evaluation of the central properties of several hypericum species from the canary islands

5
Evaluation of the Central Properties of Several Hypericum Species from the Canary Islands Begon ˜a Prado, Rosa M. Rabanal and Candelaria C. Sa ´nchez-Mateo* Departamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, c) Astrofisico Francisco Sa ´nchez s/n, 38071, La Laguna, Tenerife, Espan ˜a The infusions of the aerial parts in blossom of Hypericum canariense, H. glandulosum, H. reflexum and H. grandifolium (Hypericaceae) were evaluated for their pharmacological activity on the central nervous system in mice using various behavioural models including locomotor and muscle relaxant activity, effect on normal body temperature, pentobarbital-induced sleep, oxotremorine and tetrabenazine-induced syn- drome, apomorphine-induced hypothermia and 5-hydroxytryptophan-induced head twitches, as well as a forced swimming test. These infusions did not alter significantly the locomotor activity, pentobarbital induced sleeping time and body temperature, with the exception of H. canariense which produced a slight but significant hypothermia. Additionally, no muscle relaxant or anticholinergic activity were observed. These infusions antagonized the ptosis and/or motor depression induced by tetrabenazine as well as shortening the immobility time in the forced swimming test. The observations suggest that the infusions of these Hypericum species possess antidepressant activity in mice, without inducing muscle relaxation, anticholinergic and sedative properties. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Hypericum species; infusions; CNS activity; antidepressant effect; forced swimming test; pharmacology. INTRODUCTION The genus Hypericum (Hypericaceae) comprises a large number of species widely distributed in many countries in Middle Europe, North America and Eastern Africa. One species of this genus, Hypericum perforatum L. (popularly called St John’s wort), is a well-known medicinal plant which has been used in folk medicine for a long time for a range of indications including skin wounds, eczema, gastrointestinal and psychological disorders (Bombardelli and Morazzoni, 1995). Today, an increasing number of experimental and clinical studies performed with commercial preparations of extracts of Hypericum perforatum L. have shown that these preparations are effective for the treatment of mild to moderately severe depressive disorders with no special side effects (Linde et al., 1996; Butterweck et al., 1997; Wheatley, 1998; Stevinson and Ernst, 1999; Gaster and Holroyd, 2000). Nevertheless, it should be mentioned that recent publications have shown potentially serious drug interactions between St John’s wort extract preparations and other drugs whose metabolism is mediated by the cytochrome P450 enzymes (Johne et al., 1999; Barone et al., 2000; Piscitelli et al., 2000). These data should be taken into account when prescribing drugs for their safety with Hypericum extracts in view of the increasing use of these herbal preparations for depressive disorders. In the Canary Islands traditional medicine, infusions prepared from the flowers, leaves and fruits of various species of the genus Hypericum have been used as a vermifuge, diuretic, as well as a wound healing, sedative, antihysterical and antidepressant agent (Darias et al., 1986, 1989; Pe ´rez de Paz and Herna ´ndez Padro ´n, 1999). Since some of the indications seem to be related to central nervous system activities, the aim of the present study was to evaluate the CNS activity in mice (particularly in animal models of depression) of the infusions of the aerial parts in blossom of four species of this genus: Hypericum canariense L., H. glandulosum Ait. and H. reflexum L. fil., an endemic herbaceous plant of the Canary Islands and H. grandifolium Choisy, an endemic of the the Macaronesian region. MATERIALS AND METHODS Plant materials and extraction procedure. Aerial parts in blossom of H. canariense L., H. glandulosum Ait., H. grandifolium Choisy and H. reflexum L. fil. were collected from Pedro a ´lvarez (Tenerife, Spain). They were identified by Dr Consuelo Herna ´ndez Padro ´n, Department of Botany, University of La Laguna (Tenerife, Spain) where voucher specimens have been deposited (TFC 42211, TFC 42212, TFC 42213 and TFC 42214, respectively). After collection, the plant materials were dried in an oven at 40 °C and powdered. An infusion was prepared with 100 g of crude powder and 1000 mL of water and the volume was adjusted to a concentration of PHYTOTHERAPY RESEARCH Phytother. Res. 16, 740–744 (2002) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1060 Copyright # 2002 John Wiley & Sons, Ltd. * Correspondence to: Dr C. C. Sa ´nchez-Mateo, Departamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, c) Astrofisico Francisco Sa ´nchez s/n, 38071, La Laguna, Tenerife, Espan ˜a. Tel.: 34-922-318502; fax: 34-22-318514. E-mail: [email protected] Contract/grant sponsor: La Universided de La Laguna. Received 27 March 2001 Accepted 20 June 2001

