The Medicinal Natural Products of Cannabis sativa Linn.: A Review

Cannabis sativa is known among many cultures for its medicinal potential. Its complexity contributes to the historical application of various parts of the plant in ethno-medicines and pharmacotherapy. C. sativa has been used for the treatment of rheumatism, epilepsy, asthma, skin burns, pain, the management of sexually transmitted diseases, difficulties during child labor, postpartum hemorrhage, and gastrointestinal activity. However, the use of C. sativa is still limited, and it is illegal in most countries. Thus, this review aims to highlight the biological potential of the plant parts, as well as the techniques for the extraction, isolation, and characterization of C. sativa compounds. The plant produces a unique class of terpenophenolic compounds, called cannabinoids, as well as non-cannabinoid compounds. The exhaustive profiling of bioactive compounds and the chemical characterization and analysis of C. sativa compounds, which modern research has not yet fully achieved, is needed for the consistency, standardization, and the justified application of Cannabis sativa products for therapeutic purposes. Studies on the clinical relevance and applications of cannabinoids and non-cannabinoid phenols in the prevention and treatment of life-threatening diseases is indeed significant. Furthermore, psychoactive cannabinoids, when chemically standardized and administered under medical supervision, can be the legal answer to the use of C. sativa.


Introduction
The applications of plants as medicines predates human history. A medicinal plant refers to any plant which contains substances of therapeutic potential in one or more of its parts for the synthesis of plant-based drugs [1]. Active medicinal plant ingredients are referred to as bioactive phytochemicals. [2]. These bioactive compounds are believed to increase the ability of plants to survive or adapt to their surroundings [3] and are used as medicines, flavorings, and recreational drugs in humans. One notable medicinal plant that has continued to garner attention over the years, and in recent times, is Cannabis sativa.
Cannabis sativa L. is known for its medicinal uses since ancient times, because of its rich supply of phytochemicals [4], hence the quest for harnessing its pharmacological potential by scientists. The term "Cannabis" is used to define the products (drugs and essential oils) that are prepared or obtained from the annual herb C. sativa and its variants, which are of the family Cannabaceae [5]. The utilization of this multipurpose plant has been restrained for a long time because of the psychoactive effects of a specific cannabinoid Despite the influx of chemical-based medicines for treatments, the relevance of medicinal plants in drug development cannot be overemphasized. In recent years, commercial medicinal cannabis products with several variations in the phytocannabinoid content have been licensed and produced in Canada [7,8] and in several other countries. Several synthetic and standardized products are currently available on the market; however, patients' preferences lean towards herbal preparations, because they are easy to handle and selfadminister [7]. Thus, this review intends to highlight the phytochemicals present in the different plant parts, which potentiates their pharmacological activities, as well as the techniques for the extraction, isolation, and characterization of C. sativa compounds.

Methods
Literature on the published works of Cannabis sativa was obtained using electronic search engines, such as Google Scholar, the WSU online database (PubChem), and Science Direct. The keywords included, namely, Cannabis sativa, medicinal plants, Cannabis phytochemicals, ethnopharmacology bioactivity, and medicinal potentials, were used to source for data. An extensive review of the literature from 2011 through to 2021 (the last ten years) on Cannabis sativa L. was used to summarize its medicinal potential. Conversely, an emphasis will be placed on the isolation and characterization techniques from 1970 to 2021 to have a broadened view of the advancements in analytical techniques over the past years. Overall, twenty-nine (29) papers relating to the areas of our focus were chosen and were reviewed by all authors. The results from the search were carefully sorted, based on a general understanding, the review questions, and the related objectives.

Origin and Botanical Description of C. sativa
The genus name Cannabis means "cane-like" while sativa means "sown", which signifies that the plant is propagated from the seed and not from the roots [12]. It is believed to have originated in Asia and occurs widely in Africa [12,13]. Central and south-east Asia are the potential natural origins for the domestication of the Cannabis genus [14] and it is known by different common names in different languages (hemp, marihuana, kannabis sativa, ganja, bhang, and al-bhango) [15]. In South Africa, it is colloquially known, in Afrikaans, as "dagga"; in IsiXhoxa as "umfincafincane"; and in Isizulu as "umunyane" [16,17]. Taxonomically, Carl Linnaeus, a Swedish botanist, was the first to coin the name Cannabis sativa [18]. Other botanists stated that different types of Cannabis existed based on their size, shape, and resin content (breeding and selection). This review discusses, in particular, C. sativa.
The Cannabis phenotype (its observable traits or characteristics, such as its leaf shape and flower color) is based on two main factors: its genetic code (genotype) and the external environmental factors [19].
The roots are branched and are about 30-60 cm deep (Farag and Kayser, 2017) [12]. Cannabis inflorescence is made up of several flower heads found on long leafy stems from each leaf axil. A single brownish fruit, about 2-5mm long, is produced per flower, and it contains a single seed tightly covered with a hard shell [12]. The fruit is propagated by bird and the seed germinates after 8-12 days [18]. The leaves, bracts, and stems of the plant are rich in trichrome, which are a diverse set of structures containing the secondary metabolites (phytocannabinoids and terpenoids) responsible for the defense, plant interactions, and typical smell [18]. Figure 1, below, shows the plant parts of C. sativa.

