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Progress in Neuro-Psychopharmacology & Bio

Behavioral effects of 6-bromoflavanone and 5-methoxy-6,8-dibromoflavanone as anxiolytic compounds

Elisa Ognibene a, Paolo Bovicelli b, Walter Adriani a,Luciano Saso c, Giovanni Laviola a,⁎

a Behavioural Neuroscience Section, Department of Cell Biology & Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italyb Institute of Biomolecular Chemistry, National Research Council, Section of Rome, P.le A. Moro 5, 00185, Rome, Italy

c Department of Human Physiology and Pharmacology “Vittorio Erspamer” University of Rome “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy

Received 10 October 2006; received in revised form 29 July 2007; accepted 29 July 2007Available online 6 August 2007

Abstract

Benzodiazepines (BDZs) are the most used psychoactive drugs in the pharmacotherapy of anxiety. A large number of structurally differentclasses of ligands are also active in the modulation of anxiety, showing high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Various synthetic derivatives of natural flavonoids have been found to have very potent anxiolytic properties. This studywas undertaken to provide a behavioral characterization of two novels halogenated flavonoids, 5-methoxy-6, 8-dibromoflavanone (FV1), and6-bromoflavanone (FV2). These compounds were tested and compared to diazepam (0.5 mg/kg) and to the natural flavonoid chrysin (1 mg/kg)as a standard of activity. When injected in mice (0.5, 1 mg/kg i.p) both synthetic flavonoids increased the locomotor activity and theexploratory skills of the animals, as measured in the open-field and in the hole-board tests. Both compounds, indeed, had a clear anxiolyticactivity in the elevated plus-maze, as measured by an increased number of entries and the percentage of time spent in the open arms. At thetested doses, both compounds did not induce sedative action or compulsive behaviour. These results encourage making deeper investigationson this field.© 2007 Elsevier Inc. All rights reserved.

Keywords: Anxiety; Elevated plus-maze; Halogenated flavonoids; Locomotor activity

1. Introduction

Benzodiazepines (BDZs) are the most widely prescribedclass of psychoactive drugs, despite the important unwantedside-effects that they produce such as sedation, myorelaxation,ataxia, amnesia, ethanol and barbiturate potentiation andtolerance. (Paladini et al., 1999; Woods et al., 1992; Hamon,1997). A large number of structurally different classes ofligands, many of them sharing the main characteristics of thebenzodiazepine (BDZ) nucleus, are active in the modulation of

Abbreviations: BDZs, Benzodiazepines; CNS, Central Nervous System;DMSO, Dimethylsulphoxide; DZ, Diazepam; FV1, 6,8-dibromo-5-methoxy-flavanone; FV2, 6-bromoflavanone; GABA, Gamma-amino-butyrric acid; MA,6-methyl-apigenin; QSAR, Quantitative Structure–Activity Relationships; SAP,Stretched attending postures.⁎ Corresponding author. Tel.: +39 06 4990 2105; fax: +39 06 495 7821.E-mail address: laviola@iss.it (G. Laviola).

0278-5846/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.pnpbp.2007.07.023

anxiety, sedation, convulsion, myorelaxation, hypnotic andamnesic states in mammals (Griebel et al., 1999). Thesecompounds have high affinity for the benzodiazepine bindingsite (BDZ-bs) of the GABA(A) receptor complex (Marder et al.,2001; Kahnberg et al., 2002; Sieghart, 1989). GABA(A)receptors are transmembrane hetero-oligomeric proteins,expressed in the central and peripherical nervous system,which are ligand-gated chloride ion-channels. The benzodiaz-epine binding site is located at the interface of the alpha andgamma subunits (Mohler et al., 1995; Gallagher and Primus,1992; Barnard et al., 1998; Bowery, 1993). The currentlyaccepted receptor/pharmacophore model of the BDZ-bs (Wanget al., 1999) accounts for the general requirements that shouldbe met by this receptor for ligand recognition.