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Page 1: Evaluation of the central properties of several Hypericum species from the Canary Islands

Evaluation of the Central Properties of SeveralHypericum Species from the Canary Islands

Begona Prado, Rosa M. Rabanal and Candelaria C. Sanchez-Mateo*Departamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, c) Astrofisico Francisco Sanchez s/n, 38071, La Laguna,Tenerife, Espana

The infusions of the aerial parts in blossom of Hypericum canariense, H. glandulosum, H. reflexum andH. grandifolium (Hypericaceae) were evaluated for their pharmacological activity on the central nervoussystem in mice using various behavioural models including locomotor and muscle relaxant activity, effecton normal body temperature, pentobarbital-induced sleep, oxotremorine and tetrabenazine-induced syn-drome, apomorphine-induced hypothermia and 5-hydroxytryptophan-induced head twitches, as well as aforced swimming test. These infusions did not alter significantly the locomotor activity, pentobarbitalinduced sleeping time and body temperature, with the exception of H. canariense which produced a slightbut significant hypothermia. Additionally, no muscle relaxant or anticholinergic activity were observed.These infusions antagonized the ptosis and/or motor depression induced by tetrabenazine as well asshortening the immobility time in the forced swimming test. The observations suggest that the infusionsof these Hypericum species possess antidepressant activity in mice, without inducing muscle relaxation,anticholinergic and sedative properties. Copyright � 2002 John Wiley & Sons, Ltd.

Keywords: Hypericum species; infusions; CNS activity; antidepressant effect; forced swimming test; pharmacology.

INTRODUCTION

The genus Hypericum (Hypericaceae) comprises a largenumber of species widely distributed in many countriesin Middle Europe, North America and Eastern Africa.One species of this genus, Hypericum perforatum L.(popularly called St John’s wort), is a well-knownmedicinal plant which has been used in folk medicinefor a long time for a range of indications including skinwounds, eczema, gastrointestinal and psychologicaldisorders (Bombardelli and Morazzoni, 1995). Today,an increasing number of experimental and clinical studiesperformed with commercial preparations of extracts ofHypericum perforatum L. have shown that thesepreparations are effective for the treatment of mild tomoderately severe depressive disorders with no specialside effects (Linde et al., 1996; Butterweck et al., 1997;Wheatley, 1998; Stevinson and Ernst, 1999; Gaster andHolroyd, 2000). Nevertheless, it should be mentionedthat recent publications have shown potentially seriousdrug interactions between St John’s wort extractpreparations and other drugs whose metabolism ismediated by the cytochrome P450 enzymes (Johne etal., 1999; Barone et al., 2000; Piscitelli et al., 2000).These data should be taken into account when prescribingdrugs for their safety with Hypericum extracts in view of

the increasing use of these herbal preparations fordepressive disorders.

In the Canary Islands traditional medicine, infusionsprepared from the flowers, leaves and fruits of variousspecies of the genus Hypericum have been used as avermifuge, diuretic, as well as a wound healing, sedative,antihysterical and antidepressant agent (Darias et al.,1986, 1989; Perez de Paz and Hernandez Padron, 1999).

Since some of the indications seem to be related tocentral nervous system activities, the aim of the presentstudy was to evaluate the CNS activity in mice(particularly in animal models of depression) of theinfusions of the aerial parts in blossom of four species ofthis genus: Hypericum canariense L., H. glandulosumAit. and H. reflexum L. fil., an endemic herbaceous plantof the Canary Islands and H. grandifolium Choisy, anendemic of the the Macaronesian region.

