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Cannabidiolic Acid as a Selective Cyclooxygenase-2 Inhibitory
Component in Cannabis
Shuso Takeda, Koichiro Misawa, Ikuo Yamamoto, and Kazuhito Watanabe
Organization for Frontier Research in Preventive Pharmaceutical Sciences (S.T., K.W.) and Department of Hygienic Chemistry,
Faculty of Pharmaceutical Sciences (K.M., K.W.), Hokuriku University, Kanazawa, Japan;
and School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Japan (I.Y.)
Received February 7, 2008; accepted June 10, 2008
ABSTRACT:
In the present study it was revealed that cannabidiolic acid (CBDA)
selectively inhibited cyclooxygenase (COX)-2 activity with an IC
50
value (50% inhibition concentration) around 2
M, having 9-fold
higher selectivity than COX-1 inhibition. In contrast, ⌬
9
-tetrahydro
-
cannabinolic acid (⌬
9
-THCA) was a much less potent inhibitor of
COX-2 (IC
50
> 100
M). Nonsteroidal anti-inflammatory drugs con
-
taining a carboxyl group in their chemical structures such as
salicylic acid are known to inhibit nonselectively both COX-1 and
COX-2. CBDA and ⌬
9
-THCA have a salicylic acid moiety in their
structures. Thus, the structural requirements for the CBDA-
mediated COX-2 inhibition were next studied. There is a structural
difference between CBDA and ⌬
9
-THCA; phenolic hydroxyl groups
of CBDA are freed from the ring formation with the terpene moiety,
although ⌬
9
-THCA has dibenzopyran ring structure. It was as
-
sumed that the whole structure of CBDA is important for COX-2
selective inhibition because

