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Kavalactones from Kava (Piper methysticum) root extract as modulators of

recombinant human glycine receptors

Article  in  Biological Chemistry · May 2019

DOI: 10.1515/hsz-2019-0112

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 Biol. Chem. 2019; aop

Nada Hany Hegazy, Hans-Georg Breitinger and Ulrike Breitinger*

Kavalactones from Kava (Piper methysticum) root

extract as modulators of recombinant human
glycine receptors
https://doi.org/10.1515/hsz-2019-0112 functional similarity with other members of this super-
Received January 15, 2019; accepted May 17, 2019 family, including the nicotinic acetylcholine receptor, as
well as γ-aminobutyric acid (GABAA-R) and 5-HT3 seroto-
Abstract: Roots of kava (Piper methysticum) plant are used
nin receptors (Breitinger and Becker, 2002; Thompson
in almost all Pacific Ocean cultures to prepare a drink with
et  al., 2010). Glycine receptors (GlyRs) are anion selec-
sedative, anesthetic and euphoric properties. One of the
tive inhibitory ligand gated ion channels that are found
main active ingredients of the extract are kava lactones.
in the spinal cord, brain stem and higher brain centers
Here, kava root CO2 extract and three kavalactones, DL-
such as the forebrain, hippocampus, hypothalamus and
kavain, dihydrokavain and yangonin (isolated from whole
medial vestibular neurons as well as retina and cochlea
extract by column chromatography) were tested for their
(Breitinger and Becker, 2002; Breitinger, 2014). GlyRs are
inhibitory action on recombinant homomeric human α1
responsible for regulating sensory stimuli and important
glycine receptors expressed in HEK293 cells. Kava CO2 root
physiological functions including muscle tone, movement
extract, as well as the individual components DL-kavain,
and pain signaling. Cys-loop receptors are made of five
dihydrokavain and yangonin inhibited glycine receptor
homologous subunits forming the ion pore; each subunit
activity in a dose-dependent manner. DL-kavain was the
with a distinctive N-terminal extracellular domain (ECD),
most potent inhibitor (IC50 = 0.077 ± 0.002  mm), followed
a short extracellular C-terminus and four transmembrane
by yangonin (IC50 = 0.31 ± 0.04  mm) and dihydrokavain
segments (Breitinger et al., 2004; Breitinger, 2014). Posi-
(IC50 = 3.23 ± 0.10 mm) which were 4- and 40-fold less active
tive allosteric modulators of the inhibitory GlyR include
than DL-kavain, respectively. Application of kava root
alcohols, neurosteroids, cannabinoids, tropeines,
extract did not reduce maximum currents, but increased
general anaesthetics (Shan et al., 2001; Yévenes and Zeil-
EC50 of glycine. Simultaneous application of kava extract
hofer, 2011), as well as glucose and related saccharides
and strychnine showed additive inhibition, suggesting
­(Breitinger et  al., 2015, 2016; Breitinger and Breitinger,
that binding of kavalactones and strychnine on the recep-
2016). Strychnine, picrotoxin and several other alkaloids
tor is mutually exclusive. Overall, kavalactones exert a
are known as glycine receptor blockers (Shan et al., 2001).
moderate inhibitory effect on the human α1 glycine recep-
Some constituents from ginkgo biloba (used for treatment
tor with DL-kavain being the most potent constituent.
of senile dementia and benign forgetfulness) contain GlyR
Keywords: inhibition; ion channels; kavapyrones; modulators (Ivic et al., 2003; Jaracz et al., 2004; Maleeva
­neuronal receptors; plant extract. et al., 2015). The search for novel GlyR modulators contin-
ues to be of great interest due to the wide distribution of
GlyRs in the body and the various physiological regulatory

Introduction functions controlled by glycinergic transmission (Yévenes

The inhibitory glycine receptor belongs to the Cys-loop
ligand-gated ion channels and shares structural and
*Corresponding author: Ulrike Breitinger, Department of
Biochemistry, German University in Cairo, Main Entrance of
Al Tagamoa Al Khames, New Cairo 11835, Egypt,
e-mail: ulrike.breitinger@guc.edu.eg
Nada Hany Hegazy and Hans-Georg Breitinger: Department
of Biochemistry, German University in Cairo, Main Entrance of
Al Tagamoa Al Khames, New Cairo 11835, Egypt
and Zeilhofer, 2011).
Kava kava (Piper methysticum), also known as intoxi-
cating pepper, is a shrub commonly found in Melanesia,
Micronesia and Polynesian islands (Sarris et  al., 2011).
Native pacific islanders prepare a ritual, mildly intoxicating
beverage from the kava root (Kubatova et al., 2001), which
is ingested to induce a relaxed psychological state during
casual gatherings, ritual ceremonies, and for medicinal
reasons to alter the level of consciousness (Sarris et al., 2011).
The use of kava for relief of stress and anxiety is well docu-
mented (Singh, 1983; Baum et al., 1998; Friese and Gleitz,
1998; Cass, 2004). Phytochemically, kava root extract is

