Anti-Inflammatory, Antinociceptive, and Gastric Effects of Hypericum perforatum in Rats

The pharmacological activity of Hypericum perforatum was assessed using models of inflammation, nociception, and gastric mucosal injury in rats. H. perforatum was given systemically as well as orally. When administered systemically, H. perforatum (50–300 mg/kg, s.c.) produced a dose-related and significant inhibition of the edematogenic response to s.p. injection of carrageenan. The percentages of maximal inhibition by the above doses were 53.7, 61.3, and 75.3%, respectively (compared to 90% after 50 mg/kg fluoxetine and 60.7% after 72 mg/kg etodolac). In tests of nociception, H. perforatum, administered orally, displayed antinociceptive activity in the tail electric stimulation and hot plate tests. The antinociceptive activity was observed with 25 mg/kg and a maximal increase in hot plate latency by 50% (compared to 73.2 and 77.8% increases by 5 or 10 mg/kg fluoxetine, respectively). In contrast, the acetic acid–induced (0.6%, i.p.) writhing was significantly reduced by fluoxetine or etodolac, but not H. perforatum. Also, the nociceptive response caused by i.p. injection of capsaicin (1.6 μg/paw) was unaffected by H. perforatum, but reduced by fluoxetine. Injection of H. perforatum (50, 125, or 250 mg/kg, s.c.) to pylorus-ligated rats, decreased gastric acid secretion, but increased indomethacin-induced gastric mucosal lesions dose dependently. These results demonstrate that H. perforatum exhibits antiedematogenic and antinociceptive properties, which may be of value for the management of inflammatory painful conditions. The agent, however, causes gastric irritation and may aggravate that of NSAIDs.


INTRODUCTION
Extracts of Hypericum perforatum (St. John's wort) have gained much interest for their antidepressant effects. Studies have suggested that H. perforatum may be of comparable efficacy to the selective serotonin reuptake inhibitor (SSRI) sertraline or to the tricyclic antidepressant imipramine [1,2]. Tricyclic antidepressants are the mainstay in the management of neuropathic pain syndromes [3,4]. These agents achieve a good or moderate response, but their therapeutic utility has been limited by adverse events [5,6]. Studies have suggested anti-inflammatory and analgesic properties for H. perforatum [7,8]. In vitro, iNOS and COX-2 expression, as well as myeloperoxidase activity and the generation of reactive oxygen species, were inhibited by the agent [8,9,10].
The identification of compounds that would alleviate neuropathic pain still represents a challenging goal. It was the aim of the present study to assess the effects of H. perforatum extract in animal models of pain and acute inflammation. The effects of H. perforatum were compared with those of fluoxetine, a potent SSRI, and the selective COX-2 inhibitor etodolac. These agents displayed marked antiinflammatory and analgesic properties [11,12,13]. In addition, the gastric effects of H. perforatum in the presence of indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) commonly used in many arthritic and inflammatory conditions, were evaluated.

Animals
Sprague-Dawley strain rats weighing 120-130 g of body weight (National Research Centre, Cairo) were used. Unless otherwise indicated, food and water were provided ad libitum. All animal procedures were performed in accordance to the Institutional Ethics Committee and in accordance with the recommendations for the proper care and use of laboratory animals (NIH publication No. 85-23, revised 1985).

Carageenin-Induced Paw Edema
Paw swelling was elicited by s.p. injection of 100 μl of 1% sterile lambda carrageenan suspension in saline into the right hind paw [14]. Contralateral paw received an equal volume of saline. The edema component of inflammation was quantified by measuring the increase in paw volume (ml) with a plethysmometer (Ugo Basile, Milan, Italy) before carrageenan injection and at selected times thereafter. Edema was expressed as a percentage of change from control (predrug) values.

Tail Electric Stimulation Test
Groups of rats (n = 6/group) were given H. perforatum (50, 150, or 300 mg/kg, p.o.) or saline (control). Other groups were treated with fluoxetine (5 or 10 mg/kg, p.o., n = 6/group) or etodolac (18, 36, or 72 mg/kg, p.o., n = 6/group). The minimum current required to elicit vocalization on electrical stimulation of the tail was determined for the control and drug-treated groups [15]. Electrical stimulation of the tail was applied by means of 515 Master Shocker (Lafayette Inst. Co.). Stimulation was carried out by an alternative current of 50 cycle/s for 0.2 s.

Capsaicin-Induced Hind Paw Licking
H. perforatum (25 or 50 mg/kg), fluoxetine (5 or 10 mg/kg), etodolac (18 or 36 mg/kg), or saline was given p.o., 1 h before injection of capsaicin (1.6 μg/paw; 25 μl) under the skin of the dorsal surface of the right hind paw. Observation started after capsaicin injection and lasted for 5 min. The time the animals spent licking the injected paw was determined using a stopwatch [16].

