where available; Tr—trace (<level of quantitation).

CompoundChemotypes Rage of Average Concentrations Reported per Chemotype (mg/g Dry Weight)ReferenceAgrospirolII:Tr–0.50[45]AlloaromandreneI, II, IIII:0.004–0.08[53,60]II:0.08–0.10III:0.05–0.10AromadendreneII:0.02–0.13[61]α-BisabololI, II, IIII:Tr–1.10[34,45,46,53,60,62,63,64]II:0.57–1.22III:0.07–2.31α-BisaboleneI, II, IIII:0.13–0.50[53,61]II:0.11–0.29III:0.03–0.50β-BisaboleneI, II, IIII:0.05–0.17[53]II:0.18–0.51III:0.12–0.71BorneolI, II, IIII:0.01–0.03[34,61,63,64]II:0.05III:0.009–0.02α-bergamoteneI, II, IIII:0.024–1.18[34,53]II:0.45–0.81III:0.018–0.68Cis-bergamoteneI, IIII:0.07–0.11[61]III:0.21Trans-bergamoteneI, IIII:0.12–0.28[61]III:0.04BulnesolI, II, IIII:0.10–0.50[34,45,53]II:0.090–0.19III:0.070–0.49γ-cadineneI, IIII:0.41–0.60[61]III:0.02CampheneI, IIII:0.002–0.09[34,60,63,64]III:0.001–0.48CamphorII:0.001–0.01[61,64]P-CimeneI, IIII:0.016[64]III:0.01β-CaryophylleneI, II, IIII:0.24–8.20[34,45,46,60,61,62,63,64,65]II:0.86–3.90III:0.16–3.17β-Caryophyllene oxideI, II, IIII:0.005–0.06[60,61,63]II:0.02III:0.09Trans-β-caryophylleneI, IIII:0.02–0.06[53,61]III:0.06δ-3-careneI, II, IIII:Tr–0.60[45,46,61,64,65] II:TrIII:0.065–0.070α-CedreneI, IIII:0.038[64]III:0.023β-CitronellolI, IIII:0.002[60,64]III:0.001–0.003α-curcumeneI, IIII:0.008[60]III:0.017β -CurcumeneI, II, IIII:0.014–0.61[53,60]II:0.061–0.16III:0.016–0.09CyclounatrieneI, IIII:0.02–0.13[34]III:0.086ElemeneI, III:Tr–2.70[45,65]II:Trγ-elemeneI, IIII:0.104–1.89[34,53,61]III:0.04–0.068δ-elemeneI, IIII:Tr–0.392[34]III:0.005EucalyptolII, IIIII:0.010–0.07[53,60,63]III:0.052–0.14Eudesma-3,7(11)-dieneI, IIII:Tr–0.80[34,61,65]III:0.05EudesmaneI, IIII:0.33–0.55[34]III:0.04A-eudesmolI, III:0.02[63]II:0.26β-EudesmolI, II, IIII:Tr–0.92[45,53,61,63,64]II:0.23–0.65III:0.085–1.01γ-EudesmolI, IIII:Tr–0.80[34,45,53,61]II:0.30–0.78III:0.010–1.03α-farneseneI, II, IIII:0.02–0.06[34,63] II:0.24III:0.002β-farneseneI, II, IIII:0.019–1.96[34,53,65]II:0.73–1.6III:0.008–1.4 Trans-β-farneseneI, IIII:0.31–1.06[61,63]II:0.35III:0.05FenchoneI, II, IIII:0.005–0.03[60,63,64]II:0.02III:0.007–0.008Fenchol I, II, IIII:0.047–1.09[34,46,60,61,62,63,64]II:0.09–0.31III:0.028–0.138Germacrene BI, IIII:0.25–1.27[34]III:0.34GeraniolI, IIII:0.01[63,64]III:0.004Geranyl AcetateII:Tr–0.70[46]GuaiolI, II, IIII:Tr–1.09[34,45,53,61,63,65]II:0.27–0.87III:0.010–1.21α-guaieneI, IIII:Tr–0.50[45,65]II:TrIII:Trδ-guaieneI, III:Tr–0.80[45,61,65]II:0.8α-gurjuneneII:0.1–0.46[53]HumuleneI, II, IIII:Tr–4.00[45,46,53,64]II:0.64–1.11III:0.26–0.93α-HumuleneI, II, IIII:0.09–1.93[34,60,62,63,65]II:0.32–0.36III:0.14–0.27IsopulegolI, III:0.02–0.04[63]II:0.02LedeneI, III:0.11–0.13[63]II:0.05LimoneneI, II, IIII:Tr–9.1[34,45,46,53,60,61,62,63,64] II:0.079–1.14III:0.022–1.44LinaloolI, II, IIII:Tr–3.10[34,45,46,53,60,61,62,63,64]II:0.27–0.35III:Tr–0.36Cis-linalool oxideI, IIII:0.002[60]III:0.005Trans-linalool oxideI, IIII:0.002[60]III:0.002MentholI, IIII:0.001[60]III:0.001β-MyrceneI, II, IIII:0.12–14.8[34,45,46,53,60,61,62,63,64,65]II:0.20–3.02III:0.18–7.60NerolidolI, II, IIII:0.02[61]III:0.01Trans-nerolidolI, IIII: