Antinociceptive Activity of Pleurotus ostreatus., an Edible Mushroom, in Rats

Abstract

The antinociceptive potential of Pleurotus ostreatus. (Jacquin: Fries) P. Kummer (Tricholomataceae) was investigated in rats (doses used: 125, 500, and 1000 mg/kg). Male rats and female rats in proestrous, estrous, and diestrous stages were orally administered 1000 mg/kg of freeze-dried P. ostreatus. and the reaction times on hot-plate and tail-flick tests were recorded. In the hot-plate test, the reaction time was significantly prolonged in male rats and diestrous female rats. Marked and significant prolongation in the reaction time at 1 h in males (28% mid and 32% high dose) and up to 2 h in diestrous females (57% mid and 79% high dose after 1 h) were observed on the hot-plate test. This effect was dose-dependent. In contrast, none of the rats showed increases in reaction time in the tail-flick test. In the formalin test, in rats administered a 1000 mg/kg dose of P. ostreatus., pain was significantly suppressed in both phases (females: licking time, 23%, 48%; licking frequency, 28%, 28%; males: licking time, 32%, 43%; licking frequency, no significant change). P. ostreatus. possessed antihistamine activity (histamine wheal test). Naloxone blocked the antinociceptive activity in the hot-plate test; however, metochlopramide did not abolish the activity. P. ostreatus. also showed mild antioxidant activity. Further, a 1000 mg/kgdose of P. ostreatus. did not induce sedation (hole-board test). This dose did not cause mortality or show signs of acute toxicity or stress. It is concluded that P. ostreatus. shows antinociception against neurogenic and continuous inflammatory pain possibly by opioid mechanisms, antioxidative and antihistamine activities.

Keywords:

• Antihistamine
• antinociception
• antioxidant
• opioid mechanism
• oyster mushroom
• pain suppression
• Pleurotus ostreatus

Introduction

Oyster mushroom is regarded as one of the commercially important edible mushrooms throughout the world. The family Tricholomataceae consists of a number of different species including Pleurotus ostreatus. (Jacquin: Fries) P. Kummer, P. eryngii. (De Candolle: Fries) Quélet, P. cystidiosus. O.K. Miller, P. cornucopiae. (Paulet ex Persoon) Rolland, P. pulmonarius. (Fries) Quélet, P. tuber-regium. (Fries) Singer, P. dryinus. (Persoon) Kummer, and P. levis. (Berkeley and Curtis) Singer (Singer, 1986). The oyster mushroom is the second most important mushroom in production in the world, accounting for 25% of total world production of cultivated mushrooms.

Pleurotus ostreatus. (Jacquin: Fries) P. Kummer, commonly known as American oyster, is grown worldwide, and China is the major producer. P. ostreatus. was first cultivated in the United States in 1900. These mushrooms are wide and fleshy, usually in a semicircle shape, and caps can be up to 20 cm wide. The gills are usually decurrent, white, and have a white spore print. The stipe is either very short or there is no stipe at all and is eccentric (off center). P. ostreatus. belongs to the division Basidiomycota, order Agaricales, and the family Tricholomataceae.

P. ostreatus. was shown to possess hypocholesterolemic (Bobek et al., 19971998; Hossain et al., 2003), antioxidant (Bobek & Galbary, 2001), and antitumor (Li et al., 1994) properties. Further, mushrooms belonging to the genus Pleurotus. have been shown to possess anti-inflammatory (Jose et al., 2004) and antifungal (Wang & Ng, 2004) properties. Usually, mushrooms having anti-inflammatory activity possess antinociceptive activity as well (Sheena et al., 2003; Park et al., 2005). As such, a possibility exists that P. ostreatus. may also have antinociceptive potential, but so far this has not been investigated.

The main aim of this study was to investigate the antinociceptive potential of P. ostreatus. and to identify the possible mechanism of action of said activity. This was tested in rats with a freeze-dried preparation of P. ostreatus. using three analgesiometric methods.

Materials and Methods

Animals

Healthy adult Wistar male (150–225 g) and female rats (150–225 g) were purchased from Medical Research Institute (Borella, Colombo, Sri Lanka). They were housed under standardized animal house conditions. They had access to pelleted food (Vet House Ltd., Colombo, Sri Lanka) and tap water ad libitum..