-resorcylic acid itself did not inhibit
COX-2 activity. Methylation of the carboxylic acid moiety of CBDA
led to disappearance of COX-2 selectivity. Thus, it was suggested
that the carboxylic acid moiety in CBDA is a key determinant for
the inhibition. Furthermore, the crude extract of cannabis contain-
ing mainly CBDA was shown to have a selective inhibitory effect on
COX-2. Taken together, these lines of evidence in this study sug-
gest that naturally occurring CBDA in cannabis is a selective
inhibitor for COX-2.
Cannabis is one of the oldest known medicinal plants and produces
pharmacologically important substances. Among them, most impor-
tant are the cannabinoids that are unique components in the cannabis
plant. ⌬
9
-Tetrahydrocannabinol (⌬
9
-THC) and cannabidiol (CBD) are
known to be major cannabinoids in the plant. ⌬
9
-THC is known to
have pharmacological effects such as psychoactivity and hallucination
(Dewey, 1986; Howlett et al., 2002). Cannabinoids (i.e., ⌬
9
-THC and
CBD) are also being used as a rheumatoid arthritis agent in clinical
settings (Klein and Newton, 2007) because of their anti-inflammatory
effects (Formukong et al., 1988). In fresh plant materials, most of
⌬
9
-THC and CBD exist as their respective acid forms, ⌬
9
-tetrahydro
-
cannabinolic acid (⌬
9
-THCA) and cannabidiolic acid (CBDA) (Yam
-
auchi et al., 1967; Turner et al., 1980; Taura et al., 2007). The specific
use of acidic cannabinoids including ⌬
9
-THCA and CBDA as the
active pharmaceutical ingredients is not disclosed to date because
these acid forms of cannabinoids are recognized as pharmacologically
inactive forms (Yamauchi et al., 1967; Razdan, 1986; Burstein, 1999).
By focusing on the structures between ⌬
9
-THCA and CBDA, it was
revealed that both acidic cannabinoids have a salicylic acid moiety in
their structures (Fig. 1). Salicylic acid is known to be an inhibitor of
cyclooxygenases (COXs, also referred as prostaglandin H synthases).
Most of the conventional COX-1 and/or nonselective inhibitors con-
tain a carboxylic acid group in their structures, and the COX-2
selective inhibitors reported lack the acid group but contain a sulfo-
nyl-like group.
COX, which exists in at least two isoforms, catalyzes the first key
steps in the synthesis of all the prostaglandins (PGs) by converting
arachidonic acid (AA) into PGH
2
. Thus, COX is a bifunctional
enzyme exhibiting both cyclooxygenase (from AA to PGG
2
) and
peroxidase (from PGG
2
to PGH
2
) activities (DeWitt, 1999; Hinz and
Brune, 2002). COX-1 is constitutively expressed as a housekeeping
enzyme in nearly all the tissues and mediates physiological responses
(e.g., cytoprotection of the stomach, platelet aggregation). On the
other hand, COX-2 is expressed by cells that are involved in inflam-
mation and has emerged as the isoform primarily responsible for the
synthesis of prostanoids involved in acute and chronic inflammatory
states of pathological processes (DeWitt, 1999; Hinz and Brune,
2002). Classical nonsteroidal anti-inflammatory drugs (NSAIDs) such
as acetylsalicylic acid (aspirin) and diflunisal, which are grouped into
the salicylate derivatives of NSAIDs, were shown to inhibit both
COX-1 and COX-2 activities (DeWitt, 1999; Warner et al., 1999).
This study was supported in part by a Grant-in-Aid for Scientific Research (C)
(Research No. 20590127, recipient K.W.) and by a Grant-in-Aid for Young Sci-
entists (B) (Research No. 20790149, recipient S.T.) from the Ministry of Education,
Culture, Sport, Science, and Technology of Japan. This study was also supported
by the Academic Frontier Project for Private Universities from the Ministry of
Education, Culture, Sport, Science, and Technology of Japan.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.108.020909.
ABBREVIATIONS: ⌬
9
-THC, ⌬
9
-tetrahydrocannabinol; CBD, cannabidiol; ⌬
9
-THCA, ⌬
9
-tetrahydrocannabinolic acid; CBDA, cannabidiolic acid;
COX, cyclooxygenase; PG, prostaglandin; AA, arachidonic acid; NSAID, nonsteroidal anti-inflammatory drug; GC, gas chromatography; TMPD,
N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine.
0090-9556/08/3609-1917–1921$20.00
D
RUG METABOLISM AND DISPOSITION Vol. 36, No. 9
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 20909/3374428
DMD 36:1917–1921, 2008 Printed in U.S.A.
1917
at ASPET Journals on November 1, 2015dmd.aspetjournals.orgDownloaded from
None of the COX-2 selective inhibitors belonging to salicylates,
which show selectivity for COX-2 inhibition with low concentrations,
are reported to date (DeWitt, 1999; Warner et al., 1999). Inhibition of
COX-2-dependent PG synthesis accounts for the anti-inflammatory
and analgesic effects of NSAIDs, whereas suppression of COX-1 can
lead to many unwanted side effects (e.g., gastrointestinal ulceration
and bleeding, platelet dysfunctions). Thus, it has been thought that
specific inhibitors for the COX-2–mediated reaction might have ideal
therapeutic actions similar to those of classical NSAIDs without
causing adverse effects. Burstein et al. (1973) have reported that some
of natural cannabinoids inhibited PGE synthesis in bull seminal ves-
icles. However, there is no report whether any cannabinoid(s) selec-
tively inhibit the COX-2 isoform.
The present report describes that CBDA is a selective COX-2
inhibitor in cannabis. The mechanism of selective COX-2 inhibition
by CBDA is discussed.
Materials and Methods
Cannabinoids and Chemicals. Cannabis leaves were harvested from Can-
nabis sativa L. of ⌬
9
-THCA (Mexican) and CBDA strains grown in the
botanical garden of Hokuriku University. ⌬
9
-THC, ⌬
9
-THCA, CBD, and
CBDA were isolated and purified from the cannabis leaves according to the
methods described elsewhere (Aramaki et al., 1968). Purities of these canna-
binoids were checked to be at least 95 to 98% by gas chromatography (GC)
(Watanabe et al., 2005). The crude extract from CBDA strain was prepared by
the methods described previously (Watanabe et al., 2005) except that the crude
extract was not treated with heating to decompose the acid forms (i.e.,
decarboxylation). In fresh plant material, most of CBD has been reported to
exist as its acid form (Turner et al., 1980). The relative contents of CBDA
(77%) and CBD (23%) were determined by thin-layer chromatography anal-
ysis using Fast Blue BB salt as a coloring reagent (Watanabe et al., 2005). To
obtain more information GC analysis was used. In GC analysis, because the
applied CBDA in cannabis is subject to heating conditions causing its decom-
position into CBD (⬃100%), the apparent content of CBDA in the strain was
determined as CBD. The extract from CBDA strain contained 0.22 mg/ml of
CBD. The content of ⌬
9
-THC in the extract of CBDA strain was not deter
-
mined because ⌬
9
-THC concentration was less than the detection limit (ⱕ0.01
mg/ml). CBDA methyl ester was prepared by the methylation of CBDA with
diazomethane (Watanabe et al., 1988). 2,4-Dihydroxybenzoic acid (

