Evaluating the Metabolomic Profile and Anti-Pathogenic Properties of Cannabis Species

2.1. Search Strategies

A systematic literature search was carried out by consulting five electronic scientific databases: PubMed, Scopus, Science Direct, Semantic Scholar, and Google Scholar. A combination of the following keywords was used: “Cannabis”, “metabolomic profile” “phytocannabinoids”, “antimicrobial properties”, antiviral properties”, “antibacterial properties”, and “antifungal properties”. The publication year was used as a restriction to the retrieved articles from 2010 through April 2023 and included journal articles, review papers, and research reports published in English only.

2.2. Selection Criteria

The selection criteria were based on the PRISMA statement [28]. The search mainly focused on the original research papers published from 2010 until April 2023 papers that evaluate the analysis of Cannabis plant materials and bioactive compounds as the subject of interest. There were no limits for Cannabis plants: (herbal form—the leaves, flowering tops, and resin form—hashish, or hash oil), varieties (indica, sativa, or ruderalis), and gender (male, female, or monoecious). The search span was from the years 2010 to 2023. All articles before 2010 were excluded from the search.

3.2. Application of Metabolomics in Cannabis (Cannabinomics)

According to [46,47,48], nuclear magnetic resonance and mass spectrometry MS-based [49,50,51,52] analyzers are the main analytical platforms used in metabolomics analysis. In addition, analyzers equipped with triple quadrupole (QQQ) detectors, such as LC/QQQ/MS and GC/QQQ/MS systems, are crucial in Cannabis research due to their superior selectivity and sensitivity in quantitative analyses [53,54]. However, in the case of Cannabis-derived matrices reported to have high complex metabolome, employing various analysis tools is highly recommended as reported by [5]. The use and exploitation of Cannabis have sparked controversy; however, the recent legalization of its use for medical and other purposes in many countries within the corresponding legislative framework [1,55], in combination with the remarkable bioactivities of the plant, pose an urge for the acceleration and intensification of Cannabis research and development. Studies by [55,56] revealed that the application of cannabinomics in mapping the metabolome could assist a great deal in Cannabis research and development. More studies in cannabinomics will also aid in understanding the growth and development of Cannabis and the factors (light, water requirements, and growing conditions) that can contribute to a greater yield of Cannabis. Simultaneously, the abovementioned is likely to have a significant impact on drug discovery, medicine, food science, functional cosmetics research, and metabolic engineering of microorganisms for the biosynthesis of cannabinoids as reported by [57]. Although most studies on Cannabis have been on flowers and oils, there has been an interest in researching the level of cannabinoids and terpenoids in edibles, Cannabis sweets, medicine, and cosmetics as indicated by [58,59].

3.3. Cannabis Active Compounds

It is reported that Cannabis is one of the medicinal plants that possess a high metabolomic profile with a diverse range of essential bioactive compounds used for numerous pharmacological purposes [60]. For instance, a previous study by Fischedick et al. [61] revealed that more than 600 compounds have been discovered, with 180 belonging to the cannabinoids family and including many terpenes. In addition, Peng and Shahidi [10] indicated that their biosynthesis involves the alkylation of olivetolic acid with geranyl diphosphate. A recent study by Jin et al. [62] reported that the abovementioned reaction leads to the formation of cannabigerolic acid CBGA as shown in Figure 3, which is a precursor molecule for numerous other cannabinoids, such as ∆⁹-tetrahydrocannabinol (∆⁹-THC), which is regarded as the most important natural cannabinoid [63]; cannabidiol (CBD); cannabigerol (CBG), cannabinol (CBN); cannabichromene (CBC) [63]; Δ⁸-tetrahydrocannabinol (Δ⁸-THC) [64]; Δ⁹-tetrahydrocannabivarin (THCV) [64]; cannabivarin (CBV) [64]; and cannabidivarin (CBDV) [64]. Some researchers discovered the highest amount of THC in female inflorescences of Cannabis [65]. Another recent research study discovered that biologically active cannabinoids such as Δ⁹-THC, (25.04%) and CBD (resorcinol, 2-p-mentha-1,8-dien-4-yl-5-pentyl) (50.08%) were found in Cannabis resin in a high percentage [66].

