Antinociceptive activity of Hypericum spp Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum ( Guttiferae )

!"# $% & '( & )*& +# $% & , & & )-! ..,./ 0 1 !# &2 3 , '( 1 4"5 '( 1 6+5 )* 1 ./5 )* 1 7#5 1 /45 1 4+5 &2 .27 $% & 8 )* 8 '( 8 8 8 & 2 )* & 8 8 '( , 2 9 )*& & 8 8 2 ' , '( 8 8 & % 2 , '( 8 , 8 2 Correspondence S.M.K. Rates Faculdade de Farmácia, UFRGS Av. Ipiranga, 2752 90610-000 Porto Alegre, RS Brasil Fax: +55-51-3316-5437 E-mail: ratessmk@farmacia.ufrgs.br Presented at the XVII Annual Meeting of the Federação de Sociedades de Biologia Experimental, Salvador, BA, Brazil, August 28-31, 2002. R. Fenner, A.P. Heckler, S.M.K. Rates and A.F. Viana were the recipients of fellowships from PROPESQ-UFRGS, CNPq and CAPES, respectively. Received April 18, 2002 Accepted February 17, 2003

• Hypericum caprifoliatum The chemical investigation of the genus Hypericum (Guttiferae), which comprises approximately 400 species (1), has led to the isolation of more than 100 compounds from about 20 species with various different biological activities, especially antiviral, antimicrobial and antidepressant properties.H. perforatum extracts are widely used in Europe, in the United States, and also in Brazil, for the treatment of mild to moderate depression (2).
The Southern Brazilian Hypericum species H. brasiliensis and H. connatum are popularly used for relief of disorders such as angina, cramps and oral and pharyngeal inflammations, which suggests an analgesic property for this genus (3).
Previous reports published by our group have shown interesting biological activities for the Hypericum species native to the State of Rio Grande do Sul, Brazil.A crude lipophilic extract of H. caprifoliatum induces an anti-immobility effect in the forced swimming test (4), which is considered to indicate an antidepressant action (5), as well as an antinociceptive effect in the hot-plate test (6).H. caprifoliatum, H. piriai and H. polyanthemum showed in vitro monoamine oxidase A-inhibitory activity (7).The aim of the present study was to investigate further the antinociceptive effects of H. caprifoliatum and to start the characterization of the antinociceptive properties of H. polyanthemum.Air-dried and powdered aerial parts of H. caprifoliatum and H. polyanthemum were extracted with cyclohexane using an ultraturrax apparatus (3 x 5 min; plant/solvent ratio 1:10, w/v), yielding extracts termed CH and POL, respectively.In order to obtain an extract rich in polar substances, H. caprifoliatum was also extracted consecutively in a Soxhlet apparatus with petroleum ether, chloroform and methanol (MET) and only the MET extract was used.All solvents were evaporated to dryness under reduced pressure.The extract was dissolved in saline solution containing 2.5% (w/v) polysorbate 80.The pH of the final solutions was 6.5 to 7.0.The volume administered was 1 ml/100 g body weight for the analgesic tests.
Male Swiss CF1 mice (22-27 g) from the breeding colony of Fundação Estadual de Pesquisa e Ensino em Saúde (FEPPS, RS, Brazil) were used.The animals were housed in plastic cages, 5 to a cage, under a 12-h light/dark cycle (lights on at 7:00 h) at constant temperature (23 ± 1ºC), with free access to standard certified rodent diet and tap water.The experiments were performed according to the guidelines of the National Ethics Committee on Research, Brazilian National Health Council.Ten mice per group were used for all experiments.
Before actual testing on the hot plate, the mice were habituated to the nonfunctioning apparatus for 1 min.Thirty minutes later, the animals were placed on the functioning hot plate (Ugo Basile, Comerino, Italy) to determine baseline responsiveness 10 min before treatment with 90 mg/kg CH or MET or 180 mg/kg POL (ip and po).Treatment-induced changes in responsiveness to the hot plate were determined 30 and 45 min after ip and po administration, respectively.The negative control group received an equal volume of vehicle (saline + 2.5% (w/v) polysorbate 80).Morphine (6 mg/kg, sc) was administered to the positive control group.To determine the possible involvement of opioidmediated mechanisms, some groups of animals were pretreated with naloxone (2.5 mg/ kg, sc), a nonspecific opioid receptor antagonist, immediately after evaluating baseline responsiveness, 10 min before extract administration.