-resor-
cylic acid), indomethacin, and resorcinol were purchased from Wako Pure
Chemical Ind., Ltd. (Osaka, Japan). Diclofenac was purchased from Sigma
(St. Louis, MO). All the other reagents were of analytical grade.
Enzyme Sources. Assay of recombinant COX-1 and COX-2 activity was
performed by using a commercially available colorimetric COX (ovine) in-
hibitor screening assay kit (Cayman Chemical Company, Ann Arbor, MI; lot
184104). All the inhibitors added to the reaction system were dissolved in
ethanol and prepared just before use. In this assay, the COX activity was
measured by using N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine (TMPD) as a
cosubstrate with AA (reduction of PGG
2
to PGH
2
). TMPD oxidation was
monitored spectrophotometrically with a 96-well plate reader at 590 nm. No
colorimetric change was observed in control incubations that were performed
by omitting enzymes or with heat-denatured enzymes and inhibitors in com-
bination with TMPD.
Data Analysis. The concentration of the inhibitor that is required to produce
50% inhibition of the enzymatic activity (IC
50
) was determined from the
curves plotting enzymatic activity versus inhibitor concentrations using Origin
7.5J software (OriginLab Corp., Northampton, MA). The details of the calcu-
lations were described in our previous articles (Takeda et al., 2006; Yamaori
et al., 2007). Differences were considered significant when the p value was
calculated to be less than 0.05. All the statistical analyses were performed by
Scheffe´’s F test, which is a type of post hoc test for analyzing results of
analysis of variance testing. These calculations were done using StatView 5.0J
software (SAS Institute Inc., Cary, NC).
Results
Effects of Cannabinoids on COX Activity. The inhibitory effects
of ⌬
9
-THC and CBD and their respective acid forms ⌬
9
-THCA and
CBDA on COX-mediated TMPD oxidation activity were examined
using purified COX as enzyme sources. Although COX-1 activity was
not significantly inhibited by the addition of 100
M ⌬
9
-THC, ⌬
9
-
THCA, or CBD except for CBDA, COX-2 activity was strongly
inhibited by CBDA treatment compared with ⌬
9
-THCA (around 10%)
(Fig. 2). NSAIDs used in this study (indomethacin and diclofenac)
nonselectively inhibited COX-1/-2 (see also Table 1). Furthermore, it
should be noted that although both ⌬
9
-THCA and CBDA have the
FIG. 1. Structures of cannabinoids tested and celecoxib.
FIG. 2. Effects of cannabinoids on COX activity. CBDA was a potent inhibitor for
COX-2. Reactions were initiated with AA, and TMPD oxidation was monitored at
590 nm. Details of the assay conditions are described under Materials and Methods.
Each bar represents the mean ⫾ S.D. (triplicate determinations) of the relative
activity to the control. ⴱ, significantly different (p ⬍ 0.05) from control; #,
significantly different (p ⬍ 0.05) from ⌬
9
-THC-, ⌬
9
-THCA-, and CBD-treated
groups. N.S., not significant.
1918 TAKEDA ET AL.
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same structural moiety, namely, salicylic acid (Fig. 1), the inhibition
potency between these two acids was quite different. Based on the
results obtained in Fig. 2, the following experiments focused on the
inhibition potential of CBDA for COX-2 activity. To obtain a selec-
tivity index (COX-1/COX-2 ratio of IC
50
values), we next determined
IC
50
values for the inhibition of the two COX isoforms by CBDA.
Although CBDA inhibited both COX-1 and COX-2, with apparent
IC
50
values of 20 ⫾ 1.5 and 2.2 ⫾ 0.3, respectively (Fig. 3
, A and B),
it was revealed that CBDA was a selective inhibitor for COX-2 based
on its selectivity index of 9.1 (i.e., ⬎1) (Fig. 3; Table 1).
Structural Requirement for Inhibitory Effect of CBDA on COX
Activity. To determine key structural determinants of CBDA-medi-
ated COX-2 selective inhibition, we performed structure-activity re-
lationship analysis. Interestingly,

