* Correspof PharmaE-mail: ozg

PHYTOTHERAPY RESEARCHPhytother. Res. (2012)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.4801

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Central Nervous System Activities of Hypericumoriganifolium Extract via GABAergic andOpioidergic Mechanisms

Şahin Nuri Yaşar,1 Özgür Devrim Can,1* Nilgün Öztürk,2 Gianni Sagratini,3Massimo Ricciutelli,3 Sauro Vittori3 and Filippo Maggi31Faculty of Pharmacy, Department of Pharmacology, Anadolu University, 26470, Eskişehir, Turkey2Faculty of Pharmacy, Department of Pharmacognosy, Anadolu University, 26470, Eskişehir, Turkey3School of Pharmacy, University of Camerino, 62032, Camerino, Italy

Pharmacological effects of hydroalcoholic extract prepared from Hypericum origanifolium Willd. (Guttiferae) onbehavioral parameters and pain perceptions of mice were investigated in this study. Depression, anxiety, spontaneouslocomotor activity, and motor coordination parameters of mice were assessed by modified forced swimming, holeboard, activity cage, and rota-rod tests, respectively. In addition, antinociceptive effect was evaluated by performinghot-plate, tail-clip, and formalin tests. Reboxetine (20mg/kg), diazepam (1mg/kg), andmorphine (10mg/kg) were usedas reference antidepressant, anxiolytic, and analgesic drugs, respectively. Phytochemical analyses exhibited thatchlorogenic acid (2317.12ppm) and rutin (2108.79ppm) were the main phenolic compounds in the H.origanifoliumextract. The extract (50, 100, and 250mg/kg) induced significant antidepressant, anxiolytic, and antinociceptiveactivities following the acute administrations. Anxiolytic effect was antagonized by flumazenil (a benzodiazepinereceptor antagonist, 2.5mg/kg, i.p.) pre-treatment, which indicated the participation of GABA(A)-benzodiazepinereceptor complex in the activity. Moreover, centrally and peripherally mediated antinociception reversed by naloxone(a non-selective opioid receptor antagonist, 5mg/kg, i.p.) pre-treatment, indicating the involvement of opioid system inthe pharmacological action. These findings are the first to indicate the pharmacological effects of theH.origanifoliumextract on the emotional state and pain perceptions of mice. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: Hypericum origanifolium; chlorogenic acid; rutin; antinociceptive; antidepressant; anxiolytic.

INTRODUCTION

Several scientific papers have been published representingthe therapeutic potential of Hypericum species (fam:Guttiferae) on various central nervous system (CNS)disorders. As well as Hypericum perforatum, the mostwell-known medicinal plant belonging to this family, someother species such as Hypericum canariense, Hypericumglandulosum (Sánchez-Mateo et al., 2005), Hypericumcaprifoliatum (Viana et al., 2008), Hypericum enshiense(Wang et al., 2010), and Hypericum montbretii (Canet al., 2011), have been previously reported for theirantidepressant activities. Furthermore, some Hypericumspecies, such H. montbretii (Can and Ozkay, 2012), H.perforatum, and Hypericum hircinum (Diana et al., 2007),have been reported to have sedative activity in relation totheCNS.Another CNS-related activity, centrallymediatedantinociception, has been reported in rodents afteradministration of extracts of Hypericum grandifolium(Bonkanka et al., 2011), H. caprifoliatum, Hypericumpolyanthemum (Viana et al., 2003), H. canariense,H.glandulosum (Rabanal et al., 2005), and Hypericumreflexum (Sánchez-Mateo et al., 2006).Hypericum origanifolium Willd. is another Hypericum

genus endemic to Turkey, Georgia, and Armenia.Comprehensive phytochemical studies related to this plant

ondence to: ÖzgürDevrimCan, Faculty of Pharmacy,Departmentcology, Anadolu University, 26470 Eskişehir, Turkey.urdt@anadolu.edu.tr

© 2012 John Wiley & Sons, Ltd.

indicated that the aerial parts contain naphthodiantrones,flavonoids, xanthones, and chlorogenic acid (Öztürk et al.,2009). Most of these constituents have pharmacologicaleffects, in relation toCNS, such as antidepressant, anxiolytic,sedative, and antinociceptive activities (Beerhues, 2006;Haas et al., 2010; Lapa Fda et al., 2009; Machado et al.,2008; Marder and Paladini, 2002; Paulke et al., 2008).