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2      N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones

composed of minor amounts of amino acids, several miner- suggesting a possible effect of kava extract on GlyRs,
als such as aluminum, iron, calcium, magnesium, sodium although modulation of GlyR function by kava ingredi-
and potassium in addition to three chalcones (flavokavins ents has been suggested but not shown until now (Grunze
A, B and C) and a total of 18 kava lactones that have been et al., 2001).
identified so far (Sarris et  al., 2011). Studies made on the Here, we tested whether kavalactones isolated from
isolation and purification of kava concluded that kava lac- kava kava (P. methysticum) might also act on glycine
tones – sometimes named kavapyrones – are the major receptors. We describe the activity of a kava CO2 root
psychoactive constituents in the extract. Kava lactones are extract, containing 60% by weight of kavalactones, and
classified into substituted α-pyrones and 5,6-dihydro-α- some of its isolated constituents as inhibitors of recombi-
pyrones. The latter family of compounds includes kavain, nant human GlyR activity. The action of kava extract and
dihydrokavain, methysticin, dihydromethysticin, yan- of kavalactones DL-kavain, dihydrokavain and yangonin
gonin, desmethoxyyangonin (Kubatova et al., 2001; Sarris was tested on recombinant GlyRs using the whole-cell
et al., 2011) and tetrahydroyangonin (Kubatova et al., 2001). patch-clamp technique. DL-kavain was the most potent
Kavain, dihydrokavain and methysticin are considered to inhibitor of the glycine receptor. Co-inhibition of kava
be the most powerful central nervous system (CNS) active extract with strychnine showed simple additivity, consist-
compounds (Lebot and Levesque, 1996). ent with mutually exclusive binding of both substance
Unlike alcohol, kava was found to be non-addictive classes to the receptor.
(Lebot et al., 1997; Steiner, 2000), and unlikely to produce
tolerance, withdrawal, addiction or morning-after drowsi-
ness. In lower doses, it mostly enhances rather than
impairs cognitive function (Munte et  al., 1993). Occur-
Results
rence of hepatotoxic side effects has been reported
A 60% root extract of kava kava (P. methysticum), kindly
(Gounder, 2006; Teschke et  al., 2013) and is believed to
provided by Flavex Naturextrakte (Rehlingen-Siersburg,
be due to the method of extraction, the use of plant parts
Germany, Table 1) as well as three isolated kavalactones
other than the rhizome for extraction such as peelings or
(Figure 1), namely DL-kavain (Sigma-Aldrich, Munich,
aerial plant parts, wrong cultivation conditions (Sarris
Germany), dihydrokavain and yangonin (both isolated from
et al., 2011; Enna and Norton, 2012), and use of chemical
the whole extract by preparative column chromatography)
reagents instead of water or coconut milk, the traditional
were tested for their action on whole-cell currents mediated
extraction media (Whitton et al., 2003; Sarris et al., 2011;
by recombinant homomeric α1 glycine receptors expressed
Teschke et al., 2011).
in HEK293 cells using patch-clamp electrophysiology.
Numerous effects of kava constituents on neurotrans-
mitter signaling have been reported. Kava was found to
modulate functions of neuronal receptors, most promi- Modulation of glycine receptor currents
nently GABA-Rs (Jussofie et  al., 1994; Yuan et  al., 2002; by kava extract and kavalactones
Tawfiq et al., 2014; Chua et al., 2016; Savage et al., 2018),
as well as dopamine, histamine, opioid, and – to a lesser The effect of the kava lactones DL-kavain, dihydrokavain
extent – serotonin and benzodiazepine receptors (Dinh and yangonin, as well as the whole kava CO2 root extract
et al., 2001; Grunze et al., 2001). Yangonin was shown to on homomeric α1 GlyRs expressed in HEK293 cells was
modulate the endocannabinoid system (Ligresti et  al., examined. Glycine activated a flow of Cl− across the
2012). Anxiolytic and sedative action (Pittler and Ernst,
2003; Sarris et al., 2013), anesthetic (Raduege et al., 2004)
Table 1: Components of kava CO2 root extract.
and neuroprotective (Backhauss and Krieglstein, 1992;
Tzeng and Lee, 2015) effects of kava have been reported. Content (%) Studied here Structure
The interaction of kava constituents with other CNS drugs
Kava CO2 extract Yes
and receptor systems were studied (Dinh et al., 2001; Singh,
7,8-Dihydrokavain 17.2 Yes Figure 1A
2005), and with kava being a widely used medicinal plant, D,L-kavain 14.4 Yes Figure 1B
its physiological activities are monitored and reviewed reg- Methysticin 8.0 – Figure 1C
ularly (Kumar, 2006; Assessment Report, 2017). Yangonin 7.0 Yes Figure 1D
Kava pyrones show a marked antagonistic effect upon 1,2-Desmethoxyyangonin 6.0 – Figure 1E
the convulsant and lethal action of strychnine in doses 7,8-Dihydromethysticin 7.3 – Figure 1F
Sum of kavapyrones 59.9
up to 5 mg/kg injected in mice (Kretzschmar et al., 1970),