Research groups searching for safer BDZ-receptor (BDZ-R)ligands recently demonstrated the existence of a new family ofligands which have a flavonoid structure. First isolated fromplants used as tranquilizers in folkloric medicine, some natural

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flavonoids have shown to possess a selective and relatively mildaffinity for BDZ-bs and a pharmacological profile compatiblewith a partial agonistic action. Chrysin (5,7-di-OH-flavone) wasidentified in Passiflora coerulea L. (Medina et al., 1997; Zanoliet al., 2000). It was found to be a ligand for the benzodiazepinereceptors, both central and peripheral. Administered to mice bythe intracerebroventricular route, chrysin was able to prevent theexpression of tonic–clonic seizures induced by pentyleneter-trazol. Ro 15-1788, a central benzodiazepine receptor antago-nist, abolished this effect. The fractionation of the aqueousextract of Matricaria recutita led to the detection of severalfractions with significant affinity for the central benzodiazepinereceptor and to the isolation and identification of 5,7,4′-trihydroxyflavone (apigenin) in one of them (Viola et al.,1995). Apigenin, indeed, had a clear anxiolytic activity in micein the elevated plusmaze without evidencing sedation or musclerelaxant effects at doses similar to those used for classicalbenzodiazepines and no anticonvulsant action was detected(Avallone et al., 2000). 6-methylapigenin (MA) was recentlyisolated from Valeriana wallichii and proved that it is a benzo-diazepine binding site (BDZ-bs) ligand (Marder et al., 2003).

Further research resulted in the production of syntheticflavonoid derivatives with increased biochemical and pharma-cological activities. A QSAR regression analysis of theparameters describing the interaction with the receptordemonstrates the relevance of the substituents chemical prop-erties for the ligand binding. It shows that they are associatedwith the negatively charged oxygen atom of the carbonyl groupof the flavonoids and with the nature of the substituent inposition 3′. (Huang et al., 2001; Marder et al., 2001). Theintroduction of an electron attractive atom in position 6 of theflavonoid nucleus showed to increase affinity for the GABA(A)receptor as well (Huang et al., 2001; Haberlein, 1994).

Semisynthetic derivatives of flavone obtained by introducinghalogens, nitro groups or both in its molecule, give rise to highaffinity ligands for the benzodiazepine receptor, active in-vivo;6,3′-dinitroflavone, for example, is an anxiolytic drug 30 timesmore potent than diazepam (Viola et al., 1999). 6-Bromo-3′-nitroflavone is a synthetic flavone derivative that selectivelyrecognizes benzodiazepine receptors and has potent anxiolytic-like effects, possibly behaving as a partial agonist of thebenzodiazepine receptors (Wolfman et al., 1998; Viola et al.,2000). This study was undertaken to provide a behavioralcharacterization of the effects of two novels halogenatedflavonoids, 6-bromoflavanone and 5-methoxy-6,8-dibromofla-vanone, both derived from halogenation of flavan nucleusconduced by Bovicelli et al. (2002), starting from flavanone and5-methoxy-flavanone. Considering the chemical structure of thecompounds, related to the pharmacophore model previouslyreported, both of them were supposed to have anxiolytic effects,and to act as ligand of GABA(A) receptor. We examined indetail the pharmacological effects of acute intra-peritonealadministration of these substances in mice. The tests wereconduced comparing the novel drugs to the well known BDZdiazepam (0.5 mg/kg) and to the natural flavonoid chrysin(1 mg/kg) as a standard of activity. Levels of anxiety-related andrisk-assessment behaviours were assessed in the elevated plus-

maze (Holmes and Rodgers, 1999; Pellow et al., 1985; File,1996). Locomotor activity profile and levels of explorativitywere investigated by testing mice in the open-field andholeboard test.

