The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects

Article abstract of DOI:10.1038/sj.bjp.0705828

1 The functional characterization of hispidulin (4′,5,7-trihydroxy-6- methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. 2 After chemical synthesis, hispidulin was investigated at recombinant GABA _A/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (α _1-3,5,6β₂γ₂S) indicating positive allosteric properties. Maximal stimulation at α₁β ₂γ₂S was observed with 10 μM hispidulin. In contrast to diazepam, hispidulin modulated the α₆β ₂γ₂S-GABA_A receptor subtype. 3 When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg^-1 body weight (BW) per day) and diazepam (2 mg kg^-1 BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). 4 Permeability across the blood-brain barrier for the chemically synthesized, ¹⁴C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K_in) of 1.14 ml min^-1 g ^-1, measurements approached the values obtained with highly penetrating compounds such as diazepam. 5 Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 μM, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

Full text of DOI:10.1038/sj.bjp.0705828

British Journal of Pharmacology (2004) 142, 811–820
&
2004 Nature Publishing Group All rights reserved 0007–1188/04
$30.00
www.nature.com/bjp
The flavone hispidulin, a benzodiazepine receptor ligand with
positive allosteric properties, traverses the blood–brain barrier
and exhibits anticonvulsive effects
3
3
3
*,1Dominique Kavvadias, 2,6Philipp Sand, Kuresh A. Youdim, M. Zeeshan Qaiser, Catherine
Rice-Evans, Roland Baur, Erwin Sigel, Wolf-Dieter Rausch, Peter Riederer & Peter Schreier
4
4
5
2
1
2
1Food Chemistry, University of Wurzburg, Wurzburg D-97074, Germany; Clinical Neurochemistry, University of Wurzburg,
3
4
Wurzburg D-97080, Germany; King’s College London, London SE1 1UL; Department of Pharmacology, University of Bern,
Bern CH-3010, Switzerland; 5Institute for Medical Chemistry, Veterinary University of Vienna, Vienna, Austria and 6Department
of Psychiatry, University of Regensburg, Regensburg D-93053, Germany
Dedicated to Prof. Dr S. Ebel on his 70th birthday
1
The functional characterization of hispidulin (40,5,7-trihydroxy-6-methoxyflavone), a potent
benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of
central nervous system activity.
2
After chemical synthesis, hispidulin was investigated at recombinant GABAA/BZD receptors
expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA-
induced chloride currents at tested receptor subtypes (a1À3,5,6b2g2S) indicating positive allosteric
properties. Maximal stimulation at a1b2g2S was observed with 10 mM hispidulin. In contrast to
diazepam, hispidulin modulated the a6b2g2S-GABAA receptor subtype.
3
When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in amodel of epilepsy,
hispidulin (10 mg kgÀ1 body weight (BW) per day) and diazepam (2 mg kgÀ1 BW per day) markedly
reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of
animals in the respective treatment groups, vs 80% in the vehicle group).
4
Permeability across the blood–brain barrier for the chemically synthesized, 14C-labelled hispidulin
was confirmed by a rat in situ perfusion model. With an uptake rate (Kin) of 1.14 ml minÀ1 gÀ1
measurements approached the values obtained with highly penetrating compounds such as diazepam.
Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its
,
5
intact form. At a concentration of 30 mM, the flavone crossed the monolayer without degradation as
verified by the absence of glucuronidated metabolites.
British Journal of Pharmacology (2004) 142, 811–820. doi:10.1038/sj.bjp.0705828
Keywords: Hispidulin; GABAA receptor; partial positive allosteric modulator; epilepsy; blood–brain barrier; Caco-2
Abbreviations: BBB, blood–brain barrier; BW, body weight; BZD, benzodiazepine; CNS, central nervous system; DMEM,
Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; GABA, g-aminobutyric acid; GABAA
receptors, g-aminobutyric acid type A receptors; HBSS, Hanks’ balanced salt solution; HPLC, high-performance
liquid chromatography; Kin, rate of uptake; Poct, calculated octanol–water partition coefficient; Ro15-1788,
flumazenil; RT, retention time; TEER, transendothelial electrical resistance; TMS, tetramethylsilane
Introduction
g-Aminobutyric acid (GABA) and GABAA receptors belong
to the major inhibitory system in the central nervous system
(CNS). GABA opens achloride ion selective chnanel in
GABAA receptors, which are equally modulated by a wide
range of drugs including barbiturates, steroids and benzodia-
zepines (BZDs). These receptor modulators interact with
distinct allosteric binding sites on the GABAA receptor
complex. The BZD binding site, so-called BZD receptor, can
be occupied by a variety of substances classified as positive
allosteric modulators, antagonists or negative allosteric
modulators according to their intrinsic activity (Sigel & Buhr,
1997). BZDs, the most widely prescribed tranquilizers, are
considered classical ligands of this modulatory site. They act as
positive allosteric modulators by increasing channel-opening
frequency and confer anxiolytic, anticonvulsant, sedative-
hypnotic, muscle-relaxant effects (Sieghart & Sperk, 2002).
An intensive search has been undertaken over the past decade
with the aim to separate these diverse actions and to find new,
more selective BZD receptor ligands as drug candidates in the
treatment of epilepsy, anxiety and sleep disturbances. Natural
flavonoids count among these candidates and hold promise as
BZD receptor ligands (Medina et al., 1998). They comprise
flavones, flavonols, anthocyanins and related ubiquitous
constituents of higher plants and represent a significant part
of our daily diet. Several biological activities of flavonoids
have emerged, for example, antioxidant, antiviral, anticancer
and chemopreventive properties (Daniel & Wenzel, 2003).
*Author for correspondence; E-mail: kavva@pzlc.uni-wuerzburg.de

812
D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
Effects of plant flavonoids on the CNS are known since 1990,
when the existence of natural anxiolytic flavonoids was first
described (Medina et al., 1998). Flavones like chrysin,
apigenin, wogonin and the recently isolated 6-methylapigenin
have been shown to possess anxiolytic effects in vivo. In
contrast, their sedative, anticonvulsant and myorelaxant
effects appeared less prominent indicating a possible more
selective action of these substances (Medina et al., 1998; Hui
et al., 2002; Marder et al., 2003).
of 14C-labelled compounds was controlled by high-perfor-
mance liquid chromatography (HPLC) analysis with the
absorbance detector Spectroflow 757 (Kratos, Weiterstadt,
Germany) and the flow scintillation analyzer 500 TR (Pack-
ard, Groningen, Netherlands) using authentic references from
synthesis of unlabelled hispidulin.
Synthesis of [14C]hispidulin
Although the sources of many active flavones are well
known, information on their bioavailability and their active
forms in vivo is limited. In particular, absorption, metabolism
and CNS delivery of most flavonoid agents are poorly
understood. Recently, hispidulin (40,5,7-trihydroxy-6-methoxy-
flavone) was isolated from sage (Salvia officinalis L.) and
identified as a potent ligand of the central human BZD
receptor in vitro. With an IC50 value of 1.3 mM, hispidulin
showed the strongest binding activity to the BZD receptor in
comparison to other flavones isolated (Kavvadias et al., 2003).
