Cannabis, as a herbal medicine, is a complex mixture of compounds, including cannabinoid phenols, non-cannabinoid phenols (stilbenoids, lignans, spiro-indans, and dihydrophenanthrenes), flavonoids, terpenoids, alcohols, aldehydes, n-alkanes, wax esters, steroids, and alkaloids [6,8,11]. Over 500 chemical compounds have been isolated from the cannabis plant and have been reported [13]. The several classes of secondary metabolites are present in different parts of the plant with a wide range of applications (nutraceuticals, cosmetics, aromatherapy, and pharmacotherapy) that are beneficial for humans. However, previous studies have focused mainly on the cannabinoids, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) in particular; hence, the female flower top is only harvested, while other parts of the plant are discarded [11].

Cannabinoids are a class of terpenophenolic compounds obtained by the alkylation of an alkyl-resorcinol with a monoterpene unit [20,21]. They feature alkyl resorcinol and monoterpene moieties in their molecules [20,22]. This specific chemical class in Cannabis is present in the glandular trichomes, which are abundant in the female flower as phytocannabinoid acids, and in the vegetable matrix as neutral phytocannabinoids [6,13]. They are biosynthesized by the alkylation of olivetolic acid with geranyl-pyrophosphate by a prenyltransferase to produce cannabigerolic acid (CBGA). Decarboxylation, a chemical reaction, converts the acidic forms (Δ⁹ THCA, CBDA, CBCA, and CBGA) into their neutral forms, which are more active and efficient in terms of pharmacological activity [8,23]. To date, 125 cannabinoids have been identified and reported, in addition to five new cannabinoids reported in the past two years, 42 non-cannabinoid phenolics, 34 flavonoids, 120 terpenoids, 3 sterols, and 2 alkaloids [8,11,13]. Terpenoids are the second largest class of cannabis compounds and are responsible for their characteristic aroma [13]. 

Figure 2, Figure 3 and Figure 4 below show the structures of the different classes of bioactive compounds isolated from Cannabis sativa [13,29,41].

Many methods have been reported for the extraction of Cannabis in the literature. These include direct maceration (DM), soxhlex extraction, ultrasound-assisted extraction (UAE), supercritical fluid extraction, and microwave-assisted extraction (MAE) [41]. However, two methods of extracting Cannabis are differentiated in the literature [41]. The first is the maceration of the plant material in an organic solvent (direct maceration) and the subsequent removal of the solvent by the concentration of the extract under reduced pressure [41]. The second is the innovative supercritical fluid extraction (SFE) method, which involves the use of pressurized solvents [41]. It is necessary for cannabinoid compounds to be extracted with organic solvents instead of water, because the active compounds are less soluble in polar solvents [41]. The most commonly used solvents are ethanol, ether, chloroform, and methanol [42]. When used for extraction, various compounds, including some undesired substances, dissolve together with the cannabinoids [42]. The high solvent power of ethanol for cannabinoid compounds is the reason why it is frequently used in home-made extracts of Cannabis [41]. However, non-desired compounds (chlorophyll, lipids, and waxy materials) are also extracted which, therefore, requires further steps to remove the co-extracted impurities for a high-purity medicinal product to be obtained [41]. A patent on the method for the isolation of herbal and cannabinoid medicinal extracts stated that the solubility of non-therapeutic substances (chlorophyll and waxy materials) is reduced when the solvent is selected from a group that includes acetonitrile, benzene, dichloromethane, diethyl ether, acetone, butanol, ethanol, chloroform, ethyl acetate, hexane, pentane, propanol, tetrahydrofuran, toluene, xylene, and various combinations of these solvents [41]. The International Conference on Harmonization (ICH) recommends the use of less toxic solvents in the manufacture of drug substances and dosage forms, and sets pharmaceutical limits for residual solvents in drug products [43]. Residual solvents pose risks to human health and are classified into three classes. Class 1 solvents (including carbon tetrachloride, benzene, and methyl chloroform) are regarded as human carcinogens and are environmentally hazardous [41]. Class 2 solvents include methanol and hexane, which are generally said to be limited, and they are possible causative agents of irreversible toxicity, such as neurotoxicity or teratogenicity [41]. Class 3 solvents (ethanol and ethyl acetate) are generally regarded as having a low toxic potential to humans [41]). Above all, ethanol is generally recognized as a safe (GRAS) solvent [41]. In a study by Brighenti et al., they compared the following four extraction techniques to obtain a high yield of medicinal cannabinoids: ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and direct maceration (DM). They concluded that DM, with ethanol as the extraction solvent at room temperature for an overall time of 45 min, is the best extraction technique (in terms of a high yield) for non-psychoactive cannabinoids from hemp [44].

Over the last decade, compounds in Cannabis have been identified, isolated, and determined by various chromatographic techniques with different spectroscopic detection methods. C. sativa samples are analyzed for both legal and medicinal purposes [41]. Nevertheless, the knowledge of their exact composition remains very significant. In 2009, recommended methods for the identification and analysis of cannabis products were released by the United Nations Office on Drugs and Crime [45]. One notable technique that has been employed in identifying the diverse composition of the compounds found is high-performance liquid chromatography (HPLC) [41]. Spectroscopic approaches or methods are based on the variable absorbance or redirection of electromagnetic (EM) radiation by chemical bonds, resulting in the radiation or transition of the sample’s atoms to a higher energy state [46]. Some advantages are attributed to these spectroscopic methods, such as permitting spatial measurements of metabolites and offering a global metabolic fingerprint of a sample with rapid spectral acquisition [46]. Some of these approaches/methods include Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), HPLC, gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–mass spectrometry (LS–MS) [41,46]. Taking into account the recommended methods and the mandatory requirement of the Ministerial decree to use only chromatographic techniques coupled with mass spectrometric detection, cannabinoid concentrations and its stability in cannabis tea and cannabis oil, prepared from standardized flowering tops obtained from the Military Pharmaceutical Chemical Works of Florence, were studied by Pacifici et al. using easy and fast ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) [9,10]. 

2.3. Biological Evaluation/Potentials of C. sativa