2. Materials and methods

2.1. Subjects

Outbred CD-1 adult male mice (weight 30–33 gr.), purchasedfrom Charles River Italia (I-22050 Calco, Italy) were housed threeper cage in 33×13×14 cm Plexiglas boxes, in air-conditionedroom (temperature 21±1 °C, relative humidity 60±10%, lights onfrom 7:00 a.m. to 7:00 p.m.) with water and pellet food (Enrichedstandard diet purchased fromMucedola, Settimo Mil. Italy) beingavailable ad libitum.On dayof test, animalswere transported to thedimly illuminated testing room and left undisturbed for at least onehour. Experiments were performed between 10:00 and 16:00 h.Mice were randomly assigned for testing to one group of treatment(n=8). Animals were injected i.p. at a volume of 10 ml/kg, 20 minbefore the pharmacological tests.

2.2. Drugs

Diazepam (Roche), used as the reference anxiolitic compound,6,8-dibromo-7-methoxy-flavanone and 6-bromo-flavanone werediluted in a solution of saline 90% and dimethylsulphoxide(DMSO) 10% (Carlo Erba 445103), chrysin (Sigma AldrichC8,010-5), used as the reference natural flavonoid with anxiolyticproperties, was diluted in a solution of saline 85% (v:v) DMSO10% and 5% NaOH 0.1 N, as reported from Wolfman andcolleagues (Wolfman et al., 1994). Mice were randomly assignedfor testing to one of the following groups of treatment (n=8) :vehicle, diazepam 0.5 mg/kg, chrysin 1 mg/kg, 6,8-dibromo-5-methoxy-flavanone 0.5 mg/kg, 6,8-dibromo-5-methoxy-flava-none 1 mg/kg, 6-bromo-flavanone 0.37 mg/kg, 6-bromo-flava-none 0.73 mg/kg. For 6-bromo-flavanone equimolar doses werecalculated.

2.3. Open-field

2.3.1. Apparatus and general procedureLocomotor behaviour of mice was evaluated during the open-

field test, in a session of 10 minutes, which was performedplacing each individual animal in the centre of a square arena(50×50×50 cm) with a white floor and black walls. All sessionswere recorded by a video-camera (positioned above and at ca.60° to the maze) connected to a monitor and a VCR. To avoid un-necessary distractions, the experimenters retreated to adjacentlaboratory during testing. Videotapes were scored blind by areliable observer using dedicated software (“Observer” 3.0 fordos; Noldus, Wageningen, The Netherlands). Behaviouralparameters comprised number of crossing of 10×10-cm squareareas of the floor and the frequency of rearing of mice on itshindpaws. After each test the floor was cleaned with water andethanol, in order to avoid the potential excitatory effect onlocomotion, produced by the presence of urine residues.

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2.4. Elevated plus-maze

The elevated plus-maze comprises two open arms (27×5×0.25 cm) and two closed arms (27×5×15 cm) extended from acommon central platform (5×5 cm) (as described byMacrì et al.,2002). The apparatus is made in Plexiglas (black floor, cleanwalls) and elevated to a height of 40 cm above floor level. Open-arm exploration was encouraged by the inclusion of a slightraised edge (0.25 cm) around their perimeter, and by testingunder indirect dim white light. On day of test, animals weretransported to the dimly illuminated testing room and leftundisturbed for at least one hour.

Twenty minutes after the drug injection, mice were gentlyplaced on the central platform facing a closed arm and al-lowed to freely explore the maze for 10 min. All sessions wererecorded by a video-camera (positioned above the maze)connected to a monitor and a VCR. To avoid unnecessarydistractions, the experimenters retreated to adjacent laboratoryduring testing. Videotapes were scored blind by an expertobserver. Between subjects, the maze was thoroughly cleanedwith wet and dry cloths.

