Recent Research on Cannabis sativa L.: Phytochemistry, New Matrices, Cultivation Techniques, and Recent Updates on Its Brain-Related Effects (2018–2023)
1Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Paolo Gaifami, 18, 95126 Catania, CT, Italy
2Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei, 34, 80078 Pozzuoli, NA, Italy
*Correspondence: giuseppe.ruberto@cnr.it; Tel.: +39-0957338347; Fax: +39-0957338310Abstract
Cannabis sativa L. is a plant that humankind has been using for millennia. The basis of its widespread utilization is its adaptability to so many different climatic conditions, with easy cultivability in numerous diverse environments. Because of its variegate phytochemistry, C. sativa has been used in many sectors, although the discovery of the presence in the plant of several psychotropic substances (e.g., Δ9-tetrahydrocannabinol, THC) caused a drastic reduction of its cultivation and use together with its official ban from pharmacopeias. Fortunately, the discovery of Cannabis varieties with low content of THC as well as the biotechnological development of new clones rich in many phytochemical components endorsed with peculiar and many important bioactivities has demanded the reassessment of these species, the study and use of which are currently experiencing new and important developments. In this review we focus our attention on the phytochemistry, new matrices, suitable agronomic techniques, and new biological activities developed in the five last years.
1. Introduction
The origins of Cannabis sativa in terms of its cultivation and use date back about 12,000 years, in the regions of Central and East Asia. Its diffusion around the world has been continuous and inexorable [1], due to the easy adaptability of this plant to very different climate conditions, and thanks also to its numerous properties and uses as a fiber, food, and drug [2,3,4,5,6].
C. sativa is an herbaceous and dioecious plant belonging to the order Urticales and the Cannabaceae family [5]. The taxonomic description of the Cannabis genus is almost controversial; ‘sativa’ is considered de facto the sole species of the genus. Even though other species were suggested, ‘indica’ and ‘ruderalis’ being the most important, these different species are today considered merely varieties.
Concerning its content of secondary metabolites, C. sativa has been well studied and as of today about 750 compounds have been reported in this plant [5,7].
Cannabis has been widely cultivated due to its industrial [8], ornamental [9], nutritional [10], medicinal, and recreational potential [11]. From regulatory and application perspectives, cannabis plants are categorized based on the level of Δ9-tetrahydrocannabinol (THC), one of the most important phytocannabinoids [12]. Plants are generally classified and regulated as industrial hemp if they contain less than 0.3% THC in the dried flower (this level varies by country) or as a drug type with more than this threshold [13]. Furthermore, the study of the phytochemical profile of C. sativa has recently been demonstrated useful not only for determining the potential toxicity of C. sativa, considering the widespread use of this plant for human consumption and as animal feed, but also for traceability purposes [14,15,16]. The aim of this review is to provide an update of the studies carried out on C. sativa in the past five years, dealing with different aspects such as phytochemistry, novel cultivation techniques, and newly described biological activities especially connected with the effects of cannabis on the central nervous system.
3. Alternative Cannabis Matrices
3.1. Cannabis Roots
As mentioned above, C. sativa is a plant with a very ancient tradition. It can be used to obtain a variety of products useful in many sectors of human daily life. In the previous chapter we have shown, in a non-exhaustive selection, some of its most interesting phytochemical components. However, to obtain a more complete picture of this plant it is necessary to take into account other parts of the plant that have received scarce interest, such as roots, seeds, and seed hulls.
In the phytotherapeutic sector, the interest in this plant has been devoted to its inflorescence and leaves, where the most important bioactive components cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) are mainly concentrated. The roots have been comparatively neglected, and have not received the same attention as the aerial parts, although canapa roots have been used to fight fever, inflammation, infections, and arthritis [41,42]. Phytochemical analyses of roots did not show the presence of cannabinoids, or only in traces, whilst the most important components of this matrix resulted in a series of triterpenoids subdivided between triterpenes and phytosterols, some of which are reported in Figure 6 [1]. The presence of triterpenoidic compounds in the roots of C. sativa was recently confirmed by Ferrini et al. [43]. They suggest the application of aeroponic cultivation, which would facilitate all the relevant agronomic processes by being easy, standardizable, and favoring the growth of the plant’s root system compared with conventional soil-based cultivation methods.