MATERIALS AND METHODS

Plant materials and extraction procedure. Aerial partsin blossom of H. canariense L., H. glandulosum Ait., H.grandifolium Choisy and H. reflexum L. fil. werecollected from Pedro alvarez (Tenerife, Spain). Theywere identified by Dr Consuelo Hernandez Padron,Department of Botany, University of La Laguna(Tenerife, Spain) where voucher specimens have beendeposited (TFC 42211, TFC 42212, TFC 42213 and TFC42214, respectively). After collection, the plant materialswere dried in an oven at 40°C and powdered. An infusionwas prepared with 100 g of crude powder and 1000 mL ofwater and the volume was adjusted to a concentration of

PHYTOTHERAPY RESEARCHPhytother. Res. 16, 740–744 (2002)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1060

Copyright � 2002 John Wiley & Sons, Ltd.

* Correspondence to: Dr C. C. Sanchez-Mateo, Departamento deFarmacologia, Facultad de Farmacia, Universidad de La Laguna, c)Astrofisico Francisco Sanchez s/n, 38071, La Laguna, Tenerife, Espana. Tel.:�34-922-318502; fax: � 34-22-318514.E-mail: [email protected]/grant sponsor: La Universided de La Laguna.

Received 27 March 2001Accepted 20 June 2001

Page 2: Evaluation of the central properties of several Hypericum species from the Canary Islands

1 g/mL under reduced pressure and 40°C, cooled andstored in a freezer at �20°C. The infusions weredissolved in distilled water at the desired concentrationjust before use in pharmacological tests. In this paper, alldoses are expressed as mg of the dried plant per kg bodyweight.

Animals. Male and female albino Swiss mice (24–28 g)were purchased from the Laboratory Animal Center,University of La Laguna (Tenerife, Spain) and werehoused in groups of 10/cage for a minimum of 3 daysprior to pharmacological studies with free access tostandard laboratory food and tap water and maintained ona 12/12 h light–dark cycle (light from 8.00 a.m. to 8.00p.m.). All animals were fasted overnight before dosing,tap water being available ad libitum. Each experimentaldose group consisted of five animals unless otherwisestated. The ambient temperature was 22° � 1°C, exceptin the experiments of potentiation of barbiturate sleepingtime which were carried out in a room maintained at30° � 1°C. Behavioural observations took place between8.00 and 15.00 h and each animal was used only once.

Drugs. The following drugs were used: apomorphinehydrochloride (Sigma, Spain), atropine sulphate (Sigma,Spain), chlorpromazine hydrochloride (Rodia, Spain),fluoxetine (Lilly Indiana, Spain), L-5-hydroxytryptophan(Serva, Spain), imipramine hydrochloride (Impex,Spain), oxotremorine sesquifumarate (Sigma, Spain),sodium pentobarbital (Direccion General de Farmacia yProductos Sanitarios, Spain), tartaric acid (Merck,Spain), and tetrabenazine (Fluka, Spain).

Plant infusions and standard drugs were given orally1 h before the experiments mentioned below in a dose of0.5 mL/20 g body weight in mice. Control animalsreceived distilled water under the same conditions.

LD50 in mice. The LD50 was calculated from lethalitywithin 3 days after p.o. administration of the drugs by themethod of Litchfield and Wilcoxon (1949).

General central nervous system activity. The activityof the infusions of the four Hypericum species on theCNS was evaluated by performing assays of its effects onspontaneous motor activity, body temperature, musclerelaxant activity (traction test), barbiturate-induced sleepand oxotremorine-induced syndrome in mice.

Effect on spontaneous motor activity in mice. Loco-motor activity was recorded with a photocell activitymeter for 15 min beginning 60 and 120 min after p.o.administration of each test drug.