-resorcylic acid only significantly
inhibited COX-1 activity, although resorcinol equipotently inhibited
COX enzymes (Fig. 4A). These structural components of CBDA were
added to the reaction system at 2
M, as well as CBDA based on the
IC
50
value for COX-2 inhibition of CBDA (Fig. 3B). Thus, we
hypothesized that

-resorcylic acid moiety of CBDA is one of the key
structures of CBDA-mediated inhibition of COXs (COX-2), although

-resorcylic acid itself is insufficient for selective inhibition of
COX-2. We discussed this point under Discussion. We next studied
the effect of the methyl ester form of CBDA on COX-2 activity (Fig.
4B). The result indicated that the free carboxylic acid portion of
CBDA is important to express its full inhibitory activity. Collectively,
it was revealed that CBDA is able to inhibit COX-2 activity, which
relies on the

-resorcylic acid moiety whose 6⬘-hydroxyl residue has
to be freed from ring formation with the terpene moiety (see the
structure of ⌬
9
-THCA in Fig. 1).
Effect of the Crude Extract from CBDA Strain Cannabis on
COX Activity. This study was performed to investigate the possibility
that CBDA is a COX-2 specific inhibitory component in cannabis; the
inhibition by CBDA would be also seen even in the presence of other
constitutive components in cannabis. The extract from CBDA strain,
which contains CBDA as a major cannabinoid, inhibited both COX-1
and COX-2 activities at a concentration of 37.5
g/ml (25
Min
terms of CBDA concentration) (Fig. 5), although this inhibitory effect
was not observed at a concentration of 7.5
g/ml (5
M in terms of
CBDA concentration) (Fig. 5). The inhibitory magnitude of COX-2
by the extract from CBDA strain cannabis was clearly higher than that
of COX-1. Therefore, CBDA itself is suggested to be a selective
COX-2 inhibitory component in cannabis. However, it was also
revealed that the inhibitory potency of CBDA in the extract might be
much weaker than that of pure CBDA (Figs. 3 and 5). We discussed
this inconsistency under Discussion.
Discussion
Although it was considered that cannabinoid acids in cannabis plant
were inactive cannabinoids, in the present study it was revealed that
CBDA is a selective COX-2 inhibitor in vitro (selectivity index ⫽ 9.1;
Table 1). Burstein et al. (1973) reported that several cannabinoids
were able to suppress the biosynthesis of PGE in bull seminal vesicles,
with large IC
50
values ranging from 70 to 300
M. Because COX-2
is basically inducible by stimulations (DeWitt, 1999; Hinz and Brune,
2002), it is reasonable to understand that they only focused on the
relationship between COX-1 and cannabinoids investigated. In agree-
ment with their report, ⌬
9
-THC was also a very weak inhibitor for
COX-1 in our experiments (IC
50
value; ⬎100
M) (Fig. 2). After the
discovery of COX-2 (Kujubu et al., 1991; O’Banion et al., 1991; Xie
et al., 1991), it became a therapeutic target to avoid side effects by
nonselective COX inhibitors. Thus, we set out to discover constitu-
ent(s) that have COX-2 selectivity in cannabis, and then CBDA was
found to be an inhibitor for COX-2 (Figs. 2 and 3). Although ⌬
9
-THC
and CBD have been reported to have potential use as an analgesic for
patients with rheumatoid arthritis (Klein and Newton, 2007), it has
been suggested that the anti-inflammatory effect of ⌬
9
-THC and CBD
is not mediated by COX enzyme inhibition (Russo and Guy, 2006).
Thus, it is assumed that the inhibition mechanism between ⌬
9
-THC/
CBD and CBDA in anti-inflammation is different. However, includ-
ing this possibility, we are left with further questions that we were not
able to address in these studies, such as does CBDA have potential to
inhibit the COX-2–mediated PG production, which is able to lead to
an anti-inflammatory action in vivo? A study about this possibility is
under investigation.
It is well known that NSAIDs (salicylates) with an acidic carbox-
ylic acid moiety, such as diflunisal and salicylic acid, inhibit COX
activity via forming a salt bridge with Arg120 in COX enzymes (Picot
et al., 1994; Mancini et al., 1995; Kurumbail et al., 1996; Luong et al.,
1996). Thus, cannabinoids containing a carboxylic acid residue in
their structures (i.e., both ⌬
9
-THCA and CBDA) were expected to be
effective COX inhibitors (Fig. 1). However, this does not seem to be
TABLE 1
Comparison of IC
50
values (
M) of various COX inhibitors
The IC
50
values for each of the inhibitors are taken from the published data performed by
using ovine COX-1 and COX-2 as enzyme sources (see Materials and Methods).
Inhibitors COX-1 COX-2 COX-1/COX-2 Ratio
a
Reference
CBDA 20 2.20 9.1 This work
Celecoxib 26.61 0.44 60.48 Tsai et al., 2006
Diclofenac 0.06 0.22 0.27 Johnson et al., 1995
Indomethacin 0.004 0.34 0.01 Tsai et al., 2006
a
Ratio of the IC
50
values for COX-1 and COX-2 can be used as an indication of the COX-2
selectivity of inhibitors. A COX-1/COX-2 ratio of more than 1 indicates preferential COX-2
selectivity.
FIG. 3. Dose-dependent inhibition by CBDA on COX activity. A and B, TMPD
oxidation by two isoforms of COX enzyme (A, COX-1; B, COX-2) was examined
in the presence of indicated concentrations of CBDA. B, is composed of two parts;
left is high concentration range (2.5–100
M), right is low concentration range
(0.1–100
M). Reactions were initiated with AA, and TMPD oxidation was mon-
itored at 590 nm. Details of the assay conditions are described under Materials and
Methods. Each plot represents the mean ⫾ S.D. (triplicate determinations) of the
relative activity to the control.
1919COX-2 SELECTIVE INHIBITION BY CANNABIDIOIC ACID
at ASPET Journals on November 1, 2015dmd.aspetjournals.orgDownloaded from
the case with ⌬
9
-THCA (Fig. 2). There is only one structural differ
-
ence between these, namely, the 6⬘-hydroxyl group of CBDA is freed
from the ring formation with the terpene moiety. Based on this
information, to obtain further experimental evidence we studied the
effects of the structural moieties of CBDA, resorcinol and