Despite the noticeable psychopharmacological activ-ity potential of H. origanifolium extract due to its phyto-chemical content, no scientific report related to the CNSactivity profile of this plant is available. Informationobtained from new phytochemical and pharmacologicalstudies on different Hypericum species is believed to bebeneficial for clarifying active compound/s responsiblefrom the pharmacological activities and explaining pos-sible synergic mechanisms.

Therefore, in the present study, we aimed to analyzethe phytochemical content of the hydroalcoholic extractprepared from the aerial parts of H. origanifolium(HOE) and examine the acute and sub-acute effects ofthis extract on behavioral parameters and pain percep-tions of mice.

MATERIALS AND METHODS

Drugs. Quercetin, hypericin, hyperforin, chlorogenicacid, rutin, and quercitrin were supplied by Sigma(Milan, Italy), and hyperoside and iso-quercitrin weresupplied byApplied Biosystem (Milan, Italy). Individual

Received 07 May 2012Revised 15 July 2012

Accepted 16 July 2012

Table 1. Linear gradient program for HPLC analysis

Time (min) Solvent A (%)a Solvent B (%)b Solvent C (%)c

0 85 15 010 85 15 030 65 35 045 10 90 060 10 90 061 0 0 10070 0 0 10075 0 100 0105 0 100 0110 85 15 0130 85 15 0

aSolvent A=water and phosphoric acid, pH solution 2.7.bSolvent B=90% acetonitrile, 10% methanol.cSolvent C=10% ethyl acetate, 90% mix solvent A and B (10%A, 90% B).

Ş. N. YAŞAR ET AL.

stock solutions were prepared by dissolving each com-pound in methanol and were stored in glass-stopperedbottles at 4�C. Standard working solutions, at variousconcentrations, were prepared daily by appropriatedilution of aliquots of the stock solutions in methanol.Methanol, acetonitrile, and ethyl acetate were of gra-dient grade for liquid chromatography and purchasedfrom Merck (Darmstadt, Germany). Deionized water(>18MΩ cm resistivity) was obtained from a Milli-QSP Reagent Water System (Millipore, Bedford, MA).All of the solvents were filtered through 0.2-mmcellulose filter (RC 58) from Schleicher and Schuellbefore use.Flumazenil (Flu), naloxone hydrochloride dehydrate

(Nlx), morphine sulphate (Mor), formaldehyde solution,and ethanol used were purchased from Sigma-Aldrich(St. Louis, MO). Diazepam (Dzm) and reboxetine mesy-late (Rbx) studies were carried out with commerciallyavailable DiazemW ampul (Deva İlaç Sanayi ve TicaretA.Ş., İstanbul, Turkey) and EdronaxW tablet (PfizerPharmacia (Int.), İtaly), respectively.

Plant material and preparation of the extract. Hyperi-cumoriganifolium Willd. was collected around Tekörenvillage in Sivrihisar, Eskişehir, Turkey at 1.100m, inJune 2009, and its voucher specimen was deposited atthe herbarium of the Laboratory of Botany, AnadoluUniversity, Eskişehir, Turkey with the registry numberof OUFE 10334. Dried aerial parts were macerated in50% ethanol (1:10) for one night and extracted for 8 hat 40�C water bath, then filtered. This process wasrepeated three times; filtrates were collected andconcentrated under reduced pressure in a rotary evap-orator at 40�C to remove ethanol. The remainingaqueous part was freeze dried at �80�C and lyophilized.The extract obtained was weighed to determine theyields of soluble constituents. The yield of the extractwas calculated as percentages (2.23%).

