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Phytomedicine 14 (2007) 645–651

www.elsevier.de/phymed

Serotonin mediates beneficial effects of Hypericum perforatumon nicotine withdrawal signs

C. Mannuccia, A. Pierattia, F. Firenzuolic, A.P. Caputia,b, G. Calapaia,�

aDepartment of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, School of Medicine,

University of Messina, Via Consolare Valeria, Policlinico Universitario, Torre Biologica 51 piano, 98125 Messina, ItalybIRCCS Centro Neurolesi ‘‘Bonino-Pulejo’’, University of Messina, Messina, ItalycService of Phytotherapy, Ospedale S. Giuseppe, ASL N. 11 Empoli, Italy

Received 26 June 2006; accepted 28 March 2007

Abstract

Antidepressants may be effective treatment for smoking cessation and new evidence on relationship betweensmoking and depression is emerging. Extracts of the plant Hypericum perforatum possess antidepressant activity inhumans and reduce nicotine withdrawal signs in mice. Both nicotine and H. perforatum administration elicit changes inserotonin (5-HT) formation in the brain. On this basis, we investigated the possible involvement of 5-HT in thebeneficial effects of H. perforatum on nicotine withdrawal signs. With the aim to induce nicotine dependence, nicotine(2mg/kg, four intraperitoneal injections daily) was administered for 14 days to mice (NM). Saline (controls, M) orH. perforatum extract (Ph 50, 500mg/kg) were orally administered immediately after the last nicotine injection for 30days after nicotine withdrawal. Another group of animals treated with nicotine (14 days) and successively withH. perforatum extract was intraperitoneally co-administered with selective 5-HT receptorial antagonist WAY 100635(WAY) (1mg/kg). All animals were evaluated for locomotor activity and abstinence signs, 24 after nicotinewithdrawal. Brain 5-HT metabolism was evaluated in the cortex of mice sacrificed 30 days after nicotine withdrawalthrough evaluation of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/5-HT ratio. After nicotine withdrawalmeasurement of 5-HT metabolism in the cortex showed a reduction of 5-HT content while animals treated only withHypericum extract showed a significant reduction of total abstinence score compared to controls. WAY inhibited thereduction of total abstinence score induced by H. perforatum. Moreover, 5-HT1A expression has been evaluated 30days after nicotine withdrawal. Our results, show a significant increase of cortical 5-HT content in NM treated withH. perforatum, with a concomitant significant increase of 5-HT1A receptor. So, it is possible to suggest an involvementof 5-HT in beneficial effects of H. perforatum on suffering produced by nicotine withdrawal in dependent mice.r 2007 Published by Elsevier GmbH.

Keywords: Nicotine; Hypericum perforatum; Serotonin; 5-HT1A

e front matter r 2007 Published by Elsevier GmbH.

ymed.2007.06.005

ing author. Tel.: +3990 221 3646;

3300.

ess: gcalapai@unime.it (G. Calapai).

Introduction

Nicotine, as delivered in tobacco smoke, is one of themost widely abused drugs worldwide (Wong andLicinio, 2001) and it is responsible for profoundbehavioral effects that can contribute to ongoing

ARTICLE IN PRESSC. Mannucci et al. / Phytomedicine 14 (2007) 645–651646

smoking. Smokers report that they smoke to decreaseanxiety, improve attention, decrease appetite, relievedepression or because it is pleasurable (Picciotto, 2003).

Tobacco addiction has a considerable health impactof society and has become one of the largest healthproblems worldwide (Murray and Lopez, 2001). It hasbeen estimated that the 80% of all regular smokers wantto quit and most part of them tried to quiet and failed.Since the administration of nicotine from transdermalpatches can exert an antidepressant-like activity innonsmokers (Salin-Pascual et al., 1996) and nicotinecessation induces depressive-like symptoms, it has beenhypothesized that depressive people self-medicate withnicotine (Markou et al., 1998) and further evidenceregarding the association between depression andtobacco smoking is now emerging (Carton et al., 2002;Dierker et al., 2002). A further link between smokingand mood disorders is represented by the fact thatnicotine withdrawal in smokers may elicit a state inwhich they are more sensitive to the adverse effects ofstress (Balfour and Ridley, 2000).