A further and more recent phytochemical study on canapa roots was carried out by Oh et al. [44], who worked with a sample of hemp cultivated in Korea. Briefly, after water washing and air drying, the roots were analyzed by HPLC-DAD, which allowed researchers to establish the presence and the amounts of p-coumaric acid and ethyl-p-coumarate as the main constituents of the root extracts (Figure 7). According to their findings, the authors suggested the industrial exploitation of the hemp roots of C. sativa as a source of p-coumaric acid derivatives, in consideration of their broad biological activities.
A very recent study was carried out by an Argentinian research group, aiming to determine the antibacterial activities of extracts from inflorescences and roots of C. sativa [45]. The content of total phenolic compounds, total flavonoids, and total saponins was determined by applying previous methodologies [46,47,48,49].
Antibacterial activity against Paenibacillus larvae, Staphilococcus aureus, and Esherichia coli was measured by the MIC methodology; the recorded activities even if not very significant were associated with the high content of triterpenoids and saponins.
Many research groups have reported cannabinoids to be absent or present only at trace levels in the roots of C. sativa. However, this suggestion has recently been challenged [49]. Furthermore, a Brazilian research group recently reported the presence of a new cannabinoid called Δ9-tetrahydrocannabutol (known also as tetrahydrocannabinol-C4—THC-C4), that is structurally very similar to the main cannabinoid Δ9-THC, the sole difference being a butyl side chain instead of a pentyl side chain. Further, four nitrogenous compounds were characterized and all are reported in Figure 8 [50].
3.2. Cannabis Seeds and Seedhulls
Three different types of oil can be obtained from C. sativa:
(a) vegetable oil, obtained by extraction of seeds and constituted mainly by triglycerides, suitable for human consumption owing to a good balance of ω-6 and ω-3 essential fatty acids (EFAs);
(b) essential oil from inflorescences and flowers, obtained by hydrodistillation and steam distillation;
(c) so-called ‘hashish oil’, normally obtained by solvent extraction from plants [1].
With the aim to exploit each fraction from the workout of hemp Cannabis sativa, seed hulls from C. sativa have been also investigated for their content. Using chloroform as extracting solvent and HPLC/MS as an analytical tool, nine diacylglycerols (DAGs), six lysophatidylcholine (LPCs), five lysophosphatidylethanolamines (LPEs), eight phosphatidylethanolamine (Pes), and thirteen phosphatidylethanolamines (PCs) were recovered from the matrix [51]. These new compositional data add another input to the list of uses of this ancient plant.
Fibers represent a considerable portion (>77%) of C. sativa L. and therefore the main component usable for further industrial processes. Recently, an Australian group demonstrated a large increase in fiber solubility by applying “extrusion technology”; the authors also observed enhancement of the binding capacity of water and oil, delayed starch gelatinization, and inhibition of retro-degradation. The supplementation of this material into animal feed showed a positive effect on animal microbiota [52].
An international research team carried out a study on eight varieties of whole hemp seeds, and on eight commercial samples of dehulled hemp seeds, with the aim of determining the seeds’ chemical composition and biological properties [53]. The compounds analyzed were soluble sugar and phenolic compounds, and the tested biological properties included antioxidant, cytotoxic, and antimicrobial activity. Sugars were the main components in both samples, and whole and dehulled seeds showed similar soluble sugar profiles, containing fructose, glucose, sucrose, and raffinose. Regarding their phenolic compounds, the two studied matrices in this case showed similar profiles, with the ferulic acid hexoside and syringic acid as the main components. The biological activities were interesting and the authors concluded that hemp seed has optimal potential as a functional food.
Considering that edible seeds are today largely present in human and animal diets an Indian group has recently focused its attention on the nutritional and pro-health properties of the five most popular edible seeds: Chia (Salvia hispanica L.), Hemp (Cannabis sativa L.), Pumpkin (Cucurbita), Sunflower (Helianthus annus) and Safflower (Carthamus tinctoria) [54,55]. Concerning hemp the authors report that 100 g of seeds contain 553 kcal of energy and possesses digestible proteins, polyunsaturated fatty acids (PUFA), lipids and carbohydrate. The omega-6 compounds in the total content of fatty acids ranges from 64 to 72%. Oil from C. sativa seeds shows traces of cannabidiol, β-sitosterol, and terpenes together with the tocopherol molecular group including α-, γ-, β-, and δ-tocopherol. The presence of the three main nutrients, namely healthy fat, protein, and fiber provide to the seeds a big opportunity of success, which is also supported by a significant diffusion of vegan/vegetarian diets among the people. Always under the nutritional point of view of C. sativa seed oil it is important to underline that the seeds are particularly rich in omega-3 and stearidonic acids [56].