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 N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones      3

A B C O

O O O O O O O

OCH3 OCH3 OCH3

D OCH3 E F
O

O O O O O O O

OCH3 OCH3 OCH3

Figure 1: Structures of kavalactones in CO2 extract from kava root.

(A) Dihydrokavain, (B) kavain, (C) methysticin, (D) yangonin, (E) 1,2-desmethoxyyangonin, (F) dihydromethysticin.

Figure 2: Whole-cell currents from HEK293 cells expressing α1 glycine receptors.

(A) Application of increasing concentrations of glycine in the absence of inhibitor (concentration–response data). (B) Traces of DL-kavain
(μm), dihydrokavain (mm), yangonin (mm), kava CO2 extract (mg/ml), and strychnine (nm) with increasing concentrations in the presence of
50 μm glycine.

cell membrane, causing an inward current (Figure  2A). were determined. Currents were normalized to the
Increasing concentrations of glycine (10–1000 μm) were maximum response produced at saturating concentra-
applied and glycine concentration-response curves tions (1000  μm) of glycine of each cell (Figure 2A,B).

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4      N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones

Concentration-response data from 10 individual cells
were pooled to give an averaged concentration-response
curve (Figure 3A) with an EC50 value of 39.5 ± 2.0 μm
and an average maximum current Imax of 3023 ± 486 pA
(Table 2).
Glycine receptor-mediated currents (Figure 2B) were
recorded upon application of increasing concentrations of
kava extract (0.001–0.3 mg/ml) in the constant presence
of 50 μm glycine which is ~EC60. Whole-cell currents at dif-
ferent concentrations of pure DL-kavain (1–200 μm), dihy-
drokavain (0.05–10 mm), yangonin (0.05–3 mm), as well as
strychnine control (1–500 nm), were recorded in presence
of 50 μm glycine. Concentration-response curves were
constructed as before (Figure  3B). The resulting average
IC50 values were: DL-kavain (IC50 = 77 ± 2 μm), dihydroka-
vain (IC50 = 3230 ± 100 μm), yangonin (IC50 = 310 ± 40 μm),
kava root extract (IC50 0.060 ± 0.006  mg/ml) and strych-
nine (47.8 ± 4.5 nm). Patch-clamp electrophysiology results
are summarized in Table 2.
To test the mode of action of kava extract, a glycine
concentration-response curve was constructed in the
presence of 0.06 mg/ml kava extract and compared to the
control glycine data (Figure 3A). We observed a shift in
EC50 to larger concentrations in presence of kava extract
(EC50 = 64.5 ± 4.1 μm, n = 5) as compared to control data
without extract (EC50 = 39.5 ± 2.0 μm, n = 10) that was statis-
tically significant (Student’s t-test, p = 0.0109). Maximum
currents, on the other hand, were not altered (Table 2).

Figure 3: Summary of whole-cell patch-clamp current responses from recombinant α1 glycine receptors expressed in HEK 293 cells.
EC50 and IC50 values were determined from fits of the Hill equation to concentration response data (see Methods). Data from 5 to 19 cells per
condition were pooled, plotted as means ± standard deviation (SD) and the best fit line calculated using a non-linear last squares routine.
(A) Concentration response curves for glycine responses. Left panel: glycine alone; right panel: glycine in absence (solid squares, solid line)
and presence (open squares, dashed line) of 0.06 mg/ml kava CO2 extract. (B) Inhibition curves recorded in the presence of 50 μm glycine
(~EC60) and increasing concentrations of indicated kavalactones, kava whole extract, or strychnine. Relative currents were determined by
calculating I(50 μm Gly+Inhibitor)/I(50 μm Gly) and plotted against inhibitor concentration.

