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The Anti-Inflammatory Properties of Terpenoids from Cannabis

  • School of Pharmacy, Ein Kerem Campus, Hebrew University of Jerusalem

Abstract and Figures

Introduction: Cannabinoids are well known to have anti-inflammatory effects in mammalians; however, the Cannabis plant also contains other compounds such as terpenoids, whose biological effects have not yet been characterized. The aim of this study was to compare the anti-inflammatory properties of terpenoids with those of cannabidiol (CBD). Materials and Methods: Essential oils prepared from three monoecious nonpsychoactive chemotypes of Cannabis were analyzed for their terpenoid content and subsequently studied pharmacologically for their anti-inflammatory properties in vitro and in vivo. Results:In vitro, the three essential oils rich in terpenoids partly inhibited reactive oxygen intermediate and nitric oxide radical (NO•) production in RAW 264.7 stimulated macrophages. The three terpenoid-rich oils exerted moderate anti-inflammatory activities in an in vivo anti-inflammatory model without affecting tumor necrosis factor alpha (TNFα) serum levels. Conclusions: The different Cannabis chemotypes showed distinct compositions of terpenoids. The terpenoid-rich essential oils exert anti-inflammatory and antinociceptive activities in vitro and in vivo, which vary according to their composition. Their effects seem to act independent of TNFα. None of the essential oils was as effective as purified CBD. In contrast to CBD that exerts prolonged immunosuppression and might be used in chronic inflammation, the terpenoids showed only a transient immunosuppression and might thus be used to relieve acute inflammation.
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The Anti-Inflammatory Properties of Terpenoids
from Cannabis
Ruth Gallily,
*Zhannah Yekhtin,
and Lumı
´r Ondr
ˇej Hanus
Introduction: Cannabinoids are well known to have anti-inflammatory effects in mammalians; however, the Cannabis
plant also contains other compounds such as terpenoids, whose biological effects have not yet been characterized.
The aim of this study was to compare the anti-inflammatory properties of terpenoids with those of cannabidiol (CBD).
Materials and Methods: Essential oils prepared from three monoecious nonpsychoactive chemotypes of Can-
nabis were analyzed for their terpenoid content and subsequently studied pharmacologically for their anti-
inflammatory properties in vitro and in vivo.
Results: In vitro, the three essential oils rich in terpenoids partly inhibited reactive oxygen intermediate and nitric
oxide radical (NO
) production in RAW 264.7 stimulated macrophages. The three terpenoid-rich oils exerted
moderate anti-inflammatory activities in an in vivo anti-inflammatory model without affecting tumor necrosis
factor alpha (TNFa) serum levels.
Conclusions: The different Cannabis chemotypes showed distinct compositions of terpenoids. The terpenoid-rich
essential oils exert anti-inflammatory and antinociceptive activities in vitro and in vivo, which vary according to their
composition. Their effects seem to act independent of TNFa. None of the essential oils was as effective as purified
CBD. In contrast to CBD that exerts prolonged immunosuppression and might be used in chronic inflammation, the
terpenoids showed only a transient immunosuppression and might thus be used to relieve acute inflammation.
Keywords: cannabis; terpenoids; anti-inflammation; antinociceptive; CBD
Human beings have used Cannabis or Cannabis prod-
ucts in various forms for thousands of years
and refer-
ences to therapeutic use of the plant are found in Hieratic
script on papyri dated around 1700 BC.
More recent re-
ports have reviewed the history and characteristics of the
and determined their clinical and biological
The Cannabis plant contains hundreds
of different compounds apart from the major psycho-
active compound D
-tetrahydrocannabinol (THC).
Some of these are unique to the Cannabis plant,
while others are shared with other members of the
plant kingdom.
This century has seen a wealth of literature reports
on the therapeutic potential of Cannabis and/or its
constituents and a comprehensive review conducted
by the Committee on the Health Effects of Marijuana:
An Evidence Review and Research
considered more
than 10,700 relevant abstracts on this subject. They
concluded that there was moderate to conclusive evi-
dence for beneficial effects on chronic pain, and for a
variety of other uses in different autoimmune and in-
flammatory diseases. While studies have focused on
THC and the anti-inflammatory effects of the other
major constituent, the nonpsychoactive cannabinoid
cannabidiol (CBD),
the effects of the aromatic
The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel.
Department of Medicinal and Natural Products, Institute for Drug Research, The Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel.
*Address correspondence to: Ruth Gallily, PhD, The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of
Jerusalem, P.O.B. 11272, Jerusalem 9112102, Israel, E-mail:
ªRuth Gallily et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons
License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Cannabis and Cannabinoid Research
Volume 3.1, 2018
DOI: 10.1089/can.2018.0014
Cannabis and
Cannabinoid Research
terpene constituents have been largely neglected.
Many of the terpenoids are of pharmacological val-
About 200 terpenoids have been described in
Cannabis and constitute the essential oil of the plant,
being responsible for the characteristic odor of the
The biochemical profiles of the terpenoids
in a given plant are more closely associated to the ge-
netics than the environment.
Physiologically, they
are responsible for protecting the plant from predators
and attracting pollinating insects among other func-
tions. Pharmacologically, they have been implicated
in influencing the properties of the cannabinoids, pos-
sibly by a so-called entourage effect.
Effects on anxiety
have been noted as well as positive or negative influences
on the antibacterial, anti-inflammatory, and sedative
properties of Cannabis components.
However, there
is no consensus on the mechanism by which this is
achieved and as to whether the terpenoids themselves
possess pharmacologically significant properties.
We have previously demonstrated the ability of a tri-
ple assay, measuring swelling, pain, and tumor necrosis
factor alpha (TNFa) serum titers, to measure the anti-
inflammatory properties of CBD.
In this study, we
used a similar approach to investigate the antioxidant
and anti-inflammatory properties of three different
preparations of terpenoid-rich essential oils.