3.3. Cannabis Leaves as Functional Foods
Roots, stems, and leaves from C. sativa have recently been investigated to determine their content of tryptophan (TRP) and its metabolic products: kyurenine (KYN) and kynurenic acid (KYNA), which have biological properties (Figure 9). Since these compounds are present in higher quantities in the leaves of C. sativa, this matrix is proposed as alternative source for these substances in the field of functional foods [57].
4. Cultivation Techniques Especially Effective on Cannabis sativa—Recent Updates
4.1. The State of the Art
As previously stated, secondary metabolites in C. sativa (mainly cannabinoids, terpenoids, and polyphenols) are produced in all aerial parts of the plants, particularly in the leaves and female inflorescences. Their production, such as that of all species belonging to the vegetable kingdom, is related to genetic, environmental, and agronomic factors (plant chemotype, growth conditions, and phenological stage at harvest) [58]. This high variability of growth conditions inevitably leads to broad variability in the content of the plant’s secondary metabolites, which represents the main problem for the use of cannabis for medical purposes. To overcome this intrinsic problem, several strategies have been developed, including asexual propagation of selected varieties and highly technological indoor cultivation [59,60,61]. Asexual reproduction of cannabis requires only a single plant, allowing the multiplication of a single genotype for commercial production of individuals that meet the desirable pharmaceutical traits [62]. Since indoor cultivation in the controlled conditions of high-technology greenhouses carries significantly high manufacturing costs, attention has been directed towards biomass production and the content of bioactive substances in order to compensate for the expense [63]. An alternative approach is to improve and control the secondary metabolism of plants in open fields through the use of selected new-generation fertilizers or by controlling plant and soil microbiota. These strategies are discussed in detail in this section. It is worth mentioning that agronomic techniques can be replaced (or associated) with molecular approaches such as the sequencing of the cannabis genome, and the manipulation of the cannabis plant itself for the altered production of specific compounds, in order to produce medically relevant cannabis varieties with elevated concentrations of specific molecules [64].
4.2. Soilless Growing Technologies Applied to Cannabis: Hydroponic, Aquaponic, and Aeroponic Cultivation—Recent Updates
Because of the aforementioned issues associated with the high intrinsic variability in C. sativa secondary metabolism, the majority of medical research involving the cultivation of this species occurs in controlled environments (hi-tech greenhouses and indoor facilities) making use of soilless cultivation systems such as hydroponics, aquaponics, and aeroponics. Hydroponics systems are well-known, in which plant roots are partially and/or totally immersed in a nutrient solution. Irrigation methods in hydroponics include drip irrigation (a nutrient solution is fed into an inert growing medium used merely as physical support for the root system), deep water culture (the plant root system is completely submerged in the nutrient solution and the plants are supported by a membrane system that blocks the immersion of the aerial parts), nutrient film techniques (only the lower portion of the root system is immersed in a flowing nutrient solution, whilst the upper roots are exposed to the air), and flood and drain (plant roots are immersed in a nutrient solution for a period of time, subsequently drained and collected in a reservoir to aerate the root bed) [65]. Aquaponics is a soilless cultivation system that has recently gained scientific and commercial interest due to its circular approach and reduced environmental impact [66]. Aquaponics uses fish effluent and naturally occurring bacteria to fertilize plants in a circular ecosystem that produces fish and plant materials simultaneously. With this approach, the utilization of fertilizers and pesticides is highly reduced, providing an ecofriendly system. The comparable yields obtained from aquaponic cultivation when compared with conventional systems can be attributed to a plethora of diverse plant-growth-promoting microorganisms that can improve efficiency of nutrient use.
The third soilless approach useful for indoor C. sativa cultivation is aeroponics, in which the plant is entirely exposed to the air and the nutrient mixtures are nebulized to plant root systems in the form of droplets, typically of 10–100 µm diameter. The most commonly used aerosol generation technology in aeroponics is high-pressure atomization, where high-pressure liquids are forced through a small orifice, breaking the liquid stream into droplets [67].