In recent years, Hypericum perforatum (St. John’sWort) extracts have been studied for their therapeuticeffects on mood disorders, and its antidepressive actionhas been demonstrated in animals and in man (De Smetand Nolen, 1996; Linde et al., 1996) indicating that itsefficacy is comparable to that of tricyclic antidepressants(Phillipp et al., 1999). The plant contains severalbiologically active compounds such as napthodian-thrones including hypericin and pesudohypericin, tan-nins, proanthocyanidins, flavonoids and phloroglucinolderivatives (hyperforin) (Kasper, 2001). The exactmechanism of antidepressant action is not clearlyknown but the effects have been related to inhibi-tion of monoamino-oxydase and/or neurotransmittersreuptake.

We previosuly showed that administration ofH. perforatum can attenuate nicotine withdrawal signsin mice in which nicotine dependence has beenexperimentally induced (Catania et al., 2003); moreover,it has been shown that nicotine exposure produceschanges in serotoninergic system (Ribeiro et al., 1993).Serotonin (5-HT) involvement in mood disorders andmechanism of action of antidepressant drugs is wellknown (Cryan and Leonard, 2000). Moreover, it hasbeen reported that nicotine increases the release of 5-HTfrom striatal synaptosomes in vitro (Reuben and Clarke,2000), acute administration of high doses of nicotinestimulates in vivo the release of 5-HT in the frontalcortex of the rat (Ribeiro et al., 1993), while acutenicotine administrations decrease 5-HT biosynthesis aswell as the extracellular 5-HT levels in the hippocampus(Balfour and Ridley, 2000). Seven 5-HT receptors, havebeen identified (Peroutka, 1993), and the relationshipamong these receptors and depression is not clearbecause of the unavailability of their selective agonists

and antagonists. The 5-HT1 has been subclassified as:5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E sites(Schmidt and Peroutka, 1989), and they seem to bedifferently involved in the behavioral alteration causedby repeated nicotine administrations. Results of micro-dialysis studies indicate relative importance of 5-HT1A

autoreceptor to the mechanism of action of selective5-HT reuptake inhibitors or monoamino-oxidase in-hibitors (Gardier et al., 1996). Furthermore, recently ithas been showed that 5-HT1A receptor is one of themediators of the antidepressant-like action of otheragents (Estrada-Camarena et al., 2003).

In light of this basis, we investigated the possibleinvolvement of 5-HT in beneficial effects of H. perforatum

on nicotine withdrawal signs by studying the effects ofH. perforatum extract on serotoninergic system in micetreated with consecutive nicotine administration, and inpresence of selective 5-HT1A receptorial antagonist WAY100635 (WAY).

Materials and methods

Animals

Male Swiss 8-week-old mice, housed at a constanttemperature (2272 1C) and a light cycle of 12/12 h(7.00 a.m./7.00 p.m.) with free access to food anddrinking water, were used. Adaptation and experimentswere carried out in accordance with the internationallyaccepted principles and the national laws concerningthe care and the use of laboratory animals and withthe European Communities Council Directive of 24November 1986 (86/609/EEC).

Induction of nicotine dependence

Animals were randomly allocated in different groups(n ¼ 6 in each group). Mice were subcutaneously (s.c.)injected with saline, (M) or nicotine hydrogen tartratesalt (Sigma-Aldrich, Italy) 2mg/kg (NM), respectively,four injections daily, 4 h apart, starting at 08:00 h for 14days. This intermittent nicotine treatment more closelyresembles human use. Upon completion of the nicotinetreatment, all animals were evaluated for locomotoractivity and abstinence signs 24 h after the last saline ornicotine injection (Isola et al., 1999).

