**1. Introduction**

Plant medicine has often been used for the treatment of diverse diseases, including bacterial and fungal infections [1–8]. The plants produce a series of secondary metabolites, many of which have pharmacological as well as anti-microbial activities [4–6,9–11]. Evolutionarily, plants have developed various anti-microbial mechanisms to protect them from infectious diseases [11]. Usually, these include the production of compounds that have anti-biofilm and bacteriostatic activities rather than biocidal effect [11]. Compounds with anti-biofilm activities are believed not to induce resistance mechanisms in the microbes, since they target processes not essential for their survival. In contrast, compounds with bactericidal activity might lead to the development of resistance mechanisms in the microbe as part of the bacterial fitness adaptation process with increased probability of developing microbial plant infections.

*Cannabis sativa* L. subspecies are plants that contain a large variety of secondary metabolites, including phytocannabinoids, terpenoids and flavonoids, which have profound anti-microbial activities, in addition to possessing anti-inflammatory, anti-oxidative and neuromodulatory properties [12–14]. In mammalians, the phytocannabinoids interact with the same receptors (e.g., cannabinoid receptors CB1 and CB2) as the endocannabinoids [15], which are endogenous substances with anti-microbial, anti-inflammatory and neuromodulatory activities [16–24]. While much is known about the cannabinoid targets in mammalians, so far, little is known about the microbial targets of these compounds. It is likely that these compounds also interact with specific targets in the microbes. The present

**Citation:** Sionov, R.V.; Steinberg, D. Anti-Microbial Activity of Phytocannabinoids and Endocannabinoids in the Light of Their Physiological and Pathophysiological Roles. *Biomedicines* **2022**, *10*, 631. https://doi.org/10.3390/ biomedicines10030631

Academic Editor: Wesley M. Raup-Konsavage

Received: 17 February 2022 Accepted: 8 March 2022 Published: 9 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

review focuses on the anti-microbial activities of phytocannabinoids and endocannabinoids interwoven with selected aspects of their many physiological and pathophysiological activities.

#### **2.** *Cannabis sativa* **L.**

The hemp plant (*Cannabis sativa* L.; L = Linnaeus) belonging to the family *Cannabaceae*, originates in central-northeast Asia where it has been cultivated for more than 5000 years [15,25,26]. The Han Chinese dynasty used *Cannabis* to treat inflammatory disorders and malaria [27,28]. The Chinese pharmacopoeia of the Emperor Shen Nung, who lived approximately around 2700 BCE and is considered "The Father of Chinese Medicine", indicated *Cannabis* plant usage for the treatment of rheumatic pain, constipation, malaria, and gynecological disorders [26]. In modern times, this plant has been used for different medical conditions, including alleviating chronic pain (e.g., in cancer patients and in rheumatic diseases), muscle spasms (e.g., in multiple sclerosis), epileptic convulsion (e.g., Dravet syndrome and Lennox–Gastaut syndrome in children), nausea (e.g., following chemotherapy), intestinal inflammation (e.g., colitis, inflammatory bowel disease (IBD)), and for stimulating appetite (e.g., in devastating AIDS syndrome, anorexia, and cancer patients) [26,29,30]. It has also been used as a treatment remedy for cancer patients, since the phytocannabinoids can inhibit cell growth of certain tumor cells and enhance the efficacy of certain cancer therapeutics [31].

The phenotypes of *Cannabis* plants are highly variable and can be classified into three major subspecies: *Cannabis sativa* subsp. *sativa*, *Cannabis sativa* subsp. *indica*, and *Cannabis sativa* subsp. *ruderalis* [32]. The different subspecies have all been classified to the *Cannabis sativa* L. species [32]. There are also several chemovariants, chemotypes, or cultivars of this plant harboring different composition of chemical compounds [33–36]. Different *Cannabis* cultivars or chemotypes have been developed that contain various ratios of cannabidiol (CBD) and Δ9-tetrahydrocannabinol ( Δ9-THC), and even those containing high CBD and low Δ9-THC content, which is favorable for avoiding the psychomimetic effects of Δ9-THC [33,37]. The cannabinoids are found in most parts of the plant, with the highest concentrations in glandular trichomes on the surfaces of leaves and flowers [38–42].

