Phytomedicine, Vol. 5(2), pp.

235 -243
© Gustav Fischer Verlag 1998 _
I~~~~!~mml ..
REVIEW ARTICLE

Neuroactive flavonoids: new ligands for

the Benzodiazepine receptors
J. H. Medina", H. Viola\ C. Wolfman\ M. Marder", C. Wasowski 2 , D. Calvo!
and A. C. Paladini-
lInstituto de Biologia Celular y Neurociencias, Facultad de Medicina, UBA, Parguay, Buenos Aires Argentina
2Instituto de Quimica y Fisicoquimica Biol6gicas, (UBA-CONICET) Facultad de Farmacia y Bioquimica, UBA, Buenos Aires,
Argentina

Summary

Flavonoids isolated from plants used as tranquilizers in folkloric medicine have a selective affinity, for
central benzodiazepine receptors (BDZ-Rs) and some of them possess a pharmacological profile compat-
ible with a partial agonist action. Synthetic derivatives of the common flavone nucleus, give rise to high
affinity ligands when electronegative groups are introduced in carbons 6 and/or 3'. Representative com-
pounds such as 6,3'-dinitroflavone and, 6-bromo-3'-nitroflavone exhibit a high affinity for the BDZ-Rs
(Ki =1.5 to 30 nM) and have anxiolytic effects not associated with myorelaxant, sedative or amnesic ac-
tions.
These compounds or similar ones, could lead to improved therapeutic drugs in the treatment of anxiety.

Key words: flavonoids; anxiolysis; benzodiazepine receptors; partial agonist.

• Benzodiazepines
Benzodiazepines (BDZs) are the most widely pre-
scribed class of psychoactive drugs in current therapeu-
tic use since 1960.
Specific cerebral binding sites of BDZs in the central
GABAA receptor complex were reported by Mohler
and Okada and Squires and Braestrup, in 1977. Six dif-
ferent subunits and their isoforms (U I-6; ~1-3; YI-3; 0l;
PI-2 and f), combined in a pentameric complex give rise
to several variants of the GABAA receptor all of them
delimiting a channel permeable to chloride ions,
(McKernan and Whiting, 1996).
The BDZs act by potentiating GABA - induced Cl
currents (Olsen and Venter, 1986; Mohler et al., 1995;
but see Phillis and O'Reagan, 1988). The presence of a
and Yl or Y2 subunits is an absolute requirement for
BDZs binding and optimal modulation of CI- currents
by them (Costa and Guidotti, 1996; Sigel and Buhr,
1997).
The several variants of the GABAA receptor can be
grouped in at least two pharmacologically distinct
BDZ receptor (BDZ-R) subtypes which are known as
type 1 and type II (Siegharth and Karobath, 1980; Tri-
filetti et al., 1984; Niddam et al., 1987; Mohler et al.,
1995; McKernan and Whiting, 1996).
Type I predominates in cerebellum while the dentate
gyrus of the hippocampus, the spinal cord and the stri-
atum are enriched in type II. The cerebral cortex has
both types, approximately equally represented.
Although BDZs are the most safe psychotropic drugs
available today (Woods et al., 1992) and hence widely
prescribed as anxiolytics, their sedative and myorelax-
ant actions are often considered unwanted side effects.
Other drawbacks of these drugs are recorded instances
of amnesia, ataxia, ethanol and barbiturate increased
effects as well as their potential for drug abuse and tol-
erance. Most BDZs are also associated with antero-
grade amnesia (Thiebot, 1985; Izquierdo et al., 1990).
Pretraining administration of BDZs at subataxic doses,
impairs retention tests performance (Thiebot, 1985;
Lister, 1985, Izquierdo et al., 1990; Izquierdo and Me-
dina, 1991; Woods et al., 1992).
Several medicinal chemistry attempts made to obtain
236 J. H. Medina et al,
better ligands for the BDZ-Rs have been met with lim-
ited success:
• Bretazenil: a partial agonist (that is, a compound
with reduced efficacy compared to full agonists) is a
non-sedative anxioselective drug (Haefely at al., 1990),
which does not produce physical dependence (Moreau
et al., 1990), or tolerance (Haig and Feely, 1988).
However, bretazenil is not an ideal partial agonist be-
cause it has a relatively short half life in vivo and is rap-
idly metabolized into a full agonist (Costa and Guidot-
ti, 1996).
• Imidazenil: a BDZ derivative, fulfills some of the cri-
teria required to be considered a partial agonist: has
low intrinsic activity but is fully anxiolytic and anticon-
vulsant without evidencing sedative effects, ataxia or
muscle relaxation as well as potentiation of the de-
pressant effects of ethanol and barbiturates (Costa and
Guidotti, 1996). In addition, imidazenil blocks alpraz-
olarn-induced anterograde amnesia in monkeys (Costa
et al., 1997). This drug, however, has not yet been test-
ed in humans.
• Alpidem and zolpidem: are full agonists acting on
type I BDZ-Rs. Alpidem has a mixed pharmacological
profile with moderate intrinsic activity (Morton and
Lader, 1990) and no development of tolerance (Per-
rault et al., 1993). Zolpidem, administered subchroni-
cally, evidenced lower risk of producing withdrawal
symptoms than the full agonist alprazolam (Schoch et
al., 1992).
• Flavonoids
Flavonoids are a group of natural products, chemically
derived from the phenylbenzopyrone nucleus, isolated
from a wide variety of plants. They are found in fruits,
vegetables, nuts, seeds, stems, flowers as well as in tea
and wine and are important constituents of the human
diet (Harborne, 1994).
Flavonoids exhibit a wide range of biological activ-
ities: several members of the family were known to pos-
sess antiviral and antiinflamatory properties, vasculo-
protector and antithrombotic actions, spasmolitic ac-
tivity, estrogenic actions, antioxidant and liver protect-
ing effects (Barnard et al., 1993; Miksicek, 1993; Mid-
dleton and Kandaswami, 1994), but very little was
known before 1989 on the effects of this class of com-
pounds on the central nervous system (CNS). Some
flavonoids, like quercetin and various derivatives of it,
have recently been shown to possess sedative and anal-
gesic properties, but these effects probably have a peri-
pheric origin (see Picq et al., 1991). Furthermore, quer-
cetin does not recognize BDZ-Rs (Medina et al., 1990;
Ai et al., 1997). Another flavonoid and biflavonoid de-
rivatives isolated from Ginkgo biloba, have been
shown to increase cerebral blood flow (Kleinjnen and
Knipschild, 1992) and reduce neuronal oxidative me-
tabolism (Oyama et al., 1994). Various flavonoids are
inhibitors of enzymes like monoaminooxidase, protein
kinase C, phosphodiesterases, adenosine deaminase
and tyrosine kinase (Ruckstuhl et al., 1979; Ferriola et
al., 1989; Cushman et al., 1991; Koch et al., 1992).
Quite recently, it has been shown that several flavone
derivatives bind to three subtypes of adenosine recep-
tors in the low micromolar range (Ji et al., 1996).
Flavonoids may have existed for over a billion years
in Nature (Swain, 1975). This very long association of
plant flavonoids with various animal species through-
out evolution may account for the extraordinary range
of biochemical and pharmacological activities of these
chemicals in mammalian and other cell systems (Mid-
dleton and Kandaswami, 1994).
• Neuroactive flavonoids
The first reference to flavonoids as ligands for the
BDZ-Rs was made by Luk et al, (1983) who isolated
and identified from bovine urine 2 isoflavans which
showed low umolar affinity for the receptor (Fig. 1).
The isoflavanes where quite probably synthesized in
the gut of the cows by the microflora therein, acting on
4' -oxymethyl-?-hidroxyisoflavone (formononetin),
contributed by the ruminant's diet (Middleton and
Kandaswami, 1994, page 638). These compounds had
no in vivo pharmacological activity (Luk et al., 1983).
Using a radio receptor guided purification protocol we
were also able to isolate equol from bovine rumen con-
tents, an important natural source of BDZ-like com-
pounds (Medina et. al, 1991).
Nielsen et al. (1988), looked for substances having
binding affinity for the central BDZ-R in Karmelitter
Geist, a commercial alcoholic tincture of several
plants, used against anxiety and epilepsy or as seda-
tive hypnotic. They reported the presence of the bi-
flavonoid amentoflavone (Fig. 2) which has a high af-
finity for the BDZ-Rs in vitro. It is unknown which
of the plant constituents of the tincture contributes
the amentoflavone.
Amentoflavone was the first substance with high af-
finity for the brain BDZ-R which does not contain ni-
trogen in its chemical structure. Its affinity for this re-
ceptor (Fig. 2), is comparable to that of diazepam.
Amentoflavone does not differentiate between BDZ-Rs
type I and II since it shows similar IC so values for the
inhibition of 3H-diazepam binding to cerebellar or hip-
pocampal membranes. Its biochemical characterization
led to the proposal that it probably behaves in vivo as a
partial agonist. However, no changes in the in vivo
binding of 3H-diazepam were observed after i.p. ad-
 Neuroactive flavonoids 237