Locomotor activity

Locomotor activity was evaluated either in M (micewhich received saline) or NM (mice which receivednicotine) 24 h after the last injection of saline or nicotinein an open field apparatus subdivided in nine commu-nicating squares. The animals were placed singularly in

ARTICLE IN PRESSC. Mannucci et al. / Phytomedicine 14 (2007) 645–651 647

the apparatus and the number of squares crossed weredetermined in 6min (Calapai et al., 1995). Locomotoractivity was evaluated by observers blind to drugtreatment.

Evaluation of abstinence

Abstinence signs were evaluated by four observersblind to drug treatment at 24 h into withdrawal. Prior toeach observation session, nicotine- and saline-treatedmice were placed in clear cages to habituate for 30min,and they were returned after 60min (adaptation: 30min;observation: 30min) to their home cage upon comple-tion of the behavioral evaluation. The Nicotine Absti-nence Scale scored the frequency of the following signs:rearing, jumping, body shakes, head shakes, forelimbshakes, scratching, chewing, abdominal constrictionsand facial tremor during 30min of observation. For theevaluation of the behavioral signs, a Nicotine Absti-nence Scale was compiled (Isola et al., 1999). Theseverity of the abstinence syndrome was evaluated bycomputing the total abstinence sign score.

H. perforatum treatment

Three other groups of mice, treated with nicotine for14 days as previously described, received oral acuteadministration by gavage of an H. perforatum extract(Ph 50, 125–500mg/kg) immediately after the lastnicotine injection for 30 consecutive days after nicotinewithdrawal. A further group of mice, not treatedpreviously with nicotine, received the highest dose ofPh 50 (500mg/kg). In the second experiment, twogroups of mice treated with saline or nicotine wereevaluated for locomotor activity and abstinence signs24 h after the last saline or nicotine injection. All groupsof mice comprised six animals. Ph 50 is a solid (powder)extract of H. perforatum used in other experimentswhose results were previously published (Calapai et al.,1999, 2001a, Catania et al., 2003) containing 50%flavonoids as obtained through a modification of themethod of extraction by Kartnig T and Gobel I., 1992(Stankov and Firenzuoli, 1992), 0.3% hypericin (DAC-1986, 3rd Suppl., 1991) and 4.5% hyperforin (Calapaiet al., 2001a) (the remaining part is composed ofpolysaccharides represented by maltodextrins), pur-chased from Pharmalife-Research (Italy). Briefly, driedSt. John’s herb is added to a solution of ethanol in water(60% ethanol w/w) (continuous extraction processtemperature 40–60 1C). Then sample is concentratedunder vacuum (temperature o60%) bringing tempera-ture gradually from 140 to 70 1C. Subsequentlymaltodextrin is added (necessary for drying, stability,flowability reduction of hygroscopicity and standardiza-tion) and hypericin, hyperforin and flavonoid contents

in the St. John’s wort dry extract are controlled. Then, aquantity of flavonoids (rutin 95% purity and quercetin99% purity) was added in the sample, up to obtain a50% of total flavonoids content in the mixture. Beforeadministration, Ph 50 has been dissolved in saline.

WAY 106355 treatment

In another experiment, NM and NM+Ph 50 animalswere treated with a single subcutaneus injection of saline(2ml) or the selective 5-HT receptorial antagonist WAY(1mg/kg in 2ml ICN, Italy) 75min before the valuationof abstinence signs (Tatarczynska et al., 2002).

Serotonin and 5-hydroxyindoleacetic acid

determination

A different group of animals which received nicotinefor 14 days (NM) and their controls (M) was sacrificedafter anesthesia with chloral hydrate (400mg/kg i.p.), 30days after saline or nicotine withdrawal with the aim tomeasure the brain levels of 5-HT, and 5-hydroxyindo-leacetic acid (5-HIAA). Intact brains were removedfrom the skull, and the cortex was dissected free of theunderlying white matter and successively homogenizedin 1ml of 0.3N perchloric acid (Squadrito et al., 1993).Cell debris was removed by centrifugation at 6000g for10min. The pellet was discarded and the supernatantwas filtered. The samples obtained were frozen at�70 1C until the assay. Sample contents of 5-HT and5-HIAA were determined using an HPLC electrochemi-cal detection technique (Squadrito et al., 1991). Mea-surements of 5-HT and 5-HIAA were expressed asnanomoles per gram of protein.

