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Anti-Microbial Activity of Phytocannabinoids and Endocannabinoids in the Light of Their Physiological and Pathophysiological Roles

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Antibiotic resistance has become an increasing challenge in the treatment of various infectious diseases, especially those associated with biofilm formation on biotic and abiotic materials. There is an urgent need for new treatment protocols that can also target biofilm-embedded bacteria. Many secondary metabolites of plants possess anti-bacterial activities, and especially the phytocannabinoids of the Cannabis sativa L. varieties have reached a renaissance and attracted much attention for their anti-microbial and anti-biofilm activities at concentrations below the cytotoxic threshold on normal mammalian cells. Accordingly, many synthetic cannabinoids have been designed with the intention to increase the specificity and selectivity of the compounds. The structurally unrelated endocannabinoids have also been found to have anti-microbial and anti-biofilm activities. Recent data suggest for a mutual communication between the endocannabinoid system and the gut microbiota. The present review focuses on the anti-microbial activities of phytocannabinoids and endocannabinoids integrated with some selected issues of their many physiological and pharmacological activities.
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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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4.0/).
biomedicines
Review
Anti-Microbial Activity of Phytocannabinoids
and Endocannabinoids in the Light of Their Physiological
and Pathophysiological Roles
Ronit Vogt Sionov * and Doron Steinberg
The Biofilm Laboratory, The Institute of Biomedical and Oral Sciences, The Faculty of Dentistry,
The Hebrew University—Hadassah Medical School, Jerusalem 9112102, Israel; dorons@ekmd.huji.ac.il
*Correspondence: ronit.sionov@mail.huji.ac.il
Abstract:
Antibiotic resistance has become an increasing challenge in the treatment of various infec-
tious diseases, especially those associated with biofilm formation on biotic and abiotic materials. There
is an urgent need for new treatment protocols that can also target biofilm-embedded bacteria. Many
secondary metabolites of plants possess anti-bacterial activities, and especially the phytocannabinoids
of the Cannabis sativa L. varieties have reached a renaissance and attracted much attention for their
anti-microbial and anti-biofilm activities at concentrations below the cytotoxic threshold on normal
mammalian cells. Accordingly, many synthetic cannabinoids have been designed with the intention
to increase the specificity and selectivity of the compounds. The structurally unrelated endocannabi-
noids have also been found to have anti-microbial and anti-biofilm activities. Recent data suggest
for a mutual communication between the endocannabinoid system and the gut microbiota. The
present review focuses on the anti-microbial activities of phytocannabinoids and endocannabinoids
integrated with some selected issues of their many physiological and pharmacological activities.
Keywords:
anti-microbial activity; anti-biofilm activity; Cannabis sativa L.; endocannabinoids; gut
microbiota; pathogens; phytocannabinoids
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
]. Evolu-
tionarily, 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 pro-
found 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 endocannabi-
noids [
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
Biomedicines 2022,10, 631. https://doi.org/10.3390/biomedicines10030631 https://www.mdpi.com/journal/biomedicines
Biomedicines 2022,10, 631 2 of 48
review focuses on the anti-microbial activities of phytocannabinoids and endocannabi-
noids 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, orig-
inates 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 dis-
orders [
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), in-
testinal 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
9
-tetrahydrocannabinol (
9
-THC), and even those containing
high CBD and low
9
-THC content, which is favorable for avoiding the psychomimetic
effects of
9-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,
9
-THC and cannabinol (CBN) upon ripening of the resin [
48
]. In the intermediate ripening
state, CBD is predominant, then
9
-THC dominates in the ripened state, while CBN, the
final conversion product, is the major compound in the overripened resin [
48
]. High anti-
microbial 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 9-THC hemp breed type mar-
ijuana is that the industrial hemp contains minute amounts of
9
-THC (less than 0.2%
(w/v)), while marijuana flowers and leaves may contain as much as 17–28%
9-THC
[
49
].
Even concentrated THC products, such as oil, shatter, and dab, have been produced with
a concentration of up to 95%
9
-THC [
49
]. The use of marijuana is associated with hallu-
cinations due to the high
9
-THC content and may lead to addiction, lack of judgement,
and reduced cognition, especially during adolescence when the brain is undergoing sig-
nificant 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 get fungal infections than non-Cannabis users, which might in part be due to
fungal contamination of the Cannabis product [54].
Biomedicines 2022,10, 631 3 of 48
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 Krejˇcíwith 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 Pseu-
domonas 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
9
-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 yeast Saccharomyces cerevisiae. Zengin et al. [
68
] found that essential
oils distilled from leaves, inflorescences, and thinner stems of the hemp plant showed anti-
oxidative properties and had significant anti-bacterial activity against clinical Helicobacter
pylori strains (MIC = 16–64
µ
g/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
µ
g/mL, while 250
µ
g/mL was required to inhibit the cell proliferation of a
nonmalignant cholangiocyte cell line [
68
]. The LD
50
of hemp essential oil against larvae
of Galleria mellonella was found to be 1.56 mg/mL, which is much higher than the anti-
bacterial activity against Helicobacter pyroli, but lower than that found to be active against
Staphylococcus aureus strains [68].
Biomedicines 2022,10, 631 4 of 48
Pellegrini et al. [
69
] observed that essential oil prepared from Cannabis sativa L. cultivar
Futura 75 inflorescences with low
9
-THC content (<0.2%) cultivated in the Abruzzo terri-
tory showed anti-bacterial activity against Staphylococcus aureus and Listeria monocytogenes
with a MIC of 1.25–5
µ
L/mL, while being ineffective against Salmonella enterica. They also
showed that the essential oil possessed anti-oxidative properties [
69
]. The essential oils pro-
duced from the Cannabis sativa L. cultivar Futura 75 inflorescences was also found to have
insecticidal activity with LD
50
values of 65.8
µ
g/larva on Spodoptera littoralis, 122.1
µ
g/adult
on Musca domestica, and LC
50
of 124.5
µ
L/L on Culex quinquefasciatus larvae [
70
]. The in-
secticidal 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,
α
-
humulene, 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
9
-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 essen-
tial 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 ox-
ide
[33,7476]
.
α
-Pinene inhibited the growth of both Gram-positive bacteria (e.g., var-
ious Clostridium species, Enterococcus faecium,Streptococcus salivarius,Staphylococcus au-
reus,Staphylococcus epidermidis,Streptococcus pyogenes,Streptococcus pneumoniae) and Gram-
negative bacteria (e.g., various Pseudomonas species), as well as the fungus Candida al-
bicans [
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
µ
g/mL [
77
]. Linalool is also effective against the yeast 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,Strepto-
coccus mutans, and Streptococcus mitis [
83
].
α
-Humulene showed potent anti-fungal activity
Biomedicines 2022,10, 631 5 of 48
against Cryptococcus neoformans,Candida glabrata, and Candida krusei with MIC values of
5.0, 1.45, and 10.0
µ
g/mL, respectively, without any effect on methicillin-sensitive Staphy-
lococcus 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 β-
caryophyllene possesses anti-microbial activity against Staphylococcus aureus (MIC 2–4
µ
M),
Bacillus subtilis (MIC 6–10
µ
M), Escherichia coli (MIC 7–11
µ
M), Pseudomonas aeruginosa
(6–8
µ
M), Aspergillus niger (MIC 5–7
µ
M), and Trichoderma reesei (MIC 3–5
µ
M) 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].
