Kavalactones and the endocannabinoid system: The plant-derived yangonin is a novel CB1 receptor ligand

Article abstract of DOI:10.1016/j.phrs.2012.04.003

To investigate the possible interactions between kavalactone-based molecules and proteins of the endocannabinoid system and provide novel and synthetically accessible structural scaffolds for the design of cannabinoid receptor ligands sharing pharmacologi

Full text of DOI:10.1016/j.phrs.2012.04.003

Pharmacological Research 66 (2012) 163–169
Contents lists available at SciVerse ScienceDirect
Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs
Kavalactones and the endocannabinoid system: The plant-derived yangonin is a
novel CB receptor ligand
1
a
a
a
b,∗
a,∗∗
Alessia Ligresti , Rosaria Villano , Marco Allarà , István Ujváry , Vincenzo Di Marzo
a
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Italy
iKem BT, Budapest, Hungary
b
a r t i c l e i n f o
a b s t r a c t
Article history:
To investigate the possible interactions between kavalactone-based molecules and proteins of the endo-
cannabinoid system and provide novel and synthetically accessible structural scaffolds for the design
of cannabinoid receptor ligands sharing pharmacological properties with kavapyrones, a preliminary
SAR analysis was performed on five commercially available natural kavalactones and nine kavalactone-
analogues properly synthesized. These compounds were investigated for assessing their cannabinoid
receptor binding affinity and capability of inhibiting the activity of the two major metabolic enzymes of
the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).
Among the molecules tested, only yangonin exhibited affinity for the human recombinant CB1 receptor
with a Ki = 0.72 ␮M and selectivity vs. the CB2 receptor (Ki > 10 ␮M). None of the compounds exhibited
strong inhibitory effects on the two enzymes analyzed. The CB1 receptor affinity of yangonin suggests
that the endocannabinoid system might contribute to the complex human psychopharmacology of the
traditional kava drink and the anxiolytic preparations obtained from the kava plant.
Received 16 January 2012
Received in revised form 4 April 2012
Accepted 5 April 2012
Keywords:
Cannabinoid
Natural products
Kava
Cannabinoid receptors
CB1
CB2
Endocannabinoid
FAAH
MAGL
©
2012 Elsevier Ltd. All rights reserved.
1
. Introduction
century, kava products became available also in Europe for the
treatment of hypertension, gonorrhea and certain nervous con-
ditions [5]. In the 1990s kava organic extracts, usually sold in
encapsulated and tincture forms as over-the-counter preparations,
attracted considerable interest worldwide as a clinically proven
treatment for anxiety, depression, insomnia and stress [6]. Among
the additional subtle pharmacological and behavioral effects of
kava drinking are anesthesia and astringency on the tongue and the
inner lining of the mouth, as well as sociability, and mild euphoria
lasting for several hours. Small doses were reported to produce
mild stimulant effects while larger dosages cause muscle relax-
ation, ataxia, paraesthesia, and somnolence. Low, non-sedating
acute doses of kava do not appear to impair cognitive performance
Kava (“intoxicating pepper”; Piper methysticum Forster) is a
perennial tropical shrub widely cultivated in the South Pacific
Island Countries. Kava is also the name of the aqueous beverage
prepared from the roots of the plant; it has been used by the
islanders for social ceremonies for centuries, though recreational
kava-drinking has recently become popular [1–4]. Preparations
made from various parts of the plant have also been used in local
medicine for a range of illnesses, including fever, pain, headache
and migraines, respiratory problems, sleeping difficulties, diarrhea
or constipation, skin diseases, urogenital and menstrual prob-
lems, convulsion, and weight reduction [4]. By the early twentieth
[
7–9], though long-term studies are lacking. It must also be noted
that early reports do not contain data on the composition and dose
of the drink.
Abbreviations: 2-AG, 2-arachidonoylglycerol; AEA, arachidonoyl ethanolamine
Nevertheless, regular and heavy use of kava is not without risks:
dermopathy, liver and kidney problems, gastrointestinal distress,
as well as impaired vision have been reported [10]. Of these, of
particular concern is hepatotoxicity, which has been linked to the
poor metabolizing capacity of susceptible individuals, drug inter-
actions, extreme dosages, and mould or other impurities of kava
(
anandamide); CB, cannabinoid; COX, cyclooxygenase; GABA, ␥-aminobutyric acid;
9
MAGL, monoacylglycerol lipase; SAR, structure–activity relationship; THC, ꢀ -
tetrahydrocannabinol.