Hispidulin is a naturally occurring flavone commonly found
in several Artemisia and Salvia species. Several in vitro studies
have demonstrated its potent antioxidative, antifungal, anti-
inflammatory and antimutagenic activities (Chulasiri et al.,
1992; Gil et al., 1994; Tan et al., 1999). The lack of
pharmacokinetic data and unresolved issues with regard to
the crossing of the blood–brain barrier (BBB) prompted us to
investigate its synthesis in a radiolabelled form. In this paper,
we describe the conventional synthesis of flavones as applied
to generating 14C-labelled hispidulin, and present a compre-
hensive characterization of this natural ligand, including
effects on inhibitory hyperpolarization, BBB permeability
and anticonvulsive activity in vivo.
4-Benzyloxy-2,3-dimethoxy-6-hydroxyacetophenone (5)
Potassium carbonate (3 g, 21.7 mmol) was added to a solu-
tion of 2,4,6-trihydroxyacetophenone-monohydrate (2 g,
10.8 mmol) in acetone (30 ml). The mixture was heated to
651C. Three portions of dimethyl sulfate (3 Â 0.7 ml,
22.1 mmol) were added to the reaction solution at 3 h intervals.
The solution was further heated for 3 h, filtered and the filtrate
evaporated to give 2,4-dimethoxy-6-hydroxyacetophenone (1)
1
as a yellow solid (92%): H-NMR (250 MHz, DMSO-d6): d
2.61 (CO-CH3, s, 3H), 3.88 (2-OCH3, s, 3H), 3.93 (4-OCH3, s,
3H), 6.15 (H-3, d, J ¼ 2.1 Hz, 1H), 6.18 (H-5, d, J ¼ 2.1 Hz,
1H), 13.88 (6-OH, s, 1H). Acetophenone 1 (1.5 g, 7.7 mmol)
and dry aluminum chloride (1.5 g, 11.3 mmol) were suspended
in chlorobenzene (20 ml) and refluxed for 1 h. The organic
solvent was evaporated under reduced pressure and the residue
mixed with ice-cold water–HCl (1 : 1) solution (40 ml). The
white precipitate was filtered, dissolved in ethyl acetate and
extracted with 10% sodium hydroxide solution (3 Â 50 ml).
After acidifying the aqueous phase, the 2,4-dihydroxy-6-
methoxyacetophenone (2) was re-extracted with ethyl acetate
(3 Â 100 ml) and crystallized from ethyl acetate (45–60%).
Alternatively, column chromatography was performed accord-
1
ing to Jain et al. (1985) to give 2 as a yellow solid: H-NMR
(250 MHz, DMSO-d6): d 2.58 (CO-CH3, s, 3H), 3.88 (-OCH3,
s, 3H), 5.94 (H-3, d, 2.1 Hz, 1H), 6.04 (H-5, d, 2.1 Hz, 1H),
13.88 (2-OH, s, 1H). Compound 2 (0.5 g, 2.75 mmol) was
dissolved in dry acetone (30 ml) and the solution heated under
reflux overnight after adding potassium carbonate (3 g,
21.7 mmol) and benzyl bromide (0.43 ml, 3.6 mmol). After
evaporating the solvent, 4-benzyloxy-2-methoxy-6-hydroxy-
Methods
Animals
Seizure-prone, male Mongolian gerbils (Meriones unguiculatus)
weighing 70–102 g were kindly provided by the Leibniz-
Institute for Neurobiology (Magdeburg, Germany) and partly
bred at the Institute for Medical Chemistry (Veterinary
University, Vienna, Austria). The in situ brain perfusion study
was carried out on three male Wistar rats (Harlan UK
Limited, Oxon, U.K.) aged 6 months with a body weight (BW)
of 250 g. The animals were housed in single cages under
standard laboratory conditions (201C, 55% humidity, 12 h
light/dark cycle) with free access to food and water.
1
acetophenone (3) was obtained as a brown solid (99%): H-
NMR (250 MHz, DMSO-d6): d 2.62 (CO-CH3, s, 3H), 3.93
(-OCH3, s, 3H), 5.24 (-CH2-, s, 2H), 6.25 (H-3, d, 2.5 Hz, 1H),
6.28 (H-5, d, 2.4 Hz, 1H), 7.49 (-Ph, m, 5H). The phenolic
compound 3 (0.5 g, 1.8 mmol) and sodium hydroxide (0.4 g)
were dissolved in 4 ml water and 0.5 ml pyridine. A solution of
potassium persulfate (0.55 g, 2.0 mmol) in water (11 ml) was
added over 4 h at a constant temperature of 151C. After
stirring at room temperature for 24 h, the mixture was acidified
to pH 5 and filtered. The filtrate was extracted with diethyl
ether (2 Â 15 ml). The aqueous phase was acidified with
HClconc (2 ml) and refluxed for 1 h after addition of diethyl
ether (15 ml). The diethyl ether phase was dried over sodium
sulfate and evaporated to give 4-benzyloxy-2,5-dihydroxy-6-
methoxyacetophenone (4) as brown syrup (29%): 1H-NMR
(250 Hz, DMSO-d6): d 2.66 (CO-CH3, s, 3H), 3.91 (6-OCH3, s,
3H), 5.28 (-CH2-, s, 2H), 6.46 (Har, s, 1H), 7.48 (Ph, m, 5H).
For partial methylation of intermediate 4 (0.15 g, 0.5 mmol),
1.5 equivalents of dimethyl sulfate (0.07 ml, 0.7 mmol) and
potassium carbonate (0.8 g) were dissolved in dry acetone
(8 ml) and refluxed overnight. After filtration and evaporation
of the solvent, compound 5 was purified by silica gel column
chromatography (2.8 Â 42 cm) using chloroform as eluent. The
All experiments were performed in accordance with the UK
Home Office, Animal Procedures Act 1986 and in strict
compliance with the NIH Guide for the care and use of
laboratory animals.
Syntheses
General experimental procedures Column chromatogra-
phy for compound purification was performed with Merck 60
silicagel (0.040–0.063 mm). 1H-NMR spectraof unlbaelled
compounds were recorded on Bruker AC-250 MHz instru-
ment. Chemical shifts are reported (d) in ppm and coupling
constants (J) in Hz relative to the internal standard
tetramethylsilane (TMS). Chemical and radiochemical purity
British Journal of Pharmacology vol 142 (5)

D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
813
product 5 was obtained in fractions with 500–700 ml elution
volume (59%): 1H-NMR (250 Hz, CDCl3): d 2.66 (CO-CH3, s,
3H), 3.81 (2-OCH3, s, 3H), 4.01 (3-OCH3, s, 3H), 5.14 (-CH2-,
s, 2H), 6.30 (Har, s, 1H), 7.42 (Ph, m, 5H), 13.40 (6-OH, s, 1H).
Identity was confirmed by HPLC analysis using unlabelled
hispidulin as reference.
Synthesis of hispidulin
Unlabelled hispidulin was synthesized from 4-benzyloxy-2,3-
dimethoxy-6-hydroxyacetophenone (5) and unlabelled 4-ben-
zyloxybenzoic acid chloride (7), both prepared in separate
routes from 2,4,6-trihydroxyacetophenone and 4-hydroxyben-
zoic acid, respectively. Synthesis steps were carried out on a
large gram-scale as described above for 11. The product was
purified by silica gel column chromatography (diameter
2.2 cm, length 42 cm) and was eluted by methanol : chloroform
(3 : 97) with an elution volume of 250–370 ml. Identity was
confirmed on the basis of published spectral data using
isolated natural hispidulin as reference (UV, NMR, MS)
(Kavvadias et al., 2003).