The chemical composition of *Cannabis* is affected by the ripeness and maturation state of the plant, growth conditions, the sowing and the harvest times, as well as the storage conditions [34,38–41,43]. The plant composition of phytocannabinoids is affected by light, temperature, water supply, nutrition, heavy metals, phytohormones, soil bacteria, insects and microbial pathogens, among others [44–47]. Cannabidiolic acid (CBDA), the precursor of cannabinols, predominates in the unripen plant, while it is converted to CBD, Δ9-THC and cannabinol (CBN) upon ripening of the resin [48]. In the intermediate ripening state, CBD is predominant, then Δ9-THC dominates in the ripened state, while CBN, the final conversion product, is the major compound in the overripened resin [48]. High antimicrobial activity was found especially in unripen *Cannabis* harvested from regions with unfavorable climate for this plant, whereas ripened *Cannabis* taken from tropical areas had a more hashish-active composition [48]. For the optimal production of essential oil, the recommended stage for harvest is one to three weeks before seed maturity [43].

The difference between industrial hemp and the high Δ9-THC hemp breed type marijuana is that the industrial hemp contains minute amounts of Δ9-THC (less than 0.2% (*w*/*v*)), while marijuana flowers and leaves may contain as much as 17–28% Δ9-THC [49]. Even concentrated THC products, such as oil, shatter, and dab, have been produced with a concentration of up to 95% Δ9-THC [49]. The use of marijuana is associated with hallucinations due to the high Δ9-THC content and may lead to addiction, lack of judgement, and reduced cognition, especially during adolescence when the brain is undergoing significant development [49]. Smoking hemp may lead to decreased immune function with a consequent increase in opportunistic infections [50–53]. *Cannabis* users have a higher probability to ge<sup>t</sup> fungal infections than non-*Cannabis* users, which might in part be due to fungal contamination of the *Cannabis* product [54].

#### *2.1. Anti-Microbial Activity of Cannabis sativa* L. *Extracts*

Z. Krejˇcí, in the 1950s, observed that *Cannabis* has antibiotic activity and introduced it to the clinics in Czechoslovakia [55], a practice that was discontinued in 1990 [33]. The first compound identified by Krejˇcí with antibiotic activity was named cannabidiolic acid (CBDA) [56,57]. From then on, several other *Cannabis* components with antibiotic activities have been isolated and characterized [48,58–63], which will be further discussed below. In 1956, L. Ferenczy published a paper documenting that plant seeds from various plant species, including those from *Cannabis sativa*, exhibited antibacterial activity, especially against Gram-positive bacteria [64]. Wasim et al. [65] documented that both ethanolic and petroleum ether extracts of *Cannabis sativa* leaves showed anti-microbial activity against *Bacillus subtilis*, *Staphylococcus aureus*, *Micrococcus flavus*, *Bordetella bronchiseptica*, *Proteus vulgaris*, *Aspergillus niger*, and *Candida albicans*. Ali et al. [66] observed that the oil of the seeds of *Cannabis sativa* exerted pronounced anti-bacterial activity against *Bacillus subtilis* and *Staphylococcus aureus*, with moderate activity against *Escherichia coli* and *Pseudomonas aeruginosa*, without any activity against *Aspergillus niger* and *Candida albicans*. The petroleum ether extract of the whole plant showed high anti-bacterial activity against *Bacillus subtilis* and *Staphylococcus aureus*, moderate activity against *Escherichia coli*, while no activity against *Pseudomonas aeruginosa* or the tested fungi [66]. Thus, the extraction method and the source affect the composition of the anti-microbial content and the spectrum of responding microbes.