3. Phytocannabinoids
The Cannabis sativa L. plants produce more than 560 chemicals, including at least
144 cannabinoids and 200 terpenoids, as well as flavonoids and polyunsaturated fatty
acids [
15
,
33
,
34
,
42
,
63
,
67
,
72
,
73
,
90
107
]. The most common phytocannabinoids are
9
-tetrahy
drocannabinol (
9
-THC) and cannabidiol (CBD), which are the neutral homologs of tetrahy-
drocannabinolic acid (THCA) and cannabidiol acid (CBDA), respectively [
108
]. The phy-
tocannabinoids are terpenophenolic compounds containing a resorcinyl core with a para-
positioned isoprenyl, alkyl, or aralkyl side chain [
39
,
40
] (Figure 1). The tetrahydroben-
zochromen ring is quite unique to the genus Cannabis, although a related compound
has been found in the liverwort Radula marginata [
109
], and cannabigerol (CBG) and its
corresponding acid have been isolated from Helichrysum umbraculigerum [110].
Apart from exerting anti-microbial activities, which will be discussed in more detail
below (Section 3.3), phytocannabinoids modulate several physiological and pathophysiolog-
ical processes in humans and other mammalians, making them potential therapeutic drugs
in various settings [
12
14
,
31
,
111
115
]. Among others, these compounds have been shown
to have anti-inflammatory, anti-oxidative, anti-nausea, anti-nociceptive, anti-convulsant,
anti-neoplastic, anxiolytic, and neuroprotective properties [
14
,
111
,
112
,
114
117
]. Cannabi-
noids also affect cognition, such as learning and memory, consciousness, and emotion,
including anxiety and depression [118,119].
Some cannabinoid-based drugs (e.g., Marinol, Syndros, Cesamet, Sativex, and Epid-
iolex) have been approved by the U.S. Food and Drug Administration (FDA) for the
treatment of epilepsy, Dravet syndrome, Lennox–Gastaut syndrome, Parkinson’s dis-
ease, spasticity associated with multiple sclerosis, neuropathic pain, mental illnesses,
chemotherapy-induced nausea, and AIDS wasting syndrome [
117
,
120
122
]. Marinol and
Syndros contain the (-)-trans-
9
-THC dronabinol; Cesamet contains the synthetic cannabi-
noid nabilone that shows structural similarities to
9
-THC; and Epidiolex contains CBD.
Sativex is produced from a Cannabis-derived extract that is composed of approximately
equal quantities of
9
-THC and CBD. A major concern is the production of many psy-
chotropic synthetic cannabinoids distributed on the illicit market, which poses a potential
health treat due to their high potency and toxicity [123].
Biomedicines 2022,10, 631 6 of 48
Biomedicines 2022, 10, x FOR PEER REVIEW 6 of 50
the phosphoinositide-3 kinase (PI3K)/Akt signaling pathways and the mammalian target
of rapamycin (mTOR) [126134].
Figure 1. The chemical structures of some phytocannabinoids and the synthetic cannabinoid HU-
210.
Figure 1.
The chemical structures of some phytocannabinoids and the synthetic cannabinoid HU-210.
3.1. Cannabinoid Receptors
The effects of phytocannabinoids on humans and other mammalians are partly medi-
ated by the G
i/o
protein-coupled CB1 (encoded by the CNR1 gene) and CB2 (encoded by the
CNR2 gene) cannabinoid receptors that consist of seven transmembrane domains
[124126]
.
Biomedicines 2022,10, 631 7 of 48
The stimulation of these receptors leads to the inhibition of adenylyl cyclase with con-
sequent reduction in the intracellular cAMP levels, activation of potassium channels,
activation of mitogen-activated protein kinases (MAPKs) such as the extracellular signal-
regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as activation of the
phosphoinositide-3 kinase (PI3K)/Akt signaling pathways and the mammalian target of
rapamycin (mTOR) [126134].
The CB1 and CB2 receptors also recognize the endogenous arachidonic acid-derived
endocannabinoids, such as N-Arachidonoylethanolamine (anandamide; AEA) and 2-arach
idonoylglycerol (2-AG) [
134
136
]. Both CB1 and CB2 are expressed in various cells in
the brain and in peripheral tissues [
137
]. CB1 is especially expressed at high levels in the
neocortex, hippocampus, basal ganglia, cerebellum, and brainstem, but it is also found in
peripheral nerve terminals and some tissues, such as the vascular endothelium, spleen,
testis, and eye [
137
]. CB2 is predominantly found in cells of the immune system, and in the
central nervous system, it is primarily localized to microglia and tissue macrophages [
137
].
The CB1 receptor regulates the balance between excitatory and inhibitory neuronal
activity. The psychoactive effect is believed to be mediated through the CB1 receptor
in the brain, whereas the immunomodulatory effects are anticipated to be mediated via
the CB2 receptor expressed on immune cells [
138
,
139
]. In addition, CB1 signaling affects
metabolism and is involved in maintaining whole body energy homeostasis by increasing
appetite and stimulating feeding [
140
]. Many efforts have been made to develop CB2
specific agonists at an attempt to achieve anti-inflammatory actions without psychotropic
adverse effects [
13
,
141
143
]. The sesquiterpene (E)-
β
-caryophyllene produced by Cannabis
as well as other plants, including oregano (Origanum vulgare L.), cinnamon (Cinnamomum
spp.), and black pepper (Piper nigrum L.), was found to bind selectively to the CB2 receptor
and exert anti-inflammatory activities [144147].
Other cannabinoid receptors include transient receptor potential vanilloid 1 (TRPV1),
the G-protein-coupled receptors GPR18 and GPR55, and peroxisome proliferator activated
receptors (PPARs) [
126
,
134
,
148
152
]. The anti-nociceptive effect of Cannabis sativa extracts
was found to be mediated by the binding of CBD to TRPV1 [
153
]. A study by Ibrahim
et al. [
154
] showed that activation of the CB2 receptor by its agonist AM1241 stimulated
the release of beta-endorphin from keratinocytes, which, in turn, acted on neuronal
µ
-
opoid receptors to inhibit nociception. The Cannabis sativa extract containing multiple
cannabinoids, terpenes, and flavonoids had stronger anti-nociceptive effect than a single
cannabinoid given alone [
153
], suggesting an “entourage” effect of the various Cannabis-
containing compounds [74].
The CB1 and CB2 can form receptor heteromers [
155
]. The activity of the receptor
heteromer is affected by the agonists and antagonists that bind to each of them. A CB1
antagonist can block the effect of a CB2 agonist and vice versa; a CB2 antagonist can block
the effect of a CB1 receptor agonist [
155
]. CB1 has also been shown to form heteromers with
dopamine and adenosine receptors [
156
158
], AT1 angiotensin receptor [
159
],
µ1
-opoid
receptor [
160
,
161
], and OX1 orexin A receptor [
162
]. The many interacting partners put
CB1 signaling under strict regulation.
3.2. Pharmacological Effects of Selected Phytocannabinoids
3.2.1. 9-Tetrahydrocannibinol (9-THC)
9
-THC binds to CB1 and CB2 receptors at a more or less equal affinity
[138,163,164]
.
It also acts on CB1-CB2 receptor heterodimers [
165
].