Phytochemical analyses. High-performance liquid chro-matography (HPLC) and diode-array detector (DAD)conditions: a Hewlett Packard (Palo Alto, CA) HP-1100series, equipped with a binary solvent pump, an autosam-pler, with the volume injection set to 20mL, and aDAD coupled with an HPLC/DAD ChemStation(Rev. A. 06. 03) was used. Separation was performed ona LUNA C18 (150� 4.6mm) protected by a Securityguard cartridge C18 (4� 2mm I.D.), both fromPhenomenex USA (distributed by Chemtek Analytica,Bologna, Italy). The monitored wavelengths were210 nm for phenolic compounds, 270 nm for hyperforin,and 590 nm for hypericin. The adopted chromato-graphic method is reported in Table 1. The sampleconcentration was 3mg/mL in methanol, and theflow rate was 1mL/min. The method used for compo-nents identification was the comparison of theirretention times with respect to those of standards,chromatographed under the same conditions. Inaddition, UV spectra of both samples and standardswere compared using the DAD. Moreover, the con-firmation of the identified compounds was obtainedby injecting standard and sample solutions in anHPLC-MS equipped with an ESI interface in negativeionization mode using the same chromatographicconditions (formic acid was used instead of phosphoricacid at the same pH).

Copyright © 2012 John Wiley & Sons, Ltd.

Pharmacological studies

Animals. Adult male Swiss-albino mice weighing 30–35 gwere used for the tests. Animals were housed in a roomwith controlled temperature (25� 1�C) and a 12-h light/dark cycle (lights on 8AM–8 PM). Temperature, sound,and light conditions were not altered during the courseof the experiments. All animals were acclimatized to thelaboratory environment at least 48 h before the experi-ments. The experimental protocol was approved by theLocal Ethical Committee on Animal Experimentation ofAnadolu University, Eskişehir, Turkey.

Drug and extract administrations. Animals were ran-domly divided into groups, which are ‘control group’,‘reference groups’ (Rbx, Dzm, Mor), ‘extract-treatedtest groups’, and ‘antagonists (Flu and Nlx) pre-treatedplus extract-treated test groups’ (n= 7 in each). Thecontrol solution was physiological saline. Physiologicalsaline, reference drugs, and the extract were orally givento the animals at the same time of the day (9:00 AM).

Reference drugs Dzm, Mor, and Rbx were used at1mg/kg (Seo et al., 2007), 10mg/kg (Can et al., 2010),and 20mg/kg (Can et al., 2011) doses, respectively. Dosesof 50, 100, and 250mg/kg of the extract were tested in allbehavioral and nociceptive tests. Mechanistic studieswere conducted using Flu (2.5mg/kg), a g-aminobutyricacid-A (GABA/A)-benzodiazepine receptor complexsite antagonist (Venâncio et al., 2011), and Nlx (5mg/kg),a non-specific opioid receptor antagonist (Zapata-Sudoet al., 2010), which were applied 15min before theextract administrations.

Measurements were performed at 1st, 7th, and 14th daysof the experimental procedure.

Behavioral tests

Modified forced swimming test. The antidepressant-likeactivity of the extract was screened using the modifiedforced swimming test (MFST), as described earlier(Can et al., 2009; 2011). A time-sampling techniquewas applied to score the durations of climbing, swim-ming and immobility during 5min (Cryan et al., 2002).

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PHARMACOLOGICAL EFFECTS OF HYPERICUM ORIGANIFOLIUM

Hole-board tests. Exploratory behavior of mice wasscreened using hole-board tests (Ugo Basile, no. 6650,Varese, Italy), as described previously (Can et al., 2010;Can and Ozkay, 2012). The total number of head-dipping behavior was recorded for 5min.

Activity cage tests. Spontaneous locomotor activities ofmice were monitored in an activity cage apparatus (UgoBasile, no. 7420) (Can et al., 2010; Can and Ozkay,2012). Total number of horizontal and vertical activitywas recorded for 4min.

Rota-rod tests. The effect of the extract on motor coordin-ation of mice was examined by Rota-rod test (Ugo Basile,no. 47600) as described previously (Can et al., 2010). Thelatency to fall from the rotating mill was recorded for eachmouse tested as a criterion of motor coordination.

