**4. Non-cannabinoid phytochemicals of Cannabis: terpenes, flavonoids and anthocyanins**

double-blinded parallel-group clinical trial examined the efficacy of CBD co-treatment with the patient's existing antipsychotic medication on a range of endpoints, including positive, negative and cognitive scores and Clinical Global Impression scales (CGI, measuring illness severity, improvement and response to treatment) [32]. Results showed significant improvements in positive (not negative) symptoms and CGI scores, as well as some improvement in cognitive performance (did not reach statistical significance, *p* = 0.068 CBD vs. placebo) when

There is substantial scientific evidence to show the beneficial effects of CBD in the brain, with protection and treatment efficacy for various cognitive behaviours conferred in multiple disease states. Overall, there seems to be a general requirement for further placebocontrolled clinical trials, as well as investigation of long-term efficacy and safety in different populations of people. Evidence for illness-specific optimal dosing regimens (dose, route of administration, timing and number of daily doses, effect of concurrent medications, etc.) is also required. In addition, similar to our rodent study of CBD effects on cognition in schizophrenia [31], most studies use either isolated CBD or combined THC and CBD. While this methodology enables investigators to attribute results to a specific compound, it may not be the optimal therapeutic approach as cannabis-derived plant molecules are thought to interact and produce a synergy that enhances therapeutic effects—termed the 'entou-

The entourage effect is defined as the act by which compounds (both cannabis phytochemicals and compounds from the endogenous cannabinoid system) augment or support the effects of major cannabinoids, for example, Δ9-THC, CBD, 2-arachidonoyl-glycerol (2-AG) [33, 34]. This phenomenon has been likened to an orchestra where 'many musicians support and harmonise the melody provided by the soloists' [34]. Compounds can exert synergistic effects through several mechanisms, for example by interacting with each other to improve bioavailability of beneficial compounds, or through combined actions on different therapeutic

The concept of a cannabis entourage effect is largely based on anecdotal evidence from medicinal and recreational users attesting to the notion that cannabis 'works better' as a whole plant extract and its existence has been argued back and forth over time. However, there is evidence to suggest that the cannabis plant contains active ingredients as well as 'synergists' that boost drug effects above that of the isolated compound. Indeed, early description of a potential synergy between molecules in the cannabis plant came from a study in the 1970s that reported a 2–4 times greater deficits in parameters such as processing tasks and motor function in subjects administered Brazilian cannabis samples compared to Δ9-THC [36]. The phrase 'entourage effect' was first described in 1998 in response to the finding that certain

CBD was combined with the patient's existing antipsychotic medications [32].

**2.3. Conclusions on the use of CBD in neurological disease**

rage effect'.

targets [35].

**3. The entourage effect**

88 Recent Advances in Cannabinoid Research

The cannabis plant contains hundreds of phytochemicals, with new compounds and metabolites frequently identified. The concentration of chemicals in a cannabis plant can be influenced by multiple factors including nutrition, humidity, temperature, age of plant, strain, harvest time, plant stress, organ and storage conditions [1, 40]. Therefore, plant phytochemical composition is highly variable. Variability identified even within the same strain has led some authors to conclude that the name of a plant strain does not necessarily indicate potency or chemical composition [41]. However, others found that when grown under standardised conditions, certain cannabis strains can provide reproducible terpene and phytocannabinoid profiles that have been considered chemotaxonomic markers [42]. Furthermore, cannabinoid content can be used to classify plants into chemovars (plants with distinct photochemical profiles): Type I Δ9-THC-dominant, Type II Δ9-THC and CBD, Type III CBD-dominant and distinctions can be made outside these classes based on specific terpene profiles [43]. Therefore, it is possible to optimise plants to reproduce a distinct chemical composition and, potentially, specific medicinal characteristics.

#### **4.1. Terpenes: linalool, alpha-pinene and beta-caryophyllene**

Terpenes have been described as the most abundant class of small natural molecules by mass on Earth, undertaking innumerable structural and functional roles in most life forms on the planet (e.g., cholesterols for structural and signalling components of cell membranes, retinal in the eye for vision, carotenoids in photosynthesis) [44]. In cannabis, they create fragrances and flavours, but are also found in other plants and commonly used as safe food additives [38]. Terpenes can cross the blood brain barrier due to their lipophilic nature and studies have demonstrated a range of health benefits for some terpenes found in cannabis.