4-Benzyloxy-14COCl-benzoyl chloride (7) To asolution
of radiolabelled 4-hydroxy-14COOH-benzoic acid (55 mCi -
mmolÀ1, 250 mCi) in water–ethanol (1 : 3) mixture (450 ml),
unlabelled 4-hydroxybenzoic acid (45 mg, 0.33 mmol), aqueous
4 M potassium hydroxide solution (170 ml) and benzyl bromide
(45 ml, 0.38 mmol) were added. The reaction mixture was
heated under reflux for 6 h. After addition of 4 M potassium
hydroxide solution (0.5 ml), the mixture was further heated for
2 h, cooled down to room temperature and centrifuged at
5000 Â g. The supernatant was removed and the residue was
recrystallized in acetic acid (1 ml) to obtain 4-benzyloxy-
14COOH-benzoic acid (6) as white solid (chemical yield: 36%;
total 14C activity: 78 mCi). The dried acid 6 (27 mg, 0.12 mmol)
was suspended in dichloromethane (1 ml), and oxalyl chloride
(28 ml, 0.15 mmol) was added. The reaction mixture was stirred
at room temperature for 8 h. 4-Benzyloxy-14COCl-benzoyl
chloride (7) was obtained after evaporation of the solvent
under reduced pressure (chemical yield: 100%; total 14C
activity: 76 mCi).
Electrophysiological studies
Xenopus laevis oocytes were prepared, injected and defollicu-
lated and currents were recorded as previously described
(Sigel, 1987; Sigel et al., 1990). Briefly, oocytes were injected
with 50 nl of capped, polyadenylated cRNA dissolved in 5 mM
K-HEPES (pH 6.8). For the triple subunit combinations,
10 nM of the cRNA coding for the different a subunits (a1, a2,
a3, a5 a nda6) and the b2 subunit was used, whereas 50 nM of
the g2S subunit cRNA was used (Boileau et al., 2002). RNA
transcripts were synthesized from linearized plasmids encoding
the desired protein using the mMESSAGE mMACHINEs kit
according to the manufacturers’ recommendations. A poly(A)
tail of about 300 residues was added to the transcripts by using
yeast poly(A) polymerase. The cRNA combinations were co-
precipitated in EtOH and stored at À201C. Transcripts were
quantified on agarose gels after staining with Radiant Red
Fluorescent RNA Stain by comparing staining intensities with
various amounts of molecular weight markers. Electrophysio-
logical experiments were performed by the two-electrode
voltage clamp method at a holding potential of À80 mV. The
medium (pH 7.4) contained 90 mM NaCl, 1 mM KCl, 1 mM
CaCl2, 1 mM MgCl2 and 5 mM HEPES–NaOH (pH 7.4).
GABA, diazepam and hispidulin were applied for 20 s and
a washout period of 4–15 min was allowed to ensure full
recovery from desensitization. Hispidulin was dissolved every
day freshly in dimethyl sulfoxide (DMSO) at a concentration
of 10 mM. The stock solution of Apigenin was 10 mM in
DMSO. The final concentration of DMSO in the medium was
always adjusted to 0.5%, except when hispidulin was assayed
at 100 mM. In this case, final DMSO concentration in the
medium was 1%. These concentrations of DMSO did not by
themselves affect significantly GABA-elicited currents. Both
flavone compounds were tested for their effect on GABA
concentrations that elicited 2–5% of the maximal current
amplitude.
2-14C-hispidulin (2-14C-40,5,7-trihydroxy-6-methoxyfla-
vone) (11) Acetophenone derivative 5 (36 mg, 0.12 mmol)
was added to a solution of 14C-labelled 4-benzyloxybenzoyl
chloride (7) (29 mg, 0.12 mmol, 76 mCi) in dry pyridine (0.7 ml)
and the mixture was stirred at room temperature for 3 h. The
reaction solution was poured on ice-cold 3% HCl (11 ml) and
extracted with ethyl acetate (25 ml). The organic phase was
washed twice with aqueous saturated sodium carbonate, once
with water and then evaporated under reduced pressure to give
ester 8 (chemical yield: 81%; total 14C activity: 55 mCi). Ester 8
was dried in vacuum and redissolved in dry pyridine (0.5 ml).
After addition of freshly powdered potassium hydroxide
(175 mg), the mixture was stirred at 601C for 4 h. The reaction
was stopped by pouring on ice-cold 3% HCl (11 ml). The
diketone compound 9 was extracted with ethyl acetate (25 ml),
washed with aqueous saturated sodium carbonate and then
evaporated under reduced pressure to give the diketone 9
(chemical yield: 55%; total 14C activity: 26 mCi). Diketone 9
was dissolved in acetic acid (1.7 ml), and sulfuric acid (42 ml)
was carefully added. The solution was stirred at 601C for
90 min and then poured on ice (10 g). The resulting flavone 10
was extracted with ethyl acetate (25 ml) and dried under
reduced pressure after solvent evaporation (chemical yield:
60%; total 14C activity: 14 mCi). For selective demethylation
and deprotection of the hydroxy groups, flavone 10 was
dissolved in dichloromethane (1 ml) and cooled down to about
À651C. Boron trichloride solution (1 M in dichloromethane,
0.25 ml) was then added and the mixture was stirred at –651C
for 90 min. The reaction was stopped by addition of saturated
sodium bicarbonate (1 ml), and the aqueous solution was
extracted with diethyl ether (3 Â 5 ml). The crude 2-14C-
hispidulin (11) was purified by silica gel column chromato-
graphy (1.9 Â 18 cm) with methanol : chloroform (3 : 97) as
eluent. The major activity of pure flavone 11 (3 mg, 0.01 mmol)
was found in fractions eluted with 55–70 ml of the MeOH/
CHCl3 mixture (chemical yield: 44%; total 14C activity: 9 mCi).
Gerbil model
Only seizure-sensitive animals were used in the experiments.
They were assigned to treatment groups of 10 animals each
(with the exception of eight animals in the diazepam group).