ministration of amentoflavone, suggesting that it does HO HO

not cross the blood brain barrier (Nielsen et al., 1988).
The efforts in our laboratory were concentrated on
the screening of several plants known to contain biflav-
onoids or, alternatively, being used as tranquilizers in OH
folkloric medicine (Medina et al., 1989). In the extracts OH
analized we unequivocally detected, besides benzodiaz- 4',7 -dihydroxyisoflavan 3',7-dihydroxyisoflavan
epine-like substances, several other BDZ-Rs ligands of ICso = 80 [.1M ICso =45 [.1M
flavonoid nature. Further research with Ginkgo biloba
Fig.1. Structure and affinity for the BDZ-R of two isofla-
extracts (Marder, M., unpublished observations),
vanes isolated from bovine's urine. (Luk et aI., 1983).
pointed to the presence of a biflavonoids-containing
fraction active in our binding assays. Four authentic bi-
flavonoids: bilobetin, ginkgetin, isoginkgetin and scia-
dopitysin, structurally closely related to amentoflavone
(see Harborne, 1994), were tested in an effort to iden-
tify the active compounds in the extracts. All of them
had much lower affinity for the BDZ-Rs than amentof-
lavone. In agreement with Nielsen et al. (1988), we OH
found no clear pharmacological activity of amentofla- HO
vone in a battery of tests used routinely in our labora-
tory to characterize the pharmacological profile of clas-
sical BDZs (Medina et al., unpublished observations).
The first monoflavonoid described as a specific li-
gand for the BDZ-Rs and active in vivo, was chrysin
(5,7-dihydroxyflavone) (Medina et al., 1990). This K1 =7nM
compound (Fig. 3), isolated from Passiflora coerulea, is
a selective and competitive inhibitor of 3H-FNZ bind- Fig. 2. Structure and affinity for the BDZ-R of amentofla-
ing to the BDZ-Rs (Ki = 3 ~M), because no inhibition vone (Nielsen et aI., 1988).
was observed in the binding of 3H-prazosin, 3H-dihy-
droalprenolol, 3H-quinuclidinyl benzilate or 3H-musci-
mol to U 1 and ~ adrenoceptors, muscarinic, and GA-
BAA receptors, respectively.
Chrysin is almost equipotent to diazepam as an anx-
iolytic (Table 1), but does not exhibit sedative, myore-
laxant, analgesic or amnesic effects (Wolfman et al., R' 3

1994; Salgueiro et al., 1997). It is noteworthy that R'