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Table 2: Summary of electrophysiological data.
Compound EC50 or IC50 (μm)
Glycine 39.5 ± 2.0
Glycine + kava extract 64.5 ± 4.1
Kava CO2 extract
a
Kava CO2 extract
a
Kava CO2 extract + 30 nm strychnine
DL-kavain 77 ± 2
Dihydrokavain 3230 ± 100
Yangonin 310 ± 40
Strychnine IC50 0.048 ± 0.005
Strychnine Kib 0.021 ± 0.002
EC50 and IC50 values were determined from concentration-response analysis (Figure 3); number of cells (n) is indicated. Means ± SEM are given.
a
From ratio analysis (Figure 4B), Ki values were calculated from the slope of the current ratio regression lines in absence and presence of
30 nm strychnine.
b
Ki calculated using the Cheng-Prusoff correction.
To test whether strychnine and kava extract target
the same or different site(s) on the receptor, a control
experiment was performed where a ratio method for the
determination of receptor inhibition was used (Breitinger
et  al., 2001; Raafat et  al., 2010; Breitinger, 2012). Here,
the ratio of control currents in the absence of inhibitor,
Igly, divided by the current amplitudes recorded in the
presence of inhibitor X, Igly,X is plotted against inhibitor
concentration [X]. This results in a linear relationship
for both, non-competitive and competitive inhibitors at
I dition for a competitive inhibition mechanism. Therefore,
low concentrations for a plot of gly   vs. [X] (Figure 4B).
Igly, X
In the case of two inhibitors targeting the same site on
the receptor, one would expect an upward shift of the
ratio curve with an unchanged slope. Indeed, when
the inhibition curve for kava extract was determined in
the absence or presence of 30 nm strychnine, the slope
of both curves was the same within experimental error,
with slopes of 26.5 ± 2.5 (mg/ml)−1 for kava extract alone
and 29.7 ± 4.3 (mg/ml)−1 for kava extract in the presence
of 30 nm strychnine. The resulting inhibition constants
were Ki = 0.038 ± 0.004 mg/ml for kava extract alone, and
Ki = 0.034 ± 0.005 mg/ml in the presence of 30 nm strych-
nine. In the case of two different binding sites, the slope of
the line would be expected to increase. Thus, the expected
ratio curve for the case of two separate, independent
binding sites (equation 3, Materials and methods) was
calculated using the inhibition constant of Ki = 21.2 ± 2.3
nm for strychnine (Figure 2), calculated from the IC50 of
48.2 ± 5.0 nm and the Cheng-Prusoff correction for our
experimental conditions (50 μm glycine, EC50 = 39.5 ± 2.0
μm for glycine, Table 2). The simulated curve (dotted line,
Figure 4B) does not agree with the data. Indeed, the inhi-
bition curve for kava extract in presence of strychnine was
N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones      5
EC50 or IC50 (mg/ml) nH Imax (pA) n
1.70 ± 0.13 3023 ± 486 10
1.43 ± 0.14 3074 ± 1793 5
0.060 ± 0.006 1.60 ± 0.11 17
0.038 ± 0.004 17
0.033 ± 0.005 9
0.018 ± 0.001 1.49 ± 0.06 17
0.75 ± 0.02 1.03 ± 0.04 10
0.08 ± 0.01 1.22 ± 0.18 15
1.32 ± 0.16 9
just upward shifted (Figure 4B). This would be expected
for strychnine and kavalactones acting additively (no
synergism), thus targeting the same site on the receptor.
Given a possible common binding site for strychnine
and kavalactones on the receptor, a competitive mode
of inhibition by kava is feasible, as the binding sites for
glycine and strychnine are considered partially overlap-
ping. The observation that EC50 does not appear to be
reduced at saturating concentration of agonist is then an
important criterion and necessary but not sufficient con-
the results seen from the dose-response curve at high con-
centrations of glycine (Figure 3A) were verified in a sepa-
rate experiment. Current responses to 1 mm and 3 mm of
glycine (saturation), and 0.1 mg/ml of kava extract (~IC90)
were recorded, showing indeed that peak currents were
not reduced in the presence of kava (Figure 4C). Currents
were normalized to Imax of the glycine control to accom-
modate cell to cell variations of maximum currents, and
the effect was reproducible (n = 3 cells per condition,
Figure 4E). The maximum current spikes were indeed true
glycine-mediated currents, as is visible from an enlarge-
ment of the initial current peak (Figure 4C insert). When
glycine (1 mm) and kava extract (0.1 mg/ml) were applied
for a longer time (10 s) in a separate measurement, it was
apparent that kava extract increased the rate of desensi-
tization without reducing maximum currents (Figure 4D).
Discussion
Kava lactones are the major psychoactive constitu-
ents in kava root extract. Here, we studied the direct
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6      N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones

Figure 4: Summary of inhibitory action of tested kavalactones.