5-HT1A detection, Western blotting analysis

For western blotting analysis, animals were sacrificed30 days after nicotine withdrawal. The cortex washomogenized in 1ml of 10mM Tris–HCl (pH 7.4), 10%SDS, 1mM aprotinine and 1mM sodium orthovana-date. The homogenates were centrifuged at 15,000g for5min at 15 1C, and the supernatants were collected.Samples (50 mg of protein) were run on 10% PAGE andtransferred electrophoretically onto polyvinyl mem-brane.

The membrane was incubated with guinea anti-serotonin 5-HT1A, polyclonal antibody (1:500 dilution)in TBS 0.1% Tween 20 buffer at 4 1C overnight. Afterrinsing with TBS 0.15% Tween 20, the membrane wasincubated in 5% dried milk, with secondary antibodyhorse anti-guinea pig peroxidase coniugate (1:8000dilution) for 1 h at room temperature. After the finalrinse, the immunoreactive bands in the membrane were

ARTICLE IN PRESS

0

1

2

3

4

5

6

7

M NM NM + Ph 50

To

tal a

bs

tin

en

ce

sc

ore

*

˚

M + Ph 50

Fig. 2. Effects of oral dose of Hypericum perforatum extract

Ph 50 (500mg/kg) on total abstinence score after 14 days of

C. Mannucci et al. / Phytomedicine 14 (2007) 645–651648

visualized using ECL western blotting detection reagents(Amersham Bioscience).

Statistical analysis

All statistical procedures were performed using SPSSstatistical software package release 6.1.3. (SPSS, Chicago,IL, USA). Data analysis was performed using one-way analysis of variance with the Scheffe post hoctest for multiple comparisons. All the groups of animalswere composed of six mice. The data are expressedas the means7s.e.m. Statistical significance was set atpo0.05.

nicotine treatment (2mg/kg daily s.c.). Ph 50 was administered

after the last saline or nicotine injection for 30 days after

nicotine withdrawal. Each column represents the mean7-standard error of six animals. J ¼ po0.01 vs M; * ¼ po0.01

vs NM.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

NM + Ph 50 NM + Ph 50 + WAY (1 mg/kg)

To

tal

Ab

sti

nen

ce S

co

re

*

Fig. 3. Effects of intraperitoneal injection of WAY 100635

(1mg/kg) on total abstinence score during nicotine withdrawal

Results

Locomotor activity was estimated in mice whichreceived repeated administration of nicotine (NM), orsaline (M) and treated with Hypericum extract (Ph 50,500mg/kg). In NM there was a significant reduction oflocomotor activity compared with M.M., crossed amean (7s.e.m.) of 72.773.28 squares, NM crossed39.573.4 squares (po0.001). However, in NM+Ph 50there was an increase of locomotor activity comparedwith NM, while Ph 50 did not modify locomotoractivity. M+Ph 50 crossed 73.273.5 squares andNM+Ph 50 crossed 6675.4 squares (Fig. 1).

Total abstinence score was increased in NM com-pared with M. Treatment with H. perforatum extract(Ph 50, 500mg/kg) caused a significant reduction oftotal abstinence score in animals which received nicotine(NM), while, when given in M (M+Ph 50), Hypericum

did not modify total abstinence score (Fig. 2).

0

10

20

30

40

50

60

70

80

90

M NM M+Ph 50 NM+Ph 50

Sq

uare

s/6

min

*

˚

Fig. 1. Locomotor activity (number of squares crossed in

6min) in M or NM 24h after daily repeated injection of

nicotine (2mg/kg daily s.c.; 14 days) and treated with

Hypericum perforatum extract Ph 50 (500mg/kg) for 30 days,

after the last nicotine injection. Each column represents the

mean7standard error of six animals. J ¼ po0.01 vs M;

* ¼ po0.01 vs NM.

after 14 days of nicotine treatment (2mg/kg daily s.c.). WAY

was given 75min before the valuation of abstinence signs.