A number of spatio-temporal and ethological parameterswere collected during the session (Pellow et al., 1985;

Fig. 1. Locomotor activity measured by crossings (panel A) andwall-rearing (panelB), during a single 10-min session in an open field arena, 20min after IP injectionwithvehicle (VEH), diazepam (DZ 0.5 mg/kg), chrysin (CHRY, 1 mg/kg), 6,8dibromo-7methoxy-flavanone (FV1 at 0.5 or 1 mg/kg) or 6-bromo-flavanone (FV2 at 0.37 or0.73 mg/kg). Data are expressed as mean±s.e.m. [⁎=pb0.05 ;⁎⁎=pb0.01].

Fig. 2. Latency to enter the open arms (panel A), percentage of time spent inopen arms (panel B), closed arm entries (panel C), during a single 10-minsession in an elevated plus-maze apparatus, by mice injected as in Fig. 1. Dataare expressed as mean±s.e.m. [⁎=pb0.05 ; ⁎⁎=pb0.01].

Rodgers et al., 1995). The spatio-temporal parameterscomprised: the percentage of time in open arms (time inopen arms / total time * 100), closed arm entries (the frequencyof mouse entry with all four paws into the closed arm) and thelatency to enter into the open arms. The ethological measurescomprised frequency scores for rearing (vertical movementagainst the side and/or end of the walls), head-dipping(exploratory movement of head-shoulders over the edge of themaze) and stretched-attend posture (SAP), an exploratory

Fig. 3. Time spent in stretched attend postures (panel A), frequency of wall-rearing(panel B), and frequency of head-dipping (panel C) during a single 10-min sessionin an elevated plus-maze apparatus, by mice injected as in Fig. 1. Data areexpressed as mean±s.e.m. [⁎=pb0.05; ⁎⁎=pb0.01].

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posture in which the body is stretched forward then retractedto the original position without any locomotion.

2.5. Holeboard test

The holeboard consisted of a plastic material elevatedplatform (60×60×30 cm) with sixteen 2-cm diameter holesequidistant in the floor box. The number of head dips and thetime spent in head-dipping were counted during 10 minutes. Anincrease in the number and time spent in head-dipping implies agreater exploratory activity, a decrease of both parametersreveals a sedative behaviour (File and Andrews, 1994).

2.6. Statistics

In the Results section data are presented as mean±SEM.Data were analysed by parametric ANOVA when severaltreatments in mice were compared to vehicle. Separate analysesand post-hoc comparisons (Holm–Sidak) were performed whenallowed.

3. Results

3.1. Open-field

The locomotor activity of mice treated with different doses ofthe two halogenated flavonoids 6, 8-dibromo-7-methoxy-flavanone (FV1) and 6-bromo-flavanone (FV2), chrysin anddiazepam, was compared with that of mice treated with vehicle.ANOVA revealed a significant effect of drug [F(6, 84)=4.763,pb0.001]. As shown in Fig. 1 (panel A), diazepam, the lowdose of FV1 and both doses of FV2 induced an increment in thenumber of square crossed, when compared to vehicle injectedcontrols.

Animals treated with diazepam and the low dosage of bothFV1 and FV2 increased the frequency of exploratory wallrearing behaviour [F(6, 79)=4.796, pb0.001], compared tovehicle treated mice (see Fig. 1, panel B).

3.2. Elevated plus-maze: spatio-temporal measures

All treated animals showed a shorter latency to enter theopen arms (see Fig. 2, panel A), when compared to vehiclecontrol mice [Drug main effect F(6, 82)=5.447, pb0.001].Interestingly, the profile of diazepam treated mice was verysimilar to that of FV treated mice. In particular, the higher doseof FV1 and the lower dosage of FV2 showed comparablevalues.

A significant main effect of drug [F(6, 84)=3.713, pb0.001]indicated that treated mice spent a significantly larger amount oftime into this unprotected area of the apparatus than vehiclecontrol animals. Post hoc comparison revealed that DZ, highdose FV1 and both dosages of FV2 treated mice spent moretime than vehicle control mice in the open compartment of theapparatus (Fig. 2, panel B).

