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0Asian Pacific Journal of Tropical Medicine (2011)117-120
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Asian Pacific Journal of Tropical Medicine
journal homepage:www.elsevier.com/locate/apjtm
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Evaluation of the analgesic activity of ethyl acetate, methanol and aqueous extracts of Pleurotus eous mushroom
Suseem SR1, Mary Saral A1*, Neelakanda Reddy P2, Marslin Gregory3
'Pharmaceutical Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632 014, Tamil Nadu, India Department of Bio-organic chemistry, CLRI, Chennai 600 020, India 3Department of Pharmacology, S.A. Raja Pharmacy College, 627116, Tirunelveli, India
ABSTRACT
Objective: To evaluate the analgesic activity of the ethyl acetate, methanol and aqueous extracts of Pleurotus eous (P. eous) mushroom. Methods: The dried fruiting bodies were extracted with ethyl acetate, methanol and water. The analgesic effect of extracts of P. eous were investigated at doses 250 500 and 1 000 mg/kg body weight, using acetic-acid induced writhing, hot-plate, tail immersion and tail-clip tests. Results: P. eous extracts produced significant reduction in number of writhes induced by intraperitoneal injection of acetic-acid (P<0.05). Moreover, in hot-plate and tail immersion test, all the three extracts significantly raised the pain threshold at different time of observation (0-60 min) in comparison with control (P<0.05). In tail-clip test the extracts also caused a significant inhibition of pain at both the doses used (P<0.05). Conclusions: The results of present study suggest that extracts of P. eous possess potent analgesic property and could serve as a base for future drugs.
ARTICLE INFO
Article history: Received 12 November 2010 Received in revised form 27 November 2010 Accepted 15 December 2010 Available online 20 February 2011
Keywords: Writhing test Hotplate test Tail immersion method Tail clip test
1. Introduction
Pain is an unpleasant sensation, but it is usually beneficial to man and animal. It is mainly a protective mechanism for the body, whenever any tissues are being damaged, and it causes the individual to react to remove the pain stimulus. Typically, it is a direct response to an untoward event associated with tissue damage, such as injury, inflammation but severe pain can arise independently of any obvious predisposing cause (e.g. trigeminal neuralgia), or persistent long after the precipitating injury has healed (e.g. phantom limb pain). It can also occur as a consequence of brain or nerve injury (e.g. following a stroke or herpes infection). With many pathological conditions, tissue injury is the immediate cause of the pain, and this results in the local release of a variety of chemical agents, which are assumed to act on the nerve terminals, either activating them directly or enhancing their sensitivity to other forms of stimulation!^]. Attention is being focused on the investigation of the efficacy of mushroom-based drugs used in the traditional medicine because they are cheap and
Corresponding author: Mary Saral A, Pharmaceutical Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632 014, Tamil Nadu, India. Tel: 91-416 220 2330 Fax: 91-416 224 3092 E-mail: jaya_saral@yahoo.com
have little side effects.
Mushrooms are the fleshy and edible fruiting bodies of several species of fungi, typically produced above ground on soil or on its food source[3]. Mushrooms have high nutritive and medicinal values, and contribute to healthy diet because of their rich source of vitamins, minerals and proteins[4]. They comprise a vast and yet largely untapped source of powerful new pharmaceutical products[5,6]. They are low calorie food with very little fat and are highly suitable for obese persons[7]. Pleurotus species are commonly known as oyster mushrooms and are edible. They are reported to possess several medicinal properties. On preliminary phytochemical screening the extracts of Pleurotus eous (P. eous) were found to contain flavonoid compounds (Mg+HCl reduction test) which are known to target prostaglandins involved in the late phase of acute inflammation and pain perception. Pleurotus mushrooms are the second most important mushrooms in production in the world, 25% of total world production of cultivated mushrooms. P. eous mushroom belongs to Class Agaricomycetes and Family Pleurotaceae. This species has been of interest to researchers because its phytochemical constituents are similar to those of Pleurotus florida, Pleurotus ostreatus and Pleurotus sajor-caju, Pleurotus pulmonarious which are commonly used in medicines[4-6,8,9]. A bibliographic survey showed that there are no reports on the analgesic activity of P. eous in spite of its medicinal applications. This prompted us to investigate the effects
of pharmacological activities of P. eous mushroom in experimental models of analgesia.