∗ Corresponding author at: iKem BT, Búza u. 32, Budapest, H-1033, Hungary.
Tel.: +36 1 367 0726; fax: +36 1 367 0726.
∗
∗ Corresponding author at: Endocannabinoid Research Group, Institute of
Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,
Comprensorio Olivetti, 80078, Pozzuoli (NA), Italy. Tel.: +39 081 8675093;
fax: +39 081 8041770.
[
11,12]. Since 2002 several countries have banned the sales of kava
extracts, although there is now a rekindled interest in standardized
kava preparations as herbal medicines [13].
E-mail addresses: ujvary@iif.hu (I. Ujváry), vdimarzo@icb.cnr.it (V. Di Marzo).
1
043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phrs.2012.04.003

1
64
A. Ligresti et al. / Pharmacological Research 66 (2012) 163–169
Fig. 1. Chemical structures of the six major kavalactones.
The investigation of the chemical constituents of kava dates
two G-protein-coupled receptors, the cannabinoid type-1 (CB1)
back to the 1860s. The pharmacologically most important
compounds isolated from the root are lipid-soluble ␣-pyrone
derivatives, the kavalactones (or kavapyrones), of which 18 have
been characterized [4]. The most abundant kavalactones are
kavain, 7,8-dihydrokavain, methysticin, 7,8-dihydromethysticin,
yangonin and desmethoxyyangonin (Fig. 1). The total kavalactone-
content of the dried roots ranges from 3 to 20% and depends on
the cultivar (chemotype), geographical location, environmental
conditions, age of the plant, and time of harvest [14,15]. The phar-
macology and underlying mechanisms of action of kava extract
and kavalactones are only partially understood. Four major molec-
ular target types have been identified and characterized [4,13]:
and type-2 (CB ) receptors, the two major endogenous ligands of
2
such receptors, known as endocannabinoids, such as anandamide
(AEA) and 2-arachidonoylglycerol (2-AG), and the enzymes for
endocannabinoid metabolism (see [16] for a recent review). The
present study was initiated to examine the cannabinoid receptor
binding affinity of the five commercially available kavalactones
(desmethoxyyangonin only became available during the revision
process of this article), namely (± )-kavain, 7,8-dihydrokavain,
methysticin, 7,8-dihydromethysticin, and yangonin (Fig. 1), as well
as of a novel series of synthetic compounds, chemically related to
kavain (with various substituents at the sn-4 and sn-6 positions
of the dihydropyran ring and two open-ring molecules) (Fig. 2), in
order to perform a preliminary SAR analysis. We also evaluated the
potential indirect agonist action of these 14 compounds, i.e. their
capability to inhibit the enzymatic hydrolysis of AEA and 2-AG by
fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase
(MAGL), respectively, and hence to prolong endocannabinoid tonic
action at cannabinoid receptors.
(
a) ␥-aminobutyric acid (GABA) and benzodiazepine receptor
+
2+
sites; (b) voltage-gated Na and Ca ion channels; (c) monoamine
uptake and catabolism; (d) arachidonate cascade. Each kavalactone
has a unique pharmacological profile and the overall activity of
the root extract is more pronounced than that of the individual
constituents, which is suggestive of synergism [4]. Despite the
fact that some of the pharmacological effects (e.g. sedation, ataxia,
hyperthermia and bloodshot eyes) as well as analgesic and mood
enhancer actions of kava resemble those of Cannabis1 [1–3],
nothing is known so far of the possible interaction of kava and
kavalactones with the endocannabinoid system, which comprises
2. Materials and methods
2.1. Drugs and chemicals
7
,8-Dihydrokavain, methysticin, 7,8-dihydromethysticin and
yangonin, all isolated from Piper methysticum, were obtained from
PhytoLab GmbH & Co. KG (Vestenbergsreuth, Germany). d,l-kavain
was synthetic and also obtained from PhytoLab GmbH & Co. KG.
1
“
Auch mit der Wirkung des Lattisch und der des Haschisch ist die Kawawirkung
verglichen worden.” In Ref. [1, p. 44].