The diazepam treatment had a slight effect on the locomotoractivity of mice, as revealed by measuring of closed arms entries

in the elevated plus-maze [F(6, 74)=2.32; pb0.05] (Fig. 2panel C).

3.3. Elevated plus-maze: ethological measures

The level of risk assessment, as measured by time spent inSAP, was affected by drug treatment. In particular a significantdifference was observed in DZ and FV2 treated mice, whichexhibited this behavioural pattern approximately two fold lessthan vehicle controls or chrysin treated mice [F(6,78)=3.425,pb0.05) (Fig. 3, panel A ). An ANOVA carried out on thefrequency of the unprotected head dipping revealed a significant

Fig. 4. Time spent in head-dipping (panel A) and Number of head-dips (panel B)during a single 10-min session in an holeboard apparatus, by mice injected as inFig. 1. Data are expressed s mean±s.e.m. [⁎=pb0.05 ; ⁎⁎=pb0.01].

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difference for drug variable [F(6, 84)=3.15, pb0.01] (Fig. 3,panel B). The statistical analysis confirmed data previouslydescribed for the wall-rearing response measured in the open-field test. Animals treated with diazepam and FV2 increased thefrequency of wall rearing [F(6, 77)=3.702, pb0.001], whencompared to controls (Fig. 3, panel C).

3.4. Holeboard

As shown in Fig. 4, panel A, time spent in head-dipping wasconsistently more elevated in high dose of FV1 and bothdosages of FV2, compared to vehicle control mice, confirmingthe disinhibitory effect of the flavonoids. [Main effect of drugF(6, 80) =3.708, pb0.001]. As expected, DZ treatmentincreased the number of head dips in the holeboard test,and the same effect was induced by FV2 [Main effect of drugF(6, 80)=8.674, pb0.001] (Fig. 4, panel B). On the otherhand, treatment with the low dose of FV1 differentlymodulated this parameter.

4. Discussion

The aim of the present study was a preliminary investigationof the behavioral profile induced by 5-methoxy-6,8-dibromo-flavanone and 6-bromo-flavanone, two novel halogenatedflavonoids. The behavioral effects of acute intra-peritoneal

administration of these substances were examined in detail inmice, compared to diazepam and the natural occurringflavonoid chrysin, both tested at a dosage which is reported tobe anxiolytic but not sedative or miorelaxant (Wolfman et al.,1994; Viola et al., 1995).

The main finding of the present study was that both of thesecompounds show clear-cut anxiolytic profile, and do not inducesedation or reduce levels of exploratory behavior of animals,neither induce stereotyped behaviours. A great body ofevidence indicates that substitution at position 6 of flavonenucleus is important to increase the anxiolytic activity of thisclass of compounds (Huang et al., 2001; Marder et al., 2001),which is already active without substitutions. According toWang and colleagues, flavanone nucleus does not exert anyanxiolytic activity (Wang et al., 1999). Despite this finding, itwas observed in this study that the substitution at position 6 offlavanone nucleus with a bromine atom leads to compoundswith elevated anxiolytic properties. Considering the chemicalstructure of the two compounds, both of them were supposed toact as ligand of GABA(A) receptor, and in particular, thedisposition of substituents in the body of the molecules suggeststhat they could act as partial agonists at the BDZ receptor.Partial agonists are interesting agents, because they have severaleffects of BDZs, but do not produce CNS depression.

In the open-field task, both diazepam and 6-bromo-flavanone slightly increased levels of locomotor activity. Asexpected, all animals displayed a typical thigmotactic behav-iour, then moved to the centre of the open field. The frequencyof wall-rearing behaviour was more elevated in mice treatedwith diazepam and 6-bromo-flavanone, confirming the datapreviously obtained for crossing measurement.