Materials and Methods
Essential oil samples
Samples rich in terpenoids were prepared from three
monoecious nonpsychoactive chemotypes of hemp
(legal in Europe). Tisza is a Hungarian variety, and
Felina and Ferimon are chemotypes adapted to the cli-
mate in France.
All three chemotypes of Cannabis were harvested in
August/September 2016 in the pre-Alpine region of
Slovenia (Upper Savinja Valley), latitude NS 4620¢
29.525 and longitude E 1450¢0.777. Samples of essen-
tial oil were prepared by steam distillation of female
flowers (upper third of the plant).
Terpenoid analysis
Samples (1 lL) of essential oil were analyzed by gas
chromatography/mass spectrometry (GC/MS) in a Hew-
lett Packard G 1800B GCD system with an HP-5971
gas chromatograph, with an electron ionization detec-
tor. The software used was GCD Plus ChemStation
and the column was an Rtx
5MS Low bleed GC/MS
column (30 m ·0.25 mm ·0.25 lmfilmthickness).For
then the temperature was programmed from 50Cto
280Cat8C/min; inlet 250C; detector 280C; splitless
injection/purge time 1.0 min; initial temperature 100C;
and with initial time 4.0 min. The helium flow rate was
1 mL/min. Compound constituents were identified by
comparison with standards and by the retention times,
Kovats indices and by comparison with mass spectra
from computerized libraries (HPCH2205, Wiley7N, and
The terpenoids isolated by steam distillation
as essential oil from each of the cannabis chemotypes
gave the test samples T1 (Tisza chemotype), T2 (Felina
chemotype), and T3 (Ferimon chemotype), whose an-
algesic and anti-inflammatory properties were charac-
terized in vitro and in vivo.
Cell culture
(BALB/c) was obtained from the American Type Culture
Collection (ATCC, Rockville, MD) and cultured in Dul-
becco’s modified Eagle’s medium (DMEM) supple-
mented with 5% fetal calf serum (FCS), 1 mM sodium
pyruvate and 100 lg/L streptomycin, and 100 IU/mL
penicillin. The cell line is adherent and the cells were pas-
saged by scraping from the culture dish.
Reactive oxygen intermediate production
For reactive oxygen intermediate (ROI) assay, RAW
264.7 cells were removed from the culture dish by
scraping, and were washed and resuspended at 10
cells/mL in Hank’s balanced salt solution without phe-
nol red. Cells (5 ·10
) were added to a luminometer
tube together with various concentrations of the essen-
tial oils (5, 10, 20, or 40 lg/mL). After 5 min, 10 lL
luminol (Sigma) and 30 lL zymosan (Sigma) were
added to each tube and the chemiluminescence was
measured immediately in a luminometer (Biolumate
LB 95; Berhold, Wilbad, Germany). A second set of
samples was incubated for 24 h with the essential oils
before adding luminol and zymosan. All experiments
were done in duplicates.
Nitric oxide (NO
) determination
and MTT evaluation of viability
RAW 264.7 cells were seeded at a density of 1 ·10
well in 24-well plates and incubated overnight at 37C
and 5% CO
. On the following day, the medium was
changed to fresh DMEM without FCS, containing vari-
ous concentrations of the essential oils. The cells were
then stimulated by the addition of lipopolysaccharide
(LPS) to a concentration of 1 lg/mL. Cell supernatants
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
(SNs) were harvested after 24 h for nitric oxide radical
) assay by addition of 100 lLSNtoanequalvolume
of Griess reagent (1% sulfanilamide, 0.1% naphthalene
diamine, and 2% H
). After 10 min of incubation,
the resultant color was measured at 550 nm. The amount
of NO
produced, and any inhibition by the test materi-
als, was calculated from a standard curve prepared with
The viability of the cells after incubation with the test
materials was determined by MTT viability staining.
The absorbance was measured at 550 nm on a micro-
plate reader.
Female Sabra mice (Israel), 7–8 weeks old, were main-
tained in the specific-pathogen-free unit of the Hadas-
sah Medical School, Hebrew University, Jerusalem,
Israel. The experimental protocols were approved by
the Institutional Animal Care Ethics Committee. The
animals were maintained at a constant temperature
(20–21C) and a 12-h light/12-h dark cycle, and were
provided a standard pellet diet with water ad libitum.
Induction and treatment of paw inflammation
Inflammation was induced by injection of 40 lLofa
suspension of 1.5% w/v zymosan A (Sigma) in saline
into the subplanter surface of the right hind paw of
the mice. This was followed immediately by an injec-
tion of the sample intraperitoneally (10, 25, or 50 mg/
kg). For injection, the terpenoids were dissolved in ve-
hicle containing ethanol:Cremophore:saline at a ratio
of 1:1:18. CBD was used as a positive control. Paw
swelling and pain perception were assessed after 2, 6,
and 24 h. Blood was collected after 24 h for analysis
of TNFaserum levels.
Evaluation of edema
Calibrated calipers were used to measure paw swelling
(thickness) 2, 6, and 24 h after injection of zymosan.
Pain assay
Pain at 2, 6, and 24 h after zymosan injection was
assessed by the von Frey nociceptive filament assay,
where 1.4–60 g filaments, corresponding to 4.17–5.88 log
of force, was used to test the sensitivity of the swollen
paw. The untreated hind paw served as a control.
The measurements were performed in a quiet room
and the animals were handled for 10 s before the test.
A trained investigator then applied the filament, pok-
ing the middle of the hind paw to provoke a flexion re-
flex, followed by a clear finch response after paw
withdrawal. Filaments of increasing size were each ap-
plied for about 3–4 s. The mechanical threshold force
in grams was defined as the lowest force required to ob-
tain a paw retraction response.
FIG. 1. GC/MS spectra of essential oils from three
different chemotypes of Cannabis—Tisza (T1), Felina
(T2), and Ferimon (T3). Each chemotype displays an
individual, characteristic profile. The identity of each
peak is summarized in Table 1. GC/MS, gas
chromatography/mass spectrometry.
