1. Introduction

Cannabis (Cannabis sativa L.) is the most controversial plant ever exploited, with considerable discrepancy in the praise and disapproval it receives. It is intriguing that cannabis produces the natural substances that appear to target key protein receptors of important physiological systems quite selectively [1]. Plants containing such secondary metabolites usually belong to unique chemotaxa that induce potent pharmacological effects and have typically been used for recreational and medicinal purposes. Cannabis sativa L. has a long history as a medicinal plant and was fundamental in the discovery of the endocannabinoid system.

cannabis, because it reduces THC collateral effects. Furthermore, minor constituents such as CBC and CBG exhibit anti-inflammatory, antibacterial and antifungal activity, while CBN has strong sedative properties [5, 7]. As regards cannabidiol (CBD)-based preparations that are becoming extremely popular as CBD has been shown to have beneficial effects on human health, a recent work highlighted a wide variability in the cannabinoid profile that justifies

Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive…

http://dx.doi.org/10.5772/intechopen.79539

57

Although CBD and THC are the key molecules, the plant itself is capable of producing only their acid counterparts: cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) [9]. Decarboxylation of these forms leads to the formation of bioactive chemical species, CBD and THC, respectively. CBDA and THCA are the major components of cannabis inflorescence while among other cannabinoid acids, cannabigerolic acid (CBGA) is shown to be essential due to the fact that it is a precursor of all the other cannabinoid acids. It is worth mentioning the other minor acidic cannabinoids such as cannabichromenic acid (CBCA) which also gives

At present, the international medical and scientific community has widely recognised Cannabis sativa L. as a promising source of therapeutic agents for the treatment of certain diseases such as multiple sclerosis, HIV, epilepsy, glaucoma, chemotherapy, chronic pain, nausea/vomiting

Unfortunately, despite the emergence of a huge amount of preclinical literature that describes the actions and effects of some cannabinoids, there have, as yet, been relatively few publications describing the effects produced by cannabinoids in clinical studies performed with human subjects. Importantly, a cannabis-based medication, Sativex®, approved by the European medical association (EMA), was recently licenced in 18 European countries for the treatment of tremor and spasticity symptoms associated with multiple sclerosis [12]. Besides, other cannabinoid drugs, Cesamet® (Nabilone) and Marinol® (synthetic tetrahydrocannabinol (THC)) were successfully applied for the treatment of vomiting and nausea caused by cancer therapy. Some other cannabis-derived substances seem to be on hold. For example, Epidolex®, an experimental drug derived from cannabis-based medicine for the treatment of child epilepsy is on the brink of becoming the first of its kind to obtain FDA government approval [13]. Capsules, cannabis extracts such as mouth spray or oils, dry cannabis for inhalation or as tea are the main medical products approved by the EU, according to the European Monitoring

Within the EU there is no agreement on the legalisation of medical cannabis, but it appears to be moving toward greater use faster than in the past [15, 16]. For the time being, only Austria, the Czech Republic, Finland, Germany, Italy, Portugal, Poland, Spain and Croatia have allowed the use of cannabis in medicine in the EU, while other countries are planning to legalise it. As a confirmation of the blurred legal status of Cannabis sativa L. within the EU community, it took a 4-year trial before the Danish Parliament approved the use of medical cannabis for patients suffering from various diseases starting from January 1, 2018. Moreover, in 2017, an increasing number of EU members, such as Greece and Ireland, announced or proposed changes in legislation and the use of medical cannabis. Since November 2017,

the need for strict and standardised regulations [8].

corresponding neutral analogues upon decarboxylation.

Center for Drugs and Drug Addiction (EMCDDA) 2017 [14].

[10, 11].

Over the past decades, considerable research has been carried out to enable a clear distinction to be made between cannabis as a hazardous drug and as a beneficial medicine [2, 3].The authorised medicinal use of cannabis is still associated with doubts on its safe use due to a few ambiguous issues including quantity, dynamics and way of administration [4].

Medications based on cannabis have been used for therapeutic purposes in many cultures for centuries. In Europe, they were used at the end of the nineteenth century to treat pain, spasms, asthma, sleep disorders, depression, and loss of appetite. In the first half of the twentieth century, cannabinoid medications fell into almost complete disuse, partly because scientists were unable to establish the chemical structure of the main cannabis plant ingredients. The emergence of interest in botanical medicinal cannabis is thought by many to be a collateral effect of the opioid abuse epidemic; public perception surrounding the use of medicinal cannabis suggests that this plant-based therapy is viewed as not very different from a botanical drug product or supplement used for health or relief of symptoms if disease persists. Like some herbal preparations or supplements, however, medicinal cannabis may similarly pose health risks associated with its use, including psychoactive, intoxicating, and impairing effects, which have not been completely elucidated through clinical trials.