9
-THC is well known for its psy-
chomimetic activities that are exerted by its binding to CB1 receptor in the brain, resulting
in a calm and sedated mental state [
49
]. Besides euphoria,
9
-THC is an appetite stimula-
tor [
166
]. Oral
9
-THC (Dronabinol, Marinol) and its synthetic nabilone (Cesamet) have
been used for the treatment of nausea and appetite stimulation for people undergoing
chemotherapy and for AIDS wasting syndrome [
167
,
168
]. The activation of CB1 by
9
-THC
is believed to mediate its anti-nausea and anti-emetic effects [
169
]. Sativex, which contains
Biomedicines 2022,10, 631 8 of 48
a combination of
9
-THC and CBD, has been used for relief of neuropathic pain in multiple
sclerosis [170].
3.2.2. Cannabidiol (CBD)
The non-psychotropic cannabidiol (CBD) shows low affinity to the CB1 and CB2
receptors [
135
] and can exert antagonistic modulatory actions on these receptors [
138
,
171
].
CBD can also activate the TRPV1 channel, serotonin 1A (5-HT
1A
) receptors, and opioid
receptors [
24
,
172
]. CBD has anti-inflammatory, anti-oxidative, anti-epileptic, analgesic,
anti-neoplastic, sedative, neuroprotective, and anti-anxiety activities [
173
188
]. Moreover,
CBD inhibits sebocyte lipogenesis by activating the TRPV4 ion channel that interferes with
the pro-lipogenic ERK1/2 MAPK pathway [189].
The neuroprotective activity of CBD has been attributed in part to its anti-oxidative
activity [
190
,
191
]. Based on its immunomodulatory activities, CBD has been implicated in
the treatment of various autoimmune diseases [
14
,
21
], and its anti-nociceptive activity was
found to be beneficial in relieving chronic pain [
192
]. In addition, CBD has potential uses
in psychiatry due to its neuromodulatory activities in the brain that control recognition,
emotional and behavioral responses [
111
,
193
,
194
]. CBD has especially been reported to
have therapeutic effect for psychopathological conditions, such as substance use disorders,
chronic psychosis, and anxiety [
193
]. CBD has been shown to be well tolerated in humans
at concentrations as high as 3500–6000 mg/day [
195
197
], and the FDA-approved CBD
(marketed as Epidiolex) is indicated for preventing epileptic seizures in Lennox–Gastaut
syndrome and Dravet syndrome in children [198].
In experimental mice and rat models, CBD has been shown to have immunosup-
pressive activities [
181
], which are partly due to inhibition of TNF
α
production [
199
,
200
]
and induction of myeloid-derived suppressor cells (MDSCs) [
201
]. CBD alleviated the
symptoms of experimental autoimmune encephalomyelitis (EAE) and collagen-induced
arthritis and prevented the onset of autoimmune diabetes in experimental murine mod-
els [
199
,
200
,
202
]. In mice, the anti-inflammatory activity of CBD was found to have a
bell-shaped dose–response with an optimal dose of 5 mg/kg [
203
]. The use of a standard-
ized extract from a CBD-rich,
9
-THC
low
Cannabis indica cultivar overcame this bell-shaped
dose–response, suggesting a synergistic effect among the different compounds of the
Cannabis extract [199].
3.2.3. Cannabigerol (CBG)
CBG is another non-psychoactive Cannabis component that is produced at elevated
levels in some industrial hemps [
204
206
]. It binds to both CB1 and CB2 receptors and
modulates the signaling through these receptors, as well as the CB1-CB2 receptor het-
eromer, at concentrations as low as 0.1–1
µ
M [
207
]. CBG competes with the binding
of [
3
H]-WIN-55,212-2 to CB2, but not to CB1 [
207
]. Further studies suggest that CBG
is a partial agonist of CB1 and CB2 [
207
209
]. CBG activates TRPV1, TRPV2, TRPV3,
TRPV4, TRPA1, 5-HT
1a
receptor,
α
2-adrenergic receptor, and PPAR
γ
, while being a TRPM8
antagonist [
210
215
]. CBG has anti-inflammatory, anti-oxidative, and anti-nociceptive
activities
[117,209,213,216]
. The anti-inflammatory property is thought to be achieved by
modulating the CB2 receptor, TRP channels, and PPAR
γ
, and by inhibiting cyclooxyge-
nase 1 and 2 (COX-1/2)
[210,211,217]
, while the analgesic effect of CBG is thought to be
mediated through the
α
2-adrenergic receptor [
211
]. CBG has been shown to have potential
beneficial effects in treating inflammatory bowel disease and neurological disorders, such
as Huntington’s disease, Parkinson’s disease, and multiple sclerosis [213,215,216,218,219].
3.2.4. Cannabichromene (CBC)
CBC is a non-psychoactive phytocannabinoid that activates the CB1 and CB2 receptors,
resulting in decreased intracellular levels of cAMP [
209
]. CBC also activates the TRPA1,
TRPV3, and TRPV4 channels [
210
]. CBC has anti-inflammatory, anti-nociceptive, and
neuroprotective activities [
220
225
]. CBC reduces the activity of both the ON and OFF
Biomedicines 2022,10, 631 9 of 48
neurons in the rostral ventromedial medulla (RVM) and elevates the endocannabinoid levels
in the ventrolateral periaqueductal gray matter [
221
]. The anti-nociceptive activity of CBC
is mediated by the adenosine A1 and TRPA1 receptors [
221
]. CBC increases the viability
of neural stem progenitor cells through activation of the adenosine A1 receptor [
224
].
Moreover, it has been shown to suppress reactive astrocytes, thus offering a protective
effect against neuro-inflammation and Alzheimer’s disease [
225
]. CBC had anti-convulsant
properties in a mouse model of Dravet syndrome [
226
], and it exhibited cytotoxic activity
against some carcinoma cells [227,228].
3.2.5. Cannabidiolic Acid (CBDA)
CBDA has low affinity for both CB1 and CB2 receptors, with moderate inhibition of
adenylyl cyclase activity [
209
,
229
], and functions as an allosteric regulator on the 5-HT
1A
receptor, resulting in anti-emetic effects [
230
233
]. In addition, it activates PPAR
α
and
PPAR
γ
[
212
]. CBDA shows anti-nociceptive and anti-inflammatory effects that are in part
mediated by COX-2 inhibition and activation of the TRPV1 channel [
217
,
234
,
235
]. CBDA
has anxiolytic and anti-convulsant effects in animal models [236238].
3.2.6. Cannabigerolic Acid (CBGA)
CBGA displays low affinity for both CB1 and CB2 receptors but causes a similar de-
crease in intracellular cAMP levels as
9
-THC [
229
]. Since CBGA can activate PPARs [
212
],
it is expected to affect lipid metabolism [
117
]. A Cannabis sativa cultivar containing high
levels of CBG and CBGA inhibited the activity of the aldose reductase enzyme, which
catalyzes the reduction of glucose to sorbitol [
239
]. Since the aldose reductase level is
increased at high glucose levels and has been implicated in the development of neuropathy,
nephropathy, retinopathy, and cataract in diabetes, CBGA has been suggested as a potential
drug in preventing diabetic complications [
239
]. In the Scn1a
+/
mouse model of Dravet
syndrome, CBGA was found to have an anti-convulsant effect that was mediated by its
interaction with the GPR55, TRPV1, and GABAAreceptors [240].