Drugs (hispidulin and diazepam) were dissolved in ethanol,
diluted with water and added to a pastry manufactured with
British Journal of Pharmacology vol 142 (5)

814
D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
ground chow (Altromins Pellets). Pellets were formed to give
final concentrations of 1.08 mg hispidulin or 0.21 mg diazepam
and max. 1.4% ethanol per 5 g (daily portion). For the control
group, pellets from ground chow, ethanol and water were
manufactured in the same manner and used as vehicle. Test
substances were administered orally for 7 days at a dosage of
10 mg kgÀ1 hispidulin and 2 mg kgÀ1 diazepam per day. During
this period, the animals were left undisturbed to avoid any
unintended provocation of seizures. Epileptic seizures were
induced by a standardized handling procedure before and after
7 days of treatment. For this purpose, each animal was stroked
on the back and was separately placed outside its home cage
while its seizure activity was monitored and graded in terms
of severity as follows: no visible seizure or only minimal seizure
correlates (no noticeable changes of gross behavior, possibly
twitching of vibrissae and pinnae for a few seconds) and full
seizure (clonic–tonic seizure, loss of righting reflexes and body
rollover). Only seizures occurring within 3 min of provocation
tests were counted.
junctional) pathway to ensure that integrity was maintained
during the permeability studies. Aliquots (0.5 ml) of the
transport buffer containing 30 mM hispidulin or apigenin were
added to the donor (apical) side of the inserts. The receiver
(basolateral) chamber contained 1 ml of transport buffer
(HBSS, 25 mM HEPES and 0.1% BSA). Plates were then
incubated at 371C in a Wesbart shaking plate set at 200 rpm
(Wesbart Ltd, U.K.). After 30 min, the inserts were transferred
into new wells containing fresh transport buffer (1 ml). After
60 min, 50 ml aliquots from receiver (t ¼ 30 and 60 min) and
donor wells (t ¼ 60 min) were collected, added to scintillation
cocktail and the [3H] radioactivity was measured using a
Beckman scintillation counter. Flavonoid content in donor
and receiver wells was analyzed by HPLC with photodiode
array detection. To identify glucuronide conjugates, samples
(200 ml) were incubated with b-glucuronidase (20 ml,
10,000 U mlÀ1) for 2 h at 371C and assessed for a decrease in
the suspected glucuronide peak and concomitant increase in
the respective aglycone peak. Stability of both flavonoids was
verified by measurements following incubation of samples in
transport buffer at 371C for 60 min.
In situ rat brain perfusion model
Hemiperfusions were carried out using a modified short-
duration technique previously described by Takasato et al.
(1984). Briefly, [14C]hispidulin (1 mCi, specific activity
67.5 mCi mmolÀ1) was dissolved in ethanol (1 ml), diluted with
perfusion buffer (according to Egleton et al., 1998) to give a
final ethanol concentration of 5% and subsequently perfused
via the right carotid artery of rats for 30, 45 and 60 s (n ¼ 3
animals for each time point). The right hemisphere of the brain
was dissected and the 14C content was determined with a
scintillation counter. Results were expressed as volume of
distribution Vd (ml gÀ1) and uptake rate Kin (ml minÀ1 gÀ1). To
ensure that ethanol concentration did not disrupt the BBB and
affect permeability data, a control experiment was performed
perfusing [14C]sucrose (as a vascular marker) in perfusate
containing ethanol (5% final concentration) for 60 s.
HPLC analysis
HPLC was performed on a Waters HPLC system using a
Nova-Pak C18 column (4.6 Â 250 mm, 4 mm) and a guard
column (4.6 Â 15 mm, 4 mm) as previously described (Youdim
et al., 2003). Flavonoids were identified by comparing
retention times (RTs), photodiode array spectra and sample
spiking with reference substances.
Chemicals
The following chemicals and reagents of analytical purity were
used for the synthesis and experiments: acetic acid, acetone,
aluminum chloride, apigenin, benzyl bromide, boron trichlor-
ide solution (1 M in dichloromethane), chlorobenzene, chloro-
form, diazepam, dichloromethane, diethyl ether, dimethyl
sulfate, DMSO, DMSO-d6, ethanol, ethyl acetate, GABA, 4-
hydroxybenzoic acid, methanol, potassium carbonate, potas-
sium hydroxide, potassium persulfate, pyridine, flumazenil
(Ro15-1788), sodium hydroxide, sodium sulfate, sulfuric acid,
TMS and 2,4,6-trihydroxyacetophenone-monohydrate from
Sigma-Aldrich (Deisenhofen, Germany). 4-Hydroxy-14COOH-
benzoic acid was obtained from American Radiolabelled
Chemicals Inc. (St Louis, MO, U.S.A.). Ultima-FloTM AF
LSC-Cocktail (Packard Bioscience Company, Groningen,
Germany) was used for scintillation counting.
Caco-2 system model
Caco-2 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum, 1%
MEM non-essential amino-acid solution, GLUTAMAX-1
(2 mM) and penicillin–streptomycin (100 U mlÀ1; 100 mg mlÀ1),
and maintained at 371C in ahumidified atmosphere (5% CO 2).
For transport studies, Caco-2 cells (passage 30–50) were
seeded on Transwell inserts (polycarbonate membrane, 12 mm
diameter and 0.4 mm pore size, Corning Costar Co.) at a
density of 1 Â 105 cmÀ2. The transendothelial electrical resis-
tance (TEER) across the Caco-2 monolayers was measured
daily using an epithelial volt-ohmmeter (EVOM, World
Precision Instruments Inc., Hertfordshire, U.K.) with a planar
electrode chamber (Endohm-12, World Precision Instruments
Inc.). Monolayers used in transport studies (20–25 days
The following were used for electrophysiological studies:
mMESSAGE mMACHINEs kit (Ambion Inc.), yeast poly(A)
polymerase (Amersham Biosciences, U.K.), Radiant Red
Fluorescent RNA Stain (Bio-Rad, U.S.A.) and RNA Ladder
(Gibco BRL, Crewe-Cheshire, U.K.). Caco-2 cells were
obtained from the European Collection of Animal Cell
Cultures (ECACC, Wiltshire, U.K.). Further on, fetal bovine
serum, DMEM, MEM solution, GLUTAMAX-1, penicillin–
streptomycin and HBSS were from Invitrogen (U.K.), and
HEPES, BSA, b-glucuronidase (type L-II from Limpets) and
3H-Mannitol were from Sigma (Dorset, U.K.).
postseeding on Transwells) exhibited TEER 4500 O cmÀ2
.
Inserts were washed with Hanks’ balanced salt solution
(HBSS) pH 7.4 and were incubated at 371C for 30 min.
Hispidulin and apigenin (30 mM in DMSO) were diluted to
30 mM in transport buffer (HBSS containing 25 mM HEPES,
0.1% BSA (w vÀ1)). BSA was included to stabilize membrane
and tight junctions. 3H-Mannitol (0.1 mCi mlÀ1, specific activity
20 Ci mmolÀ1) was used as a marker for the paracellular (tight
British Journal of Pharmacology vol 142 (5)

D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
815
Data analysis
in acetic acid with small amounts of sulfuric acid (Horie et al.,
1997). The protecting groups and the labile methoxy group at
C-5 were selectively cleaved by reaction with boron trichloride
in dichloromethane at about À651C to obtain hispidulin,
which was identified on the basis of published spectral data
(UV, NMR, MS) (Kavvadias et al., 2003). For synthesis of
14C-labelled hispidulin (11), radiolabelled 4-hydroxy benzoic
acid with a total activity of 250 mCi was used. Radiolabelled
flavone 11 (total activity: 9 mCi) was obtained with 12%
Electrophysiological studies Currents were measured using
a modified OC-725 amplifier (Warner Instruments Corp.)
in combination with an XY-recorder or digitized using a
MacLab200 (AD Instruments). Current responses have been
fitted to the Hill equation [I ¼ Imax(1 þ (IC50[A]À1)n)À1], where I
is the peak current at a given concentration of GABA, Imax the
maximum current, IC50 the concentration of agonist eliciting
half-maximal current and n the Hill coefficient. The resulting
data are shown as mean7s.e.m.
isotopic incorporation and
a
specific activity of
67.5 mCi mmolÀ1 as determined by liquid scintillation counting
after silica gel column purification.
