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The Anti-Inflammatory Properties of Terpenoids from Cannabis

Authors:
  • 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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ORIGINAL RESEARCH Open Access
The Anti-Inflammatory Properties of Terpenoids
from Cannabis
Ruth Gallily,
1,
*Zhannah Yekhtin,
1
and Lumı
´r Ondr
ˇej Hanus
ˇ
2
Abstract
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
Introduction
Human beings have used Cannabis or Cannabis prod-
ucts in various forms for thousands of years
1
and refer-
ences to therapeutic use of the plant are found in Hieratic
script on papyri dated around 1700 BC.
2
More recent re-
ports have reviewed the history and characteristics of the
materials
3
and determined their clinical and biological
properties.
4–7
The Cannabis plant contains hundreds
of different compounds apart from the major psycho-
active compound D
9
-tetrahydrocannabinol (THC).
Some of these are unique to the Cannabis plant,
8
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
9
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),
10–14
the effects of the aromatic
1
The Lautenberg Center for General and Tumor Immunology, The Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel.
2
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: ruthg@ekmd.huji.ac.il
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 (http://creativecommons.org/licenses/by/4.0), 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
282
terpene constituents have been largely neglected.
15
Many of the terpenoids are of pharmacological val-
ues.
16
About 200 terpenoids have been described in
Cannabis and constitute the essential oil of the plant,
being responsible for the characteristic odor of the
Cannabis.
17
The biochemical profiles of the terpenoids
in a given plant are more closely associated to the ge-
netics than the environment.
17,18
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.
19
Effects on anxiety
have been noted as well as positive or negative influences
on the antibacterial, anti-inflammatory, and sedative
properties of Cannabis components.
19
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.
20,21
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
analysis,thecolumnwaskeptat50Cfor4minand
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
FENSC3).
22
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
Themurinemonocyte/macrophagecelllineRAW264.7
(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
6
cells/mL in Hank’s balanced salt solution without phe-
nol red. Cells (5 ·10
5
) 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
5
cells/
well in 24-well plates and incubated overnight at 37C
and 5% CO
2
. 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
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283
(SNs) were harvested after 24 h for nitric oxide radical
(NO
) assay by addition of 100 lLSNtoanequalvolume
of Griess reagent (1% sulfanilamide, 0.1% naphthalene
diamine, and 2% H
3
PO
4
). 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
NaNO
2
.
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.
Animals
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.
20
Pain assay
Pain at 2, 6, and 24 h after zymosan injection was
assessed by the von Frey nociceptive filament assay,
23
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.
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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.
Results
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
D
3
-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 —
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285
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
production.
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
produc-
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
5
/500 lLHBSS)wereeither
untreated (Control) or incubated with 20 or 40lL
essentialoilsfor5minor24hbeforeROIinduction
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
Solution.
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.
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286
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.
20,21
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
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287
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
CBD.
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
(Fig.4B).Also,theT1terpenoidsat25and50mg/kg
could significantly increase the pain threshold (Fig. 4B),
although less potent than CBD. T1 showed a correlative
doseresponseat6h,whileno significant difference
betweenthethreedosegroupscouldbeobservedat2
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
prominentat6h.
TNFaserum titer. TNFais one of the proinflamma-
tory cytokines that is produced during inflammation
and activates the nociceptive terminals that innervate
the inflamed tissue.
24
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).
Discussion
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.
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288
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.
20,21
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
functionssuchasROIandNO
production at high
concentration (40 lg/mL), which is in contrast to
6lg/mL CBD required to inhibit 90% of granulocyte-
induced ROI production
25
and 8 lg/mL CBD to in-
hibit 50% of zymosan-induced ROI in RAW 264.7
macrophages.
21
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
cannabinoids.
Conclusions
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.
Acknowledgment
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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Chapter
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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.