Each column represents the mean7standard error of six

animals. * ¼ po0.01 vs NM+Ph-50 (500mg/kg).

Moreover, NM animals receiving Ph 50, when treatedwith WAY (1mg/kg), showed a significant reduction oftotal abstinence score starting from WAY 0.1mg/kg(Fig. 3). Content of 5-HT and 5-HIAA in the cortex wasmeasured in animals sacrificed 30 days after withdrawal.NM animals showed a significant reduction in 5-HTcontent compared with M (Table 1). Treatment with Ph50 in M and NM produced a significant increase of5-HT and 5-HIAA content in the cortex compared withmice not treated with Hypericum (Table 1). Westernblotting analysis showed a great reduction of 5-HT1A

expression in NM 30 day after nicotine or salinetreatment, while after treatment with Ph 50, 500mg/kg,5-HT1A expression was significantly increased both in Mand NM (Fig. 4).

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Table 1. Effects of oral dose of Hypericum perforatum (Ph 50,

500mg/kg) extract given for 30 days after nicotine injection, on

M and NM cortex serotonin metabolism

Group 5-HT

(nmol/g)

5-HIAA

(nmol/g)

5HIAA/5-HT

M 4.8870.2 4.9270.18 1.0170.02

NM 3.0970.1** 4.7070.20 1.1670.02

M+Ph 50 5.9070.2* 670.18* 1.0270.02

NM+Ph 50 5.9370.18** 6.2070.08** 1.0470.03

M ¼ mice which received repeated administration of saline (four

injections daily for 14 days).

NM ¼ mice which received repeated administration of nicotine

(2mg/kg s.c. four injections daily for 14 days).

NM+Ph 50 ¼ mice which received oral administration of Hypericum

perforatum (500mg/kg) immediately after last nicotine injection for

30 days.

M+Ph 50 ¼ mice not treated with nicotine, which received oral

administration of Hypericum perforatum (500mg/kg) after last saline

injection for 30 days.

Each value represents the mean7s.e.m. of six animals.

* ¼ po0.05 vs M.

** ¼ po0.01 vs NM.

0

20

40

60

80

100

120

M M+Ph 50 NM NM+Ph 50

5-H

T1

A e

xp

res

sio

n

M M+Ph 50 NM NM+Ph 50

5-HT1A

Fig. 4. Western blot analysis of cortical 5-HT1A receptor, in

mice treated with repeated administration of saline (M) or

nicotine (NM) (2mg/kg s.c. for 14 days), after treatment with

Hypericum perforatum, Ph 50 (500mg/kg, given for 30 days

after nicotine withdrawal). Percentages were expressed as

arbitrary units of integrated intensity. Each value represents

the mean7s.e.m. of six animals.

C. Mannucci et al. / Phytomedicine 14 (2007) 645–651 649

Discussion

Nicotine withdrawal syndrome can be produced inthe laboratory animal. Discontinuing of nicotine,after repeated administration of high doses, causes theappearance of objective signs valuable through absti-nence scales (Isola et al., 1999). We previously showedthat administration of H. perforatum extract is able toreduce the severity of nicotine withdrawal signs in mice(Catania et al., 2003). H. perforatum extracts are widelyused as antidepressants to treat moderate depression.Their beneficial effects could be due to changes inneurotransmitters content in different brain areas