Nociceptive tests

Hot-plate test. Antinociceptive activity potential of theextract on thermal noxious stimuli was evaluated by hot-plate analgesy-meter (Ugo Basile, no. 7280), which wasset at 55� 1.0�C, as described previously (Kaplancikliet al., 2012). The time of licking the forepaws or eventuallyjumping was recorded as a parameter of nociception.Maximum latency time (cut-off time) was established as30 s to prevent probable tissue damage.

Tail-clip test. Antinociceptive activity potential of theextract on mechanical noxious stimuli was evaluatedby tail-clip test, as described previously (Can et al.,2010; Kaplancikli et al., 2012). A metal artery clampwas applied to the tail of mouse, and the time spentbefore biting the clamp was recorded by a stopwatch.A sensitivity test was carried out before the experimen-tal session, and animals that did not respond to theclamp within 10 s were discarded from the experiments.Cut-off time for the tail-clip tests was chosen as 10 s toavoid possible tissue damage.

Formalin test. Antinociceptive activity potential of theextract on chemical noxious stimuli was evaluated byformalin test, as described previously (Demir Ozkayet al., 2012); 20 mL of 2.5% formalin solution was injectedsubcutaneously into the right dorsal hind paw. Time inseconds the mouse spent for intensive licking the pawwas recorded between 0–5min (early phase) and15–30min (late phase) after the formalin injection.

Figure 1. Effects of acute Rbx (20mg/kg) and HOE (50–250mg/kg) adin the MFST. Values are given as mean�SEM. Significance againstagainst HOE-50 group, ap<0.05, bp<0.01, cp<0.001. SignificanceTukey test, n=7.

Copyright © 2012 John Wiley & Sons, Ltd.

Statistical analyses. GraphPad Prism 3.0 software(GraphPad Software, San Diego, CA) was used forstatistical analyses of the experimental data. Significantdifferences between groups were determined byone-way analysis of variance (ANOVA) followed by apost-hoc Tukey test. The results were expressed asmean� standard error of the mean (SEM). Differencesbetween data sets were considered as significant whenthe p value was less than 0.05.

RESULTS

Phytochemical analysis

Results of the phytochemical studies indicated that chloro-genic acid (2317.12ppm) and rutin (2108.79ppm)were themajor constituents in the extract. Quercitrin (271.38ppm),hyperforin (99.12ppm), hypericin (20.14ppm), hyperoside(17.23ppm), isoquercitrin (12.57ppm), and quercetin(11.23ppm) were detected in lower amounts.

Behavioral tests

Modified forced swimming test. Data obtained from theMFST tests are indicated in Fig. 1 and Table 2. Follow-ing the acute administrations, reference drug Rbx andHOE shortened both the immobility and swimmingdurations while increasing the climbing time of the ani-mals. The most effective dose was 250mg/kg (Fig. 1).In addition, sub-acute administrations for 7 and 14 dayscaused quite similar changes in climbing, immobility,and swimming parameters of animals with acute treat-ments (Table 2).

Hole-board tests. Fig. 2 and Table 3 illustrate the effects ofthe reference drug Dzm and HOE administrations on theexploratory behavior of mice in hole-board test. AcuteDzm and HOE administrations caused a significant in-crease in the number of total head-dipping behaviors.Dzm and 250mg/kg HOE-induced augmentations in thehead-dipping behavior disappearedwith Flu pre-treatment(Fig. 2).

In 7-day-long sub-acute administrations, the highestdose (250mg/kg) was not able to change the number ofhead dips, whereas 50 and 100mg/kg doses of theextract was still enhancing this parameter. Furthermore,in 14-day-long sub-acute administrations, 250mg/kg

ministrations on climbing, immobility, and swimming time of micecontrol values, *p<0.05, **p<0.01, ***p<0.001. Significanceagainst HOE-100 group, &p<0.05. One-way ANOVA, post hoc

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Table 2. Effects of sub-acute HOE (50, 100, and 250mg/kg) administrations on climbing, immobility, and swimming time (s) of mice inthe MFST

Control HOE-50 HOE-100 HOE-250

7days Climbing 49.7�7.7 87.0�14.1** 99.0�21.4*** 128.5�17.9***,b,&

Immobility 96.0�16.9 63.9�11.9* 56.2�11.1** 33.5�10.1***,a

Swimming 110.5�23.6 76.6�8.7* 66.3�14.9** 59.6�8.6***14days Climbing 46.2�9.1 77.7�16.4* 88.7�17.8** 109.9�16.6***,a