Collection of mushroom

Fresh P. ostreatus. mushroom was collected from a farmer using the spawn provided by the Mushroom Cultivation Center, Export Research Board (Ratmalana, Sri Lanka). The identification and authentication was performed by Prof. R.L.C. Wijesundera, Department of Plant Science, University of Colombo, Sri Lanka. A voucher specimen (A-T-Po, 2006) is deposited at the research laboratory, Department of Chemistry, University of Colombo.

Preparation of mushroom

Fresh Pleurotus ostreatus. (1 kg) was washed with water to remove any soil particles and was freeze-dried (LFD-600EC; Laytant Life Science Co. Ltd, Tokyo, Japan) and ground with a commercial blender (HGB-SS; Food Machine International, Osaka, Japan) to obtain 77 g of a yellowish powder and was stored air-tight at 4°C. Suspension of freeze-dried mushroom was freshly prepared with tap water (1 g in 10 mL water) prior to the feeding of rats. This is the maximum dose enabling smooth gavage of rats. In all concentrations, the volume of oral administration was 2.5 mL.

Hot-plate and tail-flick tests

Female rats at the three different stages (proestrous, estrous, and diestrous) of the estrous cycle were selected by vaginal smearing using isotonic saline (0.9% NaCl, w/v). Each group was orally treated with 1000 mg/kg (high dose) of freeze-dried mushroom suspension (n = 12). Another group of female rats (n = 12 per group) was used as a control and was orally treated with 2.5 mL tap water. As the results of the diestrous group were promising, dose-dependence activity was conducted using another two groups of rats belonging to the diestrous stage where they were orally treated with 500 mg/kg (mid-dose) of freeze-dried mushroom (n = 12) and 125 mg/kg (low dose) of freeze-dried mushroom (n = 12). Male rats were divided into three equal groups (n = 12/group), and rats in each group were orally treated with 1000 mg/kg (high dose), 500 mg/kg (mid-dose), and with 125 mg/kg (low dose) of freeze-dried mushroom, respectively. Morphine was used as the reference drug, and male rats (n = 6) were orally administered 15 mg/kg of morphine.

The reaction times of these rats were measured 1 h prior to the treatment and 1 h to 5 h after the treatment at hourly intervals using hot-plate and tail-flick test methods (Langerman et al., 1995). In the hot-plate test, the rats were placed in a hot-plate analgesia meter (Model MK 35 A; Muromachi Kikai Co., Ltd., Tokyo, Japan.) at 50°C and the time taken to lick any one of the hind paws or to jump was recorded. In the tail-flick test, the tail of the rat up to 5 cm from its tip was immersed in a water bath at 55°C and time taken to flick the tail was recorded. Rats showing a pretreatment reaction time greater than 15 s in the hot-plate test and 5 s in the tail-flick test were not used in the experiment. A cutoff time of 25 s for the hot-plate test and 10 s for the tail-flick test was set to avoid tissue damage.

Formalin test

Male rats (n = 7) and female rats of diestrous stage (n = 7) were orally treated with 1000 mg/kg freeze-dried P. ostreatus. suspension (2.5 mL), and control group of male (n = 7) and female rats in the diestrous stage (n = 7) were treated with 2.5 mL tap water. One hour later, these rats were injected with 0.05 mL 2.5% formalin (BDH Chemicals, Poole, UK) in normal saline into the subplantar surface of the left hind paw and immediately individually placed in transparent plastic cages. Rats were observed for 60 min, and the total licking time and frequency of licking of the injected paw were recorded in two phases; first phase 0–5 min and second phase 15–60 min (Farsam et al., 2000). Time per lick was computed.

Antihistamine effect

Eight rats were randomly divided into two equal groups (n = 4/group). Their fur on the posterior lateral side (abdominal area) was completely shaved under ether anesthesia. Twenty-four hours later, one group was orally administered freeze-dried 1000 mg/kg P. ostreatus. suspension and the other 2.5 mL water. One hour later, these rats were lightly anesthetized with ether and subcutaneously injected with 0.05 mL 200 µg/mL histamine dihydrochloride (Fluka, Duche, Switzerland), and the diameter of wheal formed was measured and the area was calculated (Spector, 1956).