observable also after single administration (Calapaiet al., 1999). Anti-immobility effects of Hypericum wereobserved on motor activity in the forced swimmingtest, a test used to evaluate antidepressant activity ofsubstances in laboratory. This effect suggests anantidepressant-like activity of the extracts of the plant(Calapai et al., 2001a). Such behavioral modificationsproduced by Hypericum are associated with changes in5-HT, norepinephrine and dopamine, the same neuro-transmitters involved in the mechanisms which under-lain mood disorders (Teufel-Mayer and Gleitz, 1997).Receptorial antagonists against 5-HT, norepinephrineand dopamine are able to inhibit the anti-immobilityeffects of Hypericum and the largest degree of inhibitionwas observed when 5-HT was antagonized (Calapaiet al., 2001b). The rationale for possible use ofHypericum in smoking cessation is suggested bynumerous and consistent evidences demonstrating anassociation between depressive disorders and the pre-valence of tobacco smoking. This relationship served asthe scientific substrate for the use of antidepressantssuch as bupropion a drug licensed for smoking cessation(Balfour and Ridley, 2000; Hayford et al., 1999).

In the present work, we show that the selective5-HT1A receptorial antagonist WAY abolishes thereduction of total abstinence score produced byHypericum administration in nicotine-dependent micewhen nicotine is discontinued, thus indicating thatserotoninergic system could be responsible for theappearance of withdrawal signs. This idea is reinforcedby the observation of changes of 5-HT metabolism inthe cortex of mice when nicotine was withdrawn. Inthese animals, evaluation of 5-HT metabolism showed areduction either in 5-HIAA content or 5-HIAA/5-HTratio, indicating a decreased 5-HT turnover. At the sametime, we observed that Hypericum given immediatelyafter the last nicotine injection restored a normal 5-HTturnover. So it is possible to hypothesize that normal-ization of 5-HT induced by Hypericum could beresponsible for attenuation of abstinence signs.

Neuronal nicotinic receptors are distributed with ahigh concentration in the cortex and they are predomi-nantly situated on presynaptic terminals where they actto enhance neurotransmitter release (Wevers et al.,1994). 5-HT changes observed after nicotine withdrawalin the cortex are consistent with abnormalities ofserotoninergic system associated with exposure tonicotine. It has been shown that nicotine increases therelease of 5-HT from striatal synaptosomes in vitro

(Reuben and Clarke, 2000) and that acute administra-tion of high doses stimulates in vivo the release of 5-HTin the frontal cortex of the rat (Ribeiro et al., 1993),while acute nicotine administration decreases 5-HTbiosynthesis as well as the extracellular 5-HT levels inthe hippocampus (Balfour and Ridley, 2000). Moreover,nicotine exposure produces changes in the expression of

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the 5-HT1A receptor in this brain region, giving value tothe hypothesis that nicotine may influence the post-synaptic sensitivity to 5-HT (Kenny et al., 2001). It ispossible to hypothesize that reduction of 5-HT turnoverobserved in our experiments after nicotine withdrawalcould be the sign of further adaptative events subse-quent to cessation of nicotine activity. These changescould represent one of the most important factorscontributing to the signs observed following nicotinewithdrawal.

The link between nicotine effects on 5-HT anddepression has been partially investigated. Whereaschronic exposure to nicotine elicits changes both in5-HT and adrenocortical function, which are character-istic of depression, it has been hypothesized thatsmokers are protected from the adverse consequenceof these changes while they keep to smoke, but also thatthey contribute significantly to depressive symptoms(Salin-Pascual et al., 1996). Another possible mechanismimplies the involvement of 5-HT in nicotine dependenceand brain system of reward, and it could be representedby the role that, according to different authors, theneurotransmitter seems to play a role in the modulationof nicotine stimulation on mesocorticolimbic dopamineactivity (Olausson and Engel, 2002; Salin-Pascual et al.,1996).

In conclusion, nicotine administration causes altera-tions of 5-HT metabolism and 5-HT1A receptor expres-sion. Long-term treatment with H. perforatum reducesnicotine abstinence signs bringing again to the normalvalues cortical 5-HT content and its metabolites,together with the expression of 5-HT1A receptor,suggesting that H. perforatum extract administrationmodulates one of the major biochemical systemsrelevant for antidepressant activity.

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