There is no doubt that Cannabis with the chemical diversity, unique structures (Figures 3, 4), physicochemical properties, and diverse bioactivities of its metabolites, represents an invaluable source for the development of novel applications in various sectors, such as, medicine, cosmetics, and the food industry. Although such applications are yet in their infancy, it is anticipated that Cannabis-based or Cannabis-infused products will provide solutions to major human health conditions, and lead to the development of new functional food and beverage products.

Nonetheless, the complexity of the plant’s extracts and the in-depth understanding of interactions between their components (e.g., entourage effect) and synergism, represent major challenges. The development of pharmaceuticals based on Cannabis extracts is challenging for the medicinal research, which operates according to the principle “single compound-single target” (Hazekamp et al., 2016). However, the ineffectiveness of individual compound-based medicine against multigenic diseases (e.g., cancer) or diseases that affect multiple tissues dictate the need for the discovery of drugs that will act on multiple targets (Zimmermann et al., 2007; Giordano and Petrelli, 2008). Therefore, it is of paramount importance to distinguish between the bioactivities of mixtures and those of the individual bioactive metabolites based on appropriate protocols, which could be greatly assisted by high-throughput metabolomics. Examples of Cannabis-derived pharmaceuticals are displayed in the Table 2.

Within this context, the discovery, assessment, and development of new sources of bioactivity as drugs for the treatment of various conditions, represent key priorities for the medicinal R&D (Chin et al., 2006; Dittrich and Manz, 2006; Harvey, 2008). Based on results of recent research, there is a growing amount of evidence that supports the effectiveness of various Cannabis-derived cannabinoids in the treatment of a wide range of conditions, including, among others, chronic and acute pain, epilepsy, sleep disorders, multiple sclerosis, gastrointestinal reflux disease, irritable bowel syndrome (IBS), spasticity, hypertension, and schizophrenia (Pacher et al., 2005; Hazekamp and Grotenhermen, 2010; Caraceni et al., 2014; Bruni et al., 2018).

The psychoactive and medicinal properties of Cannabis have been known for more than 5 millennia by major civilizations of the Middle East, Egypt, China, India, Ancient Greece, and the Romaine Empire (Di Marzo, 2008; Mechoulam and Parker, 2013; Farag and Kayser, 2015). Cannabinoids were the first identified group of potent Cannabis metabolites, with the medicinal properties of its major representatives being attributed to their interference with the G protein-coupled cannabinoid receptors (GPCRs) CB1 and CB2 of the endocannabinoid system (Di Marzo et al., 2004; Mechoulam and Parker, 2013). The CB1 receptors are amongst the most abundant GPCRs in the brain of mammals and are also present, to a lesser extent, in various peripheral organs, whereas the CB2 receptors have been identified throughout the central nervous system (CNS) and cells of the immune system, being part of a general protective system (Di Marzo et al., 2004; Di Marzo, 2008; Mechoulam and Parker, 2013) and modulating cytokine release (Pertwee, 2005).

Although Δ⁹-THC was isolated and synthesized in the mid 60s’ (Gaoni and Mechoulam, 1964; Mechoulam and Gaoni, 1965), the research on the mode(s)-of-action (MoA) of cannabinoids remained inconclusive for more than 20 years (Mechoulam and Parker, 2013). The similarities between the physicochemical properties and structures of cannabinoids (Figure 3), pose an obstacle toward their isolation in pure chemical form (Mechoulam and Hanuš, 2000) and the subsequent investigation of their bioactivities, MoA, and pharmacokinetics.

Furthermore, the cannabinoid interconversions during storage and heating are complex (Figure 5), which represents a major challenge for the development of new Cannabis-based products, such as drugs, cosmetics, beverages, and edibles. Additionally, of great interest is the fact that non-psychoactive Cannabis metabolites (e.g., terpenoids) can act synergistically with Δ⁹-THC, contributing to the so-called “entourage effect” of medicinal Cannabis extracts (Ben-Shabat et al., 1998; Russo, 2011, 2018), with the undergoing operating mechanism(s) being largely unexplored.

Another major challenge for the Cannabis industry related to drug development is the production of standardized extracts that will meet the standards set by the corresponding regulatory agencies (e.g., Cannabis Act, Canada)¹. The agricultural practices, plant growth conditions, and extraction processes all play key roles in achieving consistency of the extracts’ content, however, discussion on those factors are beyond the aim of the present review. The robust QC of Cannabis preparations and assessment of their consistency and potency could be achieved by applying metabolomics for the various batches of a given product. The application of metabolomics employing and integrating information acquired by various analyzers (e.g., LC and GC-based platforms) could lead to the deconvolution of the complex chemical composition of Cannabis extracts and the monitoring of the consistency across batches, facilities, and different cultivation periods. For R&D purposes, metabolomics could be employed in the optimization of agricultural practices, growth conditions, and extraction processes in order to achieve the desired composition of extracts with proven medicinal properties, as discussed below. To the best to our knowledge, such approach is in its infancy, and no reports are currently available.