Immobility 87.3�16.1 56.6�9.1* 43.3�10.8** 23.4�6.5***Swimming 127.1�19.8 81.5�7.1* 68.3�18.2** 63.5�18.2**

Values are given as mean�SEM. One-way ANOVA, post hoc Tukey test, n=7.*p<0.05 significance against control values.**p<0.01 significance against control values.***p<0.001 significance against control values.ap<0.05 significance against HOE-50 group.bp<0.01 significance against HOE-50 group.&p<0.05 significance against HOE-100 group.

Figure 2. Effects of acute Dzm (1mg/kg) and HOE (50–250mg/kg)administrations and Flu (2.5mg/kg) pre-treatments on total numberof head dips in the hole-board tests. Values are given as mean�SEM.Significance against control values, *p<0.05, **p<0.01, ***p<0.001. Significance against 1mg/kg Dzm group, ~p<0.001. Signifi-cance against HOE-50 group, ap<0.05, bp<0.01. Significanceagainst HOE-250 group, XXXp<0.001. One-way ANOVA, post hocTukey test, n=7.

Ş. N. YAŞAR ET AL.

decreased the total number of head dips compared withthat of the control values, whereas 50mg/kg dose of theextract increased the same parameter (Table 3).

Activity cage tests. As it can be seen in Fig. 3 and Table 3,data obtained from the activity cage tests following acuteand sub-acute administrations were quite similar to thedata of the hole-board tests. Acute Dzm and HOEadministrations increased the number of spontaneouslocomotor activities. In addition, Dzm and 250mg/kgHOE-induced increases in the spontaneous locomotoractivities disappeared with Flu pre-treatments (Fig. 3).On the other hand, neither horizontal nor vertical

locomotor activities were increased by 250mg/kg HOEat the seventh day of the treatment. Both of theseparameters were observed to decrease significantly bydaily 250mg/kg administrations, at 14th day of theexperiment (Table 3).

Copyright © 2012 John Wiley & Sons, Ltd.

Rota-rod tests. Following the acute and sub-acuteadministrations of Dzm or HOE in Rota-rod tests, nospecific change was observed in falling latency ofanimals from the rotating mill, compared with thecontrol values (data not shown).

Nociceptive tests

Hot-plate test. Fig. 4 displays the effect of acute Morand HOE administrations on nociception parametersof mice in the hot-plate tests. Significant increase inthe reaction time of mice was observed following theadministration of both reference drug morphine andHOE, at all of the applied doses. Mor and 250mg/kgHOE-induced increases in the reaction time of micewere antagonized with the Nlx pre-treatments.

A significant increase in the response time of micewas observed by all of the applied doses of HOE, alsoat 7th and 14th days’ measurements (Table 4).

Tail-clip test. Reaction time of the animals was signifi-cantly prolonged following the acute administrations ofMor and HOE in tail-clip tests when compared with thecontrol groups. Nlx pre-treatment antagonized the effectsof Mor and 250mg/kg dose of the HOE (Fig. 4). Reactiontime of the extract-treated mice was also significantlyhigher than those of their respective controls at 7th and14th day measurements (Table 4).

Formalin test. As can be seen in Fig. 4, acute Mor andHOE administrations significantly reduced the paw lickingtime of animals in both of the early and late phases,and Nlx pre-treatments antagonized these effects in theformalin tests. HOE administrations also caused significantdecreases in the licking time at 7th and 14th days (Table 4).