4
chrysin, in contrast to diazepam, has no amnesic effect
R7
on acquisition or retention of three different learning Rs'
tasks, even when given at doses higher than those pre-
viously reported to be anxiolytic (Salgueiro et al., Re
1997). The anxiolytic effect of chrysin as detected in 0
the elevated plus maze, was abolished by the prior ad-
R5' =R6, =H
ministration of Ro 15-1788, a specific BDZ-Rs antago-
nist. Based on these findings we postulated that this R3 Rs R6 R7 Rg R z, R3' R4 ,
natural monoflavonoid is a partial agonist of the cen- Flavone H H H H H H H H
tral BDZ-Rs. In this context, preliminary electrophys- Chrysin H OH H OH H H H H
iological experiments in Xenopus oocytes expressing a Apigenin H OH H OH H H H OH
mRNA coding for cerebral cortex GABAA receptors, Kaempferol OH OH H OH H H H OH
suggested that up to 10 ~M chrysin does not alter sig- Cirsiliol H OH OCH 3 OCH 3 H H OH OH
Skrofulein H OH OCH 3 OCH3 H H H OH
nificantly GABAAchloride ion currents. Also, biochem-
Dinatin H OH OCH 3 OH H H H OH
ical measurements performed in cerebral cortical syn- Orientin H OH H OH Glc H OH OH
aptoneurosomes with up to 30 ~M chrysin showed that Isoorientin H OH Glc OH H H OH OH
the GABA-induced 36CI- flux was not modified (Calvo,
D. and Viola, H., unpublished observations). In agree- Fig. 3. Chemical structures of several natural flavonoids de-
ment with these results the very potent anxiolytic imi- scribed as ligands for the BDZ-Rs.
238 J. H. Medina et aI.

dazobenzodiazepine bretazenil, with partial agonistic
properties, also did not modify the GABA-stimulated
Cl- flux (Schoch et aI., 1992).
More recently, we isolated apigenin (5,7,4'-trihy-
droxyflavone) (Fig. 3), from Matricaria recutita L., a
plant widely used in folkloric medicine to prepare a
tranquilizing infusion (Viola et aI., 1995). This mono-
flavonoid specifically and competitively recognizes
BDZ-Rs. Apigenin, like chrysin, inhibits 3H-FNZ bind-
ing in the low micromolar range and at doses of 3
mg/kg possesses a clear anxiolytic action in mice. At
doses similar, or greater, to those used for classical
BDZs (see Table 1), apigenin does not produce seda-
tion, muscle relaxant, anticonvulsant, amnesic or anal-
gesic effects (Viola et aI., 1995; Salgueiro et aI., 1997).
It is important to mention here that apigenin is a non-
toxic, non-mutagenic compound with anticarcinogenic
effects (Wei et aI., 1990).
The benzopyrone skeleton, common to both chrysin
and apigenin, and quite probably also found free in na-
ture as flavone (Harborne, 1994, page 285), has been
found active in several mammalian systems. For in-
stance, flavone, chrysin and apigenin decrease the
platelet cyclic adenosine monophosphate response to
prostacyclin, an effect attributed to inhibition of aden-
ylate cyclase (Middleton and Kandaswami, 1994).
For these reasons we explored the biochemical and
pharmacological properties of flavone. We found that
this compound displaces the 3H-FNZ binding to the
BDZ-Rs with a Ki = 1 [AM and exerts an anxiolytic ef-
fect in mice at a dose of 3 mg/kg (i.p.) (Table 1), as
shown by the increase in the percentage of open arm
entries in the plus maze test (vehicle: 13.1 ± 3.3; fla-
vone (3 mg/kg): 29.9 ± 4.8; p <0.05, Dunnett multiple
comparison test), without changes in the total arms en-
tries.
Up to 10 mg/kg, flavone does not evidence sedative
or myorelaxant effects as measured in the hole board
test and the wire test (Table 1). However, the i.p. injec-
tion of 1, or 3 mg/kg in mice produces a decrease in the
ambulatory locomotion measured in the Opto Varimex
apparatus (vehicle: 698 (550/831), flavone (1 mg/kg):

Table 1. Results obtained with selectednatural and synthetic flavonoids on the inhibition of 3H-FNZ binding and GABA-ra-
tio assays to rat cerebral cortical synaptosomal membranes. Plus maze, holeboard, wire, pentylenetetrazole-induced seizures
and inhibitory avoidance tests were performed to measure anxiolytic, sedative, myorelaxant, anticonvulsant and amnesic ef-
fects, respectively. Diazepam values are shown for comparison.
Effects and doses (mg/kg)
Substance Ki GABA Anxiolytic Sedative Myorelaxant Anticonvulsant Amnesic
nM ratio
Flavone 1000 N.D. 3 N.D. N.D.
(up to 10) (up to 10)
Chrysin 3000 N.D. 1 0.04 (icv)
(up to 10) (up to 30) (up to 10)
Apigenin 3000 1.4-1.5 3 30 N.D.
(up to 100) (up to 10)
Kaempferol 100,000 N.D. N.D. N.D.
(up to 10) (up to 10)
Cirsiliol 200,000 N.D. 2 N.D.
(up to 30) (up to 10)
Skrofulein- 23,000 N.D. N.D. N.D. N.D. N.D. N.D.
Dinatin" 1,000 N.D. N.D. N.D. N.D. N.D. N.D.
Orientin" N.D. N.D. 1.2 N.D. N.D. N.D. N.D.
Isoorientin" N.D. N.D. 1.2-3 N.D. N.D. N.D. N.D.
6-Methylflavone 120e 1-1.3 N.D. N.D. N.D. N.D. N.D.
6-Br flavone 70 1.6-2 0.5 3 10 N.D. N.D.
6,3'-di NO z flavone 12-30d 1.3 0.001-0.03 10
(up to 10) (up to 6) (up to 0.1)
6-Br-3'-NOz flavone 1.5 and 15< 1.4 0.01-0,1 10 0.1
(up to < 10) (at 0.3)
Amentoflavone 7 N.D. N.D. N.D. N.D. N.D. N.D.
Diazepam 7 2 0.03-0.3 1 1 0.3 1
aShen et al. (1994) e two binding sites.

b Okuyama et al. (1996) - no effect.

e ICso value (Ai et al., 1997) N. D. - not determined
d ranging values in bovine and rat cerebral membranes. icv. - intracerebroventricular.