4.3. LED Lighting Techniques
In the so-called “ecological pyramid”, plants are classified as sole producer organisms, occupying only the pyramid basement. Members of the vegetable kingdom are able to synthetize glucose, the molecule at the center of all metabolic charts, starting with carbon dioxide and water and using solar light as a catalyst. It is therefore self-evident that a close relationship exists between plants and light: plant morphology, metabolism, growth, and development are strongly influenced by circadian cycles and can be artificially manipulated by the quality and duration of light [68,69,70]. In the indoor cultivation scenario, the use of artificial lights for plant cultivation, particularly that of light-emitting diode (LED) technology, can result in significant reductions in energy consumption and in the achievement of desirable yields and phytochemical traits. In fact, it has been demonstrated that different light wavelengths have effects on different physiological features of the plant such as pigmentation, secondary metabolite production, chloroplast development, photosynthetic activity, and plant biomass accumulation, to cite just a few [71,72]. In C. sativa, due to the previously described complexity of its secondary metabolism, light quality and quantity have significant effects on the biologically active components. For example, UV-B radiation (280–315 nm) did not impact cannabinoid content in cannabis plants with the exception of Δ9-THC, whilst UV-A radiation (315–380 nm) from full-spectrum LED arrays induced an increase of several compounds including CBD, CBG, Δ9-THC, and tetrahydrocannabivarin (THCV) [73]. Recently, Moher et al demonstrated that not only the choice of light radiation but also its intensity can induce modifications in C. sativa plants’ morphology and growth processes [74]. The authors did not mention the effects of the parameters they applied on the secondary metabolic profiles of cannabis plants, which can surely represent a further step of knowledge in this field.
4.4. Cannabis Symbiotic Microorganisms: A Yet Unexplored Scenario
In the past decade, the study of microbial communities associated with higher living organisms, known as “beneficial microbes”, has gained exponential interest due to its significant effects on human health. The composition and content of microbial species hosted in several parts of the human body, particularly those residing in the gut, have been intensively studied by a plethora of multidisciplinary approaches. In spite of innumerable works in the literature dealing with human microbiota, those focused on the microbiota of agricultural plants are relatively few. Indeed, this is due to the high number of plant species involved and the fact that plant microbial communities are heavily affected by a number of factors including developmental stages, root exudation, soil type, and environmental conditions. Although not the focus of the present work, approaches involving the manipulation of C. sativa microbiota are definitely worthy of mention. For an extensive review of this specific topic, see the recent paper by Taghinasab and Jabaji [75].
Owing to its rich and variegate secondary metabolic pool, C. sativa constitutes a perfect model to explore plant–microbiome interactions and to assess how endophytes can modulate the production of secondary metabolites. Endophytes have gained a prominent role in the list of abiotic and biotic elicitors for the growth, mass yield, and metabolic performance of this plant. Pagnani et al. reported that a mixture of four bacterial species applied to the roots of cannabis seedlings in vitro efficiently colonized the entire root system of the plant, favoring plant growth and development in greenhouses as well as accumulation of secondary metabolites. Particularly, two of the main cannabinoids from cannabis, CBD and THC, were significantly improved by endophytic treatment [76]. Nowadays, even though basic information on the diversity and composition of cannabis endophytes has been published, the majority of the works deal with the isolation and identification of microbes rather than their modulating effects on the plant’s secondary metabolic pool. Therefore, systematic study of this topic is desirable.
6. Conclusions
In the time period considered by this review (2018–2023) a considerable interest in C. sativa has grown exponentially, as documented by the more than 3000 articles published on this species just in 2021.
The topics covered in this work – phytochemistry, cultivation techniques and brain-related effects of cannabis—are only apparently unrelated to each other, since a leading thread exists and it is the highly variegated secondary metabolism of this plant that gives rise to a not so usual chemodiversity. In particular, the phytochemical studies carried out on cannabis allowed for establishing the toxicity aspects normally connected to an abuse of this plant, and at the same time how, for example, CBD is recognized as the most relevant cannabinoid with therapeutic potential in several disorders.