DISCUSSION

This study was undertaken to analyze the phytochem-ical content of the HOE and investigate its putativeactivity on CNS by various psychopharmacologicalmethods. MFST and hole-board tests were performed

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Table 3. Effects of sub-acute HOE (50, 100, and 250mg/kg) administrations on total number of head dips and total number of horizontaland vertical activities of mice in the hole-board and activity cage tests, respectively

Control HOE-50 HOE-100 HOE-250

Total number of head dips 7days 24.0�3.1 32.9�5.0* 41.3�7.2*** 30.1�5.4&&

14days 25.0�3.2 33.3�4.8* 20.1�2.0c 5.1�1.9***,c, &&&

Total number of horizontal locomotoractivity

7 days 465.3�70.1 627.7�75.4* 730.1�101.8*** 464.9�55.0 a,&&&

14days 449.7�68.7 579.1�84.4* 444.7�74.6a 280.4�67.7**,c,&&

Total number of vertical locomotoractivity

7 days 87.9�13.4 147.7�26.6* 170.0�28.7*** 80.6�9.3 b,&&&

14days 83.7�8.8 133.1�26.0** 100.1�17.8 48.1�7.8*, c,&&

Values are given as mean�SEM. One-way ANOVA, post hoc Tukey test, n=7.*p<0.05 significance against control values.**p<0.01 significance against control values.***p<0.001 significance against control values.ap<0.05 significance against HOE-50 group.bp<0.01 significance against HOE-50 group.cp<0.001 significance against HOE-50 group.&&p<0.01 significance against HOE-100 group.&&&p<0.001 significance against HOE-100 group.

Figure 3. Effects of acute Dzm (1mg/kg) and HOE (50–250mg/kg) administrations and Flu (2.5mg/kg) pre-treatments on total number ofhorizontal and vertical activities of mice in the activity cage tests. Values are given as mean�SEM. Significance against control values,*p<0.05, **p<0.01, ***p<0.001. Significance against 1mg/kg Dzm group, ~p<0.001. Significance against HOE-50 group ap<0.05;bp<0.01. Significance against HOE-250 group, XXXp<0.001. One-way ANOVA, post hoc Tukey test, n=7.

PHARMACOLOGICAL EFFECTS OF HYPERICUM ORIGANIFOLIUM

to evaluate the effect of the extract on the climbing, immo-bility, swimming durations and exploratory behaviors ofthe animals, respectively. Changes induced by HOE inthe spontaneous locomotor activities and motor coordi-nation of mice were investigated by activity cage measure-ments and Rota-rod tests. Further, hot-plate, tail-clip, andformalin tests were performed to assess the effect of theHOE on the nociceptive perceptions of mice.In MFST, both acute and sub-acute administrations of

Rbx and HOE caused a significant reduction in theimmobility and swimming times as well as considerableprolongation in climbing time of animals, indicating theantidepressant-like effects. This antidepressant-like activ-ity might be related to catecholaminergic rather thanserotonergic mechanisms on the CNS, because catechola-minergic agents such as Rbx increase climbing behaviorwhen decreasing immobility (Cryan et al., 2002). However,involvement of catecholaminergic system should beconfirmed with further studies such as inhibition ofcatecholamine synthesis by alpha-methyl-para-tyrosine ormeasurement of catecholamine levels in the limbic areasof brain.In numerous earlier studies, antidepressant activities of

the Hypericum species have been attributed to naptho-dianthrones hypericin and pseudohypericin (Chavez andChavez, 1997; Holzl et al., 1989; Suzuki et al., 1984) and

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to phloroglucinol derivative hyperforin (Cervo et al.,2002; Zanoli, 2004). However, Butterweck et al. (2000;2003) have reported antidepressant activities of hypericinor hyperforin free extracts, and other studies haveexhibited the importance of flavonoid content for theantidepressant action (Can et al., 2011; Noldner andSchotz, 2002).

In this study, phytochemical analysis results indicatedquite low amounts of hyperforin and hypericin in the testedextract; instead, chlorogenic acid and rutin were the majorconstituents. Among these two compounds, rutin has beenreported for its antidepressant activity, and increasing theavailability of monoamines in the synaptic cleft has beensuggested as a responsible mechanism for the antidepres-sant action of this flavonoid (Machado et al., 2008). Inanother study, inhibition of monoamine oxidase enzymehas been suggested as a mechanism of action for rutin(Dimpfel, 2009). In addition, Noldner and Schotz (2002)have suggested that rutin is an essential constituent forthe antidepressant activity of Hypericum extracts, andother constituents may act synergistically. As well asrutin, chlorogenic acid, the second major compound ofthe tested extract, may be also associated with the anti-depressant activity because its metabolite, caffeic acid, isknown to show antidepressant activity by modulatinga1A-adrenergic receptors (Takeda et al., 2003). Therefore,