517 (429/607)*, flavone (3 mg/kg): 374 (279/651)*'f;
" P <0.02, ** P <0.002, Mann Whitney U test).
Utilizing an anticonflict test to guide the purification
procedure, Okuyama et aI. ( 1996), have isolated from
Jatropha cilliata, a peruvian medicinal plant, two C-
glucosides of the flavonoids isoorientin and orientin
(Fig. 3). These compounds were found to possess very
mild anxiolytic effects. Unfortunately, no data was pre-
sented concerning their mechanism of action.
In contrast, the flavonoids kaempferol and cirsiliol
(Fig. 3), isolated from Tilia tomentosa and Salvia guar-
anitica, respectively, exhibited a very low affinity for the
BDZ-Rs and were devoid of anxiolytic actions (Table 1)
(Viola et aI., 1994, 1997; Marder et aI., 1996b). How-
ever, cirsiliol displaced 3H-zolpidem binding to type I
BDZ-Rs with a Ki = 20 u.M and produced sedative
actions at a dose of 2 mg/kg and at doses higher than
4 mg/kg increased the percentage of sleeping mice
in the pentobarbital-induced sleep test (Viola et aI.,
1997).
Recently, two research groups have confirmed and
extended the above mentioned findings on CNS-acting
flavonoids. Haberlein et aI. (1994), have found four
flavone derivatives in extracts of the New Zealand
plant Leptospermum scoparium, with binding proper-
ties for the BDZ-Rs and affinities ranging from very
low to medium values (ICso = 2-50 flM). These com-
pounds, listed in decreasing order of affinity for the re-
ceptor, are: 5,7-dimethoxyflavone, 5-hydroxy-7-meth-
oxy-6-methylflavone, 5-hydroxy-?-methoxy-6,8-dime-
thylflavone and 5,7-dimethoxy-6-methylflavone. Some
of these compounds probably possess agonistic proper-
ties, however, 5,7-dimethoxy flavone reported by these
authors as having the highest affinity for the BDZ-Rs,
was found inactive in our binding assay (Marder et aI.,
1996). This discrepancy cannot be attributed to experi-
mental differences in the binding assays employed.
Both Haberlein et aI. (1994) and ourselves, used exten-
sively washed crude synaptosomal membranes from rat
cerebral cortices and a similar incubation protocol: ap-
proximately 0.3 mg of membrane protein suspended in
25 mM Tris-HCl buffer, (Haberlein et al (1994) uses 50
mM Tris-citrate buffer), 60 min at 4 °C with 0.4-0.6
nM 3H-FNZ and then filtered and counted.
Shen et aI. (1994) have isolated two flavones from
the egyptian plant Artemisia herba alba L.: dina tin
(5,7,4'-tryhidroxy-6-methoxyflavone) and skrofulein
(5,4'-dihydroxy-6,7-dimethoxyflavone (Fig. 1). Both
compounds inhibited 3H-diazepam binding to rat brain
membranes with Ki = 1.3 or 23 flM for dinatin and
skrofulein, respectively, and a Hill coefficient close to
unity. Dinatin did not discriminate between type I and
type II BDZ-Rs. It was suggested that both flavones are
antagonists or partial agonists of BDZ-Rs.
Quite recently, other naturally-occurring flavonoids
Neuroactive flavonoids 239
have been shown to interact with BDZ-Rs (Table 1).
Luteolin, baicalein and some related derivatives, exhib-
ited displacing activity of 3H-diazepam with ICsos in
the low micromolar range (Ai et aI., 1997).
The active flavonoids chrysin, apigenin and some of
the others listed in Table 1, have been found in plants
used in folkloric medicine. However, these compounds
are relatively common constituents in many other
plants (Harborne, 1994), where they do not determine
their therapeutic properties because they are present
only as traces. Their detection is only achievable with
appropriate fractionation methods and specific bio-
chemical assays.
• Synthetic and semi-synthetic flavone
derivatives
Studies of the structure - activity relationship per-
formed with BDZs established that the presence of
electronegative substituents (halogens, nitro groups,
etc.), in certain positions of their molecules was essen-
tial for high receptor affinity and pharmacological ac-
tivity (Sternbach, 1978; Haefely et aI., 1985).
The above-mentioned results encouraged our at-
tempts to increase the potency of the natural flavonoid
BDZ-Rs ligands by introducing electronegative substit-
uents in their molecules.
All the halo derivatives made with chrysin were un-
successful (Marder et aI., 1996a), but the presence of
bromine atoms, nitro groups, or both, in the molecule
of flavone was highly effective (Table 1). The brominat-
ed derivative 6-bromoflavone, showed a high affinity
for the BDZ-Rs (Ki = 70 nM) (Table 1), it is a compet-
itive ligand for these receptors and has a GABA ratio of
1.6-2.0. The GABA ratio (ratio between Ki values ob-
tained in the presence or in the absence of 100 u.M GA-
BA, Braestrup et aI., 1984), is a widely used biochemi-
cal parameter to characterize the intrinsic activity of
BDZ-Rs ligands. GABA increases the binding affinity
of BDZ~Rs agonists (GABA ratio >1), has no effect on
BDZ-Rs antagonists (GABA ratio == 1), and decreases
the affinity of BDZ-Rs inverse agonists (GABA ratio
<1).
The pharmacological profile of 6-bromoflavone was
quite similar to that observed for diazepam (Marder et
aI., 1996a). These data strongly suggest that 6-bromof-
lavone is a full agonist of the central BDZ-Rs.
Nitration of the flavone nucleus yielded 6,3'-dinitrof-
lavone, displaying a high affinity (Ki = 30 nM) (Table
1), for the 3H-FNZ binding to rat cerebral membranes
(Marder et aI., 1995). Furthermore, in various regions
of the rat CNS, 6,3'-dinitroflavone had different poten-
cies in displacing the binding of 3H-FNZ (cerebellum,
Ki = 16 nM, vs. spinal cord, Ki = 50 nM, n = 3). This
240 J. H. Medina et aI.
Table 2. Affinity of halogenated/nitrated derivatives of flavone or flavanone for the BDZ-Rs.
Affinity change Compound Ki" (I-lM ) Ki flavone
Ki compound
Great decrease Flavone 1 1
Flavanone 40 0.025
3,3-di Br flavanone > 100 < 0.01
3,3,6-tri Br flavanone > 100 < 0.01
3-Br flavone > 75 > 0.01
3.6-di Br flavone > 75 > 0.01
3-Br-3'-N0 2 flavone > 20 > 0.05
2'-N0 2 flavone > 20 < 0.05
4' -Br flavone > 20 < 0.05
4'-N0 2 flavone > 20 > 0.05
3',5'-di N0 2 flavone > 20 < 0.05
6,2'-di N0 2 flavone > 10 < 0.01
6,4'-di N0 2 flavone > 10 < 0.01
6-F flavone 4.5 0.22
Moderate increase 3'-Cl flavone 0.614 1.6
3'-Br flavone 0.413 2.4
3'-N0 2 flavone 0.285 3.5
6-N0 2 flavone 0.275 3.7
6-Br-2'-N0 2 flavone 0.208 4.8
6-Br-4'-N0 2 flavone 0.200 5
6-F-3'-N0 2 flavone 0.180 5.6
6-Cl flavone 0.164 6.1
6-Br flavone · 0.075 13.3
Great increase 6,3'-di N0 2 flavone 0.026 38.5
6-N0 2-3'-Br flavone 0.025 40
6,3'-di Br flavone 0.019 52.6
6-CI-3'-N0 2 flavone 0.008 125
6-Br-3'-N0 2 flavone 0.0015 and 0.015 1000 and 62.5
a The Ki values were determined by measuring the inhibition of 3H-FNZ binding to rat cerebral cortical synaptosomal mem-
branes, and are the result of 3 to 5 independent measurements. The SEM varied between 6 and 13 % of the absolute values tab-
ulated. Ki values higher than 10 are the result of duplicate measurements .