(A) Bar chart showing IC50 values of DL-kavain, dihydrokavain, yangonin and kava CO2 extract. IC50 values were converted to mg/ml for
better comparison between extract and single compounds. (B) Ratio plot of inhibition of glycine receptor-mediated currents by increasing
concentrations of kava CO2 extract in the absence (solid squares, solid line) and presence of 30 nm strychnine (open circles, broken line)
at 50 μm (~EC50) of glycine. A plot of Iglycine/Iglycine+kava or Iglycine/Iglycine+kava+strychnine vs. [kava] results in a straight line. Similar slopes of both lines
are indicative of additive (non-synergistic) inhibitory action of kava extract and strychnine on the glycine receptor. For comparison, the
expected inhibition curve for the case of superadditive inhibition (two independent binding sites) of kava extract and strychnine was
simulated using the inhibition constants obtained from IC50 analysis is drawn (dotted line), indicating that kava extract and strychnine act
additively, targeting the same site on the receptor. (C) Whole cell currents at glycine concentrations of 1000 μm and 3000 μm in the absence
or presence of kava extract (0.1 mg/ml ~ IC90). Note unchanged Imax and fast desensitization induced by kava extract. Enlargement of traces
at 3k glycine in absence (left trace) and presence of 0.1 mg/ml kava extract (right trace). Scale bars are 0.5 nA and 50 ms. (D) 10-s recordings
of whole-cell currents at saturating concentration of glycine (1 mm) in the absence and presence of kava extract, showing increased
desensitization induced by kava. (E) Summary of Imax values at saturating concentrations of glycine and kava extract. Differences were not
significant (n = 3).

action of kava extract and three isolated kavalactones,
DL-kavain, dihydrokavain and yangonin (Figure 1) on
recombinant human α1 glycine receptors expressed in
HEK293 cells. The results from patch-clamp whole-cell
current recordings (Figure 2) indicated that kava CO2
root extract, DL-kavain, dihydrokavain and yangonin
were all inhibitors of the receptor, reducing glycine-
mediated currents in a dose-dependent manner (Figures
2 and 3).
Kava constituents are well known for their activ-
ity on central nervous signal transmission, including
anxiolytic (Garrett et  al., 2003; Pittler and Ernst, 2003;
Sarris et  al., 2013), and neuroprotective (Backhauss and
Krieglstein, 1992; Tzeng and Lee, 2015) activities. Inter-
action of kavalactones with neurotransmitter systems
has often been demonstrated through indirect studies:
DL-kavain potentiated NMDA-mediated excitotoxic-
ity in hippocampal neurons (Mulholland and Prender-
gast, 2002), kavain and dihydromethysticin modulated
field potential changes in hippocampal preparations
from guinea pig (Walden et  al., 1997), and kava extract
and several of its constituents were shown to affect the