Investigation of involvement of opioid receptors

Twenty male rats were fasted overnight and randomly divided into two equal groups (n = 10/group). Those in group 1 were intraperitoneally injected with 1 mg/kg naloxone (Bodene Ltd, Port Elizabeth, South Africa), an opioid antagonist, and those in group 2 with isotonic saline. After 45 min, rats in both groups were orally administered 2.5 mL of the 1000 mg/kg freeze-dried suspension of P. ostreatus.. These rats were subjected to the hot-plate test, and reaction time was determined (Ratnasooriya & Dharmasiri, 1999) before treatment and 1 h after the administration of P. ostreatus..

Investigation of involvement of dopamine receptors

Sixteen male rats were randomly divided into two equal groups. The rats (n = 8/group) in group 1 were orally administered 1.5 mg/kg metachlopramide (Glaxo SmithKline, Pakistan Ltd, Karachi, Pakistan), a dopamine (D₂) antagonist (Ratnasooriya & Dharmasiri, 1999), in 1 mL 1% methylcellulose (Griffin and George Ltd., Middlesex, London, UK). The rats in group 2 were orally treated with 1 mL of 1% methylcellulose. One hour later, both groups of rats were orally treated with 1000 mg/kg P. ostreatus. suspension, and the reaction time was monitored in the hot-plate test.

Evaluation of antioxidant properties (TBARS method)

Into five snap-capped vials, different concentrations of freeze-dried mushroom (5.00, 2.50, 1.67, 1.25, and 1.00 mg/mL concentrations, 10 µL each) and egg yolk 50 µL were added. Distilled water (10 µL) was used as the control. Acetic acid (20% solution; 150 µL) and 0.8% thiobarbituric acid (TBA; 150 µL) were added to each snap-capped vial. Total volume was adjusted to 400 µL by adding distilled water. These mixtures were vortexed for 5 s and kept in a water bath (LCH-110 Lab Thermo Cool; Advantec, Tokyo, Japan) at 95°C for 60 min. Butanol (1 mL) was added to each tube and vortexed for 5 s. After centrifuging at 1500 × g. for 5 min, the butanol layer was separated. Absorbance values were measured at 532 nm (Dorman et al., 1995). Ascorbic acid, butylated hydroxytoluene (BHT), and vitamin E (100 µg/mL) were used as positive controls. Antioxidant index was calculated using the following equation: where T. = absorbance of test sample and C. = absorbance of fully oxidized control.

Evaluation of rectal temperature

Eight rats were randomly divided into two equal groups (n = 4/group). One group was orally administered with 1000 mg/kg freeze-dried P. ostreatus. suspension and the other 2.5 mL water. One hour later, the rectal temperature was measured (Ratnasooriya & Pieris, 2003) using a clinical thermometer (TM-II, normal glass; Focal Corporation, Tokyo, Japan).

Evaluation of sedative activity

Fourteen male rats were randomly divided into two equal groups (n = 7/group) and were fasted for 12 h. The rats in group 1 were orally administered 2.5 mL of the 1000 mg/kg freeze-dried suspension of P. ostreatus. and those in group 2 were orally administered 2.5 mL tap water. Each of these rats were then placed on a rat hole-board apparatus (File & Wardill, 1975) and observed for 7.5 min. During this period, the number of head dips, rears, locomotor activity, and number of fecal boluses expelled were counted.

Muscle strength and muscular coordination

Fourteen male rats were randomly divided into two equal groups (n = 7/group). Group 1 was orally administered 2.5 mL of the 1000 mg/kg freeze-dried P. ostreatus. suspension and those in group 2 were orally administered 2.5 mL tap water. After 1 h, rats were subjected to bar-holding test (to evaluate the muscle strength) immediately followed by the Bridge test (to evaluate the muscle coordination), and the latency to fall and slide off (in seconds) were respectively recorded (Plazinc et al., 1993).

Observation of overt signs of toxicity, stress, and aversive behaviors

The rats used in the above investigation were observed 6–8 h on the day of treatment and on day 1 posttreatment for overt signs of toxicity (such as salivation, diarrhea, yellowing of hair, loss of hair, ataxia, postural abnormalities, behavioral changes, marked impairment of food and water intake and body weight) stress (erection of fur and exopthalmia), and aversive behaviors (biting and scratching behaviors, licking of tail, paw, and penis, intense grooming behaviors, and vocalization).