compound is a selective BDZ-Rs ligand because no ef-
fect was found in several binding assays to GABAA, 5-
HTl A , muscarinic, and adrenergic receptors. However,
it must be taken into account the recent findings of ji et
al. (1996), showing that several flavonoids have mod-
erate affinity for adenosine receptors. It is important to
stress here that there are some electrophysiological,
neurochemical and pharmacological evidences indicat-
ing that purinergic receptors may be also involved in
some actions of BDZs (Phillis and O'Regan, 1988; Lo-
pez et aI., 1989).
The most striking feature of 6,3'-dinitroflavone is its
potency in eliciting anxiolytic effects (Table 1). Using
the elevated plus maze test with mice we found that
6,3'-dinitroflavone is an anxiolytic drug 30 times more
potent than diazepam (Marder et al., 1995). Recent bi-
ochemical and pharmacological findings indicate that
6,3'-dinitroflavone is an anxioselective drug with par-
tial agonistic properties at the BDZ-Rs, low selectivity
for types I and II and a GABAratio of 1.3. More impor-
tantly, 6,3'-dinitroflavone does not exhibit analgesic,
sedative, myorelaxant, anticonvulsant or amnesic ef-
fects (Wolfman et al., 1996, and Table 1), and has been
recently shown to possess anxiolytic actions when giv-
en orally (1 mglkg; unpublished observations). An on-
going detailed pharmacological characterization of this
compound will probably tell us more about its potential
as a new tool in the therapeutics of anxiety disorders.
Quite recently, we have found that the semisynthetic
flavonoid derivative, 6-bromo-3'-nitroflavone is able
to recognize with high affinity two different cerebral
cortical populations of BDZ binding sites (Kis 1.5 nM
and 15 nM; Viola et al., 1997a). A GABA ratio of 1.38
was found (Wolfman et al., 1997). Furthermore, there
is a brain regional variation in the potency of 6-bromo-
3'-nitroflavone in displacing 3H-FNZ binding. Using a
quantitative autoradiographic method in sagittal sec-
tions of the rat brain (Cammarota et aI., 1995), we