#### *2.2. Anti-Microbial Activity of Essential Oils from Cannabis sativa* L.

Novak et al. [67] analyzed the anti-bacterial effect of essential oils prepared from five different cultivars of *Cannabis sativa* L. These essential oils contained, among others, α-pinene, myrcene, trans-β-ocimene, α-terpinolene, trans-caryophyllene, and α-humulene, but undetectable levels of Δ9-THC and very poor levels of other cannabinoids [67]. They observed differences in the anti-bacterial activity between the various cultivars. All five essential oils showed anti-bacterial activity against *Acinetobacter calcoaceticus*, *Beneckea natriegens*, *Brochothrix thermosphacta* and *Staphylococcus aureus* [67]. Only one of the five essential oils had an anti-bacterial effect on *Escherichia coli*, while none affected *Enterobacter aerogenes*, *Klebsiella pneumoniae*, *Proteus vulgaris*, *Salmonella pullorum*, *Serratia marcescens*, or *Streptococcus faecalis* [67].

Nissen et al. [34] observed that essential oils of *Cannabis sativa* L., prepared from 50–70% of seed maturity, showed anti-bacterial activity against the Gram-positive bacteria *Enterococcus faecium* and *Streptococcus salivarius* at less than 1% (*v*/*v*) but were unable to inhibit the growth of the yeas<sup>t</sup> *Saccharomyces cerevisiae*. Zengin et al. [68] found that essential oils distilled from leaves, inflorescences, and thinner stems of the hemp plant showed antioxidative properties and had significant anti-bacterial activity against clinical *Helicobacter pylori* strains (MIC = 16–64 μg/mL), with lower activity against clinical *Staphylococcus aureus* isolates (MIC = 8 mg/mL) and no significant activity against *Candida* spp. and *Malassezia* spp. The minimum bacterial biofilm inhibitory concentration (MBIC) of the hemp essential oil against *Helicobacter pyroli* was similar to the MIC [68]. The hemp essential oil showed cytotoxicity against human breast cancer, cholangiocarcinoma, and colon carcinoma cell lines at 50–75 μg/mL, while 250 μg/mL was required to inhibit the cell proliferation of a nonmalignant cholangiocyte cell line [68]. The LD50 of hemp essential oil against larvae of *Galleria mellonella* was found to be 1.56 mg/mL, which is much higher than the antibacterial activity against *Helicobacter pyroli*, but lower than that found to be active against *Staphylococcus aureus* strains [68].

Pellegrini et al. [69] observed that essential oil prepared from *Cannabis sativa* L. cultivar Futura 75 inflorescences with low Δ9-THC content (<0.2%) cultivated in the Abruzzo territory showed anti-bacterial activity against *Staphylococcus aureus* and *Listeria monocytogenes* with a MIC of 1.25–5 μL/mL, while being ineffective against *Salmonella enterica*. They also showed that the essential oil possessed anti-oxidative properties [69]. The essential oils produced from the *Cannabis sativa* L. cultivar Futura 75 inflorescences was also found to have insecticidal activity with LD50 values of 65.8 μg/larva on *Spodoptera littoralis*, 122.1 μg/adult on *Musca domestica*, and LC50 of 124.5 μL/L on *Culex quinquefasciatus* larvae [70]. The insecticidal effect might in part be due to an inhibition of the enzyme acetylcholinesterase (AChE) [70]. Thomas et al. [71] found that essential oil of *Cannabis sativa* could induce 100% mortality in the mosquito larvae of *Culex tritaeniorhynchus*, *Anopheles stephensi*, *Aedes aegypti*, and *Culex quinquefasciatus* at concentrations of 0.06, 0.1, 0.12, and 0.2 μL/mL, respectively.