-resor-
cylic acid, on COX activity. As expected, both COX-1 and COX-2
activities were inhibited by the reducing agent (i.e., antioxidant)
resorcinol because it has been reported that the COX activity is
sensitive to a large number of reducing agents that act as reducing
cosubstrate for peroxidase reaction of COX enzymes (Markey et al.,
1987). On the other hand, COX-1 but not COX-2 was significantly
inhibited by

-resorcylic acid, a nonreducing agent (Seeram et al.,
2001) (Fig. 4A). It should also be noted here that COX-2 activity was
inhibited by CBDA itself, but COX-1 was not inhibited (Fig. 4A). It
appears that the inhibitory effects of pure CBDA and the crude extract
are different (Figs. 3 and 5). The reason for this discrepancy is not
clear at present, although there is a possibility that CBDA crude
extract contains other interfering component(s) for attenuating
CBDA-mediated COX-2 inhibition. The X-ray crystal structures of
the COX-1 and COX-2 enzymes have presented insight into how
COX-2 specificity is achieved. Within the hydrophobic channel of the
COX proteins, a single amino acid difference in position 523 (isoleu-
cine in COX-1, valine in COX-2) has been shown to be critical for the
COX-2 selectivity (Hood et al., 2003). Thus, the total NSAID binding
site is around 17% larger in the COX-2 isoform (Luong et al., 1996),
which allows COX-2 to bind bulky inhibitors more readily than
COX-1 (Kurumbail et al., 1996). Although the results obtained here
(Fig. 4A) are complex, at least two possibilities might be that 1)

-resorcylic acid itself can enter the catalytic site of both COX
enzymes because of its smaller molecular size compared with that of
CBDA, and 2) the whole molecule of CBDA is fitted by ideal
configuration(s) with COX-2, which leads to COX-2 selective inhi-
bition via its carboxylic acid moiety (see also Fig. 4B). Because there
are no structural similarities between CBDA and celecoxib, a highly
selective COX-2 inhibitor (selectivity index ⫽ 60.48; Table 1; see
also Fig. 1), we propose the possibility that CBDA will be a useful
“prototype” for producing COX-2 selective inhibitors different from
celecoxib.
Taking all these findings into consideration, we have shown that
CBDA in cannabis is a potent and selective inhibitor for COX-2 in
vitro. Further studies are necessary to obtain information about mo-
lecular mechanism of the inhibition.
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Address correspondence to: Kazuhito Watanabe, Department of Hygienic
Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kana-
gawa-machi, Kanazawa 920-1181, Japan. E-mail: k-watanabe@hokuriku-u.ac.jp
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