The method of its application for therapeutic purposes certainly depends on its phytocannabinoid profile: over 70 cannabinoids are defined in Cannabis sativa L. They are classified chemically into 10 most important categories where the THC, cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN)-types are recognised as the most relevant [5].

The main constituent of cannabis is THC, which is responsible for the psychoactive features of cannabis due to its high affinity to cannabinoid receptors. Most of the effects of cannabis preparations are based on the agonistic action of THC on the various cannabinoid receptors. Two primary endocannabinoid receptors have been identified: CB1 and CB2 [6]. CB1 receptors are found predominantly in the brain and nervous system, as well as in peripheral organs and tissues, and are the main molecular target of the endocannabinoid binding molecule, anandamide, as well as its mimetic phytocannabinoid, THC.

Another important component is cannabidiol (CBD) which was proven to possess several pharmacological properties (analgesic, antioxidant and antiepileptic), but not psychotropic activity as THC [7]. The presence and amount of CBD is essential in the therapeutic usage of cannabis, because it reduces THC collateral effects. Furthermore, minor constituents such as CBC and CBG exhibit anti-inflammatory, antibacterial and antifungal activity, while CBN has strong sedative properties [5, 7]. As regards cannabidiol (CBD)-based preparations that are becoming extremely popular as CBD has been shown to have beneficial effects on human health, a recent work highlighted a wide variability in the cannabinoid profile that justifies the need for strict and standardised regulations [8].

1. Introduction

56 Recent Advances in Cannabinoid Research

relevant [5].

Cannabis (Cannabis sativa L.) is the most controversial plant ever exploited, with considerable discrepancy in the praise and disapproval it receives. It is intriguing that cannabis produces the natural substances that appear to target key protein receptors of important physiological systems quite selectively [1]. Plants containing such secondary metabolites usually belong to unique chemotaxa that induce potent pharmacological effects and have typically been used for recreational and medicinal purposes. Cannabis sativa L. has a long history as a medicinal plant

Over the past decades, considerable research has been carried out to enable a clear distinction to be made between cannabis as a hazardous drug and as a beneficial medicine [2, 3].The authorised medicinal use of cannabis is still associated with doubts on its safe use due to a few

Medications based on cannabis have been used for therapeutic purposes in many cultures for centuries. In Europe, they were used at the end of the nineteenth century to treat pain, spasms, asthma, sleep disorders, depression, and loss of appetite. In the first half of the twentieth century, cannabinoid medications fell into almost complete disuse, partly because scientists were unable to establish the chemical structure of the main cannabis plant ingredients. The emergence of interest in botanical medicinal cannabis is thought by many to be a collateral effect of the opioid abuse epidemic; public perception surrounding the use of medicinal cannabis suggests that this plant-based therapy is viewed as not very different from a botanical drug product or supplement used for health or relief of symptoms if disease persists. Like some herbal preparations or supplements, however, medicinal cannabis may similarly pose health risks associated with its use, including psychoactive, intoxicating, and impairing effects,

The method of its application for therapeutic purposes certainly depends on its phytocannabinoid profile: over 70 cannabinoids are defined in Cannabis sativa L. They are classified chemically into 10 most important categories where the THC, cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN)-types are recognised as the most

The main constituent of cannabis is THC, which is responsible for the psychoactive features of cannabis due to its high affinity to cannabinoid receptors. Most of the effects of cannabis preparations are based on the agonistic action of THC on the various cannabinoid receptors. Two primary endocannabinoid receptors have been identified: CB1 and CB2 [6]. CB1 receptors are found predominantly in the brain and nervous system, as well as in peripheral organs and tissues, and are the main molecular target of the endocannabinoid binding molecule, ananda-

Another important component is cannabidiol (CBD) which was proven to possess several pharmacological properties (analgesic, antioxidant and antiepileptic), but not psychotropic activity as THC [7]. The presence and amount of CBD is essential in the therapeutic usage of

and was fundamental in the discovery of the endocannabinoid system.

which have not been completely elucidated through clinical trials.

mide, as well as its mimetic phytocannabinoid, THC.

ambiguous issues including quantity, dynamics and way of administration [4].

Although CBD and THC are the key molecules, the plant itself is capable of producing only their acid counterparts: cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) [9]. Decarboxylation of these forms leads to the formation of bioactive chemical species, CBD and THC, respectively. CBDA and THCA are the major components of cannabis inflorescence while among other cannabinoid acids, cannabigerolic acid (CBGA) is shown to be essential due to the fact that it is a precursor of all the other cannabinoid acids. It is worth mentioning the other minor acidic cannabinoids such as cannabichromenic acid (CBCA) which also gives corresponding neutral analogues upon decarboxylation.