Gerbil model Paired proportion tests
Electrophysiological studies
(http://www.ubmail.ubalt.edu/~harsham/Business-stat/
otherapplets/PairedProp.htm) were used to examine the null
hypothesis that 7 days’ pharmacological treatment had no
modulatory effect on seizure propensity. P-value of less than
0.05 was considered as significant.
In electrophysiological studies at recombinant GABAA recep-
tors expressed in X. laevis oocytes, hispidulin exhibited a
positive allosteric modulatory effect. The current elicited by
GABA in a1b2g2 GABAA receptors was enhanced in a
concentration-dependent way (Figure 2b). Maximum relative
stimulation at a1b2g2 was achieved with 10 mM hispidulin.
Interestingly, at higher concentrations, a second inhibitory
phase was observed. At 10 mM, stimulation by hispidulin
amounted to about 24% of the stimulation by 1 mM diazepam.
The potentiation of 10 mM hispidulin amounted to 4775%
(n ¼ 3, s.e.m.) and was largely blocked by co-application of the
BZD receptor antagonist Ro15-1788 with a residual stimula-
tion of 1772% (n ¼ 3, s.e.m.) (Figure 2c).
In situ rat brain perfusion model Results were expressed
as volume of distribution Vd (ml gÀ1) using the following
equation: Vd ¼ (dpm gÀ1
)
(ml dpmÀ1
)perfusate, where dpm
tissue
expresses disintegrations per minute of the sample. The uptake
rate Kin (ml minÀ1 gÀ1) was obtained as slope of the plot of Vd
(ml gÀ1) against time (min). Log Poct was calculated using the
KowWin (LogKow) software at http://www.esc.syrres.com/
interkow/logkow.htm.
Substance concentrations of about 50 nM and higher
stimulated the GABA-induced chloride current at all five
receptor subtypes (a1À3,5,6b2g2) investigated (not shown). In
contrast to diazepam, hispidulin also enhanced the GABA-
activated current at a6b2g2 GABAA receptors (Figure 2d).
In order to relate hispidulin’s effects to the effects of a
familiar flavone, apigenin was equally investigated (Figure 1d).
This compound reduced the GABA-induced chloride ion
current and therefore acted as negative allosteric modulator of
GABA (Figure 2e).
Results
Chemical synthesis
A route for the chemical synthesis of hispidulin, including its
14C-labelled form, was developed in our laboratory utilizing
the Baker Venkataraman rearrangement starting with 4-
benzyloxy-2,3-dimethoxy-6-hydroxyacetophenone (5) and 4-
benzyloxybenzoic acid chloride (7) (Figure 1c). Both educts
were prepared by separate routes from 2,4,6-trihydroxyaceto-
phenone and 4-hydroxybenzoic acid. The acetophenone 5 was
synthesized from commercially available 2,4,6-trihydroxy-
acetophenone in five steps as shown in Figure 1a. Transforma-
tion into 2,4-dimethoxy-6-hydroxyacetophenone (1) wa
achieved by reaction with dimethyl sulfate (Srivastava &
Srivastava, 1987). Subsequent selective demethylation with
aluminum chloride in chlorobenzene was performed to obtain
2,4-dihydroxy-6-methoxyacetophenone (2) (Jain et al., 1985).
After protection of the 4-hydroxy group with abenzyl group,
compound 3 was applied to Elbs persulfate oxidation (Baker
et al., 1939). In the last step of the acetophenone preparation,
the newly introduced hydroxy group at position C-5 was
methylated using dimethyl sulfate. The benzoyl chloride 7 was
prepared in two steps according to Figure 1b. Starting from
4-hydroxybenzoic acid, we protected the hydroxy group with
a benzyl group and prepared the acid chloride by reaction of
compound 6 with oxalyl chloride (Cherpeck, 1998; Bracon
et al., 1999). For hispidulin synthesis, compounds 5 and 7
(76 mCi) were converted into dry pyridine to the corresponding
benzoyl ester (Figure 1c). The crude benzoate was treated with
potassium hydroxide to induce an intramolecular Claisen
condensation resulting in formation of diketone 9. The
diketone 9 cyclized to the corresponding flavone by heating
Gerbil model
For the investigation of hispidulin effects on ictal events,
gerbils were selected for their susceptibility to the triggering
of seizures by typical environmental stressors. A visible
confirmatory seizure was inducible at baseline in 28 animals
immediately prior to treatment. Drug effects of hispidulin and
diazepam on seizure incidence are presented in Figure 3. No
ictal phenomena were observed during the period of treatment.
When re-exposed to the same stimuli on day 7, gerbils
experienced milder/fewer seizures in both the hispidulin
(10 mg kgÀ1 BW per day) and the diazepam (2 mg kgÀ1 BW
per day) groups (full seizures in 30% (Po0.04) and 25%
(Po0.04) of animals, respectively), when compared to gerbils
in the vehicle group (full seizures in 80% of animals).
s
Blood–brain barrier study
In accordance with our behavioral findings, perfusion of 14C-
labelled hispidulin into the right carotid artery of the rat
resulted in a linear flux of the flavone to the brain.
[14C]hispidulin in artificial saline was perfused into the right
carotid artery of the rat for 30, 45 and 60 s. Figure 4 shows the
British Journal of Pharmacology vol 142 (5)

816
D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
a
PhCH2Br,
OMe O
Me2SO4,
Me2CO,
K2CO3
AlCl3,
chloro-
benzene
O
OMe
OH
O
OMe O
Me2CO,
K2CO3
OH
HO
BzO
OH
OH
HO
OH
MeO
2
3
1
1. K2S2O8, pyridine,
10% NaOH/H2O
2. HCl
Me2SO4,
Me2CO,
K2CO3
OMe O
O
OMe
MeO
BzO
HO
OH
BzO
OH
5
4
b
c
oxalyl
chloride,
CH2Cl2
PhCH2Br,
HO
BzO
BzO
water/EtOH,
KOH
O
O
O
14
14
14
OH
OH
Cl
7
6
O
14
OBz
Cl
O
+
BzO
OH
O
OBz
BzO
OBz
OH
14
7
BzO
O
14
O
MeO
MeO
OMe
pyridine
KOH,
pyridine
MeO
OMe O
OMe O
5
9
8
OBz
OH
BCl3,
CH2Cl2
BzO
O
HO
O
14
14
HAc/H2SO4
MeO
MeO
O
OMe
O
OH
10
11
d
OH
HO
O
O
OH
12
Figure 1 Chemical syntheses of 14C-labelled hispidulin 11 (c) and its building blocks 5 (a) and 7 (b) and the structure of apigenin (d).