2. Materials and methods
2.1. Preparation of the extract
The fruiting bodies of the mushroom P. eous were obtained from Kerala Agricultural University, Trivandrum and authenticated by Dr. Lu Lu Das, Professor, Department of Plant Biology, College of Agriculture, Vellayani, Kerala Agricultural University, Trivandrum. Mushroom fruiting bodies were dried at 40-50 °C for 48 h and powdered. The powdered material (250 g) was extracted with petroleum ether. The defatted material was extracted with ethyl acetate and then with 70% methanol for 8-10 h using Soxhlet apparatus. For the preparation of aqueous extract defatted material was extracted with hot water (70-80 C) for 8-10 hrs. The extract was collected after filtering through Whatmann No.1 filter paper. The solvents were completely evaporated at 40 C using a rotary vacuum evaporator. The residues were designated as ethyl acetate (EA) extract and methanol extract (MeOH) and aqueous extract (Aqs), respectively.
2.2. Animals
Swiss Albino mices were purchased from Animal Breeding Centre, Kerala Agricultural University, Trivandrum. They were randomly distributed into groups and housed in cages (6 per cage) and maintained under standard conditions. All animals were fed the standard diet and water ad libitum. This project was cleared by Institutional Animal Ethical Committee (Approval No: IEAC N0.03/001/10).
2.3. Acute toxicity studies
Swiss albino mice of either sex (18-25 g of weight) were used for acute oral toxicity study. The study was carried out as per the guidelines set by OECD and no adverse effects or mortality were detected in the mice up to 4 g/kg p.o., during the 24 h observation period. Based on the results obtained from this study, the dose for analgesic activity was fixed to be 250, 500 and 1 000 mg/kg body weight, for dose dependent study.
2.4. Analgesic activity
The animals were divided into 12 groups (n=6). Group I served as control received the vehicle only (1% carboxy methyl cellulose, 10 mL/kg p.o.). Group II and III served as standard, received indomethacin (10 mg/kg b.w.) or diclofenac sodium (10 mg/kg b.w.) Groups IV-XII served as test received EA, MeOH and Aqs extracts of P. eous at doses of 250, 500 and 1 000 mg/kg b.w. p.o., respectively.
2.4.1. Writhing test
The test was carried out according to Koster et al[10]. Animals were orally administered with indomethacin and diclofenac sodium (10 mg/kg b.w.) as the standard drug, P. eous (250, 500 and 1 000 mg/kg b.w.) and vehicle. Thirty minutes after treatment, the mice were given an intraperitoneal (i.p.) injection of 0.6% v/v acetic acid in a volume of 10 mL/kg to induce the characteristic writhing. The No. of writhing occurring between 5 and 15 min. after acetic acid injection was recorded. The response of the
extract treated animals was compared with that of control[11].
2.4.2. Hot-plate test
Mice were placed on an aluminum hot plate kept at (55 ±0.5) C for a maximum time of 30 s[12]. Reaction time was recorded when the animals licked their fore and hind paws and jumped at before (0) and 15, 30, 45 and 60 min after i.p. administration of P. eous at doses of 250, 500 and 1 000 mg/kg b.w. to different groups. Indomethacin and diclofenac sodium (10 mg/kg b.w.) is the standard drug.
2.4.3. Tail clip method
A metal artery clip was applied to the root of the mouse's tail to induce pain[13]. A sensitivity test was carried out and animals that did not attempt to dislodge the clip within 10 s were discarded. The responsive mice were allotted to groups of six animals each. The tail clip was applied 60 min after oral administration of extract (500 and 1 000 mg/kg b.w.), indomethacin and diclofenac (10 mg/kg b.w.). Whereas, vehicle treated group was served as control.
2.4.5. Tail immersion method
Prior to this experiment the animals were screened for the sensitivity test by immersing the tail of mice gently in hot water maintained at 55-55.5 C. The animal dislodging the tail from hot water within 5 seconds was selected for the study[14]. After oral administration of extracts (500 and 1 000 mg/kg b.w.), indomethacin and diclofenac (10 mg/kg b.w) the reaction time was recorded at 0, 5, 10, 15, 30, 45 and 60 minutes[15].
2.5. Statistical analysis
Results are expressed as Mean±SEM. The difference between experimental groups was compared by One-way Analysis of Variance (ANOVA) followed by Dunnett's test. The results were considered statistically significant when P<0.05.
3. Results
The effects of extract on acetic-acid induced writhes in mice have been shown in Table 1. P. eous at dose (250, 500 and 1 000 mg/kg b.w.) produced a significant (P<0.05) decrease in number of writhes in comparison with the control group. Diclofenac sodium and indomethacin (10 mg/kg b.w.) also showed significant (P<0.05) decrease in number of writhes.
Table 1
Effect of EA, MeOH and Aqs extracts of P. eous on acetic-acid induced writhes in mice.