Analysis of data

Data are given as mean ± standard error of mean. Data were analyzed with Mann-Whitney U.-test.

Results

Hot-plate and tail-flick tests

The increase in the reaction time (Table 1) 1 h after treatment with freeze-dried P. ostreatus. (1000 mg/kg) in female rats in the diestrous stage (79%, p < 0.001) and male rats (32%, p < 0.05) was marked and significant. There was a marked increase in the reaction time even 2 h after treatment in both female rats in the diestrous stage (47%, p < 0.05) and male rats (29%, p < 0.05). However, in the female rats in the proestrous and estrous stages, the reaction time was not significantly altered by the high dose (1000 mg/kg) of P. ostreatus. on the hot-plate test. Further, the reaction time of the rats in the hot-plate test decreased with the decreasing doses of P. ostreatus. showing dose dependency (r² = 0.99 and 0.88 in females in diestrous stage and males, respectively) of this antinociceptive activity. The increase in the reaction time (after 1 h) in the hot-plate test upon oral administration of 500 mg/kg P. ostreatus. was 57% (p < 0.01) in female rats in the diestrous stage and 28% (p < 0.05) in male rats. There was no significant increase in the reaction time after 1 h upon treatment with 125 mg/kg dose of P. ostreatus. in female rats in the diestrous stage. The percentage increase in reaction time of the male rats administered 15 mg/kg morphine is 70% (p < 0.01) after 1 h, and the antinociceptive activity lasted up to 4 h where the increase in reaction time at 4 h is 44% (p < 0.05).

Table 1.. Effect of oral administration of freeze-dried P. ostreatus. (doses 125, 500, and 1000 mg/kg) on the hot-plate reaction time of rats.

	Hot-plate reaction time (s) (mean ± SEM)
		Treatment
Treatment (mg/kg)	Pretreatment	1 h	2 h	3 h	4 h	5 h
Female^a.
Control (n = 12)	7.9 ± 0.7	7.8 ± 0.6	8.2 ± 0.7	7.3 ± 0.5	7.6 ± 0.6	7.2 ± 0.4
125 (n = 12) FD	6.0 ± 0.6	7.8 ± 0.8	6.3 ± 0.6	6.9 ± 0.6	7.1 ± 0.9	6.2 ± 0.9
500 (n = 12) FD	6.9 ± 0.5	10.9 ± 1.0**	10.5 ± 1.1**	8.1 ± 0.4*	8.2 ± 0.8	9.2 ± 0.9*
1000 (n = 12) FD	8.7 ± 0.5	15.5 ± 1.2***	12.8 ± 1.7*	10.6 ± 1.4	8.9 ± 0.8	7.3 ± 0.6
1000 (n = 12) FO	7.6 ± 0.6	7.4 ± 0.5	6.6 ± 0.5	6.5 ± 0.5	5.8 ± 0.5	7.4 ± 0.6
1000 (n = 12) FP	7.9 ± 0.5	8.3 ± 0.7	7.7 ± 0.6	7.9 ± 0.6	8.1 ± 0.6	7.4 ± 0.8
Male
Control (n = 12)	6.2 ± 0.6	6.8 ± 0.7	4.9 ± 0.3	5.4 ± 0.4	5.6 ± 0.6	5.1 ± 0.4
125 (n = 12)	6.4 ± 0.6	5.9 ± 0.5	5.3 ± 0.6	7.1 ± 0.8	5.9 ± 0.8	5.3 ± 0.8
500 (n = 12)	8.7 ± 0.7	11.1 ± 0.9*	10.2 ± 0.5*	8.9 ± 0.7	7.8 ± 0.7	7.2 ± 0.6
1000 (n = 12)	7.0 ± 0.6	9.5 ± 0.7*	8.6 ± 0.8*	7.3 ± 0.8	7.5 ± 0.7	5.6 ± 0.6
Morphine (n = 6)	8.1 ± 0.7	13.8 ± 0.6**	17.0 ± 1.3**	11.6 ± 2.2	11.7 ± 1.4*	9.0 ± 1.2

Values are significant at *p < 0.05, **p < 0.01, and ***p < 0.001 compared with respective controls.

^a.The results of the females in the proestrous, diestrous, or estrous stages are abbreviated F_P, F_D, F_O, respectively.