A. Ligresti et al. / Pharmacological Research 66 (2012) 163–169
165
2.3. Receptor binding assays
The pharmacological activity of kavalactones was evaluated
in terms of percent inhibition of radioligand binding and K . In
i
both cases, competitive binding assays were performed using
membranes from HEK-293 cells transfected with the human
recombinant CB receptor (Bmax = 3.6 pmol/mg protein) and human
1
recombinant CB2 receptor (Bmax = 5.2 pmol/mg protein). Recep-
3
tors were incubated with [ H]CP-55,940 (0.4 nM/Kd = 0.12 nM and
0
.53 nM/Kd = 0.18 nM, respectively, for CB1 and CB2 receptors) as
the high-affinity-ligand and displaced with 10 ␮M WIN 55212-2 as
the heterologous competitor for non-specific binding (K values of
i
9
.2 nM and 2.1 nM for the CB and CB receptor, respectively). Vary-
1 2
ing concentrations of compounds (0.1, 0.5, 1.0, 5.0, and 10.0 ␮M) or
vehicle (DMSO) were tested following the procedure described by
the manufacturer (Perkin Elmer, Milan, Italy). Displacement curves
3
were generated by incubating drugs with [ H]CP-55,940 for 90 min
◦
at 30 C. K values were calculated by applying the Cheng-Prusoff
i
equation to the IC50 values (obtained using the GraphPad® pro-
gram) for the displacement of the bound radioligand by increasing
concentrations of the test compound. Data are reported as means
of n = 3 experiments.
2.4. FAAH inhibition studies
The effect of varying concentrations of compounds (0.5, 1.0,
5
.0, and 10.0 ␮M) and vehicle (DMSO) on AEA hydrolysis was
measured by incubating the membrane fractions from rat brain
◦
(
70 ␮g/sample) in Tris–HCl 50 mM, at pH 9 for 30 min at 37 C
14
in presence of [ C]-AEA properly diluted with unlabelled AEA.
After incubation, the amount of [14C]ethanolamine produced enzy-
matically was measured by scintillation counting of the aqueous
phase after extraction of the incubation mixture with 2 volume-
equivalents of CHCl /MeOH 1:1 (by volume). Data are expressed as
3
the concentration exerting 50% inhibition of AEA hydrolysis (IC50),
calculated by GraphPad . Data are reported as means of n = 3 exper-
Fig. 2. Chemical structures of the novel synthetic analogues.
®
iments.
The purity of all kavalactones was >97%, as stated by the sup-
2
.5. MAGL inhibition studies
plier. 2-AG (purity >95%) was purchased from Cayman Chemicals
3
(
Ann Arbor, MI, USA), while 2-arachidonoyl-[ H]glycerol (specific
The effect of varying concentrations of compounds (0.5, 1.0,
.0, and 10.0 ␮M) and vehicle (DMSO) on 2-AG hydrolysis was
activity 40 Ci/mmol) was from ARC, Inc. (St. Louis, MO, USA). Test
compounds were dissolved in DMSO to make serial dilutions that
were used the same day. WIN 55212-2 was obtained from Tocris
5
measured by incubating the 10,000xg cytosolic fraction of COS
cells (100 ␮g/sample) in Tris–HCl 50 mM, at pH 7.0 at 37 C for
2
◦
3
Cookson Ltd. (Avonmouth, Bristol, UK). [ H]CP-55,940 (specific
3
0 min, with synthetic 2-arachidonoyl-[ H]-glycerol properly
activity 144 Ci/mmol) was purchased by Perkin Elmer (Milan, Italy).
diluted with unlabelled 2-AG. After incubation, the amount of
3
[
H]glycerol produced was measured by scintillation counting of
2.2. Chemical synthesis of racemic kavain analogues
the aqueous phase after extraction of the incubation mixture with
volume-equivalents of CHCl /MeOH 1:1 (by volume). Data are
2
3
Synthesis of the new compounds was accomplished as depicted
expressed as the concentration exerting 50% inhibition of 2-AG
hydrolysis (IC50), calculated by GraphPad . Data are reported as
means of n = 3 experiments.