found that this compound has a potency to inhibit 3H-
FNZ binding in the cerebellum 5 -1 0 times higher than
in the dentate gyrus of the hippocampus. (Wolfman et
al., 1997).
6-bromo-3'-nitroflavone exhibits also anxioselective
properties, without evidencing sedative, myorelaxant,
anticonvulsant, amnesic or analgesic effects (Viola et
al., 1997a; Wolfman et al., 1997, and Table 1). Fur-
thermore, dose-response curves of GABA-stimulated
36CI- flux reveal that 6-bromo-3'-nitroflavone behaves
as a partial agonist at the BDZ-Rs.
An indication of the great pharmacological selectivity
of these semisynthetic derivatives is shown in Fig. 4.
The separation index, which is defined as the ratio
between the minimal sedative dose and the maximal
anxiolytic one, is 30 or 100 times higher, for 6-bromo-
3' -nitroflavone or 6,3'-dinitroflavone, respectively,
than the corresponding value for diazepam.
Several synthetic attempts to obtain other active de-
rivatives shifting the electronegative groups to various
positions other than 6 and/or 3' in the flavone nucleus
were unsuccessful (Marder et al., 1997). These posi-
tions, seem to be a strict requirement for activity. Table
2 summarizes our main recent findings on synthetic fla-
vone and flavanone derivatives. Five compounds, in-
cluding 6,3'-dinitroflavone and 6-bromo-3'-nitrofla-
vone (see above), have shown great increases (40 to
1000 times) in BDZ-Rs binding affinity in comparison
with the reference compound flavone (Marder et al.,
1997). Taken as a whole, our structure-activity rela-
tionship studies demonstrate that substitutions with
halogen atoms, or nitro groups, in positions 6 and/or 3'
of flavone give rise to very active compounds, both in
vitro and in vivo (Marder et al., 1996a, 1997 and un-
published observations).
It has been recently demonstrated that 6-methylfla-
vone is the most potent BDZ-R ligand of 32 commer-
cially available flavonoids tested (Ai et al., 1997). The
affinity of 6-methylflavone for the BDZ-Rs (ICso = 0.12
u.M) is in good accord with the results in Table 2. This
compound probably behaves as a weak partial agonist
or as an antagonist of both human and rat BDZ-Rs (Ai
et al., 1997).
Up to now the impact of plant flavonoids on mam-
malian biology was principally centered on immunity,
inflamation, lipid peroxidation, oxyradicals quenching
and cancer (Middleton and Kandaswami, 1994). The
discovery of their direct action on receptors of the CNS
controlling anxiety is then quite remarkable. Further-
more, as shown in this review, flavonoids, and their
semi-synthetic derivatives, are apparently devoid of the
unwanted side effects which are typical of the benzodi-
azepines. Further research may lead to the transforma-
tion of the compounds described, or similar ones, into
useful therapeutic drugs.
Neuroactive flavonoids 241
Separation Index
300
300
200
100
3 10
BF DZ API BNF DNF
Fig. 4. Separation indexes for 6-bromoflavone (BF), diazepam
DZ), apigenin (API), 6-bromo-3'-nitroflavone (BNF) and 6.3'-
dinitroflavone (DNF). The separation index is the ratio
between the minimal sedative and the maximal anxiolytic dos-
es of each compound.
Acknowledgements
This work was supported by National Research Council (Ar-
gentina), University of Buenos Aires (Argentina), Internation-
al Foundation for Science (Sweden), the Organization of
American States and Strathclyde Institute for Drug Research,
University of Strathclyde, Glasgow (UK).
• References
Ai, ]., Dekermendjian, K., Wang, X., Nielsen, M., Witt, M.-
R.: 6-methylflavone, a benzodiazepine receptor ligand with
antagonistic properties on rat brain and human recombi-
nant GABAA receptors in vitro. Drug Dev. Res., 41:
99-108, 1997.
Barnard, L., Smee, E, Huffman, J. H., Meyerson, L. H., Sid-
well, R. w.: Antiherpes virus activity of 59-303, a novel
plant flavonoid. Cbemother. 39: 203-211,1993.
Braestrup, c., Honore, T., Nielsen, M., Petersen, E. N., Jen-
sen, L. H.: Ligands for the benzodiazepine receptors with
positive and negative efficacy. Biocbem. Pharmacal. 33:
859-862,1984.
Cammarota, M. c., Wolfman, c., Jerusalinsky, D., Bernabeu,
R., Levi, M., Izquierdo I., Medina, J. H.: Inhibitory avoid-
ance training-induced selective changes in 3H-AMPA recep-
tor binding in the hippocamal formation. Neurobiol.
Learn. and Mem. 64: 257-264,1995.
Costa E., Anta J., Caruncho H., Guidotti A., Impagnatiello
E, Pesold c., Thompson D. M.: A search for new anticon-
vulsant and anxiolytic benzodiazepines devoid of side ef-
fects and tolerance ability, in Biggio G., Sauna E., Serra M.,
Costa E. (eds). GABA A receptors and anxiety, vol 48, Ad-
vances in Biochemical Psychopharmacology, Lippincot-Ra-
ven, Philadelphia. New York, 1997.
Costa, E. and Guidotti, A.; Benzodiazepines on trial: a re-
242 ]. H. Medina et al.
search strategy for their rehabilitation. TIPS. 17:
192-200, 1996.
Cushman, M., Nagarathnam, D., Burg, D. L., Gearhlen, R.
L.: Synthesis and protein-tyrosine kinase inhibitory activ-
ities of flavonoids analogues. j. Med Chern. 34: 798-806,
1991.
Ferriola, P. C, Coy, v., Middleton, E.: Protein kinase C inhi-
bition by flavonoids. Kinetic mechanisms and structure-ac-
tivity relationships. Biochem. Pharmacol. 38: 245 -254,
1989.
Haberlein, H., Tschiersch, K. P., Schafer, H. L.: Flavonoids
from Leptospermum scoparium with affinity to the ben-