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
Measurement of TNFa
Blood was collected 24 h after zymosan injection, and
the sera were assayed for TNFausing a mouse TNFa
ELISA kit (R&D System), according to the manufactur-
er’s instructions.
Statistical analysis
Statistical calculations used the nonparametric Mann-
Whitney Utest and Wilcoxon signed-rank test. The re-
sults are presented as average standard error.
The terpenoid content of essential oils from three
different Cannabis chemotypes
The essential oils from each of the three different che-
motypes of Cannabis—Tisza (T1), Felina (T2), and
Ferimon (T3)—were analyzed by GC/MS analyses,
and up to 50 different compounds were identified
(Fig. 1 and Table 1). The spectra show similarities
and differences between the different Cannabis chemo-
types. As the amounts of the identified terpenoids were
Table 1. Terpenoid Content in the Three Chemotypes of Cannabis: The Results Are Presented as the Relative
Ratio to the Main Terpene in the Sample, Which Was Set to 100.00%
Terpene Kovats Index Tisza chemotype (T1) % Felina chemotype (T2) % Ferimon chemotype (T3) %
a-Pinene 5.85 31.420 14.007 19.413
Camphene 6.26 0.640 0.108
b-Pinene 7.04 20.461 13.434 10.153
Myrcene 7.43 100.000 100.000 52.755
a-Phellandrene 7.85 1.316 2.693 1.163
-Carene 8.10 13.020 2.240 2.103
a-Terpinene 8.30 1.094 2.376 1.018
o-Cymene 8.59 — 0.221 0.564
p-Cymene 8.53 0.105 0.396
Limonene 8.69 10.482 3.379 7.142
b-Phellandrene 8.70 3.537 6.794 2.668
cis-b-Ocimene 8.96 4.043 4.005 2.520
trans-b-Ocimene 9.42 51.700 39.864 32.634
c-Terpinene 9.78 1.151 2.041 0.880
Terpinolene 10.98 32.842 81.256 38.728
Linalool 11.32 0.905
1,3,8-para-Menthatriene 11.86 0.449 0.377
endo-Fenchol 12.10 0.263
allo-Ocimene 12.70 1.517 1.918 1.179
Terpinen-4-ol 14.66 0.294 0.665
p-Cymen-8-ol 14.91 — 0.421
Hexyl butanoate 15.40 0.362
a-Terpineol 15.21 0.303 0.466
Eugenol 22.70 — 1.199
a-Ylangene 23.43 0.191 0.148 0.205
a-Copaene 23.49 — 0.144 0.204
Hexyl hexanoate 23.83 0.411
7-epi-Sesquithujene 24.19 0.288 0.222
Sesquithujene 24.84 — 0.231
cis-Caryophyllene 24.95 1.778 1.669 2.072
cis-a-Bergamotene 25.10 0.148 1.517 0.846
trans-Caryophyllene 25.36 75.569 68.099 100.000
trans-a-Bergamotene 25.99 3.578 11.867 11.100
a-Guaiene 26.20 0.387
trans-b-Farnesene 26.92 3.157 14.176 11.811
a-Humulene 26.82 28.338 25.453 33.158
allo-Aromadendrene 27.07 1.744 3.130
ar-Curcumene 27.96 — 0.310 0.323
b-Selinene 28.37 4.745 6.653 6.007
a-Selinene 28.74 3.785 4.654 4.747
cis-a-Bisabolene 29.09 — 1.719
trans-a-Bisabolene 1.524 —
d-Cadinene 29.72 — 0.621
b-Sesquiphellandrene 29.70 2.011
Selina-3,7(11)-diene 30.66 2.343 2.577 2.813
Caryophyllene oxide 32.16 4.437 5.523
Humulene epoxide II 33.20 1.151 1.176
allo-Aromadendrene epoxide 34.42 0.426
a-Bisabolol 36.17 0.226 —
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
not quantified, the results in Table 1 are presented as
the relative ratio to the main terpene in the sample,
which was set to 100.00%.
In vitro studies
Suppression of ROI and NO
production by RAW mac-
rophages incubated with the terpenoid-rich essential
oils. To study the effects of terpenoids on essential mac-
rophage functions, the RAW 264.7 macrophage cell line
was either untreated or incubated with the essential oils
at indicated concentrations, before stimulation with zy-
mosan to induce ROIs or LPS to induce NO
The ROI production was measured by luminol chemilu-
minescence, while NO
production was measured by
resulting nitrite concentration in the supernatant. The
generation of ROI by RAW 264.7 macrophages was sig-
nificantly suppressed following a short 5 min-incubation
with 40 lg/mL terpenoids from chemotypes T1 and T2
(Fig. 2), while lower concentrations had barely any effect.
T3 terpenoids, however, showed only a moderate inhibi-
tion at 40 lg/mL (Fig. 2). When the macrophages were
incubated with terpenoids for 24 h before zymosan in-
duction of ROI, the terpenoids had barely any inhibitory
effect (Fig. 2). This observation suggests for a transient
inhibitory effect of terpenoids.
Similar to ROI inhibition, the T1 and T2 essential
oils significantly suppressed LPS-induced NO
tion by RAW macrophages when applied at a concen-
tration of 40 lg/mL (Fig. 3). Lower concentrations of
T1 and T2 had almost no effect. The T3 essential oil
had barely any effect at the concentrations used.
The MTT assay showed that the inhibition of NO
and ROI by terpenoids was not due to cytotoxicity,
since all the cells remained over 80% viable with all
concentrations tested (data not shown).