Effect on normal body temperature. The rectal tem-perature of each mouse was measured with a thermistorthermometer (Panlab 0331) prior to the experiment and 1,2, 4, 6 and 24 h after p.o. administration of each test drug.The temperature changes at different times with respectto the temperature values before drug administrationwere calculated. Male mice with a rectal temperaturebetween 36°–38°C prior to the experiment were used.

Effect on muscle relaxant activity: traction test.Experiments were performed by the method of Boissieret al. (1961). Mice were obliged to hang with theirforelegs on a wire of 1 mm in diameter, which was

stretched horizontally at a height of 35 cm. When theyfell off the wire within 5 s or failed to grasp the wire withtheir hind legs three times successively, muscle relaxa-tion was judged to be positive. This test was conducted ingroups of five previously screened animals, 60 min afterinjection of control vehicle or test material.

Effect on barbiturate-induced sleep in mice. Sodiumpentobarbital at a hypnotic dose of 40 mg/kg was injectedi.p. to groups of eight mice 60 min after p.o. administra-tion of the drug. The latency and the duration of sleep(loss and recovery of the righting reflex) were recorded.

Effect on oxotremorine-induced syndrome in mice.Mice with a rectal temperature of 36–38°C prior to theexperiment were used and randomly assigned to testgroups of six subjects. One hour after p.o. administrationof the test drug, vehicle or standard compounds, micereceived oxotremorine sesquifumarate (0.5 mg/kg i.p.).Tremors were scored 30 min after oxotremorine using ascale of 0 to 3: 0 (no tremor), 1 (no apparent tremor, butwhen mice were hung up by the tail, tremors appearedand the forepaws crossed), 2 (intermittent tremors, buttremors always appeared when mice were moving), 3(continuous tremor). Salivation was scored 30 min afteroxotremorine as strong, moderate, weak or absentdesignated by 3, 2, 1 and 0 scores, respectively. Therectal temperature of each mouse was measured 60 minafter oxotremorine administration.

Antidepressant activity. The antidepressant activity ofthe test materials was evaluated by performing assays ofits effects on tetrabenazine-induced syndrome, apomor-phine-induced hypothermia, 5-hydroxytryptophan-in-duced head twitches and behavioural despair (forcedswimming) in mice.

Antagonism of tetrabenazine-induced ptosis, hypo-thermia and suppression of locomotor activity. Malemice with a rectal temperature of 36°–38°C prior to theexperiment were used. Mice received orally each testdrug 60 min before the administration of tetrabenazine(32 mg/kg i.p.), which was dissolved in 0.1 M tartaric acidfollowed by adjustment to pH = 6 with NaOH 10%.Thirty minutes after the intraperitoneal injection oftetrabenazine, the animals were placed at the centre ofa disk (20 cm of diameter) and the akinesia and thedegree of palpebral ptosis exhibited by each mouse wasestimated within 10 s. Mice were judged not to beakinetic if they elicited one or more of the followingresponses: (1) walk to the edge of the disk and look overthe side; (2) mice move 180° in place; (3) mice displayhead movement of 90° in one direction immediatelyfollowed by a 45° movement in the opposite direction.The degree of ptosis was rated according to the followingrating scale: 0, eyes open; 1, one-quarter closed; 2, halfclosed; 3, three-quarters closed; and 4, completelyclosed. The rectal temperature of each group wasmeasured with a thermistor thermometer (Panlab 0331)60 min after i.p. administration of tetrabenazine.

Antagonism of apomorphine (16 mg/kg)-inducedhypothermia in mice. Apomorphine (16 mg/kg) wasinjected subcutaneously to groups of six mice 60 minafter p.o. administration of the test drugs. The tempera-ture was measured with a thermistor thermometer

HYPERICUM FROM CANARY ISLANDS 741

Copyright � 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 740–744 (2002)

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(Panlab 0331) 30 and 60 min after apomorphine admin-istration. Mice with a rectal temperature between 36° and38°C prior to the experiment were used.