#### *4.1.1. Linalool*

Linalool is a monoterpene abundant in aromatic plants, such as lavender and purple basil [45]. Evidence shows that chronic administration of linalool reverses deficits in spatial memory and learning, with reduced amyloid plaque deposition and tau dysfunction in the hippocampus in rodent models of Alzheimer's disease [46, 47], using 25 mg/kg and 100 mg/ kg linalool, respectively. Linalool also prevented deficits in spatial memory, motor function, neuroinflammation and post-ischemic neurodegeneration in a rat model of global cerebral ischemia, following oral daily administration (25 mg/kg) for 1 month [48]. However, reduced short and long-term recognition memory (50 and 100 mg/kg linalool, i.p.) [49] and memory acquisition (3% preparation for inhalation) [50] were found when linalool was administered as a single dose to healthy rats. This apparent contradiction in findings could be attributed to the administration of linalool to healthy vs. cognitively impaired rats, suggesting that the compound exerts benefits in a disease state but is detrimental when not patho physiologically required; however, further investigation is necessary to confirm.

Neurological scores were improved in mice administered β-caryophyllene (24 and 72 mg/ kg, i.p.) following an induced stroke [62] and anti-depressant-like behaviour was reported in healthy mice following β-caryophyllene, through mechanisms involving catecholamine (adrenergic) neurotransmission [63]. Overall, the studies provide some evidence to support the role of β-caryophyllene as pro-cognitive, with anti-inflammatory, neuroprotective and

The United Chemicals of Cannabis: Beneficial Effects of Cannabis Phytochemicals on the Brain and Cognition

http://dx.doi.org/10.5772/intechopen.79266

91

In addition to terpenes, cannabis plants contain phenolic compounds, including flavonoids and anthocyanins [40, 64–66]. Flavonoids are commonly consumed by humans through dietary fruit, vegetable, tea and wine intake. Anthocyanins are a group of flavonoids responsible for the blue-violet and red-orange colours of plant organs. Certain strains of cannabis plants exhibit a purple phenotype (**Figure 1**), which is widely attributed to anthocyanin content in recreational cannabis culture; however, experimental data showing anthocyanin levels

Flavonoids and anthocyanins are extensively researched due to their neuroprotective, antiinflammatory and pro-cognitive characteristics and can pass the blood brain barrier [67]. For example, one study found that anthocyanin pre-treatment (200 mg/kg orally for 7 days) prevented cognitive deficits in a rat model of dementia [68]. Flavonoids improve working memory, processing speed, executive function and episodic memory in humans (reviewed in [69, 70]) and stimulate neurogenesis, synaptic plasticity and reduced neuroinflammation in the hippocampus (reviewed in [71]). Anthocyanin-rich cherry juice improved verbal fluency and short- and long-term memory performance in people with mild-to-moderate dementia during a 12 week randomised, controlled clinical trial of older people (+70 years) with mild to moderate dementia (200 ml/day cherry juice vs. control juice lacking anthocyanin) [72]. Interestingly, both cherries and cannabis plants contain phenolic acids related to flavonoid and anthocyanin biosynthesis pathways [65, 73]. Indeed, hemp seed extract can contain phenolic compound levels that are comparable to Japanese plums [74, 75]. Japanese plums are an important source of

**Figure 1.** Inflorescence of purple cannabidiol (CBD)-rich, low Δ9-tetrahydrocannabinol (Δ9-THC) medicinal cannabis

cultivar, GHM Genetic Development, Amsterdam, The Netherlands (2018).

anti-depressant effects.

**4.2. Phenolic acids: flavonoids and anthocyanins**

of purple compared to non-purple strains appear to be lacking.

#### *4.1.2. Alpha-pinene*

Alpha-pinene (α-pinene) is a highly abundant monoterpene found in coniferous trees (e.g., pine and fir) and cannabis [51] that, according to cannabis culture, provides pine-needle fragrances and tastes to cannabis. In mice with cognitive deficits caused by scopolamine-induced blockade of acetylcholine neurotransmission (apparent in advanced stages of Alzheimer's disease [52]), α-pinene (10 mg/kg, i.p.) improved working and spatial memory, and increased markers of acetylcholine synthesis in the cortex [53]. Inhalation of α-pinene can also influence major neurotransmitter signalling in the brain, for example it improved quality and duration of sleep in mice by modulating the major inhibitory neurotransmitter signalling system, gamma-aminobutyric acid (γ-aminobutyric acid, GABA)) [54], and decreased anxiety-like behaviour that was associated with increased tyrosine hydroxylase (the rate limiting enzyme for dopamine synthesis) in the midbrain [55]. Another study reported significant improvements in avoidance memory of cognitively impaired mice following administration of an essential oil obtained from a Korean fir tree containing α-pinene [56]; however, the results cannot be entirely attributed to this terpene due to the use of whole-plant extract containing other constituents.