In vivo studies
Anti-inflammatory and antinociceptive effects of
terpenoid-rich essential oils. In this study, we used
the well-accepted mouse model of zymosan-induced
inflammation to investigate the anti-inflammatory
and antinociceptive activities of the three terpenoid
preparations. The extent of hind paw swelling was de-
termined 2, 6, and 24 h following paw injection of
60 lg zymosan alone (control) or together with intra-
peritoneal injection of various concentrations of
FIG. 2. Zymosan-induced generation of ROIs by
RAW 264.7 macrophages was inhibited by essential
Cannabis oils from each of the three chemotypes
Tisza (T1), Felina (T2), and Ferimon (T3). RAW 264.7
macrophages (5 ·10
/500 lLHBSS)wereeither
untreated (Control) or incubated with 20 or 40lL
by zymosan. The ROI was measured by luminol
chemiluminescence. The percentage inhibition of
ROI production is presented. *p<0.05. ROI, reactive
oxygen intermediate; HBSS, Hank’s Balanced Salt
FIG. 3. LPS-induced generation of NO
by RAW
264.7 macrophages was inhibited by essential
Cannabis oils from each of the three chemotypes
Tisza (T1), Felina (T2), and Ferimon (T3). RAW 264.7
macrophages were incubated in serum-free
medium alone or in the presence of various
amounts of the essential oils, as indicated in the
figure. After 5 min, the macrophages were exposed
to LPS (1 lg/mL) for 24 h and the nitrite
concentration in the supernatant reflecting NO
production was measured using the Griess reagent.
*p<0.05, **p<0.01. LPS, lipopolysaccharide.
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
essential oils from each of the three chemotypes Tisza
(T1), Felina (T2), and Ferimon (T3). For comparison,
a mouse group was treated with CBD (5 mg/kg), which
is well known to exert anti-inflammatory and antino-
ciceptive effects.
Intraperitoneal injection of each
of the three terpenoid preparations significantly re-
duced zymosan-induced paw swelling at all three con-
centrations tested (10, 25, and 50 mg/kg) (Fig. 4A).
There were no significant differences between the
three concentrations, meaning that a plateau effect
FIG. 4. Anti-inflammatory (A) and antinociceptive (B) effects of intraperitoneally injected CBD or essential oils
from each of the three Cannabis chemotypes Tisza (T1), Felina (T2), and Ferimon (T3). (A) Prevention of
zymosan-induced swelling of hind paw; 1.5% zymosan in 40 lL was injected into the subplanter surface of the
right hind paw. Immediately thereafter, CBD (5 mg/kg) or essential oils (10, 25, or 50 mg/kg) dissolved in vehicle
containing ethanol:Cremophore:saline at a ratio of 1:1:18 was injected intraperitoneally. The paw thickness
indicative for paw swelling was measured 2, 6, and 24 h thereafter. The paw thickness of untreated mice was
2.3 mm, which made the baseline of the graph. N=9 in each treatment group. *p<0.05, **p<0.01. (B) The
hyperalgesia occurring after zymosan injection in control and treated mice as described in (A) was measured
by using the von Frey nociceptive filament assay. The higher the paw withdrawal threshold, the higher is the
antinociceptive effect of the drug. N=9 in each treatment group. *p<0.05, **p<0.01. CBD, cannabidiol.
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
has been reached and no further inhibition can be
achieved by increasing the dose. Also, it should be
noted that the three terpenoids were less potent than
Next, we studied the antinociceptive effects of the
three terpenoid preparations in comparison to CBD.
To this end, the same mice described above for zymosan-
induced paw swelling were used to determine the paw
withdrawal threshold by applying von Frey filaments
on the paws. Higher paw withdrawal threshold is indic-
ative for better pain-relieving effects. As expected, CBD
significantly increased the paw withdrawal threshold
could significantly increase the pain threshold (Fig. 4B),
although less potent than CBD. T1 showed a correlative
doseresponseat6h,whileno significant difference
and 24 h (Fig. 4B).
In contrast, only a moderate pain inhibition could
be achieved with the T2 and T3 terpenoid prepara-
tions (Fig. 4B). No correlative dose–response could
be seen for T2 and T3, suggesting for having reached
a maximum effect. The more potent pain-relieving ef-
fects of T1 in comparison to T2 and T3 is correlative
to the better prevention of paw swelling by T1 (com-
pare Fig. 4B with 4A). Of note, the antinociceptive ef-
fects of all compounds, including CBD, were most
TNFaserum titer. TNFais one of the proinflamma-
tory cytokines that is produced during inflammation
and activates the nociceptive terminals that innervate
the inflamed tissue.
It was therefore important to
study the effect of terpenoids on TNFaserum levels.
However, none of the terpenoid preparations had any
significant effect on the TNFaserum level 24 h after zy-
mosan injection (Fig. 5). Under the same conditions,
CBD reduced the level of TNFasignificantly by about
48% (Fig. 5).
The terpenoids provide the cannabis plant with its
characteristic fragrance and it is generally accepted
that they provide protection from marauding in-
sects. Although more than 230 different named ter-
penoids have been identified, in Cannabis, only
about 50 known terpenoids have been identified in
a single plant sample, and the profile may be character-
istic of a given chemotype (Hanus
ˇLO, unpublished
data). This variety is reflected in the differences noted
between the three cannabis chemotypes used in this
study. Despite suggestions that differences in the
FIG. 5. TNFain the sera of mice treated with zymosan and essential oils. Twenty-four hours after injecting
zymosan and/or an intraperitoneal dose of CBD (5 mg/kg) or essential oils (10, 25, or 50 mg/kg) dissolved in
vehicle containing ethanol:Cremophore:saline at a ratio of 1:1:18, the TNFaconcentration in the serum was
determined by ELISA. N=3 for each treatment group. **p<0.01. TNFa, tumor necrosis factor alpha.
Gallily et al.; Cannabis and Cannabinoid Research 2018, 3.1
pharmaceutical properties of different chemotypes may
be a consequence of the variety of terpenoids present,
there is almost no information about the biological
and medical properties of cannabis-derived terpenoids.
We have previously developed a triple assay to
demonstrate the anti-inflammatory and antinocicep-
tive properties of CBD.
This assay measures the
ability of any compound to inhibit zymosan-induced
paw swelling and to relieve zymosan-induced pain. In
addition, by collecting blood 24 h after zymosan in-
jection, the assay enables us to determine the effects
of the compounds on zymosan-induced TNFapro-
duction. We adapted this method to study the anti-
inflammatory properties of terpenoid-rich essential
oils from three different chemotypes of Cannabis.