Potentiation of 5-hydroxytryptophan (5-HTP)-in-duced head twitches in mice. Test drugs wereadministered p.o. 60 min before 75 mg/kg i.p. L-5-HTP:The mice were then placed into glass bell jars and 14 minlater the number of head twitches was counted in five2-min intervals (between 14 and 16, 24 and 26, 34 and 36,44 and 46 and 54 and 56 min).

Behavioural despair (forced swimming) test in mice.This test was performed according to the methoddescribed by Porsolt et al. (1977) with slight modifica-tions. One hour after p.o. administration of the test drugs,mice were individually forced to swim in a transparentglass vessel (25 cm high, 10 cm in diameter) filled with10 cm of water at 21°-24°C. The total duration ofimmobility (s) was measured during the last 4 min of asingle 6-min test session. Mice were considered im-mobile when they made no further attempts to escapeexcept the movements necessary to keep their headsabove water. Each experimental group consisted of eightanimals.

Statistical analysis. Data were analysed by one-wayanalysis of variance (ANOVA) followed by Student’sunpaired t-test (Tallarida and Murray, 1986). A prob-ability level of 0.05 or less was accepted as significant.The chi-square test was used for the percentage offailures in the muscle relaxant activity and the percentageof locomotor activity in tetrabenazine test. Tremor andsalivation induced by oxotremorine, and ptosis induced

by tetrabenazine were analysed using the Mann-Whitneytest for non-parametric data.

RESULTS

The four infusions of Hypericum administered p.o. didnot induce mortality up to a dose of 8 g/kg in mice up to72 h after administration. The tested animals did notpresent any toxic manifestations.

General central nervous system activity

Table 1 shows that administration of the infusions did nothave a significant effect on the spontaneous motoractivity on the sleeping time induced by a hypnotic doseof 40 mg/kg i.p. of sodium pentobarbital compared withthe control. Chlorpromazine (the reference drug), asexpected, showed an effect at the dose used. In addition,no muscle relaxant activity could be observed with thedifferent infusions when tested with the traction test (datanot shown).

Concerning the effects of infusions on the rectaltemperature (Table 2), it was found that only theHypericum canariense infusion produced a slight butsignificant ( p � 0.05) hypothermia which was main-tained up to 2 h, although without reaching the valueselicited by chlorpromazine, the reference drug.

On the other hand, these infusions presented no orscarce anticholinergic activity since they did notsignificantly antagonize the oxotremorine-induced tre-mor, salivation and hypothermia compared with thecontrol (data not shown).

Table 1. Effect of Hypericum infusions on spontaneous locomotor activity and pentobarbital (40 mg/kg i.p.) -induced sleepingtime in mice

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Table 2. Effect of Hypericum infusions on body temperature in mice (mean � SEM)

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742 B. PRADO ET AL.

Copyright � 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 740–744 (2002)

Page 4: Evaluation of the central properties of several Hypericum species from the Canary Islands

Antidepressant activity

In the tetrabenazine test (Table 3), the infusions did notantagonize the hypothermia, but they antagonized theptosis and motor depression induced by the drug in mice.In this respect, only H. glandulosum infusion antagonizedboth effects in a significant manner ( p � 0.05), withactivity values similar to those found for imipramine inthe motor depression antagonism (60%). On the otherhand, H. reflexum and H. canariense infusions werefound to only significantly antagonize the ptosis inducedby tetrabenazine compared with the control (28.57% and25.71%, respectively).

With regard to the effects of these infusions on thehypothermia induced by high-dose apomorphine(16 mg/kg s.c.), none of the infusions showed asignificant decrease of the hypothermia induced by thedrug compared with the control (Table 4), at variancewith the standard drug imipramine. Nevertheless, H.grandifolium infusion antagonized (but not significantly)in 30% of mice the hypothermia induced by apomorphine30 and 60 min after administration, indicating a possibleinhibition of norepinephrine reuptake (Puech et al.,1981).