Chemical Profile of C. sativa
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, Δ 9 -tetrahydrocannabinol (Δ 9 -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 (Δ 9 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]. Table 1 below summarizes the classes of compounds isolated from Cannabis sativa and the different plant parts in which they are present. 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, ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and cannabidiol (CBD) in particular; hence, the female flower top is only harvested, while other parts of the plant are discarded [11].

Extraction, Isolation, and Chemical Characterization of C. sativa
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].

Biological Evaluation/Potentials of C. sativa
From the biological point of view, the psychoactive cannabinoids reported include ∆ 9 THC, cannabinol (CBN), and cannabinodiol (CBND), while cannabidiol (CBD) and other cannabinoids are non-psychoactive [8,11]. THC is the major psychoactive component and the toxicity of this metabolite of Cannabis is the most studied [11,28]. Its psychoactive component decreases in the order of inflorescence (the flower), leaves, stem, roots, and seeds, respectively [8]. The interest in the potential medical use of cannabis and cannabinoids rose significantly in the 1990s, following the discovery of the endocannabinoid (eCB) system in mammals [7]. The physiological effects of cannabinoids are exerted through various receptors, such as the cannabinoid receptors (CB1 and CB2), adrenergic receptors, and the recently discovered GPCRs (GPR 55 , GPR 3 and GPR 5 ) [8]. Historically, each part of the Cannabis plant is indicated mostly for pain killing, inflammation, and for mental illnesses. For example, the Cannabis root has been recommended for treating fever, inflammation, gout, arthritis, and joint pain, as well as skin burns, hard tumors, postpartum hemorrhage, difficult child labor, sexually transmitted diseases, gastrointestinal activity, and infections [40]. Cannabis has also been used to treat asthma, epilepsy, fatigue, glaucoma, insomnia, nausea, pain, and rheumatism, as well as being used as appetite stimulant and a digestive aid [7,11,13]. Since concentrations above 0.05% are pharmacologically interesting, Cannabis inflorescence and leaf material may contain sufficient cannabinoids, mono-and sesquiterpenoids, and flavonoids for therapeutic applications [11]. Cannabis terpenoids and flavonoids, mainly myrcene, limonene, pinene, β-caryophyllene, and cannflavin A, act in synergy with cannabinoids to induce pharmacological effects [7]. It was proven that these compounds, which are synthetized in the aerial parts of the plant, enhance CBD's anti-inflammatory effects and antagonize THC dysphoric action [96]. Cannabidiol (CBD) and Cannabidavarin (CBDV) (neutral cannabinoids) have been reported to have the therapeutic potential for the treatment of epilepsy (focal seizures), as well as treating nausea and vomiting [97,98]. Conversely, THC and CBN have been found to be active in lowering intraocular pressure, and can be applied in all cases of glaucoma that are resistant to other therapies [9]. Cannflavin A and B are also notable flavonoids (prenylflavonoids) with medicinal potentials, such as their anti-inflammatory, anti-neoplastic, antioxidant, neuro-protective, anti-parasitic, and anti-viral effects [99]. Table 3, below, shows a summary of the reported bioactivities (biological potentials) of the bioactive compounds present in Cannabis sativa. Table 3. Summary of reported bioactivities associated with isolated compounds and essential oils from Cannabis sativa.
In the current COVID-19 pandemic, scientists are repurposing medicines (identifying new therapeutic use(s) of existing drugs) known for their biological potential (anti-viral or anti-inflammatory properties) to tackle the global issue and similar future viruses [140]. They have hypothesized that CBD could be used as an anti-viral agent [141] or antiinflammatory [142,143] tool, or to inhibit pulmonary fibrosis in COVID-19 patients [144]. In addition, the known growing evidence of the anxiolytic effects of CBD have also been hypothesized to be used as a therapeutic option to treat long-lasting COVID-19-related anxiety and PTSD [145], which is likely to be a significant issue of the pandemic.

Conclusions
With the recent evaluation, acceptance, and legalization of Cannabis products for therapeutic purposes, researchers, particularly in the field of natural products, are challenged to improve and standardize the extraction and characterization of the bioactive compounds from Cannabis sativa. Despite various reports of its economic and therapeutic values, it is legal in a handful of jurisdictions (Uruguay, Canada, some US states, and parts of Africa). Presently, Cannabis remains illegal in several countries. This review summarized the biological potential and the techniques for the extraction, isolation, and characterization of Cannabis sativa compounds, and it describes the effectiveness of the various parts of the herb in pharmacotherapy. The usage of C. sativa roots and stem barks in present-day medical research, and the development of new Cannabis-based medicines or products, in contrast to the flowering part only, is highly recommended because they can be exploited for medicine and other uses. In addition, Cannabis-based pharmaceutical products must undergo long purification processes to eliminate unwanted components such as chlorophyll and residual organic solvents. The use of standardized reagents is also very crucial in the analytical studies of C. sativa. Furthermore, future research should seek to clarify the factors responsible for the complexity of C. sativa extracts in terms of their chemical compositions, the physical properties of their active ingredients, and their liability to photochemical oxidation.