3.2.7. Cannabinol (CBN)
CBN is formed during the degradation of
9
-THC and has a lower binding affinity to
CB1 and CB2 receptors than
9
-THC [
117
]. CBN is an agonist of the TRPV1, TRPV2, TRPV3,
TRPV4, and TRPA1 cation channels [
210
]. CBN is a non-psychotropic phytocannabinoid
with analgesic and anti-inflammatory properties and acts as an appetite stimulant [
117
].
CBN has neuroprotective activity that is associated with its anti-oxidative actions, trophic
support, and elimination of intraneuronal
β
-amyloid in neuronal cells [
241
]. CBN preserves
mitochondrial functions, such as redox regulation, calcium uptake, mitochondrial mem-
brane potential, and bioenergetics [
242
]. CBN promotes endogenous antioxidant defense
mechanisms and triggers AMP-activated protein kinase (AMPK) signaling pathways [
242
].
3.3. Anti-Microbial Effects of Phytocannabinoids
Several phytocannabinoids have been shown to have anti-bacterial activities, especially
on Gram-positive bacteria, including various antibiotic-resistant strains [
58
,
59
,
62
,
63
,
101
,
220
,
243
247
] (Table 1). Phytocannabinoids have been shown to exert both bactericidal and
bacteriostatic effects [
61
,
62
,
244
,
247
]. Most of the studies have analyzed the half maximal
inhibitory concentration (IC
50
) or minimum inhibitory concentration (MIC) for each of
the compounds against different bacterial species, fungi, and protozoa, while only a few
studies have looked at the underlying mechanisms [61,243,244,247250] (Figure 2).
Biomedicines 2022,10, 631 10 of 48
Table 1.
Examples of Cannabis sativa constituents that have been documented to possess anti-bacterial,
anti-fungal, and/or anti-protozoal activities *.
Phytocannabinoids Anti-Microbial Activity Reference
9-Tetrahydrocannabinol
(9-THC)
MIC: 2–5 µg/mL against Staphylococcus aureus ATCC 6538
MIC: 1 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 2 µg/mL against Staphylococcus aureus SA-1199B (NorA overexpression)
MIC: 2 µg/mL against Staphylococcus aureus EMRSA-15
MIC: 0.5 µg/mL against Staphylococcus aureus EMRSA-16
MIC: 2 µg/mL against MRSA USA300
MIC: 4–8 µg/mL against MRSA ATCC 43300
MIC: 5 µg/mL against Streptococcus pyogenes
MIC: 2 µg/mL against Streptococcus milleri
MIC: 5 µg/mL against Streptococcus faecalis
MIC: 4–8 µg/mL against Neisseria gonorrhoeae ATCC 19424
IC50: 4.8 µM against Staphylococcus aureus ATCC 29213
IC50: 6.9 µM against Bacillus cereus IIIM 25
IC50: 2.8 µM against Lactococcus lactis MTCC 440
IC50: 3.5 µM against Shigella boydii NC-09357
IC50: 6.4 µM against Staphylococcus warneri MTCC 4436
No effect against Escherichia coli,Salmonella typhi or Proteus vulgaris
[58,61,245
247]
Cannabidiol (CBD)
MIC: 1–5 µg/mL against S. aureus ATCC 6538
MIC: 0.5–1 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 1 µg/mL against Staphylococcus aureus SA-1199B (NorA overexpression)
MIC: 1 µg/mL against Staphylococcus aureus EMRSA-15
MIC: 1 µg/mL against Staphylococcus aureus EMRSA-16
MIC: 1–4 µg/mL against MRSA USA300
MIC: 1–2 µg/mL against various Staphylococcus aureus isolates.
MIC: 1–2 µg/mL against Staphylococcus epidermidis.
MIC: 4 µg/mL against methicillin-resistant Staphylococcus epidermidis.
MIC: 2 µg/mL against Streptococcus pyogenes
MIC: 1 µg/mL against Streptococcus milleri
MIC: 5 µg/mL against Streptococcus faecalis
MIC: 1–4 µg/mL against various Streptococcus pneumoniae species
MIC: 0.5–4 µg/mL against various Enterococcus faecalis species
MIC: 4 µg/mL against Listereria monocytogenes
MIC: 1–2 µg/mL against Cutibacterium (Propionibacterium) acnes ATCC 6919
MIC: 2–4
µ
g/mL against Clostridioides (Clostridium) difficile M7404 human ribotype 027
MIC: 1–2 µg/mL against various Neisseria gonorrhoeae isolates.
MIC: 0.25 µg/mL against various Neisseria meningitidis ATCC 13090
MIC: 1 µg/mL against Moraxella catarrhalis MMX 3782
MIC: 1 µg/mL against Legionella pneumophila MMX 7515
IC50: 3.8 µM against Staphylococcus aureus ATCC 29213
IC50: 9.5–11.1 µM against Staphylococcus aureus ATCC 6538
IC50: 9.8 µM against Bacillus cereus IIIM 25
IC50: 2.9 µM against Lactococcus lactis MTCC 440
IC50: 4.3 µM against Shigella boydii NC-09357
IC50: 4.1 µM against Pseudomonas fluorescens MTCC 103
IC50: 5.7 µM against Staphylococcus warneri MTCC 4436
Moderate effect against Mycobacterium smegmatis (MIC 16 µg/mL) and marginal
activity against Mycobacterium tuberculosis H37Rv, Candida albicans, and
Cryptococcus neoformans with a MIC > 64 µg/mL.
No effect against Escherichia coli,Salmonella typhimurium, Shigella dysenteriae,
Proteus vulgaris,Proteus mirabilis,Klebsiella pneumoniae,Pseudomonas aeruginosa,
Acinetobacter baumannii,Serratia marcescens,Burkholderia cepacian, and Haemophilus
influenzae.
Anti-biofilm effect:
MBEC: 1–4 µg/mL against MSSA and MRSA biofilms.
BIC50: 12.5 µg/mL against Candida albicans SC5314
MBIC: 100 µg/mL against Candida albicans SC5314
[58,61,62,245
247,251253]
Biomedicines 2022,10, 631 11 of 48
Table 1. Cont.
Phytocannabinoids Anti-Microbial Activity Reference
Cannabigerol (CBG)
MIC: 0.5 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 1 µg/mL against Staphylococcus aureus SA-1199B (NorA overexpression)
MIC: 2 µg/mL against Staphylococcus aureus EMRSA-15
MIC: 1 µg/mL against Staphylococcus aureus EMRSA-16
MIC: 2 µg/mL against MRSA USA300
MIC: 2–4 µg/mL against various MRSA clinical isolates, with some
requiring > 8 µg/mL
MIC: 4–8 µg/mL against MRSA ATCC 43300
MIC: 2.5 µg/mL against Streptococcus mutans UA159 ATCC 700610
MIC: 1 µg/mL against Streptococcus sanguis ATCC 10556
MIC: 5 µg/mL against Streptococcus sobrinus ATCC 27351
MIC: 5 µg/mL against Streptococcus salivarius ATCC 25975
MIC: 1–2 µg/mL against Neisseria gonorrhoeae ATCC 19424
IC50: 15 µg/mL against Mycobacterium intracellulare
Anti-biofilm effect:
MBIC: 2–4 µg/mL against biofilm formation by MRSA
4µg/mL eradicated preformed biofilms of MRSA
MBIC: 2.5 µg/mL against biofilm formation by
Streptococcus mutans UA159 ATCC 70061
Anti-quorum sensing effect
1µg/mL CBG inhibited quorum sensing in Vibrio harveyi BB120.