Vd (ml gÀ1) for hispidulin into the right hemisphere. The rate
of uptake (Kin) 1.14 ml minÀ1 gÀ1 was obtained from the slope
of aplot of Vd (ml gÀ1) against time (min). Figure 5 shows the
relationship between log Kin (experimental) and lipophilicity
of hispidulin (log Poct 2.67, calculated octanol–water partition
coefficient) compared with the behavior of known passively
permeating compounds and efflux transport substrates.
intestinal absorption to test for any compound modification
secondary to uptake by epithelial cells. Figure 6a shows the
HPLC profile of the receiver medium following hispidulin
(30 mM) exposure at the donor side of the Caco-2 cell
monolayer for 60 min. The peak with RT at 50.93 min
represents the hispidulin aglycone, in parallel with RT and
diode array spectra of a standard solution. Apigenin (RT
43.40 min) also crossed the Caco-2 cell monolayer in its intact
form as shown in Figure 6b. Following incubation with
b-glucuronidase, no change in either peak was noted,
suggesting the absence of glucuronidated metabolites of
hispidulin or apigenin. Identification of the unknown peaks
Caco-2 cell layer permeability study
The permeation of hispidulin and apigenin aglycones across
the Caco-2 monolayers was investigated in a model of
British Journal of Pharmacology vol 142 (5)

D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
817
Figure 2 Functional properties of hispidulin and apigenin. (a) Hispidulin at 10 mM by itself did not elicit any current but stimulated
currents elicited by 4 mM GABA at recombinant a1b2g2S GABAA receptors expressed by X. leavis oocytes. (b) Concentration
dependence of allosteric stimulation by hispidulin. (c) Stimulation by hispidulin (10 mM) a ta1b2g2S GABAA receptors is partially
inhibited by co-application of the BZD receptor antagonist Ro15-1788 (1 mM). a1b2 GABAA receptors are not stimulated by 10 mM
hispidulin. (d) Allosteric stimulation by hispidulin at a6b2g2S GABAA receptors. (e) Inhibition by apigenin of GABA-induced
currents at recombinant a1b2g2S GABAA receptors. The points indicate the mean7s.e.m. (n ¼ 3–5).
100
80
vehicle group
60
hispidulin group
diazepam group
40
*
*
20
0
day 7
baseline
Figure 3 Effects of vehicle, hispidulin and diazepam on the
occurrence of epileptic seizures in the Mongolian gerbil model (M.
unguiculatus) before and after the application of test substances.
Paired proportion tests (http://www.ubmail.ubalt.edu/~harsham/
Business-stat/otherapplets/PairedProp.htm) were used to examine
the null hypothesis that 7 days’ pharmacological treatment had no
modulatory effect on seizure propensity. *Po0.04.
Figure 4 Volume of distribution (Vd) in the rat brain (right
hemisphere) for [14C]hispidulin plotted against time (n ¼ 3), as
assessed using the Takasato in situ perfusion method. R2 ¼ 0.9929.
British Journal of Pharmacology vol 142 (5)

818
D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
synthesis of hispidulin including its 14C-labelled form allowed
us to perform additional in vitro and in vivo investigations.
Electrophysiological studies
Functional effects of hispidulin were investigated in electro-
physiological studies at recombinant GABAA receptors
expressed in X. laevis oocytes. GABA was always used at
concentrations eliciting 2–5% of the maximal current ampli-
tude in the corresponding GABAA receptor. Hispidulin at
10 mM did not by itself elicit current but exhibited apositive
allosteric modulatory effect by enhancing the GABA-stimu-
lated current at a1b2g2S (Figure 2a). This stimulation was
concentration dependent (Figure 2b). Maximum relative
stimulation at a1b2g2S was achieved with 10 mM hispidulin.
Interestingly, at higher concentrations, a second inhibitory
phase was observed. The fact that action of hispidulin is two-
phasic, going through a maximum, predicts that an overdose
will result in a submaximal effect. At 10 mM, stimulation by
hispidulin amounted to about 24% of the stimulation by 1 mM
diazepam. Assuming that the optimum stimulation observed
corresponds closely to the maximum stimulation, hispidulin is
a partial positive allosteric modulator. The potentiation of
10 mM hispidulin at a1b2g2S amounted to 4775% (n ¼ 3, s.e.m.)
and was largely blocked by co-application of the BZD receptor
antagonist Ro15-1788 with a residual stimulation of 1772%
(n ¼ 3, s.e.m.) (Figure 2c). Potentiation of 10 mM hispidulin at
a1b2 amounted to 171% (n ¼ 3, s.e.m.). We conclude that
hispidulin acts predominantly through the BZD binding site.
Substance concentrations of about 50 nM and higher
stimulated the GABA-induced chloride current at all five
receptor subtypes (a1À3,5,6b2g2S) investigated (not shown). In
this study, we could not detect any receptor subtype selectivity
Figure 5 Relationship between log Kin (experimental) and log Poct
(calculated) of passively permeating compounds (diamonds), other
flavonoids (naringenin, quercetin), substrates for known uptake
(glucose) and efflux (colchicine) transport. Log Kin values with the
exception of hispidulin are taken from Qaiser et al. (in preparation;
see also Youdim et al., 2004). Log Poct was calculated using the
KowWin (LogKow) software at http://www.esc.syrres.com/interkow/
logkow.htm. This program uses fragmental analysis of the
compound structure for its prediction (values show high correlation
with quoted experimental values, P ¼ 0.98).
at RT 43.88 min (Figure 6a) and 36.81 min (Figure 6b) fell
outside the scope of the current study.
Discussion
In a previous study on novel natural ligands of the BZD
receptor from S. officinalis, we isolated the flavone hispidulin
and provided details on its affinity to the BZD binding site
in vitro (Kavvadias et al., 2003). The subsequent successful
Figure 6 HPLC traces at 320 nm obtained from receiver wells of the Caco-2 cells showing permeability of hispidulin (a) and
apigenin (b) in their intact aglycone forms.
British Journal of Pharmacology vol 142 (5)

D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
819
regarding maximal stimulation. The IC50 was 0.8–1.8 mM for
a1b2g2S, a2b2g2S and a5b2g2S, and about 5 mM for a3b2g2S
and a6b2g2S. In contrast to diazepam, hispidulin also
enhanced the GABA-activated current at a6b2g2S GABAA
receptors (Figure 2d). At these receptors, the stimulation by
10 mM hispidulin was 65717% (n ¼ 3, s.e.m.) and in the
presence of 1 mM Ro15-1788 3777% (n ¼ 3, s.e.m.). Recent
studies of Marder et al. (2001) showed absence of binding
activity of several flavonoids for GABAA receptors containing
a6-subunits. Sterical reasons owing to the small lipophilic
pocket L3 at receptors containing a6-unit were hypothesized to
explain this phenomenon. In our studies, hispidulin interacted
also with this receptor subtype. As with other ligands of the
BZD binding site, action at a6b2g2S receptors seems to depend
on ligand structure.
with either hispidulin or the classic tranquilizer diazepam aided
in the treatment of spontaneous seizures.