Since the phytocannabinoid composition is extremely inhomogeneous, and the ratios of active molecules are affected by a range of variables, the use of raw cannabis as a feedstock to reach the high-quality demands of the pharmaceutical industry is extremely challenging. For instance, to produce complex botanical medicine like Sativex®, which contains a specific THC and CBD ratio and a range of other molecules, GW Pharmaceuticals had to manage agricultural cannabis biomass selection, cultivation, and harvest/processing to ensure quality supplies for medical research to meet the prescription medicines uses for the treatment of spasticity due to multiple sclerosis. Similarly, in recent years, the production of CBD-based medicinal materials for research as potential therapeutics in childhood affected by epilepsy syndromes has come into greater attention for agricultural biomass selection and cultivation, to ensure quality supplies for medical research. To meet these requirements, an advanced Mendelian Cannabis breeding program has been exploited by pharmaceutical companies utilizing chemical markers to maximize the yield of phytocannabinoids and terpenoids with the aim to improve therapeutic efficacy and safety to produce selective chemovars with a therapeutical application for analgesic, anti-inflammatory, anticonvulsant, antidepressant, and anti-anxiety effects, while simultaneously reducing sequelae of Δ9-tetrahydrocannabinol such as panic, toxic psychosis, memory impairment. Integrated omics studies combining genomic data with metabolite profiles are now beginning to unravel the association between the expression of cannabinoid genes with THC:CBD ratio and cannabinoid content. Advanced agro-biotechnology methods could be further extended for recombinant production of cannabinoids in metabolically engineered hosts such as yeasts or bacteria. Pharmacological research coupled with rapidly evolving genome-based biotechnology applied to agriculture will further facilitate exploring cannabis plants for their remarkable potential in drug discovery.
The interest in the phytochemical composition of cannabis is not only due to the possibility to use many of its phytochemicals in the medical sectors, but also to the industrial interest towards the same products. Many industrial applications of cannabis could not exist without the development of suitable and high-performance cultivation techniques. However, notwithstanding the large number of studies carried out in the phytochemical sectors, an exhaustive and complete characterization of cannabis extracts has not yet been reached, therefore new extraction procedures, as well as novel and more sophisticated characterization methodologies still have to be applied.
In any case, cannabis phytochemistry, as well as agronomical and biotechnological techniques applied to its cultivation have benefited from this growing interest and from the trend observed in these years appears that the discovery of new specialized metabolites seems inevitable. In particular, as pointed out in this review, in the medical sector many new discoveries on the role of the metabolism of cannabis and how it interacts with the human central nervous system appear close to their discovery.
Acknowledgments
The authors sincerely thank Vincenzo Di Marzo (ICB-CNR, Italy and Université Laval, Québec City, Canada) for critically reviewing the manuscript.
Conflicts of Interest
L.C. has been a consultant for GW Pharmaceuticals; the rest of authors declare no conflict of interest.
| Code | Name | Formula | MW a |
|---|---|---|---|
| Δ9-THC | δ-9-Tetrahydrocannabinol | C21H30O2 | 314.55 |
| Δ8-THC | δ-8-Tetrahydrocannabinol | C21H30O2 | 314.52 |
| Δ9-THCP | (−)-trans-Tetrahydrocannabiphorol | C23H34O2 | 342.52 |
| THCV | Tetrahydrocannabivarin | C19H26O2 | 286.45 |