[58,61,100,
243,244,247,
248]
Cannabidiolic acid
(CBDA)
MIC: 1–2 µg/mL against Neisseria gonorrhoeae ATCC 19424
MIC: 2 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 4 µg/mL against Staphylococcus aureus USA300
MIC: 4 µg/mL against Staphylococcus epidermidis CA#71 and ATCC 51625
MIC: 16–32 µg/mL against MRSA ATCC 43300
No effect on Escherichia coli ATCC 25922 or Pseudomonas aeruginosa PA01 with a
MIC > 64 µg/mL.
[62,247]
Cannabigerolic acid
(CBGA)
IC50: 12 µg/mL against Leishmania donovani
MIC: 4 µg/mL against MRSA USA300
MIC: 2–4 µg/mL against MRSA ATCC 43300
MIC: 1–2 µg/mL against Neisseria gonorrhoeae ATCC 19424
[61,100,247]
Cannabichromene (CBC)
MIC: 1.56 µg/mL against Staphylococcus aureus ATCC 6538
MIC: 2 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 2 µg/mL against Staphylococcus aureus SA-1199B (NorA overexpression)
MIC: 2 µg/mL against Staphylococcus aureus EMRSA-15
MIC: 2 µg/mL against Staphylococcus aureus EMRSA-16
MIC: 8 µg/mL against MRSA USA300
MIC: 0.39 µg/mL against Bacillus subtilis ATCC 6633
MIC 12.5 µg/mL against Mycobacterium smegmatis ATCC 607
IC50: 5.9 µM against Staphylococcus aureus ATCC 29213
IC50: 9.2 µM against Bacillus cereus IIIM 25
IC50: 2.6 µM against Lactococcus lactis MTCC 440
IC50: 3.4 µM against Shigella boydii NC-09357
IC50: 5.6 µM against Staphylococcus warneri MTCC 4436
[58,61,220,
246]
Cannabichromenic acid
(CBCA)
MIC: 2 µg/mL against MRSA USA300
MIC: 7.8 µM against Staphylococcus aureus MSSA 34397
MIC: 3.9 µM against a clinical MRSA isolate
MIC: 7.8 µM against vancomycin-resistance Enterococcus faecalis (VRE)
[61,254]
Biomedicines 2022,10, 631 12 of 48
Table 1. Cont.
Phytocannabinoids Anti-Microbial Activity Reference
Cannabinol (CBN)
MIC: 1 µg/mL against Staphylococcus aureus ATCC 25923
MIC: 1 µg/mL against Staphylococcus aureus SA-1199B (NorA overexpression)
MIC: 1 µg/mL against Staphylococcus aureus EMRSA-15
MIC: 2 µg/mL against MRSA USA300
IC50: 7.9 µM against Staphylococcus aureus ATCC 29213
IC50: 3.2 µM against Bacillus cereus IIIM 25
IC50: 5.8 µM against Lactococcus lactis MTCC 440
IC50: 11.7 µM against Shigella boydii NC-09357
IC50: 8.3 µM against Pseudomonas fluorescens MTCC 103
IC50: 9.2 µM against Staphylococcus warneri MTCC 4436
[58,61,246]
Cannabidivarin (CBDV)
MIC: 2–4 µg/mL against MRSA ATCC 43300
MIC: 0.03–0.5 µg/mL against Neisseria gonorrhoeae ATCC 19424
IC50: 7.8 µM against Staphylococcus aureus ATCC 29213
IC50: 3.1 µM against Bacillus cereus IIIM 25
IC50: 3.2 µM against Lactococcus lactis MTCC 440
IC50: 10.4 µM against Shigella boydii NC-09357
IC50: 5.9 µM against Pseudomonas fluorescens MTCC 103
IC50: 7.9 µM against Staphylococcus warneri MTCC 4436
IC50: 11.9 µM against Candida albicans MTCC 4748
MIC: 8 µg/mL against MRSA USA300
[61,246,247]
(-)8-
Tetrahydrocannabinol
(8-THC)
MIC: 2 µg/mL against MRSA USA300
MIC: 4–8 µg/mL against MRSA ATCC 43300
MIC: 2–4 µg/mL against Neisseria gonorrhoeae ATCC 19424
[61,247]
Exo-tetrahydrocannabinol
(exo-THC) MIC: 2 µg/mL against MRSA USA300 [61]
9-Tetrahydrocannabinolic
acid A (THCA-A) MIC: 4 µg/mL against MRSA USA300 [61]
9-Tetrahydrocannabivarin
(THCV)
MIC: 4 µg/mL against MRSA USA300
MIC: 64 µg/mL against MRSA ATCC 43300
MIC: 16 µg/mL against Neisseria gonorrhoeae ATCC 19424
[61,247]
1-
Tetrahydrocannabidivarol
IC50: 6.9 µM against Staphylococcus aureus ATCC 29213
IC50: 6.9 µM against Bacillus cereus IIIM 25
IC50: 5.1 µM against Lactococcus lactis MTCC 440
IC50: 3.9 µM against Shigella boydii NC-09357
IC50: 7.8 µM against Pseudomonas fluorescens MTCC 103
IC50: 7.6 µM against Staphylococcus warneri MTCC 4436
[246]
(±)-4-
Acetoxycannabichromene
IC50: 40.3 µM against Leishmania donovani
IC50: 4–7.2 µM against Plasmodium falciparum [63]
(±)-3”-Hydroxy-(4”,5”)
cannabichromene
IC50: 24.4 µM against MRSA ATCC 33591
IC50: 29.6 µM against Staphylococcus aureus ATCC 29213
IC50: 60.5 µM against Candida albicans ATCC 90028
IC50: 60.5 µM against Candida krusei ATCC 6258
IC50: 57.5 µM against Leishmania donovani
Not active against Escherichia coli, Mycobacterium intracellulare, or Plasmodium
falciparum.
[63]
5-Acetyl-4-
hydroxycannabigerol
IC50: 53.4 µM against MRSA ATCC 33591
IC50: 10.7 µM against Leishmania donovani
IC50: 6.7–7.2 µM against Plasmodium falciparum
Not active against Staphylococcus aureus, Escherichia coli, Mycobacterium
intracellulare, or Candida albicans.
[63]
Biomedicines 2022,10, 631 13 of 48
Table 1. Cont.
Phytocannabinoids Anti-Microbial Activity Reference
4-Acetoxy-2-geranyl-5-
hydroxy-3-n-pentylphenol
IC50: 6.7 µM against MRSA ATCC 33591
IC50: 12.2 µM against Staphylococcus aureus ATCC 29213
IC50: 53.4 µM against Candida krusei ATCC 6258
IC50: 42.7 µM against Leishmania donovani
Not active against Escherichia coli, Mycobacterium intracellulare, Candida albicans, or
Plasmodium falciparum.
[63]
8-Hydroxycannabinol
IC50: 4.6 µM against Candida albicans ATCC 90028
IC50: 30.6 µM against Mycobacterium intracellulare
Not active against Escherichia coli.