®
in Fig. 1 of the Supplementary Materials. All kavain analogues 1a-
d were prepared from the appropriate aldehyde 5 in only three
steps. The iodine-promoted vinylogous aldol addition of Chan’s
diene 4 [17] to different aldehydes (5a–d), promoted by molec-
ular iodine and followed by desilylation with trifluoroacetic acid,
gave the desired ␦-hydroxy-␤-ketoesters 3a–d in high yields [18].
3
. Results
3.1. Affinity of the compounds for CB1 and CB2 receptors
Treatment of 3a–d with K CO3 in MeOH gave directly ␤-keto-␦-
2
lactones 2a–d, which could be either isolated by acidic work-up
or treated immediately with dimethyl sulfate for the synthesis of
the corresponding kavalactone analogues 1a–d. Due to its sim-
plicity, inexpensiveness, efficiency and versatility this synthetic
strategy is very attractive and, by using different classes of aldehy-
des, potentially useful in the generation of a library of structurally
diverse compounds. The spectroscopic and spectrometric charac-
terization of the purified synthetic compounds is reported in the
Supplementary Materials.
This study examined the affinity of five kavalactones and
nine synthetic derivatives for the human recombinant CB1 and
CB2 receptors. The calculated IC50 and Ki values are reported
9
in Table 1. For comparison, the corresponding values of ꢀ -
tetrahydrocannabinol (THC), the well-characterized Cannabis
component and cannabinoid CB and CB receptor agonist, are also
1
2
shown in Table 1. Among all the compounds tested, only yango-
nin exhibited the ability to displace bound radioligand, indicating
a measurable affinity for CB1 receptors. In particular, yangonin

1
66
A. Ligresti et al. / Pharmacological Research 66 (2012) 163–169
Table 1
3
Effect of five natural kavalactones and nine synthetic derivatives on [ H]-CP55940 binding to the human recombinant cannabinoid receptors. The activity, expressed as IC50,
was determined by non-linear regression of the inhibition of radioligand binding exerted by increasing concentrations of test compounds. When the IC50 was lower than
®
1
0 ␮M, the Ki values were calculated by applying the Cheng–Prusoff equation to the IC50 values (obtained by GraphPad ). Percent of binding displacement at the maximum
9
concentration tested is also reported. ꢀ -THC and WIN 55212-2, tested under the same experimental conditions, are shown as reference compound. When applicable, data
are reported as means ± SD of three experiments.
Sample
IC50, CB1
Ki, CB1
Max effect at CB1
% displacement)
IC50, CB2
Ki, CB2
Max effect at CB2
(% displacement)
(
7
7
,8-Dihydrokavain
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
1.79 ± 0.53 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
–
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
0.72 ± 0.21 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
0.0041 ␮M
10 ␮M
23.63 ± 3.21%)
10 ␮M
20.13 ± 2.15%)
10 ␮M
14.10 ± 4.13%)
10 ␮M
29.09 ± 8.22%)
25 ␮M
98.40 ± 5.63%)
10 ␮M
8.20 ± 4.17%)
10 ␮M
16.40 ± 3.33%)
10 ␮M
0 ± 1.22%)
10 ␮M
10.36 ± 3.84%)
10 ␮M
20.47 ± 5.65%)
10 ␮M
2.90 ± 0.82%)
10 ␮M
8.20 ± 2.41%)
10 ␮M
16.47 ± 2.41%)
10 ␮M
2.30 ± 1.11%)
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
–
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
0.0089 ␮M
10 ␮M
(
(8.16 ± 3.21%)
,8-Dihydromethysticin
10 ␮M
(
(12.58 ± 6.11%)
10 ␮M
d,l-Kavain
Methysticin
Yangonin
(
(16.14 ± 1.34%)
10 ␮M
(
(9.55 ± 2.21%)
10 ␮M
(
(38.24 ± 12.34%)
1
1
1
1
2
2
2
3
3
a
b
c
10 ␮M
(
(4.04 ± 2.21%)
10 ␮M
(
(45.40 ± 5.46%)
10 ␮M
(
(19.50 ± 4.17%)
10 ␮M
d
a
b
c
(
(25.67 ± 3.87%)
10 ␮M
(
(3.49 ± 0.84%)
10 ␮M
(
(6.60 ± 2.26%)
10 ␮M
(
(0 ± 0.52%)
10 ␮M
a
b
(
(8.82 ± 4.11%)
10 ␮M
(
(13.15 ± 3.52%)
ꢀ9
-THC
showed selectivity for the CB1 receptor with a K = 0.72 ␮M; its
future from the further chemical modification of these molecules.