Few studies have addressed BBB permeability for flavone
derivatives. It was recently shown in an in vitro model by
members of our group that a number of flavonoids, for
example, naringenin and hesperetin, are able to enter the brain
endothelium and cross the BBB as are several flavonoid
metabolites (Youdim et al., 2003). Other related compounds,
for example, epicatechin, possess little or no potential for
permeation. The permeability of hispidulin across the BBB has
not been investigated in cell culture or in vivo. The anti-
convulsant effects of this flavone, illustrated in seizure-prone
Mongolian gerbils, strongly suggested its delivery to the
CNS. To further confirm this important mode of distribution,
we investigated the penetration of hispidulin into the
brain in an in situ rat model. When compared to other
highly BBB-penetrating agents, hispidulin approached both
diazepam and propanolol in terms of uptake by the total right
cerebral hemisphere. When rate of uptake (Kin) by the total
right cerebral hemisphere was plotted against compound
lipophilicity (log Poct 2.67), the behavior of hispidulin was
similar to that of known highly penetrating agents such as
diazepam and propanolol. These findings suggest that
hispidulin permeates the BBB by simple passive diffusion.
However, acontrol perfusion with [ 14C]sucrose indicated that
5% ethanol did not disrupt BBB integrity, so the high
penetration of [14C]hispidulin was not due to paracellular
(junctional) flux, but likely to be transcellular, via passive
diffusion across the endothelial cell membranes. As recently
observed by Youdim et al. (2003) in an in vitro BBB
model, the potential for permeation by other flavonoids is
strongly correlated with lipophilicity. Here we show that
the lipophilicity of hispidulin appears to facilitate its permea-
tion through the BBB.
Apigenin was also investigated. Despite its strong structural
similarity to hispidulin (Figure 1d), apigenin reduced the
GABA-induced chloride ion current and therefore acted as
negative allosteric modulator of GABA in a1b2g2S (Figure 2e)
in the present electrophysiological study. Most probably it
lacks the stimulatory phase observed at low concentrations
of hispidulin and only has the inhibitory properties observed
at higher concentrations. A similar behavior was observed in
a2b2g2S, a3b2g2S and a5b2g2S. Interestingly, in a6b2g2S,
the transient stimulatory phase was also observed. A negative
allosteric effect by apigenin has previously been noted by
Avallone et al. (2000) in primary cultures of cerebellar granule
cells. Thus, its modulatory activity at the BZD receptor site
as suggested by behavioral data (Viola et al., 1995) has been
difficult to ascertain both in primary cell cultures and in
oocytes expressing recombinant receptors (Avallone et al.,
2000; Goutman et al., 2003). Sedative or other BZD-like
effects observed in vivo are therefore presumed to result from
apigenin affinity to an alternative site.
Epileptic seizures are a correlate of hypersynchronous
electrical discharges in the brain resulting from neuronal
overexcitation or deficient inhibition in a given brain area.
The GABA-mediated inhibition is readily enhanced by a large
number of BZD receptor ligands. Consequently, classical BZD
ligands such as diazepam act as effective anticonvulsants
(Treiman, 2001). We therefore hypothesized that other BZD
receptor ligands that possess an agonistic pharmacological
profile similar to that of hispidulin could also have these
effects. In this study, the diazepam-like activity of hispidulin in
vitro led us to hypothesize anticonvulsant effects of the flavone
in vivo. The Mongolian gerbil (M. unguiculatus) is well
characterized as an animal model for studying epileptiform
seizures, in particular, human myoclonic and grand mal
seizures (Bertorelli et al., 1995). A variable proportion of
Mongolian gerbils are known to suffer from clonic–tonic
seizures, which may be elicited by exposure to minor
environmental stimuli, for example, by manually stroking the
animal or by placing it in a novel environment. Seizure
sensitivity is enhanced in certain inbred strains well suited for
the study of antiepileptic drugs (Rausch et al., 1988). Owing to
their innate vulnerability, affected gerbils need not undergo
chemical or electroshock challenges to induce seizures (Rausch
et al., 1988). Intravenous or intraperitoneal injections are
mostly avoided so as not to cause immediate seizure induction.
In the present study, efficacy of hispidulin was tested in
comparison to diazepam on the basis of earlier observations
(Rausch et al., 1988). As we confirmed, antiepileptic treatment
Many flavones and their glucoside derivatives are glucuro-
nidated during the absorption process (Spencer et al., 1999).
Glucuronidation during transfer across the jejunum and ileum
is possible without the need for gut microflora. The more
highly reducing phenolics like quercetin or luteolin are
absorbed, more than 90% as glucuronidated metabolites. To
establish whether orally administered hispidulin enters the
blood circulation in its intact form, permeation across the
Caco-2 monolayer was investigated. Hispidulin and apigenin
were predominantly absorbed without any structural modifi-
cation. These studies provide evidence that the active aglycone
form of hispidulin is present after absorption.
The present study illustrates the chemical synthesis of
the flavone hispidulin. As we point out, this compound
probably acts as a partial positive allosteric modulator at
GABAA receptors, penetrates the BBB and possesses
anticonvulsant activity in the CNS. Electrophysiological data
and data on BBB permeation provide important insights
into the role of hispidulin as
a potent anticonvulsant
compound and are supported by our in vivo observations,
with the limitations of a single-dose design. Further experi-
ments will establish whether hipidulin displays other effects
typical of BZDs, such as sedative, anxiolytic and muscle
relaxant activity. Further research is warranted on the
bioavailability, distribution parameters and the putative,
active metabolites plus the effects in other epileptic
phenotypes, to put into perspective the flavone’s impact on
inhibitory brain activity.
British Journal of Pharmacology vol 142 (5)

820
D. Kavvadias et al
Hispidulin, a benzodiazepine receptor ligand
This study was supported by the Deutsche Forschungsgemeinschaft,
Bonn (Schr 211-19-3, Schr 211-21-1-2 and Sa 789-1-3). E.S. was
supported by the Swiss National Science Foundation Grant 3100-
064789.01-1 and M.Z.Q. by the King’s College London BBB
Consortium with Industry. We are most grateful to Hans Boehme
from Leibniz Institute for Neurobiology (Magdeburg, Germany) for
generously providing gerbils. We thank Prof. Joan Abbott for her
helpful suggestions on this manuscript.
References
AVALLONE, R., ZANOLI, P., PUIA, G., KLEINSCHNITZ, M.,
SCHREIER, P. & BARALDI, M. (2000). Pharmacological profile
of apigenin, a flavonoid isolated from Matricaria chamomilla.
Biochem. Pharmacol., 59, 1387–1394.
BAKER, W., BROWN, N.C. & SCOTT, J.A. (1939). The synthesis of
5-hydroxy-8-methoxyflavone (primetin monomethyl ether).
J. Chem. Soc., 2, 1922–1927.
MARDER, M., ESTIU, G., BLANCH, L.B., VIOLA, H., WASOWSKI, C.,
MEDINA, J.H. & PALADINI, A.C. (2001). Molecular modeling and
QSAR analysis of the interaction of flavone derivatives with the
benzodiazepine binding site of the GABAA receptor complex.
Bioorg. Med. Chem., 9, 323–335.
MARDER, M., VIOLA, H., WASOWSKI, C., FERNANDEZ, S.,
MEDINA, J.H. & PALADINI, A.C. (2003). 6-Methylapigenin and
hesperidin: new valeriana flavonoids with activity on the CNS.
Pharm. Biochem. Behav., 75, 537–545.
MEDINA, J.H., VIOLA, H., WOLFMAN, C., MARDER, M.,
WASOWSKI, C., CALVO, D. & PALADINI, A.C. (1998). Neuroactive
flavonoids: new ligands for the benzodiazepine receptors. Phyto-
medicine, 5, 235–243.