[63]
8-Hydroxycannabinolic
acid A
IC50: 54 µM against Candida krusei ATCC 6258
IC50: 3.5 µM against Staphylococcus aureus ATCC 29213
IC50: 54 µM against Escherichia coli
Not active against Mycobacterium intracellulare.
[63]
Non-Cannabinoid
constituents of Cannabis
sativa L.
5-Acetoxy-6-geranyl-3-n-
pentyl-1,4-benzoquinone
IC50: 15 µg/mL against MRSA ATCC 43300
IC50: 13 µg/mL against Leishmania donovani
IC50: 2.6–2.8 µg/mL against Plasmodium falciparum
[101]
Cannflavin A IC50: 4.5 µg/mL against Leishmania donovani [101]
Cannflavin B IC50: 5 µg/mL against Leishmania donovani [100]
Cannflavin C IC50: 17 µg/mL against Leishmania donovani [101]
6-Prenylapigenin
IC50: 6.5 µg/mL against MRSA ATCC 43300
IC50: 20 µg/mL against Candida albicans
IC50: 2.0–2.8 µg/mL against Plasmodium falciparum
[101]
Prenylspirodinone IC50: 49.6 µM against Bacillus thuringiensis MTCC 809 [246]
* BIC
50
= The test concentration that prevents 50% biofilm formation compared to control cells. IC
50
= The test
concentration that causes 50% growth inhibition in comparison to control cells. MBEC = Minimum biofilm eradi-
cation concentration is the lowest concentration that completely eradicates preformed biofilm.
MBIC = Minimum
biofilm inhibitory concentration is the lowest concentration that is required to completely prevent any biofilm
formation. MIC = Minimum inhibitory concentration is the lowest concentration that completely inhibits bacterial
growth (when no turbidity is observed).
3.3.1. Bacterial Growth Inhibitory Effects of Phytocannabinoids
The minimum inhibitory concentration (MIC) of
9
-THC and CBD on various Staphylo-
coccus aureus strains, including MRSA and Streptococci species (e.g., Streptococcus pyogenes and
Streptococcus. faecalis) was found to be in the range of 1–5
µ
g/mL
[58,62,245,246]
. There was
no significant difference between the anti-bacterial effect of
9
-THC and CBD
[58,245,246]
.
The anti-microbial effect was attenuated by the presence of either serum or blood, suggest-
ing that serum components can bind the compounds and prevent them from acting on the
microorganisms [
245
]. CBG shows anti-bacterial activity against Gram-positive bacteria,
including MSSA, MRSA, and the oral cariogenic Streptococcus mutans at low concentrations
similar to CBD [
58
,
61
,
244
,
247
]. CBC and CBDA showed a MIC of 1–2
µ
g/mL against
Staphylococcus aureus and Staphylococcus epidermidis [
62
,
220
]. In these studies, CBDA was
less active than CBD [
62
]. Cannabichromenic acid (CBCA) caused a rapid reduction in
the colony-forming units (CFUs) of a clinical MRSA isolate both during the exponential
and stationary growth phase, suggesting a bactericidal activity that is independent of the
metabolic state of the bacteria [
254
]. None of the phytocannabinoids had any significant
anti-bacterial activity against Gram-negative bacteria, such as Escherichia coli,Salmonella
typhi,Pseudomonas aeruginosa, and Proteus vulgaris [
61
,
62
,
220
,
245
,
247
]. This might be due to
the inability of these compounds to penetrate the outer membrane of the Gram-negative
Biomedicines 2022,10, 631 14 of 48
bacteria [
61
], or the outer membrane protects the bacteria from cell death caused by damage
to the inner membrane.
Biomedicines 2022, 10, x FOR PEER REVIEW 14 of 50
might be due to the inability of these compounds to penetrate the outer membrane of the
Gram-negative bacteria [61], or the outer membrane protects the bacteria from cell death
caused by damage to the inner membrane.
3.3.2. Outer Membrane Permeabilization of Gram-Negative Bacteria Sensitizes Them to
Phytocannabinoids
Interestingly, CBD and CBG could act on some Gram-negative bacteria (e.g., Esche-
richia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa) if the
outer membrane was permeabilized with the LPS-binding antibiotic polymyxin B
[61,247]. It was shown that an Escherichia coli ΔbamBΔtolC deletion strain that renders the
bacteria hyperpermeable to many small molecules was sensitive to CBG with a MIC of 4
μg/mL, which is in contrast to the parental Escherichia coli wild-type strain that showed a
MIC above 128 μg/mL [61]. Similarly, a lipo-oligosaccharide-deficient Acinetobacter bau-
mannii strain became sensitive to CBG with a MIC of 0.5 μg/mL compared to the parental
strain showing a MIC of 64 μg/mL [61].
Figure 2. The anti-bacterial activities of phytocannabinoids.
Figure 2. The anti-bacterial activities of phytocannabinoids.
3.3.2. Outer Membrane Permeabilization of Gram-Negative Bacteria Sensitizes Them
to Phytocannabinoids
Interestingly, CBD and CBG could act on some Gram-negative bacteria (e.g., Escherichia
coli,Acinetobacter baumannii,Klebsiella pneumoniae,Pseudomonas aeruginosa) if the outer
membrane was permeabilized with the LPS-binding antibiotic polymyxin B [
61
,
247
]. It
was shown that an Escherichia coli
bamB
tolC deletion strain that renders the bacteria
hyperpermeable to many small molecules was sensitive to CBG with a MIC of 4
µ
g/mL,
which is in contrast to the parental Escherichia coli wild-type strain that showed a MIC
above 128
µ
g/mL [
61
]. Similarly, a lipo-oligosaccharide-deficient Acinetobacter baumannii
strain became sensitive to CBG with a MIC of 0.5
µ
g/mL compared to the parental strain
showing a MIC of 64 µg/mL [61].
Biomedicines 2022,10, 631 15 of 48
3.3.3. Combined Treatment of Phytocannabinoids with Antibiotics
No synergistic or antagonistic effects of CBD were observed on MRSA strain USA300
when combined with different conventional antibiotics, such as clindamycin, ofloxacin,
meropenem, tobramycin, methicillin, teicoplanin, and vancomycin [
62
]. These authors
concluded that the membrane-perturbing effect of CBD was not sufficient to enhance the
uptake of conventional antibiotics [
62
]. However, Wassmann et al. [
251
] observed that CBD
could reduce the MIC value of bacitracin against several Gram-positive bacteria, including
Staphylococcus species, Listeria monocytogenes, and Enterococcus faecalis. The simultaneous use
of CBD and bacitracin on MRSA USA300 resulted in the formation of multiple septa during
cell division, appearance of membrane irregularities, reduced autolysis, and decreased
membrane potential [
251
]. The combined CBD/bacitracin treatment did not affect the
growth of the Gram-negative bacteria Pseudomonas aeruginosa,Salmonella typhimurium,
Klebsiella pneumoniae, and Escherichia coli [251].
3.3.4. Phytocannabinoids Also Act on Persister Cells and Do Not Induce Drug Resistance
CBG was found to be active against MRSA persister cells, which are dormant, non-
dividing bacteria [
61
]. This trait is therapeutically important, since many antibiotics require
cell division to be effective, and they are frequently unable to eradicate persister cells
that usually recover after antibiotic withdrawal [
255
257
]. Another obstacle of antibiotic
therapy is the development of drug resistance, a frequent reason for treatment failure [
258
].