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 N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones      7
concentration of neurotransmitters in rat brain areas
(Baum et  al., 1998). A strong, but indirect evidence of
action of kava constituents on GlyRs was the demonstra-
tion of antagonism of kavalactones against strychnine
toxicity in mice (Kretzschmar et  al., 1970). Thus, modu-
latory activity of kavalactones on GlyRs would not be
surprising. There are reports in the literature of a direct
action of kava and its constituents on neurotransmitter
receptors. Identified targets include transient receptor
potential (TRP) channels (Shimoda et al., 2015) as well as
vascular Ca channels (Martin et  al., 2002; Hoover et  al.,
2006), and sodium channels (Gleitz et al., 1996a,b). By far
the best-­characterized action of kava constituents is on
GABAA receptors, where positive modulation of brainstem
GABAergic signaling (Yuan et al., 2002), as well as mod-
ulation of GABAA-R binding (Jussofie et  al., 1994) were
reported. Co-application of kava enhanced the effects
of the GABAA potentiator diazepam (Tawfiq et al., 2014),
and a detailed mechanistic studied showed the direct
potentiation of GABAA receptor-mediated currents by
kavain (Chua et al., 2016), which is thus shown to be the
underlying mechanism of kava potentiation of GABAergic
signaling.
In our hands, DL-kavain was the most potent inhibi-
tor of glycine receptor activity, while the IC50s of kava
whole extract and yangonin were ~3-fold increased.
Dihydrokavain was much less active, with a ~40-fold
higher IC50 as compared to kavain (Figure 4A, Table 2). It
may be speculated that loss of the central C7-C8 double
bond of kavain and the increase in rotational freedom
may be responsible for the reduced inhibitory activity
of dihydrokavain. An additional -OCH3 group at the aro-
matic ring, as found in yangonin seemed to reduce inhib-
itory potency as well. Given the high structural similarity
of all kavalactones present in the whole extract, it seems
likely that they all bind to the same inhibitory site on the
receptor.
Our experiments covered the concentration range of
20–3000 μm for yangonin and 10–100 mg/l for whole kava
extract. Plasma concentrations of yangonin and methys-
ticin in mice were reported to be in the order of 1–10 mg/l,
corresponding to 3–40 μm (Woelk, 1995). These were
achieved after ip injection of 20–100  mg/kg of the sub-
stances. For human recreational or anxiolytic use, doses of
kava extract range from 50 to 300 mg, ~1–5 mg/kg, heavy
users may consume up to 2500  mg/day of kava extract
(see https://kava.com/kavalactones-dosage/). Thus, with
all caveats of such extrapolations, we observe effects of
kava on the glycine at or near physiological plasma levels
of kava constituents.
Mechanisms of activation and inhibition of ligand-
gated ion channels are complex, as activation com-
prises several elementary steps (ligand binding, channel
opening/closing = gating, desensitization), and each of
these steps may be modulated by inhibitors or poten-
tiators. We found that maximum currents of the glycine
receptor were not affected, EC50 was increased (shifted to
the right), and receptor desensitization markedly accel-
erated in the presence of kava extract (Figure 4C–E).
­Different mechanisms of modulation could be considered:
(i) Unchanged Imax and a right-shift of the dose-response
curve, as observed, is often associated with competitive
inhibition. However, the observed increase of the desen-
sitization rate is not consistent with this mechanism. (ii)
Open channel block – an uncompetitive mode of inhibi-
tion – would be compatible with unchanged Imax (only the
open channel can be blocked), and accelerated desensi-
tization, but not with a right-shift of the EC50 curve, since
open channel block does not alter EC50 (Gunthorpe and
Lummis, 1999). (iii) It is feasible that unliganded recep-
tor species have a higher affinity for kavalactones than the
liganded receptor, which would result in unchanged Imax
and increased EC50. Changes in desensitization rate are
not strictly required by this model, but would not argue
against it, as liganded and unliganded receptor complexes
may have different desensitization rates. This would be an
allosteric mechanism that nevertheless shows some dis-
tinct hallmarks of competitive inhibition. An allosteric
modulation of glycine receptors by kava constituents is
indeed supported by the literature, as it has been noted
that kavain is an allosteric potentiator of GABAA recep-
tors (Chua et  al., 2016), likely acting similar to benzodi-
azepines (Tawfiq et al., 2014). The action of kavain on the
GABAA-R has been identified to be mediated through a site
which is distinct from both, the agonist binding site, and
the benzodiazepine site (Chua et al., 2016).
An allosteric mode of action – mechanism (iii) as dis-
cussed – accounted for our observations. Given the simi-
larity between GABAA and glycine receptors, presence of a
distinct site for kavalactones on both receptors would not
be unexpected. Kavain and other kava constituents would
target allosteric sites on both receptors, being positive