Cannabinoids are the best-known secondary active metabolites reported extensively in modern and scientific literature due to a considerable number of studies demonstrating their effectiveness in the treatment of some infections caused by a wide range of pathogens [67]. Consequently, this is a reaffirmation of many previous research studies that asserted that cannabinoids are usually concentrated in the viscous resin produced in structures known as glandular trichomes. These findings prove that the presence of biologically active phytochemicals in high concentrations makes it a valuable source to be used in herbal preparation for different ailments. However, attention has mostly been given to cannabinoids resulting in many other compounds, such as nitrogen compounds, amino acids, hydrocarbons, carbohydrates, organics, and fatty acids, not being thoroughly investigated for their medicinal potential. In addition, the full potential of Cannabis has not been sufficiently exploited because some active molecules have not been defined and their cellular and molecular mechanisms that underlie its antimicrobial and anti-inflammatory activity are not investigated nor understood in depth. However, it is noteworthy to mention that beyond Cannabis, several plants have been reported to possess cannabinoid-like active compounds that interact with the cannabinoid system in one way or the other. For instance, studies conducted have reported the roots of Otanthus maritimus L. to be the main receptors of CB1 and CB2 of cannabinoids [68,69]. In addition, Ruta graveolens, which is used in traditional medicine for its healing properties as fresh herbs, infusions, decoctions, powders, or oils [70,71], was found to have a selective affinity to the CB2 receptors as reported by Wu et al. [72]. A recent study by Dimmito et al. [73] revealed carrots (Daucus carota, L) contain a high concentration of falcarinol (FaOH), which acts as a moderate skin irritant, aggravating histamine-induced edema skin, and showed receptor affinity to both CB1 and CB2 with a CB1 antagonist profile [74]. Catechins and epi-catechin compounds in tea (Camellia sinensis L.) were found to be receptors of CB1 [75]. Furthermore, Kava Kava (Piper methysticum forster) also known as “intoxicating pepper”, which is used as a traditional medicine to cure pains, headaches, convulsions, menstrual pains, and skin diseases [76], was reported to have the ability to activate the CB1 and CB2 receptors [77]. Some phytocannabinoids have been discovered in flowering plants, such as liverworts which are known to be prolific producers of compounds with a bibenzyl backbone as reported by Chicca et al. [78]. Furthermore, Yang et al. [79] conducted a study on several Rhododendron species (Ericaceae) which proved to produce bioactive meroterpenoids with a cannabinoid backbone which are reported to be closely associated with the CBC type decorated with an orcinol side chain. Other studies by Fuhr et al. and Pollastro et al. [80,81] showed that the edible roots of Glycyrrhiza foetida Desf. (licorice; Fabaceae) and Amorpha fruticosa L. (bastard indigo; Fabaceae) were found to contain amorfrutins—bioactive compounds with a cannabinoid backbone.

To date, about 400 of the chemical substances found in Cannabis plants are terpenes and phenolic compounds [82]. Terpenes are a chemical class of compounds acclaimed for their antioxidant, anti-inflammatory, and antimicrobial properties [83]. However, most primary, and secondary metabolites derived from Cannabis have not been sufficiently explored and studied. Therefore, an extensive investigation of other unique compounds should be extensively investigated for possible antimicrobial properties as it is becoming clear that a much wider range of Cannabis constituents may be involved in various therapeutic and/or medicinal effects. According to Ryz et al. [84], other active compounds that have been identified in Cannabis roots include triterpenoids friedelin, epifriedelanol, and the sterols β-sitosterol, stigmasterol, and campesterol (Figure 4), but neither cannabinoids such as THC nor CBD have been reported to be present. Triterpenoids and sterols have also been identified in stem barks [85].