Farha et al. [
61
] attempted to develop CBG-resistant bacteria in the hopes of finding the
target molecules. Despite rechallenging the MRSA with 2x and 16x MIC concentration of
CBG, they were unable to get any spontaneously CBG-resistant mutants [
61
]. Similarly,
MRSA that had been daily exposed to sub-lethal concentration of CBD for 20 days were still
sensitive to CBD [247]. The authors of these two studies [61,247] concluded that CBD and
CBG do not induce drug resistance. However, it should be noted that following exposure to
CBD or CBG, the surviving growth-arrested bacteria could regain growth after withdrawal
of the drug.
3.3.5. Therapeutic Anti-Microbial Potential of Phytocannabinoids
The hemolytic activity of CBD and CBG was found to be 256
µ
g/mL and 32
µ
g/mL,
respectively, which is far above the MIC of 1–4
µ
g/mL for MRSA [
61
,
247
]. Addition-
ally, the hemolytic activity of CBDA was found to be above 32
µ
g/mL [
62
]. This makes
phytocannabinoids potential drugs that can act within a reasonable therapeutic window.
Farha et al. [
61
] observed that treating MRSA-infected mice with a high dose of
100 mg/kg CBG could reduce the bacterial burden in the spleen by a 2.8 log
10
of CFU.
Blaskovich et al. [
247
] tried various CBD-containing ointment formulations that could
reduce a 2–3 log
10
of CFU of MRSA inoculated on porcine skin after 1 h and a reduction
of more than 5 log
10
of CFU after a 24 h incubation. CBD, however, failed to significantly
reduce the bacterial load of MRSA ATCC 43300 in a thigh infection mouse model [247].
3.3.6. Anti-Biofilm Activities of Phytocannabinoids
Biofilms are communities of bacteria embedded in an extracellular matrix that have
attached to a biotic surface (e.g., lung tissue, gastrointestinal tract, nasal mucosa, inner ear)
or an abiotic surface (e.g., medical devices, such as catheters, heart valves, stents, prosthe-
ses) [
259
]. The majority of infectious diseases involve bacterial biofilms that are usually
difficult to eradicate due to reduced antibiotic sensitivity [
259
,
260
]. Several studies show
that CBD and CBG can prevent biofilm formation of various Gram-positive bacteria (e.g.,
MSSA, MRSA, Streptococcus mutans) [
61
,
243
,
247
]. The extent of anti-biofilm activity of CBD
and CBG against these bacteria correlated with their anti-bacterial activity
[61,243,244,247]
.
In most cases, a similar concentration of these compounds was required to achieve both
effects, suggesting that some of the anti-biofilm effect is caused by the anti-bacterial activ-
ity [
61
,
243
,
244
]. Moreover, CBD was found to be able to eradicate preformed MSSA and
MRSA biofilms with a minimum biofilm eradication concentration (MBEC) of 1–4
µ
g/mL,
Biomedicines 2022,10, 631 16 of 48
indicating that CBD can penetrate the biofilms and act on the biofilm-embedded bacte-
ria [
247
]. Some cannabinoids (e.g., CBD, CBG, CBC, and CBN) were shown to reduce the
bacterial content of dental plaques in an
in vitro
assay where dental plaques were spread on
agar plates coated with the cannabinoids [
261
]. The anti-biofilm activity of the cannabinoids
has significant clinical importance, since the bacteria-embedded bacteria frequently show
antibiotic resistance, and some antibiotics are unable to penetrate through the extracellular
matrix of the biofilms [259,262,263].
3.3.7. Anti-Fungal Biofilm Activities of Phytocannabinoids
CBD barely affects the viability of Candida albicans with a MIC above 50–100
µ
g/mL
[247,253]
,
but it reduces biofilm formation with a biofilm inhibitory concentration 50 (BIC
50
) at
12.5
µ
g/mL and a MBIC
90
of 100
µ
g/mL [
253
]. CBD reduced the metabolic activity of
preformed Candida albicans biofilms by 50–60% at 6.25
µ
g/mL with no further reduction at
higher concentrations, even at 100
µ
g/mL [
253
]. The morphology of the Candida albicans
biofilm becomes altered in the presence of CBD. While the hyphal form was predominant
in control biofilms, the CBD (25
µ
g/mL)-treated biofilms appeared in clusters mostly in
yeast and pseudohyphal forms [
253
]. CBD caused a dose-dependent reduction in the
cell wall chitin content and the intracellular ATP level, while increasing the intracellular
reactive oxygen species (ROS) levels [
253
]. Gene expression studies showed that after
a 24 h incubation with 25
µ
g/mL CBD, there is a significant downregulation of: ADH5
(Alcohol dehydrogenase 5), involved in extracellular matrix production; BIG1, required
for synthesis of the extracellular matrix component
β
-1,6-glucan; ECE1 (extent of cell elon-
gation protein 1), involved in biofilm formation; EED1, involved in filamentous growth;
CHT1 and CHT3 chitinases, involved in the remodeling of chitin in the fungal cell wall; and
TRR1 (thioredoxin reductase) with anti-oxidant properties. On the other hand, a significant
upregulation of YWP1 (yeast-form wall protein 1) which is expressed predominantly in the
yeast form, was observed [
253
]. These changes in gene expression might explain, at least in
part, the reduced biofilm mass of Candida albicans in the presence of CBD and the increase
in oxidative stress [253].
3.3.8. Anti-Viral Activities of Phytocannabinoids
There are some lines of evidence for an anti-viral activity of phytocannabinoids
[60,264]
.
Some phytocannabinoids, especially
9
-THC and CBD, bind to the M
pro
protease of SARS-
CoV-2, which plays a role in viral replication [
60
,
264
]. CBGA and CBDA were found to
be allosteric and orthosteric ligands for the spike protein of SARS-CoV-2 and prevented
infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike
protein [
265
]. Phytocannabinoids might indirectly relieve the disease progress of COVID-19
patients through their anti-inflammatory properties [
266
]. However, CBD failed to alter
the clinical disease development of COVID-19 when given at a daily dose of 300 mg for
14 days [
267
]. Additionally, caution should be taken into account due to the immuno-
suppressive activities of phytocannabinoids that can prevent proper anti-viral immune
responses [
268
]. Notably, the use of Cannabis was increased in U.S. and Canada by 6–8%
during the COVID-19 pandemic in comparison to the pre-pandemic period [
269
], with a
special increase among people with mental health [
270
]. Vulnerability to COVID-19 was
correlated with genetic liability to Cannabis use disorder (CUD) [271].
3.4. Some Mechanistic Insight into the Anti-Bacterial Activity of Phytocannabinoids
The ability of phytocannabinoids such as CBD and CBG, to kill MRSA, NorA-overexp
ressing Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-
resistant enterococci (VRE) to a similar extent as the respective antibiotic-sensitive strains
[58,245,247]
,
suggests that its action mechanism is not hindered by the common antibiotic-resistance mecha-
nisms. Thus, phytocannabinoids can be used as an alternative drug or an antibiotic adjuvant for
infectious diseases caused by drug-resistant Gram-positive bacteria.