#### *4.1.3. Beta-caryophyllene*

Beta-caryophyllene (β-caryophyllene) is a sesquiterpene that has a weak woody-spicy characteristic, abundant in cloves, black pepper, cinnamon and thyme [57, 58]. In a mouse model of Alzheimer's disease, β-caryophyllene reversed spatial memory deficits, reduced β-amyloid deposition in the hippocampus and cortex, and reduced neuroinflammation when administered for 10 weeks (48 mg/kg, oral) [59]. In rats with chronic cerebral ischemia resembling vascular dementia, β-caryophyllene (administered in a hydroxypropyl-β-cyclodextrin inclusion complex delivery system to enhance its bioavailability) attenuated cognitive deficits and increased cerebral blood flow [60]. β-caryophyllene also prevented oxidative stress in the cortex of rats following transient global cerebral hypoperfusion/reperfusion [61]. Neurological scores were improved in mice administered β-caryophyllene (24 and 72 mg/ kg, i.p.) following an induced stroke [62] and anti-depressant-like behaviour was reported in healthy mice following β-caryophyllene, through mechanisms involving catecholamine (adrenergic) neurotransmission [63]. Overall, the studies provide some evidence to support the role of β-caryophyllene as pro-cognitive, with anti-inflammatory, neuroprotective and anti-depressant effects.

#### **4.2. Phenolic acids: flavonoids and anthocyanins**

*4.1.1. Linalool*

90 Recent Advances in Cannabinoid Research

*4.1.2. Alpha-pinene*

other constituents.

*4.1.3. Beta-caryophyllene*

Linalool is a monoterpene abundant in aromatic plants, such as lavender and purple basil [45]. Evidence shows that chronic administration of linalool reverses deficits in spatial memory and learning, with reduced amyloid plaque deposition and tau dysfunction in the hippocampus in rodent models of Alzheimer's disease [46, 47], using 25 mg/kg and 100 mg/ kg linalool, respectively. Linalool also prevented deficits in spatial memory, motor function, neuroinflammation and post-ischemic neurodegeneration in a rat model of global cerebral ischemia, following oral daily administration (25 mg/kg) for 1 month [48]. However, reduced short and long-term recognition memory (50 and 100 mg/kg linalool, i.p.) [49] and memory acquisition (3% preparation for inhalation) [50] were found when linalool was administered as a single dose to healthy rats. This apparent contradiction in findings could be attributed to the administration of linalool to healthy vs. cognitively impaired rats, suggesting that the compound exerts benefits in a disease state but is detrimental when not patho physiologically

Alpha-pinene (α-pinene) is a highly abundant monoterpene found in coniferous trees (e.g., pine and fir) and cannabis [51] that, according to cannabis culture, provides pine-needle fragrances and tastes to cannabis. In mice with cognitive deficits caused by scopolamine-induced blockade of acetylcholine neurotransmission (apparent in advanced stages of Alzheimer's disease [52]), α-pinene (10 mg/kg, i.p.) improved working and spatial memory, and increased markers of acetylcholine synthesis in the cortex [53]. Inhalation of α-pinene can also influence major neurotransmitter signalling in the brain, for example it improved quality and duration of sleep in mice by modulating the major inhibitory neurotransmitter signalling system, gamma-aminobutyric acid (γ-aminobutyric acid, GABA)) [54], and decreased anxiety-like behaviour that was associated with increased tyrosine hydroxylase (the rate limiting enzyme for dopamine synthesis) in the midbrain [55]. Another study reported significant improvements in avoidance memory of cognitively impaired mice following administration of an essential oil obtained from a Korean fir tree containing α-pinene [56]; however, the results cannot be entirely attributed to this terpene due to the use of whole-plant extract containing

Beta-caryophyllene (β-caryophyllene) is a sesquiterpene that has a weak woody-spicy characteristic, abundant in cloves, black pepper, cinnamon and thyme [57, 58]. In a mouse model of Alzheimer's disease, β-caryophyllene reversed spatial memory deficits, reduced β-amyloid deposition in the hippocampus and cortex, and reduced neuroinflammation when administered for 10 weeks (48 mg/kg, oral) [59]. In rats with chronic cerebral ischemia resembling vascular dementia, β-caryophyllene (administered in a hydroxypropyl-β-cyclodextrin inclusion complex delivery system to enhance its bioavailability) attenuated cognitive deficits and increased cerebral blood flow [60]. β-caryophyllene also prevented oxidative stress in the cortex of rats following transient global cerebral hypoperfusion/reperfusion [61].

required; however, further investigation is necessary to confirm.