Cannabinoids exert palliative effects in cancer patients by, among others, preventing nausea and pain, and stimulating appetite (Guzman, 2003). Additionally, it has been shown that they inhibit the growth of tumor cells in vitro and in vivo in animal models (Guzman, 2003) and exhibit antitumor activity (Velasco et al., 2012; Dando et al., 2013). Such bioactivities have been supported by Phase III clinical trials, however, the corresponding mechanism(s) of action remain inconclusive. In the case of pancreatic adenocarcinoma it seems that cannabinoids induce autophagy and inhibit cell growth (Dando et al., 2013).

CBD, the second-most studied cannabinoid, and various of its synthetic derivatives have attracted the interest of the pharmaceutical industry and that of academic researchers, with specific focus on the understanding of their MoA, potency, and pharmacokinetics (Morales et al., 2017). It exhibits remarkable potency, including sedative, anxiolytic, anticonvulsive, hypnotic, anti-psychotic, anti-nausea, and anti-inflammatory effects (Mechoulam et al., 2002). Preclinical studies have highlighted the inflammatory potential of CBD in mouse models (Morales et al., 2017), without causing behavioral changes (Viudez-Martínez et al., 2018). It exerts a well-documented anti-seizure and anti-epileptogenic properties against epilepsy independent of the CB1/CB2R, which is supported by Phase III clinical trials on treatment-resistant epilepsies (Rosenberg et al., 2017). Additionally, information on the action of Δ⁹-THC containing Cannabis preparations in the treatment of pediatric epilepsies remains largely fragmented (Rosenberg et al., 2017). On the other hand, Δ⁹-THC or synthetic cannabinoid-induced seizures in mice have been observed following their intraperitoneal administration, which can be prevented by a CB1-selective antagonist (Malyshevskaya et al., 2017).

In addition to the two major cannabinoids Δ⁹-THC and CBD, other cannabinoids with limited or no psychoactive properties could exhibit interesting pharmaceutical properties and bioactivities. Among those are cannabidiol and cannabinoic acids, whose MoA are yet unknown (Di Marzo et al., 2004). Several cannabinoids (e.g., Δ⁹-THC, CBD, CBC, CBG, CBN), exhibit antibiotic activity to Staphylococcus aureus, highly correlated to the stereochemistry of the molecules and the groups of substitution (Appendino et al., 2008).

Additionally, the biotransformation of cannabinoids in the human body, which determines their potency and medicinal properties, is a largely unexplored topic and could lead to the discovery of novel bioactive metabolites (Dinis-Oliveira, 2016). Due to their high lipophilicity, cannabinoids could remain in the plasma and fat tissue for prolonged periods. Focusing on the Δ⁹-THC, in a first phase (Phase I, oxidative metabolism), it is metabolized to 11-hydroxy-Δ⁹-THC, which is further metabolized to the inactive 11-nor-9-carboxy-Δ⁹-THC. The Phase II (conjugation metabolism), includes reactions such as conjugation which lead to the detoxification of the molecule (Dinis-Oliveira, 2016). More than 80 Δ⁹-THC-derived metabolites have been identified as products of its transformation (Mazur et al., 2009).

The integration of information from clinical trials in which patients provide feedback following treatments with various Cannabis chemovars and information on the corresponding metabolite profiles employing metabolomics is very important for the selection of the best varieties and their standardization for medical use and drug discovery purposes. Based on this approach, applying GC/FID/MS metabolomics, Dutch researchers evaluated 460 accessions based on their content in major cannabinoids and terpenes (Hazekamp et al., 2016). Results revealed a strong correlation between Cannabis phenotypes and their terpene content, as it can be evaluated by their smell, taste, and medicinal properties, as well the importance of gibberellic acid (GAs) in terpenoid biosynthesis.

LC/ESI/MS-based metabolomics has been employed in the study of pharmacokinetics of major cannabinoids in rat brains, following their oral administration (Citti et al., 2018). Brains were initially homogenized in deionized H₂O, followed by the addition of ACN:MeOH 70:30 (v/v) containing formic acid (0.1%, v/v). Following the removal of phospholipids, the extracts were dried and finally an ammonium acetate (2.0 mM):acetonitrile (70:30, v/v) solution was added. Analysis revealed the formation of novel, unique CBD-derived metabolites and fluctuations in the levels of several other endogenous metabolites as a result. Such application confirms the potential of metabolomics in the acquisition of fundamental knowledge related to the study of the mode(s)-of-action and bioactivity of cannabinoids for medical purposes.

The function of the endocannabinoid system and its regulation by endocannabinoids are complex, and yet relative information is largely fragmented. Their levels and relative composition vary depending and their role, which could shift from protective to deregulator of the physiological state of an individual. Therefore, compounds that could prolong the lifespan or suppress endocannabinoids could be extremely important in treating various conditions (Di Marzo, 2008).