zodiazepine receptor characterized by structure activity re-
lationships and in vivo studies of a plant extract. Pharma-
zie 49: 912-921, 1994.
Haefely, W., Kybura, E., Gerecke, M. and Mohler, H.: Recent
advances in the molecular pharmacology of benzodiaze-
pine receptors and the structure - activity relationships of
their agonists and antagonists. Adv. Drug Res., 14:
165-322,1985.
Haefely, W. E., Martin, ]. R., Schoch, P.: Novel anxiolytics
that act as partial agonists at benzodiazepine receptors.
TIPS 11: 452-456, 1990.
Haig,]. R., Feely, M. R.: 16-6028, a benzodiazepine receptor
partial agonist, does not exhibit anticonvulsant tolerance
in mice. Eur. j. Pharmacal. 147: 282-285, 1988.
Harborne, ].B. (ed.): The flavonoids, Advances in research
since 1986. Chapman and Hall, London, 1994.
Izquierdo, 1., Medina, ]. H.: GABAA modulation of memory:
the role of endogenous benzodiazepines. TIPS 12:
260-265,1991.
Izquierdo, 1., Pereira, M. E., Medina, ]. H.: Benzodiazepine
receptor ligand influences on acquisition: suggestion of an
endogenous modulatory mechanism mediated by benzodi-
azepine receptors. Behav. Neural Biol. 54: 27 -41, 1990.
Ji, X., Melman, N., Jacobson, K. A.: Interactions of flavo-
noids and other phytochemicals with adenosine receptors.
j. Med Chem. 39: 781-788, 1996.
Kleinjnen, J., Knipschild, P.: Ginkgo biloba. Lancet 340:
1136-1139,1992.
Koch, H. P.,Jager, W., Groh, D., Plank, G.: In vitro inhibition
of adenosine deaminase by flavonoids and related com-
pounds. Meth. Fin. Exp. Clin. Pharmacol. 14: 413-417,
1992.
Lister, R.: The amnesic action of benzodiazepines in man.
Neurosci. Biobehav. Rev. 9: 87-93, 1985.
Lopez, E, Miller, L. G., Greenblatt, D. ]., Kaplan, G. B.,
Shader, R. 1.: Interaction of caffeine with the GABAA recep-
tor complex: alterations in receptor function but not ligand
binding. Eur. j. Pharmacal. 172: 453-459,1989.
Luk, K. C, Stern, L., Weigele, M., O'Brien, R. A., Spirst, N.:
Isolation and identification of "diazepam-like" compounds
from bovine urine. j. Nat. Prod. 46: 852-861, 1983.
Marder, M., Viola, H., Wasowski, C, Wolfman, C Water-
man, P., Medina,]. H., Paladini, A. C: 6,3'-dinitroflavone,
a novel high affinity ligand for the benzodiazepine receptor
with potent anxiolytic properties. Bioorg. Med. Chem.
Lett. 5: 2717-2720,1995.
Marder, M., Viola, H., Wasowski, C, Wolfman, C, Water-
man, P., Cassels, B. K., Medina, ]. H., Paladini, A. C: 6
Bromoflavone, a high affinity ligand for the benzodiaze-
pine receptors is a member of a family of active flavonoids.
Biochem. Biophys. Res. Comm. 223: 384-389, 1996a.
Marder, M., Viola, H., Wasowski, C, Wolfman, C, Water-
man, P. G., Medina,]. H., Paladini, A. C: Cirsiliol and caf-
feic acid ethyl ester isolated from Salvia guaranitica, are
competitive ligands for the central benzodiazepine recep-
tors. Phytomedicine 3: 29-31, 1996b.
Marder, M., Zinczuk,]., Colombo, M. 1., Wasowski, C, Vi-
ola, H., Wolfman, C, Medina, J. H., Ruveda, E. A., Paladi-
ni, A. C: Synthesis of halogenated/nitrated flavone deriva-
tives and evaluation of their affinity for the central benzod-
iazepine receptor. Bioorg. Med Chem. Lett. 7: 2003-2008,
1997.
McKernan, R. M., Whiting, P. ].: Which GABAA receptors
subtypes really occur in the brain? TINS 19: 139-143,
1996.
Medina, ]. H., Danelon, ]. L., Wasowski, C, Levi de Stein,
M., Paladini, A. C: Production of benzodiazepine-like
molecules in bovine rumen. Biochem. Biopbys. Res.
Comm. 181: 1048-1055, 1991.
Medina, ]. H., Paladini A. C, Wolfman, C Levi, M., Calvo,
D., Diaz, L. Pefia, C: Chrysin (5,7 di-OH flavone) a natu-
rally-occurring ligand for benzodiazepine receptors, with
anticonvulsant properties. Biochem. Pharmacol. 40:
2227-2232,1990.
Medina, ]. H., Pefia, C, Levi M., Wolfman, C, Paladini A.
C: Benzodiazepine-like molecules as well as other ligands
for the brain benzodiazepine receptor are relatively com-
mon constituents of plants. Biochem. Biophys. Res.
Comm. 165: 547-55, 1989.
Middleton E., Kandaswami C: The impact of plant flavo-
noids on mammalian biology: implications for immunity,
inflammation and cancer, Pages 619-645, in Harbone
]. B. (ed.) The Flavonoids, Chapman and Hall, London,
1994.
Miksicek, R. ].: Commonly occurring plant flavonoids have
estrogenic activity. Mol. Pharmacol. 44: 37 -43, 1993.
Mohler, N., Knoflach, E, Paysan, K., Motejlek, K., Benke, D.,
Luscher, B. Fritschy, ]. M.: Heterogeneity of GABAA recep-
tors: cell-specific expression, pharmacology, and regula-
tion. Neurochem. Res. 20: 631-638,1995.
Mohler, N., Okada, T: Benzodiazepine receptors: demonstra-
tion in the central nervous system. Science 198: 849-851,
1977.
Moreau, J. L., Jenck, E, Pieri, L., Schoch, P., Martin, ]. R.,
Haefely, W. E.: Physical dependence induced in DBN2j
mice by benzodiazepine receptor full agonists, but not by
partial agonist Ro 16-6028. Eur. j. Pharmacol. 190:
269-273, 1990.
Morton, S., Lader, M.: Studies with alpidem in normal volun-
teers and anxious patients. Pharmacopsychiatry 23:
120-123, 1990.
Niddam, R. A., Dubois, A., Scatton, B., Arbilla, S., Langer, S.
Z.: Autoradiographic localization of 3H-zolpidem binding
sites in the rat CNS: comparison with the distribution of
3H-flunitrazepam binding sites. j. Neurochem. 49:
890-896, 1987.
Nielsen, M., Frokjaer, S., Braestrup, C: High affinity of nat-
urally-occurring biflavonoid, amentoflavone, to brain ben-