Biomedicines 2022,10, 631 17 of 48
3.4.1. CBD and CBG Target the Cytoplasmic Membrane, Increase Membrane Permeability,
and Reduce Metabolic Activity
There is evidence that CBD and CBG act by targeting the cytoplasmic membrane
of the Gram-positive bacteria [
61
,
247
]. Exposure of MSSA and MRSA to CBD or CBG
caused a dose-dependent increase in the fluorescence of the potentiometric probe 3,3
0
-
dipropylthiadicarbocyanine iodide [DiSC3(5)], suggesting a CBG-induced membrane de-
polarization [
61
,
247
]. CBD inhibited protein, DNA, RNA, and peptidoglycan synthesis
in a Staphylococcus aureus strain when using concentrations close to the MIC [
247
]. At
sub-MIC levels, CBD inhibited lipid synthesis [
247
]. CBG was found to inhibit the enzyme
enoyl acyl carrier protein reductase (InhA) [
272
], which is involved in type II fatty acid
biosynthesis in Mycobacterium tuberculosis. The rapid uptake of the SYTOX green dye into
Staphylococcus aureus and Bacillus subtilis by CBD at MIC, suggests that CBD causes an
increase in membrane permeability [247].
CBG prevents the growth of oral cariogenic Streptococcus mutans in a concentration and
bacterial cell density manner [
243
]. At a MIC of 2.5
µ
g/mL, CBG exhibited a bacteriostatic
effect on Streptococcus mutans, while at 2x MIC and 4x MIC, a bactericidal activity was
observed [
243
]. CBG treatment was found to alter the morphology of Streptococcus mutans
and cause intracellular accumulation of membrane-like structures [
243
]. CBG induced
an immediate membrane hyperpolarization, followed by increased uptake of propidium
iodide, suggesting increased membrane permeabilization [
243
]. At the same time, Laur-
dan incorporation into the membranes was reduced in a dose-dependent manner [
243
],
indicative of a more rigid membrane structure. The metabolic activity was decreased in a
dose-dependent manner, which might contribute to the growth inhibitory effect [243].
3.4.2. CBD Inhibits the Release of Membrane Vesicles from Escherichia coli
Kosgodage et al. [
250
] observed that CBD inhibits the release of membrane vesicles
from the Gram-negative Escherichia coli VCS257, while having negligible effect on the
membrane vesicle release from the Gram-positive Staphylococcus aureus subsp. aureus
Rosenbach. Membrane vesicles participate in inter-bacterial communication by the transfer
of cargo molecules and virulence factors [
273
]. CBD was found to enhance the anti-bacterial
effect of erythromycin, rifampicin, and vancomycin against the tested Escherichia coli
strain [250].
3.4.3. CBG Reduces the Expression of Biofilm and Quorum Sensing-Related Genes in
Streptococcus mutans
CBG inhibited sucrose-induced biofilm formation by Streptococcus mutans with a mini-
mum biofilm inhibitory concentration (MBIC) of 2.5
µ
g/mL [
243
]. Higher concentrations
(10
µ
g/mL) of CBG were required to reduce the metabolic activity of preformed Strep-
tococcus mutans biofilms [
243
]. CBG reduced the expression of various biofilm-related
genes (e.g., gtfB,gtfC,gtfD,ftf,gbpA,gbpA,brpA,wapA) with concomitant reduction in the
production of extracellular polymeric substances (EPS) [
243
]. The quorum sensing-related
genes comE,comD, and luxS were downregulated by CBG, while no effect was observed on
the gene expression of the stress-associated chaperones groEL and dnaK [
243
]. Moreover,
CBG induced reactive oxygen species (ROS) production in Streptococcus mutans, which
might be related to the reduced expression of the oxidative stress defense genes, sod and
nox [
243
]. Thus, CBG has specific anti-biofilm activity unrelated to its membrane-acting
effect. This conclusion is further supported by the study of Aqawi et al. [
248
] showing
that CBG inhibited quorum sensing, bacterial motility, and biofilm formation of the marine
Gram-negative Vibrio harveyi without affecting the planktonic growth.
3.4.4. CBG and HU-210 Inhibit Quorum Sensing in Vibrio harveyi
Quorum sensing is an inter-bacterial communication system mediated by secreted
autoinducers that interact with their respective receptors, resulting in the activation of
a signal transduction cascade that alters the gene expression repertoire in a cell-density-
Biomedicines 2022,10, 631 18 of 48
dependent manner [
274
]. CBG prevented the bioluminescence induced by the master
quorum sensing regulator LuxR of Vibrio harveyi at a concentration of 1
µ
g/mL [
248
]. Using
a
luxM,
lusS Vibrio harveyi mutant that does not produce autoinducers AI-1 and AI-2,
CBG was found to prevent the signals delivered by exogenously added autoinducers,
with a more profound inhibitory effect on the AI-2-induced than on the AI-1-induced
bioluminescence [
248
]. Further studies show that CBG prevented the expression of several
quorum sensing genes in Vibrio harveyi, including luxU,luxO,qrr1–5, and luxR, which can
explain the inhibitory effect of CBG on LuxR-mediated bioluminescence [
248
]. Altogether,
these data demonstrate that CBG can interfere with bacterial quorum sensing.
The synthetic cannabinoid HU-210, which is a dimethylheptyl analog of
8
-THC
(
Figure 1
) and acts as a high-affinity CB1 and CB2 agonist [
275
,
276
], has been shown
to inhibit quorum sensing in the Vibrio harveyi AI-1
, AI-2
+
BB152 mutant, but it had
barely any effect on the wild-type bacteria or the AI-1
+
, AI-2
MM30 mutant [
249
]. This
suggests that HU-210 specifically antagonizes the AI-2 pathway [
249
]. The concentra-
tion of HU-210 required to achieve the anti-quorum sensing activity was relatively high
(20–200
µ
g/mL) [
249
], which is 2–3 magnitudes higher than that of CBG [
248
]. HU-210
prevented biofilm formation of the AI-1
, AI-2
+
BB152 mutant with a BIC
50
of 2
µ
g/mL
and MBIC
90
of 200
µ
g/mL, while no significant effect was seen on biofilm formation by
the wild-type bacteria or the AI-1
+
, AI-2
MM30 mutant [
249
]. However, the motility of
Vibrio harveyi was reduced in all three strains at both 20 and 200
µ
g/mL HU-210 [
249
].
Gene expression studies showed that HU-210 at a concentration of 2
µ
g/mL reduced the
expression of the master regulator luxR in both wild-type and AI-1
, AI-2
+
BB152 strain,
while it had no effect on the AI-1
+
, AI-2
MM30 Vibrio harveyi mutant strain [
249
]. The
luxM gene that encodes for AI-1 was upregulated by HU-210 [249].
4. Endocannabinoids
The endocannabinoid system (ECS) modulates many physiological processes, includ-
ing the cardiovascular, gastrointestinal and immune systems, pain, learning, memory, per-
ception, mood, appetite, metabolism, emotions, and sleep [
22
,
112
,
113
,
277
285
]. The bioac-
tive endocannabinoid lipid mediators have potent anti-inflammatory activities
[286291]
.
In addition, they promote neural progenitor cell proliferation and differentiation, and have
neuroprotective effects [
20
,
292
294
]. The effect on neural cell proliferation is mediated by
both the CB1 and CB2 receptors [293,295,296].
4.1. The Endocannabinoid System
The endocannabinoid system is composed of: (1) the lipid active endogenous ligands
N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG);
(2) their biosynthetic enzymes