Palmieri et al. [72] studied the variability of *Cannabis* essential oils from various origins and observed that the time of distillation affected the chemical composition of terpenic components, sesquiterpenes, and CBD with consequent variations in the anti-microbial activities against *Staphylococcus aureus*, *Listeria monocytogenes*, and *Enterococcus faecium*. Zheljazkov et al. [73] compared the anti-microbial activity of nine wild hemp (*Cannabis sativa* spp. *spontanea* Vavilov) accessions sampled from agricultural fields in northeastern Serbia with 13 EU registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain, which showed variations in the secondary metabolites β-caryophyllene, αhumulene, caryophyllene oxide, and humulene epoxide. The CBD concentration in the essential oils of wild hemp varied from 6.9 to 52.4%, while the CBD content in the essential oils of the registered cultivars, breeding lines, and the CBD strain varied from 7.1 to 25%; 6.4 to 25%; and 7.4 to 8.8%, respectively [73]. The Δ9-THC concentration showed high variability between the different strains, with the highest concentration being 3.5% [73]. The essential oils of the wild hemp had greater anti-microbial activity compared with the essential oil of registered cultivars [73]. In general, with variations between the different essential oils, anti-microbial activity was observed toward *Staphylococcus aureus*, *Enterococcus faecalis*, *Streptococcus pneumoniae*, *Pseudomonas aeruginosa*, *Yersenia enterocolitica*, *Salmonella enterica*, *Candida albicans*, *Candida krusei*, and *Candida tropicalis* using the disc diffusion method [73]. Altogether, the data presented above show that there is high variability of the composition of hemp essential oils, which might explain the many contradictory publications of the anti-microbial activities toward the same microbial species. In general, a good anti-bacterial response is achieved on Gram-positive bacteria, with less or no effect on Gram-negative bacteria, and variable effect on fungi.

#### *2.3. Anti-Microbial Activity of Terpenoids in Cannabis Essential Oils*

Several terpenoids in the *Cannabis* essential oils have been demonstrated to have anti-microbial effect, which include the monoterpenes α-pinene, linalool, and limonene, and the bitter-tasting sesquiterpenes nerolidol, β-caryophyllene, and caryophyllene oxide [33,74–76]. α-Pinene inhibited the growth of both Gram-positive bacteria (e.g., various *Clostridium* species, *Enterococcus faecium*, *Streptococcus salivarius*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Streptococcus pyogenes*, *Streptococcus pneumoniae*) and Gramnegative bacteria (e.g., various *Pseudomonas* species), as well as the fungus *Candida albicans* [34,77–79]. Myrcene, which is also found in tea tree oil, inhibited the growth of *Staphylococcus aureus* that was associated with the leakage of K+ ions from the bacterial cells and damage to the cell membrane [80]. Linalool, a monoterpenoid alcohol, and α-terpineol, a fragrant terpene, showed anti-bacterial activity against *Propionibacterium acne* and *Staphylococcus epidermidis* with a minimum inhibitory concentration (MIC) of 0.625–1.25 μg/mL [77]. Linalool is also effective against the yeas<sup>t</sup> and hyphal forms of *Candida albicans*, where it alters the membrane integrity and induces cell cycle arrest [81]. Limonene showed anti-bacterial activity against *Staphylococcus epidermidis* [77] and *Listeria monocytogenes* [82], and exerted anti-biofilm activity against *Streptococcus pyogenes*, *Streptococcus mutans*, and *Streptococcus mitis* [83]. α-Humulene showed potent anti-fungal activity

against *Cryptococcus neoformans*, *Candida glabrata*, and *Candida krusei* with MIC values of 5.0, 1.45, and 10.0 μg/mL, respectively, without any effect on methicillin-sensitive *Staphylococcus aureus* (MSSA) 29213, methicillin-resistant *Staphylococcus aureus* (MRSA) 33591, or *Mycobacterium intracellulare* [84]. Nerolidol is a sesquiterpene with sedative properties and inhibits the growth of *Leishmania amazonensis*, *Leishmania braziliensis*, and *Leishmania chagasi* promastigotes, and *Leishmania amazonensis* amastigotes [85], as well as the growth of *Plasmodium falciparum* at the trophozoite and schizont stages [86,87]. The anti-oxidative βcaryophyllene possesses anti-microbial activity against *Staphylococcus aureus* (MIC 2–4 μM), *Bacillus subtilis* (MIC 6–10 μM), *Escherichia coli* (MIC 7–11 μM), *Pseudomonas aeruginosa* (6–8 μM), *Aspergillus niger* (MIC 5–7 μM), and *Trichoderma reesei* (MIC 3–5 μM) without any significant cytotoxic effect on normal mammalian cell lines [88]. The anti-inflammatory oxygenated sesquiterpene caryophyllene oxide exhibited anti-fungal activities against the dermatophytes *Trichophyton mentagrophytes* var. *mentagrophytes*, *Trichophyton mentagrophytes* var. *interdigitale*, and *Trichophyton rubrum* [89].