At present, the international medical and scientific community has widely recognised Cannabis sativa L. as a promising source of therapeutic agents for the treatment of certain diseases such as multiple sclerosis, HIV, epilepsy, glaucoma, chemotherapy, chronic pain, nausea/vomiting [10, 11].

Unfortunately, despite the emergence of a huge amount of preclinical literature that describes the actions and effects of some cannabinoids, there have, as yet, been relatively few publications describing the effects produced by cannabinoids in clinical studies performed with human subjects. Importantly, a cannabis-based medication, Sativex®, approved by the European medical association (EMA), was recently licenced in 18 European countries for the treatment of tremor and spasticity symptoms associated with multiple sclerosis [12]. Besides, other cannabinoid drugs, Cesamet® (Nabilone) and Marinol® (synthetic tetrahydrocannabinol (THC)) were successfully applied for the treatment of vomiting and nausea caused by cancer therapy. Some other cannabis-derived substances seem to be on hold. For example, Epidolex®, an experimental drug derived from cannabis-based medicine for the treatment of child epilepsy is on the brink of becoming the first of its kind to obtain FDA government approval [13].

Capsules, cannabis extracts such as mouth spray or oils, dry cannabis for inhalation or as tea are the main medical products approved by the EU, according to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) 2017 [14].

Within the EU there is no agreement on the legalisation of medical cannabis, but it appears to be moving toward greater use faster than in the past [15, 16]. For the time being, only Austria, the Czech Republic, Finland, Germany, Italy, Portugal, Poland, Spain and Croatia have allowed the use of cannabis in medicine in the EU, while other countries are planning to legalise it. As a confirmation of the blurred legal status of Cannabis sativa L. within the EU community, it took a 4-year trial before the Danish Parliament approved the use of medical cannabis for patients suffering from various diseases starting from January 1, 2018. Moreover, in 2017, an increasing number of EU members, such as Greece and Ireland, announced or proposed changes in legislation and the use of medical cannabis. Since November 2017, cannabis-based medicines in Poland can be sold if they are made in pharmacies with the use of an imported substance.

cannabis inflorescence is the final product. The other tactic is focused on extraction and purification procedures, which are fundamental if cannabis-derived formulations such as oils or tinctures are targeted. As recently reviewed by Citti et al. [29] and Calvi et al. [30], the choice of the analytical approach(es) employed represents a pivotal task, with particular emphasis on the need for a comprehensive chemical characterisation of the composition of cannabis and derived products. Nowadays, analytical methods based on gas chromatography-mass spectrometry (GC-MS) and/or high pressure liquid chromatography (HPLC) coupled to the recently introduced high resolution mass spectrometer HRMS-Orbitrap, represent the gold standard techniques for the investigation of the highly complex cannabis composition due to their excellent resolution, precision and sensitivity. Consequently, it is now crucial to complete the chemical and pharmacological characterisation of all phytocannabinoids known to be

Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive…

http://dx.doi.org/10.5772/intechopen.79539

59

Based on the above-mentioned considerations, in the first part of the here presented research project different analytical procedures involving the combination of headspace-solid-phase microextraction (HS-SPME) coupled to GC-MS and accelerated solvent extraction (ASE) coupled to high resolution mass-spectrometry (HPLC-Q Orbitrap®) were applied for the indepth profiling and fingerprinting of cannabinoids and terpenes in authorised medical grade varieties of Cannabis sativa L. flos (Bediol®) and in corresponding macerated oil preparation. Particular emphasis was given to the study of untargeted cannabinoids so as to investigate and obtain an exhaustive and realistic profile of medical Bediol® inflorescences and derived macerated oil preparations, since they have so far received less attention compared to target compounds (THC, THC-A, CBD, CBD-A). This approach could add new knowledge to the

All HPLC or analytical grade chemicals were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Formic acid 98–100% was from Fluka (Sigma-Aldrich, St. Louis, MO, USA). Ultrapure water was obtained through a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany). For headspace (HS) analysis, the SPME coating fibre (DVB/CAR/PDMS, 50/30 μm) was from Supelco (Bellefonte, PA, USA). Acetonitrile, 2-propanol, formic acid LC-MS grade were purchased from Carlo Erba (Milan, Italy). CBD, THC, CBN, CBG, CBNA, THCA, CBGA were purchased from Sigma Aldrich (Round Rock, Texas). High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan) was used to obtain representative ali-

Bediol® medical Cannabis chemotype that contains 6.5% THC and 8% CBD as standardised and certified by the company Bedrocan was used for all analyses. It was selected as representative

2.2. Cannabis plant material and superfine grinding (SFG) sample preparation

present in cannabis.

field of "omic" analytical applications as well.