zodiazepine receptors in vitro. Biochem. Pharmacol. 37:
3285-3287,1988.
Okuyama, E., Okamoto, Y., Yamazaki, M., Satake, M.: Phar-
macologically active components of a peruvian medicinal
plant, Huanarpo (Jatropha cilliata, M. Arg.) Chem.
Pharm. Bull. (Japan). 44: 333-336,1996.
Olsen, R. W., Venter, J. c. Benzodiazepine/GABA receptors
and chloride channels. Structural and Functional Proper-
ties. Alan R. Liss, New York, 1986.
Oyama, Y., Fuchs, P. A., Katayama, N., Noa, K.: Myricetin
and quercetin, the flavonoid constituents of Ginkgo biloba
extract, greatly reduce oxidative metabolism in both rest-
ing and Ca2+-loaded brain neurons. Brain Res. 635:
125-129,1994.
Perrault, G., Morel, E., Sanger, D. J., Zivkovic, B.: Repeated
treatment with alpidem, a new anxiolytic, does not induce
tolerance or physical dependence. Neuropharmacol. 32:
855-863, 1993.
Phillis, J. W., O'Regan, M. H.: The role of adenosine in the
central actions of the benzodiazepines. Prog. Neuropsycho-
pharmacol. & Biol. Psychiat. 12: 389-404, 1988.
Picq, M., Cheav, S. v., Prigent, A. E: Effect of two flavonoid
compounds on the central nervous system. Analgesic activ-
ity. Life Sci. 49: 1979-1988, 1991.
Ruckstuhl, L., Beretz, A., Anton, R., Laudry, Y.: Flavonoids
are selective cyclic GMP phosphodiesterase inhibitors. Bio-
chem. Pharmacal. 28: 535-538,1979.
Salgueiro, J. B., Ardenghi, P., Dias, M., Ferreira, M. B. c., Iz-
quierdo, 1., Medina, J. H.: Anxiolytic natural and synthetic
flavonoid ligands of the central benzodiazepine receptor
have no effect on memory task in rats. Pharmacol. Bio-
chem. Behav., 58: 887-891, 1997.
Schoch, P., Moreau, J. L., Martin, J. R., Haefely, W. E.: As-
pects of benzodiazepine receptor structure and function
with relevance to drug tolerance and dependence. Biochem.
Soc. Symp. 59: 121-134, 1992.
Shen, X-L., Nielsen, M., Witt, M. R., Sterner, 0., Bergen-
dorff, 0., Khayyal, M.: Inhibition of rnethyl-Il-I-diazepam
binding to rat brain membranes in vitro by dina tin and
skrofulein. Acta Pharmacol. Sinica 15: 385-388,1994.
Siegharth, W., Karobath, M.: Molecular heterogeneity of
benzodiazepine receptors. Nature 285: 826-828, 1980.
Sigel, E., Buhr A.: The benzodiazepine binding site of GABAA
receptors. TIPS: 18: 425 -429, 1997.
Squires, R. E, Braestrup, C» Benzodiazepine receptors in rat
brain. Nature 266: 732-734, 1977.
Sternbach, L. H.: The benzodiazepine story. Prog. Drug Res.
22: 229-266, 1978.
Swain, T.: pages 1096-1129, in Harborne J. B., Mabry T. J.
and Mabry M. (eds.) The Flavonoids, Chapman and Hall,
London, 1975.
Thiebot, M. H.: Some evidence for amnesic-like effects of
benzodiazepines in animals. Neurosci. Biobehav. Rev. 9:
95-100,1985.
Neuroactive flavonoids 243
Trifiletti, R. R., Lo, M. M.S., Snyder, S.H.: Kinetics differenc-
es between type I and type II benzodiazepine receptors.
Mol. Pharmacal. 26: 228-234, 1984.
Viola, H., Marder, M., Wolfman, c., Wasowski, c., Medina,
J.H., Paladini, A. C« 6-bromo-3'-nitroflavone, a new high
affinity benzodiazepine receptor agonist recognizes two
populations of cerebral cortical binding sites. Bioorg. Med.
Chem. Lett. 7: 373-378, 1997 a.
Viola, H., Wasowski, c., Levi, N., Wolfman, c., Silveira, R.,
Dajas, E, Medina, J. H., Paladini, A. c.. Apigenin, a com-
ponent of Matricaria recutita flowers is a central benzodi-
azepine receptors-ligand with anxiolytic effects. Planta
Med. 61:213-216, 1995.
Viola, H., Wasowski, c., Marder, M., Wolfman, c., Paladini,
A. c., Medina, J.H.: Sedative and hypnotic properties of
Salvia guaranitica St. Hi!. and of its active principle: Cirsil-
io!. Phytomedicine 4: 45-50, 1997b.
Viola, H., Wolfman, c., Levi, M., Wasowski, c., Pefia, c.,
Medina, J. H., Paladini, A. c. Isolation of pharmaco-
logically active benzodiazepine receptor ligands from
Tilia tomentosa (Tiliaceae). J. Ethnopharmacol. 44: 47-53,
1994.
Wei, H., Tye, L., Bresnick, E., Birt, D. E: Inhibitory effect of
apigenin, a plant flavonoid, on epidermal ornithine decar-
boxylase and skin tumor promotion in mice. Cancer Res.
50: 499-502, 1990.
Wolfman, c., Viola, H., Marder, M., Ardenghi, P., Wasowski,
c., Schroder, N., Izquierdo, 1., Ruveda, E., Paladini, A. c.,
Medina, J. H.: Pharmacological characterization of 6-bro-
mo-3'-nitroflavone, a synthetic flavonoid with high affinity
for the benzodiazepine receptor. Submitted, 1997.
Wolfman, c., Viola, H., Marder, M., Wasowski, c., Arden-
ghi, P., Izquierdo, 1., Paladini, A. c., Medina J. H.: Anxios-
elective properties of 6,3'-dinitroflavone, a high-affinity
benzodiazepine receptor ligand. Eur. J. Pharmacol. 318:
23-29, 1996.
Wolfman, c., Viola, H., Paladini, A. c., Dajas, E, Medina,
J.H.: Possible anxiolytic effects of chrysin, a central ben-
zodiazepine receptor ligand isolated from Passiflora caeru-
lea. Pharmacol. Biochem. Behav. 47: 1-4, 1994.
Woods, J. H., Katz, J. L., Winger, G.: Benzodiazepines: use,
abuse and consequences. Pharmacal. Rev. 44: 151-347,
1992.
• Address
A. C. Paladini, Instituto de Quimica y Fisicoquimica
Biol6gicas, Facultad de Farmacia y Bioquimica, junin
956, (1113) Buenos Aires, Argentina.
Phone: 00541-962-5506; Fax: 00541-962-5457;
e-mail: paladini@qb.ffyb.uba.ar