Treatment Dose(mg/kg) Number of writhes Inhibition (%)
Control - 36.83±0.60
Diclofenac sodium 10 12.17±0.31* 66.96
Indomethacin 10 14.83±0.31* 59.73
Aqs extract 250 26.33±033* 28.51
Aqs extract 500 23.33±0.21* 36.65
Aqs extract 1 000 20.83±0.31* 43.44
MeOH extract 250 21.17±0.31* 42.52
MeOH extract 500 19.33±0.21* 47.52
MeOH extract 1 000 17.17±0.31* 53.38
EA extract 250 25.67±0.21* 30.30
EA extract 500 22.33±0.21* 39.37
EA extract 1 000 19.83±0.31* 46.16
Each value is the Mean±SEM for 6 mice; *:P<0.05 vs control.
SSuseem SR et al./Asian Pacific Journal of Tropical Medicine (2011)117-120
Hot-plate test was also assayed to threshold at different time of observation (0-60 min) in comparison with control. The results presented in Table 2 showed that diclofenac sodium and indomethacin (10 mg/kg b.w.) were used as standard drug, also that the oral administration of the P. eous produced a significant (P<0.05) analgesic effect at doses 250, 500 and 1 000 mg/kg b.w. in a time-dependent way (P<0.05). The effect of P. eous on tail clip test is shown as in Table 3. The extract caused a significant inhibition of pain at both
used doses (500 and 1 000 mg/kg b.w.), diclofenac sodium (10 mg/kg b.w.), a standard drug, was highly effective (P<0.05).
The effects of extract on tail immersion test in mice have been shown in Table 4. Oral administration of P. eous produced a significant analgesic effect at both doses of 500 and 1 000 mg/kg b.w. in a time-dependent way (P<0.05). Diclofenac sodium and indomethacin (10 mg/kg b.w.) were used as standard drug.
Table 2
Effect of EA, MeOH and Aqs extracts of P. eous on mice subjected to the hot-plate test.
Treatment Dose(mg/kg) Reaction time in seconds at time (minutes)
0 min 15 min 30 min 45 min 60 min
Control - 1.50±0.22 2.00±0.26 1.83±0.31 2.17±0.17 2.00±0.00
Diclofenac sodium 10 2.17±0.31 5.83±0.31 12.00±0.63 17.33±0.33 21.83±0.47
Indomethacin 10 1.67±0.21 4.50±0.22 7.33±0.49 12.83±0.47 14.50±0.74
Aqs extract 250 1.83±0.31 3.83±0.31 5.67±0.21 8.00±0.36 10.67±0.33
Aqs extract 500 1.83±0.31 4.33±0.21 7.33±0.33 10.67±0.42 13.33±0.33
Aqs extract 1 000 1.50±0.43 4.00±0.26 6.83±0.47 11.17±0.47 14.67±0.21
MeOH extract 250 1.50±0.22 4.50±0.22 8.00±0.26 10.83±0.31 13.83±0.31
MeOH extract 500 1.83±0.31 4.83±0.31 8.50±0.43 11.50±0.43 14.67±0.42
MeOH extract 1 000 1.83±0.17 4.50±0.22 8.33±0.33 12.33±0.33 16.50±0.43
EA extract 250 1.33±0.33 3.67±0.21 6.17±0.17 8.83±0.31 11.50±0.43
EA extract 500 1.67±0.42 3.83±0.31 6.50±0.22 8.83±0.31 12.17±0.31
EA extract 1 000 2.00±0.26 4.50±0.22 6.83±0.31 9.67±0.21 12.67±0.21
Each value is the Mean±SEM for 6 mice.
Table 3
Effect of EA, MeOH and Aqs extracts of P. eous on tail-clip test in mice.
Treatment Dose (mg/kg) Reaction time in seconds at time (minutes)
0 min 5 min 10 min 15 min 30 min 45 min 60 min
Control - 1.67±0.42 2.17±0.17 2.00±0.26 2.17±0.17 2.33±0.21 2.17±0.31 2.00±0.26
Aqs extract* 500 2.17±0.17 3.33±0.21 6.00±0.26 7.50±0.22 10.00±0.26 11.67±0.21 13.33±0.33
Aqs extract* 1 000 2.00±0.26 3.17±0.17 6.50±0.22 9.33±0.21 11.50±0.22 13.50±0.22 14.67±0.21
MeOH extract* 500 2.00±0.26 3.67±0.42 7.50±0.22 9.67±0.21 12.67±0.21 15.00±0.26 16.67±0.42
MeOH extract* 1 000 2.17±0.31 4.83±0.31 8.00±0.36 11.00±0.36 13.50±0.22 15.17±0.31 17.50±0.34
EA extract* 500 2.00±0.42 4.00±0.26 6.33±0.21 9.00±0.36 11.00±0.36 12.67±0.21 14.50±0.22
EA extract* 1 000 1.83±0.42 4.00±0.00 7.17±0.17 9.50±0.22 11.67±0.21 14.00±0.36 16.50±0.22
Diclofenac sodium* 10 2.00±0.26 6.00±0.63 10.17±0.31 12.67±0.33 17.33±0.33 19.67±0.42 24.17±0.31
Each value is the Mean±S.E.M. for 6 mice; *:P<0.05 vs control.