In order to investigate their performance in theserotonin system the potentiation of 5-HTP induced headtwitches in mice was also examined. The infusions understudy did not modify significantly the head twitchesinduced by this drug (Table 4), whereas the referenceantidepressants that selectively or preferentially inhibit5-HT uptake (as fluoxetine) produced a potentiation ofthis effect (Shank et al., 1987).

Finally, it could be observed in Table 5 that themajority of the infusions assayed significantly shortened

the immobility time of mice in the forced swimming testin comparison with control animals, but without reachingthe values elicited by the antidepressant reference drugimipramine.

DISCUSSION

The results of the present study have demonstrated thatp.o. injection of the infusions of several Hypericumspecies endemic to the Canary Islands did not havesignificant effects on assays of general psychopharma-cology such as effects on spontaneous locomotor activity,muscle relaxant activity, potentiation of pentobarbitalsleeping time, antagonism of oxotremorine-inducedsyndrome and body temperature. Only Hypericumcanariense showed a slight but significant hypothermia

Table 3. Effect of Hypericum infusions on tetrabenazine (32 mg/kg i.p.)-induced ptosis, akinesia andhypothermia

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Table 4. Effect of Hypericum infusions on apomorphine (16 mg/kg s.c.)-induced hypothermia and 5-HTP (75 mg/kg i.p.)-induced head twitches in mice (mean � SEM)

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Table 5. Effect of Hypericum infusions on the behaviouraldespair (forced swimming) test

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HYPERICUM FROM CANARY ISLANDS 743

Copyright � 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 740–744 (2002)

Page 5: Evaluation of the central properties of several Hypericum species from the Canary Islands

that could involve both central and peripheral mechan-isms.

In addition to this preliminary psychopharmacologicaltest, the infusions were also assayed in several specifictests in order to evaluate their potential antidepressantactivity. In these experiments, it was found that theinfusions were incapable of producing any significantantagonism of either hypothermia induced by a high doseof apomorphine or head-twitches induced by 5-HTP, butthey caused significant effects in antagonizing the ptosisand/or motor depression induced by tetrabenazine. In thisregard, the H. glandulosum infusion antagonized sig-nificantly both effects, which suggests a certain nor-adrenergic and/or dopaminergic action whereas H.reflexum and H. canariense infusions were significantlyactive in the antagonism of ptosis, which is usuallyevidence of a alpha-adrenergic or serotonergic activity(Bourin, 1990).

It should be mentioned that these tests are based ondrug interactions and therefore are closely related to aparticular mechanism of action (mainly substancesaffecting monoamine reuptake). For this reason, it wasalso of interest to carry out the forced swimming(behavioural despair) test, which is an assay generallyused for the prediction of antidepressant activity withoutinvolving pharmacological interaction. According toPorsolt et al. (1977), immobility seen in rodents duringswimming reflects behavioural despair as seen in humandepression and it is well known that the antidepressant

drugs cause a significant decrease in the immobility timein mice. In this test, it has been shown that the majority ofthe infusions assayed significantly reduced the durationof immobility time when compared with control animals,thereby demonstrating antidepressant activity. None ofthem, however, reached the values obtained for imipra-mine, the drug chosen as the reference standard.

It must be pointed out that the antidepressant activitydetected in the forced swimming test for these infusionsis not due to CNS stimulant properties, because they haveno significant effects on the motor activity in comparisonwith the control group at the dose assayed in this test.

In summary, on the basis of the findings obtained in thepresent study it can be concluded that the Hypericuminfusions assayed appear to have a certain antidepressantactivity in mice models such as the antagonism totetrabenazine-induced ptosis and motor depression andthe forced swimming test, without inducing musclerelaxant, sedative and anticholinergic properties. Thesefindings also support the ethnomedical data concerningthe antidepressant effects of the herbal remedies preparedfrom these plant species. However, to reach a definiteconclusion, more extensive investigations are needed.

Acknowledgement

We are grateful to La Universidad de La Laguna for financial supportfor these investigations.

REFERENCES

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744 B. PRADO ET AL.

Copyright � 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 740–744 (2002)