A profound increase in the reaction time was not observed in the tail-flick test upon treatment with 1000 mg/kg mushroom. However, a significant but slight increase in reaction time after 1 h was observed in both female rats in the diestrous stage (16%, p < 0.05) and in the male rats (33%, p = 0.03) upon treatment with 500 mg/kg P. ostreatus.. Nevertheless, there is no real practical value in this enhancement.

Formalin test

The results of the formalin test shown in Table 2 indicate that oral administration of 1000 mg/kg freeze-dried P. ostreatus. to females in the diestrous stage significantly (p < 0.05) impaired the licking time (23% and 48% suppression in the first and second phases, respectively) and licking frequency (28% suppression in both phases) in the formalin test. However, the same dose in males had significantly (p < 0.05) impaired only the licking time in both phases of the formalin test (32% and 43% suppression in the first and the second phases, respectively).

Table 2.. Effect of oral administration of 1000 mg/kg freeze-dried P. ostreatus. on the licking frequency and time of rats in the formalin test.

	First phase			Second phase
Treatment (mg/kg)	Licking time (s)	Licking frequency (min^−1)	Time per lick (s)	Licking time (s)	Licking frequency (min^−1)	Time per lick (s)
Female diestrous
Control (n = 7)	75.7±4.6	4.3±0.4	3.7±0.3	568.9±24.1	1.8±0.1	7.3±0.5
Treatment (n = 7)	58.4±6.0**	3.1±0.2*	4.0±0.1	297.6±32.8***	1.3±0.2*	5.3±0.4
Male
Control (n = 7)	77.7±12.6	2.0±0.2	7.9±0.9	860.0±90.8	1.6±0.2	12.5±1.0
Treatment (n = 7)	52.7±9.6*	2.1±0.3	4.9±0.4	494.4±29.1*	1.4±0.1	8.1±0.5

Values are significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Antihistamine effect

Compared with the control, treatment with high dose of P. ostreatus. significantly (p = 0.01) reduced the area of wheal formed (50.7%) on the skin by the injection of histamine (control vs treatment: 36.3 ± 4.9 vs. 17.9 ± 1.7 mm²).

Investigation of opioid receptor mediation

In this study, the opioid receptor antagonist naloxone significantly (p = 0.0002) impaired the reaction time induced by 1000 mg/kg P. ostreatus. as shown in Table 3.

Table 3.. Effect of naloxone on the hot-plate reaction time of male rats induced by 1000 mg/kg freeze-dried P. ostreatus..

	Reaction time on hot-plate test in seconds (mean ± SEM)
Treatment (mg/kg)	Pretreatment	First hour
1000 (n = 10) no naloxone	6.0 ± 0.3	9.2 ± 0.8***
1000 (n = 10) with naloxone	6.8 ± 0.5	6.7 ± 0.7

Values are significant at ***p < 0.001.

Investigation of dopamine receptor mediation

Oral administration of metachlopramide did not significantly change the reaction time induced by 1000 mg/kg P. ostreatus..

Evaluation of antioxidant properties

As shown in Table 4, P. ostreatus. had a mild (compared with BHT, vitamin E, and ascorbic acid) but a dose-dependent (r² = 0.85, p < 0.05) antioxidant activity.

Table 4.. Antioxidant activity (TBARS method) of P. ostreatus..

Sample	Concentration (mg/mL)	Antioxidant index
BHT (n = 9)	0.1	68.37 ± 3.75
Vitamin E (n = 9)	0.1	65.32 ± 2.20
Ascorbic acid (n = 9)	0.1	80.23 ± 4.01
Freeze-dried P. ostreatus. (n = 4)	1.00	21.97 ± 3.76
	1.25	31.47 ± 1.21
	1.67	44.20 ± 13.23
	2.50	38.54 ± 5.06
	5.00	53.10 ± 6.02

Antioxidant activity was dose dependent (r² = 0.85, p < 0.05).

Evaluation of rectal temperature

Compared with controls, there was no significant (p > 0.05) change in the rectal temperature of treated rats 1 h after oral administration of 1000 mg/kg P. ostreatus. (control vs. treatment: 96.8 ± 0.5°F vs. 96.8 ± 0.2°F).