Recent findings revealed that these compounds, especially epifriedelanol, possess anticancer, anti-inflammatory, and antisenescence activities [86]; whereas, triterpenoids friedelin has been reported to display a wide spectrum of anti-inflammatory, antioxidant, antipyretic, anticarcinogenic, and antitumor effects in a variety of plants [87]. In addition, a recent study reported that sterols β-sitosterol has been shown to inhibit aromatase and 5-alpha-reductase, an activity exploited to treat pathologies such as benign prostatic hyperplasia and androgenetic alopecia [88]. Despite the historical claims of the medicinal activity of bioactive compounds in Cannabis roots, their therapeutic potential continues to be largely ignored in modern times. Currently, no research study has investigated the antimicrobial activity of friedelin or epifriedelanol and ST against antimicrobial activity (pathogenic bacteria, fungi, viruses, and nematodes) specifically isolated from Cannabis roots and stem barks. Furthermore, no information is available regarding their cellular and molecular mechanisms determined through preclinical and clinical studies. Certainly, this suggests the need for the re-examination of whole root preparations on microbes employing modern scientific techniques to provide historical and ethnobotanical claims of clinical efficacy.

3.4. Metabolomics Pathways of Cannabinoid

Cannabinoids were the first identified group of potent Cannabis metabolites, with the medicinal properties of its major representatives being attributed to their interference with the G protein-coupled cannabinoid receptors [89,90]. According to Hanuš et al. [91], about 150 cannabinoids have been identified in hemp. The two biosynthetic pathways include the polyketide pathway, which leads to olivetolic (OLA), and the plastidial 2-C-methyl-D-erytritol 4-phosphate (MEP) pathway, which leads to geranyl diphosphate (GPP). Precursors OLA and GPP form cannabigerolic acid (CBG-A), which is a precursor for different cannabinoids, as well as THC-A, CBD-A, and CBC-A as reported by Andre et al. [92]. Olivetolic is a key enzyme in the cannabinoid biosynthetic pathway as revealed by Morita et al. [93]. Studies by Carvalho et al. and Luo et al. [5,94] reported on the recent development in the biosynthesis of cannabinoids by genetically engineered organisms, which could potentially provide solutions to the large-scale production of rare cannabinoids.

Since Cannabis is an open-pollinated plant, it is prone to no-uniformity [50]. A study by Small and Antle [95] revealed that the reasons for higher heterogeneity in Cannabis varieties could be because pollen can disperse a few kilometers in relation to the wind direction. In addition, a recent study by Eržen et al. [96] reported that in Cannabis, the varieties (strains) are often not fully inbred; therefore, they have a relatively high level of heterogeneity and instability, compared to other crops.

3.5. Antibacterial Activity of Cannabis

Generally, the antibacterial activities of Cannabis against various resistant bacterial pathogens have been extensively studied, particularly against human pathogens [97]. Despite the substantial investigations undertaken about antibacterial activities, most research studies focused on the response of the same pathogenic bacteria such as Bacillus or Staphlococcus [98,99]. In addition, while inflorescence has been empirically confirmed to contain the highest concentration of active compounds, surprisingly, most studies assessing the antibacterial properties of Cannabis leaves and seeds receive much more attention when preparing extracts for their antibacterial properties [100]. The chemical characteristics of many of these active compounds, such as THC, CBD, CBN, cannabivarin (CBV), tetrahydrocannabivarin (THV), and many other compounds such as fatty acids, have been reported to exert membrane disruption on bacterial species [101]). While most studies outlined the efficacy of active compounds against several clinically relevant bacterial strains, mechanisms of membrane disruption and the potential application of these active compounds as a therapeutic against certain pathogenic bacteria is not clear nor documented [102]. Although many of these active compounds have been shown to be effective antibacterial agents, little is known about the possibility of their synergistic relationships with antibiotics against bacterial infections. Therefore, the aspect of therapeutic design such as the utilization of co-therapies whose efficacy is conferred through synergistic relationships between two or more therapeutic agents holds promising results in the development of innovative antibacterial agents [103].