THC and WIN 55212-2 produced the expected displacement in the
binding experiments (Fig. 3).
i
affinity for the CB2 receptor was significantly lower (K > 10 ␮M),
i
although a 38.2% inhibition of specific binding was noted at 10 ␮M,
the highest concentration tested (Fig. 3). The other compounds
were much weaker ligands at both receptors, their displacement
of the radioligand being always lower than 50% at 10 ␮M, the high-
est concentration tested (Table 1). However, some compounds, 1b
and 1d in particular, did produce significant displacement of radi-
3.2. Inhibition of FAAH and MAGL activities
All compounds were examined for their inhibitory activity
towards FAAH- and MAGL-like activities. As shown in Table 2, all
compounds tested were very weak inhibitors of the two enzymes,
with IC50 values >10 ␮M. However, for the natural-derived kavalac-
tones we observed a distinct activity depending on the enzyme
analyzed. In fact, except for dihydrokavain, all the molecules were
found to weakly inhibit FAAH and, in contrast, to weakly stim-
ulate MAGL activity at the highest concentration tested (10 ␮M)
oligand from CB2 but not CB , receptors (Table 1), thus raising the
1
possibility that also selective CB ligands might be developed in the
2
(Table 2).
4. Discussion
Based on some of the reported pharmacological actions of kava
drink, we have studied here the effects of five kava-derived com-
pounds and nine related synthetic compounds as direct or indirect
ligands of cannabinoid receptors. We report that one of the kava
constituents, yangonin, is a relatively good ligand of cannabinoid
CB1 receptors, although weaker than THC.
Previous studies have only partially clarified the potentially
mode of the anxiolytic, sedative and other centrally mediated or
peripheral actions of kava drink. Kavalactones, generally accepted
as the main bioactive ingredients of this preparation, bind in vitro
to several target sites with various affinities and elicit a wide
array of physiological and behavioral effects in vivo that could
account for the properties of various kava preparations with
Fig. 3. Displacement curves of yangonin and the reference compound, THC. Also the
displacement curves of WIN 55212-2, used for the determination of non-specific
binding, are shown. Data are reported as mean ± SE of three experiments.

A. Ligresti et al. / Pharmacological Research 66 (2012) 163–169
167
Table 2
Effect of five natural kavalactones and nine synthetic derivatives on the two major catabolic enzymes involved in endocannabinoid degradation. The activity, expressed as
IC50, was determined by non-linear regression of the inhibition of total activity obtained incubating the enzyme in absence of compounds. Positive signs preceding the values
indicate a stimulation of the total enzymatic activity. When applicable, data are reported as means ± SD of three experiments.
Sample
IC50, FAAH
Max effect, FAAH (% inhibition)
IC50, MAGL
Max effect, MAGL (% inhibition)
7
7
,8-Dihydrokavain
>10 ␮M
10 ␮M
>10 ␮M
10 ␮M
(
+1.80 ± 0.12%)
10 ␮M
22.20 ± 6.33%)
10 ␮M
15.64 ± 4.25%)
10 ␮M
16.37 ± 7.32%)
10 ␮M
28.34 ± 4.41%)
10 ␮M
5.40 ± 1.34%)
10 ␮M
6.63 ± 2.22%)
10 ␮M
1.40 ± 0.23%)
10 ␮M
1.91 ± 0.81%)
10 ␮M
4.55 ± 0.33%)
10 ␮M
5.28 ± 3.01%)
10 ␮M
1.85 ± 0.81%)
10 ␮M
15.29 ± 1.23%)
10 ␮M
15.18 ± 4.05%)
(+1.91 ± 0.25%)
,8-Dihydromethysticin
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
>10 ␮M
10 ␮M
(
(+3.25 ± 0.89%)
10 ␮M
d,l-Kavain
Methysticin
Yangonin
(
(+12.12 ± 2.36%)
10 ␮M
(
(+13.82 ± 4.56%)
10 ␮M
(
(+17.39 ± 4.43%)
1
1
1
1
2
2
2
3
3
a
b
c
10 ␮M
(
(15.59 ± 4.81%)
10 ␮M
(
(20.28 ± 2.45%)
10 ␮M
(
(17.82 ± 3.15%)
10 ␮M
d
a
b
c
(
(16.88 ± 1.39%)
10 ␮M
(
(23.21 ± 3.56%)
10 ␮M
(
(17.47 ± 4.73%)
10 ␮M
(
(16.06 ± 0.97%)
10 ␮M
a
b
(
(25.20 ± 6.10%)
10 ␮M
(
(0 ± 0.96%)
different chemical compositions. The anxiolytic effect of kavalac-
tones is thought to be due to modulation of GABA receptors.