Exploration of novel environments comes often in associ-ation with the experience of a certain degree of anxiety. In theelevated plus-maze paradigm, levels of anxiety-related beha-viours are directly inferred by the measure of the latency to enterand time spent in the open-unprotected areas of the apparatus(Holmes and Rodgers, 1999; Pellow et al., 1985; Macrì et al.,2002). Classical literature reports indicate that a clear-cutpreference for the closed arms as well as avoidance for the openones is usually exhibited by rats and mice (File, 1996). Thisprofile is thought to be a function of increased levels of anxietyassociated with unprotected areas of the apparatus and on itselevation from the floor. In this study both spatio–temporal andethological parameters were measured. Concerning the spatio–temporal measures, the latency to leave the central platform andenter into open arms was much lower in all treated animalscompared to controls. Interestingly the profile exhibited byflavonoid treated mice was quite similar to that of diazepamtreated animals. A short latency to enter an unprotected area isthought to be an index of less anxious behaviour, and is one ofthe main parameters used to assess the levels of anxiety inrodents. With regard to the open arm, it was observed thattreated mice spent a significantly larger amount of time into thisunprotected area of the apparatus than control animals.Diazepam treated, high dose of FV1 and both dosages of FV2treated mice, in particular, spent more time in this part of theapparatus, compared to vehicle control mice, confirming that

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the substitutions operated on the flavanone nucleus areresponsible of the anxiolytic activity. The ethological mea-surements gave analoguos results, the frequency of wall-rearing showed a profile corresponding to the one obtained inthe open field test. A factor analysis on plus-maze behaviouralprofile indicated that an elevated frequency of unprotectedhead-dipping behaviour was shown only in association withthe experience of low levels of anxiety (Rodgers et al., 1995).In the same study, a measure of risk-assessment whosedescription strictly resembles that offered for SAP in thepresent study, positively loaded on an anxiety factor. Thisindicates that the measure of SAP might be used to score levelsof perceived anxiety. The results obtained from analysis ofSAP is very interesting, because reveals that diazepam andhalogenated flavonoids significantly reduce the time spent inrisk-evaluation in mice. The natural flavonoid chrysin hadonly a slight effect on mice. The time spent in the open armswas just missing the significance, even if chrysin treated miceshowed a very short latency to enter open arms, comparable tothe one of the DZ group. Levels of exploratory behavior wereassessed in the holeboard test, by measuring the frequency andthe time spent in head-dipping. As expected, diazepamincreased the number of head dips, and the same effect wasgiven by both halogenated flavonoids. Time spent in head-dipping was more elevated in both halogenated compounds,compared to chrysin or diazepam, confirming the disinhibitoryeffect of the flavonoids.

The results obtained for 6,8-dibromo-7-methoxy-flavanoneare interesting because this compound bears substitutions indifferent positions of the flavanone nucleus, which could becomeobject of further investigations. Concerning the 6-bromo-flavanone, the demonstration that this compound has anxiolyticeffects is important, because its profile of effects is analogous orsomewhat more pronounced compared to that of 6-bromo-flavone, another flavonoid halogenated starting from flavonenucleus (Marder et al., 1996). This latter compound is reported toexert anxiolytic effects at a dose of 0.5 mg/kg (Marder et al.,1996; Paladini et al., 1999), while 6-bromo-flavanone was shownto display an anxiolytic efficacy already at 0.37 mg/kg.

Further investigations are in progress to evaluate, withspecific binding assays, wether the two flavonoids act as partialor total agonist at the BDZ-bs of the GABA(A) receptor. Anexperiment with a GABAa antagonist is also required, toconfirm the site of action of these compounds. Thus, theanxiolytic properties of these novel halogenated flavonoidsencourage to make deeper investigations on the effects of thesubstitution of flavanone nucleus.

Acknowledgements

We wish to thank the subproject “Influence of socio-environmental factors on pathogenesis and outcome in animalmodels of neurodegeneration” (PI: G.L.) within the ResearchProject “Pathogenetic processes and recovery in animal and in-vitro models of Alzheimer disease”, Italian Ministry of Health.The support by Sigma–Tau SpA (Behavioural PharmacologyLab), Pomezia, Italy is also acknowledged.

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