Many extracts of various Cannabis parts, such as roots and leaves, have also been extensively researched for their antibacterial properties. Isahq et al. [16] conducted an in vitro research study and discovered that C. indica (leaves, stems, and seeds extracts) possess strong anti-bacterial activity against multidrug-resistant bacterial strains such as S. aureus, Bacillus cereus, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis. In addition, there is a growing body of evidence indicating that multiple cannabinoids administered at 2 µg/mL are potent inhibitors of S. aureus biofilm formation, with cannabigerol (CBG) at 4 µg/mL seemingly able to disperse preformed biofilms and rapidly kill persister cells [104]. Moreover, phytocannabinoids such as cannabichromenic acid (CBCA) have proven to exert potent antibacterial activity against clinical strains of Enterobacter faecalis and both methicillin-resistant and sensitive strains of S. aureus [105]. Another study by Marini et al. [106] investigated the antimicrobial properties of “hashish” against common hospital-associated bacterial strains and found that Cannabis extract exerted the greatest antimicrobial effects on S. aureus 25923, with an inhibition zone of 14 mm, followed by MRSA, E. coli and Klebsiella pneumoniae with values of 12, 10, and 7 mm, respectively. On the other hand, essential oils of many plants have been shown to contain a wide series of secondary metabolites that can inhibit or slow the growth of pathogenic bacteria [107].

Some previous research studies reported a strong antibacterial activity exerted by seed oil (21–28 mm) against Bacillus subtilis and S. aureus and moderate activity (15 mm) against E. coli and P. aeruginosa (16 mm) [18]. Recently, when the efficacy of the essential oils from Cannabis, especially the Futura variety, was evaluated as an antimicrobial agent, it showed promising results. [108] have reported that Cannabis essential oils proved to reduce the virulence of the food contaminant Listeria monocytogenes, with a possible application in the food-processing industry. Furthermore, the inhibitory activity of C. sativa seed extracts was observed in S. aureus biofilm formation, which indicates that these extracts could have enormous potential as preservatives in both the food and cosmetics industries [109]. More recently, cannabinoids were found to be more effective in reducing the bacterial colony count in dental plaque when compared with commercial toothpaste such as Oral B and Colgate, which suggests that C. sativa-derived compounds, especially CBD, could be used for oral care applications [110]. Therefore, further extensive screening of Cannabis essential oils from different varieties is necessary and should be undertaken to identify the samples with the most promising biological value.

Nasrullah et al. [109] assessed the in vitro antimicrobial activity of methanol and n-hexane extracts of C. sativa leaves against Bacillus cereus, B. subtilis, E. coli, P. aeruginosa, and Salmonella species. Methanol extract revealed better antibacterial activity than the n-hexane extract. Other, studies discovered that crude alkaloids extracted from Cannabis leaf present antibacterial effectiveness against bacterial strains representative of skin, mouth, and ear microflora and against the β strain of E. coli [110].

3.6. Antifungal Activity of Cannabis

The continuous emerging empirical evidence regarding the antifungal properties of Cannabis and its secondary metabolites has proven to present excellent results. A few available research studies suggest that, for a long time, Cannabis has been regarded as possessing antifungal activity against a diverse range of pathogenic fungi [61]. Despite cannabinoids such as Δ⁹-THC and CBD being known to greatly contribute to antifungal activity, some studies have demonstrated that plant extracts or essential oils also present this activity [9,111]. So far, studies about the antifungal activity of Cannabis extracts and their secondary metabolites are extremely limited as compared with research studies on antibacterial activities [112]. Wanas et al. [113] showed that the n-hexane fraction of C. sativa effectively inhibits the growth and development of a life-threatening human pathogen Cryptococcus neoformans which is responsible for lung infections in humans. The results revealed modest antifungal activity with an IC50 value of 33.15 µg/mL against C. neoformans. Other findings revealed that Cannabis petroleum ether extract has effectively displayed modest activity against Candida albicans [114].