However, the results of receptor binding experiments with differ-
ent kava-extracts provided mixed results: both enhancement and
and (± )-methysticin, respectively; (± )-kavain was inactive in this
assay [27]. Finally, studies on the effects of kavalactones on
arachidonic acid metabolism in vitro indicated that (+)-kavain
inhibited arachidonic acid-induced platelet aggregation, ATP-
release, and the synthesis of prostaglandin E2 and thromboxane
A2 with IC50 values of 78, 115, 86 and 71 ␮M, respectively [28].
Another study on the effect of kavalactones on cyclooxygenase
(COX) activity showed that the most active COX-1 inhibitor was
dihydrokavain (58% inhibition at 430 ␮M), whereas the most
active COX-2 inhibitor was yangonin (34% inhibition at 387 ␮M)
[29].
In summary, the various psychoactive and physiological effects
of kavalactones, alone or in combination, are complex and not fully
understood. From in vitro studies, the GABAA receptor, affected by
sub-micromolar to low micromolar concentrations of kavalactones,
particularly kavain, methysticin and dihydromethysticin, appears
to be the most sensitive pharmacological target, whereas effects on
voltage-gated cation channels and monoamine and eicosanoid lev-
els in vitro are observed at much higher concentrations, and in vivo
could be due to interactions with other signaling systems. For these
reasons, we investigated here the possibility that these compounds,
and some of their synthetic derivatives, interact with cannabinoid
receptors.
3
inhibition of [ H]muscimol-binding were noted [19,20]. Nonethe-
less, pure kavalactones weakly but significantly enhanced in vitro
3
the GABAergic response to [ H]bicuculline methochloride, with
kavain being the most active (28% enhancement at 0.1 ␮M) [21].
Animal studies confirmed the GABAergic contribution to the
anxiolytic properties of kava. For example, in a recent mouse
behavioral test, the anxiolytic and sedative profile of a kava
extract (49.3% total kavalactone content) was similar to the GABAA
receptor modulator diazepam, albeit the natural product was sev-
eral hundred-fold less effective [22]. In a discriminative stimulus
paradigm study in rats, a root extract rich in kavain (41%) partially
substituted for chlordiazepoxide, the training anxiolytic drug [23].
Binding studies and electrophysiological experiments suggested
the involvement of voltage-gated cation channels in kavalactone
+
pharmacology [4]. For example, the binding of the Na channel
3
selective [ H]batrachotoxinin-A 20-␣-benzoate to rat brain synap-
tosomal preparation was inhibited by kavalactones with (+)-kavain
being the most active (IC50 = 52.6 ␮M), [24]. The antiseizure effects
of kava extracts and individual kavalactones observed against
electroshock- or strychnine-induced convulsions in animals and
in vitro models appear to be mediated by cation channel inhibi-
Of all the kavalactones and analogues examined in the cur-
rent study, yangonin emerged as the most interesting compound.
As reported in Table 1, the binding affinity of this kavalactone
+
tion [4,25]. Blockade of Na ion channels could account for the
local anesthetic effect of kava. The psychoactivity of kavalactones
has also been related to their effects on monoamine levels in
the central nervous system, including the mesolimbic reward sys-
tem. The extent and nature of these interactions, however, appear
to be complex and depend on the dosage of the actual chemi-
cal as well as the brain region examined [26]. Kava preparations
were also shown to interfere with monoamine metabolism by
inhibiting human platelet monoamine oxidase-B with IC50 values
of 24.0, 39.5 and 28.1 ␮M, for kava extract, desmethoxyyangonin
to the CB1 receptor is measurable (K = 0.72 ␮M), being ∼170-
i
fold lower than that of THC, the main psychoactive constituent
of Cannabis (K = 0.0041 ␮M). Nevertheless, its interaction with
i
the endocannabinoid system could be pharmacologically relevant
and contribute to the behavioral and physiological effects of kava.