Our data show that the three essential oils, which
contain various ratios of 48 identified terpenoids,
show moderate anti-inflammatory properties in an
induced paw swelling model in mice. All three prepa-
rations were much less potent than CBD. Also, no
correlative dose–response was observed, suggesting
that a maximum effect was observed already with
the lower dose. T2 was somewhat less potent than
T1 and T3 with regard to their paw swelling inhibitory
effects. T1, but not T2 and T3, exhibited moderate
antipain effects, but still, T1 was less potent than
CBD. The differences between terpenoids and CBD
might be explained by their different effect on TNFa
production. While CBD strongly reduces TNFapro-
duction in vivo, the terpenoids barely had any effect.
In vitro, the terpenoids only affected macrophage
production at high
concentration (40 lg/mL), which is in contrast to
6lg/mL CBD required to inhibit 90% of granulocyte-
induced ROI production
and 8 lg/mL CBD to in-
hibit 50% of zymosan-induced ROI in RAW 264.7
The effects were chemotype specific to a certain extent,
which is in agreement with the individuality of the es-
sential oils with terpenoids. Interestingly, in contrast to
CBD, none of the chemotype essential oil had any effect
on the levels of zymosan-induced TNFa.Thismightsug-
gest the terpenoids exert their anti-inflammatory effects
through a mechanism other than that employed by the
Different chemotypes of cannabis have a distinctive
composition of terpenoids. These essential oils do
have anti-inflammatory and antinociceptive activities
that vary according to their composition, but they
had no effect on TNFatiters. None of the essential
oils was as effective as CBD. We suggest that terpenoids
may be used to diminute acute inflammation effect,
whereas the cannabinoids to inhibit chronic inflamma-
tion symptoms.
The authors would like to thank Dr. Ronit Sionov for
her valuable editorial assistance.
Author Disclosure Statement
The authors declare no conflicts of interest.
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Cite this article as: Gallily R, Yekhtin Z, Hanus
ˇLO (2018) The anti-
inflammatory properties of terpenoids from Cannabis,Cannabis and
Cannabinoid Research 3:1, 282–290, DOI: 10.1089/can.2018.0014.
Abbreviations Used
CBD ¼cannabidiol
DMEM ¼Dulbecco’s modified Eagle’s medium
FCS ¼fetal calf serum
GC/MS ¼gas chromatography/mass spectrometry
KI ¼Kovats Index
LPS ¼lipopolysaccharide
NO ¼nitric oxide
ROI ¼reactive oxygen intermediate
SNs ¼supernatants
THC ¼tetrahydrocannabinol
TNFa¼tumor necrosis factor alpha
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... anxiolytic effect that may enhance CBD's therapeutic effect [62]. While there is a dearth of human studies comparing full spectrum and isolate products, some preclinical data suggest that full-spectrum preparations may be more useful. ...
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Purpose of Review This review will address the many uncertainties surrounding the medical use of cannabidiol (CBD). We will begin with an overview of the legal and commercial environment, examine recent preclinical and clinical evidence on CBD, explore questions concerning CBD raised by healthcare professionals and patients, investigate dosing regimens and methods of administration, and address current challenges in the accumulation of sound evidence. Recent Findings CBD has potential for relief of symptoms of pain, sleep, and mood disturbance in rheumatology patients, but sound clinical evidence is lacking. CBD is safe when accessed from a regulated source, whereas wellness products are less reliable regarding content and contaminants. Dosing for symptom relief has not yet been established. Summary As many rheumatology patients are trying CBD as a self-management strategy, the healthcare community must urgently accrue sound evidence for effect.
... Essential oils made from three monoecious nonpsychoactive chemotypes of Cannabis were analyzed for their terpenoid content and pharmacological studies were carried out for their anti-inflammatory properties in vitro and in vivo demonstrated that terpenoids in Cannabis can be used in acute inflammatory conditions [46] . Thirteen anti-inflammatory compounds were characterised and filtered out from medicinal plant species; Cannabis sativa, Prunella vulgaris and Withania somnifera and analysed them for Rheumatoid Arthritis by targeting TNF-α through in silico analyses [47] . This study revealed that these plants possess anti-inflammatory, anti-arthritic and anti-rheumatic properties. ...
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Cannabis sativa is an herbaceous plant which is mainly used as a remedy for neurological, digestive and immunological ailments in traditional medicine. Even though Cannabis is the most illicit plant around the world, its medicinal properties are beneficial in number of ways. Numerous beneficial effects of C. sativa have been demonstrated in multiple in-vitro and in-vivo studies from different parts of the world. The aim of this paper was to systematically review the literature and provide a summary on potential medicinal benefits of C. sativa. This systematic review was conducted by using the data bases; Science direct and PubMed for studies published from 1st of January 2015 to 31st of October 2020. In order to obtain further data, a manual search was also carried out from the reference lists of included articles. After removing the duplicate articles 77 total number of articles included in this present review. The beneficial health effects of C. sativa were anti-inflammatory, analgesic, anti-microbial, anti-parasitic, anti-oxidant and anti-cancer properties. In addition, it revealed that C. sativa lower blood glucose, serum cholesterol and blood pressure levels. Apart from that, the use of Cannabis in other diseases such as irritable bowel disease, renal diseases, neurofibromatosis, and leucorrhea was also identified. The wide range of medicinal effects may be due to main active ingredients of Tetrahydro cannabinol, Cannabidiol, Cannabinol and Tetrahydro cannabivarin. Available in-vitro and invivo evidence suggested that C. sativa has many favorable health effects and further randomized controlled clinical trials will be needed to determine these effects thoroughly
... A substantial amount of research has documented that C. sativa possesses hundreds of secondary metabolites including cannabinoids, terpenes and phenolic compounds [19] which have pharmacological properties in anticonvulsant therapy, appetite stimulation, neurodegenerative diseases, pain treatment, skin pathologies and infectious diseases [20]. Cannabinoids and terpenes, or essential oils (EO) enriched with these, are well known to confer anti-inflammatory effects in mammals during infectious diseases [21][22][23]. So far, 545-550 known compounds, of which about 177 phytocannabinoids, about 200 terpenes and nearly same number of phenolics, have been identified from C. sativa [20,[24][25][26]. ...