Some recent studies suggest that a 6.25 mg mL⁻¹ n-butanol leaf extract of C. sativa has proven to completely control the growth of Aspergillus versicolor [115]. Furthermore, a significantly high percentage of mycelial growth inhibition was observed in seed-borne phytopathogenic fungi Alternaria species [116]. Despite numerous research studies confirming the antifungal activities of Cannabis, most studies investigated mostly extracts from its aerial. Studies with the extracts of roots and stems have not been extensively carried out. Therefore, it is highly probable that extracts from roots and stems possess pronounced antifungal potential against a wide spectrum of resistant pathogenic fungi that cause devastating economic loss in agriculture.

More recently, there has been growing scientific evidence suggesting that the synergistic interaction of Cannabis compounds with common antibiotics has the potential to exert antimicrobial action on resistant pathogens [117]. Some previous studies have demonstrated that the synergistic effect between cannabinoids and terpenes may contribute to more effective treatment [118]. However, this synergistic interaction effect is not as extensively investigated in antifungal and cytotoxic activities. In addition, this knowledge appears to have not been given sufficient attention, especially in its application in medicine, industry, or agriculture. Moreover, it is widely known that since the emergence of diseases in fish farming, the use of herbal products to control fish pathogens in aquaculture is an alternative and current practice as chemicals or drugs used to treat infected fish are limited and have harmful effects to the environment and humans who are end consumers of fish products.

Some previous research studies observed that various plant extracts and essential oils can inhibit the growth or even cause the death of pathogenic fish fungus at comparatively low concentrations [119,120]. For instance, Origanum onites and Thymbra have been observed to possess potent antifungal properties against Saprolegnia parasitica known to cause saprolegniasis diseases and considerable economic problems on hatched salmon in aquaculture facilities [121]. In addition, Terminalia catappa has been reported to exhibit the highest antimicrobial effect of minimum inhibitory concentration (MIC) of 25 and 12.5 mg mL⁻¹ against S. parasitica [122]. However, currently, no scientific reports have been published concerning the antifungal potential of Cannabis against fish fungal pathogens and there is also no investigation on the possible synergistic interaction between standard antimicrobial drugs and Cannabis compounds. Consequently, the urgent identification of new molecules exerting novel modes of action that can work alone or in combination with synergistic compounds is required [20].

3.7. Antiviral Activity of Cannabis

One of the recent research studies shows the possibility of Cannabis-derived compounds possessing antiviral properties with the potential to reduce morbidities and slow down the progression of various types of viral illnesses [123]. However, the antiviral properties of Cannabis are the most understudied in modern medical research. Although Cannabis has proven to synthesize a wide range of bioactive compounds, only cannabinoid compounds, particularly THC and CBD, appear to have been explored for their antiviral potential, and there exists insufficient knowledge on the antiviral action modes of cannabinoids on various types of viruses. According to DeMarino et al. [124], CBD is known for its ability to induce apoptosis in mammalian cells and has been demonstrated to reduce extracellular vesicle release from HIV-infected monocytic cells and their viral cargo. Lowe et al. [125] assessed the antiviral effects of CBD in hepatitis B and hepatitis C and discovered that it was not active against HBV infection in vitro but exerted a cytotoxicity effect on the liver cell line, which was used to culture the virus.