2. Materials and methods

quots of cannabis flos samples powder.

2.1. Chemical and reagents

The current status of cannabis highlights that, since it causes "psychoactive activity," its use in medicine should follow the legal provisions of member states, including "control of the use of narcotics and psychotropic substances" [17]. European countries have an obligation to control cannabis according to the three UN Conventions on Drug Control that require them to restrict drug supplies and use it exclusively for medical and scientific purposes.

At an EU level there are no harmonised laws on the recreational and medical use of cannabis and the member states themselves decide whether to legalise them.

As an example, medical cannabis in Italy represents a multifaceted reality [16, 18]. At present varieties Bedrocan, Bediol, Bedica and Bedrolite produced by company Bedrocan from Netherlands [19] and the new strain FM2 produced by the Military Pharmaceutical Chemical Works of Florence, Italy (authorised in November 2015 with a Ministerial Decree) can be prescribed to treat a wide range of pathological conditions [16]. In relation to this, Italian galenic pharmacies are authorised to prepare precise cannabis doses for vaping, herbal teas, resins, micronised capsules and oils [20]. The latter, prepared by using European Pharmacopoeia olive oil (FU) as extraction solvent has received great attention due to the easiness with which dosage can be modulated or titrated during the treatment period. Also, oil formulations are high-steamed because of the extended bioavailability of the active compounds contained.

As regards Cannabis sativa composition, beyond and besides cannabinoids, a substantial amount of the approximately 500 compounds (terpenes, flavonoids, stilbenoids, fatty acids, alkaloids, carbohydrates, and phenols) are described [21]. Terpenes represent the volatile component of the plant and have been proven to have a synergic action with cannabinoids [19]. Cannabis plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence [22]. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties [23]. Differences between the pharmaceutical properties of different cannabis strains have been attributed to interactions (or an 'entourage effect') between cannabinoids and terpenes [24]. Terpenes themselves exhibit a wide array of pharmacological properties, including interaction with the mammalian endocannabinoid system: sesquiterpene β-caryophyllene interacts with mammalian cannabinoid receptors [25, 26]. Some terpenes like β-myrcene, limonene and linalool display anxiolytic, antibacterial, anti-inflammatory, and sedative effects, too [27].

The chemical complexity of cannabis makes its pharmaceutical standardisation challenging and must include well-defined methodologies that would characterise the plant chemotype and the herbal drug as well as extraction procedures. As a matter of fact, it was found that the concentrations of target cannabinoids obtained for the same plant chemotype originating from different suppliers varied by more than 25% [28]. This lack of standardisation could be overcome with two distinct approaches.

The first is a botanical issue and points toward strict control of varieties and strains during cultivation in order to assure the highest homogeneity in the final plants, especially if the cannabis inflorescence is the final product. The other tactic is focused on extraction and purification procedures, which are fundamental if cannabis-derived formulations such as oils or tinctures are targeted. As recently reviewed by Citti et al. [29] and Calvi et al. [30], the choice of the analytical approach(es) employed represents a pivotal task, with particular emphasis on the need for a comprehensive chemical characterisation of the composition of cannabis and derived products. Nowadays, analytical methods based on gas chromatography-mass spectrometry (GC-MS) and/or high pressure liquid chromatography (HPLC) coupled to the recently introduced high resolution mass spectrometer HRMS-Orbitrap, represent the gold standard techniques for the investigation of the highly complex cannabis composition due to their excellent resolution, precision and sensitivity. Consequently, it is now crucial to complete the chemical and pharmacological characterisation of all phytocannabinoids known to be present in cannabis.

Based on the above-mentioned considerations, in the first part of the here presented research project different analytical procedures involving the combination of headspace-solid-phase microextraction (HS-SPME) coupled to GC-MS and accelerated solvent extraction (ASE) coupled to high resolution mass-spectrometry (HPLC-Q Orbitrap®) were applied for the indepth profiling and fingerprinting of cannabinoids and terpenes in authorised medical grade varieties of Cannabis sativa L. flos (Bediol®) and in corresponding macerated oil preparation. Particular emphasis was given to the study of untargeted cannabinoids so as to investigate and obtain an exhaustive and realistic profile of medical Bediol® inflorescences and derived macerated oil preparations, since they have so far received less attention compared to target compounds (THC, THC-A, CBD, CBD-A). This approach could add new knowledge to the field of "omic" analytical applications as well.