modulators of GABAA-R (Chua et al., 2016), and negative
modulators of glycine receptors.
Co-application of the well-established GlyR inhibitor
strychnine and kava whole extract indicated only addi-
tive inhibition, but no synergy (Figure 4B). This would
indicate mutually exclusive binding of strychnine and
kavalactones to the receptor, consistent with both com-
pounds targeting the same binding site (Breitinger et al.,
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8      N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones
2001), or binding of one modulator would induce a con-
formational change that would prevent the second one
from binding.
In the literature, strychnine toxicity was reported to be
alleviated by kava extract (Kretzschmar et al., 1970), and
potentiated by flavonoids (Raafat et al., 2010), while kava
extract and flavonoids both are inhibitors of the receptor.
Thus, one would expect that both, kava and flavonoids
would enhance strychnine toxicity. However, flavonoids
act synergistically with strychnine (Raafat et  al., 2010),
while kava extract did not, so the mechanism of GlyR mod-
ulation would be different for the two substance classes.
Two separate physiological activities of kavalactones have
to be considered: (i) inhibition of glycine receptors, and
(ii) potentiation of GABAA-R signalling (Chua et al., 2016).
It is known that GABAA agonists and potentiators such as
clonazepam can be used to compensate loss of function
of glycine receptors in hyperekplexia (Zhou et al., 2002).
Thus, kava may increase strychnine toxicity at glycine
receptors while its concomitant potentiation of compen-
satory GABA signaling would alleviate its overall toxicity
at the same time.
Kava extract and kavalactones constitute a class of
neuroactive substances with a broad spectrum of activ-
ity and several molecular targets within the CNS, which
include the inhibitory glycine receptor.
Materials and methods
Materials
Kava kava extract (CO2 extract of kava roots with a standardized
content of 60% kavalactones) was kindly provided by Flavex Natu-
rextrakte (Rehlingen-Siersburg, Germany). The extract contained
six kavapyrones, namely kavain, 7,8-dihydrokavain, methysticine,
7,8-dihydromethysticine, yangonin and 1,2-desmethoxyyangonin
(Table 1). DL-kavain was purchased from Sigma-Aldrich (Deisen-
hofen, Germany).
Column chromatography (CC)
Column chromatography (Heftmann, 2004) was used to separate out
individual kavalactones from the 60% root extract. A 60  cm glass
column with 3 cm diameter was used, packed with 75 g of silica as
stationary phase (pore size: 60 Å; particle size: 63–200 μm). Six
hundred milligrams of kava CO2 extract dissolved in acetone were
adsorbed on a minimum amount of silica, placed on top of the col-
umn and eluted using hexane – isopropanol (70:30 ratio) as eluent
and collecting 4-ml fractions. Column separation was repeated with
a second 600 mg sample of kava extract. Fractions were combined,
solvent removed in a rotavap and fractions examined by thin layer
chromatography (TLC).
Thin layer chromatography (TLC)
TLC was carried out using 2.5  cm × 10  cm silica on TLC aluminum
foils. Silica plates were pre-treated with in an impregnation reagent
(8 g of caffeine dissolved in 200 ml of methylene chloride) and dried
at room temperature for 5 min. TLC sheets were heated on a hot plate
(80°C for 5 min) before application of samples (dissolved in acetone).
The protocol used for TLC visualization and detection of separated
compounds was adopted from the herbal medicine compendium
published by the U.S. Pharmacopeial Convention (USP). Briefly, TLC
plates were treated with a derivatization reagent (170 ml of ice cold
methanol with 20 ml of glacial acetic acid, 10 ml sulfuric acid, and
1 ml anisaldehyde), heated at 100°C for 4 min and viewed under UV
light at 366  nm and visible light (Herbal Medicines Compendium,
2015, https://hmc.usp.org/). Kavalactones were identified according
to Rf values and colour on the TLC plates (Lebot and Levesque, 1996;
Compendium, 2015), and structures verified by MS (not shown).
Cell culture and transfection
HEK293 cells were grown in 10 cm tissue culture Petri dishes in Eagle
Minimal Essential Medium (EMEM) (Sigma-Aldrich, Munich,  Ger-
many) supplemented with 10% FBS (Invitrogen, Karlsruhe, G ­ ermany)
and penicillin/streptomycin (Sigma-Aldrich, Munich,  Germany) at
5% CO2 and 37°C in a water saturated atmosphere. For electrophysi-
ological experiments, cells were plated on poly-L lysine treated glass
coverslips in 6 cm plates. Transfection was performed 1 day after cell
passage using Gene Carrier (Epoch Life Science, Sugar Land, TX,
USA) as transfection agent. Briefly, 1 μg of receptor DNA and 1 μg of
green fluorescent protein DNA were mixed in 100 μl EMEM; in a sec-
ond Eppendorf tube, 3 μl Gene Carrier was added to 100 μl EMEM,
mixed and combined with the DNA solution. After 20 min incuba-
tion at room temperature, the transfection solution was added
drop wise to the cells. Measurements were performed 1–3 days after
transfection.
Electrophysiological recordings and data analysis
Whole-cell recording experiments were performed using a HEKA