In another clinical trial, Cannabis treatment was associated with lower levels of pro-inflammatory biomarkers in cerebral fluid (CSF) of HIV patients [126]; whereas, some studies reported that the treatment of Kaposi sarcoma-associated herpesvirus (KSHV) with various concentrations of CBD significantly reduced VEGFR-3 levels, suggesting another mechanism whereby CBD may affect the proliferation and viability of Kaposi sarcoma through the VEGFR-3 signaling pathway [127]. Similarly, in an HCV assay, CBD was reported to have successfully inhibited the virus by 84.5% with minimal cytotoxicity effect. Therefore, the direct antiviral activity of CBD towards HCV has demonstrated that the compound affects hepatitis, which is caused by activated T cells and macrophages as reported by Lowe et al. [125]. A previous research study by Adejumo et al. [128] reported that the use of Cannabis is related to a lower incidence of liver cirrhosis in chronic HCV patients. Further, longitudinal studies are still needed to ensure the effects of Cannabis extracts and bioactive compounds to verify their effectiveness.

Scientists have recently observed cannabinoids, especially THC and CBD, as possible drugs for SARS-CoV-2 [129]. Reviews and investigations on SARS-CoV-2 are currently dominating the research, resulting in fewer studies on some viruses or the possible re-emergence of other viruses. This is primarily due to empirical evidence revealing that cannabinoids such as CBD may be an efficient inhibitor of SARS-CoV-2 (strain 229E) replication in human lung fibroblasts (MRC-5) through enhancement of antiviral terpene efficacy [130]. In addition, cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA) have been proven to interact with the SARS-CoV-2 spike protein S1 subunit and prevent the entry of several live viral variants into human epithelial cells [131]. The mechanism of action of cannabinoids in SARS-CoV-2 inhibition has been reported to include inhibition of viral cell entry, inhibition of viral proteases, and stimulation of cellular innate immune responses [129]. There are also reports of CBD’s ability to inhibit TMPRSS2 in several models of human epithelia. Additionally, CBD and THC, acting as CB2 receptor agonists, reduce the level of pro-inflammatory cytokines in lung cells [123].

Other recent studies have shown the ability of ∆⁹-THC to inhibit viral 3CL pro caused by SARS-CoV-2 at IC50 3.62 μM [132] while others demonstrated a promising effect of ∆⁹-THCA-A in molecular modeling, thus, predicting inhibition of human angiotensin-converting enzyme 2 (ACE2) [133]. CBGA has been noted to bind orthosterically and allosterically to the SARS-CoV-2 spike protein S1subunit (Kd = 5.6 μM) and prevent the cell entry of human epithelial cells and Vero cells by SARS-CoV-2 and early variants [131].

Moreover, it has been proven that cannabinoids can effectively produce a heightened pharmacological effect than individual isolated compounds by working synergistically with other specialized metabolites via the “entourage effect” [134,135]. It is, however, worth noting that the interaction of cannabinoids with other bioactive metabolites against the viruses has not been adequately studied nor investigated. Concurrently, there exist no reported studies on the antiviral effects of Cannabis plant extracts (roots, leaves, flowers, and seeds) and essential oils. Therefore, further studies should be conducted in the quest to find a therapeutic agent that will provide alternative treatments to prompt sensitive, cost-effective management of viruses. Furthermore, whilst it is widely known that infectious viral diseases remain an important problem all over the world, most research studies have focused on the viruses affecting humans, thus de-emphasizing research on the antiviral potential of Cannabis against livestock animals and crop viruses. Many studies have begun to look at the possible use of feed additives (FA) such as essential oils, extracts, and by-products from medicinal plants. These could be included in an animal’s diet to control pathogens [136] at the molecular level. In addition, the use of feed additives, especially in South Africa, is still limited to experiments, and there is also a lack of application at the farm level. Furthermore, a systematic review of the literature retrieved little information on the use of Cannabis feed additives in fish farming. Consequently, thorough investigations concerning the tolerance or effects of graded levels of Cannabis-derived compounds, especially CBD and THC, in animals, especially livestock, should be undertaken to ensure their safety as there are currently less well-controlled studies, most of which focus on companion animals and poultry [137]. Research by Mahmoudi et al. [138] on poultry demonstrated significant changes in blood parameters, particularly cholesterol levels.