Evaluation of sedative activity

In the rat hole-board test, none of the parameters investigated was significantly (p > 0.05) altered by 1000 mg/kg P. ostreatus. (control vs. treatment: number of rears, 31.6 ± 4.1 vs. 30.4 ± 3.0; number of head dippings, 9.0 ± 1.6 vs. 9.6 ± 1.3; dipping time, 11.9 ± 3.3 vs. 12.9 ± 2.2; time per dip, 1.2 ± 0.2 vs. 1.3 ± 0.1; number of crossings, 34.9 ± 4.2 vs. 32.3 ± 4.8; number of fecal boluses, 2.6 ± 0.8 vs. 2.3 ± 0.8).

Muscle strength and muscular coordination

The latency to fall in the bar-holding test and latency to slide off in the Bridge test did not show a significant (p > 0.05) change upon administration of 1000 mg/kg P. ostreatus. (control vs. treatment: latency to fall in the bar-holding test, 12.7 ± 1.7 s vs. 16.1 ± 4.2 s; latency to slide off in the Bridge test, 31.7 ± 6.5 s vs. 32.1 ± 6.4 s).

Observation of overt signs of toxicity, stress, and aversive behaviors

No overt signs of toxicity, stress, or aversive behaviors were observed in both female and male rats during this study. Further, none of the treated or control rats died (up to day 1 posttreatment).

Discussion

This study examined the antinociceptive potential of P. ostreatus., a worldwide popular edible mushroom. The results showed, for the first time, that it has acute, orally active, moderate to strong pain-relieving properties against both neurogenic pain (as shown by the hot-plate test and the first phase of the formalin test) and continuous inflammatory pain (as shown by the second phase of the formalin test) (Farsam et al., 2000). This effect is genuine and not a false-positive result springing from muscle relaxation (evident by the bar-holding test), impaired muscle coordination (as judged by the Bridge test), or hypothermia (revealed by rectal temperature). This is an important finding that may have therapeutic potential as a home remedy for mild body aches and pains. In addition, P. ostreatus. may contain a lead compound (s) that may be developed as a potent analgesic.

Antinociceptive activity of P. ostreatus. was not gender dependent. Gender differences in analgesic response exists with certain antinociceptives (Gear et al., 1996a), women being more sensitive than men (Gear et al., 1996b). However, it is of particular interest to note that the antinociceptive action of P. ostreatus. on females was dependent on the phase of the estrous cycle: most potent in diestrous and least potent in proestrous and estrous. Such estrous cycle stage–dependent changes in sensitivity of antinociception have been shown previously. For example, Ratnasooriya et al. (1994) have shown the antinociceptive activity of the leaf extract of Murraya koenigii. was highest in the proestrous stage of the female rats, and Terner et al. (2005) have shown that morphine and buprenorphine were most potent in metestrous and proestrous and least potent in estrous stage of rats. The observation of antinociceptive activity by P. ostreatus. only in the diestrous female and males could possibly be due to the influence of progesterone (predominant in diestrous females) and testosterone (predominant in adult males) in the antinociception mechanism. Progesterone and testosterone have identical steroidal skeleton except at the C-17 substituent where the substituent is a –COCH₃ in progesterone and an –OH in testosterone (Finar, 1991).

The antinociceptive activity of P. ostreatus. had rapid onset (within 1 h) and a short duration of action (1 h in males and 2 h in females). This is presumably due to rapid absorption and equally rapid degradation and/or equally rapid clearance of the active component. Alternatively, the short duration of action could be due to the formation of a metabolite that is antagonistic to the production of antinociceptive effect (Hernandez et al., 2000).

Antinociceptive activity of P. ostreatus. was dose-dependent. This possibly indicates a receptor mediation. When evaluated in the tail-flick test, antinociceptive activity was minimal indicating that it is not mediated centrally via spinal mechanisms: tail flick predominately measures spinal reflexes (Wong et al., 1994). In contrast, the hot-plate test reaction time was greatly prolonged with P. ostreatus. suggesting a centrally mediated supraspinal mechanism of action: hot plate predominaetly measures supraspinally organized responses (Wong et al., 1994). Dopamine is known to play a role in pain mediation, and dopamine receptor antagonist impairs pain (Rang et al., 2003); however, P. ostreatus.–induced antinociception was not blocked by metachlopramide, a D₂-dopamine receptor antagonist. Therefore, this antinociceptive activity is unlikely to be mediated via dopamine receptors. Though there are five dopamine receptor types, pain modulation and activation appears to be mediated mainly by receptors of the D₂ family (Gao et al., 1998), hence an antagonist for the D₂ family was used in this study.