EPC10 amplifier (HEKA Electronics, Lambrecht, Germany) controlled
by Pulse software (HEKA) as described before (Breitinger et al., 2018).
Briefly, recording pipettes were pulled from borosilicate glass (World
Precision Instruments, Berlin, Germany) using a Sutter P-97 horizon-
tal puller (Sutter, Novato, CA, USA). Pipettes routinely had an open
tip resistance of 3–4 MΩ. Current responses were measured at room
temperature (21–23°C) at a holding potential of −60  mV. Solutions
were applied using an Octaflow system (NPI electronics, Tamm, Ger-
many), where cells were bathed in a laminar flow of buffer, giving a
time resolution for solution exchange and re-equilibration of about
100  ms. The external buffer consisted of 135  mm NaCl, 5.5  mm KCl,
2 mm CaCl2, 1.0 mm MgCl2 and 10 mm Hepes (pH adjusted to 7.4 with
NaOH); the internal buffer was 140 mm CsCl, 1.0 mm CaCl2, 2.0 mm
MgCl2, 5.0 mm EGTA and 10 mm Hepes (pH adjusted to 7.2 with CsOH).
All agonist-, antagonist- and kava-containing solutions were freshly
prepared from stock solutions which could be kept at −20°C for sev-
eral months without loss of activity. Glycine (100 mm) and strychnine
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(10 mm) stock solutions were prepared in extracellular buffer while
kava extract (15  mg/ml) and kava lactone (40  mm) stock solutions
were prepared in DMSO.
Glycine evoked currents were measured in the absence of any
other glycine receptor modulator as a control, followed by increas-
ing concentrations of strychnine (control), kava CO2 root extract,
DL-kavain, dihydrokavain or yangonin in presence of 50 μm glycine.
Control experiments using 50 μm glycine dissolved in external buffer
with increasing concentrations of DMSO (0.5–2%) was performed on
four cells to verify that DMSO at these concentrations does not inter-
fere with glycine receptor function.
Dose response data was fitted to the Hill equation
n
[glycine] H
Iglycine = I sat × n n
using a nonlinear algorithm in Micro-
EC50 +  [glycine] H
H
cal Origin. Here, Iglycine is the current amplitude at a given glycine
concentration, Isat is the maximum current amplitude at saturating
concentrations of glycine, EC50 is the glycine concentration at half-
maximal current responses, and nH is the Hill coefficient. Currents
from each individual cell were normalized to the maximum response
at saturating glycine concentrations. IC50 curves were fitted using
n
IC50H
the equation I inh = Iglycine × n n
, where Iinh is the current in
IC50H + [inh] H
­ resence of inhibitor, Iglycine is the current in absence of inhibitor, IC50
p cum G. Forst rhizoma. European Medicinal Agency, Committee
is the inhibitor concentration that causes 50% inhibition, and nH is
the coefficient for inhibition. EC50 and IC50 values were determined
from fitting average concentration values of all investigated cells;
errors from the fitting procedure are given.
Simultaneous application of strychnine and kava extract was
performed to test for possible synergistic effects. An inhibition curve
for kava extract was measured using (i) kava extract alone, and
(ii)  kava extract in the presence of 30 nm strychnine. Glycine con-
centration was 50 μm at all times. Data were analyzed using a ratio
method (Breitinger et al., 2001; Raafat et al., 2010; Breitinger, 2012),
were the ratio of control currents in the absence of inhibitor, Igly, was
divided by the current amplitudes recorded in the presence of inhibi-
tor X, Igly,X. If Igly/Igly,X is plotted against inhibitor concentration [X], a
linear relationship is obtained for non-competitive inhibitors and
competitive inhibitors at low concentrations. For the application of
one inhibitor, inhibition is described by the equation
Igly [X]
 =  1  +   
Igly,X KX
where constants are as described before. The slope of the regression
line is 1/KX.
In the case of two inhibitors, X and Y, competing for the same
site on the receptor, the equation becomes
Igly [ X ] [Y ]
= 1+ + (2)
Igly,X K X KY
where [Y] is the concentration of the second inhibitor, and KY its cor-
responding inhibition constant. In the case of two inhibitors binding
to different sites of the receptor (a necessary condition for synergistic
inhibition), the equation is
Igly [ X ] [Y ]  [ X ]
= 1+ + 1 +  (3)
Igly,X K X KY  K X 
Thus, if inhibitor X is applied at a constant concentration, and
the concentration of the second inhibitor, Y, is varied, one would
N.H. Hegazy et al.: Glycine receptor inhibition by kavalactones      9
expect an upward shift of the ratio curve with an unchanged slope.
In the case of two different binding sites, the slope of the line would
be expected to increase (Breitinger, 2012).
Acknowledgments: We thank Flavex Naturextrakte
(Rehlingen-Siersburg, Germany) for a gift of Kava kava
extract, and Mohamed Abdelhalim (Department of Phar-
maceutical Chemistry, German University in Cairo) for
gas chromatography-mass spectrometry (GC-MS) analy-
sis. The excellent technical support by Mousa Abdalla is
gratefully acknowledged.
Conflict of interest statement: The authors report no con-
flict of interest.
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