Indeed, the K of yangonin is commensurate with the reported value
i
of 1.0 ␮M as the concentration of this kavalactone necessary to
produce maximal enhancement of bicuculline methochloride bind-
ing to GABAA receptor complex [21]. However, further studies are

1
68
A. Ligresti et al. / Pharmacological Research 66 (2012) 163–169
needed to establish the functional, i.e. agonistic or antagonistic,
nature of the interaction of yangonin with the CB1 receptor. Sur-
prisingly, all compounds were nearly inactive at inhibiting FAAH
and MAGL activities, the two major catabolic enzymes involved
in endocannabinoid degradation. FAAH and MAGL are typical
serine-hydrolase presenting slight differences in their catalytic
mechanism. In fact, while AEA hydrolysis is activated by a lysine
residue, which acts as base [30], the hydrolysis of 2-AG is facili-
tated by proximal cysteine amino acids, as demonstrated by crystal
structure studies [31]. Accordingly, lipophilic sulfhydryl-reagents
such as maleimides [32,33] and isothiazolinones [34], as well as
disulfiram [35], are effective inhibitors of this enzyme. Recently,
an analogue of the known ␤-lactone serine hydrolase inhibitor
tetrahydrolipstatin was also found to inhibit MAGL in vitro and to
possess antinociceptive activity in vivo [36]. On the other hand, the
detoxification of kavalactones in humans is postulated to involve
an electrophilic opening of the lactone ring by the sulfhydryl group
of glutathione (GSH) [37,38]. In fact, the observed hepatotoxic-
ity of commercial kava extracts has been explained by the lack
of plant-derived GSH in such preparations. Whether the reactiv-
ity of kavalactones towards critical sulfhydryl groups accounts for
their slight enhancement of the MAGL-catalysed hydrolysis of 2-
AG, observed here, requires further investigation. During the assay,
such a reaction, if reversible, could protect the enzyme from par-
tial oxidative inactivation by contaminants in the assay buffer and
cell homogenate. Such a protective mechanism, involving redox-
sensitive cysteine thiol groups, has recently been proposed for
kavalactones [39].
5. Conclusions
In conclusion, through the screening of five major natu-
ral kavalactones and nine synthetic derivatives on cannabinoid
receptors and endocannabinoid metabolic enzymes, we identi-
fied yangonin as a unique and selective, though not very potent,
CB1 receptor ligand. The sub-micromolar affinity concentration
determined here is within the range found for the GABAA affin-
ity of some of the other kavalactones. The significance of our
results is twofold. First, the CB receptor affinity of yangonin might
1
indicate the involvement of the endocannabinoid system in the
complex human psychopharmacology of the traditional kava drink
and also in the anxiolytic preparations obtained from the kava
plant. Indeed, if at all due to CB -mediated effects, these anxi-
1
olytic effects would indicate for yangonin an agonist action, since
CB1 inverse agonists/antagonists such as rimonabant and tarana-
bant have been shown to cause anxiogenic effects in humans [16].
Secondly, yangonin provides a novel, versatile and synthetically
accessible structural scaffold for the design of new cannabinoid
receptor ligands. Further pharmacological, including functional and
behavioral, studies on the cannabimimetic activity of yangonin and
other kavalactones as well as with a larger number of synthetic
analogues, are thus warranted.
Acknowledgement
We thank PhytoLab GmbH for the supply of the natural kavalac-
tones.
One interesting issue is the question of whether or not the
in vitro micromolar concentrations at which yangonin interacts
Appendix A. Supplementary data
with CB , determined here, might be relevant in vivo. Among
1
kavalactones, only the human pharmacokinetics of kavain has been
studied so far [40]. One and four hours after oral administration of a
single dose of 800 mg (± )-kavain, the blood serum concentration of
this compound was 0.043 ␮M and 0.17 ␮M, respectively. However,
the effective total daily dose reported from clinical trials with com-
mercial kava extract formulations is 60–240 mg of kavalactones
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phrs.2012.04.003.
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