In addition to terpenes, cannabis plants contain phenolic compounds, including flavonoids and anthocyanins [40, 64–66]. Flavonoids are commonly consumed by humans through dietary fruit, vegetable, tea and wine intake. Anthocyanins are a group of flavonoids responsible for the blue-violet and red-orange colours of plant organs. Certain strains of cannabis plants exhibit a purple phenotype (**Figure 1**), which is widely attributed to anthocyanin content in recreational cannabis culture; however, experimental data showing anthocyanin levels of purple compared to non-purple strains appear to be lacking.

Flavonoids and anthocyanins are extensively researched due to their neuroprotective, antiinflammatory and pro-cognitive characteristics and can pass the blood brain barrier [67]. For example, one study found that anthocyanin pre-treatment (200 mg/kg orally for 7 days) prevented cognitive deficits in a rat model of dementia [68]. Flavonoids improve working memory, processing speed, executive function and episodic memory in humans (reviewed in [69, 70]) and stimulate neurogenesis, synaptic plasticity and reduced neuroinflammation in the hippocampus (reviewed in [71]). Anthocyanin-rich cherry juice improved verbal fluency and short- and long-term memory performance in people with mild-to-moderate dementia during a 12 week randomised, controlled clinical trial of older people (+70 years) with mild to moderate dementia (200 ml/day cherry juice vs. control juice lacking anthocyanin) [72]. Interestingly, both cherries and cannabis plants contain phenolic acids related to flavonoid and anthocyanin biosynthesis pathways [65, 73]. Indeed, hemp seed extract can contain phenolic compound levels that are comparable to Japanese plums [74, 75]. Japanese plums are an important source of

**Figure 1.** Inflorescence of purple cannabidiol (CBD)-rich, low Δ9-tetrahydrocannabinol (Δ9-THC) medicinal cannabis cultivar, GHM Genetic Development, Amsterdam, The Netherlands (2018).

anthocyanins, with particularly high levels in darker purple, blue and black coloured fruits [75]. Similar to cannabis plants, the phytochemical profile of Japanese plum varieties is influenced by horticultural practices, processing and storage conditions [75]. Other commercial plants, such as violet cauliflower and Thai purple basil, gain their unusual purple colouring through modifications to anthocyanin regulatory genes [76, 77]. Therefore, it is possible that plants can be manipulated naturally and artificially (i.e., genetically) to maximise anthocyanin content.

pro-cognitive effects of CBD in a rodent model of schizophrenia. Thanks to Mr. Heiko Hampsink, GHM Genetic Development, The Netherlands, for supplying the purple cannabis photo. I extend gratitude to Mr. Thomas Forrest (Indicated Technology, Australia) and Mr.

The United Chemicals of Cannabis: Beneficial Effects of Cannabis Phytochemicals on the Brain and Cognition

http://dx.doi.org/10.5772/intechopen.79266

93

Heiko Hampsink for generously sharing their knowledge of cannabis horticulture.

I dedicate this book chapter to my husband, M. Green, for his tireless support.

1 Neuropharmacology and Molecular Psychiatry Research Laboratory, School of Medicine,

2 Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong,

[1] Citti C, Pacchetti B, Vandelli MA, Forni F, Cannazza G. Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA). Journal of Pharmaceutical and Biomedical Analysis. 2018;**149**:532-540. DOI:

[2] ElSohly MA, Gul W. Constituents of Cannabis. In: Pertwee R, editor. Handbook of Cannabis. Oxford, UK: Oxford University Press; 2014. DOI: 10.1093/acprof:oso/97801

[3] Laprairie RB, Bagher AM, Kelly ME, Denovan-Wright EM. Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor. British Journal of Pharmacology.

Faculty of Science, Medicine and Health, University of Wollongong, NSW, Australia

3 Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia

4 Australian Centre for Cannabinoid Clinical and Research Excellence, New Lambton

\*Address all correspondence to: katrina\_green@uow.edu.au

**Conflict of interest**

**Author details**

NSW, Australia

**References**

Heights, NSW, Australia

10.1016/j.jpba.2017.11.044

2015;**172**(20):4790-4805. DOI: 10.1111/bph.13250

99662685.001.0001

Katrina Weston-Green1,2,3,4\*

There are no conflicts of interest to declare.

**Notes/Thanks/Other declarations**

#### **4.3. Conclusions on the effects of terpenes and flavonoids on the brain**

The terpenes linalool, α-pinene and β-caryophyllene, as well as flavonoids and anthocyanins confer pro-cognitive, neuroprotective and anti-inflammatory effects in models of cerebral ischemia and Alzheimer's disease, as well as some anxiolytic effects. Most studies have been conducted in pre-clinical (rodent) models; however, pro-cognitive effects of flavonoids and anthocyanins have been shown in human clinical studies of dementia. Overall, combinations of CBD with other key phytochemicals found in cannabis could confer benefits on brain health through a multi-target synergy (entourage effect); however, further research is required.