3.8. Anti-Nematicidal Activities of Cannabis

The ability of plant-derived extracts and compounds to either paralyze or kill nematodes has spurred more interest in the discovery of plants that can be used as control agents for parasitic nematodes, and thus far, studies relating to this have received substantial attention [139,140]. This use of plant extracts and compounds is already being greatly exploited commercially in pest management and a rising trend towards organic farming [141]. This is because several pesticides and nematicides are linked to human health and environmental hazards, which have become a worrisome public health concern [142]. This has propelled a need to find alternative methods that are safe and suitable for the environment [143]. Extracts of various plants and pure cannabinoids contained in Cannabis have been found to possess nematicidal properties and have proven to be effective for the control of a wide range of parasitic nematodes [144]. Some previous research demonstrated a significant reduction in several galls, egg masses, nematode fecundity, and build-up caused by C. sativa, and the maximum reductions in these variables were recorded with a 20 g dosage [145].

Similarly, some research studies confirmed that the use of dried flowers and leaves of Cannabis is effective in killing or repelling plant pathogenic nematodes [146]. However, the current body of knowledge on the investigation of Cannabis against pathogenic nematodes remains quite limited. In addition, this is probably because few researchers have focused only on phytopathogenic nematodes, most notably root-knot nematodes, and thus far, there are no studies that have investigated the nematicidal properties of Cannabis against nematodes causing diseases in humans. Although many plants are recommended for use against gastrointestinal nematodes, especially in animals, and related problems in many parts of the world, it is surprising that, to date, no studies have investigated the medicinal activity of Cannabis against gastrointestinal nematodes affecting livestock animals. Consequently, questions on the role of psychoactive, medicinal, or other constituents in human nematode enhancement or suppression also must greatly be addressed in experimental studies.

Most publications on Cannabis nematicidal properties usually are reports involving leaves and flowers as extracts and provide little or no useful data on the other parts of the Cannabis plant, such as roots, stem, and seeds [147]. Despite the little information on the nematicidal properties of Cannabis, the continuous display of in vitro nematicidal activity by other plants, such as Eucalyptus globulus, inconsistent suppression of the Meloidogyne incognita population on tomato roots is evidence enough that a lot more work still needs to be completed in relation to nematicide properties of Cannabis given its copious bioactive metabolites [100]. Likewise, the essential oils of Eucalyptus citriodora and Pelargonium asperum L. proved to be nematicidal against M. incognita [148]. Some recent studies revealed that gall formation and multiplication of both M. incognita and M. hapla on tomato roots have been found to have been significantly reduced by all soil treatments with Artemisia annua meal powder and water extract [149]. On the other hand, a study by Bawa et al. [150] conducted on the neem plant (Azadirachta indica), red-bell pepper (Capsicum annuum), ginger (Zingiber officinale), and African locust bean has shown to have completely prevented attack and hatching of M. incognita eggs and destroyed 100% of the juveniles at 1000 ppm concentrations (at 10% and above). Leela et al. [142] reported that aqueous leaf extracts of Strychnos nuxvomica caused 100% mortality of second-stage juveniles of M. incognita at a 2% concentration. Another study reported that extracts from Nicotiana tabacum and Acorus calamus were found more effective in killing M. incognita, with an EC50 that was 5–10 times lower than the EC50 of the synthetic pesticides chlorpyrifos, carbosulfan, and deltamethrin [151]. This, therefore, suggests that nematicidal effects may be more potent in plant extracts than synthetic nematicides.

3.9. Acaricidal Activities of Cannabis

Since a large number of research studies confirmed various plant-derived compounds possessing powerful pharmacological potential, few researchers investigated the acaricidal properties of Cannabis and its secondary metabolites. However, a systematic review of the literature retrieved extremely little information on the therapeutic use of Cannabis against parasitic mites and ticks. These mites and ticks classified in a taxon of arachnids known as the acari have been frequently reported to cause deleterious effects on humans, animal health, and crops, subsequently resulting in significant economic losses [152]. For instance, ticks have been observed to economically impact cattle production by reducing weight gain and milk production [153]. In addition, mites are greatly reported as a major pest for attacking crops of worldwide economic importance and numerous agricultural production systems [154].