Table 4
Effect of EA, MeOH and Aqs extracts of P. eous on tail-immersion test in mice.
Treatment Reaction time in seconds at time (minutes)
Dose(mg/kg) 0 min 5 min 10 min 15 min 30 min 45 min 60 min
Control - 2.00±0.26 3.17±0.17 2.33±0.21 3.00±0.00 2.83±0.17 2.00±0.26 2.00±0.00
Aqs extract 500 2.67±0.21 4.00±0.26 5.00±0.26 7.00±0.26 9.33±0.33 11.00±0.26 12.33±0.21
Aqs extract 1 000 2.17±0.31 4.00±0.26 6.00±0.26 7.67±0.21 10.17±0.31 12.17±0.31 13.67±0.21
MeOH extract 500 2.00±0.25 3.83±0.17 5.50±0.22 7.67±0.21 10.67±0.21 13.00±0.26 15.33±0.33
MeOH extract 1 000 2.00±0.00 5.00±0.00 6.33±0.21 8.67±0.21 11.83±0.31 14.50±0.22 17.00±0.26
EA extract 500 2.17±0.31 4.17±0.31 6.00±0.36 7.83±0.40 9.33±0.21 11.00±0.26 12.50±0.22
EA extract 1 000 2.50±0.22 4.17±0.17 6.33±0.21 8.17±0.17 10.33±0.21 12.00±0.26 14.00±0.26
Diclofenac sodium 10 2.00±0.36 5.17±0.40 9.17±0.40 15.17±0.47 22.17±0.79 25.67±0.42 27.67±0.42
Indomethacin 10 2.67±0.26 5.17±0.31 9.17±0.31 12.33±0.21 16.00±0.36 20.50±0.43 24.17±0.31
Each value is the Mean±SEM for 6 mice.
4. Discussion
The data presented here suggests that the P. eous possess anti-nociceptive activity. The extract at the doses tested
was shown to possess anti-nociceptive activity in all the nociceptive models, signifying it possess both central and peripherally mediated activities. The abdominal constriction response induced by acetic acid is a sensitive procedure to
evaluate peripherally acting analgesics!16]. In general, acetic acid causes pain by liberating endogenous substances such as serotonin, histamine, prostaglandins (PGs), bradykinins and substance P, which stimulate nerve endings. Local peritoneal receptors are postulated to be involved in the abdominal constrictions response[17]. The method has also been associated with prostanoids in general, that is, increased levels of PGE2 and PGF2 a in peritoneal fluids[18], as well as lipoxygenase products!19,20]. The significant reduction in acetic acid-induced writhes by P. eous suggests that the analgesic effect may be peripherally mediated via the inhibition of synthesis and release of PGs and other endogenous substances. The hot-plate, tail-immersion and tail-clip tests are useful in elucidating centrally mediated antinociceptive responses, which focuses mainly on changes above the spinal cord level[2i,22]. The significant increase in pain threshold produced by P. eous in these models suggests involvement of central pain pathways. Pain is centrally modulated via a number of complex processes including opiate, dopaminergic, descending noradrenergic and serotonergic systems[23-25].The analgesic effect produced by the extract may be via central mechanisms involving these receptor systems or via peripheral mechanisms involved in the inhibition of prostaglandins, leucotrienes, and other endogenous substances that are key players in inflammation and pain[26].
In conclusion, the present study demonstrates that extracts of P. eous has potent analgesic property, which are mediated via peripheral and central inhibitory mechanisms. This could provide a rationale for the use of this mushroom in pain and inflammatory disorders in folk medicine.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
The authors wish to acknowledge Dr. Lu Lu Das, Professor, Dept of Plant Biology, College of Agriculture, Vellayani, Kerala Agricultural University, Trivandrum for identification and authentification of P. eous. The authors are thankful to Mr. Philip Jacob, Research Scholar, Pharmaceutical Chemistry Division, School of Advanced Sciences, VIT University, Vellore, for Statistical analyses. The authors would like to thank the VIT University management for their generous support and facilities.
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