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Abstract: Antimicrobial resistance has emerged as a global health crisis and, therefore, new drug discovery is a paramount need. Cannabis sativa contains hundreds of chemical constituents produced by secondary metabolism, exerting outstanding antimicrobial, antiviral, and therapeutic properties. This paper comprehensively reviews the antimicrobial and antiviral (particularly against SARS-CoV-2) properties of C. sativa with the potential for new antibiotic drug and/or natural antimicrobial agents for industrial or agricultural use, and their therapeutic potential against the newly emerged coron-avirus disease (COVID-19). Cannabis compounds have good potential as drug candidates for new antibiotics, even for some of the WHO's current priority list of resistant pathogens. Recent studies revealed that cannabinoids seem to have stable conformations with the binding pocket of the M pro enzyme of SARS-CoV-2, which has a pivotal role in viral replication and transcription. They are found to be suppressive of viral entry and viral activation by downregulating the ACE2 receptor and TMPRSS2 enzymes in the host cellular system. The therapeutic potential of cannabinoids as anti-inflammatory compounds is hypothesized for the treatment of COVID-19. However, more systemic investigations are warranted to establish the best efficacy and their toxic effects, followed by preclinical trials on a large number of participants.
... During exudation stage, cannabinoids inhibit the angiogenesis (Wietecha and DiPietro, 2013). Cannabinoids also actively contribute to the proliferation stage by suppressing chronic inflammation (Costa et al., 2007;Gallily et al., 2018), myofibroblast activation (Garcia- Gonzalez et al., 2009), the excessive deposition of ECM components (Garcia- Gonzalez et al., 2009) and normalization of TIMP-MMP imbalance (Garcia- Gonzalez et al., 2009). miRNAs, including pro-inflammatory miRNAs miR-21, miR-141 and miR-222 and miR-204, that directly targets matrix metalloproteinase 8 (MMP-8), an enzyme that degrades collagen (Kumar et al., 2019). ...
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Fibrosis is a condition characterized by thickening or/and scarring of various tissues. Fibrosis may develop in almost all tissues and organs, and it may be one of the leading causes of morbidity and mortality. It provokes excessive scarring that excels the usual wound healing response to trauma in numerous organs. Currently, very little can be done to prevent tissue fibrosis, and it is almost impossible to reverse it. Anti-inflammatory and immunosuppressive drugs are among the few treatments that may be efficient in preventing fibrosis. Numerous publications suggest that cannabinoids and extracts of Cannabis sativa have potent anti-inflammatory and anti-fibrogenic properties. In this review, we describe the types and mechanisms of fibrosis in various tissues and discuss various strategies for prevention and dealing with tissue fibrosis. We further introduce cannabinoids and their potential for the prevention and treatment of fibrosis, and therefore for extending healthy lifespan.
... Terpenes are a family of organic compounds found especially in essential oils produced by plants and are not only known to be valuable penetration enhancers by causing disorders on the integrity of the stratum corneum structure [155], but also to have important antiinflammatory properties [156]. In the work of Pivetta and collaborators (2018), thymol was incorporated into NLC, and a biphasic release was obtained. ...
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The main function of the skin is to protect the body from the external environment. However, the skin can undergo inflammatory processes, due to genetic, hormonal, or environmental factors. When the defense system is overloaded, there is an increase in pro-inflammatory mediators and reactive oxygen species (ROS), which results in skin disorders. Among the substances used to treat these inflammatory processes, many natural substances with anti-inflammatory and antioxidant properties are being studied: nature is yet an abundant source to obtain diverse pharmacological actives. The treatment of skin diseases is usually focused on topical application, as it reduces the risk of systemic side effects and prevents drug degradation by first-pass metabolism. Thus, the properties of drug delivery vehicles can facilitate or inhibit its permeation. Due to the hydrophobic nature of the skin, a promising strategy to improve dermal drug penetration is the use of lipid-based nanoparticles, such as nanostructured lipid carriers (NLC). Therefore, in this review, we present NLC as a tool to improve dermal administration of natural substances with anti-inflammatory properties.
Supplementation with cannabidiol (CBD) may expedite recovery when consumed after exercise. The purpose of this study was to determine if supplementation with CBD reduces inflammation and enhances performance following strenuous eccentric exercise in collegiate athletes. Twenty-four well-trained females (age = 21.2 ± 1.8 years, height = 166.4 ± 8 cm, weight = 64.9 ± 9.1 kg) completed 100 repetitions of unilateral eccentric leg extension to induce muscle damage. In this crossover design, participants were randomized to receive 5 mg/kg of CBD in pill form or a placebo 2 h prior to, immediately following, and 10 h following muscle damage. Blood was collected, and performance and fatigue were measured prior to, and 4 h, 24 h, and 48 h following the muscle damage. Approximately 28 days separated treatment administration to control for the menstrual cycle. No significant differences were observed between the treatments for inflammation, muscle damage, or subjective fatigue. Peak torque at 60°/s (p = 0.001) and peak isometric torque (p = 0.02) were significantly lower 24 h following muscle damage, but no difference in performance was observed between treatments at any timepoint. Cannabidiol supplementation was unable to reduce fatigue, limit inflammation, or restore performance in well-trained female athletes.