Sedation is known to impair pain (Nadeson & Goodchild, 1997), and several sedatives have antinociceptive action (Lawrence & Bennett, 1997). A sedative mode of action is unlikely to be operative in this study as none of the parameters in the rat hole-board experiment was altered with the highest dose of P. ostreatus.. This test is widely used to evaluate potential sedatives (Dharmasiri et al., 2003). In rats, food deprivation induces antinociception (McGivorn et al., 1979), but such a mode of action is unlikely in this study as food was available ad libitum. and there was no apparent suppression of food intake. Stress can provoke analgesia (Rang et al., 2003); however, P. ostreatus. was not stressogenic (in terms of fur erection, exopthalmia, and aversive behaviors). Therefore, antinociception due to stress can be ruled out.

In contrast, naloxone, an opioid receptor antagonist, blocked the P. ostreatus.–induced antinociception indicating opioid receptor mediation. Further, opioids are known to inhibit both phases of the formalin test (Sevostianova et al., 2005) and a similar effect was seen with P. ostreatus.. This provides additional evidence for the opioid receptor mediation; however, the antinociceptive potential of P. ostreatus. was low compared with strong opioids like morphine. This is not surprising as the magnitudes of the antinociceptive potency of opioids are known to depend on the type of opioids (Terner et al., 2005). This opioid receptor mediation of antinociceptive activity of P. ostreatus. could operate at region-specific sites in the brain and also peripherally. Impairment of both phases of the formalin test also indicates peripheral antinociceptive activity. This effect could be brought about in two ways: the chemical constituents in the P. ostreatus. may act directly as an opioidomimetic on the opioid receptors or they may act indirectly by inducing the release of endogenous peptides such as endorphins and enkephalins to bring about the antinociceptive activity. As suggested earlier, progesterone and testosterone may play a vital role in bringing about the opioidomimetic effects and/or release of endogenous peptides as the antinociceptive activity of P. ostreatus. was seen only in the males and in females in the diestrous stage. In this respect, it is most interesting to note that Terner et al. (2005) have recently provided evidence in rats to suggest that gonadal hormones can modulate sensitivity to nociception and opioid antinociception.

In the formalin test, the late phase is mainly due to the release of inflammatory mediators like histamine, prostaglandins, serotonin, and bradykinin (Murray et al., 1988; Tjolsen et al., 1992). P. ostreatus. suppresses the late phase suggesting the involvement of one or more of the above inflammatory mediators. P. ostreatus. has antihistamine activity as revealed by the histamine wheal formation test. Such an action could inhibit the activity of histamines released due to injury of the paw resulting from formalin injection.

Free radicals are linked to a number of diseases and conditions, including pain and inflammation (Kim et al., 2004), and antioxidants have antinociceptive activity (Nishiyama & Ogawa, 2005; Kim et al., 2006). P. ostreatus. had a mild antioxidant action as judged by the TBARS assay. This mild antioxidant activity shown by P. ostreatus. may have an auxiliary effect on its antinociceptive activity. Morphine, a well-known opioid agonist, was also provend to possess antioxidant properties (Gülcin et al., 2004).

Acute oral administration of a high dose of a P. ostreatus. showed no overt signs of toxicity and stress or aggressive behavior. Furthermore, a previous study (Abeyweera, 2005) has shown subchronic oral administration of an even higher dose of P. ostreatus. (3000 mg/kg per day consecutively for 14 days) did not alter the glutamic-oxaloacetic transaminase and glutamic pyruvic transaminase levels in the rat serum. Collectively, these observations suggest that P. ostreatus. may be devoid of any undesirable side effects.

In conclusion, this study shows for the first time that P. ostreatus. can act as an orally active, safe, short-acting, moderate to strong antinociceptive agent. This may be used as a dietary supplement for day-to-day relief of body aches and pains. Further studies are, however, warranted before firm recommendations are made.

Acknowledgments

We appreciate the technical support given by Mr. J.R.A.C. Jayakody, Department of Zoology, University of Colombo, in conducting the animal experiments and the financial support of the NSF (grant RG/2004/C/02).