| Δ9-THCA-A | δ-9-Tetraidrocannabinolic acid A | C22H30O4 | 358.47 |
| NA b | Anhydrocannabimovone | C21H28O3 | 328.51 |
| CBN | Cannabinol | C21H26O2 | 310.43 |
| 7-OH-CBD | 7-Hydroxycannabinol | C21H30O3 | 330.46 |
| CBNA | Cannabinolic acid | C22H30O4 | 358.47 |
| CBD | Cannabidiol | C21H30O2 | 314.46 |
| CBDD | Cannabidiol dimethyl ether | C23H34O2 | 342.52 |
| CBD-C2 | Cannabidiorcol | C17H22O2 | 258.35 |
| CBD-DER1 | 1,2-Dihydroxycannabidiol | C21H33O4 | 348.36 |
| CBD-DER2 | 3,4-Dehydro-1,2-dihydroxycannabidin | C21H31O3 | 330.37 |
| CBD-DER3 | Hexocannabitriol | C21H3104 | 348.64 |
| CBC | Cannabichromene | C21H30O2 | 314.46 |
| CBG | Cannabigerol | C21H32O2 | 316.48 |
| CNM | Cannabimovone | C21H30O4 | 346.46 |
| CBND | Cannabinodol | C21H26O2 | 310.43 |
| CBE | Cannabielsoin | C21H30O4 | 346.46 |
| CBF | Cannabifuran | C21H26O2 | 310.24 |
| CBL | Cannabicyclol | C21H30O2 | 314.46 |
| CBT | Cannabitriol | C21H30O3 | 330.46 |
| CBR | Cannabiripsol | C21H32O4 | 348.23 |
| CBCA | Cannabichromenic acid | C22H30O4 | 358.21 |
| CBGA | Cannabigerolic acid | C22H32O4 | 360.49 |
| CBDA | Cannabidiolic acid | C22H30O4 | 358.47 |
| CBDP | Cannabiphorol | C23H34O2 | 342.52 |
| CBDD | Cannabitwinol | C43H60O4 | 64033 |
| CBDV | Cannabidivarin (Cannabidivarol) | C19H22O2 | 282.38 |
| CBTC | Cannabicitran | C21H30O2 | 314.46 |
| THCA/CBDA | >1 | Chemotype I |
| <1 | Chemotype III | |
| >1–<1 | Chemotype II | |
| High CBGA | Chemotype IV | |
| No cannabinoids | Chemotype V |
| Monoterpenes | |||||
| α-Thujene | Sabinene | α-Pinene | Camphene | α-Terpinene | β-Myrcene |
| β-Pinene | α-Phellandrene | 1,8-Cineole | (Z)-β-Ocimene | (E)-β-Ocimene | p-Cymene |
| Limonene | Δ3-Carene | α-Terpinene | γ-Terpinene | Fenchone | Terpinolene |
| Terpinen-4-ol | Linalool | Fenchol | Camphor | Isoborneol | Borneol |
| Menthol | α-Terpineol | Citronellol | Pulegone | Geranyl acetate | p-Cymenene |
| Geraniol | Nerol | Geranial | Neral | 2-Heptnone | Heptanal |
| 2-Pinen-10-ol | p-Cymen-8-ol | trans-Pinocarveol | Myrtenol | Linalool acetate | Safranal |
| Sesquiterpenes | |||||
| α-Ylangene | α-Copaene | β-Elemene | β-Longipinene | Z-Caryophyllene | β-Caryophyllene |
| α-Longipinene | α-Humulene | β-Guaiene | β-Acoradiene | γ-Himachalene | α-Selinene |
| β-Himacalene | δ-Amorphene | γ-Cadinene | δ-Cadinene | α-Cadinene | E-Nerolidol |
| Germacrene B | Spathunelol | Viridiflorol | Ledol | Humulene epoxide | epi-α-Bisabolol |
| α-Cedrene | Cedrol | β-Eudesmol | α-Bisabolol | Valencene | α-Calacorene |
| β-Patchoulene | γ-Gurjunene | α-Curcumene | β-Selinene | γ-Gurjunene | Globulol |
| Sativene | α-Santalene | Sesquithujene | α-Zingiberene | β-Curcumene | Palustrol |
| Iso-Caryophyllene | α-Bulnesene | Aromadendrene | 6,9-Guaiadiene | Iso-Valencenol | γ-Muurolene |
| Dehydro-aromadendrene | β-cis-Farnesene | Caryophyllene oxide | α-cis-Bergamotene | α-trans-Bergamotene | Allo-aromadendrene |
| Not Terpenoidic Compounds | |||||
| 2-Heptanone | Heptanal | Hexyl hexanoate | Nonanal | Eugenol | Estragol |
| Cannabinoids | |||||
| Cannabidivarol | Cannabicitran | Cannabidiol | Cannabichromene | Cannabigerol | Δ-9-THC |
| Code | Name | Formula |
|---|---|---|
| Epilepsy (Lennox–Gastaut and Dravet syndromes) | Cannabidiol (Epidiolex®) | Phase III, Regulatory approval |
| Chronic pain | THC, nabiximols | Phase II RCTs |
| Schizophrenia | CBD | Phase II |
| Sleep disturbance | THC, nabilone, nabiximols | Phase II–III |
| Tourette syndrome | THC, cannabis | Phase II, observational studies |
| Parkinson’s disease symptoms | THC, CBD, cannabis | Observational studies |
| Post-traumatic stress disorder | Cannabis | Observational studies |
| Dementia with agitation | THC, cannabis | Observational studies |
| Social anxiety | CBD | Phase II, observational studies |