Background Marijuana’s putative anti-inflammatory properties may benefit HIV-associated comorbidities. How recreational marijuana use affects gene expression in peripheral blood cells (PBC) among youth with HIV-1 (YWH) is unknown. Approach YWH with defined substance use (n = 54) receiving similar antiretroviral therapy (ART) were assigned to one of four analysis groups: YWH with detectable plasma HIV-1 (> 50 RNA copies/ml) who did not use substances (H+V+S−), and YWH with undetectable plasma HIV-1 who did not use substances (H+V−S−), or used marijuana alone (H+V−S+[M]), or marijuana in combination with tobacco (H+V−S+[M/T]). Non-substance using youth without HIV infection (H−S−, n = 25) provided a reference group. PBC mRNA was profiled by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Differentially expressed genes (DEG) within outcome groups were identified by Significance Analysis of Microarrays and used for Hierarchical Clustering, Principal Component Analysis, and Ingenuity Pathways Analysis. Results HIV-1 replication resulted in > 3000 DEG involving 27 perturbed pathways. Viral suppression reduced DEG to 313, normalized all 27 pathways, and down-regulated two additional pathways, while marijuana use among virally suppressed YWH resulted in 434 DEG and no perturbed pathways. Relative to H+V−S−, multiple DEG normalized in H+V−S+[M]. In contrast, H+V−S+[M/T] had 1140 DEG and 10 dysregulated pathways, including multiple proinflammatory genes and six pathways shared by H+V+S−. Conclusions YWH receiving ART display unique transcriptome bioprofiles based on viral replication and substance use. In the context of HIV suppression, marijuana use, alone or combined with tobacco, has opposing effects on inflammatory gene expression.
Background: Clerodendrum petasites S. Moore predominantly contains hispidulin (His) and nepetin (Nep) which are immunosuppressive potentials. Although the effect of individual compounds was previously confirmed, a cumulative suppression of these flavonoid mixtures is unknown. This study thus investigated their inhibitory effects and cytotoxicity on T cells by using His:Nep ratios following a naturally-occurring dose (3:1) and optimized doses (1:1 and 1:3). Materials and methods: Anti-CD3/28 stimulated peripheral blood mononuclear cells were treated with individual compounds and their mixtures. Changes in early cell activation markers in activated T cells and apoptosis were analyzed by a flow cytometer. Results: Mixtures at 3:1 suppressed CD69 and CD25 expression in CD4+ and CD8+ T cells in a dose-dependent manner. At the highest concentration of 200 µM His +66.7 µM Nep, over 90% inhibition was observed for CD25 expression in CD4+ and CD8+ T cells, whereas a lesser effect was observed for CD69 expression. A dose-dependent inhibition was still observed when using 1:1 and 1:3 ratios. Interestingly, 80-97% inhibition were observed in CD69 and CD25 expression without inducing cell death after treated with the highest doses of each ratio (66.7 µM His +200 µM Nep and 200 µM His +200 µM Nep). These mixtures were also exhibited a better suppression than individual compounds. Conclusions: The optimized mixture of His and Nep at 66.7:200 µM is suggested for further study due to a greater suppressive effect than a single compound or a naturally-occurring dose.
The first evidence that cannabinoids may have in vitro and in vivo antineoplastic activity against tumor cell lines and animal tumor models was published in the Journal of the National Cancer Institute nearly 50 years ago. Cannabinoids appear to induce apoptosis in rodent brain tumors by way of direct interaction with the cannabinoid receptor. They may inhibit angiogenesis and tumor cell invasiveness. Despite preclinical findings, attempts to translate the benefits from bench to bedside have been limited. This session provides a review of the basic science supporting the use of cannabinoids in gliomas, paired with the first randomized clinical trial of a cannabis-based therapy for glioblastoma multiforme. Another preclinical presentation reports the effects of cannabinoids on triple-negative breast cancer cell lines and how cannabidiol may affect tumors. The session’s second human trial raises concerns about the use of botanical cannabis in patients with advanced cancer receiving immunotherapy suggesting inferior outcomes.
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Oral and dental diseases are a major global burden, the most common non-communicable diseases (NCDs), and may even affect an individual’s general quality of life and health. The most prevalent dental and oral health conditions are tooth decay (otherwise referred to as dental caries/cavities), oral cancers, gingivitis, periodontitis, periodontal (gum) disease, Noma, oro-dental trauma, oral manifestations of HIV, sensitive teeth, cracked teeth, broken teeth, and congenital anomalies such as cleft lip and palate. Herbs have been utilized for hundreds of years in traditional Chinese, African and Indian medicine and even in some Western countries, for the treatment of oral and dental conditions including but not limited to dental caries, gingivitis and toothaches, dental pulpitis, halitosis (bad breath), mucositis, sore throat, oral wound infections, and periodontal abscesses. Herbs have also been used as plaque removers (chew sticks), antimicrobials, analgesics, anti-inflammatory agents, and antiseptics. Cannabis sativa L. in particular has been utilized in traditional Asian medicine for tooth-pain management, prevention of dental caries and reduction in gum inflammation. The distribution of cannabinoid (CB) receptors in the mouth suggest that the endocannabinoid system may be a target for the treatment of oral and dental diseases. Most recently, interest has been geared toward the use of Cannabidiol (CBD), one of several secondary metabolites produced by C. sativa L. CBD is a known anti-inflammatory, analgesic, anxiolytic, anti-microbial and anti-cancer agent, and as a result, may have therapeutic potential against conditions such burning mouth syndrome, dental anxiety, gingivitis, and possible oral cancer. Other major secondary metabolites of C. sativa L. such as terpenes and flavonoids also share anti-inflammatory, analgesic, anxiolytic and anti-microbial properties and may also have dental and oral applications. This review will investigate the potential of secondary metabolites of C. sativa L. in the treatment of dental and oral diseases.