For the hot-plate test, mice were placed on a metal surface kept at 53 ± 1ºC.The time elapsed until the animal licked one of its hind paws or jumped was recorded (latency time, in s) and considered to be the reaction time in both exposures.Mice that presented baseline reaction times of more than 15 s in the first session were not used.In the second session, a maximum latency time of 30 s was imposed in order to avoid tissue damage.
The data were analyzed by the paired Student t-test, considering the animal as its own control (second measure vs first measure).The results obtained in the hot-plate test are reported as the mean ± SEM absolute latency time or as the percent of antinociceptive effect relative to morphine (6 mg/kg, sc) according to the following formula: % analgesia = (test after -test before )/(morphine aftermorphine before ) x 100.
The animals were treated with CH, MET (90 mg/kg, po) or POL (180 mg/kg, po) for the writhing test 45 min before receiving an ip injection of 0.8% acetic acid.Mice were then placed individually in glass observation chambers and the number of abdominal writhes was counted over a period of 15 min.The control group received an equal volume of vehicle (saline + 2.5% (w/v) polysorbate 80, po).Dipyrone (150 mg/kg, po) was the positive control treatment.Previous experiments carried out in our laboratory have revealed that none of the extracts caused any signs of pain or writhes per se, in mice, when injected ip (8).The results obtained in the writhing test are reported as median values and their respective interquartile intervals, and were analyzed by the Kruskal-Wallis test.
All extracts displayed antinociceptive effects in the hot-plate test (Table 1).Pretreatment with naloxone abolished the effects of CH and POL, indicating that these effects are produced by opioid-mediated mechanisms.Conversely, antinociception produced by MET administered ip was only partially prevented by naloxone, whereas the po antinociceptive activity was not modified, indicating that opioid-like substances present in this extract were not absorbed by the gastrointestinal tract or suffered singlepass inactivation by the liver.In addition, the percent of analgesia was higher when MET was administered by the ip route compared to the po route.
Administration of CH and POL significantly reduced the number of abdominal writhes induced by acetic acid, whereas MET did not have a significant effect (Table 2).Interestingly, the magnitude of the antinociceptive effect of CH in the writhing test (oral route) was similar to that observed in the The CH extract is rich in phloroglucinol (7) and the POL extract contains benzopyrans as its main constituent (9).Phloroglucinol derivatives may be responsible for the opioidlike effect since Simmen et al. (10) have reported that hyperforin -a phloroglucinol isolated from H. perforatum -inhibited binding to opioid receptors.With respect to the benzopyrans as well as the flavonoid derivatives, which are present in MET (DallAgnol R, Ferraz A, Schapoval ES and von Poser G, unpublished data), we are unaware of any previous reports on their influence/action on opioid systems.None of the extracts (CH, POL or MET) contains hypericin (11).
In conclusion, extracts obtained from both species, H. caprifoliatum and H. polyanthemum, contain compounds with substantial antinociceptive properties related, at least in part, to activation of opioid-mediated mechanisms.Further studies are in progress in order to elucidate the mechanisms underlying the antinociceptive effects of these species.

Table 1 .
Antincociceptive effect of cyclohexane (CH) and methanol (MET) extracts of the aerial parts of Hypericum caprifoliatum and of the cyclohexane extract of aerial parts of H. polyanthemum (POL) in mice submitted to the hot-plate test.
Data are reported as mean ± SEM.MOR: morphine; NAL: naloxone.*P<0.005compared to the latency of the same mouse before treatment (paired Student t-test).

Table 2 .
Analgesic effects of methanol (MET) and cyclohexane (CH) extracts of aerial parts of Hypericum caprifoliatum (90 mg/kg, po) and of the cyclohexane extract of aerial parts of H. polyanthemum (POL, 180 mg/kg, po) on writhing induced by 0.8% acetic acid (ip) in mice.