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Figure 4. Effects of acute Mor (10mg/kg) and HOE (50–250mg/kg) administrations and Nlx (5mg/kg) pre-treatments on nociceptionparameters of mice in the hot-plate (I), tail-clip (II), and formalin (IIIa, early phase; IIIb, late phase) tests. Values are given as mean�SEM. Sig-nificance against control values, *p<0.05, **p<0.01, ***p<0.001. Significance against 10mg/kg Mor group, ~p<0.001. Significanceagainst HOE-50 group, ap<0.05. Significance against HOE-250 group, Xp<0.05. One-way ANOVA, post hoc Tukey test, n=7.

Table 4. Effects of sub-acute HOE (50, 100, and 250mg/kg) administrations on nociception parameters of mice in the hot-plate, tail-clip,and formalin tests

Response latencies Hot-plate test Tail-clip test

Formalin test

Early phase Late phase

7days Control 9.1�1.6 1.7�0.4 103.3�15.2 108.5�16.2HOE-50 13.5�2.0* 3.6�1.0* 77.6�10.9* 84.8�15.1*HOE-100 15.2�2.7** 4.5�0.8*** 58.5�6.2*** 63.6�12.0***,a

HOE-250 18.2�3.5***,a 6.0�1.1***,b 46.2�5.2***,b 41.3�8.0***,c,&

14days Control 10.8�1.3 1.9�0.5 100.7�19.2 100.0�14.6HOE-50 14.6�2.7* 3.9�0.6* 75.0�8.5* 70.7�8.9**HOE-100 16.4�2.8** 4.4�1.0** 51.7�9.7***,a 48.0�10.9***,a

HOE-250 20.3�3.2***,b,& 6.7�0.8***,c,&& 25.9�6.7***, c,& 20.8�3.3***,c,&&

Values are given as mean�SEM. One-way ANOVA, post hoc Tukey test, n=7.*p<0.05 significance against control values.**p<0.01 significance against control values.***p<0.001 significance against control values.ap<0.05 significance against HOE-50 group.bp<0.01 significance against HOE-50 group.cp<0.001 significance against HOE-50 group.&p<0.05 significance against HOE-100 group.&&p<0.01 significance against HOE-100 group.

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it can be suggested that the antidepressant effect exhibitedin this study may probably be related to high rutin and/orchlorogenic acid concentrations in HOE.Acute Dzm and HOE administrations not only caused

a significant rise in the number of total head-dippingbehaviors in the hole-board tests but also increasedthe number of horizontal and vertical locomotor activitiesof mice, in the activity cage tests. Increase in the

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exploratory behavior as well as the spontaneouslocomotor activities of animals suggested the presence ofan anxiolytic activity induced by the extract. Chlorogenicacid and rutin, two major components of the testedextract, have been previously reported for their anxiolyticactivities by different research groups. Furthermore,activation of GABA/A-benzodiazepine receptors hasbeen suggested as mode of action for these two flavonoids

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PHARMACOLOGICAL EFFECTS OF HYPERICUM ORIGANIFOLIUM

(Bouayed et al., 2007; Nassiri-Asl et al., 2008). On thebasis of these reports, a possible contribution of theGABA/A-benzodiazepine receptors to the observedactivity was assessed by the Flu pre-treatment. Flu antag-onized all of the alterations induced by the acute extractadministration in hole-board and activity cage tests,suggesting the involvement of GABA/A-benzodiazepinereceptor complex in the activity.On the other hand, long-term administrations of the

extract at the highest dose induced a reduction in bothhead-dipping behaviors and spontaneous locomotoractivity of mice. Inhibition of the exploratory behaviorin hole-board tests as well as reductions in the spon-taneous locomotor activities of animals in activity cagetests indicates a general inhibition of neuronal activityin the CNS (Fernández et al., 2006). This neurodepres-sant effect, arise by the high-dose/long-term adminis-trations, may be related to the accumulation of somesedative components of HOE due to repetitive admin-istrations or desensitization of the GABA/A-benzodi-azepine receptors playing role in the anxiolytic activity.Further studies are needed to clarify this issue.In the nociceptive tests, both acute and sub-acute