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Rodents are commonly used to study the pathophysiological mechanisms of pain as studies in humans may be difficult to perform and ethically limited. As pain cannot be directly measured in rodents, many methods that quantify “pain-like” behaviors or nociception have been developed. These behavioral methods can be divided into stimulus-evoked or non-stimulus evoked (spontaneous) nociception, based on whether or not application of an external stimulus is used to elicit a withdrawal response. Stimulus-evoked methods, which include manual and electronic von Frey, Randall-Selitto and the Hargreaves test, were the first to be developed and continue to be in widespread use. However, concerns over the clinical translatability of stimulus-evoked nociception in recent years has led to the development and increasing implementation of non-stimulus evoked methods, such as grimace scales, burrowing, weight bearing and gait analysis. This review article provides an overview, as well as discussion of the advantages and disadvantages of the most commonly used behavioral methods of stimulus-evoked and non-stimulus-evoked nociception used in rodents.
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Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono-and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myr-cene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.
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Cannabidiol (CBD), a major constituent of Cannabis, has been shown to be a powerful anti-inflammatory and anti-anxiety drug, without exerting a psychotropic effect. However, when given either intraperitoneally or orally as a purified product, a bell-shaped dose-response was observed, which limits its clinical use. In the present study, we have studied in mice the anti-inflammatory and anti-nociceptive activities of standardized plant extracts derived from the Cannabis sativa L., clone 202, which is highly enriched in CBD and hardly contains any psychoactive ingredients. In stark contrast to purified CBD, the clone 202 extract, when given either intraperitoneally or orally, provided a clear correlation between the anti-inflammatory and anti-nociceptive responses and the dose, with increasing responses upon increasing doses, which makes this plant medicine ideal for clinical uses. The clone 202 extract reduced zymosan-induced paw swelling and pain in mice, and prevented TNFα production in vivo. It is likely that other components in the extract synergize with CBD to achieve the desired anti-inflammatory action that may contribute to overcoming the bell-shaped dose-response of purified CBD. We therefore propose that Cannabis clone 202 (Avidekel) extract is superior over CBD for the treatment of inflammatory conditions.
Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB2) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if non-psychotropic cannabinoids like cannabidiol (CBD) produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C) and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as CBDA, CBDV, CBDVA, CBC, CBG, CBGA, CBGV, THCV, THCVA) and antagonists of cannabinoid type-1 (CB1), CB2 or transient receptor potential vanilloid type 1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol (2-AG)] and related molecules [palmitoylethanolamide (PEA), oleoylethanolamide (OEA)] were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We showed that in poly-(I:C)-stimulated HaCaT cells, CBD elevated the levels of AEA and dose-dependently inhibited poly-(I:C)-induced release of MCP-2, IL-6, IL-8 and TNF-α in a manner reversed by CB2 and TRPV1 antagonists, AM630 and I-RTX, respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.
An advanced Mendelian Cannabis breeding program has been developed utilizing chemical markers to maximize the yield of phytocannabinoids and terpenoids with the aim to improve therapeutic efficacy and safety. Cannabis is often divided into several categories based on cannabinoid content. Type I, Δ 9-tetrahydrocannabinol-predominant, is the prevalent offering in both medical and recreational marketplaces. In recent years, the therapeutic benefits of cannabidiol have been better recognized, leading to the promotion of additional chemovars: Type II, Cannabis that contains both Δ 9-tetrahydrocannabinol and cannabidiol, and cannabidiol-predominant Type III Cannabis. While high-Δ 9-tetrahydrocannabinol and high-myrcene chemovars dominate markets, these may not be optimal for patients who require distinct chemical profiles to achieve symptomatic relief. Type II Cannabis chemovars that display cannabidiol- and terpenoid-rich profiles have the potential to improve both efficacy and minimize adverse events associated with Δ 9-tetrahydrocannabinol exposure. Cannabis samples were analyzed for cannabinoid and terpenoid content, and analytical results are presented via PhytoFacts, a patent-pending method of graphically displaying phytocannabinoid and terpenoid content, as well as scent, taste, and subjective therapeutic effect data. Examples from the breeding program are highlighted and include Type I, II, and III Cannabis chemovars, those highly potent in terpenoids in general, or single components, for example, limonene, pinene, terpinolene, and linalool. Additionally, it is demonstrated how Type I – III chemovars have been developed with conserved terpenoid proportions. Specific chemovars may produce enhanced analgesia, anti-inflammatory, anticonvulsant, antidepressant, and anti-anxiety effects, while simultaneously reducing sequelae of Δ 9-tetrahydrocannabinol such as panic, toxic psychosis, and short-term memory impairment.
This chapter reviews the enzymatic pathways involved in the biosynthesis of cannabis constituents and the variety of phytocannabinoids and terpenoids that are produced. While phytocannabinoids are often referred to as the “active” ingredients in cannabis, the other chemical constituents have a broad spectrum of pharmacological properties and can contribute to the effects seen upon cannabis ingestion or combustion and inhalation, and may also be contained within and contribute to the activity of extracts, tinctures, and other cannabis formulations. The chapter concludes with a focus on the detailed ways in which these processes are manifested in vitro, in laboratory animals, and in the therapeutic use of cannabis preparations.
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The endocannabinoid system (ECS) regulates multiple physiological processes, including cutaneous cell growth and differentiation. Here, we explored the effects of the major nonpsychotropic phytocannabinoid of Cannabis sativa, (-)-cannabidiol (CBD), on human sebaceous gland function and determined that CBD behaves as a highly effective sebostatic agent. Administration of CBD to cultured human sebocytes and human skin organ culture inhibited the lipogenic actions of various compounds, including arachidonic acid and a combination of linoleic acid and testosterone, and suppressed sebocyte proliferation via the activation of transient receptor potential vanilloid-4 (TRPV4) ion channels. Activation of TRPV4 interfered with the prolipogenic ERK1/2 MAPK pathway and resulted in the downregulation of nuclear receptor interacting protein-1 (NRIP1), which influences glucose and lipid metabolism, thereby inhibiting sebocyte lipogenesis. CBD also exerted complex antiinflammatory actions that were coupled to A2a adenosine receptor-dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of the NF-κB signaling. Collectively, our findings suggest that, due to the combined lipostatic, antiproliferative, and antiinflammatory effects, CBD has potential as a promising therapeutic agent for the treatment of acne vulgaris.