RAUSCH, W.-D., GOLECH, S., RIEDERER, P. & WEISER, M. (1988).
Inhibitory activity of tranquilizers, antiepileptics, calcium antago-
nists, sedatives and other compounds on the epileptiform seizures in
the Mongolian gerbil. New Trends in Clinical Neuropharmacol.
London: John Libbey Co Ltd, London, pp. 55–58.
SIEGHART, W. & SPERK, G. (2002). Subunit composition, distribution
and function of GABAA receptor subtypes. Curr. Top. Med. Chem.,
2, 795–816.
SIGEL, E. (1987). Properties of single sodium channels translated by
Xenopus oocytes after injection with messenger ribonucleic acid.
J. Physiol. (Lond.), 386, 73–90.
SIGEL, E. & BUHR, A. (1997). The benzodiazepine binding site of
GABAA receptors. Trends Pharmacol. Sci., 18, 425–429.
SIGEL, E., BAUR, R., TRUBE, G., MOHLER, H. & MALHERBE, P.
(1990). The effect of subunit composition of rat brain GABAA
receptors on channel function. Neuron, 5, 703–711.
SPENCER, J.P.E., CHOWRIMOOTOO, G., CHOUDHURY, R.,
DEBNAM, E.S., SREI, S.K. & RICE-EVANS, C. (1999). The small
intestine can both absorb and glucuronidate luminal flavonoids.
FEBS Lett., 458, 224–230.
SRIVASTAVA, S.D. & SRIVASTAVA, S.K. (1987). Synthesis of anew
flavone. Indian J. Chem., 26B, 57–58.
TAKASATO, Y., RAPOPORT, S.I. & SMITH, Q.R. (1984). An in situ
brain perfusion technique to study cerebrovascular transport in the
rat. Am. J. Physiol., 247, H484–H493.
TAN, R.X., LU, H., WOLFENDER, J.L., YU, T.T., ZHENG, W.F.,
YANG, L., GAFNER, S. & HOSTETTMANN, K. (1999). Mono- and
sesquiterpenes and antifungal constituents from Artemisia species.
Planta Med., 65, 64–67.
TREIMAN, D.M. (2001). GABAergic mechanisms in epilepsy. Epilepsia,
42 (Suppl. 3), 8–12.
VIOLA, H., WASOWSKI, C., LEVI DE STEIN, M., WOLFMAN, C.,
SILVEIRA, R., DAJAS, F., MEDINA, J.H. & PALADINI, A.C. (1995).
Apigenin, acomponent of Matricaria recutita flowers, is acentral
benzodiazepine receptors-ligand with anxiolytic effects. Planta
Med., 61, 213–216.
BERTORELLI, R., ADAMI, M.
&
ONGINI, E. (1995). The
Mongolian gerbil in experimental epilepsy. Ital. J. Neurol. Sci.,
16, 101–106.
BOILEAU, A.J., BAUR, R., SHARKEY, L.M., SIGEL, E.
&
CZAJKOWSKI, C. (2002). The relative amount of cRNA coding
for g2 subunits affects stimulation by benzodiazepines in GABAA
receptors expressed in Xenopus oocytes. Neuropharmacology, 43,
695–700.
BRACON, F., GUITTARD, F., TAFFIN DE GIVENCHY, E.
&
CAMBON, A. (1999). Highly fluorinated monomeres precursors of
side-chain liquid cristalline polysiloxanes. J. Polym. Sci. A, 37,
4487–4496.
CHERPECK, R.E. (1998). Preparation and use of aromatic amides of
poly(oxyalkylene) carbamates as fuel additives, especially gasoline
deposit inhibitors. US Patent 5786499.
CHULASIRI, M., BUNYAPRAPHATSARA, N. & MOONGKARNDI, P.
(1992). Mutagenicity and antimutagenicity of hispidulin and
hortensin, the flavonoids from Millingtonia hortensis L. Environ.
Mol. Mutagen., 20, 307–312.
DANIEL, H. & WENZEL, U. (2003). Flavonoids and their possible role
in colon cancer prevention and therapy. Exogenous factors in
colonic carcinogenesis. In: Falk-Symposium, ed. Scheppa ch, W. &
Scheurlen, M. Vol. 128, pp. 264–274. Dordrecht: Kluwer Academic
Publisher.
EGLETON, R.D., ABBRUSCATO, T.J., THOMAS, S.A. & DAVIS, T.P.
(1998). Transport of opioid peptides into the central nervous
system. J. Pharm. Sci., 87, 1433–1439.
GIL, B., SANZ, M.J., TERENCIO, M.C., FERRANDIZ, M.L., BUSTOS,
G., PAYA, M., GUNASEGARAN, R. & ALCARAZ, M.J. (1994).
Effects of flvaonoids on Njanjanad humna recombinnat
synovial phospholipases A2 and inflammatory responses in mice.
Life Sci., 54, 333–338.
GOUTMAN, J.D., WAXEMBERG, M.D., DONATE-OLIVER, F.,
POMATA, P.E. & CALVO, D.J. (2003). Flavonoid modulation of
ionic currents mediated by GABA(A) and GABA(C) receptors. Eur.
J. Pharmacol., 461, 79–87.
HORIE, T., SHIBATA, K., YAMASHITA, K., KAWAMURA, Y.
TSUKAYAMA, M. (1997). Studies of the selective O-alkylation and
dealkylation of flavonoids. XXII. convenient method for
&
A
synthesizing 3,5,7-trihydroxy-6-methoxyflavones. Chem. Pharm.
Bull., 45, 446–451.
HUI, K.M., HUEN, M.S.Y., WANG, H.Y., ZHENG, H., SIGEL, E.,
BAUR, R., REN, H., LI, Z.W., WONG, J.T.-F. & XUE, H. (2002).
Anxiolytic effect of wogonin, abenzodizaepine receptor lignad
isolated from Scutellaria baicalensis Georgi. Biochem. Pharmacol.,
64, 1415–1424.
YOUDIM, K.A., DOBBIE, M.S., KUHNLE, G., PROTEGGENTE, A.,
ABBOTT, N.J.
& RICE-EVANS, C. (2003). Interaction between
flavonoids and the blood–brain barrier: in vitro studies.
J. Neurochem., 85, 180–192.
JAIN, A.C., GUPTA, S.M. & BAMNAH, P. (1985). Aromatic benzhy-
drylation: part III – synthesis of chromones of triphenylmethane
type. Indian J. Chem., 24B, 393–397.
YOUDIM, K.A., QAISER, M.Z., BEGLEY, D.J., RICE-EVANS, C.A. &
ABBOTT, N.J. (2004). Flavonoid permeability across an in situ model
of the blood–brain barrier. Free Radic. Biol. Med., 36, 592–604.
KAVVADIAS, D., MONSCHEIN, V., SAND, P., RIEDERER, P.
&
(Received November 27, 2003
Revised February 11, 2004
Accepted April 5, 2004)
SCHREIER, P. (2003). Constituents of sage (Salvia officinalis L.)
with in vitro affinity to human brain benzodiazepine receptor.
Planta Med., 69, 113–117.
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