Statistical Analyses
Data are expressed as mean ± S.E. Differences between vehicle control and treatment groups were tested using one-and two-way ANOVA followed by multiple comparison by the Duncan's multiple comparison test. When there were only two groups a two-tailed Student's t test was used. A two-tailed probability value less than 0.05 was considered statistically significant.

Hot Plate Assay
The reaction time on the hot plate was delayed by H. perforatum. The antinociceptive effect of the agent was produced with a 25 mg/kg and maximal increase in hot plate latency by 50% 2 h after injection. In comparison, 73.2 and 77.8% increases in hot plate latency were observed after 5 or 10 mg/kg fluoxetine, respectively. Meanwhile, an increase in hot plate latency by about 38.8% was seen after treatment with etodolac at 36 or 72 mg/kg (Table 1).

Tail Electric Stimulation Test
H. perforatum (50, 150, or 300 mg/kg) produced a significant rise in electrical current threshold in the tail stimulation test in rat by 16.1, 24.9, and 27.1% and 19.9, 36.9, and 37.7% vs. control values, 1 and 2 h postdrug, respectively. A significant rise in nociceptive thresholds by 31.3 and 42.5% was obtained with fluoxetine 2 h following administration. Etodolac at the highest dose examined of 72 mg/kg significanly increased the nociceptive threshold by 21.8% for 1 h postinjection ( Table 2).

Capsaicin-Induced Hind Paw Licking
The duration of paw licking following i.p. capsaicin injection was unaffected by prior administration of H. perforatum or etodolac, but reduced by 18.9 and 20.4% after 5 or 10 mg/kg fluoxetine, respectively (Table 3) .  Shown are control and drug-induced (1-and 2-h measurements) changes for the nociceptive reaction. Data are expressed as means and S.E.M. (n = 6/group). Asterisks indicate significant rise in electrical current threshold (microA) (p < 0.05) compared with the control level of nociceptive reaction (one-way ANOVA, Duncan test).

Acetic Acid-Induced Writhing Test
Writhing was unaffected by H. perforatum, but significantly reduced by fluoxetine or etodolac (Table 3).

Indomethacin-Induced Gastric Lesions
H. perforatum increased the number and severity of gastric mucosal lesions evoked by indomethacin, in a dose-dependent manner. Gastric acid secretion decreased by 17.7, 29.2, and 53.1% in rats treated with H. perforatum (Table 4).

DISCUSSION
The present study confirms and extends other work that H. perforatum extract exerts both antiinflammatory and antinociceptive effects. In this respect, the agent inhibited paw edema caused by the injection of carrageenan in rat. The effect was dose dependent, with a maximal reduction in edema of 75.3%, a value that is even higher than that obtained with the COX-2 inhibitor etodolac in maximal doses. The antiedema effect of H. perforatum was marked in the first hour following drug administration. This could suggest interference with the actions of histamine, serotonin, or kinins, the inflammatory mediators implicated in the early phase of the carrageenan-induced inflammatory response [19]. H. perforatum showed, in addition, marked antinociceptive properties when examined in the hot plate assay and tail electric stimulation test. These effects were also exhibited by fluoxetine and etodolac. The former, an SSRI, has been shown to possess marked anti-inflammatory and analgesic properties [11,12,13]. In a model of neuropathic pain in rat, etodolac, a COX-2 inhibitor, alleviated heat-evoked hyperalgesia [12].
H. perforatum was ineffective, however, in the acetic acid-induced writhing response, which was significantly reduced by fluoxetine or etodolac. The writhing response to acetic acid is brought about by the release of prostacyclin synthesized by cyclo-oxygenase in the abdominal cavity of the mice [20]. It is reduced by cyclo-oxygenase inhibitors such as meloxicam or diclofenac [21], by morphine [22], and also by fluoxetine [13]. H. perforatum, also and in contrast to fluoxetine, did not have antinociceptive properties against neurogenic pain induced by C-fiber excitation with i.p. capsaicin.
Antidepressants increase noradrenaline and serotonin concentrations at the synapse, an action thought to involved their pain-alleviating properties [23]. The antinociceptive action of systemically administered antidepressants can be inhibited by the opioid receptor antagonist naloxone, suggesting the involvement of opioid sensitive pathways [24]. Extracts of H. perforatum, on the other hand, do not fit into the action of classic tricyclic antidepressants or the SSRIs. The drug acts to inhibit the reuptake of several neurotransmitters, including serotonin, norepinephrine, dopamine, gamma-aminobutyric acid, and Lglutamate [25,26].