administrations caused significant increases in reactiontime of animals against mechanical noxious stimuli intail-clip and thermal noxious stimuli in the hot-platetests. These administrations decreased the chemicalnoxious stimulus-induced paw licking time of mice inthe formalin tests. These findings clearly indicated thatHOE has antinociceptive actions on all mechanical,thermal, and chemical nociceptive neuronal pathways.Reference drug morphine also exhibited its antinocicep-tive action in all of the performed nociceptive tests,as expected.Hot-plate and tail-clip tests have been reported as a

measure of centrally mediated transient pain. Hot-platetest predominantly measures responses organizedsupraspinally, whereas the tail-clip test mainly measuresspinal reflexes (Gabra and Sirois, 2003). As HOE exhib-ited significant antinociceptive activities in both tail-clipand hot-plate tests, it may be suggested that bothsupraspinal and spinal mechanisms were involved inthe exhibited analgesic action.In a formalin-induced nociception test, HOE signifi-

cantly shortened the licking time of animals in bothearly and late phases. Antinociceptive activity observedin the early phase confirmed the results of tail-clip andhot-plate tests and supported the presence of centrallymediated antinociceptive effect as early phase of for-malin test presents centrally mediated pain (Tjølsenet al., 1992). Moreover, antinociceptive activity of HOEin the late phase indicated the participation of someadditional peripheral mechanisms as the second phaseinvolves some peripheral nociceptive mechanisms aswell as central ones (Tjølsen et al., 1992). Opioidsare known to show their antinociceptive activities viaacting on both central and peripheral nociceptivepathways (Le Bars et al., 2001). Therefore, involve-ment of the opioid system in the antinociceptive

Copyright © 2012 John Wiley & Sons, Ltd.

activity of HOE in hot-plate, tail-clip, and formalintests was investigated with naloxone pre-treatment. Itwas observed that the antinociceptive activity ofHOE was antagonized by the pre-treatment of nalox-one in all of the nociceptive tests performed in thisstudy, as clear evidence for the involvement of opioidmechanisms in the analgesia. These effects could bedue directly to opioid receptor agonistic activities ofthe constituents in the extracts and/or stimulation ofendogenous opioid release. Rutin, one of the mainconstituents of the extract, has been suggested as ananalgesic agent when assessed in hot-plate test (Rylskiet al., 1979). Furthermore, Rad et al. (2008) have demon-strated an opioid-mediated mode of action for rutin.These previous papers support our findings indicatingthe opioid-mediated antinociceptive activity for HOE.Studies related to antinociceptive activities of chloro-genic acid are quite limited. There are only a few papersreporting its peripheral antinociceptive (dos Santos et al.,2006; Yonathan et al., 2006) and especially antiinflamma-tory (dos Santos et al., 2006) activities.

Absence of alterations in the rota-rod activity ofanimals has an important experimental indication thatthe results of the behavioral and nociceptive testsperformed in this study were not affected by means ofany changes in the motor activity of the animals.

CONCLUSION

Results of this study exhibited the phytochemicalcontent of the HOE and clearly pointed out the anti-depressant, anxiolytic, and antinociceptive activitiesof this extract, for the first time. The contribution ofthe GABA/A-benzodiazepine receptor complex to theobserved anxiolytic activity, whereas participation ofopioid receptors to both centrally and peripherallymediated antinociceptive action was also displayed.Therefore, by activating GABA/A-benzodiazepinereceptors, HOE may be a new drug source for treat-ment of patients suffering from anxiety and sleep disor-ders. Moreover, opioid-mediated antinociceptive effectof HOE may make it a natural drug candidate fortreating some painful diseases.

Acknowledgements

This research received no specific grant from any funding agency in thepublic, commercial, or not-for-profit sectors.

Conflict of Interest

There is no conflict of interest. The authors alone are responsible forthe content and writing of the paper. All the authors have approvedthe final article.

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