For a very long time, Cannabis has been traditionally employed as a repellent and pesticide to protect crops from phytophagous arthropods without an in-depth knowledge of the bioactive compounds possessing this effect, and most publications were anecdotal [155]. A few research investigations on selected crops have been tested, predominantly on terpenoids which constitute the plant’s essential oil [156]. Several other classes of natural products known to be minor constituents of Cannabis, such as flavonoids, phenols, polyphenols, phytosterols, amines, lignanamides, and fatty acids, are poorly investigated regarding acaricidal properties, and only a few have been exploited commercially [156]. Although in human medicine, the focus has always been on phytocannabinoids, some papers reported that a great number of highly volatile terpenoids from Cannabis have the potential to act as an insect repellent and antifeedant against grazing animals [157].

A recent study by Nasreen et al. [158] investigated the efficacy of C. sativa against Rhipicephalus microplus ticks and the high lethal effect of C. sativa on egg laying with an index of egg laying of 0.26 and 0.24, respectively, egg hatching (33.5 and 37.1, respectively), and total larval mortality (100%, both), at 40 mg/mL. In addition, when this was applied to cattle that had been inoculated with larvae ticks, it was observed that a 45% solution of both herbal extracts significantly reduced the number of ticks by 96 h post-treatment. Several terpenoids and cannabinoids of Cannabis also propelled the investigation of acaricidal effects against Dermanyssus gallinae and Hyalomma dromedarii, also known as the poultry red mite [153]. The active molecules, notably (E)-caryophyllene and α-humulene, alone revealed a higher toxic effect than the whole essential oil and a lower toxic effect of myrcene. Although, it can be concluded that (E)-caryophyllene and α-humulene were responsible for the acaricidal effect of hemp essential oil, further investigation on the comprehensive understanding of the mechanisms of action underlying this effect as well as longitudinal studies with these active molecules needs to be established or documented as many active ingredients from Cannabis, which has shown to deter acari, have not been confidently ascertained.

In addition, this method continues to be advocated as a potential substitute for synthetic pesticides because of it being an environmentally friendly, low-cost, sustainable, and effective option in the fight against mites and ticks [153]. There are still very few rigorous studies undertaken concerning the acaricidal properties of Cannabis against ectoparasites of veterinary importance. Thus far, R. microplus remains the most studied tick across all the reviewed papers.

Furthermore, even though roots and stem barks of Cannabis are ignored for their antimicrobial activities, they are characterized by common active compounds such as triterpenoids friedelin and epifriedelanol, which have been observed to be effective against a broad range of insect pest species on various plants such as Neem (Azadirachta indica), Gynandropsis gynandra and Lavandula angustifolia [102].

A laboratory experiment carried out on A. indica against Tetranychus urticae revealed the acaricidal properties attributed to triterpenoids [159]. Despite this empirical evidence from A. indica, no studies so far have evaluated the acaricidal properties of triterpenoids friedelin and epifriedelanol from Cannabis. Moreover, although the occurrence of the mites, such as the oribatid mite Trhypochthoniellus longisetus, has long been reported to colonize weak or stressed fishes, attaching to the mucous membranes and causing serious damage to farmed fish such as tilapia Oreochromis niloticus, it is very surprising that, to date, there are neither in vitro nor in vivo research studies documented which have investigated the acaricidal potential of Cannabis extracts and bioactive metabolites against fish mites [160]. This evidence proves that Cannabis remains one of many medicinal plants that are insufficiently tapped for their acclaimed pharmacological properties.

The authors would like to thank the University of South Africa M and D for the bursary and scholarship granted towards the study.